HR-deficient Cells Research Articles

EP-2286: CHK1-mediated replication fork stabilization confers radioresistance in HR deficient tumor cells

EP-2286: CHK1-mediated replication fork stabilization confers radioresistance in HR deficient tumor cells

Abstract B189: New class of TOP1 inhibitors selective for HR deficiencies and SLFN11 expression

Abstract To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication-induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives are plagued by limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (the LMPs: LMP400, LMP776, and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2, and PALB2 are needed to repair DSB induced by TOP1 inhibitors. It has been shown that BRCA1 is a determinant of response to camptothecins in addition to olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives. To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival and cell cycle modifications after treatment with the LMPs. We found that SLFN11-negative cells are 10 times hypersensitive to LMPs compared to WT cells. Also, BRCA1-, BRCA2-, and PALB2-deficient cells are 3 to 5 times hypersensitive to the LMPs. Adding olaparib led to a greater cell death, especially in HR-deficient cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers. Citation Format: Laetitia Marzi, Yves G. Pommier. New class of TOP1 inhibitors selective for HR deficiencies and SLFN11 expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B189.

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

BackgroundProper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism.MethodsWe hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells.ResultsOur data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival.ConclusionThis study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.

Redundancy between nucleases required for homologous recombination promotes PARP inhibitor resistance in the eukaryotic model organism Dictyostelium.

ADP-ribosyltransferases promote repair of DNA single strand breaks and disruption of this pathway by Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) is toxic to cells with defects in homologous recombination (HR). Here, we show that this relationship is conserved in the simple eukaryote Dictyostelium and exploit this organism to define mechanisms that drive resistance of the HR-deficient cells to PARPi. Dictyostelium cells disrupted in exonuclease I, a critical factor for HR, are sensitive to PARPi. Deletion of exo1 prevents the accumulation of Rad51 in chromatin induced by PARPi, resulting in DNA damage being channelled through repair by non-homologous end-joining (NHEJ). Inactivation of NHEJ supresses the sensitivity of exo1− cells to PARPi, indicating this pathway drives synthetic lethality and that in its absence alternative repair mechanisms promote cell survival. This resistance is independent of alternate-NHEJ and is instead achieved by re-activation of HR. Moreover, inhibitors of Mre11 restore sensitivity of dnapkcs−exo1− cells to PARPi, indicating redundancy between nucleases that initiate HR can drive PARPi resistance. These data inform on mechanism of PARPi resistance in HR-deficient cells and present Dictyostelium as a convenient genetic model to characterize these pathways.

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.

Abstract 5863: Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer

Abstract Ovarian cancer is the seventh-most common cancer with a high mortality rate among women. Radiation therapy (RT) is used in ovarian cancer for local relapse, but doses are limited due to concern for small bowel obstruction in patients who have had multiple surgeries and chemotherapeutic regimens. Therefore, it’s an ideal setting to improve the therapeutic window by combining RT and a radiosensitizer. Poly (ADP-ribose) polymerase-1 (PARP-1) is involved in the recognition of DNA damage and the facilitation of DNA repair by recognizing DNA single-strand breaks (SSB). Inhibition of PARP1, especially in tumors driven by BRCA1 or 2 mutations, has been shown to potentiate the DNA-damaging effects of radiation in vitro and in vivo and to increase tumor vasculature perfusion and oxygenation. Approximately half of ovarian cancers have germline or somatic BRCA gene mutation, epigenetic silencing or other mutations that affect homologous recombination (HR) competency resulting in DNA repair defects, making this tumor particularly susceptible to the radiation-sensitizing effects of PARP inhibitors. Here, we investigated the therapeutic effects of the PARP inhibitor olaparib in preclinical models of ovarian cancer. Basic BRCA1 and PARP1 protein and mRNA levels were characterized in a panel of ovarian cancer cell lines. The radiosensitizing effects of olaparib were tested on both HR-proficient and HR-deficient ovarian cancer cells by evaluating the colony formation, DNA damage, apoptosis, and PARP1 activity. Survival benefit of olaparib was also examined in xenograft models of ovarian cancer. Our results demonstrate that combination of olaparib with IR decreased colony formation, inhibited DNA damage (γH2AX) repair, and induced more apoptosis compared to olaparib or IR alone. In addition, HR-deficient cells were more sensitive to IR than HR-proficient cells in the presence of olaparib. Furthermore, olaparib inhibited PARP1 activity in vivo, significantly decreased tumor growth and increased overall survival when combined with IR in mice bearing subcutaneous xenografts of HR-deficient OVCAR8 cells while producing a relative modest effect on overall survival of mice bearing xenografts of HR-proficient SKOV3 cells. These results provide a preclinical rationale for improved treatment modalities using olaparib as an effective radiosensitizer in ovarian cancer, particularly in tumors with HR-deficiencies. Citation Format: Yue Bi, Ioannis Verginadis, Souvik Dey, linlang Guo, Lilie Lin, Yanfang Zheng, Constantinos Koumenis. Identifying the radiosensitizing effects of PARP inhibitor in 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 5863. doi:10.1158/1538-7445.AM2017-5863

