RB1 deficiency as a predictive biomarker for treating cancer with wee1 inhibitors

Abstract

The present disclosure provides, among other things, methods for treating cancer comprising administering an effective dose of a WEE1 inhibitor such as azenosertib, or a pharmaceutically acceptable salt thereof, to a subject selected to have a cancer associated with deficiency in the retinoblastoma transcriptional corepressor 1 (RB1) gene and / or the RB1 protein.

Description

ZENO.190WO PATENTRBI DEFICIENCY AS A PREDICTIVE BIOMARKER FOR TREATING CANCER WITH WEE1 INHIBITORSINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby expressly incorporated by reference under 37 CFR 1.57, and Rules 4.18 and 20.6, including U.S. Provisional Application No. 63 / 776,497, filed March 24, 2025, the content of which is hereby incorporated by reference in its entirety including any drawings.SEQUENCE STATEMENT

[0002] This application contains a Sequence Listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing, was created on March 19, 2026, is named ScqucnccListing_ZENO190.xml and is 2,925 bytes in size.BACKGROUNDField

[0003] The present application relates generally to methods of using a WEE1 inhibitor to treat diseases or conditions, such as a cancer associated with a deficiency.Description

[0004] Triple -negative breast cancer (TNBC) is a heterogeneous group of tumors characterized by aggressive behavior, high risk of distant recurrence and poor survival. Chemotherapy is still the main therapeutic approach for this subgroup of patients, therefore, progress in the treatment of TNBC remains an important challenge. Data derived from molecular technologies have identified TNBCs with different gene expression and mutation profiles that may help developing targeted therapies. So far, however, only a few of these have shown to improve the prognosis and outcomes of TNBC patients. Likewise, small cell lung cancer (SCLC) is also characterized by genetic diversity i.e., heterogeneity. While it responds well to chemotherapy initially, it soon develops drug resistance. Late diagnosis, rapid tumor growth, and drug resistance, among other things, lead to poor overall survival. Thus, robust predictive biomarkers to accelerate clinical progress in treating TNBC and SCLC are needed.ZENO.190WO PATENTSUMMARY

[0005] Provided herein is the discovery that WEE1 inhibitors (e.g., azenosertib or ZN-c3, including pharmaceutically acceptable salts thereof) can effectively treat cancers associated with deficiency in the retinoblastoma transcriptional corepressor 1 (RBI) gene and / or the RBI protein, including small cell lung cancer (SCLC) and / or triple negative breast cancer (TNBC). In other words, it is found that cancers that are associated with deficiency in the RBI gene and / or the RB 1 protein exhibit better overall response to treatment with a WEE1 inhibitor in the clinic, as compared to cancers that lack such deficiency.

[0006] Accordingly, in a first aspect, provided herein is a method of treating a cancer comprising administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, to a subject selected to have a cancer associated with deficiency in the retinoblastoma transcriptional corepressor 1 (RBI) gene and / or the RBI protein, wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC). In a second aspect, provided herein is use of azenosertib, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer in a subject with an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, wherein the subject is / was / has been selected to have a cancer associated with deficiency in the RBI gene and / or the RBI protein, and wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC). In a third aspect, provided herein is azenosertib, or a pharmaceutically acceptable salt thereof, for use in treating cancer in a subject with an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, wherein the subject is / was / has been selected to have a cancer associated with deficiency in the RBI gene and / or the RBI protein, and wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC).

[0007] Other features, objects and advantages are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Drawings are for illustration purposes only and not for limitation.ZENO.190WO PATENT

[0009] FIGS. 1A-1D are graphs showing cell viability and growth rate inhibition of various Bl-wl or RR7-mutated small cell lung cancer (SCLC) and triple negative breast cancer (TNBC) cells being treated with azenosertib in vitro.

[0010] FIGS. 2A-2D are graphs showing in vivo tumor volume changes in mice inoculated with RBl-wt or RBI -mutated SCLC or TNBC cells and treated with azenosertib.

[0011] FIGS. 3A-3D are graphs showing cell viability and growth rate inhibition of shCtrl (i.e., RBI-v.l) or shRBl-1 (i.e., RBI gene knockdown) SCLC and TNBC cells being treated with azenosertib in vitro. shRBl -1 indicates human small cell lung cancer (SCLC) RB1-wt DMS53 and triple negative breast cancer (TNBC) R I-wi MDA-MB-231 cells that were transduced with lentiviral particles expressing a short-hairpin ribonucleic acid (shRNA) specific for the knockdown of the RBI gene. shCtrl indicates human small cell lung cancer (SCLC) RBJ-wt DMS53 and triple negative breast cancer (TNBC) RBl-wt MDA-MB-231 cells that were transduced with non-silencing shRNA (i.e., control shRNA).

[0012] FIG. 4A shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBl-wt or RBI gene knockdown SCLC cells in response to azenosertib treatment at three different concentrations. FIG. 4B shows Western blot of RBI protein expression confirming expression and lack of expression in RBl-wt or RBI knockdown SCLC cells, respectively, in response to azenosertib treatment at the three different concentrations. FIG. 4C shows quantification of pCHKl, yH2AX and c-Casp-3 / 7 protein levels of RBl-wt or RBI gene knockdown SCLC cells in response to azenosertib treatment at the three different concentrations.

[0013] FIG. 4D shows Western blot of RB 1 protein expression in RBI -wt or RBI gene knockdown TNBC cells in response to azenosertib treatment at the three different concentrations. FIG. 4E shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBl-wt or RBI gene knockdown TNBC cells in response to azenosertib treatment at three different concentrations. FIG. 4F shows quantification of pCHKl, yH2AX and c-Casp-3 / 7 protein levels of RBl-wt or RBI gene knockdown TNBC cells in response to azenosertib treatment at the three different concentrations.

