Breast cancer therapies

Abstract

Disclosed herein, in some aspects, are methods and compositions for treating breast cancer. In some aspects, a method comprises administering a combination therapy comprising one or more PARP inhibitors and one or more additional cancer therapies. The methods of the disclosure may be particularly applicable to treat individuals with ESR1-mutant breast cancers. Other aspects relate to methods for providing one or more cancer therapies to an individual having a specific gene signature.

Description

BAYM.P0443WO / BLG 25-012BREAST CANCER THERAPIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 724,685, filed 25 November 2024, the contents of which are hereby incorporated by reference in their entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under R01CA207270 and R01CA072038 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on 17 November 2025, is named BAYMP0443WO_Sequence_Listing.xml and is 2,460 bytes in size.TECHNICAL FIELD AND BACKGROUNDI. Technical Field

[0004] Aspects of this disclosure relate at least to the fields of cancer biology and medicine.II. Background

[0005] Nearly 4 million women currently live with breast cancer in the United States, and up to 13% of women will be diagnosed with breast cancer in their lifetime.1While mortality rates have decreased in recent years, up to 20% of breast cancer will recur or metastasize.1,2For patients with breast cancer expressing the estrogen receptor (ER), one of the leading causes of metastatic progression is the acquisition and enrichment of mutations in the ER gene (ESRI) conferring ligand independence and resistance to ER-targeting endocrine therapy (ET).3’7Genomic surveillance of metastatic breast cancer has revealed the presence of ESRI mutations in up to 50% of ER-positive (ER+) metastatic breast cancer.8Acquisition of these mutations contributes to both decreased therapeutic benefit and reduced overall survival.7,8Though acquisition and enrichment of ESRI mutations is a significant clinical issue in metastatic breast cancer, only one therapeutic option, the selective estrogen receptor degrader (SERD) elacestrant, has been approved specifically for ESRI mutant (ESRlm) metastatic breast cancer.9,10Therefore, identification of additional therapeutic options for the treatment of ESRlm metastatic breast cancer is a top clinical priority.300527252.1 - 1 -BAYM.P0443WO / BLG 25-012SUMMARY

[0006] Aspects of the present disclosure address certain needs by providing at least combination therapies and methods to treat cancer (e.g., breast cancer) useful in the field of cancer biology and medicine.

[0007] In certain aspects, there is a method of treating a cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a poly ADP ribose polymerase (PARP) inhibitor and one or more cancer therapies, wherein cells of the cancer comprise one or more mutations in an estrogen receptor 1 (ESRI) gene. In some aspects, one or more cancer therapies comprises, consists of, consists essentially of, or excludes a checkpoint inhibitor, an endocrine therapy, a growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof. In some aspects, a checkpoint inhibitor comprises, consists of, consists essentially of, or excludes a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof. In some aspects, a checkpoint inhibitor comprises, consists of, consists essentially of, or excludes ribociclib, abemaciclib, palbociclib, PF00477736, PF-477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes an estrogen receptor antagonist, aromatase inhibitor, a selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof. In some aspects, a GFR antagonist comprises, consists of, consists essentially of , or excludes lapatinib. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI-002, EPI-7170, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes masofaniten, bavdegalutamide, or a combination thereof. In some aspects, a translesion inhibitor comprises, consists of, consists essentially of, or excludes JH- RE-06. In some aspects, a SUMOylation inhibitor comprises, consists of, consists essentially of, or excludes TAK981. In some aspects, a PARP inhibitor comprises, consists of, consists essentially of, or excludes Olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof. In some aspects, a PARP inhibitor and one or more cancer therapies may be used at a300527252.1 - 2 -BAYM.P0443WO / BLG 25-012 ratio between 1:1 to 1:100,000, respectively. In some aspects, a PARP inhibitor and one or more cancer therapies may be used at a PARP inhibitor to cancer therapy ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:100000, or any range or value derivable therebetween. In some aspects, a PARP inhibitor and cancer therapy may be used at a ratio between or 1:1 to 100,000:1, respectively. In some aspects, a PARP inhibitor and one or more cancer therapies may be used at a PARP inhibitor to cancer therapy ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000: 1, 70000:1, 80000:1, 90000:1, 100000:1, or any range or value derivable therebetween. In some aspects, a PARP inhibitor may be used at 7.5 to 200 mg / kg. In some aspects, a PARP inhibitor may be used at 7.5, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg / kg, or any range or value derivable therein. In some aspects, one or more cancer therapies may be used at 7.5 to 200 mg / kg. In some aspects, one or more cancer therapies may be used at 7.5, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg / kg, or any range or value derivable therein. In some aspects, a PARP inhibitor comprises, consists of, or consists essentially of olaparib used at 10, 20, 30, 40, 50, 60, 70, 80, 90 mg / kg, or any range or value derivable therebetween, and the cancer therapy comprises, consists of, or consists essentially of fulvestrant used at 160, 170, 180, 190, 200, 210, 220, 230, 240 mg / kg, or any range or value derivable therebetween. In some aspects, a PARP inhibitor comprises, consists of, or consists essentially of olaparib used at 10, 20, 30, 40, 50, 60, 70, 80, 90 mg / kg, or any range or value derivable therebetween, and the cancer therapy comprises, consists of, or consists essentially of PF00477736 used at 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11 mg / kg, or any range or value derivable therebetween. In some aspects, a PARP inhibitor and / or cancer therapy may be comprised in a pharmaceutically acceptable carrier. In some aspects, one or more mutations in the ESRI gene comprises, consists of, consists essentially of, or excludes E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1. In some aspects, one or more mutations in the ESRI gene are homozygous. In some aspects, one or more mutations in the ESRI gene are heterozygous. In some aspects, a cancer comprises functional genomic homologous recombination pathways. In some aspects, a cancer exhibits or excludes enhanced stem-cell activity, enrichment of epithelial-to-mesenchymal300527252.1 - 3 -BAYM.P0443WO / BLG 25-012 transition (EMT) genes, and / or increases in GFR activity. In some aspects, an individual previously received one or more cancer therapies. In some aspects, an individual previously received endocrine therapy, fulvestrant, elacestrant, a CDK4 / 6 inhibitor, an mTOR inhibitor, everolimus, an aromatase inhibitor (Al), or a combination thereof. . In some aspects, an individual previously did not receive one or more cancer therapies. In some aspects, an individual previously did not receive endocrine therapy, fulvestrant, elacestrant, a CDK4 / 6 inhibitor, an mTOR inhibitor, everolimus, an aromatase inhibitor (Al), or a combination thereof. In some aspects, a cancer is resistant to one or more cancer therapies. In some aspects, a cancer comprises, consists of, consists essentially of, or excludes breast cancer (BC), primary BC, and / or metastatic BC. In some aspects, a cancer comprises, consists of, or consists essentially of luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, luminal androgen receptor BC, or a combination thereof.

[0008] In some aspects, there is a method of treating cancer in an individual comprising the steps of: i) identifying a gene expression signature in a sample from the individual comprising: increased expression of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof; and ii) administering an effective amount of one or more cancer therapies to the individual. In some aspects, there is a method comprising: providing a therapeutically effective amount of one or more cancer therapies to an individual in need thereof having, wherein a sample of the individual has been determined to have increased expression levels of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof. In some aspects, there is a method comprising: providing a therapeutically effective amount of one or more cancer therapies to an individual in need thereof having a cancer with an increased gene expression signature. In some aspects, a gene expression signature comprises, consists of, consists essentially of, or excludes increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, or a combination thereof. In some aspects, gene expression signature comprises, consists of, consists essentially of, or excludes increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, MEPH, XPOT, or a combination thereof. In some aspects, a gene expression signature comprises, consists of, consists essentially of, or excludes increased expression of ABCG1, AFF3, ALG8, AMFR, APH1B, ARFGEF2, ASCE2, BBS2, BCAS4, BMP7, BRIP1, CA12, CCDC117, CDC45, CDC6, CD24, CHEK2, CUX1, E2F6, FOXA1, GAB2, GPATCH4, HDAC11, HNRNPAB, IL24, IQGAP3, ITPR1, ITGB5, KLF4, MAN1A2, MCCC2, MLPH, N4BP3, NCOA3, NDUFC2, NFYA, NHP2, NUDT21, OGFOD1, PARD6B, PDE6A, PFKM, PPARGC1B, PREXI, RMND5B, RSPH1, SENP1, SEC25A17,300527252.1 - 4 -BAYM.P0443WO / BLG 25-012SMARCE1, SNX9, SPAG9, SYNJ2, TBK1, TFF1, TFF3, TH, TOBI, TOP2A, UHRF1, UMPS, VDR, WDR35, WDR77, XPOT, ZNF385B, ZNRF3, or a combination thereof. In some aspects, a gene expression signature further comprises, consists of, consists essentially of, or excludes increased expression of PARP1. In some aspects, an increase in gene expression comprises, consists of, consists essentially of, or excludes a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9,12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6,13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3,15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0,17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7,18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0 fold increase, or any range or value derivable therebetween, relative to wild type cells, non-cancerous cells, primary tumor from the individual, or cells without ESRI mutations. In some aspects, a sample comprises, consists of, consists essentially of, or excludes blood, plasma, a primary tumor biopsy, a metastatic tumor biopsy, or a combination thereof. In some aspects, identifying a gene expression signature comprises, consists of, consists essentially of, or excludes measuring RNA and / or protein from the sample. In some aspects, a cancer comprises, consists of, consists essentially of, or excludes BC, primary BC, and / or metastatic BC. In some aspects, a cancer comprises, consists of, consists essentially of, or excludes luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, or luminal androgen receptor BC. In some aspects, a method further comprises, consists of, consists essentially of, or excludes the step of identifying a mutation in the ESRI gene. In some aspects, a mutations in the ESRI gene comprises, consists of, consists essentially of, or excludes E380Q, Y537S, Y537C, Y537N, E536P, E536Q, E536R, D538G, or a combination thereof in reference to SEQ ID NO: 1. In some aspects, a mutation is identified with one or more of the following probes: dHsaMDS732897750 for Y537C, dHsaMDS296069817 for Y537N, dHsaMDS975379796 for Y537S, dHsaMDS460485301 for D538G. In some aspects, one or more cancer therapies comprises, consists of, or consists essentially of a PARP inhibitor, one or more cancer therapies, or a combination thereof. In some aspects, one or more cancer therapies comprises, consists of, consists essentially of, or excludes a checkpoint inhibitor, an endocrine therapy, a300527252.1 - 5 -BAYM.P0443WO / BLG 25-012 growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof. In some aspects, a checkpoint inhibitor comprises, consists of, consists essentially of, or excludes a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof. In some aspects, a checkpoint inhibitor comprises, consists of, consists essentially of, or excludes ribociclib, abemaciclib, palbociclib, PF00477736, PF- 477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes an estrogen receptor antagonist, aromatase inhibitor, an selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof. In some aspects, a GFR antagonist comprises, consists of, consists essentially of, or excludes lapatinib. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI- 002, EPI-7170, or a combination thereof. In some aspects, an endocrine therapy comprises, consists of, consists essentially of, or excludes masofaniten, bavdegalutamide, or a combination thereof. In some aspects, a translesion inhibitor comprises, consists of, consists essentially of, or excludes JH-RE-06. In some aspects, a SUMOylation inhibitor comprises, consists of, consists essentially of, or excludes TAK981. In some aspects, a PARP inhibitor comprises, consists of, consists essentially of, or excludes olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof. In some aspects, a PARP inhibitor is used at 7.5 to 200 mg / kg, or any range or value derivable therein. In some aspects, one or more cancer therapies is used is used at 7.5 to 200 mg / kg, or any range or value derivable therein. In some aspects, a PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 1:100,000, respectively, or any range or value derivable therein. In some aspects, a PARP inhibitor and cancer therapy are used at a ratio between or 1:1 to 100,000:1, respectively, or any range or value derivable therein. In some aspects, one or more cancer therapies comprises olaparib used at 50 mg / kg and fulvestrant used at 200 mg / kg. In some aspects, cancer therapy comprises olaparib used at 50 mg / kg and PF00477736 used at 7.5 mg / kg. In some aspects, a PARP inhibitor and / or cancer therapy are comprised in a pharmaceutically acceptable carrier. In some aspects, one or more cancer therapies comprises, consists of, or consists essentially of tamoxifen if the gene300527252.1 - 6 -BAYM.P0443WO / BLG 25-012 signature comprises increased expression of at least CD24 and / or NC0A3. In some aspects, one or more cancer therapies comprises, consists of, or consists essentially of etoposide, methotrexate, SI- 10, SI- 12, or a combination thereof if the gene signature comprises increased expression of at least NCOA3. In some aspects, one or more cancer therapies comprises, consists of, or consists essentially of prednisone, cytarabine, daunorubicin, doxorubicin or a combination thereof if the gene signature comprises increased expression of at least BMP7. In some aspects, one or more cancer therapies comprises, consists of, or consists essentially of pentoxifylline if the gene signature comprises increased expression of at least PDE6A.

[0009] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0010] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0011] As used herein, “or” is inclusive and not exclusive, unless expressly indicated otherwise or indicated otherwise by context, and can have the same meaning as “and / or.” To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0012] The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), and “include” (and any form of include, such as “includes” and “including”) are open-ended linking verbs. As a result, a composition that “comprises,” “has,” or “includes” one or more elements possesses those one or more elements, but is not limited to possessing only those elements. Likewise, a method that “comprises,” “has,” or “includes” one or more steps possesses those one or more steps, but is not limited to possessing only those one or more steps.

[0013] Any configuration of any of compositions or methods can consist of or consist essentially of - rather than comprise / include / have - any of the described steps, elements, and / or features. Thus, in any of the claims, the term “consisting of’ or “consisting essentially of’ can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb. The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.300527252.1 - 7 -BAYM.P0443WO / BLG 25-012

[0014] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, small molecule, composition, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present. In specific aspects, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered, e.g., through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0015] The terms “subject,” “host,” “individual,” and “patient” are used interchangeably herein to refer to human and veterinary subjects, for example, humans, animals, nonhuman primates, dogs, cats, sheep, mice, horses, and cows. In some aspects, the subject is a human.

[0016] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0017] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1 % to about 5%” should be interpreted to include not just about 0.1 % to about 5%, but also the individual values (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1 % to 0.5%, 1.1 % to 2.2%, 3.3% to 4.4%) within the indicated range.

[0018] “Combination therapy” refers to either a fixed combination in one dosage unit form, or a nonfixed combination (or kit of parts) for the combined administration where a compound and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agent” or “second therapy”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic, effect. The terms “combined administration,” “co-administration,” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a 300527252.1 - 8 -BAYM.P0443WO / BLG 25-012 patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. A combination therapy may be a “fixed combination” meaning that the active ingredients, e.g. a PARP inhibitor and an additional cancer therapy, are both administered to a patient simultaneously in the form of a single entity or dosage. A combination therapy may be a “non-fixed combination” or “kit of parts” meaning that the active ingredients, e.g. a PARP inhibitor and an additional cancer therapy, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.

[0019] “Therapeutically effective” as used herein refers to an amount of a therapeutic agent that is therapeutically or in a broader sense also prophylactically effective against the progression of a disease, such as cancer.

[0020] The term “biomarker” is widespread in the art and commonly broadly denotes a biological molecule, more particularly an endogenous biological molecule, and / or a detectable portion thereof, whose qualitative and / or quantitative evaluation in a tested object (e.g., in or on a cell, cell population, tissue, organ, or organism, e.g., in a biological sample of a subject) is predictive or informative with respect to one or more aspects of the tested object’s phenotype and / or genotype. Biomarkers as intended herein may be nucleic acid-based or peptide-, polypeptide- and / or protein-based. For example, a marker may be comprised of peptide(s), polypeptide(s) and / or protein(s) encoded by a given gene, or of detectable portions thereof. Further, whereas the term “nucleic acid” generally encompasses DNA, RNA and DNA / RNA hybrid molecules, in the context of markers the term may typically refer to heterogeneous nuclear RNA (hnRNA), pre-mRNA, messenger RNA (mRNA), or complementary DNA (cDNA), or detectable portions thereof. Such nucleic acid species are particularly useful as markers, since they contain qualitative and / or quantitative information about the expression of the gene. Particularly preferably, a nucleic acid-based marker may encompass mRNA of a given gene, or cDNA made of the mRNA, or detectable portions thereof. Any such nucleic acid(s), peptide(s), polypeptide(s) and / or protein(s) encoded by or produced from a given gene are encompassed by the term “gene product(s)”.

[0021] As used herein a “signature” or “gene signature” is used to refer to any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is associated with a specific cell type, sub-type, or cell state of a specific cell type or sub-type within a population of cells (e.g., cancer cell subtype or state). A gene signature may be comprised of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,300527252.1 - 9 -BAYM.P0443WO / BLG 25-01223, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or more biomarkers.

[0022] The terms “increased” or “increase” or “upregulated” or “upregulate” as used herein generally mean an increase by a statically significant or practical (e.g., for the purposes of characterization) amount relative to a reference. For avoidance of doubt, “increased” may mean an increase, statistical significant or otherwise, of at least 10% as compared to a reference level, including an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, including, for example at least 1.1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold increase or greater as compared to a reference level, or any value therebetween.

[0023] The term “reduced” or “reduce” or “decrease” or “decreased” or “downregulate” or “downregulated” as used herein generally means a decrease by a statistically significant or practical (e.g., for the purposes of characterization) amount relative to a reference. For avoidance of doubt, “reduced” may mean a decrease, statistically significant or otherwise, of at least 10% as compared to a reference level, for example a decrease by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, at least 90% or more, up to and including a 100% decrease (i.e., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level, or any value therebetween.

[0024] The term “inhibitor” as used herein can refer to an agent, molecule, or compound that decreases, suppresses, or prevents the activity or function of a specific target, such as an enzyme, protein, receptor, or signaling pathway. For example, a PARP inhibitor is a compound that reduces or blocks the enzymatic activity of poly ADP ribose polymerase (PARP). Inhibitors may act through various mechanisms, including direct binding to the target, interference with substrate binding, or modulation of regulatory sites, thereby resulting in a reduction of the biological activity associated with the target.

[0025] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0026] Certain aspects of the present invention are characterized through the following enumerated aspects.

[0027] Aspect 1 is a method of treating a cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a poly ADP 300527252.1 - 10 -BAYM.P0443WO / BLG 25-012 ribose polymerase (PARP) inhibitor and one or more cancer therapies, wherein cells of the cancer comprise one or more mutations in an estrogen receptor 1 (ESRI) gene.

[0028] Aspect 2 is the method of aspect 1, wherein the cancer therapy comprises a checkpoint inhibitor, an endocrine therapy, a growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof.

[0029] Aspect 3 is the method of aspect 2, wherein the checkpoint inhibitor comprises a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof.

[0030] Aspect 4 is the method of aspect 2, wherein the checkpoint inhibitor comprises ribociclib, abemaciclib, palbociclib, PF00477736, PF-477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof.

[0031] Aspect 5 is the method of aspect 2, wherein the endocrine therapy comprises an estrogen receptor antagonist, aromatase inhibitor, a selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof.

[0032] Aspect 6 is the method of aspect 2 or aspect 5, wherein the endocrine therapy comprises elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof.

[0033] Aspect 7 is the method of aspect 2 or aspect 5, wherein the endocrine therapy comprises letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof.

[0034] Aspect 8 is the method of aspect 2, wherein the growth factor receptor (GFR) antagonist comprises lapatinib.

[0035] Aspect 9 is the method of aspect 2 or aspect 5, wherein the endocrine therapy comprises a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI- 506, EPI-002, EPI-7170, or a combination thereof.

[0036] Aspect 10 is the method of aspect 2 or aspect 5, wherein the endocrine therapy comprises masofaniten, bavdegalutamide, or a combination thereof.

[0037] Aspect 11 is the method of aspect 2, wherein the translesion inhibitor comprises JH-RE-06.

[0038] Aspect 12 is the method of aspect 2, wherein the SUMOylation inhibitor comprises TAK981.

[0039] Aspect 13 is the method of any one of aspects 1-12, wherein the PARP inhibitor comprises olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof.300527252.1 - 11 -BAYM.P0443WO / BLG 25-012

[0040] Aspect 14 is the method of any one of aspects 1-13, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 1:100,000, respectively.

[0041] Aspect 15 is the method of any one of aspects 1-13, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 100,000:1, respectively.

[0042] Aspect 16 is the method of any one of aspects 1-15, wherein the PARP inhibitor is used at 7.5 to 200 mg / kg.

[0043] Aspect 17 is the method of any one of aspects 1-16, wherein the cancer therapy is used at 7.5 to 200 mg / kg.

[0044] Aspect 18 is the method of any one of aspects 1-17, wherein the PARP inhibitor comprises olaparib used at 50 mg / kg and the cancer therapy comprises fulvestrant used at 200 mg / kg.

[0045] Aspect 19 is the method of any one of aspects 1-17, wherein the PARP inhibitor comprises olaparib used at 50 mg / kg and the cancer therapy comprises PF00477736 used at 7.5 mg / kg.

[0046] Aspect 20 is the method of any one of aspects 1-19, wherein the PARP inhibitor and / or cancer therapy are comprised in a pharmaceutically acceptable carrier.

[0047] Aspect 21 is the method of any one of aspects 1-20, wherein the one or more mutations in the ESRI gene comprise E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1.

[0048] Aspect 22 is the method of any one of aspects 1-21, wherein the one or more mutations in the ESRI gene are homozygous.

[0049] Aspect 23 is the method of any one of aspects 1-22, wherein the one or more mutations in the ESRI gene are heterozygous.

[0050] Aspect 24 is the method of any one of aspects 1-23, wherein the cancer comprises functional genomic homologous recombination pathways.

[0051] Aspect 25 is the method of any one of aspects 1-24, wherein the cancer exhibits enhanced stem-cell activity, enrichment of epithelial-to-mesenchymal transition (EMT) genes, and / or increases in GFR activity.

[0052] Aspect 26 is the method of any one of aspects 1-25, wherein the individual previously received one or more cancer therapies.

[0053] Aspect 27 is the method of any one of aspects 1-26, wherein the individual previously received fulvestrant, elacestrant, a CDK4 / 6 inhibitor, an mTOR inhibitor, everolimus, an aromatase inhibitor (Al), or a combination thereof.300527252.1 - 12 -BAYM.P0443WO / BLG 25-012

[0054] Aspect 28 is the method of any one of aspects 1-27, wherein the cancer is resistant to one or more cancer therapies.

[0055] Aspect 29 is the method of any one of aspects 1-28, wherein the cancer is breast cancer (BC), primary BC, and / or metastatic BC.

[0056] Aspect 30 is the method of any one of aspects 1-29, wherein the cancer is luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, or luminal androgen receptor BC.

[0057] Aspect 31 is a method of treating cancer in an individual comprising the steps of: i) identifying a gene expression signature in a sample from the individual comprising increased expression of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof; and ii) administering an effective amount of one or more cancer therapies to the individual.

[0058] Aspect 32 is a method comprising providing a therapeutically effective amount of one or more cancer therapies to an individual in need thereof, wherein a sample of the individual has increased expression levels of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof.

[0059] Aspect 33 is the method of aspect 31 or aspect 32, wherein the gene expression signature comprises increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, or a combination thereof.

[0060] Aspect 34 is the method of any one of aspects 31-33, wherein the gene expression signature comprises increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, MLPH, XPOT, or a combination thereof.

[0061] Aspect 35 is the method of any one of aspects 31-34, wherein the gene expression signature comprises increased expression of ABCG1, AFF3, ALG8, AMFR, APH1B, ARFGEF2, ASCL2, BBS2, BCAS4, BMP7, BRIP1, CA12, CCDC117, CDC45, CDC6, CD24, CHEK2, CUX1, E2F6, FOXA1, GAB2, GPATCH4, HDAC11, HNRNPAB, IL24, IQGAP3, ITPR1, ITGB5, KLF4, MAN1A2, MCCC2, MLPH, N4BP3, NCOA3, NDUFC2, NFYA, NHP2, NUDT21, OGFOD1, PARD6B, PDE6A, PFKM, PPARGC1B, PREXI, RMND5B, RSPH1, SENP1, SLC25A17, SMARCE1, SNX9, SPAG9, SYNJ2, TBK1, TFF1, TFF3, TH, TOBI, TOP2A, UHRF1, UMPS, VDR, WDR35, WDR77, XPOT, ZNF385B, ZNRF3, or a combination thereof.

[0062] Aspect 36 is the method of any one of aspects 31-35, wherein the gene expression signature further comprises increased expression of PARP1.300527252.1 - 13 -BAYM.P0443WO / BLG 25-012

[0063] Aspect 37 is the method of any one of aspects 31-36, wherein the increase in gene expression comprises a 1.1-20.0 fold increase relative to wild type cells, non-cancerous cells, primary tumor from the individual, or cells without ESRI mutations.

[0064] Aspect 38 is the method of any one of aspects 31-37, wherein the sample comprises blood, plasma, a primary tumor biopsy, a metastatic tumor biopsy, or a combination thereof.

[0065] Aspect 39 is the method of any one of aspects 31-38, wherein identifying the gene expression signature comprises measuring RNA and / or protein from the sample.

[0066] Aspect 40 is the method of any one of aspects 1-39, wherein the cancer is BC, primary BC, and / or metastatic BC.

[0067] Aspect 41 is the method of any one of aspects 1-40, wherein the cancer is luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, or luminal androgen receptor BC.

[0068] Aspect 42 is the method of any one of aspects 1-41, further comprising the step of identifying a mutation in the ESRI gene.

[0069] Aspect 43 is the method of any one of aspects 1-42, wherein the mutations in the ESRI gene comprise E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1.

[0070] Aspect 44 is the method of aspect 42 or aspect 43, wherein the mutation is identified with one or more of the following probes: dHsaMDS732897750 for Y537C, dHsaMDS296069817 for Y537N, dHsaMDS975379796 for Y537S, dHsaMDS460485301 for D538G.

[0071] Aspect 45 is the method of any one of aspects 31-44, wherein the cancer therapy comprises a PARP inhibitor, one or more cancer therapies, or a combination thereof.

[0072] Aspect 46 is the method of aspect 45, wherein the cancer therapy comprises a checkpoint inhibitor, an endocrine therapy, a growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof.

[0073] Aspect 47 is the method of aspect 46, wherein the checkpoint inhibitor comprises a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof.

[0074] Aspect 48 is the method of aspect 46, wherein the checkpoint inhibitor comprises ribociclib, abemaciclib, palbociclib, PF00477736, PF-477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof.

[0075] Aspect 49 is the method of aspect 46, wherein the endocrine therapy comprises an estrogen receptor antagonist, aromatase inhibitor, a selective androgen receptor degrader300527252.1 - 14 -BAYM.P0443WO / BLG 25-012(SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof.

[0076] Aspect 50 is the method of aspect 46 or aspect 49, wherein the endocrine therapy comprises elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof.

[0077] Aspect 51 is the method of aspect 46 or aspect 49, wherein the endocrine therapy comprises letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof.

[0078] Aspect 52 is the method of aspect 46, wherein the growth factor receptor (GFR) antagonist comprises lapatinib.

[0079] Aspect 53 is the method of aspect 46, wherein the endocrine therapy comprises a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI- 002, EPI-7170, or a combination thereof.

[0080] Aspect 54 is the method of aspect 2 or aspect 5, wherein the endocrine therapy comprises masofaniten, bavdegalutamide, or a combination thereof.

[0081] Aspect 55 is the method of aspect 46, wherein the translesion inhibitor comprises JH-RE-06.

[0082] Aspect 56 is the method of aspect 46, wherein the SUMOylation inhibitor comprises TAK981.

[0083] Aspect 57 is the method of any one of aspects 45-56, wherein the PARP inhibitor comprises olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof.

[0084] Aspect 58 is the method of any one of aspects 45-57, wherein the PARP inhibitor is used at 7.5 to 200 mg / kg.

[0085] Aspect 59 is the method of any one of aspects 31-58, wherein the cancer therapy is used at 7.5 to 200 mg / kg.

[0086] Aspect 60 is the method of any one of aspects 45-59, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 1:100,000, respectively.

[0087] Aspect 61 is the method of any one of aspects 45-59, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 100,000:1, respectively.

[0088] Aspect 62 is the method of any one of aspects 45-61, wherein cancer therapy comprises olaparib used at 50 mg / kg and fulvestrant used at 200 mg / kg.

[0089] Aspect 63 is the method of any one of aspects 45-61, wherein the cancer therapy comprises olaparib used at 50 mg / kg and PF00477736 used at 7.5 mg / kg.

[0090] Aspect 64 is the method of any one of aspects 45-63, wherein the PARP inhibitor and / or cancer therapy are comprised in a pharmaceutically acceptable carrier.300527252.1 - 15 -BAYM.P0443WO / BLG 25-012

[0091] Aspect 65 is the method of any one of aspects 31-64, wherein the cancer therapy comprises tamoxifen if the gene signature comprises increased expression of at least CD24 and / or NCOA3.

[0092] Aspect 66 is the method of any one of aspects 31-65, wherein the cancer therapy comprises etoposide, methotrexate, SI- 10, SI- 12, or a combination thereof if the gene signature comprises increased expression of at least NCOA3.

[0093] Aspect 67 is the method of any one of aspects 31-66, wherein the cancer therapy comprises prednisone, cytarabine, daunorubicin, doxorubicin, or a combination thereof if the gene signature comprises increased expression of at least BMP7.

[0094] Aspect 68 is the method of any one of aspects 31-67, wherein the cancer therapy comprises pentoxifylline if the gene signature comprises increased expression of at least PDE6A.

