Methods and compositions for identifying and treating olaparib resistance in cancers

Abstract

This disclosure relates to methods for identifying and treating olaparib resistance in a subject, the methods comprising, in part, measuring expression levels of SMYD3 and / or SAMHD1 in the subject.

Description

[0001] Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0002] METHODS AND COMPOSITIONS FOR IDENTIFYING AND TREATING

[0003] OLAPARIB RESISTANCE IN CANCERS

[0004] CLAIM OF PRIORITY

[0005] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 736,079, filed on December 19, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under Grant Nos. CA217662 and GM138778 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Provided herein are compositions and methods for diagnosing and treating olaparib resistance in cancers.

[0010] BACKGROUND

[0011] Ovarian cancer, with High-Grade Serous Carcinoma (HGSC) being its most aggressive form, accounts for 5% of all cancer-related deaths among women worldwide [Worzfeld et al., 2017], Olaparib, a Poly(ADP-ribose) polymerase inhibitor (PARPi), has emerged as an attractive alternative to treat ovarian cancer [Maiorano et al., 2023], Despite the encouraging initial effects, olaparib resistance is a known challenge. Approximately 40-70% of patients are anticipated to develop resistance when administered as first-line treatment [Kim and Nam, 2022], Additionally, about 20-30% of patients undergoing a maintenance regimen will experience recurrence within five years [DiSilvestro et al., 2023],

[0012] SUMMARY

[0013] The present disclosure relates to methods of identifying a subject resistant to olaparib and methods of treating the subject. For example, the method comprises measuring expression levels of one or more biomarkers, including, for example, SMYD3 and / or SAMHD1, in a biological sample obtained from the subject. The present disclosure has provided evidence of identifying differentially expressed Attorney Docket No. 29539-0857WO1 / MGH 2025-144 biomarkers between olaparib -resistant and non-resistant cells that are linked to resistance pathways and for distinguishing olaparib resistance. Furthermore, the present disclosure has provided evidence showing that olaparib-treated patients may retain sensitivity to niraparib, indicating a therapeutic strategy to counter olaparib resistance.

[0014] Accordingly, in some aspects, provided herein is a method comprising: providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; and identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control.

[0015] In some embodiments, the expression level of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0016] In some embodiments, the expression level of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0017] In some embodiments, the subject was previously treated with olaparib.

[0018] In some embodiments, the subject has never been treated with olaparib.

[0019] In some embodiments, the method described herein further comprises administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

[0020] In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib if the subject is identified to be resistant to olaparib.

[0021] In some embodiments, the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib. In some embodiments, the PARPi is selected from the group consisting of niraparib, rucaparib, and talazoparib.

[0022] In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective amount of an anti-cancer agent Attorney Docket No. 29539-0857WO1 / MGH 2025-144 if the subject is identified to be resistant to olaparib, wherein the anti-cancer agent is not a PARPi.

[0023] In some embodiments, the subject has a cancer.

[0024] In some embodiments, the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer.

[0025] In some embodiments, the subject has a homologous recombination deficiency (HRD) mutation.

[0026] In some embodiments, the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.

[0027] In some embodiments, the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

[0028] In some aspects, provided herein is a method of treating a cancer in a subject, the method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control; and administering a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib to the subject if the subject is identified to be resistant to olaparib, or administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

[0029] In some embodiments, the expression levels of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0030] In some embodiments, the expression levels of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0031] 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0032] In some embodiments, the subject was previously treated with olaparib.

[0033] In some embodiments, the subject has never been treated with olaparib.

[0034] In some embodiments, the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib.

[0035] In some embodiments, the PARPi that is not olaparib is selected from the group consisting of niraparib, rucaparib, and talazoparib.

[0036] In some embodiments, the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer.

[0037] In some embodiments, the subject has a homologous recombination deficiency (HRD) mutation.

[0038] In some embodiments, the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.

[0039] In some embodiments, the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

[0040] In some aspects, provided herein is a method of identifying a subject to be resistant to olaparib, the method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; and identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control.

[0041] In some embodiments, the expression levels of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0042] In some embodiments, the expression levels of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0043] 30%, 50%, 70%, 100%. 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

[0044] In some embodiments, the subject was previously treated with olaparib.

[0045] In some embodiments, the subject has never been treated with olaparib.

[0046] In some embodiments, the method further comprises administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

[0047] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib if the subject is identified to be resistant to olaparib.

[0048] In some embodiments, the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib.

[0049] In some embodiments, the PARPi is selected from the group consisting of niraparib, rucaparib, and talazoparib.

[0050] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an anti -cancer agent if the subject is identified to be resistant to olaparib, wherein the anti -cancer agent is not a PARPi.

[0051] In some embodiments, the subject has a cancer.

[0052] In some embodiments, the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer.

[0053] In some embodiments, the subject has a homologous recombination deficiency (HRD) mutation.

[0054] In some embodiments, the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.

[0055] In some embodiments, the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended Attorney Docket No. 29539-0857WO1 / MGH 2025-144 to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0057] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0058] DESCRIPTION OF DRAWINGS

[0059] FIGS. 1A-1E show establishment of olaparib resistance in vitro models.

[0060] FIG. 1A is schematic illustration showing the process of drug -resistant cell establishment (created with Biorender). Cells were seeded and subjected to olaparib treatment with gradual dose escalation to foster recovery and resistance development in parental tumoral cells. FIG. IB shows dose-response curves of UWV1.289 parental and olaparib resistance (REOL) cell lines. FIG. 1C shows comparison of the LC50 values between UWV1.289 parental and resistant cell lines. **P = 0.003 in unpaired t-test. FIG. ID shows dose-response curves of OVCAR429 parental and olaparib resistance (REOL) cell lines. FIG. IE shows comparison of the LC50 values between OVCAR429 parental and resistant cell lines. **P = 0.004 in unpaired t-test. Error bars represent mean ± standard deviation (SD) from three independent measurements.

[0061] FIGS. 2A-2B show changes in cell morphology after olaparib treatment. Impact of chronic olaparib administration on the morphology of cultured human ovarian cancer cells: UWB 1.289 parental and UWB 1.289 REOL. Morphological analysis by phase contrast microscopy. Small images show fixated cells and nuclei stained with DAPI.

[0062] FIGS. 3A-3D show comparative growth profiles between parental and olaparib-resistant cell subtypes. FIG. 3A shows absolute cell counts per well at designated time points with corresponding doubling time (in days) for each cell line. FIG. 3B shows comparison of the calculated doubling time between parental and olaparib-resistant (REOL) cell lines. FIG. 3C shows cell cycle distribution analysis results. Each set of cells was treated with three different concentrations of olaparib (0.1 pM, 1 pM, and 10 pM) for 24 hours. The colored bars represent the percentage of cells in G2, S, and G1 determined by flow cytometry. FIG. 3D shows the G2 fraction of parental and REOL cells. All values are presented as mean ± SD. *P < Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0063] FIGS. 4A-4C show DNA damage response marker expression in S phase cells. DNA damage markers in nuclei after 24-hour olaparib treatment in parental (UWB 1.289 and OVCA429) and their corresponding olaparib-resistant subtypes (UWB 1.289 REOL and O VC A429 REOL) determined by immunofluorescence assays for (FIG. 4A) yH2AX, (FIG. 4B) RAD51, and (FIG. 4C) XRCC1. DAPI was utilized for nuclear segmentation, classifying cells into Gl, S, and G2 cell cycle phases. Normalization of marker intensity was performed relative to the DMSO control. Each dot on the scatter plot represents the normalized intensity per cell, with squares indicating the average intensity of cells per well.

[0064] FIGS. 5A-5F show molecular profiling of DNA damage response markers on parental and resistant cell subtypes. DNA damage markers in nuclei after 24-hour olaparib treatment in parental (UWB 1.289 and OVCA429) and their corresponding olaparib-resistant subtypes (UWB 1.289 REOL and OVCA429 REOL) determined by Immunofluorescence. (FIGS. 5A and 5B) yH2AX, (FIGS. 5C and 5D) RAD51, and (FIGS. 5E and 5F) XRCC1. DAPI was utilized for nuclear segmentation, classifying cells into Gl, S, and G2 cell cycle phases. Normalization of marker intensity was performed relative to the DMSO control.

[0065] FIGS. 6A-6D show differentially expressed proteins in parental and resistant cell subtypes. FIGS. 6A-6B show volcano plot illustrating differential protein expression in each subtype of parental and resistant cells of (FIG. 6A) UWB 1.289 and (FIG. 6B) OVCA429. Orange and green symbols depict up- and down-regulated proteins, respectively, while gray symbols denote proteins with no significant expression changes. FIGS. 6C-6D show gene ontology term enrichment analysis exhibiting the 15 most enriched terms (-log 10 ( / ?-value)) within the most informative nodes for (FIG. 6C) UWB 1.289 and (FIG. 6D) OVCA429.

[0066] FIGS. 7A-7C show analysis of differentially expressed proteins in olaparib- resistant vs. parental cell subtypes. FIG. 7A shows a heatmap illustrating the expression patterns of 19 proteins consistently differentially expressed in both parental (UWB 1.289 and OVCA429) and olaparib-resistant subtypes (UWB 1.289 REOL and OVCA429 REOL). Protein abundance data were log2 -transformed for enhanced visualization of expression disparities, with a gradient from white (lower expression) to blue (higher expression). FIG. 7B shows diagrammatic representation of the pathways involving SMYD3 and SAMHD1 that contribute to olaparib Attorney Docket No. 29539-0857WO1 / MGH 2025-144 resistance. SMYD3 facilitates oncogenic pathways via transcriptional activation of genes crucial for cell proliferation and epithelial-mesenchymal transition. Conversely, SAMHD1 is instrumental in homologous recombination-mediated double-strand break repair, aiding DNA end resection. FIG. 7C shows impact of SAMHD1 and SMYD3 inhibition on olaparib sensitivity.

[0067] FIGS. 8A-8B show annotation analysis of 19 differentially expressed proteins. FIG. 8A shows subcellular localization chart of differentially expressed proteins. FIG. 8B shows WikiPathways analysis chart of differentially expressed proteins.

[0068] FIGS. 9A-9F show cross-resistance of olaparib-resistant cells. Comparison of the LC50 values of carboplatin, doxorubicin, niraparib, rucaparib, paclitaxel, and topotecan between parental (UWB 1.289 and OVCAR429) and resistant cell lines (UWB 1.289 REOL and OVCAR429 REOL). Error bars represent mean ± SD from three independent measurements. **P < 0.005, *P < 0.05 in unpaired t-test.

[0069] FIGS. 10A-10B show niraparib and olaparib sensitivity measurements using patient-derived organoids. Dose-response curves depicting the drug sensitivity of five patient-derived organoid cells (passage 7 / 8) upon exposure to (FIG. 10A) olaparib and (FIG. 10B) niraparib. The x-axis illustrates drug concentrations, while the y-axis represents growth rate inhibition values (GR) post-six cycles of drug exposure. Each data point represents the mean of six replicates from two biological experiments, each conducted with three technical replicates. Error bars indicate standard error.