[0014] FIGS. 5A-5F are graphs showing cell viability and growth rate inhibition of RBI -mutated SCLC cells with forced overexpression of wild-type RBI (induced with varying concentrations of doxycycline) being treated with azenosertib.ZENO.190WO PATENT

[0015] FIGS. 5G-5L are graphs showing cell viability and growth rate inhibition of RBI -mutated TNBC cells with forced overexpression of wild-type RBI (induced with varying concentrations of doxycycline) being treated with azenosertib.

[0016] FIG. 6A shows Western blot of RB 1 protein expression of RBI inducible overexpression RB7-mutated SCLC in response to azenosertib treatment at the different concentrations of azenosertib and doxocycline. FIG. 6B shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBI inducible overexpression (IOE) RBI-mutated SCLC cells in response to azenosertib treatment at different concentrations of azenosertib and doxocycline (Dox). FIG. 6C shows quantification of pCHKl, yH2AX and c-Casp-3 / 7 protein levels of RBI inducible overexpression RBI -mutated SCLC cells in response to azenosertib treatment at the different concentrations of azenosertib and doxocycline.

[0017] FIG. 6D shows Western blot of RB 1 protein expression of RBI inducible ovcrcxprcssion RBI -mutated TNBC in response to azenosertib treatment at the different concentrations of azenosertib and doxocycline. FIG. 6E shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBI inducible overexpression RBI -mutated TNBC cells in response to azenosertib treatment at different concentrations of azenosertib and doxocycline. FIG. 6F shows quantification of pCHKl, yH2AX and c-Casp-3 / 7 protein levels of RBI inducible overexpression RBI -mutated TNBC cells in response to azenosertib treatment at the different concentrations of azenosertib and doxocycline.

[0018] FIGS. 7A-7D show 2D heatmaps of in vitro synergy and inhibition of cell viability between azenosertib and docetaxel in RBl-v (FIGS. 7A and 7B) or RBI gene knockdown (FIGS. 7C and 7D) TNBC cells. FIGS. 7E-7H show 2D heatmaps of in vitro synergy and inhibition of cell viability between azenosertib and carboplatin in RBl-wt (FIGS.7E and 7F) or RBI gene knockdown (FIGS. 7G and 7H) TNBC cells. FIGS. 7L7L show 2D heatmaps of in vitro synergy and inhibition of cell viability between azenosertib and irinotecan in RBJ-wt (FIGS. 71 and 7J) or RBJ gene knockdown (FIGS. 7K and 7L) TNBC cells.

[0019] FIG. 8A shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBl-v / t or RBI gene knockdown TNBC cells in response to treatment with azenosertib and docetaxel. FIG. 8B shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RBI -wt or RBI gene knockdown TNBC cells in response to treatment with azenosertib and carboplatin. FIG.8C shows Western blot of protein expression of various pharmacodynamic (PD) biomarkers of RB7-wt or RBI gene knockdown TNBC cells in response to treatment with azenosertib and irinotecan.ZENO.190WO PATENTDETAILED DESCRIPTIONDefinitions

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0021] As used herein, the term “about” has its usual meaning as understood by those skilled in the art and thus indicates that a value includes the inherent variation of error (i.e., ±10%) for the method being employed to determine a value, or the variation that exists among multiple determinations.

[0022] As used herein, the terms “function” and “functional” have their usual meaning as understood by those skilled in the art and thus refer to a biological, enzymatic, or therapeutic function.

[0023] The term “mutation” has its usual meaning as understood by those skilled in the art and refers to an alteration of genetic sequence, by nucleotide substitution, nucleotide insertion and / or nucleotide deletion. In some embodiments, cells have mutation(s), such as 1, 2, 3, 4, 5. 6 or more than 6 mutations. In some embodiments, mutations are in coding regions of the genome. Mutations can range in size from a single base-pair, to a large segment of the chromosome that includes multiple genes, such as 1, 2, 3, 4, 5, 6 or more than 6 genes. In some embodiments, at least one mutation is silent. In some embodiments, the mutation may have no significant impact on gene expression or function. In some embodiments, at least one mutation has an impact on gene expression or function, such as gene amplification, overexpression or enhanced copy number. In some embodiments, at least one mutation is silent (e.g. , not changing the coding sequence). In some embodiments, at least one mutation has a small impact on protein expression or function. In some embodiments, at least one mutation has a moderate impact on protein expression or function. In some embodiments, at least one mutation has a large impact on protein expression or function. In some embodiments, at least one mutation prevents protein expression or function. Non-limiting examples of mutations include insertions, deletions, truncations, substitutions, duplications, translocations and inversions. In some embodiments, mutations are “somatic,” or occurring in body cells and are not inheritable. In some embodiments, a subset of somatic cells in an organism have at least one mutation that other somatic cells do not have. In some embodiments, mutations are “germline,” or occurringZENO.190WO PATENTin germ cells and are inheritable. Mutations can also be categorized as pathogenic or likely pathogenic, which means that such mutations are likely to cause disease, such as cancer.

[0024] As disclosed herein, mutations, including mutations in the RBI gene, can be monitored through a variety of sequencing, expression or functional assays. Non-limiting examples include DNA sequencing, RNA sequencing, DNA hybridization, protein sequencing, targeted genomic sequencing, whole exome sequencing, whole genome sequencing, ATAC-sequencing, Sanger sequencing, PCR, qPCR, RT-PCR, RT-qPCR, Next Generation Sequencing, protein truncation test, DNA microarrays, heteroduplex analysis, denaturing gradient gel electrophoresis, nucleotide sequencing, single strand conformational polymorphism, restriction enzyme digestion assay, fluorescence in situ hybridization (FISH), comparative genomic hybridization, restriction fragment length polymorphism, amplification refractory mutation system PCR, nested PCR, multiplex ligation-dependent probe amplification, single strand conformational polymorphism and oligonucleotide ligation assay. Mutations can also be monitored through a variety of antibody-based methods using biological samples including, but are not limited to, Western blotting, fluorescence activated cell sorting, immunofluorescence, immunohistochemistry, immunocytochemistry, immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassays and electrochemiluminescence assays.