[0095] It is specifically contemplated that any limitation discussed with respect to one aspect of the invention may apply to any other aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Any aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0096] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better300527252.1 - 16 -BAYM.P0443WO / BLG 25-012 understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0098] FIG. 1A-1J. ESRI mutant cells exhibited defective replication stress response (RSR) and DNA damage response (DDR). FIG. 1A shows a Western blot analysis of cell cycle checkpoint proteins in Y537S and Y537C cell line models. Cells were maintained in charcoal stripped serum media for 48 hours before harvest. Densitometry is indicated under each band and relative to WT cells normalized to loading control (GAPDH). FIG. IB shows a gene set enrichment analysis of defective RSR genes in Y537S and Y537C models. Down and up-regulated gene signatures were determined by fold change (FC)>1.25 and p-value<0.05 in siATR or siChkl models compared to siControls. For each group, from top to bottom, the bars represent “Y537S / WT” and “Y537C / WT.” FIG. 1C shows a Venn diagram of differentially expressed up-regulated genes in siChkl and siATR versus siControl, Y537C versus WT, and Y537S versus WT cells. Differentially expressed genes in Y537S and Y537C models were determined by a step-up p-value<0.05 and FO1 via microarray analysis. siChkl and siATR versus siControl differential expression was determined by p-value<0.05 and fold change>1.25. FIGs. 1D-1E show Kaplan-Meier plots of disease specific survival (DSS) (FIG. ID) and overall survival (OS) (FIG. IE) for the ESRlm defective RSR signature in the top 50% versus bottom 50% of signature expressing ER+ patients from the METABRIC cohort (n=1508 ER+ patients). Low signature is labeled “(i)” and high signature is labeled “(ii)”. FIG. IF shows KEGG pathway enrichment via overrepresentation analysis of the ESRlm defective RSR signature. MMR=mismatch repair, NER=nucleotide excision repair, NHEJ=non- homologous end joining, HR=homologous recombination, BER=base excision repair. For each group, from left to right, the bars represent “All Mutant Overlap,” “Y537S Overlap,” and “Y537C overlap.” FIG. 1G shows molecular signature database (MsigDB) HALLMARK overrepresentation analysis of the ESRlm defective RSR signature. For each group, from left to right, the bars represent “All Mutant Overlap,” “Y537S Overlap,” and “Y537C overlap.” FIG. 1H shows Western blot analysis of DDR genes RAD51 and RPA2 in Y537S and Y537C versus WT cell line models. Cells were grown in charcoal stripped media for 48 hours before harvest. Densitometry is indicated under each band relative to WT cells and normalized to loading control (P-Actin). FIGs. 1I-1J show representative images (FIG. II) and quantitation (FIG. 1 J) of alkaline comet assays of WT, WT+Cisplatin (CP, 10 pM) positive control, Y537S, and Y537C cell lines after 48 hours of estrogen deprivation with charcoal stripped serum media. Significance was determined utilizing two-way ANOVA and a minimum of 150 comet300527252.1 - 17 -BAYM.P0443WO / BLG 25-012 tails per condition. p<0.05 was considered statistically significant for all panels (****p<0.0001).

[0099] FIG. 2A-2G. Therapeutic efficacy of RSR and DDR inhibitors in ESRlm cells. FIG. 2A-2C show MTT growth assays of cells treated with Chkl inhibitor PF00477736 (Chkli) (FIG. 2A), PAPR inhibitor Olaparib (Olap) (FIG. 2B), and Chkli+Olap (FIG. 2C) in WT, Y537S, and Y537C models. Percent survival was normalized to untreated controls. Graphs represent mean+standard deviation with minimum n=4. Statistics comparing % Survival of ESRlm versus WT cells at indicated concentrations are denoted by asterisks in corresponding colors (Y537S=red, Y537C=gold). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 FIG. 2D shows cell cycle quantitation of propidium iodide flow cytometry in WT, Y537S, and Y537C cell lines treated with indicated drugs for 48 hours after estrogen deprivation for 48 hours in charcoal stripped serum media. Percent values were estimated utilizing the FlowJo Cell Cycle Watson univariate algorithm, therefore p-values were not calculated. Graphs represent the average estimated percent between two biological replicates in each condition. For each bar, the groups represented from bottom to top are Gl, S, and G2 / M. FIG. 2E shows percent BrdU uptake analyzed via flow cytometry. Bars represent the average percentage of cells with BrdU uptake relative to total propidium iodide positive cells from two biological replicates. Indicated drug treatments (500 nM Chkli or 500 nM Chkli+5 pM Olap) were administered for 48 hours with hydroxurea (HU) treatment at 0.5 mM administered 24 hours into treatments for an additional 24 hours as indicated. All treatments were performed in charcoal stripped serum media after an initial 48 hours of estrogen deprivation with charcoal stripped serum media. Groups are labeled as follows: i=WT, ii=Y537S, iii=WT + HU, iv=Y537S + HU. Each set of three bars represents a group as denoted above the bars. FIG. 2F shows Western blot analysis of representative in vivo Y537S xenograft tumors for select cell cycle / DNA damage repair and ER-regulated proteins. Densitometry is indicated under each band and relative to lane 1 control (Ctrl) cells normalized to loading control (GAPDH). FIG. 1G shows lung micrometastatic frequency plots as lung foci / mouse with n indicated on the plot for each group. Statistical significance was evaluated utilizing one-way ANOVA corrected for multiple comparisons, *p<0.05.

[0100] FIG. 3A-3G. PARP inhibition reduced ER-regulated gene expression and increased ER-PARP1 genomic interactions in ESRlm cells. FIG. 1A shows Western blot analysis of siPARPl knockdown compared to siControl (siCtrl) in WT and Y537S cell lines with or without 48 hours of Olap (5 pM) treatment after 48 hours of estrogen deprivation in charcoal stripped serum media. Densitometry is indicated under each band and relative to WT 300527252.1 - 18 -BAYM.P0443WO / BLG 25-012 siCtrl cells shown in the left-most column and normalized to loading control (GAPDH). FIG. IB shows Western blot analysis of WT or Y537S cells after 48 hours of estrogen deprivation in charcoal stripped serum media followed by Olap (5 pM) treatment harvested at indicated timepoints in hours. Progesterone receptor A (PR-A; PR in figure) and estrogen receptor (ER) densitometry are relative to untreated controls within each cell line due to differences in protein expression between cell lines. ER protein is represented at different exposures for WT (low exposure) and Y537S (high exposure) cell lines due to cell line differences in ER protein expression to effectively visualize differences relative to treatments. Densitometry is indicated under each band and relative to WT cells with the exception of PR-A and ER. PR-A was not quantitated in WT cells due to low expression and is instead relative to untreated Y537S control. ER was quantitated relative to each cell line’s untreated control. All densitometry was normalized to loading controls (GAPDH). FIG. 3C shows Western blot analysis of WT, Y537S, and Y537C cell lines following 24 hour treatment with Fulvestrant (Fulv; 100 nM), Olap (5 pM) or Fulv+Olap compared to untreated controls. All cells were estrogen deprived with charcoal stripped serum media for 48 hours before treatment. The dashed line separates different protein gels. Densitometry is indicated under each band relative to WT untreated control (Ctrl) cells and normalized to loading control (GAPDH). FIGs. 3D-3G show representative images and quantitation of proximity ligation assays for WT (FIGs. 3D and 3F) or Y537S (FIGs. 3E and 3G) cells. Cells were grown in charcoal stripped media for 5 days followed by 48 hours of indicated treatments of Fulv (100 nM), Olap (5 pM) or Fulv+Olap with untreated cells as control (Ctrl). Graphs represent foci per nucleus from three images per condition. Statistical significance was determined utilizing two-way ANOVA to compare mean foci per nucleus, **p<0.01,****p<0.0001.

[0101] FIG. 4A-4J. ESRlm cells treated with first-line metastatic therapy Fulv plus Abema exhibited defective RSR and DDR and were vulnerable to DDR inhibition. FIG.4A shows a GSEA enrichment plot of the siATR & siChkl defective RSR signature in Fulvestrant+Abemaciclib (F+A) treated cells. Significance was determined by a false discovery rate<0.25 and normalized enrichment score (NES)>1. FIG. 4B shows overrepresentation analysis of selected MSigDB HALLMARK pathways from RNA-seq of Abema and F+A cells compared to WT. 3% Y537S F+A cells were sampled at 8 months of treatment and 30% Y537S cells at 12 months of treatment. Plots represent -logio(p-value) with a significance cutoff of p<0.05. For each group, the bars from left to right represent Abema, F+A (3% Y537S), and F+A (30% Y537S). FIG. 4C shows RNA-seq fold change of Abema and F+A cells compared to WT. All bars represent fold change with a false discovery rate<0.05.300527252.1 - 19 -BAYM.P0443WO / BLG 25-012For each group, the bars from left to right represent Abema, F+A (3% Y537S), and F+A (30% Y537S). FIGs. 4D-4E show representative images (FIG. 4D) and quantitation (FIG. 4E) of alkaline comet assay analysis of Abema and F+A (30% Y537S) cell lines compared to WT untreated and WT+Cisplatin (CP, 10 pM) positive control cell lines. All cells were grown for 48 hours in charcoal stripped serum media before embedding in agarose for subsequent comet assay preparation. Statistical significance was determined utilizing one-way ANOVA corrected for multiple comparisons, ****p<0.0001. FIGs. 4F-4G show MTT growth assays of Chkli+Olap (FIG. 4F) and Fulv+Olap (FIG. 4G) treatment of F+A (30% Y537S) cells versus WT. Plots represent mean+standard deviation at indicated treatments with a minimum of 4 replicates at each point. Individual plot lines represent varying concentrations of Chkli plus Olap (1 pM) (FIG. 4F) or Fulv plus Olap (1 pM, 5 pM, 10 pM) (FIG. 4G) as indicated on the x-axis and legend. Statistics calculated utilizing the student’s t-test are indicated with asterisks at each concentration comparing Chkli monotherapy (FIG. 4F) or Fulv monotherapy (FIG. 4G) %Survival between F+A and WT cell lines. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 FIG. 4H shows Western blot analysis of F+A versus WT cells treated for 12 hours with indicated treatments of Fulv (100 nM), Olap (5 pM) or Fulv+Olap after estrogen deprivation for 48 hours in charcoal stripped serum media. Densitometry is displayed under each band relative to Ctrl WT cells and normalized by loading control (GAPDH). FIG. 41 shows representative proximity ligation assay images of PARP1-ER co-localization in F+A (30% Y537S) cells. Cells were estrogen deprived in charcoal stripped serum media for 5 days followed by 48 hours of indicated treatments of Fulv (100 nM), Olap (5 pM), or Fulv+Olap with untreated cells as control. FIG. 4J shows quantitation of proximity ligation assays represented as foci per nucleus from a minimum of 3 images per condition. Statistical significance was determined utilizing two-way ANOVA to compare mean foci per nucleus, **p<0.01, ***p<0.001, ****p<0.0001.

[0102] FIGs. 5A-5H. PARP1 inhibition in combination with endocrine therapy (ET) was an effective and novel therapeutic strategy in ESRI mutant breast cancer models. FIGs. 5A-5B show MTT growth assay results for Y537S (FIG. 5A) and Y537C (FIG. 5B) cell lines treated with Fulv+Olap compared to Fulv monotherapy in WT cells. Individual lines represent varying concentrations of Olap in combination with Fulv as indicated on the x-axis and legend. Points represent mean+standard deviation of a minimum 4 replicates. Statistical comparisons of %Survival for Fulv monotherapy in ESRlm versus WT cells calculated utilizing two-way ANOVAs are indicated with asterisks, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FIGs. 5C-5D show heat maps of combination index values calculated utilizing 300527252.1 - 20 -BAYM.P0443WO / BLG 25-012CompuSyn software for non-constant drug ratios at indicated concentrations for Fulv+Olap in Y537S cells (FIG. 5C) or Y537C cells (FIG. 5D) corresponding to MTT assays in FIGs. 5A- 5B. FIGs. 5E-5F show organoid counts after 2 weeks of indicated drug treatments (Fulv 1 pM, Olap 5 pM) quantitated via GelCount software for WHIM20 Y537S PDX primary tumor (FIG. 5E) or lung metastasis (FIG. 5F) organoids plated in sextuplicate. Significance was determined via one-way ANOVA corrected for multiple comparisons, **p<0.01. FIG. 5G shows Western blot analysis of representative tumors from the Ctrl and Fulv+Olap WHIM20 in vivo tumors. The dashed line separates different protein gels. Densitometry is indicated under each band and is relative to the left-most column representing Ctrl tumor normalized to P-Actin loading controls. FIG. 5H shows a Kaplan-Meier plot representing time to tumor tripling relative to size at tumor randomization of WHIM20 PDX in vivo primary tumors with indicated treatments. Growth was censored at time of tumor resection if tumor tripling was not achieved indicated by hash marks. P-values were determined utilizing the log-rank test adjusted for multiple comparisons. *p<0.05, **p<0.01.

[0103] FIG. 6 shows longitudinal switching experimental design to mimic clinical metastatic progression to determine the positioning and clinical utility of PARP inhibitor therapy during ET of metastatic breast cancer (MBC).

[0104] FIGs. 7A-7D. Activation of checkpoint and DDR pathways in ESRlm models. FIG. 7A shows Western blot analysis showing activation of pATR and pChkl, and DNA damage markers RAD51 and RPA2. FIG. 7B shows GSEA analysis showing upregulation of Hallmark DNA repair pathway. Y537S NES=1.68; LTED NES=1.71. FIG. 7C shows quantitation of DNA damage markers using immunofluorescent staining. FIG. 7D shows results of Comet assays of cells treated with the specified conditions. ****p<0.0001

[0105] FIGs. 8A-8B. ESRlm transcriptome overlaps with RS signature depleted of Chkl / ATR function. FIG. 8A shows mutant overlap of KEGG pathways. For each group, the bars represent, from left to right, “All Mutant Overlap,” “Y537S Overlap,” and “Y537C Overlap”. FIG. 8B shows probability of survival of patients from the SCAN-B dataset2with mutant gene RS overlap. Top quartile is labeled “(i)” and bottom quartile is labeled “(ii)”.

[0106] FIGs. 9A-9C. ET in combination with the PARP inhibitor olaparib (Olap) enhanced PARP trapping at ER genomic sites. FIG. 9A shows PLA quantitation in MCF7 parental (P) or Y537S ESRlm cell lines. FIG. 9B shows Western blots after PARP1 knockdown or Olap treatment. FIG. 9C shows Western blots of a time course treatment of cells with Olap.300527252.1 - 21 -BAYM.P0443WO / BLG 25-012

[0107] FIGs. 10A-10C. Significant redistribution of AR or ER-AR binding sites in ESRlm cells. FIG. 10A shows overlap with ChromHMM chromatin states in MCF7 P WT vs. MCF-7 Y537S cells. For each bar, the groups from left to right represent heterochromatin, promoters, enhances, and other regions. FIG. 10B shows overlap with ATAC-Seq peaks in matching MCF7 WT and ESRlm models. FIG. 10C shows ATAC-Seq signal (marker of open chromatin) in matching MCF7 WT and ESRlm models.

[0108] FIGs. 11A-11D show that ER-AR co-localize in Y537S (YS1) ESRlm cells at enhancers (FIG. 11 A), but not promoters (FIG. 11B), and are enriched in metastatic patients (FIG. 11C); PARP1 is also preferentially co-bound at ER-AR sites in ESRlm cells (FIG. 11D).

[0109] FIG. 12 shows nuclear colocalization of ER and AR with +E2 using fluorescence imaging. Parental and mutant cells were grown in control (charcoal serum stripped media - E2) for 5 days, and +E2 for 2h.

[0110] FIG. 13 shows ER-PARP1 and AR-PARP1 PLA assays in MCF-7 P and Y537S mutant without or with E2 for 45 minutes.

[0111] FIGs. 14A-14C show co-binding of ER, AR, or PARP on promoter sites of N4BP3 and BMP7 in Y537S model. FIG. 14A shows ER ChIP, FIG. 14B shows AR, and FIG. 14C shows PARP1 binding on the ER-AR co-binding promoter sites in N4BP3 and BMP7. Where indicated, the AR degrader ARV110 was included for 24 hrs3. ** p<.01, ***p<0.001,**** p<0.0001.

[0112] FIGs. 15A-15B. E2 treated Y537S ESRlm AR / ER co-binding sites identified a poor prognostic expression signature. FIG. 15A shows elevated gene expression of signature genes in a cohort of metastatic tumors (GSE12467).1For each condition, the left group represents “Primary” and right group represents “Metastases”. FIG. 15B shows Kaplan-Meier analysis of bone or lung metastases as first site of metastasis (EMC-MSK) with low or high expression signature. In each graph, low signature is labeled “(i)” and high signature is labeled “(ii)”.

[0113] FIGs. 16A-16C. The 8 gene ESRlm signature had prognostic association. FIG. 16A shows, in the TCGA BC cohort ER+ positive tumor samples, that the two signatures had prognostic association with worse survival, p=4xl(F5for 10 genes and p=4xl0-5for 8 genes. Bottom 50% is labeled “(i)” and top 50% is labeled “(ii)”. FIG. 16B shows that both predict 10-year survival using a stringent cross-validation with 100 iterations, in which both gene signatures achieved an AUC of 0.8 or above using kNN (k-nearest neighbor) method; random forest (RF) and Support Vector Machines (SVM) achieved comparable performance. FIG. 16C300527252.1 - 22 -BAYM.P0443WO / BEG 25-012 shows overall survival of patients with overexpressed or underexpressed ESRlm signature from the BostonGene RNA-Seq database of NST ductal cancers. Overexpressed in labeled “(i)” and underexpressed is labeled “(ii)”.

[0114] FIGs. 17A-17B. Olaparib single agent significantly decreased tumor growth and metastasis of mutant models. FIG. 17A shows dissemination of Y53S to the lung, and FIG. 17B shows growth of tumor xenografts grown as ex vivo organoid cultures. *p<0.05, **p<0.01, ***p<0.001.

[0115] FIGs. 18A-18D. ET in combination with the PARP inhibitor olaparib (Olap) enhanced PARP trapping at ER genomic sites. FIGs. 18A-18B show quantitation of PLA assays in parental WT MCF7 P (FIG. 18A) and Y537S ESRlm (FIG. 18B) cells. FIG. 18C shows Western blot analysis of PARP1 and ER levels after PARP1 knockdown or Olap treatment. FIG. 18D shows Western blot analysis of an Olap treatment time course.

[0116] FIGs. 19A-19C. ESRlm transcriptome overlapped with RS signature depleted of Chkl / ATR function. FIG. 19A shows mutant overlap with Hallmark pathways, and FIG. 19B with KEGG pathways. For each group, the bars represent, from left to right, “All Mutant Overlap,” “Y537S Overlap,” and “Y537C Overlap”. FIG. 19C shows overlap of mutant gene RS predicted poor outcomes in a SCAN-B dataset. Bottom 50 percent of samples is labeled “(i)” and top 50 percent of samples is labeled “(ii)”.

[0117] FIG. 20 shows divergent transcriptional profiles between WT and Y537S ESRlm cells. Signature correlations over ER+ samples from the EMC-MSK ER+ primary breast cancer cohort (N=371) showing comparison of patient’s gene expression with YS1 E2 / P E2 and P E2 / P CSS. CSS-charcoal stripped serum devoid of hormones.

[0118] FIGs. 21A-21B shows that mutant (YS1) ER / AR binding was enriched in metastatic tumors (FIG. 21A), and PARP1 binding overlapped at mutant ER- AR sites (FIG.21B).

[0119] FIGs. 22A-22D show overlay of ER / AR mutant binding sites with published ER ChlP-Seq data.

[0120] FIGs. 23A-23D show prognostic significance of the mutant 8 gene signature in Metabric database (FIG. 23A), ILC BostonGene database (FIG. 23B), and correlation with Molecular Grade (FIG. 23C), and TME (FIG. 23D). For FIG. 23A, bottom 25 percent of samples is labeled “(i)” and top 25 percent of samples is labeled “(ii)”. For FIG. 23B, overexpressed is labeled “(i)” and underexpressed is labeled “(ii)”. For FIG. 23C, the bars represent, from bottom to top, Gl-like and G3-like. For FIG. 23D, the bars represent from300527252.1 - 23 -BAYM.P0443WO / BLG 25-012 bottom to top, Fibrotic, Highly Vascularized, Immune Desert, Immune-Enriched Fibrotic, Immune-Enriched Non-fibrotic.

[0121] FIGs. 24A-24D. ChlP-qPCR of co-regulators at ER binding sites on 4 mutant regulated genes. FIG. 24A shows PARP ChIP on N4BP3. FIG. 24B shows PARP ChIP on BMP7. FIG. 24C shows SRC-3 ChIP on BMP7. FIG. 24D shows FOXA1 ChIP on BMP7.

[0122] FIGs. 25A-25B. Olaparib single agent significantly decreased tumor growth and metastasis of mutant models. FIG. 25A shows dissemination of Y53S to the lungs. FIG. 25B shows primary tumor growth treatment of the Y537S WHIM 20 PDX model. *p<0.05, **p<0.01. E2 is labeled “(i),” Olap is labeled “(ii),” Fulv is labeled “(iii),” and Fulv+Olap is labeled “(iv)”.

[0123] FIG. 26 shows that ChlP-SICAP and MS chromatin proteomics identified AR binding ubiquitin enzymes in ETRbreast cancer.

[0124] FIGs. 27A-27C shows NTD, but not LBD, binding AR antagonists inhibited constitutive AR activity on ARE luciferase reporter (FIG. 27A) and spheroid numbers (FIG. 27B) and size (FIG. 27C) of ETRbreast cancer cells.

[0125] FIGs. 28A-28B show that high SUMO inhibits proteasomal targeting K48 ubiquitination of AR in TNBC MDA-MB-231 cells as observed in immunoblots (FIG. 28A) and densitometry (FIG. 28B) from 2 independent biological replicate experiments.

[0126] FIGs. 29A-29D show that high SUMO3 is associated with worse prognosis and has higher expression in AA patients with TNBC. In FIGs. 29A-29B TCGA PanCancer Atlas RNA-Seq datasets were evaluated for SUMO isoforms in 4 groups (FIG. 29A) or SUMO3 in AA with TNBC vs. other subtypes (FIG. 29B). FIG. 29C shows Kaplan-Meier curves for high / low SUMO3 generated with Sabatier TNBC datasets. FIG. 29D shows protein lysates from noncancerous (“MCF10-2A”), 5 HR+ breast cancer, 1 TNBC, and 2 prostate cancer cell lines evaluated for SUMO2 / 3 levels.

[0127] FIG. 30 shows that high SUMO3 increases AR modification in TNBC cells. Protein lysates MCF7 ETRlines (TamR, GH01, GILM2, EDR-7, and YS1) and MDA-MD-231 with and without high SUMO3. Top arrows show modified AR.

[0128] FIGs. 31A-31B show selective degradation of AR populations by AR PROTAC. FIG. 31A shows representative immunoblots of AR protein 24 hours after the indicated treatment; the bottom and upper arrows show unmodified and modified AR, respectively. FIG. 31B shows densitized AR blots from 2 independent experiments that were normalized to GAPDH and subject to nonlinear regression analysis to determine DC50 values (in table).300527252.1 - 24 -BAYM.P0443WO / BLG 25-012

[0129] FIGs. 32A-32B show that AR SUMOylation dictates protein stability and drug response. FIG. 32A shows that AR SUMOylation (arrows) is lost in 3 CRISPR / Cas9 edited sumo-deficient (sd) clones compared to parental ETRTamR7 cells. Equal loading of protein lysates in the input reveals changes in AR protein levels. FIG. 32B shows that ETRbreast cancers cells treated for 24 hours with the indicated drugs or vehicle (labeled “Control”) were evaluated using AR-luciferase reporter assay.

[0130] FIGs. 33A-33C show divergent transcriptional profiles between WT and Y537S ESRI mutant MCF-7 cells. FIG. 33A shows gene set enrichment analysis of (GSEA) in Y537S ESRI mutant (YS1) compared to parental (P) cells in E2 stimulated condition. FIG. 33B shows signature correlations over ER-positive samples from the EMC-MSK ER+ primary breast cancer cohort (N=371). FIG. 33C shows a comparison of patient’s gene expression with YS1 E2 / P E2 and P E2 / P CSS. CSS-charcoal stripped serum devoid of hormones.

[0131] FIG. 34 shows significant redistribution of AR or ER / AR binding sites in ESRI mutant cells. ChlP-Seq data were mapped using Bowtie 26 to the human genome build UCSC hg38. AR only, ER only, and ER-AR peak overlaps were determined using BEDTools.8For each group, the bars represent, from top to bottom, Heterochromatin, Repeat, Repressed, Transcribed, CTCF, CTCF Enhancer, Enhancers, CTCF Promoter, Poised Promoter, and Promoters.

[0132] FIGs. 35A-35D show that ER / AR binding sites co-localized in Y537S (YS1) ESRlm cells at enhancers (FIG. 35A), but not promoters (FIG. 35B), and are enriched in metastatic patients (FIG. 35C). PARP1 was also preferentially co-bound at ER / AR sites (FIG. 35D).

[0133] FIGs. 36A-36B show AR, ER, and PARP1 co-localization determined by coimmunofluorescence (FIG. 36A) or PLA (FIG. 36B). The mutant LTED model is described in1.

[0134] FIGs. 37A-37B shows that the 10 gene mutant gene signature had significant prognostic association. FIG. 37A shows that, in the TCGA BC cohort, ER+ positive tumor samples, the two signatures had prognostic association with worse survival, p=4xl0-5for 10 genes at 50:50 split. FIG. 37B shows that using a stringent cross-validation with 100 iterations, the 10 gene signatures achieve an AUC of 0.8 or above using KNN (k-nearest neighbor) method; random forest (RF) and Support Vector Machines (SVM) achieved comparable performance.

[0135] FIGs. 38A-38C shows Kaplan-Meir analysis of METABRIC cohorts with a 4 gene LAR signature of ER negative by immunohistochemistry (IHC; FIG. 38A), a subset of this300527252.1 - 25 -BAYM.P0443WO / BLG 25-012 cohort classified as LAR (FIG. 38B), and ML classification of LAR DSS (FIG. 38C). In FIG. 38A, bottom 50% of samples is labeled “(i)” and top 50% of samples is labeled “(ii)”. In FIG. 38B, bottom 25% of samples is labeled “(i)” and top 25% of samples is labeled “(ii)”.

[0136] FIG. 39 shows Western blot analysis of PARP1 and AR levels in WT vs Y537S ESR1.

[0137] FIG. 40 shows an MTT growth assay of CAL- 148 cell line treated with olaparib.

[0138] FIG. 41 shows ER / AR co-bound sites overlapped with FOXA1 in LAR MDA-MB-453 cells.

[0139] FIGs. 42A-42B. Confirmation of Y537C mutation in LTED cell line. FIG. 42A shows a scatter plot of individual droplets for Y537C or WT ESRI confirmed by ddPCR. FIG. 42B shows quantitation of ddPCR droplets for Y537C and WT alleles. Positive mutations were detected at a threshold of 3 per 1000 droplets.

[0140] FIG. 43 shows ESRI mutant defective replication stress response signature predictions of overall survival in the SCAN-B cohort. Kaplain-meier curve of the bottom and top quartiles of patients expressing the ESRI mutant defective replication stress response signature in the SCAN-B ER-positive patient cohort. P-values were determined utilizing the log-rank test. Top quartile is labeled “(i)” and bottom quartile is labeled “(ii)”.

[0141] FIGs. 44A-44B. The ESRI mutant defective replication stress response signature did not predict disease specific or overall survival in the METABRIC ERnegative breast cancer cohort. FIG. 44A shows a Kaplan-Meier plot of disease specific survival and FIG. 44B shows a Kaplan-Meier plot of overall survival for the ESRlm defective RSR signature in the top 50% versus bottom 50% of signature expressing ER-negative patients (n=440) from the METABRIC cohort. For each graph, low signature is labeled “(i)” and high signature is labeled “(ii)”.

[0142] FIGs. 45A-45B. GSEA enrichment of the DNA repair MSigDB Hallmark in Y537S and Y537C cells compared to WT MCF-7 cells. Enrichment plots of the DNA repair MSigDB Hallmark for Y537S (FIG. 45A) and Y537C (FIG. 45B) cell lines compared to WT MCF-7 cells.

[0143] FIGs. 46A-46C. Synergy heatmaps of Chkli and Olap treatments of Y537S, Y537C, and WT cell lines. Drug synergy heatmaps of individual synergy calculations at all tested drug ratios represented by CompuSyn calculated combination indices in Y537S (FIG. 46A), Y537C (FIG. 46B), and WT MCF-7 cells (FIG. 46C).

[0144] FIG. 47. Bromodeoxyuridine (BrdU) uptake was attenuated in Y537C cells by Chkl inhibitor (Chkli) alone and in combination with Olap under replication stress300527252.1 - 26 -BAYM.P0443WO / BLG 25-012 conditions. FIG. 47 shows percent BrdU uptake in Y537C cells analyzed via flow cytometry. Bars represent the average percentage of BrdU-positive cells relative to total propidium iodide positive cells with two biological replicates. Cells were treated with 24 hours of Chkli (500 nM) or Chkli+Olap (5 pM) followed by 24 hours of 0.5 mM HU in combination with indicated drug treatments as applicable. All treatments were performed in charcoal stripped media after 48 hours of estrogen deprivation in charcoal stripped media followed by 36 hours of Lovastatin (10 pM) to synchronize cell cycles. Ctrl groups are labeled “(i)” and +HU groups are labeled “(ii)”.

[0145] FIG. 48. Time to tumor doubling of Y537S xenograft primary tumors in vivo. FIG. 48 shows Kaplan-Meier plots representing time to tumor doubling of Y537S cell line xenograft tumors in vivo. Curves were tested for significance utilizing the log-rank test corrected for multiple comparisons. Tumor doubling was measured from time of treatment randomization at approximately 300 mm3and tumors not achieving tumor doubling before tumor resection were censored at times indicated by filled circles. Ctrl is labeled “(i),” Olap is labeled “(ii),” and Olap+Chkli is labeled “(iii)”.

