Peterson, Kevin L.

Abstract BRCA2 mutations contribute to the pathogenesis and treatment sensitivity of a subset of ovarian, breast, prostate, and pancreatic cancers. When these cancers become therapy resistant, secondary mutations that restore the BRCA2 open reading frame are found in half the cases, but other causes of resistance remain incompletely understood. Here, we identified translational readthrough of a premature termination codon (PTC) as a cause of resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) and cisplatin in cells derived from the BRCA2-mutated ovarian cancer line PEO1 by PARPi selection. Despite persistence of the signature 4965C > G (p.Y1655X) BRCA2 mutation, low-level expression of full-length BRCA2 protein was detectable in these cells by immunoblotting and tandem mass spectrometry. Either BRCA2 knockdown or gene interruption 5′ or 3′ to the PTC restored treatment sensitivity, implicating BRCA2 in the resistance. Reporter assays demonstrated UAG-selective readthrough in the resistant clones but not parental cells. Moreover, custom searching of global proteomic data indicated readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. Finally, multi-omic analysis identified multiple changes in the nonsense-mediated decay and termination machineries that favor readthrough. Accordingly, the present results identify PTC readthrough as a potential mechanism of drug resistance in cells with BRCA2 nonsense mutations.

Abstract DAC combinations serve as frontline therapies for AML. DAC forms DNA-DNMT1 crosslinks and activates the ATR-CHK1-WEE1 DDR. Combinations of DNA-protein crosslinking agents with ATR, CHK1, or WEE1 inhibitors (i) have shown efficacy in solid tumors. This study evaluates the cytotoxic synergy of DAC plus ATRi, CHK1i, or WEE1i in AML. Public datasets were queried for mRNA expression and dependency. Commercial cell lines (U937, KG1a, Molm13, MV-4-11) were genomically validated. Molm13 TP53-/- clones were generated via CRISPR. Cells were cultured in complete RPMI. To treat, cells were cultured for 48 h in RPMI with drugs or diluent (0.2% v/v DMSO). Protein expression was evaluated by Western blotting (WB). Apoptosis was assayed by flow cytometry with annexin V or propidium iodide. Primary AML samples were obtained at study centers after informed consent. Isolated mononuclear cells (Ficoll) were used in colony forming unit (CFU) assays with DMSO or drug for 12-16 days. Synergy was determined by median-effect (CalcuSyn) where a combination index <1 is synergistic and >1 antagonistic. DNMT1 expression (TCGA) was highest in AML vs all other tumor types (p<0.001). In CRISPR screens (DepMap), AML cell lines were increasingly dependent on DNMT1, ATR, CHK1, and WEE1 (ANOVA p<0.001). WB of AML cell lines treated with DAC (25 - 200 nM) for 24 h showed phosphorylations of H2AX and CHK1 S296/S345 that were most pronounced in TP53MT lines. In apoptosis assays, ATRi, CHK1i, and WEE1i were synergistic with clinical concentrations of DAC (Table 1). In isogenic Molm13 lines, TP53 loss conferred DAC resistance that was overcome with combination treatment. Selective CHK1i were generally more synergistic than CHK1/2i, consistent with a CHK1 DDR. CFU assays of primary AML samples also showed synergy between DAC and ATRi, CHK1i, and especially WEE1i. Table 1. Synergy between decitabine and replication checkpoint inhibitors in preclinical models of AML. Cell line data are derived from subdiploid apoptosis assays. U937 data were replicated using annexin V. Prexasertib showed no activity in KG1a cells (suggesting drug efflux) requiring the use of the alternate CHK1 inhibitor, rabusertib. Abbreviations: ATR, ataxia telangiectasia and Rad3 related; CHK1/2, checkpoint kinase 1 and/or 2; CI, combination index; Conc, concentration; DNMT1, DNA methylatransferase 1; IQR, interquartile range; MT, mutated; ND, not determined; SD, standard deviation; WT, wild type. Drug Target Conc. (nM) Molm13 (TP53WT) Molm13 (TP53-/-) U937 (TP53G187fs*/-) KG1a (TP53V225fs*/-) Primary AML (TP53WT orTP53MT) Units Decitabine DNMT1 6.25 – 200 25 ± 11 350 ± 260 310 ± 180 6.1 ± 1.6 ND EC50, nM (mean, SD) Ceralasertib ATR 250 – 500 0.93 (0.66 – 1.24) 0.90 (0.84 – 0.96) 0.15 (0.08 – 0.21) 0.74 (0.63 – 1.02) 0.66 (0.43 – 2.43) CI with decitabine (median, IQR) Camonsertib ATR 10 – 100 0.27 (0.19 – 0.59) 0.24 (0.22 – 0.34) 0.34 (0.21 – 0.39) ND ND Prexasertib CHK1/2 1.0 – 6.0 0.89 (0.82 – 1.06) 0.78 (0.68 – 0.90) 0.95 (0.93 – 1.08) ND 0.88 (0.29 – 1.28) MK-8776 CHK1 250 – 1, 000 0.57 (0.48 – 0.80) 0.76 (0.73 – 1.19) 0.89 (0.57 – 1.39) ND ND Rabusertib CHK1 600 – 1, 000 ND ND ND 0.51 (0.48 – 0.56) ND Adavosertib WEE1 100 – 400 0.70 (0.63 – 1.11) 0.99 (0.78 – 1.14) 0.67 (0.60 – 0.74) 0.67 (0.37 – 0.93) 0.65 (0.43 – 0.88) DAC synergizes with ATRi, CHK1i, and WEE1i in AML. Future work will test these combinations in vivo and incorporate BH3 mimetics. Citation Format: Clifford M. Csizmar, Antoine N. Saliba, Olivia K. Rossman, Kevin L. Peterson, X. Wei Meng, B. Douglas Smith, Gabriel Ghiaur, Jonathan A. Webster, Mrinal M. Patnaik, Aref Al-Kali, Scott H. Kaufmann. Decitabine (DAC) induces a DNA damage response (DDR) and synergizes with replication checkpoint inhibitors in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1170.