[0025] The terms “individual”, “subject” or “patient” as used herein have their usual meaning as understood by those skilled in the art and thus includes a human or a nonhuman mammal. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea or pigs. In some embodiments, the subject can be human. In some embodiments, the subject can be a child (i.e., > 1 year and < 18 years old) and / or an infant (i.e., < 1 year old). In other embodiments, the subject can be an adult (i.e., > 18 years old).

[0026] The term “pharmaceutically acceptable salt” refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), a sulfuric acid, a nitric acid and a phosphoric acid (such as 2,3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, forZENO.190WO PATENTexample formic, acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, trifluoroacetic, benzoic, salicylic, 2-oxopentanedioic or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium, a potassium or a lithium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of a carbonate, a salt of a bicarbonate, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine and salts with amino acids such as arginine and lysine.

[0027] It is to be understood that where compounds disclosed herein have unfilled valencies, then the valencies are to be filled with hydrogens or isotopes thereof, e.g. , hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0028] It is understood that the compounds described herein include crystalline forms (also known as polymorphs, which include the different crystal packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol or the like. In other embodiments, the compounds described herein exist in unsolvated form. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol or the like. Hydrates are formed when the solvent is water or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated fomis for the purposes of the compounds and methods provided herein.

[0029] The term “platinum-resistant” refers to a cancer that responds at first to treatment with a drug that contain the metal platinum (e.g., cisplatin and carboplatin), but the cancer requires treatment again (such as with a drug that does not contain the metal platinum) within a certain period of time. For example, a cancer that has a platinum-free interval (PFI) of 3 to 6 months is considered platinum-resistant. Platinum-free interval (PFI) is the time elapsed between after the last treatment dose a dmg that contain the metal platinum and detection of recurrence of the cancer. Subjects whose cancer progress while on platinum-based therapy (e.g., a tumor progresses or does not shrink) or whose cancer progresses less than 3 months after the last treatment dose a drug that contain the metal platinum are referred to as having “platinum-refractory” cancer.ZENO.190WO PATENT

[0030] The term “platinum-sensitive” refers to a cancer that responds at first to treatment with a drug that contain the metal platinum (e.g., cisplatin and carboplatin), but the cancer requires treatment again (such as with a drug that does not contain the metal platinum) within a certain period of time. For example, a cancer that has a platinum-free interval (PFI) of more than 6 months is considered platinum-sensitive. Of this group, those subjects who require treatment again more than 6 months to 12 months are referred to as having “partially platinumsensitive” cancer, and those subjects who requirement treatment again more than 12 months are referred to as having “fully platinum-sensitive” cancer.

[0031] The term “PARP inhibitor-resistant” refers to a cancer that fails to respond to treatment with a PARP inhibitor (including pharmaceutically acceptable salts thereof), including cancers that are BRCA 7 / 2-mutated or BRCA 1 / 2 -deficient.

[0032] Various aspects are described in detail in the following sections. The use of sections is not meant to limit the present disclosure. Each section can apply to any aspect of present disclosure. In this application, the use of “or” means “and / or” unless stated otherwise.Treatment of Cancers Associated with RBI Gene and / or RBI Protein Deficiency

[0033] In a first aspect, provided herein is a method of treating a cancer comprising administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, to a subject selected to have a cancer associated with deficiency in the retinoblastoma transcriptional corepressor 1 (RBI) gene and / or the RB 1 protein, wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC). In a second aspect, provided herein is use of azenosertib, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer in a subject with an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, wherein the subject is / was / has been selected to have a cancer associated with deficiency in the RBI gene and / or the RBI protein and wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC). In a third aspect, provided herein is azenosertib, or a pharmaceutically acceptable salt thereof, for use in treating cancer in a subject with an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, wherein the subject is / was / has been selected to have a cancer associated with deficiency in the RBI gene and / or the RBI protein and wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC). Collectively, all of the foregoing aspects of this disclosure are referred to as “methods and uses described herein including all aspects and embodiments thereof’ in the ensuing paragraphs.ZENO.190WO PATENT

[0034] As used herein, azenosertib, also called ZN-c3, is a compound represented by the following structure:NNH\ '\VO:H

[0035] In one embodiment, in the methods and uses described herein including all aspects and embodiments thereof, the cancer treated is SCLC. In one embodiment, in the methods and uses described herein including all aspects and embodiments thereof, the cancer treated is TNBC.The retinoblastoma transcriptional corepressor 1 RBI) gene

[0036] The loss of function of the RBJ gene has been considered to be a cause or a promoting factor of retinoblastoma. However, in recent years, a decrease in function such as loss-of-function mutations or suppression of expression of the RBI gene is reported to be observed in many human cancers, and involvement in the occurrence and progression of a tumor is attracting attention in a wide variety of cancers. The RBI protein is considered to be involved in the biology of various cancers, such as cell cycle, inflammation, metabolism, autophagy, apoptosis, differentiation, aging, DNA repair and stability of genomes.

[0037] As used herein, a cancer associated with “deficiency in the RBI gene and / or the RB 1 protein” refers to a cancer, including SCLC and TNBC, whereby one or more genetic alterations (e.g., mutations) are present in the RBI gene and / or one or more alterations in function are present in the RBI gene and / or the RB 1 protein.

[0038] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the deficiency in the RBI gene is selected from a mutation in the RBI gene, a deletion of the RBI gene, a decrease in function of the RBJ gene, a decrease or suppression of expression of the RBI gene and an epigenetic change in the RBI gene. In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the mutation in the RBI gene is selected from a nonsense mutation, a missense mutation, a frameshift mutation, a splicing mutation and a regulatory mutation. In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the deficiency in the RBI protein is selected from a truncated RBIZENO.190WO PATENTprotein, an inactive RBI protein and a decrease or suppression of expression of the RBI protein.

[0039] As used herein, a “deletion of the RBI gene” or alternatively "RBI gene deletion” refers to an alteration in which a portion of the RBI gene (e.g., a single nucleotide or a section of the RBI gene) or the entire RBI gene is lost.

