Ewa Gogola

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX–MDC1–RNF8–RNF168–53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Replication fork stability confers chemoresistance in BRCA-deficient cells

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Significance Poly(ADP-ribose) polymerase (PARP) inhibitors hold promise for patients with breast cancer 1 (BRCA1)-associatated cancers but are anticipated to give rise to resistance. We show that mouse mammary tumors resembling BRCA1-associated metaplastic breast carcinoma display intrinsic resistance to the PARP inhibitor olaparib as a result of increased P-glycoprotein drug efflux transporter expression. These findings may have implications for ongoing clinical trials. Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.

Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells

Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death. PARP-inhibitor-induced multinucleated cells fail clonogenic outgrowth, and high percentages of multinucleated cells are found in vivo in remnants of PARP inhibitor-treated Brca2−/−;p53−/− and Brca1−/−;p53−/− mammary mouse tumours, suggesting that mitotic progression promotes PARP-inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogates PARP-inhibitor-induced cytotoxicity. These findings provide insight into the cytotoxic effects of PARP inhibition, and point at combination therapies to potentiate PARP inhibitor treatment of HR-deficient tumours.

BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells

Summary Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.

PO-496 Loss of parg drives parp inhibitor resistance in brca2-deficient mouse mammary tumours

Introduction Inhibitors of poly(ADP-ribose) polymerase (PARP) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, resistance to PARP inhibitors (PARPi) is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. Material and methods To tackle this question, we generated matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumours. By combining next-generation sequencing with functional genetic screens, we identified loss of poly(ADP-ribose) glycohydrolase (PARG) as a major resistance mechanism. Results and discussions We demonstrate that PARG depletion restores PAR formation, rescues controlled DNA replication fork progression and promotes the recruitment of downstream DNA repair factors. The potential relevance of PARG in clinical PARPi resistance is underscored by the presence of PARG-negative clones in a subset of human triple-negative breast and serous ovarian cancers. Importantly, acquisition However, the gain of PARPi resistance comes at a cost, as PARG inactivation results in new vulnerabilities that can be exploited therapeutically. Conclusion We conclude that loss of PARG should be assessed as a potential cause of clinical PARPi resistance. In this case, measurement of PARG activity should further improve clinical decision making for patients with tumours that lack homology-directed DNA repair.

Abstract IA09: Replication fork stability confers chemoresistance in BRCA-deficient cells

Brca1 - and Brca2 -deficient cells have reduced capacity to repair DNA double strand breaks (DSBs) by homologous recombination (HR) and consequently are hypersensitive to DNA damaging agents including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein PTIP protects Brca1/2 -deficient cells from DNA damage and rescues the lethality of Brca2 -deficient embryonic stem cells. However, PTIP deficiency does not restore HR activity at DSBs. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn, protects nascent DNA strands from extensive degradation. More generally, acquisition of PARPi and cisplatin resistance is associated with replication fork (RF) protection in Brca2 -deficient tumor cells that do not develop Brca2 -reversion mutations. Disruption of multiple proteins including PARP1 and CHD4 leads to the same end point of RF protection, highlighting the complexities by which tumor cells evade chemotherapeutic interventions and acquire drug resistance. Citation Format: Arnab Ray Chaudhuri, Elsa Callen, Xia Ding, Ewa Gogola, Alexandra A. Duarte, Ji-Eun Lee, Nancy Wong, Vanessa Lafarga, Jennifer A. Calvo, Nicholas J. Panzarino, Sam John, Amanda Day, Anna Vidal Crespo, Binghui Shen, Linda M. Starnes, Julian R. de Ruiter, Jeremy A. Daniel, Panagiotis A. Konstantinopoulos, David Cortez, Sharon B. Cantor, Oscar Fernandez-Capetillo, Kai Ge, Jos Jonkers, Sven Rottenberg, Shyam K. Sharan, Andre Nussenzweig. Replication fork stability confers chemoresistance in BRCA-deficient cells [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA09.

Abstract LB-329: MicroRNA profiling to identify novel determinants of platinum resistance in BRCA1/2-mutated high-grade serous ovarian cancer

Ovarian cancer is the fifth leading cause of mortality in women and the most lethal of all gynecologic tumors. Patients with high grade serous ovarian carcinoma (HGSOC) have poor prognosis due to a combination of factors including lack of early detection and failure of therapeutic regimens. Approximately 50% of all ovarian tumors have germline/somatic mutations or epigenetic alterations in genes making up the homologous recombination (HR) pathway. The resultant “BRCAness” phenotype pre-disposes these tumors to an improved response from current ovarian cancer therapies i.e. platinum therapy and PARP inhibitors. Despite the predicted synthetic lethality, a majority of patients with recurrent ovarian cancer eventually develop resistance. A detailed understanding of clinically relevant mechanisms of chemotherapy resistance is an important step in improving disease outcomes. To systematically identify microRNA (miRNA) mediators of chemo-resistance in HGSOC, we performed small RNA sequencing of 38 BRCA mutated tumors with known response to platinum chemotherapy and identified three miRNA’s: miR-139-5p, miR-493-5p and miR-494-3p that were significantly overexpressed in platinum resistant compared to the platinum sensitive tumors. Overexpression of these candidate miRNA’s was associated with worse overall outcome among BRCA1/2-mutated tumors in the TCGA dataset. Interestingly, overexpression of miR-493-5p only correlated with poor progression free survival in BRCA2 mutant tumors and not BRCA1 mutant tumors. Importantly, overexpression of miR-493-5p was also identified in 13 out of 40 olaparib resistant tumors in KB2P BRCA2 deficient mouse models. Additionally, overexpression of miR-493-5p in BRCA2 mutated HGSOC cell-lines induced resistance to cisplatin and olaparib in vitro. Mechanistically, resistance mediated by miR-493-5p was not associated with restoration of HR (as evaluated by restoration of Rad51 foci) but was rather related to replication fork stabilization. Identification of specific targets of miR-493-5p is currently underway. In summary, overexpression of miR-493-5p may be a novel mechanism of resistance to platinum and PARP-inhibitors in BRCA2-mutated ovarian cancers and may serve as a biomarker of response to these agents in BRCA2 mutated ovarian tumors. Inhibition of miR-493-5p may be a novel strategy to resensitize chemo-resistant BRCA2 mutated tumors to platinum therapy and PARP-inhibitors. Citation Format: Khyati Meghani, Ewa Gogola, Sven Rottenberg, Jos Jonkers, Ursula Matulonis, Elizabeth Swisher, Panagiotis Konstantinopoulos, Dipanjan Chowdhury. MicroRNA profiling to identify novel determinants of platinum resistance in BRCA1/2-mutated high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-329. doi:10.1158/1538-7445.AM2017-LB-329