[0146] FIG. 49. Individual growth curves of Y537S xenograft tumors in vivo treated with Olap or Olap+Chkli. Primary tumor growth of individual Y537S xenograft tumors from first measurable tumor volume for Ctrl (FIG. 49A) Olap (FIG. 49B) and Olap+Chkli (FIG. 49C) treatment groups.

[0147] FIG. 50. Proximity ligation assay of ER-PARP1 co-localization in Y537C cells after treatment with Fulv, Olap, or Fulv+Olap. FIG. 50A shows representative images of proximity ligation assays for detecting ER-PARP1 co-localization as fluorescent puncta in Y537C cells treated for 48 hours with Fulv (100 nM), Olap (5 pM), or Fulv+Olap compared to untreated control (Ctrl) following 5 day estrogen deprivation in charcoal stripped media. FIG. 50B shows quantitation of puncta per nucleus for each treatment condition. Individual nuclei were represented as dots with bar and errors representing mean and standard deviation of nuclei from at least 3 representative images. Statistical significance was determined utilizing ANOVA relative to Ctrl with correction for multiple comparisons, **p<0.01, ****p<0.0001.

[0148] FIGs. 51A-51B. Confirmation and enrichment of the Y537S ESRI mutation in F+A cells. FIG. 51A shows scatter plots of individual droplets for Y537S or WT ESRI after indicated lengths of treatment with Fulv+Abema confirmed by ddPCR in F+A cells. FIG. 51B shows quantitation of ddPCR droplets for Y537S and WT alleles. Positive mutations were detected at a threshold of 3 per 1000 droplets.300527252.1 - 27 -BAYM.P0443WO / BLG 25-012

[0149] FIG. 52. Response of F+A cells to Olap monotherapy. FIG. 52 shows results from an MTT growth assay of Olap monotherapy treatment on F+A versus WT cells. Graphs represent mean+standard deviation at indicated concentrations of Olap with a minimum of 4 replicates per condition. Statistics comparing % Survival between F+A and WT cells at each concentration were calculated utilizing the student’s t-test and are represented by asterisks, *p<0.05, **p<0.01, ***p<0.001, *p<0.0001.

[0150] FIGs. 53A-53B. Heatmap of synergy of Olap with Chkli or Fulv in F+A cells. Drug synergy heatmaps of individual synergy values at all tested drug ratios represented by CompuSyn combination indices in F+A cells for Olap+Chkli (FIG. 53A) or Olap+Fulv (FIG. 53B).

[0151] FIG. 54. An Abema cell line exhibited reduced ER-regulated and checkpoint protein expression in response to Fulv+Olap. FIG. 54 shows Western blot analysis of checkpoint and ER-regulated proteins in response to Fulv (100 nM), Olap (5 pM), or Fulv+Olap treatments for 12 hours after 48 hours of estrogen deprivation in charcoal stripped media. Densitometry was normalized to loading control GAPDH and relative to control (Ctrl) conditions labeled under each representative band. PR was not quantified due to low expression in all conditions.

[0152] FIGs. 55A-55D. Individual tumor growth curves of WHIM20 PDX in vivo. Primary tumor growth of individual WHIM20 PDX tumors from first measurable tumor volume groups by -E2 (FIG. 55A), Olap (FIG. 55B), Fulv (FIG. 55C), and Fulv+Olap (FIG. 55D) treatments.

[0153] FIG. 56A shows a western blot analysis of DDR genes RAD51, RPA2 and cell cycle protein Cyclin A2 in Y537S and Y537C versus WT cell line models. Cells were grown in charcoal stripped media for 48 hours before harvest. Densitometry is indicated under each band relative to WT cells after normalization to loading control (GAPDH). GAPDH densitometry is displayed relative to WT cells. FIG. 56B shows quantitation of pH2AX foci in MCF-7 WT, Y537S and Y537C cells (representative images found in FIG. 62). Cells were grown in charcoal stripped media for two days. Dots represent foci per nucleus from four representative images per condition (n=3). Statistical significance was determined utilizing two-way ANOVA to compare mean foci per nucleus, **p<0.01, ****p<0.0001. FIG. 56C shows representative images of neutral comet assays of WT, WT+Cisplatin (CP, 5 pM) positive control, Y537S, and Y537C cell lines after 48 hours of estrogen deprivation with charcoal stripped serum media. Significance was determined utilizing two-way ANOVA and a minimum of 150 comet tails per condition. p<0.05 was considered statistically significant for 300527252.1 - 28 -BAYM.P0443WO / BLG 25-012 all panels (****p<0.0001). FIG. 56D shows quantitation of alkaline comet assays of WT, WT+Cisplatin (CP, 10 pM) positive control, Y537S, and Y537C cell lines after 48 hours of estrogen deprivation with charcoal stripped serum media. Significance was determined utilizing two-way ANOVA and a minimum of 150 comet tails per condition. p<0.05 was considered statistically significant for all panels (****p<0.0001). FIG. 56E shows quantitation of neutral comet assays of WT, WT+Cisplatin (CP, 10 pM) positive control, Y537S, and Y537C cell lines after 48 hours of estrogen deprivation with charcoal stripped serum media. Significance was determined utilizing two-way ANOVA and a minimum of 150 comet tails per condition. p<0.05 was considered statistically significant for all panels (****p<0.0001).

[0154] FIG. 57A shows MTT growth assay of Chkli+Olap at a constant drug ratio of 1:5 respectively in WT, Y537S and Y537C models (concentration range: Olap 0.0625 pM- 16 pM; Chki 12.5 nM- 3200 nM). Percent viability was normalized to untreated controls. Graphs represent mean+standard deviation with minimum n=4 at each concentration. Combination IC50s (WT: 0.38 pM Chki+ 1.53 pM Olap, Y537S: 0.34 pM Chki+ 1.37 pM Olap and Y537C: 0.51 pM Chki+ 2.04 pM Olap) and summary combination index (CI) (Fa=0.5) score were determined using CompuSyn. Statistics comparing %Viability of ESRlm versus WT cells at indicated concentrations are denoted by asterisks in corresponding colors (Y537S=red, Y537C=gold). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FIG. 57B shows drug synergy heatmaps at all tested drug ratios represented by CompuSyn combination indices. FIG. 57C shows cell cycle quantitation of propidium iodide flow cytometry in WT, Y537S and Y537C cell lines treated with 500 nM Chki and indicated Olaparib concentrations, for 48 hours after estrogen deprivation for 48 hours in charcoal stripped serum media. Percent values were estimated utilizing the FlowJo Cell Cycle Watson univariate algorithm. Cells positive for propidium iodide but not called by the Watson algorithm are plotted as undefined. Bars represent average % cells in each phase of the cell cycle + standard deviation (n=2). Significance was calculated between treatment and Ctrl within each cell line for G2 / M phase cells utilizing two-way ANOVA corrected for multiple comparisons **p<0.01, ***p<0.001, ****p<0.0001. FIG. 57D shows percent BrdU uptake analyzed via flow cytometry. Bars represent the average + standard deviation of the percentage of cells with BrdU uptake relative to total propidium iodide positive cells (n=2). Indicated drug treatments (500 nM Chkli or 500 nM Chkli + 5 pM Olap) were administered for 48 hours with hydroxyurea (HU) treatment at 0.5mM administered 24 hours into treatments for an additional 24 hours as indicated. All treatments were performed in charcoal stripped serum media after an initial 48 hours of estrogen deprivation with charcoal stripped serum media.300527252.1 - 29 -BAYM.P0443WO / BLG 25-012

[0155] FIG. 58A shows quantitation of pH2AX foci in MCF-7 WT, Y537S and Y537C cells (representative images found in FIG. 68). Cells were grown in charcoal stripped media for 48 hours, followed by 48 hours of treatment with 10 nM Fulvestrant, 5 pM Olaparib, and combination treatment with both. Dots represent foci per nucleus from four representative images per condition (n=2). Statistical significance was determined utilizing two-way ANOVA to compare mean foci per nucleus, **p<0.01, ****p<0.0001. FIG. 58B shows quantitation of PLA in Y537C cells (representative images found in FIG. 66). Cells were grown in charcoal stripped media for 5 days followed by 48 hours of indicated treatments of Fulv (100 nM), Olap (5 pM) or Fulv+Olap with untreated cells as control (Ctrl). Dots represent foci per nucleus from three representative images per condition. Statistical significance was determined utilizing two-way ANOVA to compare mean foci per nucleus, **p<0.01, ****p<0.0001.

[0156] FIGs. 59A-59D show MTT growth assays and synergy heatmaps of Chkli+Olap (FIGs. 59A-59B) or Fulv+Olap treatment (FIGs. 59C-59D) of F+A (30% Y537S) cells versus WT. Plots represent mean+standard deviation at indicated treatments with a minimum of 4 replicates at each point. Individual plot lines represent varying concentrations of Chkli plus Olap (FIG. 59A) at constant ratio 1:5 (concentration range: Chki 12.5 - 3200 nM, Olap 0.0625 - 16 mM; combination IC50s: WT: 0.38 pM Chki+ 1.53 pM Olap and F+A: 162 nM Chkil+ 648 nM Olap) or Fulv plus Olap (FIG. 59C) at constant ratio 1:250 (concentration range: Fulv 0.25 nM - 64 nM, Olap 0.0625 - 16 mM; combination IC50s: WT: 0.28 nM Fulv + 70.90 nM Olap) nM Fulv + F+A: 9 nM Fulv+ 2.24 pM Olap) as indicated on the x-axis and legend. Statistics calculated utilizing the student’s t-test are indicated with asterisks at each concentration comparing combination treatment Chkli + Olap (FIG. 59A) or single agent Fulv (FIG. 59C) % viability between F+A and WT cell lines. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IC50s for single and combination agents, as well as the summary CI (Fa=0.5) score were determined using CompuSyn. Drug synergy heatmaps represent CompuSyn combination indices at increasing concentrations of the drugs at constant ratios.

[0157] FIG. 60 shows ESRlm defective RSR signature showing prognostic association with disease specific survival (DSS) in the METABRIC ER+ breast cancer cohort. Kaplan- Meier plot of high (top 50%) versus low (bottom 50%) of patients expressing the ESRI mutant defective replication stress response signature in the METABRIC ER+ patient cohort. P-values were determined utilizing the log-rank test.

[0158] FIGs. 61A-61B show that ESRlm defective RSR signature did not predict disease specific or overall survival in the METABRIC ER-negative breast cancer cohort. FIG. 61A shows Kaplan-Meier plots of disease specific survival (DSS) and FIG. 61B shows overall 300527252.1 - 30 -BAYM.P0443WO / BLG 25-012 survival (OS) for the ESRlm defective RSR signature in the top 50% versus bottom 50% of signature expressing ER-negative patients (n=440) from the METABRIC cohort.

[0159] FIG. 62 shows that the ESRlm cell line models exhibited enhanced DNA damage and repair activity. Representative images of immunofluorescent staining of pH2AX foci in WT, Y537S and Y537C after cells were cultured in charcoal stripped medium for four days.

[0160] FIGs. 63A-63B show MTT growth assays of single treatments with Chkli (treated with concentrations ranging from 12.5 nM - 5 pM) and Olap (treated with concentrations ranging from 0.25 pM- 64 pM) in WT, Y537S and Y537C models. Percent viability was normalized to untreated controls. Graphs represent mean + standard deviation with minimum n=4 at each concentration. IC50s were determined using CompuSyn. Statistics comparing % viability of ESRlm versus WT cells at indicated concentrations are denoted by asterisks in corresponding colors (Y537S=red, Y537C=gold). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0161] FIG. 64 shows western blot analysis of cell cycle checkpoint protein Chkl and p- Chkl in Y537S and Y537C cell line models in control (Ctrl) treated vs 24 h treatment with 0.5 and 1 pM Chki (PF00477736). Cells were maintained in charcoal stripped serum media for 48 hours before harvest. Densitometry is indicated under each band and relative to WT cells after normalization to loading control (GAPDH). GAPDH densitometry is displayed relative to WT cells.

[0162] FIG. 65 shows representative gating for flow cytometry analyzing percent BrdU uptake relative to PI in MCF-7 cells analyzed via flow cytometry. All treatments were performed in charcoal stripped media after 48 hours of estrogen deprivation in charcoal stripped media followed by 36 hours of Lovastatin (10 pM) to synchronize cell cycles. Gating for BrdU is shown for both WT and Y537S cells untreated or treated with 0.5 mM Hydroxyurea (HU).

[0163] FIG. 66 shows western blot analysis of cell cycle checkpoint and DDR proteins in Y537S and Y537C cell line models control (Ctrl) vs 24 h treatment with 0.5 mM HU. Cells were maintained in charcoal stripped serum media for 48 hours before harvest. Densitometry is indicated under each band and relative to WT cells after normalization to loading control (GAPDH). GAPDH densitometry is displayed relative to WT cells.

[0164] FIG. 67 shows that lower concentrations of Olaparib treatment inhibited PARylation but did not show effect on estrogen regulated proteins in ESRlm models. Western blot analysis of WT or Y537S cells after 48 hours of estrogen deprivation in charcoal stripped serum media followed by Olap (300 nM, 1 pM, 2 pM) treatment harvested at indicated 300527252.1 - 31 -BAYM.P0443WO / BLG 25-012 timepoints in hours. Densitometry is relative to untreated controls within each cell line due to differences in protein expression between cell lines. Densitometry is indicated under each band and was normalized to lane 1 WT control.

[0165] FIG. 68 shows Fulv plus Olap treatment increased DNA damage in ESRlm. Representative images of immunofluorescent staining of pH2AX foci in WT, Y537S and Y537C after cells were cultured in charcoal stripped medium for 48 hours, followed by treatment with 10 nM Fulvestrant, 5 pM Olaparib, or combination treatment.

[0166] FIGs. 69A-69F show development of FulvR and AbemaR ER+ breast cancer cell line models. FIG. 69A shows MTT survival analysis of FulvR versus parental MCF-7 cells treated with indicated concentrations of Fulv. FIG. 69B shows MTT survival analysis of AbemaR versus parental MCF-7 cells treated with indicated concentrations of Abema. FIG. 69C shows MTT survival analysis of F+A versus parental MCF-7 cells treated with indicated concentrations of Fulv or FIG. 69D Abema. Bars indicate mean+ / - standard deviation of % survival normalized to untreated control at indicated drug concentrations. FIG. 69E shows MTT survival analysis of FulvR versus parental ZR75-1 cells treated with indicated concentrations of Fulv. FIG. 69D shows MTT survival analysis of AbemaR versus parental ZR75-1 cells treated with indicated concentrations of Abema. All MTT analyses were performed in minimum technical quadruplicate at each drug concentration. Significance was determined utilizing 2-way ANOVA comparing long-term treated cells to parental at indicated treatments. *p-val<0.05, **p-val<0.01, ***p-val<0.001, ****p-val<0.0001.

[0167] FIGs. 70A-70F show evaluation of CDK4 / 6 inhibitor cross-resistance in FulvR and AbemaR breast cancer models. FIG. 70A shows MTT survival analysis of FulvR vs parental MCF-7 cells treated with indicated concentrations of Abema and FIG. 70B shows MTT survival analysis of CDK4 / 6 inhibitors ribociclib (1.5 pM), palbociclib (200 nM) or Abema (500 nM). FIG. 70C shows MTT survival analysis of AbemaR versus parental MCF-7 cells; FIG. 70D shows MTT survival of F+A versus parental MCF-7 cells; FIG. 70E shows MTT survival of FulvR versus parental ZR75-1 cells; and FIG. 70F shows MTT survival of AbemaR versus parental ZR75-1 cells treated with ribociclib (1.5 pM), palbociclib (200 nM) or Abema (500 nM). Bars represent mean+standard deviation of % Survival normalized to untreated controls within each cell line. All MTT assays were performed in minimum technical quadruplicate at all concentrations in all cell lines. Cell lines are labeled by biological replicate. Statistical analysis was performed utilizing 2-way ANOVA corrected for multiple comparisons comparing all resistant cell lines to parental controls. *p-val<0.05, **p-val<0.01, ***p- vaKO.001, ****p-val<0.0001.300527252.1 - 32 -BAYM.P0443WO / BLG 25-012

[0168] FIGs. 71A-71L show ddPCR confirmation and enrichment of the Y537S ESRI mutation in F+A cells, and WT ER in AbemaR and FulvR cells. Scatter plot and quantitation of ddPCR droplets for Y537S or WT ESRI after 8 months (FIGs. 71A-71B) or 12 months (FIGs. 71C-71D) of treatment with Fulv+Abema confirmed by ddPCR in F+A cells. FIG. 71E shows a scatter plot and FIG. 71F shows a quantitation of ddPCR droplets for Y537S and WT alleles in the MCF-7 AbemaR cell line after 17 months of treatment. FIG. 71G shows a scatter plot and FIG. 71H shows a quantitation of ddPCR droplets for Y537S and WT alleles in the MCF-7 FulvR cell line after 13 months of treatment. FIG. 711 shows a scatter plot and FIG. 71J shows a quantitation of ddPCR droplets for Y537S and WT alleles in the ZR75-1 AbemaR cell line after 8 months of treatment. FIG. 71K shows a scatter plot and FIG. 71L shows a quantitation of ddPCR droplets for Y537S and WT alleles in the ZR75-1 FulvR cell line after 8 months of treatment. Positive mutations were detected at a threshold of 3 per 1000 droplets.

[0169] FIGs. 72A-72B show Response of ESRlm cells to Olap monotherapy. MTT growth assay of Olap monotherapy treatment on ESRlmversus WT cells. Graphs represent mean+standard deviation at indicated concentrations of Olap (concentrations used 0.25-64 pM) with a minimum of 4 replicates per condition. FIG. 72A shows MTT for single agent Olap treatment comparing %Viability of F+A and WT cells, with stars indicating statistically significant differences. FIG. 72B shows MTT for single agent Olap treatment comparing %Viability of Y537S, Y537C and WT cells, with stars indicating statistically significant differences between WT and Y537S. Statistics comparing %Viability between ESRlmand WT cells at each concentration were calculated utilizing the student’ s t-test and are represented by asterisks, *p<0.05, **p<0.01, ***p<0.001, *p<0.0001.

[0170] FIGs. 73A-73B show that Olap increased chromatin retention of PARP. FIG. 73A shows Western blot analysis showing the chromatin bound fraction of MCF-7 WT, Y537S, Y537C and F+A cells after 48 hours of estrogen deprivation in charcoal stripped serum media followed by 48 hours of combination treatment with 10 nM Fulvestrant and 5 uM Olaparib compared to untreated controls. FIG. 73B shows Western blot analysis showing the chromatin bound fraction of MCF-7 WT, Y537S, Y537C and F+A cells after 48 hours of estrogen deprivation in charcoal stripped serum media followed by 72 hours of treatment with 5 uM Olaparib or 5 uM Veliparib, isolated with the ThermoFisher subcellular protein fractionation kit.300527252.1 - 33 -BAYM.P0443WO / BLG 25-012DETAILED DESCRIPTION

[0171] It is to be understood that the present disclosure is not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0172] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0173] The practice of the present disclosure may employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and / or recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition.300527252.1 - 34 -BAYM.P0443WO / BLG 25-012

[0174] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely illustrative and that equivalents of such are known in the art. It is to be inferred without explicit recitation and unless otherwise intended, that when the present technology relates to a polypeptide, protein, polynucleotide, cell, inhibitor, small molecule, cancer therapy, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.A. Cancer

[0175] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is a Stage I cancer. In some aspects, the cancer is a Stage II cancer. In some aspects, the cancer is a Stage III cancer. In some aspects, the cancer is a Stage IV cancer.

[0176] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous300527252.1 - 35 -BAYM.P0443WO / BLG 25-012 carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0177] In some aspects, the cancer is breast cancer. In some aspects, the cancer is HER2- positive breast cancer. In some aspects, the cancer is HER2-negative breast cancer. In some aspects, the cancer is estrogen receptor(ER)-positive breast cancer. In some aspects, the cancer is ER-negative breast cancer. In some aspects, the cancer is progesterone receptor (PR)-positive 300527252.1 - 36 -BAYM.P0443WO / BLG 25-012 breast cancer. In some aspects, the cancer is PR-negative breast cancer. In some aspects, the cancer is triple negative breast cancer, i.e., lacks detectable expression of PR, ER, and HER2. In some aspects, the cancer is BRCA1 -positive breast cancer. In some aspects, the cancer is BRCA1 -negative breast cancer. In some aspects, the cancer is metastatic breast cancer. In some aspects, the cancer is ESRI mutant breast cancer. In some aspects, the cancer comprises one or more mutations in the ESRI gene, such as, for example Y537S, Y537C, Y537N, L536P, D538G. In some aspects, the cancer is luminal breast cancer, ductal breast cancer, lobular breast cancer, invasive lobular breast cancer, triple-negative breast cancer, and / or luminal androgen receptor breast cancer.

[0178] In some aspects, the cancer is a breast cancer resistant to one or more cancer therapies. A cancer “resistant to one or more cancer therapies” refers to a cancer or tumor that either fails to respond favorably to treatment with prior cancer therapy, or alternatively, recurs or relapses after responding favorably to the therapy. The cancer or tumor may be resistant or refractory at the beginning of treatment or it may become resistant or refractory during treatment. In some aspects, the cancer is a breast cancer resistant to treatment with hormone therapy (e.g., estrogen antagonist or progesterone antagonist). In some aspects, the cancer is a breast cancer resistant to treatment with a kinase inhibitor (e.g., Chkl inhibitor, ATR inhibitor, and / or CDK4 / 6 inhibitor). In some aspects, the cancer is a breast cancer resistant to treatment with a kinase inhibitor and a hormone therapy.B. Gene Signature

[0179] Gene signatures and gene signature biomarkers derived using the methods described herein may be useful to identify cancer patients who are most likely to achieve a clinical benefit from treatment with any of the compositions, combinations therapies, or methods of the disclosure. This utility supports the use of such biomarkers in a variety of research and commercial applications, including but not limited to, clinical trials in which patients are selected on the basis of whether they test positive or negative for a gene signature, diagnostic methods and products for determining a patient’s gene signature score or for classifying a patient as positive or negative for a gene signature, personalized treatment methods which involve tailoring a patient’s drug therapy based on the patient’s gene signature score or status, as well as pharmaceutical compositions and drug products comprising a composition or combination therapy of the disclosure for use in treating patients who test positive for a gene signature.300527252.1 - 37 -BAYM.P0443WO / BLG 25-012

[0180] The utility of any of the research and commercial applications claimed herein does not require that 100% of the patients who test positive for a gene signature achieve an antitumor response based on the administration of a composition, combination therapy, or method of the disclosure; nor does it require a diagnostic method or kit to have a specific degree of specificity or sensitivity in determining the presence or absence of a gene signature in every subject; nor does it require that a diagnostic method claimed herein be 100% accurate in predicting for every subject whether the subject is likely to have a beneficial response to a composition, combination therapy, or method of the disclosure. Thus, the inventors herein intend that the terms “determine”, “determining” and “predicting” should not be interpreted as requiring a definite or certain result; instead these terms should be construed as meaning either that a claimed method provides an accurate result for at least the majority of subjects or that the result or prediction for any given subject is more likely to be correct than incorrect. Preferably, the accuracy of the result provided by a diagnostic method of the invention is one that a skilled artisan or regulatory authority would consider suitable for the particular application in which the method is used, such as, for example treatment of cancer.

[0181] Similarly, the utility of the claimed compositions, combination therapies, and / or methods does not require that the claimed or desired effect is produced in every cancer patient; all that is required is that a clinical practitioner, when applying his or her professional judgment consistent with all applicable norms, decides that the chance of achieving the claimed effect of treating a given patient according to the claimed method or with the claimed composition or combination therapy is more likely to be correct than incorrect.

[0182] A gene signature is determined from a sample, e.g., cancer tissue, removed from a subject. The cancer may be primary or recurrent, and may be of any type (as described above), any stage (e.g., Stage I, II, III, or IV or an equivalent of other staging system), and / or histology. The subject may be of any age, gender, treatment history and / or extent and duration of remission. The sample may be solid tumor tissue. The sample may be circulating cancer cells.

[0183] The cancer sample can be obtained by a variety of procedures including, but not limited to, surgical excision, aspiration, or biopsy. The sample may be sectioned and assayed as a fresh specimen; alternatively, the sample may be frozen for further sectioning. The sample may be preserved by fixing and / or embedding in paraffin or the like. The sample may be lysed and further processed for assaying for a gene signature. The sample may be refrigerated or frozen.300527252.1 - 38 -BAYM.P0443WO / BLG 25-012

[0184] Once a suitable sample of cancer tissue has been obtained, it may be analyzed to quantitate the RNA expression level for each of the genes in Table 1, or for a gene signature derived therefrom, using methods known in the art.Table 1. 65 Gene Signature300527252.1 - 39 -BAYM.P0443WO / BLG 25-012300527252.1 -40-BAYM.P0443WO / BLG 25-012300527252.1 -41 -BAYM.P0443WO / BLG 25-012300527252.1 -42-BAYM.P0443WO / BLG 25-012300527252.1 -43-BAYM.P0443WO / BLG 25-012

[0185] Such methods may include separating, detecting and / or quantifying markers at the nucleic acid level, more particularly RNA level, e.g., at the level of hnRNA, pre-mRNA, mRNA, or cDNA. A person skilled in the art will appreciate that a number of methods can be used to isolate RNA from the tissue sample for analysis. For example, RNA may be isolated from frozen tissue samples by homogenization in guanidinium isothiocyanate and acid phenolchloroform extraction. RNA may be isolated from fresh tissue samples by methods known in the art. Commercial kits are available for isolating RNA from frozen, fixed, or fresh tissue samples.300527252.1 - 44 -BAYM.P0443WO / BLG 25-012

[0186] Persons skilled in the art are also aware of several methods useful for detecting and quantifying the level of RNA transcripts within the isolated RNA, or products thereof, or whole cell lysates, for example. Quantitative detection methods include, but are not limited to, arrays (i.e., microarrays), PCR (e.g., RT-PCR, RE-qPCR, end-point PCR, digital PCR, droplet digital PCR, or the like), multiplex assays, nuclease protection assays, Northern blot analyses, RNA in situ hybridization, and sequencing assays (e.g., pyro sequencing, polony cyclic sequencing by synthesis, simultaneous bi-directional sequencing, single-molecule sequencing, single molecule real time sequencing, true single molecule sequencing, hybridization-assisted nanopore sequencing, sequencing by synthesis, single-cell RNA sequencing (sc-RNA seq), or the like). In some aspects, methods to detect RNA employ labeled probes that are complimentary to a portion of each transcript to be detected. Probes for use in these methods can be readily designed based on the known sequences of the genes and the transcripts expressed thereby. Suitable labels for the probes are well-known and include, e.g., fluorescent, chemilumnescent and radioactive labels.

[0187] In some aspects, a biomarker comprises the estrogen receptor 1 (ESRI) gene. ESRI is also known as ER; ESR; Era; ESRA; ESTRR; NR3A1. ESRI protein reference sequences may comprise one of the following NCBI accession numbers: NP_000116; NP_000116.2; NP_001116212; NP_001116212.1; NP_001116213; NP_001116213.1; NP_001116214; NP_001116214.1; NP_001278159; NP_001278159.1; NP_001278170; NP_001278170.1; NP_001315029; NP_001315029.1; NP_001372497; NP_001372497.1; NP_001372498; NP_001372498.1; NP_001372499; NP_001372499.1; NP_001372500; NP_001372500.1; NP_001372501; or NP_001372501.1. In some aspects, a biomarker comprises a mutant form of ESRI. In some aspects, a cell population, e.g., a cancer biopsy, may comprise about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of cells that have a mutation in an ESRI allele. In some aspects, a cell population, e.g., a cancer biopsy, may comprise a mutated ESRI allele frequency of about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, an ESRI mutation comprises Y537S, Y537C, Y537N, L536P, D538G, or a combination thereof. In some aspects,300527252.1 - 45 -BAYM.P0443WO / BLG 25-012 an ESRI mutation comprises E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof relative to SEQ ID NO: 1. In some aspects, an ESRI mutant is identified by one or more of the following COSMIC mutation entries: COSV52782264 (E380Q), COSV52783938 (Y537S), COSV52782924 (Y537C), COSV52784978 (Y537N), COSV52785937 (L536Q), COSV52787207 (L536R), accessible at https: / / cancer.sanger.ac.uk / cosmic. In some aspects, a probe for detecting a mutation of the ESRI gene comprises probe dHsaMDS732897750 for detecting a Y537C mutation in the ESRI gene, probe dHsaMDS296069817 for detecting a Y537N mutation in the ESRI gene, probe dHsaMDS975379796 for detecting a Y537S mutation in the ESRI gene, and / or probe dHsaMDS460485301 for detecting a D538G mutation in the ESRI gene.SEQ ID NO: 1 - ESRI Polypeptide Sequence:MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEF NAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPH GQQVP YYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCA VCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEV GMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSL TADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTL HDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSI ILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLT LQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNWPLYDLLLEMLDAHRLHAPTSRGGA SVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV ( SEQ ID NO : 1 )

[0188] By way of further example, DNA microarrays can be used to measure gene expression. In brief, a DNA microarray, also referred to as a DNA chip, is a microscopic array of DNA fragments, such as synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (see Schena, BioEssays 18:427 (1996)). Exemplary microarrays and methods for their manufacture and use are set forth in T. R. Hughes et al., Nature Biotechnology 9:342-347 (2001). A number of different microarray configurations and methods for their production are known to those of skill in the art and are disclosed in U.S. Pat. Nos. 5,242,974; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,556,752; 5,405,783; 5,412,087; 5,424,186;5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071;5,571,639; 5,593,839; 5,624,711; 5,700,637; 5,744,305; 5,770,456; 5,770,722; 5,837,832;5,856,101; 5,874,219; 5,885,837; 5,919,523; 6,022,963; 6,077,674; and 6,156,501; Shena, et al., Tibtech 6:301-306, 1998; Duggan, et al., Nat. Genet. 2:10-14, 1999; Bowtell, et al., Nat.300527252.1 - 46 -BAYM.P0443WO / BLG 25-012Genet. 21:25-32, 1999; Lipshutz, et al., Nat. Genet. 21:20-24, 1999; Blanchard, et al., Biosensors and Bioelectronics 77:687-90, 1996; Maskos, et al., Nucleic Acids Res. 2:4663-69, 1993; and Hughes, et al., Nat. Biotechnol. 79:342-347, 2001. Patents describing methods of using arrays in various applications include: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,848,659; and 5,874,219; the disclosures of which are herein incorporated by reference. For example, an array of oligonucleotides targeting mRNA products from gene from Table 1 may be synthesized on a solid support. Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc. Using chip masking technologies and photoprotective chemistry, it is possible to generate ordered arrays of nucleic acid probes. These arrays, which are known, for example, as “DNA chips” or very large scale immobilized polymer arrays (“VLSIPS®” arrays), may include millions of defined probe regions on a substrate having an area of about 1 cm2to several cm2, thereby incorporating from a few to millions of probes (see, e.g., U.S. Pat. No. 5,631,734). To compare expression levels, labeled nucleic acids may be contacted with the array under conditions sufficient for binding between the target nucleic acid and the probe on the array. In one aspect, the hybridization conditions may be selected to provide for the desired level of hybridization specificity; that is, conditions sufficient for hybridization to occur between the labeled nucleic acids and probes on the microarray. The methods described above will result in the production of hybridization patterns of labeled target nucleic acids on the array surface. The resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid. Representative detection means may include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.