[0040] As used herein, a “decrease in function of the RBI gene” refers to a genetic or epigenetic change that reduces or eliminates the normal activity of the RBI gene, which results in a diminished or absent RBI protein product or a RBI protein with impaired function.

[0041] As used herein an “epigenetic change in the RBI gene” refers to a modification to DNA (e.g., promoter), its associated non-coding RNA or its associated proteins (e.g., histones) that can influence expression of the RBI gene without altering the DNA sequence of the RBI gene. Examples of an epigenetic change in the RBI gene include methylation of the RBI promoter (including hypermethylation), misregulation of noncoding RNA and histone modification.

[0042] As used herein, a “nonsense mutation” refers to a mutation that causes the protein product to stop translating early, which occurs when nucleotide (or base pair) in a gene is substituted, which creates a stop codon where an amino acid codon should be.

[0043] As used herein, a “missense mutation” refers to a genetic alteration where a nucleotide (or base pair) is changed, resulting in the substitution of the corresponding amino acid for another in the resulting protein.

[0044] As used herein, a “frameshift mutation” refers to a genetic mutation that occurs when one or more nucleotides are inserted or deleted from a DNA sequence, which shifts the reading frame of the DNA and can lead to the production of an incorrect protein.

[0045] As used herein, a “splicing mutation” refers to a genetic change that typically occurs at the boundaries between exons (coding regions) and introns (non-coding regions), thereby disrupting the recognition sequences for splicing machinery and resulting in incorrect removal of introns or inclusion of exons in the mature mRNA.

[0046] As used herein, a “regulatory mutation” is a change in the DNA sequence that controls gene expression, rather than the coding sequence of the gene itself.

[0047] As used herein, a “loss-of-function (LOF) mutation” is a genetic alteration that impairs or eliminates the normal function of a gene or its protein product.

[0048] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the mutation in the RBI gene is a nonsense mutation.ZENO.190WO PATENT

[0049] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the mutation in the RBI gene is a loss-of-function (LOF) mutation.

[0050] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the deficiency in the RBI gene is an epigenetic change in the RBI gene. In one embodiment, the deficiency in the RBI gene or the epigenetic change in the RBI gene is methylation of the RBI promoter.

[0051] In one embodiment, the RBI protein has an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% similarity to SEQ ID NO: 1 :MPPKTPRKTAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTA LCQKLKIPDHVRERAWLTWEKVSSVDGVLGGYIQKKKELWGICIFIAAVDLDEMSFT FTELQKNIEISVHKFFNLLKEIDTSTKVDNAMSRLLKKYDVLFALFSKLERTCELIYLT QPSSSISTEINSALVLKVSWITFLLAKGEVLQMEDDLVISFQLMLCVLDYFIKLSPPML LKEPYKTAVIPINGSPRTPRRGQNRSARIAKQLENDTR11EVLCKEHECNIDEVKNVYF KNFIPFMNSLGLVTSNGLPEVENLSKRYEEIYLKNKDLDARLFLDHDKTLQTDSIDSF ETQRTPRKSNLDEEVNVIPPHTPVRTVMNTIQQLMMILNSASDQPSENLISYFNNCTV NPKESILKRVKDIGYIFKEKFAKAVGQGCVEIGSQRYKLGVRLYYRVMESMLKSEEE RLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTSQNLDSGTDLSFPWILNVLNLK AFDFYKVIESFIKAEGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDREGPT DHLESACPLNLPLQNNHTAADMYLSPVRSPKKKGSTTRVNSTANAETQATSAFQTQ KPLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEIIPELEIIIIWTLFQIITLQNEYELMRD RHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLIKEEEYDSIIVF YNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISE GLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKL RFDIEGSDEADGSKHLPGESKFQQKLAEMTSTRTRMQKQKMNDSMDTSNKEEK.

[0052] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, wherein the mutation in the RBI gene occurs at one or more nucleotides in a codon encoding an amino acid selected from E31, C61, K265, R552, S576, C706 and Q850 in SEQ ID NO:1.

[0053] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the subject is not further selected to have a cancer associated with any oncogene alteration or status, any oncoprotein expression level or statusZENO.190WO PATENTand / or any signaling pathway level or alteration, other than the deficiency in the RBI gene and / or the RBI protein.

[0054] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the subject is further selected to have a cancer associated with a TP53 mutation.Azenosertib Monotherapy, Combination Therapies, Prior Treatments

[0055] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered as monotherapy.

[0056] In alternative embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered as combination therapy, for example, azenosertib, or a pharmaceutically acceptable salt thereof, in combination with a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include altretamine, bendamustine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, mechlorethamine, melphalan, oxaliplatin, procarbazine, temozolomide, thiotepa, trabectedin, carmustin, lomustine, strepozocin, 5 -fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine / tipiracil combination, etoposide, irinotecan, imitecan liposomal, mitoxantrone, teniposide, topotecan, cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, vinorelbine, daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, mitoxantrone, valrubicin, etc. (including pharmaceutically acceptable salts of any of the foregoing). In one embodiment, in the methods and uses described herein including all aspects and embodiments thereof, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered in combination with docetaxel, or a pharmaceutically acceptable salt thereof. In one embodiment, in the methods and uses described herein including all aspects and embodiments thereof, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered in combination with carboplatin, or a pharmaceutically acceptable salt thereof. In one embodiment, in the methods and uses described herein including all aspects and embodiments thereof, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered in combination with irinotecan, or a pharmaceutically acceptable salt thereof.ZENO.190WO PATENT

[0057] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the subject has received no more than 1, at least 1, 1 or 2, 1 to 3, or 1 to 4 prior line(s) of therapy. In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the therapy is selected from surgery, radiation, chemotherapy, immunotherapy and targeted drug therapy.