[0070] DETAILED DESCRIPTION

[0071] Ovarian cancer, with high-grade serous carcinoma (HGSC) being its most aggressive form, accounts for 5% of all cancer-related deaths among women worldwide [Worzfeld et al., 2017], Platinum -based chemotherapy, particularly involving drugs like cisplatin and carboplatin, has been considered the gold standard for initial treatment of ovarian cancer for many years [Zhang et al., 2022], However, the development of resistance and the side effects associated with platinum-based therapies have driven the search for alternative treatments [Zhang et al., 2022], Since 2014, olaparib, a Poly(ADP-ribose) polymerase inhibitor (PARPi), has emerged as an attractive alternative to treat ovarian cancer[Maiorano et al., 2023], The mechanism of action of this PARPi is rooted in the concept of synthetic lethality [Farmer et al., 2005], While exact mechanism of action of olaparib continues to be studied, it includes inhibiting the repair mechanism of DNA single-strand breaks, preferably in Attorney Docket No. 29539-0857WO1 / MGH 2025-144 tumors with homologous recombination (HR) deficiencies [Farmer et al., 2005], Emerging evidence strongly supports the use of olaparib not only in inherited cancers associated with BRCA mutations but also in sporadic cancers that harbor deficiencies in HR repair mechanisms [Yap et al., 2011], Despite the encouraging initial effects, olaparib resistance is a known challenge. Approximately 40-70% of patients are anticipated to develop resistance when administered as first-line treatment [Kim and Nam, 2022], Additionally, about 20-30% of patients undergoing a maintenance regimen will experience recurrence within five years [Di Silvestro et al., 2023], The HR restoration, DNA replication fork stabilization, BRCA reversion mutations, increased drug efflux, dissociation of PARP1 and poly(ADP-ribose) glycohydrolase (PARG), and epigenetic molecular modifications are only some of the established mechanisms contributing to olaparib resistance in BRCA mutant tumors [Desai et al., 2022 ; Tobalina et al., 2021; Langelier et al., 2018; Mirman et al., 2022; Rondinelli et al., 2017; Rottenberg et al., 2008; Taglialatela et al., 2017], These mechanisms, occurring individually or in combination, contribute to the complex landscape of olaparib resistance, highlighting a significant challenge in ovarian cancer management.

[0072] Recent advances in molecular oncology have shifted the focus toward identifying unique potential biomarkers to be exploited for alternate treatment strategies or drug development. Alterations in focal distribution or levels like RAD51, ATM, ATR, and CHK2 are known to impact the efficacy of olaparib, yet their precise roles in the development of drug resistance remain less understood [Kim et al., 2021; Schlacher et al., 2011; Huntoon et al., 2013; Carrassa et al., 2004], This uncertainty underscores the need for a comprehensive approach to identifying olaparib resistance markers [Kim and Nam, 2022], However, large-scale assessments of protein biomarkers in ovarian cancer cell lines exposed to chronic olaparib are not yet available.

[0073] In overcoming olaparib resistance, niraparib presents a viable second-line treatment option due to a combination of molecular and pharmacological factors. One key distinction is niraparib's differential PARP trapping ability compared to olaparib [Murai et al., 2012; Giudice et al., 2022], Alternatively, niraparib and olaparib may differ in their affinity for PARP1 and / or PARP2 [Lord and Ashworth, 2017], A recent study from a multi-institutional, retrospective analysis of ovarian cancer patients Attorney Docket No. 29539-0857WO1 / MGH 2025-144 treated with >2 lines of PARPi indicates that previous treatment with a PARPi does not inherently lead to resistance against subsequent PARPi therapies, suggesting that such treatment remains a viable option in recurrent scenarios [Giudice et al., 2022],

[0074] A systematic analysis of olaparib resistance is presented herein, including the development of resistance models with chronic exposure to olaparib, comparative proteomic analysis to identify potential biomarkers associated with olaparib resistance, and investigation of cross-resistance with other PARPis (e.g., rucaparib and niraparib) using patient-derived organoid models. The proteomic analysis described herein identified 19 differentially expressed proteins between parental and resistant cells, including SMYD3 and SAMHD1, linked to resistance pathways and potential biomarkers for distinguishing olaparib resistance. Furthermore, crossresistance investigation using patient-derived organoid models from olaparib-treated patients showed retained sensitivity to niraparib, indicating a strategy to counter olaparib resistance. This finding indicates a novel method to better inform clinicians for sequential or combination therapies involving different PARPi or other chemotherapeutic agents.

[0075] Certain terms employed within this document are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below to aid in describing particular embodiments.

[0076] Definitions

[0077] As used herein, the word “a” before a noun represents one or more of the particular noun. For example, the phrase “a genetic alteration” encompasses “one or more genetic alterations.”

[0078] As used herein, the term “about” means approximately, in the region of, roughly, or around. When used in conjunction with a numerical range, the term “about” modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%.

[0079] As used herein, the term “biomarker” as used herein refers to “a biological molecule found in blood, other bodily fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease”, e.g., as defined by the National Cancer Institute, (see, e.g., the URL www.cancer.gov / publications / dictionaries / cancer- terms?CdrID=45618). A biomarker can include a genetic biomarker such as, without Attorney Docket No. 29539-0857WO1 / MGH 2025-144 limitation, a nucleic acid (e.g., a DNA molecule, an RNA molecule (e.g., a microRNA, a long non-coding RNA (IncRNA) or other non-coding RNA). A biomarker can include a protein biomarker such as, without limitation, a peptide, a protein, or a fragment thereof.

[0080] As used herein, the phrases “protein biomarker”, “protein marker”, “peptide biomarker”, and “peptide marker” refer to a protein that is characteristic, alone in combination with other protein or other biomarkers, of resistance to olaparib in a subject. In some embodiments, a protein biomarker includes an elevated level of the protein in a subject as compared to a reference subject that does not have resistance to olaparib.

[0081] As used herein, the terms “mutation”, “genetic modification”, and “genetic alteration” are used interchangeably to indicate a change in a wild type nucleic acid sequence.

[0082] As used herein, the term “sensitivity” refers to the ability of a method to correctly identify or diagnose the presence of a disease in a subject (e.g., the sensitivity of a method can be described as the ability of the method to identify the true positive rate or probability of detecting a condition in a subject). For example, when used in reference to any of the variety of methods described herein that can detect the presence of cancer or the presence of resistance to olaparib in a subject, a high sensitivity means that the method correctly identifies the presence of cancer in the subject a large percentage of the time. For example, a method described herein that correctly detects the presence of cancer in a subject 95% of the time the method is performed is said to have a sensitivity of 95%. In some embodiments, a method described herein that can detect the presence of cancer in a subject provides a sensitivity of at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher). In some embodiments, methods provided herein that include detecting the presence of one or more members of two or more classes of biomarkers (e.g., genetic biomarkers and / or protein biomarkers) provide a higher sensitivity than methods that include detecting the presence of one or more members of only one class of biomarkers.

[0083] As used herein, the term “specificity” refers to the ability of a method to correctly reject the presence of a disease in a subject (e.g., the specificity of a method can be described as the ability of the method to identify the true negative rate or Attorney Docket No. 29539-0857WO1 / MGH 2025-144 probability of correctly determining that a condition does not exist in a subject. For example, when used in reference to any of the variety of methods described herein that can detect the presence of cancer in a subject, a high specificity means that the method correctly identifies the absence of cancer or the absence of resistance to olaparib in the subject a large percentage of the time (e.g., the method does not incorrectly identify the presence of cancer or the absence of resistance to olaparib in the subject a large percentage of the time). For example, a method described herein that correctly detects the absence of cancer or the absence of resistance to olaparib in a subject 95% of the time the method is performed is said to have a specificity of 95%. In some embodiments, a method described herein that can detect the absence of cancer or the absence of resistance to olaparib in a subject provides a specificity of at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher). In some embodiments, methods provided herein that include detecting the presence of one or more members of two or more classes of biomarkers (e.g., genetic biomarkers and / or protein biomarkers) provide a higher specificity than methods that include detecting the presence of one or more members of only one class of biomarkers.

[0084] As used herein, the term “subject” is used interchangeably with the term “patient” and means a vertebrate, including any member of the class mammalia, including humans and non-human animals, e.g., domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle, horse, and higher primates. In some embodiments, the subject is a human. In some embodiments, the subject has a disease. In some embodiments, the subject has cancer, e.g., ovarian cancer, e.g., HGSC. In some embodiments, the subject is a human harboring a cancer cell. In some embodiments, the subject is a human harboring a cancer cell, but is not known to harbor the cancer cell. In some embodiments, the subject can be a patient or a subject in a clinical setting. The subject can be suspected of, or at risk for, having or developing a disease or disorder, or may have already been diagnosed as having a disease or disorder. The subject may be a patient undergoing treatment.

[0085] A “biological sample” is derived or obtained from a living organism. The organism can be a whole organism or can be cells or organs grown in culture. In one embodiment, a “biological sample” also refers to a cell or population of cells or a quantity of tissue or fluid from a subject. Most often, a sample has been removed Attorney Docket No. 29539-0857WO1 / MGH 2025-144 from a subject. Often, a “biological sample” will contain cells from a subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine. In one embodiment, a biological sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary, secondary, or metastatic tumor, e.g., an ovarian tumor.

[0086] Biological samples also include explants and primary and / or transformed cell cultures derived from patient tissues. A biological sample can be provided by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells or cellular extracts (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as those having treatment or outcome history may also be used. Biological samples include, but are not limited to, tissue biopsies, scrapes (e.g., buccal scrapes), whole blood or other bodily fluids, such as bile, plasma, serum, urine, saliva, cell culture, urine, ascites, pleural fluid, or cerebrospinal fluid.

[0087] A “nucleic acid,” as described herein, can be RNA or DNA, and can be single or double stranded, and can be, for example, a nucleic acid encoding a protein of interest, a polynucleotide, an oligonucleotide, a nucleic acid analogue, for example, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Nucleic acid sequences include, for example, but are not limited to, nucleic acid sequences that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.

[0088] The term “polypeptide” as used herein refers to a polymer of amino acids.

[0089] The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a non -polypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide.” Examples of modifications include glycosylation and palmitoylation. Polypeptides Attorney Docket No. 29539-0857WO1 / MGH 2025-144 can be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc.

[0090] As used herein, “expression level” refers to the number of mRNA molecules and / or polypeptide molecules encoded by a gene of interest that are present in a cell or sample.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0092] Methods of Identifying Olaparib Resistance

[0093] In some aspects, provided herein is a method comprises measuring expression levels of one or more biomarkers, including, for example, SMYD3 and / or SAMHD1, in a biological sample obtained from the subject. The present disclosure has provided evidence of identifying differentially expressed biomarkers between olaparib-resistant and non-resistant cells that are linked to resistance pathways and for distinguishing olaparib resistance. Furthermore, the present disclosure has provided evidence showing that olaparib-treated patients may retain sensitivity to other PARPi, such as niraparib, indicating a therapeutic strategy to counter olaparib resistance.

[0094] Accordingly, in some aspects, disclosed herein is a method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; and identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control.

[0095] In some embodiments, the expression level of SMYD3 (e.g., expression level of a SMYD3 protein or a SMYD3 mRNA) in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 20%, 30%, 50%, 70%, Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0096] 100%, 150%, 200%, 250% 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% higher than the reference control.

[0097] In some embodiments, the expression level of SAMHD1 (e.g., expression level of a SAMHD1 protein or a SAMHD1 mRNA) in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 250% 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% higher than the reference control.

[0098] As used herein with reference to biomarkers (e.g., protein biomarkers or nucleic acid biomarkers), the phrase “reference level” refers to the level of the biomarker that is typically present in a subject that does not have resistance to olaparib. Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. For example, for a biomarker associated with resistance to olaparib, a reference sample can be a sample obtained from a subject that does not have resistance to olaparib. As another example, a reference level of a biomarker can be a level that is present in a subject who has resistance to olaparib. As another example, reference level of a biomarker can be a level that is present in a reference subject that does not exhibit a resistance to olaparib or can be a level that is present in a subject prior to the onset of resistance to olaparib. In some embodiments, a disease or condition can be identified in a subject when the measured or detected level of one or more biomarkers is higher than reference level(s) of the one or more biomarkers.