Abstract 2794: Indotecan (LMP400), imidotecan (LMP776) and LMP744: A new class of non-camptothecin Top1 inhibitors selective for homologous recombination deficient (HRD) cells

Abstract To relax DNA supercoiling during transcription and replication, topoisomerase I (Top1) induces transient DNA cleavage complexes, which are trapped by anticancer drugs, leading to DNA double-strand breaks (DSB) that need to be repaired by homologous recombination (HR). BRCA1, BRCA2 and PALB2, which are key components for HR, lead to “synthetic lethality” with PARP inhibitors. Topotecan and irinotecan (camptothecin derivatives) are the only FDA-approved Top1 inhibitors. In spite of their wide usage they are plagued by their chemical instability, being drug efflux substrates, having short half-life, and dose-limiting bone marrow and gastro intestinal toxicity. It is now possible to overcome those limitations with the non-camptothecin indenoisoquinolines (LMP400, LMP776 and LMP744), which are in clinical trials. To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether the LMPs present a “synthetic lethality” toward BRCA1, BRCA2 or PALB2 deficiency, and whether this selectivity could be enhanced by combining them with the recently approved PARP inhibitor, olaparib.Using isogenic DT40 cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, we assessed the role of HR in the cellular responses to the LMPs. Survival and cell cycle modifications were tested after treatment with the LMPs as single agents, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times hypersensitive to the LMPs (IC50’s for LMP400 and LMP744= 10 nM for HR-deficient cells vs. 45 nM for WT cells, and IC50 for LMP776 around 5 nM vs. 18 nM for WT cells). Cell cycle analyses confirmed the death of these HR-deficient cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib (Combination index<0.7). The HR-deficient cells being markedly more sensitive than WT cells (taken as surrogate for normal tissues) to both treatments, led to a better response to the combination in those cells.Our results show that the LMPs are active at nanomolar concentrations, selectively in HR-deficient cells, indicative of a “synthetic lethality” of the indenoisoquinolines (LMPs) with HRD genotypes. They also demonstrate that the LMPs synergize with olaparib. These findings provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient cancers. Citation Format: Laetitia Marzi, Keli Agama, Zoe Weaver Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Muthana Al Abo, Yves Pommier. Indotecan (LMP400), imidotecan (LMP776) and LMP744: A new class of non-camptothecin Top1 inhibitors selective for homologous recombination deficient (HRD) cells [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 2794. doi:10.1158/1538-7445.AM2017-2794

The homologous recombination protein RAD51D protects the genome from large deletions.

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that protects the genome from chromosomal instability. RAD51 mediator proteins (i.e. paralogs) are critical for efficient HR in mammalian cells. However, how HR-deficient cells process DSBs is not clear. Here, we utilized a loss-of-function HR-reporter substrate to simultaneously monitor HR-mediated gene conversion and non-conservative mutation events. The assay is designed around a heteroallelic duplication of the Aprt gene at its endogenous locus in isogenic Chinese hamster ovary cell lines. We found that RAD51D-deficient cells had a reduced capacity for HR-mediated gene conversion both spontaneously and in response to I-SceI-induced DSBs. Further, RAD51D-deficiency shifted DSB repair toward highly deleterious single-strand annealing (SSA) and end-joining processes that led to the loss of large chromosomal segments surrounding site-specific DSBs at an exceptionally high frequency. Deletions in the proximity of the break were due to a non-homologous end-joining pathway, while larger deletions were processed via a SSA pathway. Overall, our data revealed that, in addition to leading to chromosomal abnormalities, RAD51D-deficiency resulted in a high frequency of deletions advancing our understanding of how a RAD51 paralog is involved in maintaining genomic stability and how its deficiency may predispose cells to tumorigenesis.

Poly(ADP-ribose)polymerase (PARP) inhibition and anticancer activity of simmiparib, a new inhibitor undergoing clinical trials

Poly(ADP-ribose)polymerase (PARP)1/2 inhibitors have been proved to be clinically effective anticancer drugs. Here we report a new PARP1/2 inhibitor, simmiparib, displaying apparently improved preclinical anticancer activities relative to the first approved inhibitor olaparib. Simmiparib inhibited PARP1/2 approximately 2-fold more potently than olaparib, with more than 90-fold selectivity over the other tested PARP family members. Simmiparib and olaparib caused similar cellular PARP1-DNA trapping. Simmiparib selectively induced the accumulation of DNA double-strand breaks, G2/M arrest and apoptosis in homologous recombination repair (HR)-deficient cells. Consistently, simmiparib showed 26- to 235-fold selectivity in its antiproliferative activity against HR-deficient cells over the corresponding isogenic HR-proficient cells. Notably, its antiproliferative activity was 43.8-fold more potent than that of olaparib in 11 HR-deficient cancer cell lines. Simmiparib also potentiated the proliferative inhibition of several conventional anticancer drugs. Simmiparib reduced the poly(ADP-ribose) formation in HR-deficient cancer cells and xenografts. When orally administered to nude mice bearing xenografts, simmiparib revealed excellent pharmacokinetic properties. Simmiparib caused approximately 10-fold greater growth inhibition than olaparib against HR-deficient human cancer cell- or tissue-derived xenografts in nude mice. Collectively, these findings support the undergoing clinical trials of simmiparib.