[0058] In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the cancer treated is platinum-resistant or platinum-refractory. In some embodiments, in the methods and uses described herein including all aspects and embodiments thereof, the cancer treated is PARP inhibitor-resistant.Effective Doses and Dosing Schedules

[0059] As used herein, the terms “treat,” “treating,” “treatment,” “therapeutic,” and “therapy” do not necessarily mean total cure or abolition of the disease and / or condition, such as osteosarcoma. Any alleviation of any undcsircd signs or symptoms of the disease and / or condition (such as osteosarcoma), to any extent can be considered treatment and / or therapy. Eurthermore, treatment may include acts that may worsen the subject’s overall feeling of wellbeing or appearance.

[0060] The term “effective dose” is used to indicate an amount of azenosertib, or a pharmaceutically acceptable salt thereof, that elicits the biological or medicinal response indicated. For example, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, can be the amount needed to prevent, alleviate or ameliorate symptoms of cancer, or prolong the survival of the subject being treated. This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the cancer being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein. The effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, required as a dose will depend on the route of administration and the physical characteristics of the human subject under consideration. The effective dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. In one embodiment, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, refers to an amount, including, but not limited to, a minimum amount of azenosertib, or a pharmaceutically acceptable salt thereof, that is sufficient to result in >10% (e.g., >15%, >20%, >25%, >30%, >35%, >40%) of a statistically significant population of subjects (e.g., at leastZENO.190WO PATENT30 subjects) achieving an at least 16-week event-free survival (EES) per RECIST vl.l (i.e., time from treatment initiation until disease progression or death due to any cause).

[0061] In one embodiment, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, refers to an amount, including, but not limited to, a minimum amount, of azenosertib, or a pharmaceutically acceptable salt thereof, that is sufficient to result in a statistically significant population of subjects (e.g., at least 30 subjects) achieving and maintaining a stable disease (SD) status for at least 16 weeks.

[0062] In one embodiment, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, refers to an amount, including, but not limited to, a minimum amount, of azenosertib, or a pharmaceutically acceptable salt thereof, that is sufficient to result in a statistically significant population of subjects (e.g., at least 30 subjects) achieving a disease control rate (DCR) of >40% (e.g., >45%, >50%, >55%, >60%, >65%).

[0063] In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 350 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 400 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 450 mg, or an equivalent thereof, in one or more dosing weeks. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 500 mg, or an equivalent thereof, in one or more dosing weeks.ZENO.190WO PATENT

[0064] In some embodiments of the methods described herein, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered to the subject once a day (QD). In alternative embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered to the subject twice a day (BID). The QD and / or BID dosing regimens may be varied from one dosing week to another.

[0065] In some embodiments of the methods described herein, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on a continuous dosing schedule (e.g., 7 days a week with no intermission). In some embodiments of the methods described herein, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on an intermittent dosing schedule, during which one or more dosing parameters, such as dosage amount and / or dosage interval are varied or changed. For example, an intermittent dosing phase may comprise a period of continuous administration (e.g., 6 days, 5 days, 4 days, 3 days, 2 days in a week) followed by a “rest” or intermission phase (e.g., 1 day, 2 days, 3 days, 4 days, 5 days in the same week) during which azenosertib, or a pharmaceutically acceptable salt thereof, is not administered or is administered at a reduced dosage amount and / or less frequently. A dosing regimen may further comprise one or more repeated cycles of intermittent dosing regimens. In one embodiment, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on an intermittent dosing schedule comprising 5 days with dosing and 2 days without dosing in each of the one or more dosing weeks. In one embodiment, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on an intermittent dosing schedule comprising 5 consecutive days with dosing and 2 consecutive days without dosing in each of the one or more dosing weeks. In one embodiment, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on an intermittent dosing schedule comprising 4 days with dosing and 3 days without dosing in each of the one or more dosing weeks. In one embodiment, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is provided on an intermittent dosing schedule comprising 4 consecutive days with dosing and 3 consecutive days without dosing in each of the one or more dosing weeks. In one embodiment, the methods described herein include administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 6 days on / 1 day off, 5 days on / 2 days off or 4 days on / 3 days off intermittent dosing schedule in one or more dosing weeks. In one embodiment, the methods described herein include administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 5 days on / 2 days off intermittent dosing schedule in one or more dosing weeks. In one embodiment, the methods described herein include administering theZENO.190WO PATENTeffective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 4 days on / 3 days off intermittent dosing schedule in one or more dosing weeks. As used herein “on” day(s) means receiving an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, and “off’ means not receiving an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof.EXAMPLES

[0066] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.Example 1RBI gene mutations sensitize SCLC and TNBC models to azenosertib in vitro and in vivo Table 1. SCLC and TNBC models described in Examples 1-4.Indi Model RBI gene TP53 gene -cation Variation Function Variation Function - S241F Likely LOF DMS53 wtE56Ter Termination SW1571 wt - C277F Likely LOF H209 C706F Likely LOF V225fs Frameshift H526 E31Ter Termination S33fs Frameshift R273C Likely LOF SCLC Hl 048 R552Ter TerminationDEL P47RfsTer76 H1963 C61Ter Termination V147D Likely LOF H446 SNV - G154V Likely LOF H510 K265Ter Termination R282G Likely LOF H524 S576Ter Termination T155N Likely LOF H146 Q850Ter Termination DEL P318LfsTer21 MDA-MB- wt - R280K LOF 231TNBC MDA-MB- c. 265_2787del2523 Termination R273H LOF 468BT549 c. 265_607del343 Termination R249S Likely LOF

[0067] To assess the in vitro efficacy across different cell lines, 9 SCLC cell lines and 3 TNBC cell lines were treated with azenosertib as a single agent, and cell viability and growth rate (GR) inhibition were measured. To compare the in vitro efficacy between cancer models having RBI gene deficiency versus cancer models having wild-type RBI gene, two ofZENO.190WO PATENTthe 9 small cell lung cancer (SCLC) cell lines (i.e., DMS53 and SW1271) and one of the 3 triple negative breast cancer (TNBC) cell lines (i.e., MDA-MB-231) studied in this Example each contained the native RBI gene (R57-wt). Details of the RBI gene status of all models described in Examples 1 -4 are provided in Table 1.