[0099] The reference level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing resistance or presence of resistance in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8- fold, 16-fold or more) than the risk or presence of resistance in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with Attorney Docket No. 29539-0857WO1 / MGH 2025-144 the lowest risk or likelihood of having or developing resistance to olaparib, and the highest quartile being subjects with the highest risk or likelihood of having or developing resistance to olaparib, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk or likelihood of having or developing resistance to olaparib, and the highest of the n- quantiles being subjects with the highest risk or likelihood of having or developing resistance to olaparib,.

[0100] In some embodiments, the reference level is a level in the same subject, e.g., at a different time point, e.g., an earlier time point, e.g., a time point before the subject developed resistance to olaparib.

[0101] The term “SMYD3” or “SET And MYND Domain Containing 3” herein refers to a gene encoding a histone methyltransferase which functions in RNA polymerase II complexes by an interaction with a specific RNA helicase as well as the proteins encoded by that gene. The human SMYD3 gene region covers approximately 757.933 kb. Nucleotide and amino acid sequences of SMYD3 may be found, for example, at GenBank Accession No. NC_000001. ll (Homo sapiens chromosome 1, GRCh38.pl4 Primary Assembly); GenBank Accession No. NM_001167740.2 (Homo sapiens SMYD3, transcript variant 1, mRNA); GenBank Accession No. NM_022743.3 (Homo sapiens SMYD3, transcript variant 2, mRNA); GenBank Accession No. NM_001375962.1 (Homo sapiens SMYD3, transcript variant 3, mRNA); GenBank Accession No. NM_001375963.1 (Homo sapiens SMYD3, transcript variant 4, mRNA); GenBank Accession No. NM_001375965.1 (Homo sapiens SMYD3, transcript variant 5, mRNA); or GenBank Accession No. NM_001375966.1 (Homo sapiens SMYD3, transcript variant 6, mRNA).

[0102] The term “SAMHD1” or “SAM And HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase 1” refers to a gene asscoated with Chilblain Lupus 2 and Aicardi-Goutieres Syndrome 5 as well as the proteins encoded by that gene. This gene may play a role in regulation of the innate immune response. The encoded protein is upregulated in response to viral infection and may be involved in mediation of tumor necrosis factor-alpha proinflammatory responses. The human SAMHD1 gene region covers approximately 61.936 kp. Nucleotide and amino acid sequences of SAMHD1 may be found, for example, at GenBank Accession No. NC_000020.ll (Homo sapiens chromosome 20, GRCh38.pl4 Primary Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0103] Assembly); GenBank Accession No. NM_015474.4 (Homo sapiens SAMHD1, transcript variant 1, mRNA); GenBank Accession No. NM_001363729.2 (Homo sapiens SAMHD1, transcript variant 2, mRNA); or GenBank Accession No. NM_001363733.2 (Homo sapiens SAMHD1, transcript variant 3, mRNA).

[0104] Olaparib has the chemical name 4-[(3-[(4-cyclopropylcarbonyl)piperazin-l- yl]carbonyl) -4-fluorophenyl]methyl(2H)phthalazin-l-one and the structure is

[0105] Olaparib is a PARP inhibitor. Poly(ADP -ribose) polymerase (PARP) is a family of enzymes crucial for detecting DNA damage and initiating repair. PARP1 is best known for its role in DNA SSB repair (SSBR). PARP1 is best known for its role in DNA SSB repair (SSBR). PARPl has been implicated in DNA repair pathways other than SSBR, including the repair of DNA double- strand breaks (DSBs) by non- homologous end joining (NHEJ) and alternative end joining. Cancer cells that are defective in homologous recombination DNA repair (due to mutation in BRCA1, BRCA2, or PALP2) can be sensitive to targeted inhibition of PARP.

[0106] In some embodiments, the subject was previously treated with olaparib. In some embodiments, the subject has never been treated with olaparib.

[0107] In some embodiments, the method described herein is for treatment of cancer in a subject. In some embodiments, the subject has a homologous recombination deficiency (HRD) mutation. In some embodiments, the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation. In some embodiments, the method described herein is for treatment of cancer in a subject, wherein the subject has a HRD mutation, wherein the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

[0108] In some embodiments, the subject has a cancer (e.g., ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer).

[0109] In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib if the subject is identified to be Attorney Docket No. 29539-0857WO1 / MGH 2025-144 resistant to olaparib. In some embodiments, the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib. In some embodiments, the PARPi is selected from the group consisting of Nicotinamide, 3- aminobenzamide, PD128763, DPQ, NU1025, 4-ANI, ISO, PJ-34, rucaparib, niraparib, talazoparib, veliparib, INO-1001, 2x-121, CEP-8983, pamiparib, fluzoparib, and amelparib (JPI-289). The structures of these PARPi are known in the art and can be found in, for example, Scott CL et al., Poly (ADP -ribose) polymerase inhibitors: recent advances and future development. J Clin Oncol. 2015 Apr 20;33(12): 1397-406, the entire contents of each of which are hereby incorporated herein by reference. In some embodiments, the PARPi is selected from the group consisting of niraparib, rucaparib, and talazoparib.

[0110] In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective amount of an anti-cancer agent if the subject is identified to be resistant to olaparib, wherein the anti-cancer agent is not a PARPi.

[0111] The anti-cancer agent can include any appropriate cancer treatments. A cancer treatment can include surgery. A cancer treatment can include radiation therapy. A cancer treatment can include administration of a pharmacotherapy such chemotherapy, hormone therapy, targeted therapy, and / or cytotoxic therapy. Examples of cancer treatments include, without limitation, platinum compounds (such as cisplatin or carboplatin), taxanes (such as paclitaxel or docetaxel), albumin bound paclitaxel (nab-paclitaxel), altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, luteinizing-hormone-releasing hormone (LHRH) agonists (such as goserelin and leuprolide), anti-estrogen therapy (such as tamoxifen), aromatase inhibitors (such as letrozole, anastrozole, and exemestane), angiogenesis inhibitors (such as bevacizumab), poly(ADP)-ribose polymerase (PARP) inhibitors (such as olaparib, rucaparib, and niraparib), external beam radiation therapy, brachytherapy, radioactive phosphorus, and any combinations thereof.

[0112] In some embodiments, the anti -cancer agent is selected from the group consisting of an SMYD3 inhibitor and an SAMHD1 inhibitor. Accordingly, in some embodiments, the method described herein further comprises administering a Attorney Docket No. 29539-0857WO1 / MGH 2025-144 therapeutically effective amount of olaparib and an SMYD3 inhibitor and / or an SAMHD1 inhibitor, if the subject is identified to be resistant to olaparib. In some embodiments, the SMYD3 inhibitor and / or the SAMHD1 inhibitor is administered prior to, concurrently, or after administration of olaparib. In some embodiments, the SMYD3 inhibitor and the SAMHD1 inhibitor can be small molecules, polypeptides (e.g., antibodies or ligands), polynucleotides (e.g., siRNAs, shRNAs, or dsRNAs), or gene-editing systems (e.g., CRISPR).

[0113] In some aspects, provided herein is a method of treating a subject resistant to olaparib or reversing olaparib resistance in subject, wherein the method comprises administering a therapeutically effective amount of olaparib and a therapeutically effective amount of an SMYD3 inhibitor and / or an SAMHD1 inhibitor. In some embodiments, the SMYD3 inhibitor and / or the SAMHD1 inhibitor is administered prior to, concurrently, or after administration of olaparib. In some embodiments, the subject has ovarian cancer. In some embodiments, the expression levels of SMYD3 and / or SAMHD1 in a biological sample from the subject are higher than a reference control, prior to administration of the SMYD3 inhibitor and / or the SAMHD1 inhibitor.

[0114] Cancers and Drug Resistance

[0115] As used herein, the terms “cancer,” “tumor” or “tumor tissue” has the meaning as understood by one skilled in the art. A cancer, tumor, or tumor tissue can include tumor cells that are neoplastic cells with abnormal growth properties. Tumors, tumor tissue, and tumor cells can be benign or malignant. Cancer can include primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject’s body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).

[0116] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Additional examples of such cancers are noted below and include: squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including Attorney Docket No. 29539-0857WO1 / MGH 2025-144 gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, cholangiocarcinoma, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

[0117] One of the benefits of monitoring or identifying olaparib-resistance using the methods described herein is that olaparib-resistance in a subject can be detected prior to an observable increase in size of the subject’s cancer (e.g., tumor). With an early detection of olaparib-resistance, a toxic treatment can be terminated early to reduce side effects or minimize unnecessary treatment. Other benefits of the longitudinal monitoring of olaparib-resistance of monitoring or identifying olaparib-resistance using the methods described herein include predicting drug treatment efficacy, minimizing the detection of residual diseases, and facilitating early detection of disease recurrence.

[0118] Therapeutic and Diagnostic Use

[0119] Any appropriate method can be used to detect the presence or absence of one or more biomarkers (e.g., genetic biomarkers) as described herein. In some embodiments, one or more genetic biomarkers can be detected independently (e.g., via singleplex peptide tools). In some embodiments, one or more genetic biomarkers can be detected simultaneously (e.g., via multiplex DNA tools such as “chips” or microarrays). Examples of methods for detecting genetic biomarkers include, without limitation, sequencing, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, PCR-based multiplex methods, digital PCR methods, droplet digital PCR (ddPCR) methods, PCR-based singleplex PCR methods, Sanger sequencing methods, next-generation sequencing methods (e.g., singlemolecule real-time sequencing, nanopore sequencing, and Polony sequencing), quantitative PCR methods, ligation methods, and microarray methods.

[0120] Any appropriate method can be used to detect the level (e.g., an elevated level) of one or more biomarkers (e.g., peptide biomarkers) as described herein. In some embodiments, the levels of one or more peptide biomarkers can be detected independently (e.g., via singleplex peptide tools). In some embodiments, the levels of one or more peptide biomarkers can be detected simultaneously (e.g., via multiplex Attorney Docket No. 29539-0857WO1 / MGH 2025-144 peptide tools such as “chips” or microarrays). Examples of methods for detecting peptide levels include, without limitation, spectrometry methods (e.g., high- performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC / MS)), antibody dependent methods (e.g., enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, western blotting, and protein immunostaining), and aptamer dependent methods. In some embodiments, the level of one or more peptide biomarkers can be detected as described in the Examples. For example, the level of one or more peptide biomarkers can be detected by multiplex immunoassay.

[0121] The disclosure provides methods of treatment that include administering to a subject a composition disclosed herein.

[0122] In some embodiments, the composition disclosed herein can be used for treating a cancer in a subject, wherein the subject is identified to be resistant to olaparib. For example, a PARPi that is not olaparib or an anti-cancer agent that is not PARPi can be used to treat a cancer in a subject, wherein the subject is identified to be resistant to olaparib.

[0123] In some embodiments, the composition disclosed herein can be used for treating a cancer in a subject, wherein the subject is identified to be resistant to olaparib, and wherein the composition comprises an SMYD3 inhibitor and / or an SAMHD1 inhibitor. In some embodiments, the SMYD3 inhibitor and / or the SAMHD1 inhibitor is administered prior to, concurrently, or after administration of olaparib. In some embodiments, the SMYD3 inhibitor and the SAMHD1 inhibitor can be small molecules, polypeptides (e.g., antibodies or ligands), polynucleotides (e.g., siRNAs, shRNAs, or dsRNAs), or gene-editing systems (e.g., CRISPR). In some embodiments, the subject has ovarian cancer.

[0124] In one aspect, the disclosure provides a kit comprising one or more agents as described herein for therapeutic and / or diagnostic purposes.