Introduction Acute myeloid leukemia (AML) with mutated TP53 (TP53MT) is resistant to chemotherapy and has a poor prognosis. Prior reports suggest that DNA methyltransferase inhibitors (DNMTi), such as decitabine and azacitidine, form covalent DNA-DNMT1 adducts and invoke a DNA damage response (DDR) characterized by activation of the ATR-CHK1 pathway, including in TP53MT AML samples. This study evaluates whether decitabine exhibits cytotoxic synergy with replication checkpoint modulators (ATRi, CHK1/2i, WEE1i) in pre-clinical models of AML. Methods Parental cell lines (U937, KG1a, Molm13, MV-4-11) were obtained commercially and genomically validated. The Molm13 TP53-/- cell line was generated via CRISPR/Cas9 knockout. Cells were maintained in RPMI-1640 medium with 10% FBS, 2 mM glutamine, and antibiotics at 37 ºC, 5% CO2. Decitabine, ceralasertib (ATRi), prexasertib (CHK1/2i), MK-8776 (CHK1i), and adavosertib (WEE1i) were reconstituted at 10 mM in DMSO. For drug treatments, cells were cultured for 48 h in medium with 0.2% (v/v) DMSO or drug. Western blots were stained with commercial antibodies followed by peroxidase-coupled secondary antibodies and imaged by chemiluminescence. Apoptosis assays were performed via flow cytometry (BD FACSCanto II) with annexin V-APC (BD Biosciences) and/or propidium iodide (Sigma) staining. Peripheral blood and bone marrow samples were obtained from AML patients after informed consent at Mayo Clinic and Johns Hopkins University. Mononuclear cells were isolated via Histopaque-1077 (Sigma) centrifugation and used immediately. Colony forming assays were performed in Methocult H4435 (Stem Cell Technologies) supplemented with vehicle or drug; leukemic colonies were counted after 12-16 days. Combination indices (CI) were determined by the Chou-Talalay method (CalcuSyn) where values <1 indicate synergy and >1 antagonism. This work was supported in part by R01 CA289285. Results Western blots of TP53MT AML cell lines (U937, KG1a) and primary samples treated with decitabine (≤200 nM) for 24 h demonstrated increased phosphorylation of H2AX and S296/S345 CHK1, consistent with activation of the ATR-CHK1 DDR pathway. These changes were less pronounced in TP53 wild type cell lines (Molm13, MV-4-11). In subdiploid (SubG1) assays, U937 cells exhibited resistance to monotherapy with decitabine (EC50 = 330 ± 200 nM) and variable sensitivity to ceralasertib (250 - 500 nM), prexasertib (1 - 10 nM), MK-8776 (250 - 1,000 nM), and adavosertib (50 - 400 nM). When combined, however, marked synergy was observed between decitabine and both ceralasertib (mean CI 0.15) and adavosertib (CI 0.59). Synergy was also observed with the selective CHK1i, MK-8776 (CI 0.56), but less-so with the dual CHK1/2i, prexasertib (CI 0.93), consistent with the hypothesis of an ATR-CHK1 DNA damage response. Similar results were obtained from decitabine (EC50 = 300 ± 100 nM) and ceralasertib (CI 0.54), adavosertib (CI 0.67), MK-8776 (CI 0.52), and prexasertib (CI 0.81) in annexin V assays. These combinations were further evaluated (SubG1 assays) in an isogenic pair of Molm13 and Molm13 TP53-/- cell lines. In Molm13 cells, decitabine (EC50 = 23 ± 11 nM) was again synergistic with adavosertib (CI 0.60), but not ceralasertib (CI 1.3) or prexasertib (CI 1.2). In Molm13 TP53-/- cells, however, very little synergy was observed between decitabine (EC50 = 53 ± 22 nM) and ceralasertib (CI 1.5), prexasertib (CI 4.3), or adavosertib (CI 1.0) except for at higher concentrations of adavosertib (400 nM, CI 0.89). Thus, while biallelic TP53 inactivation conferred resistance to DNMTi and checkpoint inhibitors, the degree of resistance was context-dependent and could be partially overcome by WEE1 inhibition. The combinations of decitabine with ceralasertib (n = 2), prexasertib (n = 4), or adavosertib (n = 3) were evaluated in colony forming assays using primary AML samples from newly-diagnosed patients with diverse clinicomolecular profiles. Combination treatment was antagonistic with ceralasertib (CI 2.2), additive with prexasertib (CI 1.0), and synergistic with adavosertib (CI 0.88). Conclusions In multiple cell lines and diverse primary AML samples, decitabine activated the ATR-CHK1 DDR pathway and synergized with ATRi, CHK1/2i, and WEE1i. Amongst these, only the combination of decitabine plus adavosertib exhibited synergy across all three models, highlighting WEE1 as a potential target in TP53MT AML.