[0068] The results in FIGS. 1A-1D indicate that the cytotoxic effect of azenosertib as a single agent was present in all of the SCLC and TNBC models, but the cytotoxic effect in terms of both cell viability and growth rate inhibition was consistently higher in both SCLC and TNBC models where the RBI gene is deficient (i.e., one form of RBI gene deficiency). The IC50 and GR50 values derived from the cell viability and growth rate inhibition curves in FIGS. 1C and ID were lower in RBI -mutated TNBC cells as compared to the RBl-wt TNBC cells, thereby corroborating the observation that RBI -mutated TNBC cells were more sensitive to azenosertib treatment.

[0069] 6-8 weeks old BALB / c nude (DMS53 that is RB 1 -wt SCLC; MDA-MB-231 that is RBl-wt TNBC), NOD / SCID (NCI-H146 that is RB7-mutatcd SCLC), or SCID (MDA-MB-468 that is A7i / -mulatcd TNBC) mice were inoculated subcutaneously on the right flank with the single cell suspension of 95% viable tumor cells. Mice were quarantined and acclimatized to the laboratory environment for 7 days before the study and housed in an environmentally monitored, well-ventilated room maintained at a temperature of (23 ± 3 °C) and relative humidity of 40% to 70%, were exposed to 12 h of light and dark cycles and were supplied with food and water ad libitum. Mice were injected subcutaneously with tumor cells in 200 pL of media and Matrigel mixture (1:1 ratio) in the right flank with 2 x 107cells. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumors were measured twice a week, and tumor volume (TV) measurements were determined utilizing the formula A x B2 / 2 in which A and B are long and short diameters of a tumor, respectively. The TVs are used for the calculation of the tumor growth inhibition (TGI, an indicator of antitumor effectiveness) value using the formula: (l-(Td - To) / (Ca - Co)) x 100%. Ta and Ca are the mean tumor volumes of the treated and control animals, and To and Co are the mean tumor volumes of the treated and control animals at the start of the experiment. When tumors reached an average tumor volume of approximately 200 mm3, mice were randomized into treatment groups. Mice were treated with drugs orally or intraperitoneally. The experiment was terminated when the mean tumor volume exceeded 2,000 mm3or severe body weight loss was observed.

[0070] Mice bearing DMS53 (RBJ- / t) and NCLH146 (RB7-mut) SCLC cell line-derived xenografts (CDXs) were treated with clinically relevant dosing regimens.ZENO.190WO PATENTMonotherapy treatment with azenosertib once daily (QD) in mice bearing RBI -mutated cells (FIG. 2B) resulted in strong and statistically significant tumor growth inhibition (90%; p < 0.001), as compared to 49% tumor growth inhibition (p < 0.005) in mice bearing RBJ-wt cells (FIG. 2A).

[0071] The observations in mice bearing the TNBC CDXs were similar and consistent in that mice bearing RBI -mutated TNBC cells (FIG. 2D) resulted in strong and statistically significant tumor growth inhibition (87%; p < 0.01), as compared to 50% tumor growth inhibition (p < 0.001) in mice bearing RBl-vrt TNBC cells (FIG.2C).Example 2RBI gene deletion sensitizes SCLC and TNBC models to azenosertib

[0072] Human small cell lung cancer (SCLC) RBI -wt DMS53 and triple negative breast cancer (TNBC) RBl-wt MDA-MB-231 cell lines were transduced with lentiviral particles expressing a short-hairpin ribonucleic acid (shRNA) specific for the knockdown of the RBI gene (i.e., shRBl-1). Cells transduced with control (non-silencing) shRNA served as controls (i.e., shCtrl). After selection with puromycin (2 pg / mL) to enrich cells that had stable expression of the shRNA, cells were analyzed for expression of the RBI protein by Western blot system.

[0073] Cells with RBI knockdown were deposited on a 6 cm plate. Plates were incubated at 37 °C with 95% oxygen and 5% CO2 for 24 h to allow for cell attachment. After 24 h, azenosertib monotherapy or in combination with a chemotherapeutic agent (docetaxel, carboplatin or irinotecan) in DMSO was deposited into the plates. Plates were incubated at 37 °C with 95% oxygen and 5% CO2 for 16 h for monotherapy and / or combination treatment with irinotecan, 24 h for combination treatment with docetaxel, and 48 h for combination treatment with carboplatin. Protein expression was determined for the indicated markers using the JESS Simple Western blot instrument or traditional Western blot system. Quantification of intensity was calculated using ImageJ and represented as fold change compared to DMSO. Phospho-CHK1, yH2AX, and cleaved caspase 3 or cleaved caspase 7 were measured by JESS and quantified using ImageJ.

[0074] The effect of RBI gene silencing (mimicking RBI gene deletion, a form of RBI gene deficiency) on azenosertib sensitivity m A7J / -WI SCLC cells and RBl-wt TNBC cells: Compared to cells transduced with sh-Ctrl, RBI knockdown cells (i.e., transduced with respective shRBl-1) in both RBl-wt SCLC (FIGS.3A and 3B) and RBl-wt TNBC (FIGS.3C and 3D) models exhibited lower IC50 but higher Ymax and GRmax values, thereby indicating thatZENO.190WO PATENTsilencing of the RBI gene enhances sensitivity of the cancer cells to azenosertib. Ymax (Maximum Response): The highest response observed in a dose-response experiment, typically representing the control or untreated condition (e.g., 100% viability in a cell viability assay). GRmax (Maximum Growth Rate Inhibition): The maximum level of growth inhibition achieved at the highest drug concentration, where -1 indicates complete cell death, 0 indicates a cytostatic effect, and 1 indicates no effect on proliferation.