[0125] Methods of Treatment

[0126] Treatment of a disease (e.g., cancer, e.g., ovarian cancer) or individual (e.g., a subject having cancer) according to the methods described herein is an approach for obtaining beneficial or desired medical results, including clinical results, but not necessarily a cure. For purposes of the methods described herein, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or Attorney Docket No. 29539-0857WO1 / MGH 2025-144 more symptoms, diminishment of extent of disease (e.g., number or size of tumors), stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression (e.g., tumor growth or appearance of new tumors), amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment or if receiving a different treatment. A treatment can include administration of one or more therapeutic agents as described herein, e.g., a Parpi other than olaparaib.

[0127] The present disclosure also provides for methods of treating a cancer and / or preventing an increase or spread of cancer. In some embodiments, the method described herein kills the cancer cells. In some embodiments, the method described herein results in delayed growth, inhibited growth, and / or reduced size of a tumor. In some embodiments, the growth of the tumor is inhibited or delayed by at least about 10%, 20%, 25%, 30%, 40%, or 50% and / or the size of the tumor is decreased by at least about 10%, 20%, 25%, 30%, 40%, or 50%, in comparison to a reference control (e.g., a subject resistant to olaparib that is not treated or identified using the method described herein). In some embodiments, the growth of the tumor is inhibited or delayed by at least about 10%, 20%, 25%, 30%, 40%, or 50% and / or the size of the tumor is decreased by at least about 10%, 20%, 25%, 30%, 40%, or 50%, in comparison to a reference control (e.g., untreated control).

[0128] In some embodiments, the size of the tumor is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% from the size of the tumor before treatment with the method described herein. In certain aspects, the size of the tumor is reduced from any of about 10%, 20%, 25%, 30%, 40%, or 50% to any of about 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% from the size of the tumor before treatment with the method described herein. In certain aspects, the size of the tumor is reduced by at least about 25%, 30%, 40%, or 50% from the size of the tumor before treatment with the method described herein. It is understood that the time during which any of the above is determined is generally measured over the time that one of ordinary skill in the art would expect the subject cancer treatment to be administered and / or exhibit an effect and can be over, for example, any of about or between any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 month, 3 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 month, 4 month, 5 month 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years.

[0129] In some embodiments, a cancer treatment can include an anti -cancer treatment, e.g., a PARPi other than olaparib, and / or cancer-preventative, therapy, drug, protocol, and / or chemotherapy.

[0130] In some embodiments, the administration of inhibitors and / or anticancer drugs can be by any known delivery route, for example, oral, mucosal, intravenous, intramuscular, enteral (gastrointestinal), parenteral, local, topical, and / or inhalation. It will be understood by one of ordinary skill in the art that delivery can be accomplished by any known pharmaceutical delivery method, for example, via pill, tablet, capsule, enema, suppository, injection, surface application, insufflation, and / or infusion.

[0131] An “effective amount” or a “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount or a therapeutically effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of an active agent (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.

[0132] Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed Attorney Docket No. 29539-0857WO1 / MGH 2025-144 as the ratio LD50ZED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0133] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0134] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0135] EXAMPLES

[0136] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0137] Materials and Methods

[0138] The following materials and methods were used in the following examples.

[0139] Chemicals and reagents

[0140] All drugs were purchased from SelleckChem, USA. Olaparib (AZD2281), niraparib (MK-4827), doxorubicin, topotecan, and paclitaxel stock solutions were prepared in DMSO. Carboplatin stock solution was prepared in water. All stocks were stored at -20 °C, and serial dilutions were performed with a culture medium before use. Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0141] Cell lines

[0142] Two cell lines with distinct genomic backgrounds were utilized to enrich the understanding of PARP inhibitor resistance mechanisms, described as multifaceted and varied across cellular strategies. The UWB 1.289 cell line, characterized by a BRCA mutation, and the OVCA429 cell line, devoid of this mutation, were selected. These two cell lines were chosen to explore differential cellular responses arising from diverse genetic contexts.

[0143] The human ovarian carcinoma cell line UWB 1.289 was purchased from the American Type Culture Collection (ATCC). The cell line OVCA429 was kindly provided by Dr. Mathew Dubach. UWB 1.289 was maintained in RPMI-1640 (Gibco™). OVCA429 was cultured in Dulbecco’s modified Eagle’s medium (Coming™). All complete media were supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific) and 100 units / ml penicillin-streptomycin (Millipore Sigma) at 37 °C in 5% CO2. All cell lines were tested and confirmed to be free of mycoplasma using the Universal Mycoplasma Detection Kit (Invivogen).

[0144] Establishment of olaparib-resistant subtypes from parental (control) cell lines

[0145] To develop olaparib resistance cell lines subtypes, cells were continually exposed to stepwise increases in the concentration of olaparib over 16 to 18 weeks. Briefly, cells were seeded at a density of ~5 * 105 / mL in a T75 cell culture flask with 10 mL complete growth medium. After 4-6 hours of incubation, relatively low concentrations of olaparib (ranging from 0.01 to 0.1 pg / ml), dissolved in phosphate- buffered saline (PBS) without Ca2+and Mg2+, were added into the medium. Cells were left in olaparib for three days or until a stable cell re-population formed. Regular medium replenishment was performed throughout this period. The olaparib concentration was then increased by 1.5 to 2 folds. This stepwise dose escalation continued for 16 to 18 weeks until the olaparib concentration reached at least ten times the starting concentration. After that, all olaparib-resistant cell lines (UWB 1.289 REOL and O VC A429 REOL) were maintained in the same medium as their parental cell line. Resistant cell subtypes were authenticated by STR profile at Dana-Farber Cancer Institute (Boston, MA) and used within six months. Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0146] Drug sensitivity

[0147] Drug sensitivity was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT) colorimetric assay. First, cells were counted with a hemocytometer, seeded in a 96-well plate, and cultured for 24 hours. After that, eight drug concentrations were added and left in contact with the cells for 72 hours: olaparib (range: 10'3- 1000 / / M), carboplatin (10‘2- 250 / / M), doxorubicin (10‘3- 100 / M), niraparib (10‘3- 500 / M), rucaparib (10‘3- 500 / M), paclitaxel (10‘6- 10 «M), topotecan (10‘3- 104nM). Subsequently, MTT dye (Sigma-Aldrich; Merck KGaA) at a final concentration of 0.5 mg / ml was added for 3 hours. This was followed by replacing the medium and dissolving the formazan crystals with DMSO. The optical density (OD; absorbance at 540 nm) was measured using a plate reader (Tecan). Data (mean ±SD of at least three independent experiments performed in triplets) are presented as the relative proliferation as a function of time after seeding.

[0148] Doubling time and cell cycle distribution analysis

[0149] To determine the doubling time of each parental and resistant cell subtype, cells were cultured in 48-well plates, with initial seeding densities of 5 * 103cells per well. After 24, 48, 72, and 96 hours, viable cells were determined in three wells per cell line with the Trypan Blue exclusion using a hemocytometer. The cell doubling times were calculated according to the formula: Doubling time (hours) = Incubation time (hours) x (1 / (Log2 (Cellsf / Celli))). Cellsf and Celli indicate the initial and final number of cells, respectively.

[0150] Cell cycle distributions of each pair of parental and resistant sub-types were tested for cell cycle analysis. Cell lines were seeded in their respective complete medium in 6-well plates (1.5 x io5cells per well) and allowed to attach overnight. Then, cells were synchronized with serum-free medium for 24 hours and then incubated with 10% FBS medium for 24 hours. Finally, each cell line was harvested and fixed with 70% ethanol, followed by staining using the cell cycle analysis kit (Abeam) according to the manufacturer's instructions. The LSRII flow cytometer was used to analyze the samples. The percentage of cells in the Gl, S, and G2 phases was determined (Flow Jo™ Software v 10.6.2). Attorney Docket No. 29539-0857WO1 / MGH 2025-144 yH2AX, RAD51, and XRCC1 nuclei staining and analysis

[0151] Immunofluorescence was used to compare RAD51, yH2AX, and XRCC1 nuclear intensity between parental and resistant cell subtypes. The cells were initially counted using a hemocytometer, seeded into a 384-well plate, and cultured for 24 hours. Following fixation and permeabilization, primary antibodies (Table 5) were diluted 1 :500 with antibody diluent (TBS, 2% BSA, 0.05% Sodium Azide, and 100 mM Trehalose (Sigma-Aldrich) and incubated Overnight. Subsequently, secondary antibodies (Table 5), diluted 1 : 1000 in PBS IX, were added after washing steps with PBS IX. Secondary antibodies were incubated for 1 hour at room temperature in a dark and humid chamber. After three washes, the cells were stained with DAPI (2.5 pg / ml, Sigma-Aldrich) in darkness for 15 minutes.

[0152] Image acquisition was promptly performed using a Nikon Ti2 inverted microscope (Nikon Instruments) equipped with an LED light source (pE-4000 CoolLED) to capture multicolor widefield fluorescence images. Emitted light was captured using a CMOS camera (Dual ORCA Flash 4.0 Digital CMOS camera Cl 3440) set to a 16-bit scale detection mode. The optimal exposure time for each fluorescence channel was determined by maximizing the dynamic range and preventing saturation based on preliminary observations from randomly selected cores. Cells were stained with DAPI to visualize nuclei, and secondary antibodies were conjugated with two distinct fluorophores, Alexa Fluor 555 and Alexa Fluor 647, to visualize all the target antigens. The data acquisition was managed using the NIS Elements software version 5.30.07 (Nikon Instruments). The confocal imaging settings were kept the same across each dataset to provide comparable measurements per dataset. Image analysis was performed in MATLAB as previously described [Gopal et al., 2024, 38262416], The acquired images enabled nuclear segmentation and classified the cell lines into three distinct phases: Gl, G2, and S. The expression of each specific marker (RAD51, yH2AX, and PCNA) was then assessed in correlation with each specific cell phase.

[0153] Proteomic analysis

[0154] For the proteomic analysis, cells (5* 106UWB 1.289, OVCA429, UWB 1.289 REOL, and OVCA429 REOL) were collected at an 80%~ confluence, and harvested in cold PBS, washed three times with PBS, and lysed in lysis buffer (50 mM Tris- Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0155] HCL (pH 8.0), 150 mM NaCl, 0.1% SDS, lOmM NaF, lmM Na3VO4, 0.5% sodium deoxycholate, 1% Triton X-100, and 1 / 12 (v / v) protease inhibitor cocktail, Sigma- Aldrich). The lysates were vortexed for 15 minutes, centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatants were collected. Protein samples were precipitated by adding four times the sample volume of ice-cold (-20 °C) acetone, followed by incubation at -20 °C for 60 minutes and centrifugation (11,000 x g) at 4°C for 10 minutes. After acetone evaporation, protein concentrations were determined using a BCA assay kit (Thermo-Fisher Scientific). Each 200 pg protein sample was trypsin-digested (12.5 ng / pL trypsin proteomic grade, Sigma-Aldrich) in an enzyme: protein ratio of 1 : 100 (w / w) at 37 °C overnight. 5 pL formic acid (Fluka) was added to each sample, centrifuged, and the supernatants were dried using a Speed Vac for 30 minutes. Pellets were solubilized in 20 pL 0.5% formic acid and loaded into a C18 Stage Tip. After elution with 10 pL 80% ACN and 0.1% formic acid and drying in a speedVac for 10 min, samples were resuspended in 10 pL 0.1% formic acid for LC-MS / MS analysis.