Abstract Background: Ovarian cancer ranks fifth in cancer deaths among women. The gold standard of care for advanced disease includes cytoreductive surgery along with adjuvant or neoadjuvant platinum/taxane-based chemotherapy. Although response to this treatment is high, chemotherapy-induced nausea and vomiting due to platinum agents and allergic reactions due to both agents are common. For nearly half a century, the glucocorticoid (GC) dexamethasone (DEX) has been administered as part of supportive care. However, there have periodically been questions about the possible deleterious effects of DEX on chemotherapy response, especially in solid tumors. Methods: Platinum-sensitive, glucocorticoid receptor (GRa) expressing ovarian cancer cells were exposed to DEX prior to chemotherapy and clonogenic survival was assessed. The effects of DEX on cell cycle and apoptosis were assessed by flow cytometry. Changes in the tumor cell transcriptome after acute DEX treatment were determined by RNA sequencing. Mice bearing orthotopic ovarian cancer patient-derived xenografts (PDXs) were treated with chemotherapy ± DEX to assess changes in chemotherapy response in vivo. Findings: DEX pre-treatment decreased platinum cytotoxicity in ovarian cancer cell lines in vitro and diminished platinum response in some PDXs analyzed for chemotherapy response ex vivo. RNA-seq analysis revealed changes in transcripts encoding proteins involved in platinum detoxification and TNFa pro-survival signaling. In ovarian cancer PDXs, DEX decreased platinum response in one model and had limited impact on both platinum and platinum/taxane chemotherapy in three distinct studies. Interpretation: DEX appears to modulate chemotherapy response in some settings in vitro and ex-vivo. The impact of DEX on chemotherapy response in vivo appears to be limited. However, further investigation of this question in vivo in non-immunosuppressed murine models appears warranted. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Cordelia Mcgehee, Sarah Finstuen-Magro, Paula A. Schneider, Rachel Hurley, Kevin L. Peterson, Ethan Heinzen, Melissa C. Larson, Xiaonon Hou, Ann L. Oberg, Saravut Weroha, Scott H. Kaufmann. Impact of dexamethasone on chemotherapy response in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B098.

