Schneider, Paula A.

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 Background: Replication checkpoint modulators (RCMs), including inhibitors of CHK1, ATR, and WEE1, have been widely studied for their ability to inhibit cell cycle checkpoints and enhance the toxicity of agents that stall replication forks. In preclinical studies each of these inhibitors also demonstrates notable activity as a single agent against relapsed/refractory acute myeloid leukemia (R/R AML). In particular, our prior study (PMID: 33414171) demonstrated that several myeloid cell lines underwent cell death at nanomolar concentrations, with a median IC50 of 9 nM irrespective of the TP53 status. Examination of pathways involved in RCM-induced leukemia cell killing demonstrated that CHK1 inhibition induces CDC25A upregulation accompanied by activation of a death program that involves a CDK2-initiated 10- to 50-fold upregulation of TNFα and downstream engagement of the extrinsic apoptotic pathway. Here we have comprehensively characterized the processes that occur between the interruption of replication checkpoints and leukemic cell death in these sensitive cells versus cells selected for RCM resistance. Methods: Using the CHK1 inhibitors MK8776 and prexasertib, ATR inhibitors berzosertib and ceralasertib, and WEE1 inhibitor adavosertib as paradigm drugs, we examined signaling in two AML cell lines, U937 and THP.1, that were selected for CHK1 inhibitor resistance, examined cross-resistance patterns, and assessed the biochemical basis for leukemic cell death. Changes in gene expression and signaling induced by RCMs were analyzed in isogenic pairs using RNA sequencing, flow cytometry and immunoblotting. Results: In contrast to parental U937 and THP.1 cells, CHK1 inhibitor-selected U937 and THP.1 cell exhibited a lack of TNFα mRNA induction and did not undergo cell death in response to multiple CHK1 inhibitors. RNA sequencing of CHK1 inhibitor-selected AML lines revealed an almost complete absence of induction of CDK2-activated genes following prexasertib treatment. Interestingly, unlike solid tumors, AML cell lines that were resistant to CHK1 inhibitors exhibited hypersensitivity to WEE1 or ATR inhibition. Further studies demonstrated that CHK1 inhibitor response was varied in primary AML samples, with 50% of primary AML specimens resistant to CHK1i monotherapy, again without any relationship to TP53 status. The common features among sensitive vs. resistant leukemias was the successful inhibition of CDK2 phosphorylation and the associated induction of TNFα. Conclusions: Our findings reveals critical new insights into the factors that influence the response to CHK1 inhibitors in AML. Importantly, we found no evidence of cross-resistance to inhibitors of other replication checkpoint proteins, enabling alternative options. Citation Format: Annapoorna Venkatachalam, Husheng Ding, Nicole Vincelette, Cordelia McGehee, Kevin Peterson, Paula Schneider, Cristina Correia, Hu Li, Scott Kaufmann. CDK2 activation mediates response of acute myeloid leukemia to CHK1, ATR and WEE1 inhibitors [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 423.

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.