[0156] LC-MS / MS analysis

[0157] For peptide analysis, an LTQ-Orbitrap XL instrument (Thermo-Fisher Scientific) coupled with an Ultimate 3000 Dionex nanofl ow LC system (Dionex, Sunnyvale, CA, USA) was utilized. High mass resolution enabled peptide identification, while high energy collision dissociation (HCD) facilitated reporter ion quantification. The RP-LC system comprised a peptide Cap-Trap cartridge (0.5 x 2 mm, Michrom BioResources, Auburn, CA, USA) and a pre-packed BioBasic C18 PicoFrit analytical column (75 pm i.d. x 15 cm length, New Objective, Woburn, MA, USA) with a FortisTip emitter tip. Peptides were loaded onto the trap cartridge and concentrated using mobile phase A (98% H2O, 2% acetonitrile, 0.1% formic acid) for desalting. Following this, peptides were eluted over 180 minutes from the analytical column using a linear gradient of 6 to 100% mobile phase B (20% H2O, 80% acetonitrile, 0.1% formic acid) at a flow rate of 0.3 pL / minute, employing specific gradient steps.

[0158] The LTQ-Orbitrap tandem mass spectrometer was operated in a data- dependent mode. Full MS scans (60,000 resolving power) were succeeded by six MS / MS scans, where the three most abundant molecular ions were dynamically Attorney Docket No. 29539-0857WO1 / MGH 2025-144 chosen and fragmented by collision-induced dissociation (CID) using normalized collision energies. Additionally, these three ions were scanned three times via HCD- MS2. LTQ-Orbitrap settings included specific parameters such as spray voltage, resolution, mass ranges, and the selection of ions based on charge state and precursor information. The Mascot protein search algorithm (Matrix Science, Boston, MA, USA) was used, generating Mascot format files through Proteome Discoverer 1.2 software (Thermo-Fisher Scientific). Abundance differences were assessed by comparing each resistant cell subtype with its respective parental line, enabling the analysis of protein levels.

[0159] Organoid culture and sensitivity evaluation

[0160] This study was performed following all relevant guidelines and regulations. Olaparib-resistant patient-derived ovarian cancer organoids, 17-39, 17-121, 17-116, and 18-47, were provided by Dr. Sarah Hill, Dana Farber Cancer Institute, and reestablished. The treatment-naive patient-derived organoid (PDO), VCRB357 PDXO, was generated from samples recovered by the Vincent Center for Reproductive Biology Gyn Sample repository at the Massachusetts General Hospital with the approval of the Institutional Review Board (DFHCC 07-049). Tissue processing, organoid generation, and organoid culture were performed as described previouslyfZhang et al., 2020], To determine the sensitivity of the organoid models to olaparib and niraparib cells were plated in 96 well plates (Thermo-Fisher Scientific) in organoid media cocktail: Advanced DMEM / F12 (Thermo-Fisher Scientific) supplemented with 2 mM HEPES (Thermo-Fisher Scientific), 1 x GlutaMAX-I (Thermo-Fisher Scientific), 1X B27 supplement (Thermo-Fisher Scientific), 10 nM Leul5-Gastrin I (Sigma-Aldrich), 1 mM N-acetylcystein (Sigma-Aldrich), 100 ng / mL recombinant human IGF-1 (R&D Systems), 50 ng / mL recombinant human FGF-2 (PeproTech), 20% Afamin / Wnt3a CM (JSR Life Sciences), Ipg / mL humanR-spondin (R&D Systems), 100 ng / mL Noggin (PeproTech), 500 nM A-83-01 (Tocris Bioscience), 200 U / mL penicillin / streptomycin (Thermo-Fisher Scientific), and lOpM Y-27632 (FUJIFILM Wako Pure Chemical Corporation). Media with the drug was added to cells on day one, and this was sustained for three days. Following this period, the medium was replaced with fresh medium, also containing the drug. This replenishment process was carried out three times in total. After the final treatment, Attorney Docket No. 29539-0857WO1 / MGH 2025-144 the cells were incubated in a fresh medium for an additional 48 hours. Subsequently, a Cell Titer-Gio assay was performed to evaluate cell viability. Dose-response curves were generated at concentrations of 0 pM (vehicle control with the highest DMSO volume), 2, 10, 50, 100, and 200 pM for both Olaparib and 1.28, 3.2, 8, 20 and 50 pM Niraparib.

[0161] Inhibition Assay Sensitivity

[0162] Cephalosporin C zinc salt and SMYD3-IN-1 (MedChem) were used to selectively inhibit SAMHD1 and SMYD3, respectively. To evaluate the effect of this inhibition on olaparib sensitivity, we employed the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) colorimetric assay. Initially, OVCA429 parental and REOL cells were counted using a hemocytometer, seeded in a 96-well plate, and cultured for 24 hours. Subsequently, the cells were treated with three concentrations of each inhibitor: Cephalosporin C zinc salt at 0.5 pM, 1 pM, and 2 pM, and SMYD3-IN-1 at 5 nM, 10 nM, and 50 nM. The cells were exposed to the inhibitors for 1 hour, after which each cell subtype was treated with either 7 pM or 150 pM of olaparib and maintained in contact with the drug for 72 hours. After this period, MTT dye (Sigma-Aldrich; Merck KGaA) at a final concentration of 0.5 mg / ml was added for 3 hours. The medium was then replaced, and the formazan crystals were dissolved with DMSO. The optical density (OD; absorbance at 540 nm) was measured using a plate reader (Tecan).

[0163] Statistical analysis

[0164] The LC50 values were calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, USA v 10.0.03). Comparison of two samples was done using unpaired two-tailed Student’s t-test assuming equal variances from at least three independent experiments unless stated otherwise. Statistical significance for three or more samples was calculated via one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons tests.

[0165] Protein statistical analyses were performed using the Perseus software (version 1.4.0.6) [Tyanova et al., 2016, 27348712], Only proteins present and quantified in at least two out of three technical repeats were considered as positively identified in a sample. The required false positive rate was set to 1% at the peptide and 1% at the Attorney Docket No. 29539-0857WO1 / MGH 2025-144 protein level, and the minimum required peptide length was set to six amino acids. Statistically significant differences were computed by ANOVA and false discovery rate (P < 0.05) followed by Tukey post hoc test (P < 0.01).

[0166] EXAMPLE 1. Generation and characterization of olaparib resistance in ovarian cancer cell lines

[0167] Olaparib resistance (REOL) in vitro models were established using two ovarian cancer cell lines, the high-grade serous carcinoma UWB 1.289 and OVCA429 [DelloRusso et al., 2007, 17259345; Beaufort et al., 2014, 25230021], through chronic exposure to olaparib. UWB 1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele [DelloRusso et al., 2007, 17259345], OVCA429 is a sporadic HGSC [Langland et al., 2010, 20204287], Treatment was initiated with a lower dose followed by dose escalation over time (FIG. 1A). Simultaneously, regular passage of the parental cell lines was performed in parallel with the establishment of olaparib resistance. This yielded two resistant sublines, UWB 1.289 REOL and OVCA429 REOL, which showed no significant size or shape differences from their parental counterparts (FIGS. 2A-2D). The lethal concentration 50 (LC50) of olaparib, representing the concentration required to inhibit 50% of cell growth, increased significantly in resistant sub-cell lines compared to parental cell lines (FIGS. 1B-1E). UWB 1.289 REOL and OVCA429 REOL showed LC50 values of 94.4 ± 24.3 pM and 151.9 ± 22.5 pM, while their parental types showed LC50 values of 0.84 ± 0.3 pM and 7.11 ± 6.2 pM, respectively. This approach established olaparib-resistant cell line models.

[0168] Next, the paired UWB 1.289 and OVCA429 cell lines were characterized for their doubling times and cell cycle distributions. If the acquired resistance alters cell dynamics, a longer growth doubling time would appear for the resistant cell subtypes than their corresponding parental subtypes. FIG. 3A shows the number of cell counts in culture monitored over 96 hours. These counts were used to calculate the growth rate over 96 hours. The mean doubling times for REOL subtypes were notably longer (FIG. 3B) compared to their parental counterparts. The mean doubling time increased by 28% for UWB 1.289 (P = 0.13) and 32% for OVCA429 (P = 0.01). The cell cycle distributions of these paired cell lines (see the Methods for details) were also estimated. After 24 hours of olaparib treatment at three different concentrations (0.1 pM, 1 pM, and 10 pM), the resistant cell subtypes exhibited a higher proportion of Attorney Docket No. 29539-0857WO1 / MGH 2025-144 cells in the G2 stage of the cell cycle and a reduced number of cells in the G1 phase compared to parental cell lines (FIG. 3C). This trend was more pronounced at the higher olaparib concentration (10 pM) than at the lower doses; however, it was significant only for the OVCA429 model. The mean portions of the G2 cycle were increased by 24% for UWB 1.289 (P = 0.24) and by 42% for OVCA429 (P = 0.04, Figure 2D). These results could indicate that resistant cells spend an extended duration attempting to repair DNA damage.

[0169] EXAMPLE 2. Evaluation of DNA damage responses

[0170] Olaparib's inhibition of PARP disrupts DNA repair and replication, prompting cells to adapt by potentially upregulating DNA repair mediators or activating alternative, less efficient repair mechanisms. To evaluate DNA damage responses, the next experiment investigated changes in key DNA repair response markers (y-H2AX, RAD51, and XRCC1) at nuclear distribution. y-H2AX marks the initial response to DNA double-strand breaks and marks stalled replication forks [Sharma et al., 2012], RAD51 is crucial for homologous recombination repair, and nuclear foci are used as a surrogate for HR proficiencyfKolinjivadi et al., 2017; Su et al., 2008], XRCC1 plays a major role as a scaffold protein that interacts with and coordinates the activities of various enzymes necessary for base excision repair [Caldecott, 2019, 31324530], The nuclear local accumulation of these three proteins was evaluated in each cell cycle phase (Gl, S, and G2) in both parental (UWB 1.289 and OVCA429) and REOL subtypes (UWB 1.289 REOL and OVCA429 REOL). FIG. 4 highlights notable changes in the S phase, while all cycle data are presented in FIG. 5. It showed increases in y-H2AX intensity in all parental and REOL cell lines at the S cell phase, corresponding to an escalation in olaparib concentration (FIG. 4A). This elevation in y-H2AX abundance supports responses to DNA double-strand breaks from olaparib treatments [Rogakou et al., 1998], Interestingly, different changes in the nuclear abundance of RAD51 between parental and resistant subtypes were obaseved. Both parental cells (UWB 1.289 and OVCA429) showed a decrease in the intensity of RAD51 corresponding to escalating doses of olaparib. In contrast, the nuclear intensity of RAD51 remained consistent across all concentrations for resistant subtypes (UWB 1.289 REOL and OVCA429 REOL, FIG. 4B). The prolonged presence of RAD51 potentially contributes to the resistance of olaparib by directly enhancing HR-mediated repair or by safeguarding and stabilizing replication forks Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0171] [Su et al., 2008; Kolinjivadi et al., 2017], Finally, a dose-dependent decrease in XRCC1 content in parental subtypes compared to a constant level in their REOL counterparts was observed, particularly pronounced in the UWB 1.289 parental compared with the UWB 1.289 REOL subtype (FIG. 4C). XRCC1 is known to act as an “anti -trapper,” where it prevents or resolves the trapping of PARP on DNApreventing PARPi trapping during DNA base excision repair [Demin et al., 2021, 34102106], These findings indicate that the relatively increased level of XRCC1 on the REOL subtypes facilitates the efficient completion of base excision repair and possibly assists in the removal or recycling of trapped PARP, contributing to reduced olaparib sensitivity.

[0172] EXAMPLE 3. Proteomics analysis for proteins exhibiting enrichment between parental and resistant cell subtypes

[0173] Next, a whole-proteomic sequencing was performed to explore proteins associated with the acquisition of resistance, investigating the underlying mechanisms of olaparib resistance, and identify potential markers. Among 5,023 and 5,061 total proteins detected in the UWB 1.289 and OVCA429 groups, a total of 297 differential expressed proteins, including 112 downregulated and 185 upregulated proteins, were identified between UWB 1.289 parental and REOL subtypes (FIG. 6A, Table 2). For OVCA429, a total of 289 differentially expressed proteins, including 173 downregulated and 116 upregulated proteins, were identified between parental and REOL subtypes (FIG. 6B, Table 3).