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1, and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing, and HR, little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high-grade serous ovarian cancer (HGSOC) cell lines and patient-derived xenografts (PDXs) to understand the predominant apoptotic players and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: HGSOC cell lines were examined for responses to ceralasertib (ATRi), prexasertib (CHK1i), or adavosertib (WEE1i) using colony forming assays, immunoblotting, and assays for apoptosis. To characterize the cell death networks after prexasertib treatment, RNA-seq analysis was performed in two representative cell lines. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib- resistant HGSOC cell lines. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines, and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. CDC25A-CDK2 signaling was predictive of responsiveness in vitro and in vivo. The subsequent apoptotic response was c-Jun N-terminal kinases (JNK) dependent and involved engagement of mitochondrial pathways rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (H. Ding, N. Vincelette, et al., Cancer Res. 81:2666-2678, 2021). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i but were sensitive to front-line therapy options, including carboplatin and PARP inhibitor. Conclusions: Unlike AML, death receptor-mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Our findings identify the key cell death-initiating events that serve as measures of response to replication checkpoint inhibitors in ovarian cancer. Acquired resistance to these therapies is multifactorial and mainly involves failure to exacerbate the replication stress response. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Kevin L. Peterson, Xiaonan Hou, Annabella R. Strathman, Paula A Schneider, Karen S. Flatten, Laura N. Duffield, Cordelia D. McGehee, Melissa C. Larson, Xue Meng, Nicole D Vincelette, Ann L. Oberg, Hu Li, John Weroha, Scott H Kaufmann. Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B012.

AbstractBackgroundDespite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK‐228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL.MethodsWe conducted a single‐arm multi‐center Phase II study of sapanisertib monotherapy (3 mg orally daily of the milled formulation for 21 days every 28 days) in patients with ALL through the Experimental Therapeutics Clinical Trials Network (NCI‐9775).ResultsSixteen patients, 15 of whom were previously treated (median 3 prior lines of therapy), were enrolled. Major grade 3–4 non‐hematologic toxicities included mucositis (3 patients) and hyperglycemia (2 patients) as well as hepatic failure, seizures, confusion, pneumonitis, and anorexia (1 patient each). Grade >2 hematological toxicity included leukopenia (3), lymphopenia (2), thrombocytopenia, and neutropenia (1). The best response was stable disease in 2 patients (12.5%), while only 3 patients (19%) were able to proceed to Cycle 2. Pharmacokinetic analysis demonstrated drug exposures similar to those observed in solid tumor patients. Immunoblotting in serially collected samples indicated limited impact of treatment on phosphorylation of mTOR pathway substrates such as 4EBP1, S6, and AKT.ConclusionIn summary, single‐agent sapanisertib had a good safety profile but limited target inhibition or efficacy in ALL as a single agent. This trial was registered at ClinicalTrials.gov as NCT02484430.