[0189] For aspects in which hybridization may be carried out, the conditions permitting essentially specific hybridization may be determined based on the length and GC content of the nucleic acids to be hybridized, i.e., the thermal melting point. These factors are well known to a person of skill in the art, and may also be tested in assays. An extensive guide to nucleic acid hybridization may be found in Tijssen, et al. (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed.; Elsevier, N.Y. (1993)).

[0190] An example assay method to measure transcript abundance for the genes listed in Table 1 includes the nCounter® Analysis System marketed by NanoString® Technologies300527252.1 - 47 -BAYM.P0443WO / BLG 25-012(Seattle, Wash. USA). This system, which is described by Geiss et al., Nature Biotechnol. 2(3):317-325 (2008), utilizes a pair of probes, namely, a capture probe and a reporter probe, each comprising a 35- to 50-base sequence complementary to the transcript to be detected. The capture probe additionally includes a short common sequence coupled to an immobilization tag, e.g. an affinity tag that allows the complex to be immobilized for data collection. The reporter probe additionally includes a detectable signal or label, e.g., is coupled to a color- coded tag. Following hybridization, excess probes are removed from the sample, and hybridized probe / target complexes are aligned and immobilized via the affinity or other tag in a cartridge. The samples are then analyzed, for example using a digital analyzer or other processor adapted for this purpose. Generally, the color-coded tag on each transcript is counted and tabulated for each target transcript to yield the expression level of each transcript in the sample. This system allows measuring the expression of hundreds of unique gene transcripts in a single multiplex assay using capture and reporter probes designed by NanoString.

[0191] In measuring expression of the biomarkers described herein, the absolute expression of each of the biomarkers in a tumor sample may be compared to a control. For example, the control can be the average level of expression of each of the biomarkers, respectively, in a pool of subjects. To increase the sensitivity of the comparison, however, the expression level values are preferably transformed in a number of ways.

[0192] Raw expression values of the clinical response genes in a gene expression platform described herein may be normalized by any of the following: quantile normalization to a common reference distribution, by the mean RNA levels of a set of housekeeping genes, by global normalization relying on percentile, e.g., 75th percentile, or other biologically relevant normalization approaches known to those skilled in the art.

[0193] For example, the expression level of each biomarker can be normalized by the average RNA expression level of all of the biomarkers in a sample, or by the average expression level of a set of normalization biomarkers, e.g., housekeeping genes. Thus, in one aspect, biomarkers may be represented by a set of probes, and the RNA expression level of each of the biomarkers is normalized by the mean or median expression level across all of the represented biomarkers. The normalization may be carried out by dividing the median or mean level of RNA expression of all of the biomarkers in the sample. Alternatively, the RNA expression levels of the biomarkers may be normalized by the mean or median level of expression of a set of normalization biomarkers. The normalization biomarkers may comprise housekeeping genes. The normalization of a measured RNA expression level for a biomarkers may be300527252.1 - 48 -BAYM.P0443WO / BLG 25-012 accomplished by dividing the measured level by the median or mean expression level of the normalization biomarkers.

[0194] The sensitivity of a gene signature score may be increased if the expression levels of individual genes in the gene signature are compared to the expression of the same genes in a pool of samples. Preferably, the comparison is to the mean or median expression level of each signature gene in the pool of samples. This has the effect of accentuating the relative differences in expression between genes in the sample and genes in the pool as a whole, making comparisons more sensitive and more likely to produce meaningful results than the use of absolute expression levels alone. The expression level data may be transformed in any convenient way; preferably, the expression level data for all genes is log transformed before means or medians are taken.

[0195] In performing comparisons to a pool, two approaches may be used. First, the expression levels of the signature genes in the sample may be compared to the expression level of those genes in the pool, where nucleic acid derived from the sample and nucleic acid derived from the pool are analyzed during the course of a single experiment. Such an approach requires that a new pool of nucleic acid be generated for each comparison or limited numbers of comparisons, and is therefore limited by the amount of nucleic acid available. Alternatively, and preferably, the expression levels in a pool, whether normalized and / or transformed or not, are stored on a computer, or on computer-readable media, to be used in comparisons to the individual expression level data from the sample (i.e., single-channel data).

[0196] When comparing a subject’s cancer sample with a standard or control, the expression value of a particular gene in the sample may be compared to the expression value of that gene in a standard or control. For each gene in a gene signature, the log(10) ratio may be created for the expression value in the individual sample relative to the standard or control. Thus, a score for a gene signature may be calculated by determining the mean log(10) ratio of the genes in the signature. If the gene signature score for the test sample is equal to or greater than a pre-determined threshold for that gene signature, then the sample may be considered to be positive for the gene signature biomarker. The pre-determined threshold may also be the mean, median, or a percentile of scores for that gene signature in a collection of samples or a pooled sample used as a standard or control.

[0197] It will be recognized by those skilled in the art that other differential expression values, besides log(10) ratio, may be used for calculating a signature score, as long as the value represents an objective measurement of expression, e.g., transcript abundance, of the genes.300527252.1 - 49 -BAYM.P0443WO / BEG 25-012Examples include, but are not limited to: xdev, error- weighted log (ratio), and mean subtracted log(intensity).

[0198] Each of the steps of obtaining a tissue sample, preparing the tissue sample for assaying gene expression, performing the assay, and scoring the results may be performed by separate individuals at separate locations. For example, a surgeon may obtain by biopsy a tissue sample from a cancer patient’s tumor or cancer and then send the tissue sample to a pathology lab, and a technician in the lab may process the tissue sample for the assay. The samples(s) or processed sample(s) may be assayed soon after preparation, or stored for future assaying. The laboratory that prepared a tissue sample may conduct the assay or send the samples(s) to a different laboratory to conduct the assay. A technician who scores sample(s) for a gene signature may work for the diagnostic lab, or may be an independent contractor. Alternatively, a single diagnostic laboratory may obtain the tissue sample(s) from the subject’s physician or surgeon and then performs all of the steps involved in preparing the sample(s), assaying the sample(s), and calculating the gene signature score for the tissue sample(s).

[0199] In some aspects, the individual(s) involved with preparing and / or assaying the tissue sample(s) for a gene signature do not know the identity of the subject whose sample is being tested, i.e., the sample received by the laboratory is made anonymous in some manner before being sent to the laboratory. For example, the sample(s) may be merely identified by a number or some other code (a “sample ID”) and the results of the assay are reported to the party ordering the test using the sample ID. In preferred aspects, the link between the identity of a subject and the subject's tissue sample is known only to the individual or to the individual's physician.

[0200] In some aspects, after the test results have been obtained, the diagnostic laboratory may generate a test report, which may comprise any one or both of the following results: the tissue sample was positive or negative for one or more biomarkers; and / or the gene signature score for the sample and the reference score for that gene signature. The test report may also include a list of genes whose expression was analyzed in the assay.

[0201] In other aspects, the test report may also include guidance on how to interpret the results for predicting if a subject is likely to respond to one or more cancer therapies, combination therapy, or method of the disclosure. For example, in one aspect, if the tested sample is from an ESRI mutant breast cancer and has a gene signature score that is at or above a prespecified threshold, the test report may indicate that the subject has a score that is associated with response or better response to treatment with a PARP inhibitor in combination with one or more cancer therapies, while if the gene signature score is below the threshold,300527252.1 - 50 -BAYM.P0443WO / BLG 25-012 then the test report may indicate that the patient has a score that is associated with no response or poor response to treatment with a PARP inhibitor in combination with one or more cancer therapies.

[0202] In some aspects, a test report may be a written document prepared by the diagnostic laboratory and sent to the patient or the patient's physician as a hard copy or via electronic mail. In other aspects, the test report may be generated by a computer program and displayed on a video monitor, e.g., at the physician's office. The test report may also comprise an oral transmission of the test results directly to the patient or the patient's physician or an authorized employee in the physician’s office. Similarly, the test report may comprise a record of the test results that the physician makes in the patient’s file.

[0203] Assaying samples for expression of biomarkers or gene signatures described herein may be performed using a kit that has been specially designed for this purpose. In one aspect, the kit comprises a set of oligonucleotide probes capable of hybridizing to one or more set of target transcripts listed in Table 1. In another aspect, the kit comprises a set of oligonucleotide probes capable of hybridizing to the set of target transcripts listed in Table 12. In another aspect, the kit comprises a set of oligonucleotide probes capable of hybridizing to the set of target transcripts listed in Table 11. The set of oligonucleotide probes may comprise an ordered array of oligonucleotides on a solid surface, such as a microchip, silica beads (such as BeadArray technology from Illumina, San Diego, Calif.), or a glass slide (see, e.g., WO 98 / 20020 and WO 98 / 20019). In some aspects, the set of oligonucleotides probes may comprise probes suitable for other assaying methods, e.g., PCR. In some aspects, the oligonucleotide probes are provided in one or more compositions in liquid or dried form. In some aspects, the kit may comprise components for other assaying methods, such as sequencing methods.C. Cancer Therapy

[0204] In some aspects, the disclosed methods comprise administering one or more cancer therapies to a subject, individual, or patient. The cancer therapy may be chosen based on an expression level measurement, alone or in combination with a clinical risk score calculated for the subject. The cancer therapy may be chosen based on a genotype of a subject. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy comprises a systemic cancer therapy. In some aspects, the cancer therapy comprises one or more PARP inhibitors, one or more hormone therapies, one or more checkpoint inhibitors, one or more one or more mTOR inhibitors, one or more chemotherapies, one or300527252.1 - 51 -BAYM.P0443WO / BLG 25-012 more immunotherapies, one or more oncolytic viruses, one or more T-cell engagers, one or more surgeries, one or more radiotherapies, or a combination thereof. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered. a. PARP inhibitors

[0205] Poly(ADP-ribose)polymerase (PARP), previously known as poly(ADP- ribose)synthase and poly(ADP-ribosyl)transferase, is a nuclear and cytoplasmic enzyme that cleaves NAD+ to nicotinamide and ADP-ribose to form long and branched ADP-ribose polymers on target proteins, including topoisomerases, histones, and PARP itself. PARPs constitutes a super family of proteins containing PARP catalytic domains. These proteins include PARP-1, PARP-2, PARP-3, vaultPARP, and TiPARP. PARP-1, for example, consists of an amino (N)-terminal DNA-binding domain (DBD) containing two Zinc fingers; an automodification domain; and a carboxy (C)-terminal catalytic domain. PARP has been implicated in several biological processes, including DNA repair, gene transcription, cell cycle progression (including proliferation and differentiation), cell death, chromatin functions, genomic (e.g., chromosomal) stability and telomere length.

[0206] In some aspects, one or more cancer therapies comprises a PARP inhibitor (PARPi). A PARPi may bind any PARP enzyme (e.g., PARP-1, PARP-2, PARP-3, vaultPARP, and TiPARP), at any location of the PARP enzyme (e.g., N-terminus, DBD, auto-modification domain, or C-terminus). A PARPi may inhibit PARP enzyme activity directly or indirectly. A PARPi may trap a PARP enzyme at an intracellular site, such as DNA, thereby preventing PARP from performing its functions. A PARPi may comprise olaparib, talazoparib (BMN- 673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AGO 14699, BSI-201, CEP-8983, PF00477736, 3-aminobenzamide, AZD5303 (Sanuparib) or a combination thereof. b. Endocrine therapy

[0207] In some aspects, one or more cancer therapies is an endocrine therapy. Various endocrine therapies are known in the art and contemplated herein.

[0208] One type of endocrine therapy is known as aromatase inhibitors. Aromatase inhibitors work by inhibiting the action of the enzyme aromatase, which converts androgens into estrogens by a process called aromatization. As some breast tumor tissue is stimulated by estrogens, decreasing their production is a way of suppressing recurrence of breast cancer. The main source of estrogen is the ovaries in premenopausal women, while in post-menopausal300527252.1 - 52 -BAYM.P0443WO / BLG 25-012 women most of the body’s estrogen is produced in peripheral tissues (outside the CNS), and also a few CNS sites in various regions within the brain. Estrogen is produced and acts locally in these tissues, but any circulating estrogen, which exerts systemic estrogenic effects in men and women, is the result of estrogen escaping local metabolism and spreading to the circulatory system. There are two types of aromatase inhibitors: (1) steroidal inhibitors, such as exemestane (Aromasin) which forms a permanent and deactivating bond with the aromatase enzyme; and (2) non-steroidal inhibitors, such as anastrozole (Arimide or Letrozole (Femara) which inhibit the synthesis of estrogen via reversible competition for the aromatase enzyme.

[0209] Another type of anti-hormonal agent is estrogen receptor (ER) modulators (SERMs) or degraders (SERDs). ERs, whether activated by binding to estrogen, or through mutations that induce constitutive activation, can cause tumors to grow. By inhibit or degrading ER, the cancer may be inhibited from growing and / or metastasizing. Endocrine therapy targeting ER may bind to and block estrogen receptors from binding to a ligand or induce ER degradation to reduce ER signaling in ER-positive cancers. Examples of endocrine therapy targeting ER include, for example, elacestrant, fulvestrant, tamoxifen, toremifene, camizestrant, imlunestrant.

[0210] An endocrine therapy may comprise, consist of, or consist essentially of an estrogen receptor antagonist, aromatase inhibitor, a selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, letrozole, tamoxifen, anastrozole, exemestane, a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI-002, EPI-7170, masofaniten, bavdegalutamide, or a combination thereof. Any of these endocrine therapies may be excluded. Combinations of these endocrine therapies may also be administered. c. Kinase Inhibitors

[0211] Kinase inhibitors are agents that inhibit kinases involved in abnormally activated intracellular signaling to exert an antitumor effect. Examples of such kinase inhibitors include a CDK4 / 6 inhibitor, an mTOR inhibitor, a PI3K inhibitor, an AKT inhibitor, an ERK inhibitor, an MEK inhibitor, an RAF inhibitor, a CDK1 inhibitor, a CDK2 inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, an PLK1 inhibitor, an Aurora kinase inhibitor, a Bcr-Abl inhibitor, an Src inhibitor, an EPH inhibitor, a VEGFR inhibitor, a KIT inhibitor, an RET inhibitor, a PDGFR300527252.1 - 53 -BAYM.P0443WO / BLG 25-012 inhibitor, an FGFR inhibitor, a BTK inhibitor, an FLT3 inhibitor, an ALK inhibitor, a JAK inhibitor, an MET inhibitor, a CSF-1R inhibitor, and an NTRK inhibitor.

[0212] The function of CDKs is to phosphorylate and thus activate or deactivate certain proteins, including e.g. retinoblastoma proteins, lamins, histone Hl, and components of the mitotic spindle. Tumor development is closely associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful anti-cancer therapeutics. Several groups of compounds (reviewed in e.g. Fischer, P. M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623-634) have been found to possess anti-proliferative properties by virtue of CDK- specific ATP antagonism.

[0213] The catalytic step mediated by CDKs involves a phospho-transfer reaction from ATP to the macromolecular enzyme substrate. At a molecular level mediation of CDK / cyclin complex activity requires a series of stimulatory and inhibitory phosphorylation, or dephosphorylation, events. CDK phosphorylation is performed by a group of CDK activating kinases (CAKs) and / or kinases such as Weel, Mytl, and Mikl. Dephosphorylation is performed by phosphatases such as CDC25A, CDC25C, PP2A, or KAP. CDK / cyclin complex activity may be further regulated by two families of endogenous cellular proteinaceous inhibitors: the Kip / Cip family, or the INK family. The INK proteins specifically bind CDK4 and CDK6. pl6ink4 (also known as MTS1) is a potential tumour suppressor gene that is mutated, or deleted, in a large number of primary cancers. The Kip / Cip family contains proteins such as p21 Cipl ,Wafl, p27Kipl and p57kip2, where p21 is induced by p53 and is able to inactivate the CDK2 / cyclin(E / A) complex. Atypically low levels of p27 expression have been observed in breast, colon and prostate cancers. Conversely over expression of cyclin E in solid tumours has been shown to correlate with poor patient prognosis. Over expression of cyclin DI has been associated with oesophageal, breast, squamous, and non-small cell lung carcinomas. The pivotal roles of CDKs, and their associated proteins, in coordinating and driving the cell cycle in proliferating cells has been described. Some of the biochemical pathways in which CDKs play a key role have also been described.

[0214] mTOR is a kinase protein predominantly found in the cytoplasm of the cell. It acts as a central regulator of many biological processes related to cell proliferation, angiogenesis, and cell metabolism. mTOR exerts its effects primarily by modulating the cell’s translational machinery, which includes ribosomes, and is responsible for modulating protein synthesis. mTOR is a key intracellular point of convergence for a number of cellular signaling pathways, which are activated by a variety of growth factors (including vascular endothelial growth factors (VEGFs), platelet-derived growth factor (PDGF), epidermal growth factor (EGF),300527252.1 - 54 -BAYM.P0443WO / BLG 25-012 insulinlike growth factor 1 (IGF-1)), hormones (estrogen, progesterone), and the presence or absence of nutrients (glucose, amino acids) or oxygen. One or more of these signaling pathways may be abnormally activated in patients with many different types of cancer, resulting in deregulated cell proliferation, tumor angiogenesis, and abnormal cell metabolism.

[0215] In some aspects, a kinase inhibitor comprises abemaciclib, palbociclib, ribociclib, trilaciclib, G1T38, PF-06873600, TP-1287, FN-1501, KRX-0601, everolimus, sirolimus, temsirolimus, TAK-228, CC-223, AZD8055, dactolisib, apitolisib, gedatolisib, LY3023414, PF-04691502, NVP-BGT226, PQR309, regorafenib, sorafenib, lenvatinib, nintedanib, axitinib, pazopanib, gefitinib, erlotinib, afatinib, osimertinib, dacomitinib, lapatinib, neratinib, pyrotinib, or poziotinib, or a pharmacologically acceptable salt thereof, or a combination thereof. d. Chemotherapies

[0216] In some aspects, a therapy of the present disclosure comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon- a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0217] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.300527252.1 - 55 -BAYM.P0443WO / BLG 25-012

[0218] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21-day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week.

[0219] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria, is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred in certain cases. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0220] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0221] The amount of the chemotherapeutic agent delivered to a patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutic s of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example,300527252.1 - 56 -BAYM.P0443WO / BLG 25-012 such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples. e. Immunotherapies(1) Immune Checkpoint Inhibitors

[0222] Aspects of the disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy,” “checkpoint blockade therapy,” “immune checkpoint therapy,” “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer.(a) PD-1, PDL1, and PDL2 inhibitors

[0223] PD- 1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0224] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0225] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7- 1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509,300527252.1 - 57 -BAYM.P0443WO / BLG 25-012 and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0226] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO20 11 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0227] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0228] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value between any two therein) variable region amino acid sequence identity with the above-mentioned antibodies.(b) CTLA-4, B7-1, and B7-2300527252.1 - 58 -BAYM.P0443WO / BLG 25-012

[0229] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CTLA4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7- 1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0230] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0231] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W0200 1 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0232] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0233] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1,300527252.1 - 59 -BAYM.P0443WO / BLG 25-012CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value between any two therein) variable region amino acid sequence identity with the above-mentioned antibodies.(c) LAG3

[0234] Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has the Genbank accession number NM_002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+ T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity.

[0235] In some aspects, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0236] Anti-human-LAG3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG3 antibodies can be used. For example, the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as LAG3.1); WO 2016 / 028672 (MK-4280, also known as 28G-10); WO 2017 / 019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11:6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO 2016 / 028672, WO 2017 / 106129, WO 2017062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846,300527252.1 - 60 -BAYM.P0443WO / BLG 25-012WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 220569, WO 2018 / 071500, WO2017 / 015560, WO 2017 / 025498, WO 2017 / 087589, WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271, WO 2017 / 086367, WO2017 / 086419, WO 2018 / 034227, and WO 2014 / 140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.

[0237] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value between any two therein) variable region amino acid sequence identity with the above-mentioned antibodies.(d) TIM-3

[0238] Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782. TIM-3 is found on the surface IFNy- producing CD4+ Thl and CD8+ Tel cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates T-cell exhaustion. TIM-3 has also been shown as a CD4+ Thl -specific cell surface protein that regulates macrophage activation. Inhibitors of the disclosure may block one or more functions of TIM-3 activity.

[0239] In some aspects, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0240] Anti-human-TIM-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the300527252.1 - 61 -BAYM.P0443WO / BLG 25-012 claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015 / 0218274, and US 2016 / 0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 also can be used.

[0241] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies.(2) Activator of co-stimulatory molecules

[0242] In some aspects, the immunotherapy comprises an activator (also “agonist”) of a co-stimulatory molecule. In some aspects, the agonist comprises an agonist of CD3, B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Agonists include activating antibodies, polypeptides, compounds, and nucleic acids.(3) Dendritic cell therapy

[0243] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0244] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0245] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.300527252.1 - 62 -BAYM.P0443WO / BLG 25-012

[0246] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0247] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.(4) Chimeric Immune Receptors (CIR)

[0248] Chimeric immune receptors (CIRs), including Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors), and chimeric T cell receptors (cTCRs; also known as artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of an antigen binding domain, e.g., an antibody, onto a T cell, natural killer (NK) cell, or other immune cell. The receptors are called chimeric because they are fused of parts from different sources. CIR- immune cell therapy, including CAR-T cell therapy and cTCR-T cell therapy, refers to a treatment that uses such transformed cells for cancer therapy, where the transformed cells are immune cells. Similar therapies include, for example, CAR-NK or cTCR-NK cell therapy, which uses engineered NK cells.

[0249] The basic principle of CIR cell design involves recombinant receptors that combine antigen-binding and immune cell activating functions, e.g., T-cell activating functions. The general premise of CIR cells is to artificially generate immune cells targeted to markers found on cancer cells. For example, immune cells can be removed from a patient, e.g., T or NK cells, genetically altered to attack cancer cells, and then infused back into the patient. Once the immune cell has been engineered to become a CIR-immune cell, it acts as a “living drug”. CIR- immune cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates immune cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CIR-immune cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CIR-immune cells is determined by the choice of molecule that is targeted.300527252.1 - 63 -BAYM.P0443WO / BEG 25-012

[0250] Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).

[0251] While CARs are encouraging therapies against cancer, CARs are limited to surface antigens and have limited stimulatory capabilities. TCRs have properties that may help overcome some of the deficiencies of CARs. For example, TCRs can recognize any peptide that is processed for antigen presentation, and TCR signaling is 10 to 100 times more sensitive than CAR signaling (Harris et al., J Immunol. 2018 Feb l;200(3): 1088- 1100.). For at least these reasons, TCR-based immunotherapy is a promising anti-cancer therapy, especially towards solid tumors.

[0252] TCRs are composed of aP (or y5) chain heterodimers that assemble at the cell membrane with the CD3 signaling complex (CD3ey, CD3e6, and CD3 Q. The a and P chains each comprise an extracellular immunoglobulin (Ig)-like domain comprising a variable region that provides antigen binding specificity and a constant domain, a transmembrane domain, and a short cytoplasmic region that lacks intracellular signaling motifs. Upon TCR binding to an antigen, immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 complex undergo phosphorylation and activate a downstream T cell signaling cascade.

[0253] Chimeric TCRs (cTCRs) combine the specificity of antigen binding domains, e.g., antibody scFv, with the ability of TCRs to engage endogenous signaling complexes (e.g., CD3 complex) to activate immune cells, e.g., T cells. cTCRs may comprise various structural configurations. In general, the endogenous variable region of a and / or p chains are replaced with an antigen binding domain of interest (e.g., a scFv targeting a cancer antigen) that is linked directly to the constant region of the a or p chains. In some instances, the entire antigen binding domain may be linked to one or both a and P chains, although the antigen binding domain may also be split amongst the two chains (e.g., an antibody VE chain may be linked to the a-chain while the VH may be linked to the P chain, or vice versa). In some instances, the a and / or p chains may be engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8): 3898- 3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety. The antigen binding domain may also be linked to other components of the TCR complex, such as the y6 chains or the CD3 complex subunits, including the CD3y, CD36, and / or CD3e subunits.300527252.1 - 64 -BAYM.P0443WO / BLG 25-012(5) Adoptive cell therapy

[0254] Adoptive cell therapy is a form of passive immunization by the transfusion of immune cells, such as T cells, NK cells, or other immune cells (also called “adoptive cell transfer”). Immune cells used for adoptive cell therapy include those found in normal tissue and those found in tumor tissue (where they are known as tumor infiltrating immune cells or tumor infiltrating lymphocytes). Although tumor infiltrating immune cells can attack a tumor, the environment within the tumor is generally highly immunosuppressive, preventing immune- mediated tumor death.

[0255] Multiple ways of producing and obtaining tumor targeted immune cells have been developed. Immune cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing may be performed ex vivo, with the results reinfused. Activation can take place through gene therapy, by exposing the immune cells to tumor antigens, or by other methods known in the art. f. Oncolytic virus

[0256] In some aspects, the cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy. One example of oncolytic virus therapy is teserpaturev (Delytact®). g. T-cell Engager (TCE)

[0257] T-cell engagers (TCEs) are chimeric antibodies comprising a target cell binding domain comprising an antigen binding domain such as a single-domain antibody (e.g., a VHH) that specifically binds to an antigen on a target cell, and an immune effector cell binding domain that specifically binds to an antigen on an immune effector cell. In this way, TCEs can redirect T cell to recognize and kill tumor cells. In certain aspects, bispecific antibodies comprising two scFvs targeting CD3 on T cells and a tumor antigen on cancer cells may be used.300527252.1 - 65 -BAYM.P0443WO / BLG 25-012 h. Surgery

[0258] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0259] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0260] Additional cancer therapies

[0261] Therapeutic methods disclosed herein may comprise one or more additional cancer therapies. One or more cancer therapies of the disclosure may comprise, for example, cryoablative therapy, high-intensity ultrasound (also “high-intensity focused ultrasound”), photodynamic therapy, laser ablation, and / or irreversible electroporation. One or more cancer therapies of the disclosure may comprise 1, 2, 3, 4, 5, or more distinct therapeutic methods.

[0262] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, aspects of the present disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the patient is one that has been determined to be resistant to a therapy described herein. In some aspects, the patient is one that has been determined to be sensitive to a therapy described herein. i. Radiotherapy

[0263] In some aspects, a radiotherapy, such as ionizing radiation, is administered to a subject. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A non-limiting example of ionizing radiation is x- radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.300527252.1 - 66 -BAYM.P0443WO / BLG 25-012In some aspects, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some aspects, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some aspects, the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some aspects, the radiotherapy is administered to a primary tumor. In some aspects, the radiotherapy is administered to a metastatic tumor.

[0264] In some aspects, the amount of ionizing radiation is greater than 20 gray (Gy) and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, or is 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range or value between any two therein). In some aspects, the ionizing radiation is administered in at least, at most, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range or value between any two therein). When more than one dose is administered, the dose may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range or value between any two therein.

[0265] In some aspects, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of radiation is at least, at most, or about 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range or value between any two therein). In some aspects, the total dose is administered in fractionated doses of at least, at most, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range or value between any two therein). In some aspects, at least, at most, or is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,300527252.1 - 67 -BAYM.P0443WO / BLG 25-01240,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range or value between any two therein). In some aspects, at least, at most, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range or value between any two therein) fractionated doses are administered per day. In some aspects, at least, at most, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range or value between any two therein) fractionated doses are administered per week.D. Pharmaceutical composition

[0266] In certain aspects, the compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e., to a specific location of the body, such as a tumor) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration.

[0267] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer’s solutions, dextrose solution, Hank’s solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0268] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.300527252.1 - 68 -BAYM.P0443WO / BLG 25-012

[0269] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0270] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0271] In certain aspects, the pharmaceutical compositions are administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0272] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer’s dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0273] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0274] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively,300527252.1 - 69 -BAYM.P0443WO / BLG 25-012 administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0275] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0276] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.E. Combination Therapy

[0277] A therapeutically effective amount of each of the cancer therapies disclosed herein may be administered as a combination therapy. A combination therapy may be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination. For example, the method of preventing or treating a cancer according to the disclosure may comprise: (i) administration of a first cancer therapy in free or pharmaceutically acceptable form; and (ii) administration of a second cancer therapy in free or pharmaceutically acceptable form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in synergistically effective amounts, e.g., in daily or intermittently dosages corresponding to the amounts described herein. The individual combination partners of the combination of the disclosure may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant disclosure is therefore to be understood as embracing all such regimens of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.300527252.1 - 70 -BAYM.P0443WO / BLG 25-012

[0278] The effective dosage of each of combination partner agents employed in the combination of the disclosure may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated, the severity of the condition being treated. Thus, the dosage regimen of the combination of the disclosure is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy requires a regimen based on the kinetics of the drug’s availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

[0279] A benefit of combination therapy, for example, is that lower doses of cancer therapies can be used, e.g., that the dosages need not only often be smaller but are also applied less frequently, or can be used in order to diminish the incidence of side effects. This is in accordance with the desires and requirements of the patients to be treated. The combination of the agents can be combined in the same pharmaceutical preparation or in the form of combined preparations “kit of parts” in the sense that the combination partners can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.