[0174] Using the proteomics data, the functional ontology enrichment analysis of differentially expressed proteins was examined based on gene ontology annotations. Some common nodes were enriched among the two cellular models. For biological processes, many differentially expressed proteins were involved in cellular component organization or biogenesis and metabolic processes; for cellular components, they were associated with cytoplasm, cytosol, organelle, extracellular vesicles, etc. As for molecular function, they were commonly enriched in RNA-binding and protein binding.

[0175] The UWB 1.289 cell model exhibits an enrichment in proteins involved in key biological processes encompassing cell migration, angiogenesis regulation, and cellular localization regulation. For metabolic processes, these findings noted enrichment in nodes related to cell adhesion molecule binding and structural cellular Attorney Docket No. 29539-0857WO1 / MGH 2025-144 organization activity (FIG. 6C). The OVCA429 cell model showed enrichment in proteins that are known to influence different biological processes, including regulation of DNA replication, double-strand break repair via break-induced replication, and cytoskeleton organization. The most enriched metabolic process nodes encompassed nucleotide and purine binding (FIG. 6D). This can be interpreted to means that each cell line may deploy distinct strategies in response to olaparib toxicity.

[0176] Nineteen differentially expressed proteins commonly enriched in both cell lines (UWB 1.289 and OVCA429) when comparing parental and resistant subtypes were further interrogated better to appreciate their subcellular localization (FIG. 7A). The result shows 10 enriched proteins that were consistently up-regulated in the REOL subtypes DPYSL3, AKR1C3, ALB, T0GARAM2, SAMHD1, FADS3, CAP2, CP, SMYD3, and LTF; and 9 proteins were down-regulated including LCN2, TAGLN, RPL36AL, ARHGDIB, KRT17, TRIM16, PTMA, RPL39 and EPCAM (Table 1)

[0177] To elucidate the functional context of these differentially expressed proteins, their predominant subcellular locations were assessed. The subcellular localization annotation revealed that most of them were localized to the cytoplasm, nucleus, and endoplasmic reticulum (FIG. 8A). The diverse subcellular localization of the differentially expressed proteins suggests their involvement in a wide range of cellular functions, including but not limited to signaling, gene expression regulation, and the management of olaparib-induced stress or could also just be non-specific. Pathway analysis through WikiPathway [Slenter et al., 2018, 29136241] showed that those differentially expressed proteins were involved in ferroptosis, eicosanoid synthesis, estrogen metabolism, histone modifications, and the aryl hydrocarbon receptor pathway (AhR), among others (FIG. 8B). In particular, the AhR pathway's influence on detoxification enzymes such as cytochrome P450 suggests a potential effect on olaparib responses, given its primary metabolism by cytochrome P450 (CYP) 3 A4 [Bruin et al., 2022, 36219340], Notably, while rucaparib and niraparib, both FDA- approved PARPi for ovarian cancer, are metabolized by CYP3 A4, the contribution of this enzyme to niraparib metabolism is minimal [Bruin et al., 2022, 36219340], Instead, niraparib is primarily transformed into its inactive metabolite Ml by carboxylesterases and UDP-glucuronosyltransferases [Valabrega et al., 2021, Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0178] 33921561], This implies a possible distinct impact of AhR pathway activation on the sensitivity of each PARP inhibitor.

[0179] Proceeding with the analysis and focusing on identifying individual players, potential biomarkers for olaparib resistance were selected among the 19 differentially expressed proteins. The criteria for prioritization included their known association with ovarian cancer pathophysiology, relevance to PARP mechanisms, and consistent expression across the two cellular models (UWB1.289 / UWB 1.289 REOL and OVCA429 / OVCA429 REOL). Following this analysis, two candidates as initial biomarkers were identified: SET and MYND domain-containing protein 3 (SMYD3) and Sterile Alpha Motif and Histidine-Aspartate Domain-Containing Protein 1 (SAMHD1). These proteins are involved in cell proliferation and epithelial- mesenchymal transition (SMYD3) and promotion of the HR pathway (SMYD3, SAMHD1) [Fenizia et al., 2019, 30544196; Kueck et al., 2018] (FIG. 7B). SAMHD1 is primarily known for its role as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase enzyme, involved in regulating the cellular pool of deoxynucleotide triphosphates (dNTPs) by hydrolyzing them into deoxynucleosides and inorganic triphosphate [Mauney and Hollis, 2018], While SAMHDl's role in dNTP metabolism is well-established, emerging research suggests its involvement in regulating DNA repair mechanisms, which is crucial for olaparib efficiency [Jiang et al., 2019, 31417652], Dysregulation of SAMHD1 has been positively associated with various cancers, including leukemia, colorectal cancer, and breast cancer [Mauney and Hollis, 2018, 29583030], Of interest is the observation that AhR activation can lead to the repression of CDK1 / 2 activity [Kueck et al., 2018], CDK1 / 2 phosphorylates SAMHD1, resulting in the degradation of SAMHD1 and, subsequently, higher dNTP concentrations, which are necessary for DNA synthesis during the S phase of the cell cycle [Stillman, 2013], Thus, the upregulation of SAMHD1 could serve as a resistance mechanism in olaparib-resistant cells by compensating for the decreased activity of CDK1 / 2. This compensation may allow cells to maintain sufficient dNTP levels for DNA repair and replication, contributing to resistance against PARP inhibitors like olaparib. In vitro studies in prostate cancer have demonstrated that inhibiting DNA repair via CDK1 inhibition enhances DNA damage, thereby resensitizing multi-treated cells to olaparib [Lombard et al., 2022], SMYD3 is a lysine methyltransferase that catalyzes the methylation of histone and Attorney Docket No. 29539-0857WO1 / MGH 2025-144 non-histone proteins [Jarrell et al., 2021], SMYD3 can methylate histone H3 at lysine 4 (H3K4) and non-histone proteins such as p53 and MAP3K2 [Jarrell et al., 2021], It has been implicated in various cellular processes, including transcriptional regulation, cell cycle progression, and tumorigenesis. SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in colorectal and breast cancer cells [Sanese et al., 2020; Sanese et al., 2021],

[0180] The next experiment evaluated the effects of inhibiting SAMHD1 and SMYD3 on olaparib sensitivity. In this study, cell viability in OVCA429 parental and REOL subtypes was assessed after treatment with the IC50 of olaparib alone or in combination with inhibitors of SAMHD1 and SMYD3. As demonstrated in FIG. 7C, none of the inhibitor concentrations affected cell viability compared with the PBS control. Cephalosporin C zinc salt not only enhanced the sensitivity of olaparib by 74.24% in the REOL subtype but also showed no significant effect on the parental cell subtype. Similarly, the SMYD3-IN-1 inhibitor did not significantly improve olaparib sensitivity in the parental cell subtype but did increase sensitivity by 71.25% when combined with olaparib compared to olaparib alone. These results show that both inhibitors effectively target their respective proteins in a concentration- dependent manner and indicate an interaction between these proteins and olaparib sensitivity.

[0181] Specifically, SMYD3 is a histone methyltransferase that primarily targets histone H3 (methylating H3K4). It's involved in chromatin remodeling and gene expression regulation and has been implicated in the progression and metastasis of various cancers. SMYD3 may indirectly influence DNA repair processes or the expression of DNA repair genes through its chromatin-modifying activities. SMYD3's involvement in the NHEJ pathway suggests that it contributes to the repair of DNA double-strand breaks. Since PARP inhibitors, such as olaparib, primarily target the HR repair pathway, the upregulation of an alternative repair pathway like NHEJ could potentially compensate for the inhibited HR pathway. This suggests that higher levels of SMYD3 might be associated with increased capacity for DNA repair through NHEJ, potentially leading to resistance against PARP inhibitors. A recent paper highlighted SMYD3's role in recruiting and activating the LIG4 / XRCC4 / XLF complex, essential for effective NHEJ repair. In cancers, where HR is already Attorney Docket No. 29539-0857WO1 / MGH 2025-144 compromised, reliance on NHEJ for DNA repair could be heightened. In such cases, targeting SMYD3 to disrupt NHEJ might enhance the effectiveness of PARP inhibitors. SAMHDl's function in dNTPase activity, which hydrolyzes dNTPs, suggests it could influence DNA repair pathways. By regulating the pool of available dNTPs, SAMHD1 might affect DNA synthesis during repair processes.

[0182] EXAMPLE 4. Cross-resistance with other drugs

[0183] This study investigated if olaparib resistance shows cross-resistance with other commonly used drugs in ovarian cancer by using established in vitro models. In particular, if different PARP inhibitors may share the resistance mechanism(s) was investigated (i.e., if cells resistant to olaparib also show resistance to other drugs or PARP inhibitors). The efficacy of four commonly administered chemo drugs (carboplatin, doxorubicin, paclitaxel, and topotecan) and the two FDA-approved PARP inhibitors for ovarian cancer (rucaparib and niraparib) was examined across the paired cell lines: UWB 1.289 and OVCA429, along with their respective REOL subtypes (UWB 1.289 REOL and OVCA429 REOL, FIG. 9). The LC50 values summarized in Table 4 indicate no significative increase on the resistant cell subtypes compared with the parental cell lines for carboplatin, doxorubicin, paclitaxel, rucaparib, and niraparib. However, the REOL subtypes showed a significant increase in LC50 for topotecan, with the UWB 1.289 REOL subtype displaying a 3-fold higher LC50 (P = 0.009) and the OVCA429 REOL subtype a 6-fold increase (P = 0.013) compared to their respective parental counterparts. DNA repair proteins' enhanced activity, or expression, may play a pivotal role in mitigating the damage induced by topotecan and olaparib, underpinning the cross-resistance observed between these drugs.

[0184] EXAMPLE 5. Patient-derived organoid sensitivity to sequential PARPi treatment

[0185] Building on preclinical and clinical data that underscored the unique trapping capacities of niraparib and its potential effectiveness in cells resistant to olaparib, as well as studies indicating no inherent resistance to subsequent PARPi treatments [Giudice et al., 2022], the efficacy of PARPi combinations for ovarian cancer treatment were investigated. The absence of cross-resistance observed in the in vitro

[0186] 31 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 assays reinforced the decision to assess the feasibility of sequential PARPi using patient-derived organoids (PDOs).

[0187] To validate this strategy, the next experiment assessed the sensitivity of PDOs derived from a patient naive to olaparib (VCRB357 PDXO), deemed sensitive, against PDOs derived from patients with olaparib resistance (17-121, 18-47, 17-116, and 17-39) [Hill et al., 2018, 30213835], Initially, all organoid cultures were evaluated for sensitivity to the PARPi olaparib (FIG. 10A). The VCRB357 PDXO culture exhibited the highest sensitivity, with an LC50 of 4.39±3.10 pM. Organoids previously identified as olaparib-resistant displayed LC50 values of approximately 38.98 ± 17.35 pM (17-39), -200 pM (17-121), 25.34 ± 3.14 pM (17-116), and 16.94 ± 7.42 pM (18-47). Despite varying olaparib IC50 values across all PDOs, all models exhibited lower LC50 values for niraparib, ranging from 3.63±0.35 pM (VCRB357 PDXO) to 10.93±0.14 pM for (17-121) (FIG. 10B). The consistent sensitivity of all PDOs to niraparib aligns with prior observation of no crossresistance between these two PARPi agents, reinforcing the potential benefits of considering an alternating PARPi treatment regimen.