Abstract Background: Ovarian cancer ranks fifth in cancer deaths among women. The gold standard of care for advanced disease includes cytoreductive surgery along with adjuvant or neoadjuvant platinum/taxane-based chemotherapy. Although response to this treatment is high, chemotherapy-induced nausea and vomiting due to platinum agents and allergic reactions due to both agents are common. For nearly half a century, the glucocorticoid (GC) dexamethasone (DEX) has been administered as part of supportive care. However, there have periodically been questions about the possible deleterious effects of DEX on chemotherapy response, especially in solid tumors. Methods: Platinum-sensitive, glucocorticoid receptor (GRα) expressing ovarian cancer cells were exposed to DEX prior to chemotherapy and clonogenic survival was assessed. The effects of DEX on cell cycle and apoptosis were assessed by flow cytometry. Changes in the tumor cell transcriptome after acute DEX treatment were determined by RNA sequencing. Mice bearing orthotopic ovarian cancer patient-derived xenografts (PDXs) were treated with chemotherapy ± DEX to assess changes in chemotherapy response in vivo. Findings: DEX pre-treatment decreased platinum cytotoxicity in ovarian cancer cell lines in vitro and diminished platinum response in some PDXs analyzed for chemotherapy response ex vivo. RNA-seq analysis revealed changes in transcripts encoding proteins involved in platinum detoxification and TNFα pro-survival signaling. In ovarian cancer PDXs, DEX decreased platinum response in one model and had limited impact on both platinum and platinum/taxane chemotherapy in three distinct studies. Interpretation: DEX appears to modulate chemotherapy response in some settings in vitro and ex-vivo. The impact of DEX on chemotherapy response in vivo appears to be limited. However, further investigation of this question in vivo in non-immunosuppressed murine models appears warranted. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Cordelia D. Mcgehee, Sarah Finstuen-Magro, Jamison VanBlaricom, Paula A. Schneider, Kevin L. Peterson, Ethan P. Heinzen, Melissa C. Larson, Xiaonon Hou, Ann L. Oberg, Saravut John Weroha, Scott H. Kaufmann. Impact of dexamethasone on chemotherapy response in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5058.

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1832.

The poly(ADP-ribose) binding protein CHFR regulates cellular responses to mitotic stress. The deubiquitinase UBC13, which regulates CHFR levels, has been associated with better overall survival in paclitaxel-treated ovarian cancer. Despite the extensive use of taxanes in the treatment of ovarian cancer, little is known about expression of CHFR itself in this disease. In the present study, tissue microarrays containing ovarian carcinoma samples from 417 women who underwent initial surgical debulking were stained with anti-CHFR antibody and scored in a blinded fashion. CHFR levels, expressed as a modified H-score, were examined for association with histology, grade, time to progression (TTP) and overall survival (OS). In addition, patient-derived xenografts from 69 ovarian carcinoma patients were examined for CHFR expression and sensitivity to paclitaxel monotherapy. In clinical ovarian cancer specimens, CHFR expression was positively associated with serous histology (p = 0.0048), higher grade (p = 0.000014) and higher stage (p = 0.016). After correction for stage and debulking, there was no significant association between CHFR staining and overall survival (p = 0.62) or time to progression (p = 0.91) in patients with high grade serous cancers treated with platinum/taxane chemotherapy (N = 249). Likewise, no association between CHFR expression and paclitaxel sensitivity was observed in ovarian cancer PDXs treated with paclitaxel monotherapy. Accordingly, differences in CHFR expression are unlikely to play a major role in paclitaxel sensitivity of high grade serous ovarian cancer.

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.