[0280] In some aspects, a combination therapy comprises administration of each cancer therapy in proportion to another. For example, a first cancer therapy and second cancer therapy may be administered at a ratio of 100,000:1, 50,000:1, 25,000:1, 10,000:1, 5,000:1, 2,500:1, 1,000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1,000, 1:2,500, 1:5,000, 1:10,000, 1:25,000, 1:50,000, 1:100,000, respectively. In some aspects, a first cancer therapy is administered at 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 0 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 300527252.1 - 71 -BAYM.P0443WO / BLG 25-012 mg / kg, or any value therebetween, in combination with a second cancer therapy administered at 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 0 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 mg / kg, or any value therebetween. In some aspects, a combination therapy comprises 2, 3, 4, 5, or more cancer therapies administered in combination each at a concentration of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 0 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 mg / kg, or any value therebetween.

[0281] In some aspects, a combination therapy comprising two or more cancer therapies administered concurrently. In some aspects, the combination therapy is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day. In some aspects, the combination therapy is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more hours, days, weeks, months, or years.

[0282] In some aspects, the combination therapy comprises two or more cancer therapies administered separately. In some aspects, the time between administration of each cancer therapy in a combination therapy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more hours, days, weeks, months, or years.EXAMPLES

[0283] The following examples are included to demonstrate preferred aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate300527252.1 - 72 -BAYM.P0443WO / BLG 25-012 that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Methods

[0284] Cell Culture and Maintenance: MCF-7 cells were cultured in MEM (Corning MT10-010-CV) media supplemented with 10% fetal bovine serum (GeminiBio 100-500-500), lOOU / mL penicillin and lOOug / mL streptomycin (GeminiBio 400-109-100), IX MEM non- essential amino acids (Coming MT-25-025-CI), 50ug / mL Gentamicin (ThermoFisher, 15750060), and 5ug / mL Plasmocin Prophylactic (Invivogen, NC9886956). For studies with estrogen reduced conditions, cells were cultured in phenol-free MEM media (Corning, MT 17- 305-CV) supplemented with 5% charcoal stripped fetal bovine serum (GeminiBio 100-119- 500) instead of 10% fetal bovine serum, the additions above, and 200pM L-glutamine (Coming, MT-25-005-C1). Cells were cultured at 5% CO2 in a 37°C incubator. Cells with longterm treatment exposure to Fulv (lOOnM) or Abemaciclib (Abema) (500nM) were treated twice per week. All cells were passaged weekly and tested for mycoplasma every 30-60 days utilizing the MycoAlert Detection Kit (Lonza, LT07-318). Detailed information for dmg sources can be found in TABLE 2.TABLE 2: Drug Sources

[0285] Immunoblot Assays: Cells were harvested utilizing cell scraping on ice and washed with PBS. Cells were lysed for 30 minutes utilizing RIPA buffer (Boston BioProducts, BP- 115) supplemented with 1:100 phosphotase and protease inhibitor cocktail sets I, II, and III (EMD Millipore, 539134, 524625, 524624) followed by centrifugation at 14000rpm for 15 minutes at 4°C. Protein concentrations were determined utilizing the Pierce BCA Protein Assay kit (ThermoFisher 23225) per manufacturer’s instructions. Cell extracts were resolved by electrophoresis in 4-15% polyacrylamide gels (BioRad 5671085) in IX Tris / Glycine / SDS buffer (BioRad 1610772) at 80V for 20 minutes followed by 120V until appropriate sample300527252.1 - 73 -BAYM.P0443WO / BLG 25-012 separation was reached. Proteins were transferred utilizing Trans-Blot Turbo Nitrocellulose Transfer Packs (BioRad 1704159) in IX Tris / Glycine Buffer (BioRad 1610771) at 1A, 25V for 30 minutes. Membranes were stained with Ponceau solution (Sigma, P7170) to confirm protein transfer. Blots were blocked overnight at 4°C in 5% milk in IxTBST solution (Fisher Scientific AAJ77500K8). Antibodies were incubated with membranes overnight in 5% BSA (Sigma, A7030) in IxTBST for phosphorylation antibodies or 5% milk in IxTBST for other antibodies. Membranes were washed with IxTBST and secondary antibodies were incubated for Ihr at room temperature in 5% milk in IxTBST. Membranes were imaged utilizing ChemiGlow reagents (BioTechne PN 60-12596-00) on the BioRad ChemiDoc instrument. Gels re-probed with multiple antibodies (such as phosphorylated and total protein) were stripped after imaging with stripping buffer (Thermo Fisher 21063) followed by 3x 10-minute washes with IxTBST followed by membrane blocking overnight in 5% milk in IxTBST. Primary and secondary antibody incubation for re-probes and imaging were repeated as described above. Antibody details and concentrations can be found in TABLE 3.TABLE 3: Antibody Usage and Sourcing300527252.1 - 74 -BAYM.P0443WO / BLG 25-012

[0286] Microarray Analysis: Microarray analyses were performed as described in Gu et al.12and Dustin et al.11

[0287] Gene Set Enrichment Analysis: Gene set enrichment analysis (GSEA) was performed utilizing the GSEA software from the Broad Institute as described in Dustin et al. with a significance cutoff of FDR<0.25.nGenes in microarray (Y537S and Y537C cell lines)300527252.1 - 75 -BAYM.P0443WO / BLG 25-012 or RNAseq (F+A and Abema cell lines) analyses were sorted by high to low fold change as ranked lists. These ranked lists were analyzed for enrichment of the HALLMARK MSigDB pathways or RSR defect signatures. ATR and Chkl RSR defect signatures were developed utilizing differentially expressed genes (FO1.25 or <-1.25, Pval<0.05) from siRNA knockdown of Ataxia telangiectasia and Rad3 related (ATR), or checkpoint kinase 1 (Chkl) in a hyperplastic cell line, as described in McGrail et al.28

[0288] Survival Analysis: McGrail et al. utilized siRNA to individually knockdown key replication checkpoint proteins ATR, Chkl, Ataxia- telangiectasia mutated (ATM), or checkpoint kinase 2 (chk2) in a hyperplastic cell line.28These authors used the overlap of differentially expressed genes shared between the four knockdown cell lines compared to siCtrl (FO1.25, FDR<0.05) to identify defective RSR. We utilized only the siChkl and siATR differentially expressed genes to develop a defective RSR signature specific to Chkl or ATR knockdown. Shared siATR and siChkl differentially expressed genes were overlapped with differentially expressed genes in Y537S or Y537C cells versus parental WT from microarray analyses (FO1, p-value<0.05). 355 genes exhibited shared differential gene up-regulation in the Y537S and Y537C ESRlm models and siChkl and siATR McGrail et al.28knockdown cell lines. These 355 genes were converted into gene signature scores in patient expression data utilizing the sum of z-scores method as described in Gu et al.12High vs low patient signatures were evaluated utilizing cut points as indicated in figure legends. We assessed disease specific survival and overall survival using the METABRIC dataset29(1508 ER+ samples) and overall survival using the SCAN-B cohort (1747 ER+ samples).30Log-rank p-values were calculated utilizing the “survdiff” command in the “survival” package of R. Kaplan-Meier curves were drawn with the “survfit” command in the same R package. Plots were generated utilizing the ggplot2 package.

[0289] Alkaline Comet Assays: Comet assays (R&D Systems 4250-050) were performed with minor modifications to the manufacturer’ s protocol. Cells were cultured for 48 hours in MEM charcoal stripped serum media. Cells were resuspended at IxlO5cells / mL in charcoal stripped serum media. Cell suspensions were mixed with 1% low melt agarose at a 1:10 ratio and plated on 2-well comet slides Cells were lysed overnight at 4°C in lysis solution. DNA was unwound in alkaline unwinding solution (200mM NaOH, ImM EDTA in distilled water) for 20 minutes at 25°C. Slides were electrophoresed in alkali unwinding solution at 20V for 30 minutes and washed in distilled water and 70% ethanol before drying at 37°C for 30 minutes. DNA was stained with SYBR gold solution according to manufacturer’s directions (Thermo Fisher Scientific, SI 1494). Fluorescence microscopy to quantitate comet tails was performed 300527252.1 - 76 -BAYM.P0443WO / BLG 25-012 at 10X magnification using the Keyence BZ-X800 microscope. Analysis of tail moments was performed using the Comet Assay IV software from Instem with quantitation of at least 150 comet tails per sample. Statistical significance was determined utilizing one-way ANOVA in GraphPad PRISM with a significance cutoff of p-value<0.05.

[0290] MTT Growth Assays and Synergy: Cells were plated at 1000 cells per well in 96 well plates with all treatments in minimum quadruplicate. Cells were treated for 7 days with media and drug replenished every 48 hours. At day 7 cells were treated with MTT solution (1:2 dilution of 1 part 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Thermo Fisher M6494) in PBS to 2 parts cell culture media) for 2 hours followed by DMSO for 20 minutes. Absorbances were read at 570nM and background subtracted from 655nM. Viability was determined relative to no treatment control utilizing GraphPad PRISM software to perform two-way ANOVA with a significance cutoff of p<0.05. Synergy values were calculated utilizing the CompuSyn software for non-constant ratios.

[0291] Cell Cycle Analysis: Cells were starved for 48 hours before treatment with lOpM Lovastatin for 36 hours. Media was replaced after Lovastatin treatment and cells were treated with additional drugs for 48 hours (PF00477736 (500nM, Chkli) or Olap (IpM or 5pM). Cells were harvested via trypsinization and washed twice with IX PBS. Cells were manually single cell suspended by repeat pipetting in 70% ethanol overnight for fixation. Approximately IxlO6cells per condition were pelleted and washed with IX PBS to remove ethanol after fixation. Cells were resuspended in Propidium Iodide staining solution (0.1% Triton X-100 (Sigma 93443), 0.2mg / mL DNA-free RNase A (Invitrogen 12091021), 0.02mg / mL Propidium Iodide (Molecular Probes P4864) in IX PBS) for 15 minutes protected from light. Propidium iodide positive cells were analyzed utilizing the BD FACS CantoIIYG flow cytometer with 10,000 events per condition. Cell cycle analysis was performed utilizing FlowJo software (BD BioSciences).

[0292] siRNA Knockdown: Transfections with siRNA were performed as directed by the manufacturer (ThermoFisher). Cells were starved for 48 hours utilizing charcoal stripped serum media, and transfections were performed for an additional 48 hours with the following siRNAs as indicated (Silencer Select Negative Control No.l 4390844 ThermoFisher (50nM) or PARP1 sl099 Silencer Select siRNA (25nM) 4390824 Thermo Scientific plus PARP1 sl097 Silence Select siRNA (25nM) 4390824 Thermo Fisher) and Lipofectamine RNAiMAX (Invitrogen 13778). Cells treated with Olaparib (Olap) (5pM) received treatment halfway through transfection at 24 hours. Cells were harvested at the end of the 48 hour transfection for downstream analysis via immunoblot assay.300527252.1 - 77 -BAYM.P0443WO / BLG 25-012

[0293] Digital Droplet Polymerase Chain Reaction: ddPCR was performed utilizing the BioRad platform as described in Dustin et al.11Probes IDs were as follows, with 3 / 1000 droplets defined as positive detection of ESRI mutations: Y537C dHsaMDS732897750, Y537N dHsaMDS296069817, Y537S dHsaMDS975379796, D538G dHsaMDS460485301.

[0294] In Vivo Tumor Growth and Metastasis Experiments: Xenograft tumor methods for the WHIM20 and MCF-7 CRISPR Y537S in vivo experiments were performed as described in detail in Gu et al.12Removal of estrogen supplementation and treatment randomization was performed at 200mm3for WHIM20 experiments and 350mm3for CRISPR Y537S experiments. WHIM20 experimental treatment specifications: Olap 50mg / kg 5x / week via oral gavage, Fulvestrant 200mg / kg Ix / week via subcutaneous injection. MCF-7 CRISPR Y537S xenograft treatment specifications: Olap 50mg / kg 5x / week via oral gavage, Chkl inhibitor (Chkli) PF004777367.5mg / kg 2x / week via i.p. injection. Tumor resections were performed at 800mm3and mice were harvested when exhibiting signs of moribund behavior or 6 months postresection.

[0295] Statistical analysis of metastases was conducted by comparing lung foci / mouse between treatment groups utilizing two-way ANOVA. Primary tumor growth of all xenograft experiments was compared utilizing Kaplan-Meier analysis in the PRISM software (GraphPad) with growth determined relative to volume at time of randomization.

[0296] RNA-Sequencing: Library preparation and RN A- sequencing analysis was conducted by the Baylor College of Medicine Genomic and RNA Profiling Core (GARP) using the Illumina NovaSeq 6000 platform. Paired-end reads were trimmed using TrimGalore and mapped to the UCSC hg38 genome build using HISAT2.31Aligned reads were counted against the Gencode gene model annotation32to obtain expression values by using FeatureCounts.33Differential gene expression was evaluated using the R package edgeR34, with TMM (trimmed means of m values) normalization. Significance was achieved with an adjusted p-value of <0.05.

[0297] Over-representation Analysis: Overrepresentation analysis was performed to detect enrichment of gene sets corresponding to pathways and biological processes based on differential expressed genes (DEGs). Pathway enrichment was performed with DEGs from ESRlm models (fold change>1.5, p-value<0.05) using Hallmark and KEGG compendia from MSigDB (v7.5.1) and the Molecular Signature Database methodology (MSigDB).35A hypergeometric test was used to assess the enrichment with significance achieved at an adjusted p-value<0.05.300527252.1 - 78 -BAYM.P0443WO / BLG 25-012

[0298] BrdU Assays: Cells were treated with indicated drugs for 48 hours, with hydroxyurea treatment to induce replication stress introduced 24 hours into relevant drug conditions. After treatment, cells were washed lx with PBS before incubation with 20pM BrdU (BD Pharmigen 550891) for 2 hours at 37 °C. Cells were washed with PBS, trypsinized, and fixed in 70% ethanol in PBS and stored at -20 °C until analysis. Approximately 1.5xl06cells were collected per sample and resuspended in 2M HC1 (Sigma 1.09063) denaturing solution for 20 minutes at room temperature. Cells were washed with 0.5% bovine serum albumin solution in PBS before incubation in 0.1M sodium tetraborate (pH 8.5) (ThermoFisher J62902.AP) for 2 minutes at room temperature. Cells were again washed in 0.5% bovine serum albumin solution in PBS. Cells were incubated with FITC anti-BrdU antibody (BioLegend, 364104) for 1 hour protected from light. Cells were again washed then labeled with propidium iodide as described above. Cells were resuspended in lx PBS and analyzed utilizing the BD FACS CantoIIYG flow cytometer with 10,000 events per condition. Cells were gated for BrdU and propidium iodide staining and counted utilizing the FlowJo software (BD BioSciences).

[0299] Proximity Ligation Assays: Cells were plated in 10cm cell culture dishes and treated with 5% charcoal stripped serum media for 5 days. Cells were trypsinized and plated in Ibidi IBItreated 8 well glass slides (Ibidi 80826) at 20K cells per well in 5% charcoal stripped serum media and treated with indicated drugs for 48 hours. Cells were washed with PBS and fixed in 4% paraformaldehyde solution (Fisher Scientific 043368.9M, diluted in ultrapure H2O) for 10 minutes at room temperature. Fixed cells were washed with PBS 3x followed by 10 minutes in permeabilization solution (0.2% triton-lOOx, 0.2% bovine serum albumin in PBS). Permeabilized cells were washed with PBS and blocked with 1% bovine serum albumin in PBS for 30 minutes. Primary antibodies (ER6F11 1:100, PARP1 ActiveMotif, 1:100) were incubated overnight at 4°C diluted in 1% bovine serum albumin in PBS. Cells were washed 3x with PBS and incubated with 1:5 diluted PLA probes (Duolink In Situ PLA Probes anti-rabbit DU092002 and anti-mouse DU092004) in 1% bovine serum PBS for 1 hour at 37°C. Ligation and amplification steps were performed per manufacturer instructions (Wash buffers: DUO82049, Duolink In Situ Detection Reagents Green: DUO 92014). Slides were rinsed with PBS and cells stained with 300nM DAPI (ThermoFisher DI 306) in PBS for 10 minutes. Slides were washed in PBS 2x and covered with Vectashield Mounting Medium (Vector Laboratories H- 1000- 10) for long-term storage.

[0300] PLA were imaged utilizing the Olympus 1X83 epifluorescence deconvolution microscope with the 40x dry objective for quantitation. Quantitation was performed utilizing CellProfiler36in consultation with the Baylor College of Medicine Integrated Microscopy Core.300527252.1 - 79 -BAYM.P0443WO / BLG 25-012Graphs are depicted as foci / nucleus from representative images. Statistical significance was determined via one-way ANOVA relative to 5% charcoal stripped serum media control.

[0301] Organoid Growth Assays: Organoids were plated, treated, and imaged as described in Dustin et al.11.

[0302] Statistical Analysis: Statistical tests are noted in above relevant methods sections. Significance is set at p<0.05 unless otherwise noted. Replicates of samples are indicated in relevant figure legends.Example 2 - Targeting the DNA damage response as a therapeutic vulnerability in ESRI mutant breast cancer

[0303] Abstract: ESRI mutations are the leading cause of endocrine therapy resistance and progression in ER-positive metastatic breast cancer. ESRI mutations are detected in up to -50% of metastatic breast cancer patients, and identification of effective targeted therapeutics are critically needed. The inventors identified enrichment of dysregulated replication stress and DNA damage responses in multiple ESRI mutant models. Targeting the replication stress response utilizing checkpoint inhibition in combination with PARP inhibition was synergistic and resulted in cell cycle arrest and attenuation of DNA replication. PARP inhibition blocked metastatic development in vivo and reduced both PARP and ER-regulated protein expression. PARP trapping with olaparib treatment with or without endocrine therapy resulted in a significant increase of co-localized DNA-bound PARP and ER protein in ESRI mutant cells, indicating ER-PARP1 co-regulation in ESRI mutant breast cancer. The inventors observed acquisition of the Y537S ESRI mutation in a cell line long-term treated with endocrine therapy plus the CDK4 / 6 inhibitor abemaciclib. This acquired ESRI mutant model also exhibited dysregulation of replication stress and enhanced DNA damage as well as synergistic responses to inhibitors of these pathways. PARP inhibition was synergistic with endocrine therapy in ESRI mutant models and resulted in reduced tumor growth both ex vivo and in vivo. Our results identify replication stress and DNA damage responses as key dysregulated pathways in ESRI mutant breast cancer with significant clinical potential for PARP inhibition in this subset of ER-positive breast cancer without genomic homologous recombination deficiency.

[0304] Statement of Significance: ESRI mutant breast cancer exhibited enriched replication stress and DNA damage creating a therapeutic vulnerability to PARP inhibition in ER+ breast cancer without genomic homologous recombination defects.

[0305] Introduction: While therapeutics specifically targeting mutant ERs have yet to be approved, extensive pre-clinical investigation has identified a variety of potentially targetable phenotypes associated with ESRlm acquisition. Acquired phenotypes include enhanced stem-300527252.1 - 80 -BAYM.P0443WO / BLG 25-012 cell activity, enrichment of the epithelial to mesenchymal transition (EMT), and increases in growth factor receptor (GFR) expression.11 15Unfortunately, while evaluation of GFR targeting drugs is underway in ER+ metastatic breast cancer11 16 17, the other phenotypes lack effective targeted therapeutics to date. Therefore, the inventors sought to identify a unique “druggable phenotype” with a variety of therapeutic options. In this example, the inventors identified an enrichment of the replication stress response (RSR) and DNA damage response (DDR) pathways controlled by poly ADP ribose polymerase 1 (PARP1) in multiple ESRlm models. The RSR induces cell cycle checkpoints and recruits DDR proteins to ensure effective DNA replication and facilitate DNA repair through activation of ataxia telangiectasia and Rad3 related (ATR) and checkpoint kinase 1 (Chkl) proteins.18 19PARP1 is a critical protein for the effective repair of DNA strand breaks, and fidelity of DNA replication.20,21PARP inhibitors are synthetic lethal in breast cancer with defective DNA repair such as breast cancer gene (BRCA) mutations.22The clinical efficacy of disrupting the DDR with PARP inhibitors has led to significant efforts in the clinical development of cell cycle checkpoint inhibitors of ATR and Chkl to target the RSR.23-25Cancer is highly proliferative which can lead to genomic instability and accumulation of DNA damage26,27. Herein, the inventors proposed that the constitutively active and highly proliferative ESRlm breast cancers are a unique subset of breast cancer that exhibit a novel sensitivity to both PARP and cell cycle checkpoint inhibitors due to defective RSR and enriched DDR.

[0306] The inventors identified significant enrichment of defective RSR in ESRlm models resulting in elevated intrinsic DNA damage. Inhibition of the RSR and DDR utilizing Chkl and PARP inhibitors was synergistic in cell line models and significantly reduced the progression of ESRlm metastatic lesions. PARP inhibition in combination with the SERD fulvestrant (Fulv) reduced primary tumor growth in ESRI mutant models and was synergistic in ESRlm cell lines. Thus, defective RSR in ESRlm breast cancer was found to drive both metastatic and primary tumor growth, and inhibition of the RSR and downstream DDR utilizing Chkl and PARP inhibitors was found to be an effective therapeutic strategy.

[0307] ESRI mutant cells exhibited defective RSR and DDR: The inventors previously developed two ESRlm cell lines, a CRISPR / Cas9 MCF-7 cell line expressing the homozygous ligand binding domain activating Y537S ESRlm (clone YS1), and a long-term estrogen deprived (LTED) MCF-7 cell line with spontaneously acquired ESRI mutations Y537C and Y537N.11,12Extended culture of the LTED cell line resulted in an outgrowth of the Y537C ESRlm at an allele frequency of 50% (FIG. 43). The CRISPR Y537S and LTED Y537C cell lines exhibited both enhanced metastatic potential and up-regulation of epithelial-300527252.1 - 81 -BAYM.P0443WO / BLG 25-012 mesenchymal transition (EMT), growth factor and proliferative cell cycle-driven pathways.11 12These cell lines were also estrogen independent, and exhibited constitutive ER-regulated activity and hyperproliferation compared to WT ER cells.11 12

[0308] The inventors first investigated activation of the RSR in the ESRlm models due to their hyperproliferative phenotype. RSR activation is a critical pathway for maintaining DNA integrity and preventing lethal genomic instability in response to enhanced proliferation,26’27 37Key RSR proteins ATR and Chkl initiate cell cycle checkpoints to facilitate critical DNA repairs, particularly at single strand DNA breaks.18ATR and Chkl exhibited enhanced phosphorylation and total protein levels in the Y537S and Y537C ESRlm cell lines (FIG. 1A). While the RSR canonically induces cellular senescence, defective RSR can enhance tumor proliferation and promote tumorigenesis in cancer models.28Thus, the inventors next evaluated the ESRlm cell lines for a defective RSR phenotype. The inventors investigated a defective RSR signature driven by ATR or Chkl knockdown in a hyperplastic cell line as described in Example 2. ATR and Chkl defective RSR phenotypes were enriched in the ESRlm models using gene set enrichment analysis (GSEA) (FIG. IB, TABLE 4). These results suggested that ESRlm cells activate defective RSR genes to tolerate their intrinsic replication stress induced by hyperproliferation.300527252.1 - 82 -BAYM.P0443WO / BLG 25-012TABLE 4: GSEA results in ESRlm models of differentially expressed siATR and siChkl signatures300527252.1 - 83 -BAYM.P0443WO / BLG 25-012

[0309] To investigate the clinical impact of defective RSR in ESRlm breast cancer, the inventors developed a prognostic gene signature from up-regulated differential gene expression shared between four models: the Chkl and ATR knockdown cell lines compared to siControl from McGrail et al28and the Y537S and Y537C ESRlm models compared to WT cells (FIG. 1C, TABLE 5). The 355 gene signature developed from up-regulated gene expression shared between these four models was named the ESRlm defective RSR signature. The inventors utilized the METABRIC ER+ breast cancer cohort29to evaluate survival outcomes of the ESRlm defective RSR signature in primary breast cancer patient tumors. Primary tumor enrichment of the ESRlm defective RSR signature was associated with both worse disease specific survival and overall survival in the METABRIC cohort (FIG. 1D-1E, respectively, p<0.0001 and FIG. 60, p=0.00569). The signature was validated using the SCAN-B ER+ primary breast cancer cohort,30signature enrichment in this cohort predicted significantly reduced overall survival (FIG. 44, p=0.03). The ESRlm defective RSR signature was not prognostic in the METABRIC ER-negative cohort (FIG. 45 and FIG. 61), though therapeutics targeting the RSR and DDR are currently utilized in ER-negative tumors with homologous recombination deficiency, especially BRCA-mutant patients. Therefore, the ESRlm defective RSR signature must require ER-signaling. Thus, the ESRlm defective RSR signature developed by the inventors was prognostic and identified defective RSR activation as a potential pathway influenced by ER-signaling and driving poor survival outcomes in ER+ breast cancer.TABLE 5: ESRlm Replication Stress Response Defect Signature Genes300527252.1 - 84 -BAYM.P0443WO / BLG 25-012TABLE 5: Cont.300527252.1 -85-BAYM.P0443WO / BLG 25-012300527252.1 - 86-BAYM.P0443WO / BLG 25-012

[0310] The inventors utilized overrepresentation analysis of the ESRlm defective RSR genes to identify specific DNA repair and replication pathways driving defective RSR in mutant models. The top enriched KEGG pathway was cell cycle (FIG. IF, TABLE 6) followed by DNA replication, mismatch repair, nucleotide excision repair, non-homologous end joining, homologous recombination, and base excision repair pathways (FIG. IF). Overrepresentation analysis of MSigDB HALLMARK pathways included enrichment of DNA repair, G2M Checkpoint, and E2F Targets (FIG. 1G, TABLE 6). Thus, it was found that defective RSR significantly impacts numerous DNA repair and replication pathways in ESRlm breast cancer. These results identified a reliance on DNA repair pathways to tolerate replication stress in ESRlm models.TABLE 6: Over-representation analysis enrichment of MSigDB KEGG and HALLMARK pathways in the ESRlm RSR defect signature300527252.1 - 87 -BAYM.P0443WO / BLG 25-012TABLE 6 cont.300527252.1 -88-BAYM.P0443WO / BLG 25-012300527252.1 - 89-BAYM.P0443WO / BLG 25-012

[0311] Disruption of the RSR significantly impacts DNA repair and can lead to downstream accumulation of DNA damage resulting in cell death.18 26 27Therefore, the inventors next evaluated activation of key DNA repair proteins, including replication protein A2 (RPA2) and RAD51 recombinase (RAD51), which promote single strand38and double strand39break repair, respectively and are both essential for DNA repair pathways addressing single stranded and double stranded breaks. Since the protein level of these DNA repair proteins can be increased during DNA replication, the inventors also evaluated the cell cycle protein Cyclin A2 which is enriched in S- and G2-M-phase. RAD51 exhibited increased protein expression levels in Y537S and Y537C cell lines (FIG. 56A), and RPA2 was elevated in the Y537C cell line indicating that ESRlm cells exhibit enhanced DNA repair activation at the protein level (FIG. 1H). The inventors discovered significantly higher levels of intrinsic DNA damage in both Y537S and Y537C ESRlm cell lines at comparable levels to WT cells treated with the DNA intercalating chemotherapeutic agent cisplatin (FIGs. 1I-1J, p<0.0001). These data taken together identified significant activation of DNA repair pathways driven by defective RSR. The inventors reasoned that activation of RSR and DDR pathways in ESRlm breast cancer may be essential to repair high levels of intrinsic DNA damage and promote tumor survival. These results suggested that Cyclin A2 protein was also elevated in the mutant cells which is consistent with the hyperproliferation phenotype previously reported for ESRlm and could contribute to elevated RAD51 protein levels.

[0312] The inventors further assessed DNA damage and repair activation in the ESRlm cells using quantification of pH2AX foci and found significantly elevated pH2AX foci in nuclei of both Y537S and Y537C cells. Elevated pH2AX foci are a known marker for both induction of DNA ds-breaks and activation of DNA repair (FIG. 56B, FIG. 62).43The inventors found significantly higher levels of endogenous DNA damage in mutant cells using alkaline comet assays for the detection of both ss- and ds-DNA breaks (FIGs. II and 56D, p<0.0001), and neutral comet assays that reliably identify ds-DNA breaks and are insensitive to replication intermediates (FIGs. 56C and 56E, p>0.0001).44Treatment with the DNA intercalating chemotherapeutic agent cisplatin was used as positive controls. These results together demonstrate significant activation of DNA repair pathways driven by defective RSR in mutant cells. Activation of RSR and DDR pathways may be essential to repair the high levels of intrinsic DNA damage present in ESRlm cells to promote tumor survival.