[0188] Conclusion

[0189] This study addresses the pivotal issue of olaparib resistance in ovarian cancer, utilizing proteomic techniques to characterize a cellular context altered by olaparib treatment while assessing alternative therapeutic strategies' effectiveness. This investigation explores the impact of chronic olaparib exposure on protein expression in ovarian cancer cells by developing resistant sublines UWB 1.289 REOL and OVCA429 REOL. These subtypes exhibit significant changes in LC50 values, doubling times, and cell cycle distribution, providing insights into the cellular adjustments induced by prolonged olaparib treatment.

[0190] The differential expression of proteins involved in DNA replication and repair, including y-H2AX, RAD51, and XRCC1, between resistant and parental cell lines elucidates a complex network of repair processes and cell cycle management in response to olaparib-induced DNA damage. Importantly, the upregulation of y-H2AX in resistant cells signifies the DNA damage induced by olaparib. In the absence of efficient PARP activity due to inhibitors like olaparib, the need for effective DSB repair becomes paramount. The stable expression of RAD51 in resistance cell Attorney Docket No. 29539-0857WO1 / MGH 2025-144 subtypes suggests the activation of an adaptive mechanism that preserves HR repair efficiency.

[0191] Concurrently, XRCCl's role in reducing PARP trapping complements this mechanism by smoothing the base excision repair process, indirectly supporting HR. This coordination minimizes replication disruptions and genomic instability, enhancing the overall integrity of DNA repair facilitated by RAD51 [Orhan et al., 2021, 34208195; Kolinjivadi et al., 2017; Su et al., 2008; Gonzalez-Magana and Blanco, 2020; Cardano et al., 2020], The critical need for therapeutic alternative targeting strategies becomes apparent, emphasizing the necessity to inhibit PARP activity or disrupt other DNA repair mechanisms of resistant cells. The exploration of PARPi combinations represents an active area of research. Studies have underscored the efficacy of BET inhibitors, such as I-BET762, are acknowledged for their capacity to downregulate DNA damage response genes, thereby inducing an HR-deficient phenotype in cells initially proficient in HR [Fiorentino et al., 2020], Significantly, the synergistic application of BET inhibitors in conjunction with PARP inhibitors has been shown to enhance anti-tumor effects beyond the use of either treatment in isolation across various cell lines. This includes those with BRCA1 / 2 wild-type or deficiencies in 53BP1 or PARPI, thus presenting a promising path for circumventing various mechanisms of PARPi resistance [Sun et al., 2018; Fiorentino et al., 2020], In this study, we chose to analyze the nuclear distribution of these DNA damage markers, allowing us to evaluate the prevalence across a broader population of cells. Incorporating foci counting and colocalization analysis is investigated, providing a more detailed map of DNA repair activity.

[0192] The proteomic analysis described herein identified differentially expressed proteins in cells with acquired resistance to olaparib and their associated pathways related to the chronic administration of olaparib. While the direct correlation between the altered pathways and olaparib efficiency has not yet been completely understood, mechanistic studies on other pathologies suggest an interesting potential relationship. For instance, the enrichment in the ferroptosis pathway, an intracellular irondependent form of cell death, has been extensively reported as associated with lipid peroxide accumulation and is implicated in chemotherapy resistance [Zhang et al., 2022], Eicosanoid synthesis pathways could influence drug responses, particularly through the modulation of inflammation and immune responses [Wang and Dubois, Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0193] 2010], Histone modifications, which may impact gene expression patterns, could affect genes involved in the HR pathway, such as BRCAlfAudia and Campbell, 2016], Moreover, the AhR pathway's direct involvement in regulating the metabolism of PARPi may impact drugs such as olaparib, rucaparib, and niraparib differently due to their varying substrate affinities [Larigot et al., 2018; Bruin et al., 2022], The potential interactions among these pathways and their impact on olaparib's metabolism, bioavailability, and efficacy encourage new research and exploration avenues.

[0194] Among the 19 identified differentially expressed proteins, SMYD3 and SAMHD1 can be key in the landscape of olaparib resistance. SMYD3, as a transcriptional activator for oncogenesis-associated genes [Jiang et al., 2019], and its promotion of HR repair, contributes to genomic stability in cancer cells and aids tumor progression [Sanese et al., 2021], Similarly, SAMHD1, associated with tumor histology and grade [Felip et al., 2022], plays a critical role in the response to DNA damage-inducing agents, such as platinum derivatives [Daddacha et al., 2017], Cells with SAMHD1 knockout (KO) exhibit heightened susceptibility to DNA damage and apoptosis, providing a compelling basis for exploring the impact of SAMHD1 knockout on restoring sensitivity to olaparib [Gutierrez-Chamorro et al., 2023],

[0195] The results herein provide insights into the dynamics of resistance mechanisms within the context of ovarian cancer treatment. In this context, patient- derived organoids (PDOs) have emerged as indispensable tools due to their ability to replicate critical aspects of clinical scenarios more faithfully compared to traditional 2D cultures [Graham et al., 2023; Tao and Wu, 2021], However, it is noteworthy that recent co-clinical trials have revealed disparities between organoid responses and patient outcomes, particularly concerning DNA damage drugs. Notably, our observation that olaparib resistance does not necessarily confer cross-resistance to other chemotherapeutic agents or PARP inhibitors, such as niraparib, challenges conventional assumptions regarding treatment strategies. It is well -documented that both niraparib and rucaparib exhibit superior trapping of PARP -DNA complexes compared to olaparib [Zhou et al., 2020], The fact that niraparib is still capable of exerting a cytotoxic effect in olaparib-resistant cells may be attributed to its heightened ability to stabilize PARP -DNA adducts, underscoring the importance of further studies to explore the potential benefits of switching between PARP inhibitors. Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0196] More validation studies and clinical trials are warranted to bridge the gap between organoid models and patient outcomes

[0197] Table 1. Representative relative expression levels of protein between parental and REOL cells, UP, up-regulated; DOWN, down-regulated

[0198] Table 2 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0199] Table 3 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144 Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0200] Table 4. In Vitro cross-resistance analysis Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0201] Table 5. List of antibodies used in the experiments.

[0202] Reference:

[0203] 1. Worzfeld, T. et al. The Unique Molecular and Cellular Microenvironment of Ovarian Cancer. Front Oncol 7, 24 (2017).

[0204] 2. Zhang, C., Xu, C., Gao, X. & Yao, Q. Platinum-based drugs for cancer therapy and anti -tumor strategies. Theranostics 12, 2115-2132 (2022).

[0205] 3. Maiorano, B. A., Maiorano, M. F. P. & Maiello, E. Olaparib and advanced ovarian cancer: Summary of the past and looking into the future. Front Pharmacol 14, 1162665 (2023).

[0206] 4. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917-921 (2005).

[0207] 5. Yap, T. A., Sandhu, S. K., Carden, C. P. & de Bono, J. S. Poly(ADP-ribose) polymerase (PARP) inhibitors: Exploiting a synthetic lethal strategy in the clinic. CA Cancer J Clin 61, 31-49 (2011).

[0208] 6. Kim, D. & Nam, H. J. PARP Inhibitors: Clinical Limitations and Recent Attempts to Overcome Them. Int J Mol Sci 23, 8412 (2022).

[0209] 7. DiSilvestro, P., Banerjee, S. & Colombo..., N. Overall survival with maintenance olaparib at a 7-year follow-up in patients with newly diagnosed advanced ovarian cancer and a BRCA mutation: the SOLO1 .... Journal of Clinical ... (2023).

[0210] 8. Desai, C., Pathak, A., Limaye, S. & Maniar. . ., V. A review on mechanisms of resistance to PARP inhibitors. Indian Journal of ... (2022).

[0211] 9. Tobalina, L., Armenia, J., Irving, E. & O’Connor. . ., M. J. A meta-analysis of reversion mutations in BRCA genes identifies signatures of DNA end-joining repair mechanisms driving therapy resistance. Annals of ... (2021). Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0212] 10. Langelier, M. F., Eisemann, T. & Riccio. . ., A. A. PARP family enzymes: regulation and catalysis of the poly (ADP-ribose) posttranslational modification. Current opinion in ... (2018).

[0213] 11. Mirman, Z., Sasi, N. K., King, A. & Chapman..., J. R. 53BPl-shi el dindependent DSB processing in BRC Al -deficient cells requires CST-Pola-primase fill- in synthesis. Nature cell ... (2022).

[0214] 12. Rondinelli, B., Gogola, E., Yiicel, H. & Duarte. . ., A. A. EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation. Nature cell ... (2017).

[0215] 13. Rottenberg, S. et al. High sensitivity of BRC Al -deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proceedings of the National Academy of Sciences 105, 17079-17084 (2008).

[0216] 14. Taglialatela, A., Alvarez, S., Leuzzi, G. & Sannino..., V. Restoration of replication fork stability in BRC Al -and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers. Molecular cell (2017).

[0217] 15. Kim, D. S., Camacho, C. V. & Kraus, W. L. Alternate therapeutic pathways for PARP inhibitors and potential mechanisms of resistance. Experimental & molecular medicine (2021).

[0218] 16. Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529- 542 (2011).

[0219] 17. Huntoon, C. J. et al. ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status. Cancer Res 73, 3683-3691 (2013).

[0220] 18. Carrassa, L., Broggini, M., Erba, E. & Damia, G. Chkl, but not Chk2, is Involved in the Cellular Response to DNA Damaging Agents: Differential Activity in Cells Expressing, or not, p53. Cell Cycle 3, 1175-1179 (2004).

[0221] 19. Murai, J., Huang, S. N., Das, B. B., Renaud, A. & Zhang. . ., Y. Trapping of PARP 1 and PARP2 by clinical PARP inhibitors. Cancer research (2012).

[0222] 20. Giudice, E. et al. PARP Inhibitors Resistance: Mechanisms and Perspectives. Cancers (Basel) 14, 1420 (2022).

[0223] 21. Lord, C. J. & Ashworth, A. PARP inhibitors: Synthetic lethality in the clinic. Science 355, 1152-1158 (2017). 22. DelloRusso, C. et al. Functional Attorney Docket No. 29539-0857WO1 / MGH 2025-144 characterization of a novel BRCAl-null ovarian cancer cell line in response to ionizing radiation. Mol Cancer Res 5, 35-45 (2007).

[0224] 23. Beaufort, C. M. et al. Ovarian cancer cell line panel (OCCP): clinical importance of in vitro morphological subtypes. PLoS One 9, el03988 (2014).

[0225] 24. Langland, G. T. et al. Radiosensitivity profiles from a panel of ovarian cancer cell lines exhibiting genetic alterations in p53 and disparate DNA-dependent protein kinase activities. Oncol Rep 23, 1021-1026 (2010).

[0226] 25. Sharma, A., Singh, K. & Almasan, A. in Methods in Molecular Biology: DNA Repair Protocols 613-626 (Humana Press, Totowa, NJ, 2012).

[0227] 26. Kolinjivadi, A. M., Sannino, V., Antoni, A. D. & Zadorozhny..., K. Smarcall -mediated fork reversal triggers Mrel 1 -dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Molecular cell (2017).

[0228] 27. Su, X., Bernal, J. A. & Venkitaraman, A. R. Cell-cycle coordination between DNA replication and recombination revealed by a vertebrate N-end rule degron-Rad51. Nature structural & molecular ... (2008).

[0229] 28. Caldecott, K. W. XRCC1 protein; Form and function. DNA Repair (Amst) 81, 102664 (2019).

[0230] 29. Rogakou, E. P., Pilch, D. R., Orr, A. H. & Ivanova. . ., V. S. DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139. Journal of biological ... (1998).

[0231] 30. Demin, A. A. et al. XRCC1 prevents toxic PARP1 trapping during DNA base excision repair. Mol Cell 81, 3018-3030. e5 (2021).

[0232] 31. Slenter, D. N. et al. WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research. Nucleic Acids Res 46, D661-D667 (2018).