[0075] To understand the mechanism of these results, pharmacodynamic (PD) biomarker changes in response to azenosertib were analyzed after RBI gene knockdown in RBl-wt SCLC (FIGS. 4A-4C) and RBl-wt TNBC (FIGS. 4D-4F) cell lines. WEE1 phosphorylates CDK1 at tyrosine 15 (p-CDKl Y15) and loss of this phosphorylation signal is a measure of target engagement by azenosertib. It was found that p-CDKl Y15 was reduced in response to azenosertib, which is indicative of suppression of WEE1 kinase activity. A moderate to significant increase in levels of p-CHKl at S345 (p-CHKl), a DNA damage marker, and H2AX at SI 39 (yH2AX), a DNA damage marker and proxy for replication stress (RS), was observed with increasing doses of azenosertib in control cells (transduced with shCtrl), and the level of signals were significantly enhanced in RBI gene knockdown cells (transduced with shRBl-1), indicating that knockdown of the RB J gene further upregulates the level of DNA damage and RS. Furthermore, azenosertib treatment resulted in a much stronger upregulation of apoptosis as shown by cleaved caspase 3 (c-Caspase 3) and cleaved caspase 7 (c-Caspase 7).Example 3Induced RBI overexpression desensitizes RBI -mutated SCLC and TNBC models to azenosertib

[0076] Isogenic cell lines with inducible RBI overexpression were established using lentiviral plasmid vectors. Lentiviruses carrying wild-type RBI and empty vectors were purchased from Cellecta and used to transduce RBLmutated SCLC NCLH1048 and RB1-mutated TNBC MDA-MB-468 cells. IxlO6cells were seeded in growth media in T25 flasks, and lentiviruses were added at MOI (multiplicity of infection) = 1. Polybrene (8 pg / mL) was added simultaneously, followed by incubation at 37 °C with 95% oxygen and 5% CO2 for 24 h. The media were removed and replaced with fresh growth media containing puromycin (2 pg / mL). After selection, cells were treated with doxycycline (0.1 pM, 1 pM, 10 pM for RB1-mutated SCLC NCLH1048; 0.5 pM, 1 pM, 10 pM for RBI -mutated TNBC MDA-MB-468) to induce expression of RBI, and RBI overexpression was confirmed by Western blotting. AllZENO.190WO PATENTRBI inducible overexpression (IOE) and vector control cells were maintained on puromycin (2 pg / mL) for in vitro assays.

[0077] 5 x 105SCLC or TNBC cells with inducible RBI expression were seeded on a 6 cm plate in the presence of doxycycline (0, 0.5, 0.1, and 0.5 pM). Plates were incubated at 37 °C with 95% oxygen and 5% CO2 for 24 h to allow cell attachment and induction of RBI expression. After 24 h, azenosertib in DMSO was deposited into the plates. Plates were incubated at 37 °C with 95% oxygen and 5% CO2 for 16 h. Protein expression was determined for the indicated markers using the JESS Simple Western blot instrument (Bio-Techne) or traditional Western blot system. Quantification of intensity was calculated using Image.! and represented as fold change compared to DMSO. Phospho-CHKl, yl I2AX, and cleaved caspase 3 or cleaved caspase 7 were measured by JESS and quantified using ImageJ.

[0078] The effect of inducible RBI overexpression on azenosertib sensitivity in isogenic RBI -mutated SCLC cells and RBI -mutated TNBC cells was evaluated. As indicated in Table 1, these two cancer models each contain an RBI gene mutation that leads to termination of the RBI gene and consequently, no detectable protein expression. Compared to control cells with no RBJ expression, and at all tested concentrations of doxycycline, inducible RBI overexpression in both RBI -mutated SCLC (FIGS. 5A-5F) and RBI -mutated TNBC (FIGS. 5G-5L) cell lines exhibited higher IC50 but lower Ymaxand GRmax values, thereby indicating that the induced overexpression of RBI decreases the sensitivity of the cancer cells to azenosertib. Moreover, in both the RBJ-mutated SCLC and TNBC models, the higher the level of RBI protein expression, the more resistant the cells were to azenosertib.

[0079] To understand the mechanism of these results, RBJ -mutated SCLC (FIGS.6A-6C) and RBJ -mutated TNBC (FIGS.6D-6F) cell lines with RBJ inducible overexpression were analyzed for PD biomarker changes in response to azenosertib. In both cell lines, RBJ expression was confirmed to be upregulated after treating with increasing doses of doxycycline. It was also confirmed that p-CDKl Y15 was reduced in response to azenosertib, demonstrating suppression of WEE1 kinase activity. Azenosertib treatment caused increased DNA damage and RS, as indicated by elevated p-CHKl and yH2AX levels, in the control (no doxycycline) cells, however these biomarkers were significantly decreased when RBI overexpression was induced by doxycycline treatment. Furthermore, azenosertib treatment led to strong induction of apoptosis, indicated by upregulation of c-Caspase 3 and c-Caspase 7, in the control (no doxycycline) cells, but this effect was significantly decreased upon RBJ overexpression. Together these data indicate that RBJ overexpression suppresses the levels of DNA damage, RS, and apoptosis that would normally be induced by azenosertib in RBJ-mutated cell lines.ZENO.190WO PATENTExample 4RBI deletion sensitizes a TNBC model to combination therapies with azenosertib + chemotherapeutic agents (docetaxel / carboplatin / irinotecan)

[0080] FIGS. 7 A to 7L depict cell viability and synergy heatmaps generated from the SynergyFinder tool in MDA-MB-231 RBl-wt control (z'.e., transduced with shCtrl as described in Example 2) and RBI gene knockdown (i.e., transduced with shRBl-1 as described in Example 2) cells. Cells were treated in a matrixed format with escalating doses of azenosertib as a single agent or in combination with escalating doses of docetaxel (FIG.7B for control and FIG. 7D for RBI gene knockdown), carboplatin (FIG. 7F for control and FIG. 7H for RBI gene knockdown), or irinotecan (FIG. 7J for control and FIG. 7L for RBI gene knockdown). The Loewe synergy score was determined for each combination, where a Synergy Score > 10 is considered synergistic. The data indicates that, for each of the combinations azenosertib + docetaxel (FIG. 7 for control and FIG. 7C for RBI gene knockdown), azenosertib + carboplatin (FIG. 7E for control and FIG. 7G for RBI gene knockdown), or azenosertib + irinotecan (FIG. 71 for control and FIG. 7K for RBI gene knockdown), synergy scores of > 10 were obtained at multiple different concentration combinations, indicating that each of the three combination therapies was synergistic in vitro. Overall, greater synergistic effects were observed in RBI -knockdown cells as indicated by larger and darker areas in the synergy map, except with azenosertib + carboplatin treatment.