[0313] Defective RSR and DDR pathways revealed new therapeutic vulnerabilities in ESRlm breast cancer: The inventors investigated the therapeutic potential of Chkl and PARP inhibitors in ESRlm breast cancer. The inventors found a significant transcriptional increase in 300527252.1 - 90 -BAYM.P0443WO / BLG 25-012PARP1 expression in the Y537S and Y537C cell lines (Table 7). PARP1 facilitates DNA repair through multiple pathways21making it an ideal target for the DNA repair pathways enriched in the ESRlm defective RSR phenotype. ESRlm cells were significantly less sensitive to single agent Chkli with higher IC50s for both Y537S (3.8pM) and Y537C (905nM) cell lines compared to WT (75nM) (FIG. 2A). The Y537S ESRlm cell line was also significantly less sensitive to single agent treatment with the PARP inhibitor Olap compared to WT cells (FIG. 2B). Combination treatment with both Chkli and Olap, however, was synergistic resulting in substantial cell death at Chkli and Olap concentrations below single agent IC50s (FIG. 2C, FIG. 46). Inhibition of PARP has been reported to enhance cellular reliance on the RSR pathway to facilitate DNA damage repair, particularly for single strand breaks.37,41Thus, enrichment of defective RSR in ESRlm cells may enhance DNA repair, and underly the reduced efficacy of PARP inhibitors as single agent therapeutics. Inhibition of Chkl and PARP as a combination therapy may prohibit the compensatory activity of the RSR, resulting in significant cytotoxicity.

[0314] PARP1 facilitates DNA repair through multiple pathways making it an ideal target with the activated RSR and DNA repair phenotype in ESRlm cells. Therefore, the inventors investigated the therapeutic potential of Chkl and PARP inhibitors in combination, and found that Chkli treatment was synergistic when used with the PARP inhibitor Olap, especially at the higher clinically relevant doses in ESRlm breast cancer, resulting in substantially decreased cell viability at Chkli and Olap concentrations below single agent IC50s in ESRlm cells (Combination treatment shown in FIGs. 57A-57B, single agents in FIG. 63). ESRlm cells were significantly less sensitive to single agent Chkli but demonstrated increased sensitivity to Olap treatment compared to WT cells (FIG. 63). Chkl inhibition was evaluated at the concentrations used by demonstrating a decrease in Chkl protein levels, and an increase in p- Chkl, both of which are used as pharmacodynamic markers of Chkl inhibition (FIG. 64). These results indicated that inhibition of both Chkl and PARP in combination blocks compensatory RSR DNA repair activity in mutant cells resulting in enhanced toxicity.Table 7: PARP1 Expression in ESRlm models300527252.1 - 91 -BAYM.P0443WO / BLG 25-012

[0315] The inventors evaluated cell cycle and DNA replication dynamics to interrogate effects of Chkl and PARP inhibitors in ESRlm breast cancer. Cells accumulated in the G2 / M phase of the cell cycle after Chkli treatment as a single agent and in combination with Olap indicating potential premature mitotic entry37,43in ESRlm cells after Chkl inhibition (FIG. 2D and FIG. 57C). ESRlm cells also exhibited reduced BrdU uptake after Chkli single agent and after combination treatment with Olap (FIG. 2E, FIG. 47, Table 8, FIG. 57D, and FIG. 65). Mutant cells also tolerated higher levels of RS induced with Hydroxyurea (HU) treatment resulting in high levels of DNA replication and increased DDR protein RAD51 (FIG. 2E, FIG. 57D, FIG. 66). This DNA replication was greatly reduced with addition of Chkli as a single agent and in combination with Olap, but not in WT cells (FIG. 57D) suggesting that both Chkl and PARP activities are necessary to maintain high levels of DNA replication induced by RE in ESRlm cells (FIG. 2E). Therefore, ESRlm cells were tolerant to HU induction of replication stress, but Chkl and PARP activity were necessary to maintain these high levels of DNA replication. Disruption of DNA replication and cell cycle progression significantly reduce DNA repair efficacy and induce DNA damage44, identifying a potential mechanism for the synergistic cytotoxicity of Chkli and Olap combination therapy in ESRI models.

[0316] Since Chkli and Olap treatments induced cell death and disrupted both cell cycle and DNA replication in ESRlm models, the inventors evaluated the effects of these agents on in vivo tumor growth and metastatic progression using Y537S tumor xenograft models. Olap treatment reduced levels of multiple ER-regulated proteins including progesterone receptor (PR), trefoil factor 1 (TFF1), and the proto-oncogene c-Myc in addition to DDR proteins (H2AX, PARP, cleaved PARP (c-PARP))45,46and RSR proteins (ATR and Chkl) in Y537S primary tumors (Figure 2F). Olap plus Chkli increased time to primary tumor doubling (Control 5.5 weeks, Olap plus Chkli 8 weeks median time to tumor doubling), though it was not statistically significant (FIG. 48, FIG. 49). The inventors discovered a significant reduction of lung metastases after Olap treatment as a single agent or combination with Chkli (FIG. 2G, p<0.05). These results identified PARP as a key regulator of ESRlm cell and tumor growth with significant impacts on ER-regulated gene expression. More importantly, the inventors observed significant effects on metastatic progression to the lung with Olap treatment preventing micrometastatic formation in the lungs, identifying PARP as a potential driver of ESRlm metastasis.Table 8: BrdU Cell Cycle Quantification300527252.1 - 92 -BAYM.P0443WO / BLG 25-012

[0317] PARP inhibition reduced ER-regulated gene expression and increased genomic ER- PARP interactions in ESRlm cells: PARP inhibition significantly reduced ER-regulated protein levels in ESRlm tumors. Therefore, the inventors used siRNA to knockdown PARP1 and evaluate estrogen-regulated gene expression in the ESRlm models. PARP1 knockdown reduced expression of PR-B and ER (FIG. 3A). This result identified PARP as a key protein required for the expression of ER. PR- A and ER were reduced with Olap treatment both with and without PARP1 knockdown. Thus, abrogation of PARP activity with Olap resulted in reduction of both ER protein and ER-regulated protein expression. The primary function of300527252.1 - 93 -BAYM.P0443WO / BLG 25-012PARP is PARylation of proteins, facilitating protein recruitment for effective DNA repair and replication.21The inventors also investigated endogenous levels of PARylation in Y537S cells and identified a more than 5-fold increase confirming significant activation of DNA repair (FIG. 3B) . Olap treatment reduced levels of both PARP 1 and PR proteins in Y537 S cells (FIG. 3B). c-Myc was reduced at a later time point (FIG. 3B). In contrast, in WT ER-expressing cells reduction of these proteins was not sustained for long periods of treatment. Thus, PARP inhibition exhibited a prolonged and greater reduction of ER-regulated protein expression in mutant cells. While treatment with Olap inhibited PARylation at 300 nM (FIG. 67), reduction of estrogen-regulated proteins was observed only at higher concentrations (FIG. 3B, 5uM), that have been used to inhibit growth of various cancer models.

[0318] Next, the inventors evaluated the efficacy of Olap in combination with the SERD Fulv as a novel clinically relevant therapeutic strategy in ESRlm metastatic breast cancer. Fulv plus Olap treatment significantly reduced PR and ER levels in both mutant models (FIG. 3C). Olap treatment as a single agent also decreased PR levels, but only in Y537S cells not the Y537C model with co-expressed WT ER. Olap treatment induced higher levels of phosphorylated and total Chkl proteins in all models. PARP inhibition has been reported to enhance RSR activation to promote DNA repair37,41Thus, defective RSR may function as a compensatory pathway in ESRlm cells to promote effective DNA repair and replication. Fulv blocked Chkl activation (FIG. 3C). Fulv is the preferred ET for ESRlm metastatic breast cancer in combination with targeted therapeutics such as CDK4 / 6 and mTOR inhibitors.10Thus, reduction of ER-regulated protein levels and reduction of Chkl activation utilizing Fulv plus Olap treatment targeted two tumor promoting pathways (e.g., metastatis)12,47in ESRlm models. To confirm that combined treatments induced increased DNA damage and also activated DNA repair, the inventors quantified pH2AX foci that are a hallmark of DNA double strand (ds) breaks (FIG. 58A, and FIG. 68). Olap increased the number of pH2AX foci / nucleus for both the WT and mutant cells, but only Y537S cells demonstrated a significant increase in pH2AX foci compared to WT with combination treatment. Y537C cells with 50% WT allele frequency showed only a moderate increase. These results were consistent with DDR activation in mutant cells to promote DNA repair with Olap treatment.

[0319] The inventors investigated genomic interactions of PARP1 and mutant ER to elucidate PARPl’s role in ER-regulated protein expression. The inventors quantitated PARP1 and mutant ER co-localization in the nucleus in response to Olap and Fulv as single agent and combination therapies. WT cells exhibited no changes in nuclear co-localization with Olap treatment (FIGs. 3D-3F). WT cells exhibited a reduction of ER- PARP 1 foci with Fulv as a300527252.1 - 94 -BAYM.P0443WO / BLG 25-012 single agent (p<0.0001) or in combination with Olap (p=0.0011) (FIGs. 3D-3F). These results were likely due to ER degradation by Fulv. The inventors discovered enrichment of PARP1- mutant ER nuclear co-localization after all treatments in Y537S cells, with the highest increase of ER-PARP1 foci per nucleus with Fulv plus Olap combination treatment (p<0.0001) (FIGs. 3E-3G). Olap as a single agent also enhanced ER-PARP1 foci in Y573C cells (FIG. 50, and FIG. 58B), though Fulv significantly reduced co-localization foci likely due to the presence of WT ER in the these cells. PARP inhibitors trap PARP1 on the DNA to enhance cytotoxicity in addition to blocking enzymatic PARylation activity.48These results indicated that Olap may trap both PARP1 and mutant ER on the DNA, which provides a mechanistic explanation of Olap’s reduction of ER-regulated proteins in ESRlm cells. The identification of enhanced endogenous PARylation in the Y537S model and increased PARP 1 -mutant ER nuclear interactions after Olap treatment provided mechanistic data of PARPl’s role as a transcriptional regulator of ER. Thus, combination treatment increased DNA damage in mutant cells.

[0320] ESRlm cells treated with first-line metastatic therapy Fulv plus Abema exhibited defective RSR and DDR and were vulnerable to DDR inhibition: ESRlm metastatic breast cancer patients receive Fulv plus CDK4 / 6 inhibition as first-line therapy.10The addition of CDK4 / 6 inhibitors Abema, ribociclib, or palbociclib to Fulv significantly improves progression-free survival in metastatic breast cancer patients. Clinical evaluation of Abema in the adjuvant setting significantly increased invasive disease free survival, however adjuvant palbociclib did not significantly improve patient outcomes. Thus, a significant proportion of women with high-risk primary or metastatic ER+ breast cancer will be treated with Abema in combination with ET.10 The PADA-1 trial identified ESRlm emergence as a significant driver of resistance to aromatase inhibitors (AIs) plus CDK4 / 6 inhibition. Switching patients with emergent ESRlm from AIs to Fulv plus CDK4 / 6 inhibition significantly improved progression-free survival. Patients switched to Fulv at the time of rising ESRlm frequency rather than at time of progression with AIs also exhibited increased efficacy with Fulv. These results highlight the importance of monitoring ESRlm emergence for the selection of ET for combination therapy with CDK4 / 6 inhibitors. The PALOMA-3 and MONARCH-2 trials identified significant acquisition and enrichment of ESRI mutations in 20-50% of metastatic breast cancer patients after treatment with Fulv plus CDK4 / 6 inhibition. Therefore, investigation of the effects of Fulv plus Abema on ESRlm breast cancer is highly clinically relevant to identify effective new therapeutic approaches.300527252.1 - 95 -BAYM.P0443WO / BLG 25-012

[0321] The inventors reported for the first time a novel MCF-7 cell line long-term treated with the metastatic therapy combination Fulv plus the CDK4 / 6 inhibitor Abema (F+A). The inventors used long-term treatment of MCF-7 and ZR75-1 cell lines with Abema (AbemaR) or Fulv (FulvR) as single agent controls int eh development of the F+A MCF-7 model (FIG. 69, and FIG. 70). The F+A cell line acquired the Y537S ESRI mutation at 3% allele frequency after 8 months of treatment (FIG. 51) which was enriched to 30% allele frequency after an additional 4 months of treatment (FIG. 71). The PALOMA-3 and MONARCH-2 trials investigated the efficacy of Fulv plus the CDK4 / 6 inhibitors palbociclib or Abema respectively and identified acquisition and enrichment of ESRI mutations in metastatic breast cancer patients after treatment.8,51The PADA-1 trial identified ESRlm enrichment as a significant driver of resistance to aromatase inhibitiors (AIs) plus CDK4 / 6 inhibition.52This study demonstrated a significant improvement in progression free survival by switching patients with rising ESRlm frequency to Fulv plus CDK4 / 6 inhibition. Thus, ESRI mutations are a significant driver of resistance to ET plus CDK4 / 6 inhibitors.53The F+A cell line is an ideal model to investigate therapeutic vulnerabilities in ESRlm breast cancer with previous exposure to Fulv plus CDK4 / 6 inhibitors. An MCF-7 cell line treated for 17 months with Abema only did not acquire ESRI mutations (FIG. 51). The inventors utilized this Abema cell line to investigate the effects of long-term treatment with CDK4 / 6 inhibition as a control. The inventors did not observe emergence of ESRlm with single agent treatment controls.

[0322] The inventors observed significant enrichment of the ATR / Chkl defective RSR signature using GSEA in the F+A resistant model after 12 months of treatment (NES=1.56, FIG. 4A). The F+A resistant and MCF-7 AbemaR cell lines exhibited transcriptional enrichment of CHEK1, CHEK2, and PARP1 using ORA, and transcriptional up-regulation of HALLMARK G2 / M checkpoint, E2F targets, and DNA repair (FIGs. 4B-4C). The F+A line exhibited significantly increased endogenous DNA damage concomitant with the emergence of ESRlm (FIGs. 4D-4E). Thus CDK4 / 6 inhibitor resistance also enriches for a defective RSR phenotype in breast cancer cells.

[0323] The inventors evaluated the cytotoxicity of Chkli and Olap in the long-term treated cell lines to explore the potential of these inhibitors as targeted therapeutics after resistance to Fulv and Abema. Both Y537S and F+A mutant models were sensitive to ~5 pM Olap (FIG. 72), similar with that reported for other cancer cell lines.54,66Combination of Chkl and Olap inhibitors were synergistic at concentrations near the single agent and combined IC50s in F+A mutant cells (FIGs. 4F-4G). Similarly, Fulv plus Olap inhibitor treatments were also synergistic at higher concentrations in mutant cells (FIGs. 4H-4I). Endogenous PARylation 300527252.1 - 96 -BAYM.P0443WO / BLG 25-012 levels were elevated and levels of ER and c-Myc proteins were reduced in F+A resistant cells (FIG. 4H) similar to that seen in Y537S and Y537C models. Together these results showed that tumors resistant to the CDK4 / 6 inhibitor Abema also exhibited a defective RSR and were sensitive to combination therapies.

[0324] The inventors investigated PARP1 and ER genomic interactions in the F+A cell line using PLA. Increased ER-PARP1 nuclear co-localization was observed after treatment with Olap and Fulv as single agents or in combination (FIGs. 4I-4J, Fulv+Olap p<0.001, Olap p<0.01). Thus, both the CRISPR engineered and acquired ESRlm mutant models exhibited increased mutant ER-PARP1 genomic interactions in response to PARP inhibitor therapy.

[0325] Retention of ER on chromatin is crucial for its role as a nuclear transcription factor. To explore whether Olap treatment enhances chromatin retention of ER or PARP1, which can function as an ER co-regulator, the inventors isolated chromatin bound protein fractions and compared PARP and ER levels after Fulv plus Olap treatment (FIG. 73) and found higher levels of PARP1 were retained on chromatin in the mutant models. The inventors also used Veliparib (Velip) treatment as a control because of its low trapping ability,58and found increased levels of PARP1 protein with Olap single treatment compared to Velip. The inventors also detected increased levels of chromatin bound ER protein for both the single agent Olap and Velip treatment in WT and mutant models (FIG. 73B). These results indicated that Olap traps both PARP1 and ER on DNA, which is associated with the reduction of ER-regulated proteins after Olap treatment in mutant cells. Additional studies examining ER protein half-life will be required to address this possibility. Previous reports identified PARP1 as a transcriptional regulator of WT ER through active enhancer formation and ER PARylation. This identification of enhanced endogenous PARylation in the Y537S model and increased ER-PARP1 -nuclear interactions after Olap treatment provides novel mechanistic data supporting PARPl’s role as an important transcriptional regulator of mutant ER.

[0326] PARP1 inhibition in combination with ET was an effective and novel therapeutic strategy for ESRI mutant breast cancer: Fulv significantly improves patient outcomes in ESRlm metastatic breast cancer compared to AIs both alone and in combination with CDK4 / 6 inhibitors.52,54Therefore, the inventors investigated the cytotoxic effects of Olap in combination with Fulv in the ESRlm models. Both Y537S (IC50: 90 nM) and Y537C (IC50: 0.35 nM) cell line models were less sensitive to Fulv treatment than WT cells (IC50: 0.03 nM) in agreement with previous reports (FIGs. 5A-5B).11,12Addition of Olap to Fulv induced cell death to levels similar to single agent Fulv treatment in WT cells (FIGs. 5A-5B). Fulv plus Olap was synergistic in both Y537S and Y537C cell lines at both physiological (10nM)55and300527252.1 - 97 -BAYM.P0443WO / BLG 25-012 supraphysiological (lOOnM) levels of Fulv (FIGs. 5C-5D), identifying this combination as efficacious in ESRlm breast cancer.

[0327] Next, the inventors investigated the impact of Fulv plus Olap on tumor growth in the WHIM20 Y537S ESRlm patient derived xenograft (PDX) model. Fulv plus Olap significantly reduced growth of organoids derived from WHIM20 primary tumors and lung metastases (FIGs. 5E-5F). Single agent treatment with Fulv or Olap did not significantly reduce organoid growth. Finally, the inventors evaluated the effects of Fulv plus Olap on in vivo tumor growth utilizing the WHIM20 model. Fulv plus Olap reduced PARylation, ER, and ER-regulated protein levels in the WHIM20 tumor model, strengthening the previous observation that Olap inhibited these pathways in ESRlm cell lines (FIG. 5G). Fulv plus Olap treatment also reduced phosphorylated ATR and total Chkl protein, confirming reduction of the RSR in tumors as was observed with Fulv plus Olap in the ESRlm cell lines (FIG. 5G). Fulv as both a single agent and in combination with Olap significantly reduced tumor growth, with the greatest decrease in tumor growth observed in the combination treatment group (FIG. 5H, FIG. 55). The combination of Olap plus Fulv was a “triple threat,” inhibiting PARP, Chkl, and ER-regulated proteins, all of which were enriched in ESRlm breast cancer models. Taken together, these results strongly supported further investigating PARP inhibitors in clinical trials in combination with Fulv for the treatment of ESRlm metastatic breast cancer.

[0328] Discussion: The inventors discovered enrichment of defective RSR in ESRlm breast cancer. Enriched expression of RSR proteins and PARP1 in ESRlm models compared to WT ER models identified significant dysregulation of the RSR and downstream DDR. ESRlm cells exhibited significantly elevated intrinsic DNA damage, and activation of DNA repair due to defective RSR. Enrichment of defective RSR is a novel therapeutic vulnerability in ER+ breast cancer, and inhibition of Chkl in combination with Olap reduced ESRlm tumor growth and significantly reduced distant lung metastasis in ESRlm tumor models. Primary patient tumor enrichment of defective RSR expression was also prognostic with significant reduction in overall survival in multiple ER+ patient cohorts. The identification of defective RSR in ER+ breast cancer is a novel discovery as previous literature reported this phenotype only in homologous-recombination deficient or oncogene-driven cancer models.27'28'37'40'42,47Thus, the inventors demonstrated for the first time that metastatic breast cancer patients with ESRlm are an ER+ subtype without genomic homologous recombination defects that are responsive to RSR and PARP inhibition as single or combination agents.

[0329] Aberrant activation of the RSR has also been reported as a therapeutic vulnerability in cancer models enriched for EMT56and stem-cell phenotypes.28The inventors previously300527252.1 - 98 -BAYM.P0443WO / BLG 25-012 reported that ESRlm breast cancer models also exhibit enhanced activation of EMT and stemcell phenotypes.12 14Both stem-cell and EMT phenotypes promote tumor plasticity, which is a key hallmark of cancer metastasis.57The inventors demonstrated a significant reduction in the progression of ESRlm metastatic lesions by targeting the RSR and downstream DDR with Chkl and PARP inhibitors. Thus, the inventors provided strong evidence that inhibition of the RSR disrupts the metastatic capabilities of ESRlm cells. This was a critical discovery, since currently there are no effective therapeutics to block EMT and stem-cell phenotypes. Therefore, the inventors reasoned that inhibition of the RSR and DDR may be an alternative approach to targeting metastasis driver phenotypes in ER+ breast cancer.

[0330] The inventors demonstrated synergistic responses to RSR and PARP inhibitors in multiple ESRlm cell line models. The findings by the inventors were the first to identify enhanced cell death in ESRlm ER+ breast cancer models utilizing this therapeutic combination. The inventors also identified cell cycle arrest and reduction of actively replicating DNA after treatment with Chkl inhibition as a single or combination agent with Olap. These data suggested that inhibition of the RSR and PARP prevents effective DNA replication in addition to disruption of DNA repair. Both Chkl and PARP are critical players in DNA replication by facilitating fork progression. PARP1 regulates fork reversal58, and the RSR promotes fork stalling59-61to ensure a high level of DNA fidelity. These pathways are complementary, ensuring cell cycle is delayed and aberrant DNA replication is corrected.20'61,62Accumulation of ESRlm cells in the G2 / M phase of the cell cycle suggested that these inhibitors force premature mitotic entry which may enhance DNA damage and promote cell death.37Disruption of DNA replication through PARP or RSR inhibition promoted significant genomic instability and cell death in a multitude of cancer models.26'27'62,63Thus, future investigation of the impacts of Chkl and PARP inhibitors on replication fork dynamics will provide mechanistic insights into the efficacy of RSR and DDR inhibitors in ESRlm breast cancer.

[0331] The results presented herein were the first to identify enrichment of PARP 1 -mutant ER DNA co-localization with PARP inhibition. PARP inhibitors block PARylation and trap PARP protein on the DNA to enhance cytotoxicity.48The PARP trapping experiment presented herein revealed that PARP and mutant ER must interact at the DNA regulatory level. Reduced expression of both ER and ER-regulated proteins in ESRlm cell lines and tumors after PARP inhibition identified PARP as a critical regulator of mutant ER-driven gene transcription. The inventors previously published that ESRlm cells significantly enhanced recruitment of PARP1 to co-activator protein complexes.64Investigation of WT ER identified PARP as a promoter of active enhancer formation at ER-regulated genes.49Thus, protein recruitment and regulation of300527252.1 - 99 -BAYM.P0443WO / BEG 25-012 chromatin structure are two potential mechanisms of PARP’s genomic regulation of mutant ER. PARylation also promoted resistance to the ER modulator tamoxifen by modifying ER to enhance ER-regulated gene expression.50The results presented herein extended these observations to Fulv resistance. The inventors identified enhanced endogenous PARylation in ESRlm cell lines which harbor both intrinsic (Y537S, Y537C) and acquired (F+A) Fulv resistance. Addition of Olap to Fulv was synergistic, significantly increasing cell death. The inventors also identified a significant reduction in ESRlm tumor growth with the combination of Fulv plus Olap. Taken together, these results identified PARP1 as a critical co-regulatory protein for SA / m-driven gene transcription and PARP activity as a promoter of resistance to ET. Future investigation of the ESRlm genomic landscape, including chromatin accessibility and identification of gene loci with PARP 1 -mutant ER co-localization, will provide mechanistic insight into PARPl’s regulatory role in ESRlm transcriptional regulation.

[0332] Herein, the inventors provided strong evidence for the inclusion of RSR or DDR inhibitors, particularly PARP inhibitors, in the treatment of ESRlm breast cancer. Addition of Olap to Fulv significantly reduced tumor growth of both primary and metastatic ESRlm tumors and decreased ER-regulated protein expression in multiple ESRlm models. When to introduce PARP inhibitors to ESRlm metastatic breast cancer patients remains a critical question. Fulv plus Olap was synergistic in F+A Y537S cell line, indicating that PARP inhibition is a viable therapeutic option for ESRlm patients with previous exposure to Fulv and CDK4 / 6 inhibitors. The PADA-1 trial recently highlighted the importance of monitoring ESRI mutations for the selection of second-line therapeutics.52Utilizing a novel “crossover” design, patients with rising baseline ESRI mutations were switched from AIs to Fulv with continued CDK4 / 6 inhibition. The Fulv crossover cohort exhibited significantly increased progression free survival compared to the Al cohort. Patients switched to Fulv after detection of ESRlms also remained on Fulv without disease progression longer than patients switched to Fulv at the time of progression with AIs. Thus, selection of ET for targeted therapy combinations is a critical challenge for ESRlm breast cancer. The approval of the oral SERD elacestrant for ESRlm metastatic breast cancer9and promising preliminary clinical results of the oral SERDs camizestrant65and imlunestrant66'67could provide multiple opportunities to introduce new targeted therapeutic combinations, such as PARP inhibitors, to ER+ breast cancer. Therefore, utilizing the PADA-1 trial design, the inventors propose that enrichment of ESRI mutations in patients could be used as a novel trigger to “crossover” to PARP inhibitors in combination with new oral SERDs or Fulv after treatment with ET plus CDK4 / 6 inhibitors.Example 3300527252.1 - 100 -BAYM.P0443WO / BLG 25-012

[0333] Endocrine Therapy (ET) and ESRI mutations (ESRlm) Mutations in the ESRI gene are common (25-59% frequency) in ER+ metastatic breast cancer (MBC) patients who progress after therapy and are especially prevalent in aromatase-inhibitor (Al) treated tumors.4,5The inventors were the first to discover the tyrosine 537 to asparagine (Y537N) mutation using highly sensitive, manual exome sequencing of MBC tumors, and originally hypothesized an important role for constitutively active ESRlm in both ET resistance and metastatic progression,7,8These hypotheses have proven correct with the majority of identified ESRlm being localized to two hot spot residues — tyrosine 537 (Y537S, Y537C and Y537N) and aspartic acid 538 (D538G) that render tumors estrogen independent.9It is now known that ESRlm represent a key mechanism of resistance to ET in MBC.10Clinical data has demonstrated that patients with ESRlm have an inferior progression-free survival (PFS) and overall survival (OS) when treated with an Al versus the ER degrader Fulvestrant (Fulv).11The PADA-1 clinical trial demonstrated that early switching of the ET backbone from an Al to Fulv with combination CDK4 / 6 inhibitor treatment based on rising ESRlm levels in blood circulating tumor DNA (ctDNA), doubled patient PFS.12In the MONARCH 2 trial of Fulv + Abemaciclib (Abema) the median PFS of patients with ESRlm was only 20,7 months.5The phase III EMERAED trial demonstrated superiority of the next generation oral ER degrader elacestrant (Elac) compared to standard ET with a significant PFS improvement in patients with ESRlm, resulting in FDA approval earlier this year for patients with ESRlm. These recent clinical results were exciting, and the inventors decided to explore the utility of Elac and Fulv in combination with CDK4 / 6 (control treatments) or PARP inhibitors in preclinical models as presented herein. Treatment with the combination of Elac and PARP inhibitors was novel and undoubtedly more therapeutic options are needed to accomplish longer durations of effective therapy for MBC since a significant percentage of MBC patients can acquire ESRlm during therapy.

[0334] Transcriptional Reprogramming in ESRlm models: ESRlm tumors display a unique transcriptome that is associated with multiple metastatic phenotypes.14ESRlm are enriched for stem cell and epithelial mesenchymal transition (EMT) properties, both of which are associated with enhanced metastasis.15,16Unfortunately, there are no clinically effective EMT inhibitors identified to date. RNA expression analyses of MCF-7 cells genetically engineered using CRISPR correlated with established EMT and PAM50 basal-like signatures, and these signatures were associated with shorter PFS in primary breast cancer patients.16Importantly, ESRlm exhibited enhanced metastatic potential with a greater metastatic frequency in vivo compared to cells with wild-type (WT) ER, and xenograft experiments300527252.1 - 101 -BAYM.P0443WO / BLG 25-012 mixing different proportions of mutant versus WT cells resulted in metastases with 100% mutant frequency regardless of the proportion of mutant cells injected.16These preclinical studies established ESRlm as key drivers of metastasis. Others have recently validated the inventors’ findings.5The inventors also found simultaneous activation of several growth factor receptors, such as RON and IGF1R, using proteomic kinome profiling of ESRlm models.17 18Although selective inhibitors were significantly effective in these preclinical models, the inventors reasoned that this therapeutic approach would eventually fail in patients, either due to the toxicity of combining small molecule inhibitors or due to the heterogeneity and loss of some of these targets during spontaneous metastases to different sites in vivo. Thus, the inventors proposed that a greater understanding of how ESRlm induce transcriptional reprogramming and cistrome plasticity could lead to a discovery of foundational therapeutic vulnerabilities that would result in impactful, not just incremental clinical benefit.

[0335] Replication Stress (RS) and the DNA damage response (DDR). The inventors reasoned that acquisition of ESRlm act as an activated “oncogene” inducing hyperproliferation, which triggers replication stress (RS) and excessive DNA damage. ESRlm tumors escape from this stress by inducing a signaling network termed the DDR that coordinates cell cycle progression and DNA repair.19The inventors showed that the RS and DDR are compromised in ESRlm, which affords a novel therapeutic opportunity to block metastatic dissemination using polyADP-ribose polymerase (PARP) inhibitors. PARP inhibitors are currently approved for BRCA-related breast, ovarian, and prostate cancer with specific genomic alterations,20and these agents are generally well-tolerated.21The inventors’ proteomic studies demonstrated that PARP1 is the top enriched ER DNA binding protein in ESRlm cells,22suggesting that PARP1 may play a critical role in regulating ER mutant gene expression at the chromatin level. There is literature demonstrating that PARPs can bind and regulate both WT ER and the androgen receptor (AR), although the data are limited.23,24The inventors acquired solid preliminary in vivo preclinical data indicating that inhibition of the DDR through combined ET with PARP inhibition was an effective therapeutic option for ER mutant tumors. These novel data suggested that MBC patients with ESRlm may be an ER+ subtype without genomic homologous recombination defects (HRD) that will be responsive to PARP inhibition as a single or combination agent.