[0233] 32. Bruin, M. A. C., Sonke, G. S., Beijnen, J. H. & Huitema, A. D. R. Pharmacokinetics and Pharmacodynamics of PARP Inhibitors in Oncology. Clin Pharmacokinet 61, 1649-1675 (2022).

[0234] 33. Valabrega, G., Scotto, G., Tuninetti, V., Pani, A. & Scaglione, F. Differences in PARP Inhibitors for the Treatment of Ovarian Cancer: Mechanisms of Action, Pharmacology, Safety, and Efficacy. IntJMol Sci 22, 4203 (2021). Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0235] 34. Fenizia, C. et al. SMYD3 promotes the epithelial-mesenchymal transition in breast cancer. Nucleic Acids Res 47, 1278-1293 (2019).

[0236] 35. Kueck, T., Cassella, E., Holler, J., Kim, B. & Bieniasz, P. D. The aryl hydrocarbon receptor and interferon gamma generate antiviral states via transcriptional repression. Elife 7 , e38867 (2018).

[0237] 36. Mauney, C. H. & Hollis, T. SAMHD1 : Recurring roles in cell cycle, viral restriction, cancer, and innate immunity. Autoimmunity 51, 96-110 (2018).

[0238] 37. Jiang, Y. et al. Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20. J Cancer 10, 4072-4084 (2019).

[0239] 38. Stillman, B. Deoxynucleoside triphosphate (dNTP) synthesis and destruction regulate the replication of both cell and virus genomes. Proc Natl Acad Sci USA 110, 14120-14121 (2013).

[0240] 39. Lombard, A. P. et al. Olaparib-Induced Senescence Is Bypassed through G2-M Checkpoint Override in Olaparib-Resistant Prostate Cancer. Mol Cancer Ther 21, 677-685 (2022).

[0241] 40. Jarrell, D. K., Hassell, K. N., Alshiraihi, I., Crans, D. C. & Brown, M. A. Structural Analysis of SMYD3 Lysine Methyltransferase for the Development of Competitive and Specific Enzyme Inhibitors. Diseases 10, 4 (2021).

[0242] 41. Sanese, P. et al. Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP -Mediated Synthetic Lethality. iScience 23, 101604 (2020).

[0243] 42. Sanese, P., Fasano, C. & Simone, C. Playing on the Dark Side: SMYD3 Acts as a Cancer Genome Keeper in Gastrointestinal Malignancies. Cancers (Basel) 13, 4427 (2021).

[0244] 43. Hill, S. J. et al. Prediction of DNA Repair Inhibitor Response in Short- Term Patient Derived Ovarian Cancer Organoids. Cancer Discov 8, 1404-1421 (2018).

[0245] 44. Orhan, E., Velazquez, C., Tabet, I., Sardet, C. & Theillet, C. Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy. Cancers (Basel) 13, 2930 (2021).

[0246] 45. Gonzalez-Magana, A. & Blanco, F. J. Human PCNA structure, function, and interactions. Biomolecules (2020). Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0247] 46. Cardano, M., Tribioli, C. & Prosperi, E. Targeting proliferating cell nuclear antigen (PCNA) as an effective strategy to inhibit tumor cell proliferation. Current Cancer Drug ... (2020).

[0248] 47. Fiorentino, F. P. et al. BET-Inhibitor I-BET762 and PARP -Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells. IntJMol Set 21, 9595 (2020).

[0249] 48. Sun, C. et al. BRD4 Inhibition Is Synthetic Lethal with PARP Inhibitors through the Induction of Homologous Recombination Deficiency. Cancer Cell 33, 401-416. e8 (2018).

[0250] 49. Zhang, C., Liu, X., Jin, S., Chen, Y. & Guo, R. Ferroptosis in cancer therapy: a novel approach to reversing drug resistance. Mol Cancer 21, 47 (2022).

[0251] 50. Wang, D. & Dubois, R. N. Eicosanoids and cancer. Nat Rev Cancer 10, 181-193 (2010).

[0252] 51. Audia, J. E. & Campbell, R. M. Histone Modifications and Cancer. Cold Spring Harb Per sped Biol 8, aO 19521 (2016).

[0253] 52. Larigot, L., Juricek, L., Dairou, J. & Coumoul, X. AhR signaling pathways and regulatory functions. Biochim Open 7 , 1-9 (2018).

[0254] 53. Felip, E. et al. Modulation of DNA Damage Response by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase (SAMHDl) Determines Prognosis and Treatment Efficacy in Different Solid Tumor Types. Cancers (Basel) 14, 641 (2022).

[0255] 54. Daddacha, W. et al. SAMHDl Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination. Cell Rep 20, 1921-1935 (2017).

[0256] 55. Gutierrez-Chamorro, L. et al. SAMHDl expression modulates innate immune activation and correlates with ovarian cancer prognosis. Front Immunol 14, 1112761 (2023).

[0257] 56. Graham, O., Rodriguez, J. & Biljon. . ., L. V. Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids. JoVE (Journal of ... (2023).

[0258] 57. Tao, M. & Wu, X. The role of patient-derived ovarian cancer organoids in the study of PARP inhibitors sensitivity and resistance: from genomic analysis to functional testing. Journal of Experimental & Clinical Cancer Research 40, (2021). Attorney Docket No. 29539-0857WO1 / MGH 2025-144

[0259] 58. Zhou, P., Wang, J., Mishail, D. & Wang, C. Y. Recent advancements in PARP inhibitors-based targeted cancer therapy. Precis Clin Med 3, 187-201 (2020).

[0260] 59. Gopal, A. A., Fernandez, B., Delano, J., Weissleder, R. & Dubach, J. M. PARP trapping is governed by the PARP inhibitor dissociation rate constant. Cell Chem Biol S2451-9456(23)00476 (2024).

[0261] 60. Zhang, L., Kwan, S. Y., Wong, K. K., Soliman, P. T. & Lu. . ., K. H. Pathogenesis and clinical management of uterine serous carcinoma. Cancers (2020).

[0262] 61. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13, 731-740 (2016). OTHER EMBODIMENTS

[0263] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Attorney Docket No. 29539-0857WO1 / MGH 2025-144WHAT IS CLAIMED IS:

1. A method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; and identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control.

2. The method of claim 1, wherein the expression level of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

3. The method of claim 1, wherein the expression level of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

4. The method of any one of claims 1-3, wherein the subject was previously treated with olaparib.

5. The method of any one of claims 1-3, wherein the subject has never been treated with olaparib.

6. The method of any one of claims 1-5, further comprising administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

7. The method of any one of claims 1-5, further comprising administering to the subject a therapeutically effective amount of a poly (ADP-ribose) polymeraseAttorney Docket No. 29539-0857WO1 / MGH 2025-144 inhibitor (PARPi) that is not olaparib if the subject is identified to be resistant to olaparib.

8. The method of any one of claims 1-5 and 7, wherein the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib.

9. The method of claim 7 or 8, wherein the PARPi is selected from the group consisting of niraparib, rucaparib, and talazoparib.

10. The method of any one of claims 1-9, further comprising administering to the subject a therapeutically effective amount of an anti -cancer agent if the subject is identified to be resistant to olaparib, wherein the anti -cancer agent is not a PARPi.

11. The method of any one of claims 1-10, further comprising administering to the subject a therapeutically effective amount of an SMYD3 inhibitor and / or an SAMHD1 inhibitor if the subject is identified to be resistant to olaparib.

12. The method of claim 11, wherein the SMYD3 inhibitor and / or the SAMHD1 inhibitor is administered prior to, concurrently, or after administration of olaparib.

13. The method of any one of claims 1-12, wherein the subject has a cancer.

14. The method of claim 13, wherein the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer, optionally, wherein the cancer is ovarian cancer.

15. The method of any one of claims 1-14, wherein the subject has a homologous recombination deficiency (HRD) mutation.

16. The method of claim 15, wherein the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.Attorney Docket No. 29539-0857WO1 / MGH 2025-14417. The method of claim 15 or 16, wherein the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

18. A method of treating a cancer in a subject, the method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control; and administering a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib to the subject if the subject is identified to be resistant to olaparib, or administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

19. The method of claim 18, wherein the expression levels of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

20. The method of claim 18, wherein the expression levels of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

21. The method of any one of claims 18-20, wherein the subject was previously treated with olaparib.

22. The method of any one of claims 18-20, wherein the subject has never been treated with olaparib.Attorney Docket No. 29539-0857WO1 / MGH 2025-14423. The method of any one of claims 18-22, wherein the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib.

24. The method of any one of claims 18-23, wherein the PARPi that is not olaparib is selected from the group consisting of niraparib, rucaparib, and talazoparib.

25. The method of any one of claims 18-24, wherein the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer, optionally, wherein the cancer is ovarian cancer.

26. The method of any one of claims 18-25, wherein the subject has a homologous recombination deficiency (HRD) mutation.

27. The method of claim 26, wherein the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.

28. The method of claim 26 or 27, wherein the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

29. A method of identifying a subject to be resistant to olaparib, the method comprising providing a biological sample from a subject; measuring expression levels of SMYD3 and / or SAMHD1 in the biological sample; and identifying the subject to be resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are higher than a reference control, or identifying the subject to be less likely resistant to olaparib if the expression levels of SMYD3 and / or SAMHD1 are not higher than a reference control.Attorney Docket No. 29539-0857WO1 / MGH 2025-14430. The method of claim 29, wherein the expression levels of SMYD3 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

31. The method of claim 29, wherein the expression levels of SAMHD1 in the biological sample from the subject who is identified to be resistant to olaparib is at least 10%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, or 1000% higher than the reference control.

32. The method of any one of claims 29-31, wherein the subject was previously treated with olaparib.

33. The method of any one of claims 29-31, wherein the subject has never been treated with olaparib.

34. The method of any one of claims 29-33, further comprising administering a therapeutically effective amount of olaparib to the subject if the subject is identified to be less likely to be resistant to olaparib.

35. The method of any one of claims 29-33, further comprising administering to the subject a therapeutically effective amount of a poly (ADP-ribose) polymerase inhibitor (PARPi) that is not olaparib if the subject is identified to be resistant to olaparib.

36. The method of any one of claims 29-33 and 35, wherein the subject who is identified to be resistant to olaparib is sensitive to a PARPi that is not olaparib.

37. The method of claim 35 or 36, wherein the PARPi is selected from the group consisting of niraparib, rucaparib, and talazoparib.Attorney Docket No. 29539-0857WO1 / MGH 2025-14438. The method of any one of claims 29-37, further comprising administering to the subject a therapeutically effective amount of an anti -cancer agent if the subject is identified to be resistant to olaparib, wherein the anti -cancer agent is not a PARPi.

39. The method of any one of claims 29-37, further comprising administering to the subject a therapeutically effective amount of an SMYD3 inhibitor and / or an SAMHD1 inhibitor if the subject is identified to be resistant to olaparib.

40. The method of claim 39, wherein the SMYD3 inhibitor and / or the SAMHD1 inhibitor is administered prior to, concurrently, or after administration of olaparib.

41. The method of any one of claims 29-40, wherein the subject has a cancer.

42. The method of claim 41, wherein the cancer is ovarian cancer, prostate cancer, pancreatic cancer, or breast cancer, optionally, wherein the cancer is ovarian cancer.

43. The method of claim 29-42, wherein the subject has a homologous recombination deficiency (HRD) mutation.

44. The method of claim 43, wherein the HRD mutation is selected from the group consisting of a BRCA1 mutation, a BRCA2 mutation, a RAD51C mutation, a RAD51D mutation, and a PALB2 mutation.

45. The method of claim 44, wherein the HRD mutation comprises a BRCA1 mutation or a BRCA2 mutation.

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