[0081] To understand the mechanism of observed synergy in the combination therapies, PD biomarker changes in the R l-v control and RBI gene knockdown cells were analyzed in response to treatment with azenosertib + docetaxel (FIG. 8A), azenosertib + carboplatin (FIG. 8B), or azenosertib + irinotecan (FIG. 8C), as well as with their respective monotherapies. It was found that p-CDKl Y15 was reduced in response to all of the azenosertib combination treatments, confirming suppression of WEE1 kinase activity. In control cells, the combination treatments led to moderate (azenosertib + docetaxel) / moderate to significant (azenosertib + carboplatin) / strong (azenosertib + irinotecan) increases in vII2AX. a DNA damage marker and proxy for replication stress (RS). In RBI gene knockdown cells, however, yII2AX levels were significantly (azenosertib + docetaxel; azenosertib + carboplatin) / mildly increased (azenosertib + irinotecan), indicating that knockdown of the gene further upregulates DNA damage and RS. Furthermore, all three combination treatments resulted in a much stronger upregulation of apoptosis in RBI gene knockdown cells as shown by increased c-Caspase 3 and c-Caspase 7.ZENO.190WO PATENT

[0082] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

ZENO.190WO PATENTCLAIMS1. A method of treating a cancer comprising :administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, to a subject selected to have a cancer associated with deficiency in the retinoblastoma transcriptional corepressor 1 (RBI) gene and / or the RBI protein;wherein the cancer is selected from small cell lung cancer (SCLC) and triple negative breast cancer (TNBC).

2. The method of claim 1 , wherein the deficiency in the RBI gene is selected from the group consisting of a mutation in the RBI gene, a deletion of the RBI gene, a decrease in function of the RBI gene, a decrease or suppression of expression of the RBI gene and an epigenetic change in the RBI gene.

3. The method of claim 2, wherein the mutation in the RBI gene is selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splicing mutation and a regulatory mutation.

4. The method of claim 2 or claim 3, wherein the mutation in the RBI gene is a nonsense mutation.

5. The method of any one of claims 2 to 4, wherein the mutation in the RBI gene is a loss-of-function (LOF) mutation.

6. The method of any one of claims 2 to 5, wherein the mutation in the RBI gene occurs at one or more nucleotides in a codon encoding an amino acid selected from the group consisting of E31, C61, K265, R552, S576, C706 and Q850 in SEQ ID NO:1.

7. The method of any one of claims 1 to 6, wherein the deficiency in the RBI protein is selected from the group consisting of a truncated RBI protein, an inactive RBI protein and a decrease or suppression of expression of the RBI protein.

8. The method of any one of claims 1 to 7, wherein the subject is not further selected to have a cancer associated with any oncogene alteration or status, any oncoprotein expression level or status and / or any signaling pathway level or alteration, other than the deficiency in the RBI gene and / or the RB 1 protein.

9. The method of any one of claims 1 to 7, wherein the subject is further selected to have a cancer associated with a TP53 mutation.

10. The method of any one of claims 1 to 9, wherein the cancer is small cell lung cancer (SCLC).

11. The method of any one of claims 1 to 9, wherein the cancer is triple negative breast cancer (TNBC).ZENO.190WO PATENT12. The method of any one of claims 1 to 11, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks.

13. The method of any one of claims 1 to 12, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks.

14. The method of any one of claims 1 to 13, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, or an equivalent thereof, in one or more dosing weeks.

15. The method of any one of claims 1 to 14, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 300 mg or an equivalent thereof, in one or more dosing weeks.

16. The method of any one of claims 1 to 14, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 350 mg or an equivalent thereof, in one or more dosing weeks.

17. The method of any one of claims 1 to 14, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 400 mg or an equivalent thereof, in one or more dosing weeks.

18. The method of any one of claims 1 to 14, wherein the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, comprises a daily dose of at least about 450 mg or an equivalent thereof, in one or more dosing weeks.

19. The method of any one of claims 1 to 14, wherein the effective dose of azenosertib or a pharmaceutically acceptable salt thereof comprises a daily dose of at least about 500 mg or an equivalent thereof, in one or more dosing weeks.

20. The method of any one of claims 1 to 19, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a continuous dosing schedule in one or more dosing weeks.

21. The method of any one of claims 1 to 19, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on an intermittent dosing schedule in one or more dosing weeks.ZENO.190WO PATENT22. The method of claim 21, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 6 days on / 1 day off, 5 days on / 2 days off, or 4 days on / 3 days off intermittent dosing schedule in one or more dosing weeks.

23. The method of claim 22, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 5 days on / 2 days off intermittent dosing schedule in one or more dosing weeks.

24. The method of claim 22, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, on a 4 days on / 3 days off intermittent dosing schedule in one or more dosing weeks.

25. The method of any one of claims 1 to 24, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable thereof, as monotherapy.

26. The method of any one of claims 1 to 25, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with docetaxel, or a pharmaceutically acceptable salt thereof.

27. The method of any one of claims 1 to 25, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with carboplatin, or a pharmaceutically acceptable salt thereof.

28. The method of any one of claims 1 to 25, wherein the method comprises administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with irinotecan, or a pharmaceutically acceptable salt thereof.

29. The method of any one of claims 1 to 28, wherein the subject has received no more than 1, at least 1, 1 or 2, 1 to 3, or 1 to 4 prior line(s) of therapy.

30. The method of claim 29, wherein the therapy is selected from the group consisting of surgery, radiation, chemotherapy, immunotherapy and targeted drug therapy.

31. The method of any one of claims 1 to 30, wherein the cancer is platinum-resistant or platinum-refractory.

32. The method of any one of claims 1 to 31 , wherein the cancer is PARP inhibitorresistant.

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