[0336] Innovation: Recurrences after combination inhibitor therapy in MBC can be clinically aggressive, presenting a challenge in clinical decision-making for how best to treat patients.25ET (Fulv or Elac) or ET plus CDK4 / 6 inhibition are the preferred first-line therapies for patients with ESRlm, and as OS results have matured clinical trials have shown that Fulv300527252.1 - 102 -BAYM.P0443WO / BLG 25-012 plus either ribociclib (Ribo) or Abema have significantly improved OS.26Based on these clinical data the inventors developed an innovative longitudinal experimental design to address this clinical challenge (FIG. 6). As a first innovation, the inventors propose to use ER+ xenograft and patient derived xenograft (PDX) transplants from MBC patients for experiments to simulate a first treatment, then establish ex vivo organoid growth assays from primary and metastatic tumors to evaluate post CDK4 / 6 progression therapy. Since the PDX models were from MBC patients most were relatively resistant to ET with Fulv. The inventors published the utility of this approach in therapeutic studies in a recent publication evaluating RON and PI3K inhibitors,17thus the proposed experimental design was innovative and feasible. The inventors will evaluate both mechanism of action and therapeutic efficacy using this experimental design in Aims 1 and 3, respectively. To the inventors’ knowledge only one Phase II clinical trial in Korea is planned to test Fulv+Olaparib (Olap) in ER+ advanced breast cancer post CDK4 / 6 inhibitors (NCT05536128), however the study was “not yet recruiting” as of June 2024. Currently PARP inhibitors are only recommended in ER+ patients with genomic BRCA mutations.25Clinical studies suggested that early adaption of PARP inhibitors is better compared to later lines of therapy,21which supports the inventors’ evaluation of PARP inhibitors as first and then second treatment after progression on CDK4 / 6 inhibitors

[0337] A second innovation is unpublished data demonstrating activation of G2 / M checkpoint and DNA repair pathways in mutant tumors concomitant with enhanced intrinsic DNA damage. Third, the inventors will show therapeutic efficacy of the PARP inhibitor Olap in ESRlm tumors with remarkable reduction of metastatic dissemination in vivo. Mechanistically the inventors will evaluate PARP’s role in chromatin remodeling and transcriptional regulation of ER and / or AR (Aspect 2). Fourth, the inventors had compelling data demonstrating down-regulation of ER and AR along with PARP inhibitor treatment that may further augment PARP inhibitor efficacy in mutant tumors.Aspect 1. To elucidate mechanisms underlying PARP inhibitor vulnerability.

[0338] Introduction and Preliminary Data. RS and DDR. It is known that oncogene activation induces hyper-proliferation that can trigger a RS repair response,32however whether constitutive activation of ESRlm induces RS has not been shown. Defects in the RS response kinases ATR / Chkl enable cells to evade oncogene-induced senescence and continue proliferating.33,34The biologic rationale for Aspect 1 was founded in preliminary data showing that ESRlm cells (Y537S, Y537C, and D538G) expressed constitutively elevated levels of300527252.1 - 103 -BAYM.P0443WO / BLG 25-012 pATR and pChkl checkpoint proteins, and increased levels of DNA damage markers RAD51 and RPA2 (FIG. 7A). GSEA analyses of RNA expression data demonstrated induction of the Hallmark DNA repair pathway in Y537S and Y537C mutant models (FIG. 7B). DNA damage markers RPA1 and yH2AX were elevated in Y537S mutant cells (FIG. 7C). The inventors observed significantly increased constitutive DNA damage suggesting a defect in DDR in ESRlm cells (FIG. 7D). These data strongly suggested a role for RS activation in a defective RS / DDR in mutant cells.

[0339] Defective RS repair response in ESRlm models. A gene signature that predicts defective RS repair responses in breast tumors has been developed.34The signature was developed by stably depleting the key RS signaling factors ATR, Chkl, ATM, Chk2 in MCF- 10A mammary epithelial cells. The inventors overlapped RNA-Seq from a CRISPR Y573S and a Y537C LTED models with genes up-regulated in their ATR / Chkl knockdown signatures and identified 355 shared genes (FIG. 8 . Gene set enrichment of the overlap demonstrated enrichment of specific KEGG pathways involved in cell cycle, DNA replication, and DNA repair (FIG. 8A) supporting a defective RS repair response in mutant. Kaplan-Meier analysis using the publicly available SCAN-B ER+ data cohort (GSE96058) showed that the mutant RS gene signature significantly identified ER+ patients with poor outcomes at 6.8 years of followup (FIG. 8B). These data provided solid evidence for a defective RS repair response in mutant cells; the molecular basis associated with this defect will be evaluated in Aspect 1.

[0340] Enrichment of DNA damage phenotype in mutant cells. Two objectives in Aspect 1 are to determine if and how the DDR is compromised in PDX models vs cell line models and the role of PARP1 in ER / AR turnover. To address these questions, in addition to the inventors’ published CRISPR models (MCF-7 Y537S and D538G),16the inventors recently developed models with acquired resistance to long-term estrogen deprivation (LTED / Y537C), the CDK4 / 6 inhibitor palbociclib (PalboR),17or Fulv plus the CDK4 / 6 inhibitor Abema (Fulv+AbemaR) (Table 9). The LTED and Fulv+AbemaRmodels acquired ESRlm during selection therapy mimicking the rapid clonal evolution and expansion of ESRlm in patients treated in the Monarch 2, SOFeA, and PADA-1 trials.5 11'35Using RNA-Seq analyses the inventors detected elevated PARP1 RNA levels during selection, with the highest levels in the Fulv+AbemaRline with the longest therapy exposure. Similar to the CRISPR genetically engineered models,16Pathway analysis showed enrichment for Hallmark E2F targets, G2M checkpoint, and DNA repair in the Fulv+AbemaRlines with elevated RNA levels of Chkl and PARP1 similar to that seen in FIG. 7. Of note, the Fulv+AbemaRline with an acquired Y537S300527252.1 - 104 -BAYM.P0443WO / BLG 25-012 mutation also exhibited significantly increased levels of endogenous DNA damage as measured by Comet assays (data not shown).Table 9. ETRand CDK4 / 6RCell Line Models:

[0341] PARP1 as a transcriptional regulator of ER: Expression of PARP1 is enriched in the luminal subtype of breast cancer and high PARP1 expression correlates with poor OS in ER+ patients.23PARP1 acts as a sensor of DNA damage modulating downstream mediators of the DDR. A major mechanism of how PARP inhibitors kill cancer cells is through “trapping” at sites of DNA damage.36PARP inhibitors complex with PARP1 / 2 and “locks” onto damaged DNA preventing DNA repair, replication and transcription thus leading to cell death. Therefore, the inventors evaluated quantitative PARP trapping in MCF-7 Y537S cells and parental (P) cells using ER- PARP 1 Duolink proximity ligation assays (PLA) and cofluorescence imaging (FIG. 9) after treatment with single agents (Fulv or Olap) or the combination. Fulv reduced PARP1 trapping onto sites bound by WT ER probably due to effective degradation of ER, whereas the opposite was observed in Y537S mutant cells- both single and combination treatments significantly trapped PARP1 at ESRlm bound sites on chromatin (FIG. 9A). These results were consistent with the known relative resistance of ESRlm to degradation by Fulv, as well as data showing that Fulv treatment increases ER levels in chromatin-enriched fractions, and induces engagement with ER at canonical estrogen response element (ERE) binding sites.37The inventors will explore if enhanced recruitment to300527252.1 - 105 -BAYM.P0443WO / BLG 25-012 selective mutant bound chromatin sites is associated with the efficacy of PARP inhibitors in mutant models. To further define PARPl’s effects on ER protein itself, the inventors transfected a pool of two PARP1 knockdown siRNAs and performed Western blot (WB) analysis (FIG. 9B). siPARPl knockdown and Olap treatment significantly decreased both ER and AR protein levels in mutant cells. FIG. 9C shows enhanced PARylation activity in mutant cells, and down-regulation of E2-regulated gene expression (PR, c-Myc) after 24h Olap treatment. These results suggested that PARylation might affect transcription of ER itself or protein levels in mutant cells. Thus, the inventors reasoned that PARP inhibition might provide increased selective benefit through additive interactions on mutant ER protein degradation / turnover.

[0342] Another objective of Aspect 1 is to explore novel functions of PARP1 as a transcriptional regulator,2438including post-translational modifications of ER using Western blot analysis, recruitment of co-regulators, or protein-protein interactions using immunoprecipitation.23Poly-ADP-Ribosylation (PARylation) of WT ER increases with antiestrogen (tamoxifen) treatment and PARylation activity mediates resistance to tamoxifen.39Activation of AR in prostate cells initiates K48 ubiquination and 26S proteosomal degradation.40PARylation can recruit E3 ligases to regulate protein turnover.41The inventors will evaluate these potential mechanisms for effects on ER and AR in the mutant models.41

[0343] Research Design: The inventors will address three essential questions in Aspect 1: 1) Are RS response pathways activated and the DDR compromised in ESRlm tumors? 2) What is the role of PARP1 in regulating ER or AR levels? 3) How does PARP1 function as a transcriptional regulator of ER? The impact of these studies will be both a mechanistic understanding of the DDR defect in mutant cells, along with insight into how best to block mutant tumor progression.

[0344] Methods: Are RS response pathways activated and the DDR compromised in ESRlm tumors? ETRmetastatic models: The inventors have MCF-7 and T47D WT (P) and Y537S and D538G mutant CRISPR models,16PalboRderivatives of MCF-7 WT and Y537S,42LTED models with acquired Y537C mutations,42and the new Fulv+AbemaRmodels. (Table9). In addition, the inventors will utilize four patient derived xenograft (PDX) models (Table10); the number of models is limited due to budget constraints. The inventors had Western blot data demonstrating that all, except the WHIM20 Y537S PDX model previously proposed, expressed high levels of the 8 gene signature proteins (to be discussed in Aspect 3). The inventors will thus examine a minimum of 8 cell lines and 4 PDX models to evaluate activation of ESRlm and defects in RS / DDR pathways. Western blot analysis of ATR / Chkl, ATM / Chk2,300527252.1 - 106 -BAYM.P0443WO / BLG 25-012 and downstream mediators (pCdc25c) will evaluate activation of the cell cycle checkpoint axis, and the inventors will also evaluate levels of ER, AR, and PARP1. Details of Western blot analyses are published.16As a control the inventors will use the MDA-MB-436 breast cancer cell line with a mutation in exon 20 splice donor site accompanied with loss of the other BRCA allele and is BRCA1 protein negative.4 JTable 10. ET-Resistant Metastatic PDX Cohort:

[0345] In vitro PARP1 knockdown and growth studies: Stable PARP1 silencing in cell line models will use the MISSION shRNA f pLOKI.l-Puro-CMV-tGFP vector (MILLIPORE SIGMA®) according to manufacturer's protocol. MTT growth assays will be done on the control and PARP1 knockdown cell lines in vitro. Control and PARP1 knockdown cell lines will also be grown in vivo as primary tumors in athymic nude mice. Control parental cells require E2 supplementation to grow; mutant cell lines will be grown in the absence of E2. At approximately 3.5-4 weeks when tumors reach 2-300 mm3, tumors will be processed for organoid growth assays. Experimental details for growth assays and knockdown experiments are described in17. Treatments for MTT and organoid growth assays include single agent controls (Fulv, Elac), and combinations shown in FIG. 6. The first treatment in FIG. 6A simulates the EMERALD trial.13The first treatment in FIG. 6B is designed to simulate ongoing clinical studies to evaluate Elac in various combinations (NCT05563220). In FIG. 6C the first treatment is designed to study the use of PARP1 inhibitors as initial treatment. All secondary treatments are designed as treatment switching arms where cell lines and PDX are assigned onto an alternative treatment. Ex vivo growth data will be represented by mean number of organoids plated in sextuplets and standard deviation. Student’s t-test or one-way ANOVA will be used for statistical analysis. To test whether inhibitors have synergistic or additive effects ex vivo, the inventors will also use MTS endpoint viability assays with digested organoids as described by Sachs et al,44because these assays are more sensitive and measure quantitative metabolic activity associated with organoid growth for computation of combination index statistics (CalcuSyn software). Continued knockdown of PARP1 will be monitored in cell lines300527252.1 - 107 -BAYM.P0443WO / BLG 25-012 and organoid cultures using Western blot. The focus of these studies is to identify mechanisms associated with therapeutic response.

[0346] RNA-sequencing of PARP1 knockdown cell lines and organoid experiments: This is a novel analysis in the inventors’ experimental plan. The inventors will perform RNA-Seq analyses on the shPARP knockdown cell lines and PDX organoids described above to determine if the defective DDR phenotype is targeted by knockdown and provide an initial assessment of mechanistic utility of treatments to block mutant phenotypes. The inventors are confident that early passages of knockdown cells will maintain PARP1 inhibition and they will monitor for knockdown before inclusion in RNA-Seq analysis. To the inventors’ knowledge there are limited datasets with this approach. The RNA-Seq analyses on inhibitor treated cell lines and organoids will be used to evaluate global effects of the therapeutics on mutantspecific Hallmark pathways, such as Hallmark DNA damage response, E2F targets, and downstream DDR signaling. The inventors will follow standard protocols for RNA-Seq library preparation and sequencing.16The inventors will assess data quality with FastQC and then map sequence data onto the human genome build hg38 using STAR. Gene expression will be assessed using featureCounts and the GENCODE reference. Differential gene expression will be determined using the DESeq245 46R package, with significance achieved for FDR-adjusted p-value<0.05 and fold change exceeding 1.25. The inventors will generate genome- wide maps using BEDTools,47and visualize data using the Integrative Genomics Viewer or the UCSC Genome Browser. Enriched pathways will be inferred using Gene Set Enrichment Analysis (GSEA) and enrichR. Gene signatures will be used to identify networks of candidate interacting proteins using STRING (Search Tool for the Retrieval of Interacting Genes / Proteins).

[0347] Comet alkaline assays: Comet assays are a sensitive technique for detection of DNA damage and DNA repair.48Assays will be performed as described using cisplatin as a control DNA damaging agent and the inhibitor combinations shown in FIG. 7. The inventors will also include the shPARP knockdown models, and Talazoparib (Tala) in addition to Olap due to observed significant improvement of OS benefit with the use of Tala vs chemotherapy in the 1stline setting.49Analysis of tail moments will be performed using the Comet Assay IV software (Instem) with quantitation of at least 150 comet tails per sample. Statistical significance will be determined utilizing one-way ANOVA in GraphPad (PRISM®), with significance at FDR-adjusted p-value<0.05.

[0348] DNA fiber analysis: In this assay, progressing replication forks are sequentially labeled with two consecutive thymidine analogs — e.g., 5-iodo-2-deoxyuridine (IdU) and 5- chloro-20-deoxyuridine (CldU). Immuno staining allows for microscopic visualization of both300527252.1 - 108 -BAYM.P0443WO / BLG 25-012 tracts to examine changes in replication fork progression, fork symmetry, and origin firing, as well as nucleolytic degradation of nascent DNA with RS. Methodological details are published.50,51

[0349] What is the role of PARP1 in regulating ER or AR protein levels? While the effects of PARP1 on the DDR is well understood, the impact of PARP1 on regulation of specific proteins is limited. Key data shown in FIGs. 9B-9C showed significant reduction of ESRlm and AR protein with Olap treatment and elevated PARylation in mutant cells was observed. PARylation has been identified as a signal for triggering protein degradation through the ubiquitin-proteasome pathway,41and Fulv is known to utilize ubiquitination mechanisms to induce proteasomal degradation of ER.52The NCOA3 co-regulator recruits transcription factors for ER transcription, but also regulates ER degradation mediated by the ubiquitin- protein machinery.53The inventors will address the effects of PARP inhibitors on ubiquitin- mediated degradation of ER and AR, and address whether NCOA3 is involved since NCOA3 is required for E2 stimulated WT ER degradation.53The Y537 mutation site is important for ER ubiquitylation and degradation.54AR is also a substrate for ubiquitination at K845 and 847.55The inventors will measure ubiquitin-mediated proteasomal degradation rates of ER and AR using cycloheximide-chase Western blot experiments in cell lines treated with ET and PARP inhibitors and the 20S proteasome inhibitor MG13256,57Levels of ER and AR will be determined relative to P-actin quantitated by densitometry of triplet replicates using an Odyssey imaging system. Half-life will be calculated using one-phase exponential decay curve using GraphPad software (PRISM®). The inventors will employ site directed mutagenesis of relevant sites in ER and / or AR to confirm ubiquitin-proteasome degradation mechanisms with PARP1 treatment.

[0350] How does PARP1 function as a transcriptional regulator of ER? Cells and organoids will be treated with estradiol (+E2) for 2 hrs for ER, AR, PARP1, FOXA1, NCOA3, and H3K27AC ChlP-Seq analysis to identify inhibitor effects on genome-wide binding of these transcription factors. Antibodies to ER, AR, PARP1, FOXA1, NCOA3, and H3K27ac will be used for ChlP-Seq with three biological replicates. The inventors will assess ChlP-Seq data quality with FastQC, then map it with BOWTIE258to human genome build hg38; enriched regions (peaks) will be called using MACS2.59Differential peaks between different models and treatments will be determined using BEDTools.47ChlP-Seq data will be visualized using deepTools60and Circos platforms.61The shRNA PARP1 silencing effects on binding sites will be integrated with RNA-Seq expression data. The integration of RNA-Seq and ChlP-Seq data will be done using Binding and Expression Target Analysis (BETA) software to infer direct300527252.1 - 109 -BAYM.P0443WO / BLG 25-012 transcriptional targets of transcription factor binding sites. The +E2 studies will also facilitate mechanistic insight into the ER-AR-PARP1 functional interactions to be studied in Aspect 2. The inventors will determine enriched transcription factors using HOMER62and integrate the results with other breast cancer ChlP-Seq data sets using the CisromeDB resource,63by analyzing gained or lost peaks alone, or by analyzing gained or lost peaks that associate with transcription changes at nearby genes. The inventors will determine and localize pattern of cobinding or complexes of the regulators studies (ER, AR, PARP1, F0XA1, NC0A3) as well as determine other co-regulators of potential functional relevance.

[0351] Expected Outcomes and Statistical Plan: The inventors expect to demonstrate how PARP1 inhibitors block the RS / DDR pathways and enhance ER / AR protein turnover. For ChlP-Seq and ATAC-Seq, the inventors will follow Encode consortium guidelines for sequencing read depth to achieve a high resolution of peak detection. For RNA-Seq, using n=3 per group and assuming a variance of 20% in the population, the inventors can detect a fold change of 2 with a power of over 82%. For qRT PCR, ChIP qPCR, and organoid growth, using an n=6-7 per group and assuming a variance of 20% in the population, the inventors can detect a fold change of 1.5 with a power of over 86%.

[0352] Potential Problems and Alternative Strategies: It is possible that defects in RS and the DDR may result in mitotic catastrophe with PARP targeting.64Alternatively, the DDR, and in particular ATR, can be activated independently by DNA damage through oxidative stress.65These redox-dependent mechanisms then participate in a number of signaling pathways involved in metabolic regulation. ESRlm’s confer a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis.65,66Aspect 2. To define epigenetically encoded programs that facilitate metastasis of ESRlm tumors.

[0353] Introduction and Preliminary Data: The inventors’ premise is that if ER function is blocked at both the chromatin and protein levels, metastatic evolution can be more effectively stopped or slowed. Multi-omics analyses performed on primary and metastatic tumors from the AURORA US trial found that in addition to known somatic genetic alterations, changes in the epigenetic landscape were critical for metastatic progression.67The inventors previously reported that ESRlm tumors changed from PAM50 luminal tumor subtype to basal-like.16Similarly, the AURORA US study reported -30% of metastatic tumors exhibited changes in300527252.1 - 110 -BAYM.P0443WO / BLG 25-012 their PAM50 expression subtypes. Thus, in addition to specific metastasis-causing mutations like ESRlm, more widespread chromatin alterations that affect aggressive phenotypes must occur for progression.

[0354] The inventors published that AR overexpression is associated with endocrine therapy resistance (ETR).68-70AR can also compete with ER for binding to estrogen response elements (EREs) thereby acting to repress ER signaling in ER+ / AR+ breast cancer.71,72Agonist activation of AR alters E2-bound ER’s interaction with chromatin, with a reduction in AR- overlapping ER binding sites and ER transcriptional activity in preclinical models. E2 can also induce AR genomic binding, but only a fraction of these sites overlap with canonical ligand- activated AR, suggesting that AR can facilitate binding to a unique set of ER / AR binding sites depending on the hormonal milieu.73The inventors recently reported elevated levels of AR protein in engineered ESRI mutant lines and PDX tumors and a role for AR in metastatic dissemination of ESRlm PDX models.74

[0355] PARP1 is enriched in promoter regions of actively transcribed genes where its PARylation function assures an open chromatin configuration and spatiotemporal interaction scaffold for transcription factors.75PARP1 enzymatic activity is critical for AR occupancy on chromatin and AR activity.24In castration-resistant prostate cancer, AR inhibitors promote “BRCA-ness” via reduction of BRCA1 and RAD54 levels.76The inventors reasoned that a loss of negative AR regulatory function may help drive metastasis via AR’ s collaboration with ESRlm oncogenic activity. How PARP1 participates at the chromatin level in ER / AR driven transcription at specific mutant unique binding sites is an objective of Aspect 2. The biologic rationale for this is detailed next.

[0356] Significant redistribution of AR and ER / AR binding sites in ESRlm cells using integration of ChlP-Seq with ATAC-Seq analyses: The inventors employed transcriptional profiling of MCF-7 WT P and the Y537S ESRlm CRISPR clone YS1 in cells treated with +E2 and conducted parallel AR and ER ChlP-Seq analyses.74ER only binding sites were similar between P and YS1 ESRlm cells, but a reduction in the number of AR only sites was observed in mutant (FIG. 10A). In the YS1 mutant, AR only binding sites shifted from promoters to transcriptionally inactive heterochromatin in the presence of E2 (first column), and ER / AR cobinding sites moved from gene promoters to enhancers (third column from left to right in “YS1 E2”). These results suggested that AR is reprogrammed in ESRlm cells with E2 treatment, and potentially suppressed via redistribution of AR to heterochromatin.

[0357] To confirm the shift of overlapping ER / AR peaks to heterochromatin the inventors performed ATAC-Seq, a marker of open chromatin, using the MD Anderson Cancer Center300527252.1 - I l l -BAYM.P0443WO / BLG 25-012 core. The inventors generated ATAC-Seq profiles, and computed overlap of ER / AR and AR alone peaks with ATAC-Seq peaks (FIG. 10B) and then plotted the distribution of ATAC-Seq signal (FIG. IOC). The movement of AR only binding sites to closed chromatin is apparent in YS1 E2 (arrow in FIG. IOC). Thus, AR colocalizes with ER at enhancers in WT and ESRlm cells, but AR alone shifts to closed chromatin in ESRlm treated with E2. These results demonstrated that ESRlm promotes cooperative function between ER and AR, but silences function of AR independent of ER, Thus, ESRlm epigenetically silences AR activity independent of ER. ChlP-Seq data were mapped using Bowtie 2 to the human genome build UCSC hg38. AR only, ER only, and ER / AR peak overlaps (number of sites is shown) were determined using BEDTools.

[0358] It is known that actively transcribed genes are more likely to associate with enhancer-related epigenetic chromatin modifications such as H3K4mel and H3K4me3.77Therefore, the inventors also overlapped the binding sites data with a ChlP-Seq dataset (GSE8515878) of H3K4mel enhancer-related (FIG. 11A) and H3K4me3 promoter-related (FIG. 11B) chromatin modifications generated from 6 breast cancer cell lines. ER / AR sites in E2 treated YS1 mutant cells moved from promoters to enhancers. These collective results confirm that the ER / AR E2 co-bound sites (dashed line peaks) were centered at regions of active enhancer chromatin regions and the inventors will pursue this lead in Aspect 2.

[0359] Only a few ER ChlP-Seq analyses have been done on breast tumors, however Ross- Ines et al. mapped 18 ER+ clinical samples with outcomes data including three MBC tumors.79They reported differential ER binding in patients with poor outcomes due to FoxAl mediated reprogramming of ER. To explore the ER / AR cooperative binding hypothesis, the inventors overlaid ER only, AR only, and ER+AR co-binding data onto a published ChlP-Seq dataset (GSE322222). The highest signal density of ER from MBC patients was observed in the overlap of ER / AR binding in the YS1 mutant E2 (FIG. 11C, dashed line peak). These results confirmed Ross-Innes’ conclusions regarding high ER recruitment in breast cancer patients with poor outcomes,79but expands the observation to mutant ER / AR E2 co-binding as well. The inventors also detected preferential binding of PARP1 to ER / AR binding sites using ChlP- Seq data published by80(FIG. 11D). These data showed that PARP1 was highly bound at ER / AR mutant chromatin bound sites (dashed line peak). These data demonstrated that the mutant ER / AR E2 co-bound sites best discriminated the ER genomic binding events in metastatic tumors and supported a potential high translational impact of this experimental plan for MBC patients.300527252.1 - 112 -BAYM.P0443WO / BLG 25-012

[0360] To confirm direct interactions, the inventors performed ER-AR PLA and coimmunofluorescence imaging in 4 ESRlm models compared to P cells with WT ER (FIG. 12). These preliminary results showed ER-AR nuclear co-localization with +E2 treatments in all mutant models. These data were consistent with the chromatin binding data of ER / AR interactions at the chromatin level.

[0361] An important question is the role of PARP1 in regulation of chromatin binding of ER / AR? First, the inventors performed ER-PARP1 and AR-PARP1 PLA assays and determined that +E2 increases PARP1 binding to both (FIG. 13). Next, the inventors used chromatin immunoprecipitation (ChlP-qPCR) to assess recruitment of ER (FIG. 14A), AR (FIG. 14B), or PARP-1 (FIG. 14C) to the regulatory loci of two ER / AR co-binding target genes N4BP3 and BMP7 (details of these mutant regulated genes will be provided in Aspect 3). Binding of ER was minimally affected by treatment with Olap (FIG. 14A), potentially due to PARP1 trapping, however significant reduction in AR promoter occupancy was seen with treatment (FIG. 14B). PARP binding was significantly decreased in mutant cells with the use of an AR degrader ARV110 (FIG. 11C), which is currently in Phase 1, 2 clinical trials in castration resistant prostate cancer.3These data suggested that PARP1 or its PARylation function regulates AR association with chromatin and that AR plays a functional co-regulator role for ESRlm.

[0362] Research Design. The inventors will study if PARP1 regulates chromatin structure or facilitates the exchange of co-regulators such as AR, FOXA1 or NCOA3 on chromatin using ATAC-Seq analyses. The inventors will test whether AR and / or PARP1 are required for ER- AR binding and transcription of mutant unique ER-AR regulated genes that are associated with poor prognosis and metastasis. The invenotrs will test whether PARP inhibitors can block colocalization of ER, AR or PARP1 using PLA and co-fluorescence imaging.

[0363] Methods: Does PARP1 regulate chromatin structure or facilitate the exchange of co-regulators? ATAC-Seq: Epigenetic reprogramming at ER binding sites occurs in ETRbreast cancer models which alters the 3D chromatin landscape,83and chromatin accessibility is central to inducible gene transcription, like +E2. Thus, the inventors will perform ATAC- Seq to examine chromatin accessibility in the aforementioned models to determine protein- DNA interactions with ER, AR, and PARP1 that regulate gene transcription. The inventors will use the engineered models with PARP1 knockdown treated with control CSS or E2 for 2 h, and subject extracts to ATAC-Seq using biological duplicates with published protocols.84ATAC- Seq and RNA-Seq data will be integrated to correlate chromatin accessibility with gene expression changes, promoter, and enhancer regions. FoxAl is a pioneer factor for both ER300527252.1 - 113 -BAYM.P0443WO / BLG 25-012 and AR, but it was shown not to be enriched at S' / / m-selective binding sites, or essential for proliferation of mutant cells.85However, these authors also showed that F0XA1 upregulation can promote transcriptional reprogramming in ETRbreast cancer.86The inventors will evaluate for changes in FOXA1 and NCOA3 accessibility and localization; both of these are uniquely E2 regulated in mutant cells, and are contained in the mutant gene signature to be described in Aspect 3 (Table 11). The inventors will also perform ATAC-Seq on selected cell line models after PARP inhibitor treatment (1 week). ATAC-Seq data will be mapped and peaks called as described above for ChlP-Seq. Differential peaks will be determined using diffBind and diffReps and mapped to nearby genes using BEDTools. Using published datasets of peaks for F0XA1, NC0A3, and the insulator protein CTCF77and random genomic background matching the chromosome and region size distribution, the inventors will determine significant overlaps using Chi-Square with significance achieved at p<0.05. Enriched motifs at differentially bound sites will be determined using HOMER,62and the inventors will integrate the ATAC-Seq results with published BC ChlP-seq datasets using the CistromeDB63platform. By analyzing increased or decreased peaks that associate with transcription changes in matching direction at nearby genes, the inventors will determine potential transcription regulators. The inventors expect to confirm the presence and enrichment of the regulators studied via ChlP-Seq in Aspect 1, e.g., ER, AR, PARP1, FOXA1, NCOA3, as well as determine novel co-regulators of potential functional relevance.

[0364] Are AR and / or PARP1 required for ER-AR binding and transcription of mutant unique ER-AR regulated genes? PLA trapping: The inventors’ preliminary data demonstrated that ER and AR co-localized with PARP1. The inventors will use engineered cell lines and the PDX tumor models to perform PLA assays using Duolink kits per vendor instructions (OLINK BIOSCIENCE®, Uppsala Sweden).70Experiments were designed to assess the effect of treatments on binding, and include Fulv, Elac, with AR PROTAC (ARV- 110 AND EBL101) and PARP inhibitors. Quantitation methods will be the same as for comet assays.

[0365] ChlP-qPCR and qRT: These assays will be performed using the mutant models with - / +E2 treatment for 2 hrs. Cell line models with shAR silencing, or treatment with ARV-110 or PARP inhibitors will be used in ER, AR, PARP ChlP-qPCR of classic E2 regulated genes (PR, pS2, myc) as well as the 8 mutant signature genes to be described in Table 11 of Aspect 3. For statistical power, five replicates will be used for each assay. Since E2 has differential effects on PARP occupancy dynamics in mutant cells, the inventors will concurrently perform300527252.1 - 114 -BAYM.P0443WO / BLG 25-012 qRT PCR assays to determine effects of AR silencing or PARP inhibitor treatment on RNA expression of the mutant unique E2-regulated targets.

[0366] Do PARP inhibitors block ER-AR co-localization in mutant models? AR / ER / PARP1 co-localization: The inventors’ preliminary data showed that the majority of co-localization in WT P cells was seen between ER-PARP. AR co-localized with the heterochromatin marker H3K9me3 in mutant cells (YS1 and YS30 MCF-7 Y537S clones) with E2 treatment. In mutant cells (YS1, YS30 and LTED) the inventors also observed elevated levels of PARP bound to AR (data not shown). These intriguing results suggested a redistribution of a subpopulation of AR and PARP in mutant cells with E2 treatment. The inventors will use the mutant engineered models to extend these preliminary data. The inventors will also test whether a cytoskeletal (CSK) buffer for removal of soluble cytoplasmic proteins and loosely held nuclear proteins will reveal more stable associations of some transcription factors with chromatin.87Imaging will be performed in an automated fashion capturing a large number of cells to increase statistical power (>1000 cells / condition across a minimum of 4 technical replicates and three biological replicates). CellProfiler will be used to analyze individual z-planes for co-localization. Statistical analysis will be performed using non-parametric tests depending on the number of experimental groups to compare. For AR / ER / heterochromatin the primary H3K9me3 antibody allows for segmenting nuclear subregions (heterochromatin vs euchromatin) at high precision.

[0367] Expected Outcomes. The inventors reasoned that ER-AR collaborate at the cistrome level to drive metastatic phenotypes in mutant tumors. The results from the experimental plan pose to discover new insights in the epigenetic modulation of ESRlm, such as a role for PARP1.

[0368] Potential Problems and Alternative Strategies. Motif enrichment using HOMER and MEME and mining of a large compendium of human chip-seq datasets using CistromeDB might reveal other co-factors bound at areas of chromatin altered by PARP1 inhibitors. Alternative assays include PARP1 ELISA experiments and co-immunoprecipitation assays. The inventors also plan to assess if AR sumoylation might be involved in chromatin reprogramming in mutant cells as an alternative approach using recombinant in vitro PARylation assays on mutant ER and AR.Aspect 3. To evaluate mutant-specific genes as therapeutic targets, a gene signature for prognosis of survival after metastatic progression, and therapeutic300527252.1 - 115 -BAYM.P0443WO / BLG 25-012 efficacy of ET combined with PARP inhibitors to block spontaneous metastatic dissemination.

[0369] Introduction and Preliminary Data: The inventors’ goals for Aspect 3 are to evaluate ER / AR mutant-regulated genes for their role in predicting MBC outcomes and as new therapeutic targets. A second major goal is to test the therapeutic efficacy of ET + PARP inhibitors to block metastatic dissemination in vivo.

[0370] Integration of ChlP-Seq with mutant transcriptomes to generate a mutant gene signature predicting metastatic outcomes: To determine the relationship between the ER / AR E2 co-bound sites (FIG. 10A), gene expression and clinical outcomes in breast cancer, the inventors integrated the YS1 ER / AR co-bound sites with the differentially-expressed E2- treated mutant transcriptome12and identified a 65 gene signature that predicted poor diseasespecific survival and overall survival in ER+ primary breast cancer patients using the METABRIC database88(not shown). Next, the inventors determined which of the 65 genes exhibited elevated gene expression in a cohort of metastatic tumors (N=97), compared to primary tumors (N=276).89Significantly elevated expression of 10 genes was seen in the metastatic tumors; representative genes (NCOA3, BMP7, N4BP3, and FOXA1 are shown in FIG. 15A. This analysis allowed for refinement to a 10 gene signature that predicted primary breast cancer metastasis-specific metastasis to bone and lung6(FIG. 15B). These results demonstrated that the gained ER / AR E2 co-bound sites occured at transcriptionally active regions enriched in MBC patients with poor outcomes. Two of the genes were not uniquely upregulated in mutant cells and were removed, resulting in a novel 8 gene mutant unique E2- regulated signature (Table 11). Kaplan-Meier analysis using the TCGA data cohort with IHC+ ER staining showed that the 8 gene mutant signature significantly identified patients with poor outcomes (FIG. 16A). Further, the signature enabled prediction of 10-year survival with an area under the curve (AUC) of 80% or higher (FIG. 16B). In a new collaboration with BostonGene, the inventors validated the mutant signature in >3,000 primary invasive breast cancer patients of no specific type (NST) and found highly significant association with poor outcomes. Some of these genes, NCOA390and FOXA1,86have established roles in metastasis. CD24 is an immune checkpoint protein that is regulated by PARP I.91BMP7 increases metastatic potential.92N4BP3 promotes breast cancer metastasis via E-cadherin.93PDE6A is associated with bone, brain and lung metastasis of breast cancer.94Since the mutant 8 gene signature was developed using only the Y537S mutant, the inventors wanted to extend the analyses to the D538G and other Y537 mutants as well. The goals in Aspect 3 are to first300527252.1 - 116 -BAYM.P0443WO / BLG 25-012 evaluate the signature using RNA-Seq from additional L536P, D538G and Y537C cell lines and PDX organoid (PDXO) models, to investigate 4 of the genes as therapeutic targets, correlate the inventors’ RNA-Seq data with CPTAC proteomics, and then determine whether the mutant gene signature is a significant prognostic factor in available MBC cohorts as a potential companion diagnostic for PARP inhibitor use in MBC.Table 11. AR-ER mutant 8 gene signature:

[0371] Therapeutic efficacy of Fulv+ PARP inhibitors in vivo: The inventors determined the effect of PARP inhibitors on mutant growth in an in vivo experiment testing Olap single agent treatment in the Y537S MCF7 CRISPR YS1 model (FIG. 17A); metastasis was completely blocked with Olap. Next, MCF7 P PalboRand Y537S PalboRcells17were grown as tumor xenografts, treated with Palbo in vivo as first treatment, primary tumors excised, and then grown ex vivo as organoids for high-throughput therapeutic studies (FIG. 17B). Both P and YS1 PalboRcells were resistant to Fulv. Surprisingly, Fulv in combination with Olap significantly and dramatically reduced organoid growth of PalboRmodels. These results not only highlighted the inventors’ unique model resources, but also suggested that Olap may be beneficial to block metastasis and also useful in tumors that have acquired resistance to first- line treatment with a CDK4 / 6 inhibitor. These novel results showed significant potential clinical relevance. The inventors will use these new resources to evaluate therapeutic efficacy of PARP inhibitors in vivo in Aspect 3.

[0372] Research Design. The inventors will investigate the mutant metastatic signature as targets using approved therapeutics and integrate their RNA-Seq data with CPTAC to uncover proteomic correlates. The inventors will evaluate the mutant signature using data from additional metastatic cohorts to define a clinically useful (sensitive and specific) prognostic300527252.1 - 117 -BAYM.P0443WO / BLG 25-012 signature associated with OS from metastatic diagnosis. The inventors will explore the use of PARP inhibitors in combination with Fulv or Elac in ESRlm PDX models determining effects on primary tumor growth and metastatic outcomes.

[0373] Methods: Evaluation of mutant gene signatures as therapeutic targets: The inventors utilized the Drug Gene Interaction database to identify approved drugs for 4 of the signature genes. Tamoxifen is approved for CD24 and NCOA3; etoposide and methotrexate are approved for NCOA3; and the inventors also have access to NCOA3 small molecule inhibitors.22Prednisone, cytarabine, daunorubicin and doxorubicin are approved for BMP7. Pentoxifylline is approved for PDE6A. The inventors will evaluate the effects of these inhibitors on established PDXOs as described in Aspect 1 and confirm effects on targets using Western blots. The inventors have also obtained cultured PDXOs UCD4 (D538G), UCD12 (WT) from the University of Colorado and HCI005 and HCI007 (both L536P) from the University of Utah. The inventors will test for negative effects on organoid growth and if effective will transplant in vivo to test for effects on growth and spontaneous metastasis using transduced pLenti CMV Puro luciferase (ADDGENE®) for imaging. Treatments will include controls (tamoxifen, Fulv or Elac), since all 4 genes are E2-regulated, and the approved agents listed above.

[0374] Clinical Proteomics Tumor Analysis Consortium (CPTAC) to Uncover Proteomic Correlates: CPTAC has mass-spectrometry-based proteomics integrated with RNA-Seq on primary breast cancer samples.95CPTAC proteomic analyses have revealed pathways associated with chemotherapy resistance in triple negative breast cancer.96The inventors will explore proteogenomic and matched transcriptomic pathway signatures associated with the 8 gene signature using CPTAC databases. Proteomic analyses could identify novel clinical targets for intervention of ER+ metastatic progression based on the mutant signature genes.

[0375] Signature evaluation in MBC cohorts to predict further progression and survival: In the metastatic breast cancer setting only circulating tumor cells (ctDNA) have been shown to be prognostic for OS; thus no prognostic signatures exist for MBC survival.97NCCN Guidelines Version 2.2024 Breast Cancer recommend biopsy of at least first recurrence and next generation sequencing for biomarker evaluation, thus tissues should become available for analysis. First, the inventors will validate overexpression of the signature in primary vs. metastatic tumors using the AURORA US RNA-Seq cohort67. The inventors will then use the published MET500 (N=16% ESRlm) and POG570 metastatic datasets to further evaluate the signatures for significance in the metastatic setting.98’99Finally, through access to a number of primary and MBC RNA-Seq datasets, signature scores in patients will be calculated utilizing300527252.1 - 118 -BAYM.P0443WO / BLG 25-012 the sum-of-z scores method. Stratification of patients will compare the top vs bottom 50% of signature expression as high vs low expression respectively.100P-values will be calculated utilizing the log-rank calculation. Finally, patients will be stratified based on the gene signature activity score to assess survival using the log-rank test and Cox proportional hazards test as implemented in the survival101R package.

[0376] Data Analysis Plan: 1) Elucidate a protein-level role for each of the 8 genes: the inventors will leverage the primary breast cancer transcriptomic and proteomic data compiled by CPTAC95For each of the 8 genes, the inventors will first stratify all CPTAC breast cancer samples in top and bottom 50% based on its gene expression, then will use the matching proteomics profiles and compute differential protein expression. The inventors will use a t-test with significance at FDR<0.05. The inventors will further analyze the proteomic signatures for enriched pathways and for protein-protein network interactions using STRING. 2) Based on integration ofATAC-Seq and RNA-Seq (Aims 1 and 2) over multiple models, the inventors will rank the 8 genes based on the number of cell lines where they display both gene and chromatin changes. 3) By using small molecule inhibitors and approved drugs. The inventors determined for 4 of the genes potential drug to target said genes. The inventors will target each gene in multiple breast cancer PDXO models. The inventors will score and rank the 4 genes based on the number of breast cancer models where they respond to drug targeting. 4) Use MBC datasets to determine expression of the 8 genes. Using the AURORA and the Symmans datasets, the inventors will determine how many MBC biopsies overexpress each of the 8 genes compared to primary BC, whereas in the MET500 and POG570 the inventors will determine how many biopsies express each of the 8 genes. Overall, the inventors will rank the 8 genes based on how many patients ’ biopsies across all cohorts over-express or express each of the genes. 5) Use machine learning to develop an overall survival (OS) classifier in the AURORA MBC dataset. The ivnentors will model OS at 1 year, 3 years, 5 years and 10 years. The inventors will use RandomForest to train a model, with 100 cross-validation iterations with data split in 80% training and 20% testing and use Area Under the Receiver Operating Characteristics (AUROC) performance metric. Based on the ranking of genes determined above in points 2-4, the inventors determine a reduced set of genes needed for classification, then will deploy the reduced and best performing model as an R package via GitHub and a user-friendly R / Shiny application.

[0377] Testing of Drug Combinations In Vivo for Therapeutic Effects. Determination of effects on primary tumor growth: The in vivo experimental design is published.22 102The overall objectives of these experiments are to determine whether treatment with Ctrl, Fulv or 300527252.1 - 119 -BAYM.P0443WO / BEG 25-012Elac, or combinations with Olap or Tala (7 treatment arms) will decrease primary tumor and distant metastatic frequency in 4 PDX models (Table 10). Transplantation has been described.35. Primary tumor growth will proceed until tumors reach 350 mm3. Mice (N=10 per group) will then be randomized to treatment arms until tumors reach 800 mm3. Time to various events such as a fold change in tumor size (i.e. doubling or tripling), or treatment-induced regression will be analyzed using survival analysis methods, such as Kaplan-Meier survival curves and generalized Wilcoxon test. The main objective for the primary tumor phase of the experiment is whether any of the treatments will significantly block tumor growth and affect proliferation, cell cycle arrest, or apoptosis. Metastatic outcomes of in vivo experiments: When primary tumors reach 800 mm3(-30-60 days post randomization), primary tumors will be surgically removed using survival surgery (SS), treatments will continue, and the animals observed for the development of distant metastases by following their weight, image analysis (fluorescently tagged expression vectors are employed), or moribund behavior. Primary tumors and internal tissues will be obtained, and examined for metastatic deposits by H&E, fluorescent imaging, and IHC staining for Ki67, annexin V, ER, AR, PARP-1, FOXA1, NCOA3, N4BP3 and CD24. The inventors will evaluate markers of DNA damage using immunofluorescence to RAD-51 and yH2AX3. These experiments are applicable for translation into a clinical trial in MBC patients.

[0378] Expected Outcomes and Future Directions. The inventors assert that these preclinical studies will provide clinically relevant treatment information and advance the translation of these studies to the clinic. The inventors anticipate building on the preclinical data from Aspect 3 and propose a clinical trial to include a 6 patient lead-in safety cohort followed by a single-arm phase II study of Olap+Fulv in ER / PR+ MBC following progression on CDK4 / 6 inhibitors, regardless of gBRCAl / 2 mutation status. The primary endpoint will be 6-month PFS rate with second endpoints including objective response rate (ORR), clinical benefit rate (CBR), safety, and toxicity. Exploratory endpoints will include blood and tissue samples for potential biomarkers of response. The inventors reasoned that the combinations of Olap+Fulv will increase the PFS rate at 6 months compared to historical trials with Fulv alone following progression on CDK4 / 6 inhibitors while resulting in an acceptable safety and toxicity profile.

[0379] Potential Problems and Alternative Strategies. If machine learning for metastasis-free survival at fixed time intervals does not reveal informative genes, the inventors will employ models for both even and time to event, including CoxBoost and RandomForest- survival.300527252.1 - 120 -BAYM.P0443WO / BLG 25-012Example 4

[0380] Lobular breast cancer (LBC). LBC is an understudied subtype of breast cancer, representing 12-15% of all breast cancer cases.1Unfortunately lobular cancer incidence rates are steadily increasing.2Most LBCs are ER+ and have features of good prognosis; however, they tend to have a higher risk of late recurrence.3LBCs also have a clinically challenging metastatic pattern at recurrence with a propensity to spread to unusual sites with worse outcomes, including peritoneum, gynecological organs, and the GI tract.4These tumors thus represent a biological disparity for outcomes. The molecular profile of metastatic LBC is also different from ductal carcinomas with high prevalence of E-cadherin, HER2, FOXA1 and ESRI mutations (ESRlm).5,6ESRlm were found to be mutually exclusive with these other mutations in LBC, and their mechanistic role underlying a new therapeutic vulnerability in metastatic LBC will be investigated through this experimental plan. Although LBCs are a unique subset, they are treated with endocrine therapy (ET) like other ER+ tumors in both the adjuvant and metastatic settings, but are known to be relatively resistant to tamoxifen treatment.1

[0381] ESRlm are associated with a defective replication stress (RS) repair response. The major mechanism of ET resistance in MBC is the acquisition of ER gene mutations.7,8The inventors were the first to discover the acquisition of ligand- independent ESRlm in metastatic breast cancer (MBC) patient tumor biopsies.9It is now known that ESRI mutations occur in approximately 20-60% of patients, especially those who have received an aromatase inhibitor (Al) for treatment of MBC; Al is also the preferred ET for LBC.3The inventors reasoned that acquisition of ESRlm in metastatic LBC induces hyper-proliferation which triggers replication stress (RS) and excessive DNA damage. ESRlm tumors escape from this stress by inducing a signaling network termed the RS response that coordinates cell cycle progression and DNA repair. The inventors will demonstrate that the RS repair response is defective in ESRlm tumors, which affords a novel therapeutic opportunity to block metastatic dissemination using polyADP-ribose polymerase (PARP) inhibitors.

[0382] PARP inhibitors. PARP inhibitors are currently approved for BRCA1 / 2 mutated breast, ovarian, and prostate cancer, and these agents are generally well tolerated. The inventors previously demonstrated that PARP1 is the top enriched ER DNA binding protein in ESRlm cells using proteomics analyses,10suggesting that PARP1 may play a critical role in regulating ESRlm gene expression at the chromatin level. PARP1 levels are highly expressed in ESRlm models. There is also literature demonstrating that PARPs can bind and regulate WT ER although the data are limited.11,12The inventors have solid in vivo preclinical data showing that300527252.1 - 121 -BAYM.P0443WO / BLG 25-012 inhibition of ESRlm by combining PARP inhibition and ET with fulvestrant is an effective therapeutic option for ESRlm tumors. Recently the oral selective ER downregulator (SERD) elascestrant13was FDA-approved for the ET treatment of MBC with ESRlm. The inventors’ novel data suggested that MBC patients with ESRlm may be an ER+ subtype without genomic homologous recombination defects that will be responsive to PARP inhibition as a single or combination agent. The inventors will test this question using elascestrant plus PARP inhibitors in novel LBC patient derived xenografts (PDX). patient derived organoid (PDO) models, and LBC cell lines based on preliminary data showing an ESRlm-like metastatic signature in LBCs.

[0383] Data also demonstrated that LBC cell lines can exhibit a dysfunctional DNA damage response through loss of the mediator of DNA damage checkpoint (MDC1) protein.14The inventors will show activation of the G2 / M checkpoint and DNA damage markers in ESRlm tumors concomitant with enhanced intrinsic DNA dama...

Claims

BAYM.P0443WO / BLG 25-012WHAT IS CLAIMED IS:

1. A method of treating a cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a poly ADP ribose polymerase (PARP) inhibitor and one or more cancer therapies, wherein cells of the cancer comprise one or more mutations in an estrogen receptor 1 (ESRI) gene; wherein,(a) the PARP inhibitor comprises Olaparib and / or talazoparib;(b) the cancer therapy comprises fulvestrant, PF00477736, elacestrant, and / or bavdegalutamide; and(c) the one or more mutations in an ESRI gene comprise Y537S, Y537C, Y537N, E380Q, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1.

2. A method of treating a cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a poly ADP ribose polymerase (PARP) inhibitor and one or more cancer therapies, wherein cells of the cancer comprise one or more mutations in an estrogen receptor 1 (ESRI) gene.

3. The method of claim 2, wherein the cancer therapy comprises a checkpoint inhibitor, an endocrine therapy, a growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof.

4. The method of claim 3, wherein the checkpoint inhibitor comprises a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof.

5. The method of claim 3, wherein the checkpoint inhibitor comprises ribociclib, abemaciclib, palbociclib, PF00477736, PF-477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof.

6. The method of claim 3, wherein the endocrine therapy comprises an estrogen receptor antagonist, aromatase inhibitor, a selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof.

7. The method of claim 3 or 6, wherein the endocrine therapy comprises elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof.

8. The method of claim 3 or 6, wherein the endocrine therapy comprises letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof.

9. The method of claim 3, wherein the GFR antagonist comprises lapatinib.300527252.1 - 161 -BAYM.P0443WO / BLG 25-01210. The method of claim 3 or 6, wherein the endocrine therapy comprises a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI-002, EPI- 7170, or a combination thereof.

11. The method of claim 3 or 6, wherein the endocrine therapy comprises masofaniten, bavdegalutamide, or a combination thereof.

12. The method of claim 3, wherein the translesion inhibitor comprises JH-RE-06.

13. The method of claim 3, wherein the SUMOylation inhibitor comprises TAK981.

14. The method of any one of claims 1-13, wherein the PARP inhibitor comprises Olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof.

15. The method of any one of claims 1-14, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 1:100,000, respectively.

16. The method of any one of claims 1-14, wherein the PARP inhibitor and cancer therapy are used at a ratio between or 1:1 to 100,000:1, respectively.

17. The method of any one of claims 1-16, wherein the PARP inhibitor is used at 7.5 to 200 mg / kg.

18. The method of any one of claims 1-17, wherein the cancer therapy is used is used at 7.5 to 200 mg / kg.

19. The method of any one of claims 1-18, wherein the PARP inhibitor comprises olaparib used at 50 mg / kg and the cancer therapy comprises fulvestrant used at 200 mg / kg.

20. The method of any one of claims 1-18, wherein the PARP inhibitor comprises olaparib used at 50 mg / kg and the cancer therapy comprises PF00477736 used at 7.5 mg / kg.

21. The method of any one of claims 1-20, wherein the PARP inhibitor and / or cancer therapy are comprised in a pharmaceutically acceptable carrier.

22. The method of any one of claims 1-21, wherein the one or more mutations in the ESRI gene comprise E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1.

23. The method of any one of claims 1-22, wherein the one or more mutations in the ESRI gene are homozygous.

24. The method of any one of claims 1-23, wherein the one or more mutations in the ESRI gene are heterozygous.

25. The method of any one of claims 1-24, wherein the cancer comprises functional genomic homologous recombination pathways.300527252.1 - 162 -BAYM.P0443WO / BLG 25-01226. The method of any one of claims 1-25, wherein the cancer exhibits enhanced stem-cell activity, enrichment of epithelial-to-mesenchymal transition (EMT) genes, and / or increases in GFR activity.

27. The method of any one of claim 1-26, wherein the individual previously received one or more cancer therapies.

28. The method of any one of claims 1-27, wherein the individual previously received fulvestrant, elacestrant, a CDK4 / 6 inhibitor, an mTOR inhibitor, everolimus, an aromatase inhibitor (Al), or a combination thereof.

29. The method of any one of claims 1-28, wherein the cancer is resistant to one or more cancer therapies.

30. The method of any one of claims 1-29, wherein the cancer is breast cancer (BC), primary BC, and / or metastatic BC.

31. The method of any one of claims 1-30, wherein the cancer is luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, or luminal androgen receptor BC.

32. A method of treating cancer in an individual comprising the steps of: i) identifying a gene expression signature in a sample from the individual comprising: increased expression of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof; and ii) administering an effective amount of one or more cancer therapies to the individual.

33. A method comprising: providing a therapeutically effective amount of one or more cancer therapies to an individual, wherein a sample from the individual has a gene expression signature comprising increased expression levels of one or more genes comprising FOXA1, NCOA3, RMND5B, ARFGEF2, or a combination thereof.

34. The method of claim 32 or 33, wherein the gene expression signature comprises increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, or a combination thereof.

35. The method of any one of claims 32-34, wherein the gene expression signature comprises increased expression of CD24, NCOA3, BMP7, N4BP3, FOXA1, PDE6A, RMND5B, ARFGEF2, MEPH, XPOT, or a combination thereof.

36. The method of any one of claims 32-35, wherein the gene expression signature comprises increased expression of ABCG1, AFF3, ALG8, AMFR, APH1B, ARFGEF2, ASCE2, BBS2, BCAS4, BMP7, BRIP1, CA12, CCDC117, CDC45, CDC6, CD24, CHEK2, CUX1, E2F6, FOXA1, GAB2, GPATCH4, HDAC11, HNRNPAB, IL24, IQGAP3, ITPR1,300527252.1 - 163 -BAYM.P0443WO / BLG 25-012ITGB5, KLF4, MAN1A2, MCCC2, MLPH, N4BP3, NC0A3, NDUFC2, NFYA, NHP2, NUDT21, 0GF0D1, PARD6B, PDE6A, PFKM, PPARGC1B, PREXI, RMND5B, RSPH1, SENP1, SLC25A17, SMARCE1, SNX9, SPAG9, SYNJ2, TBK1, TFF1, TFF3, TH, TOBI, TOP2A, UHRF1, UMPS, VDR, WDR35, WDR77, XPOT, ZNF385B, ZNRF3, or a combination thereof.

37. The method of any one of claims 32-36, wherein the gene expression signature further comprises increased expression of PARP1.

38. The method of any one of claims 32-37, wherein the increase in gene expression comprises a 1.1-20.0 fold increase relative to wild type cells, non-cancerous cells, primary tumor from the individual, or cells without ESRI mutations.

39. The method of any one of claims 32-38, wherein the sample comprises blood, plasma, a primary tumor biopsy, a metastatic tumor biopsy, cancer cells, or a combination thereof.

40. The method of any one of claims 32-39, wherein identifying the gene expression signature comprises measuring RNA and / or protein from the sample.

41. The method of any one of claims 1-40, wherein the cancer is BC, primary BC, and / or metastatic BC.

42. The method of any one of claims 1-41, wherein the cancer is luminal BC, ductal BC, lobular BC, invasive lobular BC, triple-negative BC, or luminal androgen receptor BC.

43. The method of any one of claims 1-42, further comprising the step of identifying a mutation in the ESRI gene.

44. The method of any one of claims 1-43, wherein the mutations in the ESRI gene comprise E380Q, Y537S, Y537C, Y537N, L536P, L536Q, L536R, D538G, or a combination thereof in reference to SEQ ID NO: 1.

45. The method of claim 43 or 44, wherein the mutation is identified with one or more of the following probes: dHsaMDS732897750 for Y537C, dHsaMDS296069817 for Y537N, dHsaMDS975379796 for Y537S, dHsaMDS460485301 for D538G.

46. The method of any one of claims 32-45, wherein the cancer therapy comprises a PARP inhibitor, one or more cancer therapies, or a combination thereof.

47. The method of claim 46, wherein the cancer therapy comprises a checkpoint inhibitor, an endocrine therapy, a growth factor receptor (GFR) antagonist, a translesion inhibitor, a SUMOylation inhibitor, or a combination thereof.

48. The method of claim 47, wherein the checkpoint inhibitor comprises a CDK4 / 6 inhibitor, Chkl / 2 inhibitor, ATR inhibitor, and / or Chkl inhibitor, or a combination thereof.300527252.1 - 164 -BAYM.P0443WO / BLG 25-01249. The method of claim 47, wherein the checkpoint inhibitor comprises ribociclib, abemaciclib, palbociclib, PF00477736, PF-477736, gartisertib, prexasertib, ceralasertib, berzosertib, or a combination thereof.

50. The method of claim 47, wherein the endocrine therapy comprises an estrogen receptor antagonist, aromatase inhibitor, an selective androgen receptor degrader (SARD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a combination thereof.

51. The method of claim 47 or 50, wherein the endocrine therapy comprises elacestrant, fulvestrant, toremifene, raloxifene, camizestrant, imlunestrant, giredestrant, vepdegestrant, or a combination thereof.

52. The method of claim 47 or 50, wherein the endocrine therapy comprises letrozole, tamoxifen, anastrozole, exemestane, or a combination thereof.

53. The method of claim 47, wherein the GFR antagonist comprises lapatinib.

54. The method of claim 47, wherein the endocrine therapy comprises a PROTAC, bicalutamide, enzalutamide, apalutamide, darolutamide, EPI-001, EPI-506, EPI-002, EPI- 7170, or a combination thereof.

55. The method of claim 3 or 6, wherein the endocrine therapy comprises masofaniten, bavdegalutamide, or a combination thereof.

56. The method of claim 47, wherein the translesion inhibitor comprises JH-RE-06.

57. The method of claim 47, wherein the SUMOylation inhibitor comprises TAK981.

58. The method of any one of claims 46-57, wherein the PARP inhibitor comprises olaparib, talazoparib, AZD5303 (Sanuparib), or a combination thereof.

59. The method of any one of claims 46-58, wherein the PARP inhibitor is used at 7.5 to 200 mg / kg.

60. The method of any one of claims 32-59, wherein the cancer therapy is used is used at 7.5 to 200 mg / kg.

61. The method of any one of claims 46-60, wherein the PARP inhibitor and cancer therapy are used at a ratio between 1:1 to 1:100,000, respectively.

62. The method of any one of claims 46-60, wherein the PARP inhibitor and cancer therapy are used at a ratio between or 1:1 to 100,000:1, respectively.

63. The method of any one of claims 46-62, wherein cancer therapy comprises olaparib used at 50 mg / kg and fulvestrant used at 200 mg / kg.

64. The method of any one of claims 46-62, wherein the cancer therapy comprises olaparib used at 50 mg / kg and PF00477736 used at 7.5 mg / kg.300527252.1 - 165 -BAYM.P0443WO / BLG 25-01265. The method of any one of claims 46-64, wherein the PARP inhibitor and / or cancer therapy are comprised in a pharmaceutically acceptable carrier.

66. The method of any one of claims 32-65, wherein the cancer therapy comprises tamoxifen if the gene signature comprises increased expression of at least CD24 and / or NCOA3.

67. The method of any one of claims 32-66, wherein the cancer therapy comprises etoposide, methotrexate, SI- 10, SI- 12, or a combination thereof if the gene signature comprises increased expression of at least NCOA3.

68. The method of any one of claims 32-67, wherein the cancer therapy comprises prednisone, cytarabine, daunorubicin, doxorubicin or a combination thereof if the gene signature comprises increased expression of at least BMP7.

69. The method of any one of claims 32-68, wherein the cancer therapy comprises pentoxifylline if the gene signature comprises increased expression of at least PDE6A.300527252.1 - 166 -

Images