Mechanism-Based Drug Combinations with the DNA Strand-Breaking Nucleoside Analog CNDAC.

CNDAC (2'-C-cyano-2'-deoxy-1-β-d-arabino-pentofuranosyl-cytosine, DFP10917) and its orally bioavailable prodrug, sapacitabine, are undergoing clinical trials for hematologic malignancies and solid tumors. The unique action mechanism of inducing DNA strand breaks distinguishes CNDAC from other deoxycytidine analogs. To optimize the clinical potentials of CNDAC, we explored multiple strategies combining CNDAC with chemotherapeutic agents targeting distinct DNA damage repair pathways that are currently in clinical use. The ability of each agent to decrease proliferative potential, determined by clonogenic assays, was determined in paired cell lines proficient and deficient in certain DNA repair proteins. Subsequently, each agent was used in combination with CNDAC at fixed concentration ratios. The clonogenicity was quantitated by median effect analysis, and a combination index was calculated. The c-Abl kinase inhibitor imatinib had synergy with CNDAC in HCT116 cells, regardless of p53 status. Inhibitors of PARP1 that interfere with homologous recombination (HR) repair or base excision repair (BER) and agents such as temozolomide that cause DNA damage repaired by the BER pathway were also synergistic with CNDAC. The toxicity of the nitrogen mustards bendamustine and cytoxan, or of platinum compounds, which generate DNA adducts repaired by nucleotide excision repair and HR, was additive with CNDAC. An additive cell killing was also achieved by the combination of CNDAC with taxane mitotic inhibitors (paclitaxel and docetaxel). At concentrations that allow survival of the majority of wild-type cells, the synergistic or additive combination effects were selective in HR-deficient cells. This study provides mechanistic rationales for combining CNDAC with other active drugs. Mol Cancer Ther; 15(10); 2302-13. ©2016 AACR.

PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response.

There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be “synthetic lethal” in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs.

Abstract IA04: Targeting DNA repair in ovarian cancer therapy.

Abstract Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability and replicative stress of EOCs and for their cellular hyperdependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. On the basis of this hyperdependence, in 2014, the FA approved the PARP inhibitor, olaparib, for patients with advanced ovarian cancer associated with BRCA1 and BRCA2 germline mutations, whose tumors are HR deficient. Previous studies have implicated the DNA polymerase POLQ in an error-prone pathway required for the repair of DNA double-strand breaks, referred to as alternative endjoining (alt-EJ). In Drosophila melanogaster and Caenorhabditis elegans, POLQ functions in alt-EJ and in the maintenance of genome stability at sites of G-quadruplex structures. Whether POLQ interacts with canonical DNA repair pathways to prevent genomic instability at stalled replication forks has remained unknown. We have recently demonstrated an inverse correlation between HR activity and POLQ expression in EOCs (Ceccaldi R. et al, Nature, 518: 258-262, 2015). While knockdown of POLQ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, knockdown of POLQ in HR-deficient EOCs results in enhanced cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, POLQ contains a repeat of RAD51 binding motifs, and it blocks D-loop formation and recombination. Importantly, PARP1 activity is required for the recruitment of POLQ to sites of DNA repair, accounting, in part, for the PARP1 hyperdependence of HR-deficient ovarian tumor cells. Taken together, these results reveal a synthetic lethal relationship between the HR pathway and PARP1/POLQ-mediated alternative end-joining in EOCs, and identify POLQ as a novel druggable target for ovarian cancer therapy. Such inhibitors may be useful in the treatment of PARP inhibitor-resistant ovarian cancers. Citation Format: Alan D. D'Andrea. Targeting DNA repair in ovarian cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA04.

Homologous Recombination Deficiency: Exploiting the Fundamental Vulnerability of Ovarian Cancer.

Approximately 50% of epithelial ovarian cancers (EOC) exhibit defective DNA repair via homologous recombination (HR) due to genetic and epigenetic alterations of HR pathway genes. Defective HR is an important therapeutic target in EOC as exemplified by the efficacy of platinum analogues in this disease, as well as the advent of PARP inhibitors, which exhibit synthetic lethality when applied to HR-deficient cells. Here, we describe the genotypic and phenotypic characteristics of HR-deficient EOCs, discuss current and emerging approaches for targeting these tumors, and present challenges associated with these approaches, focusing on development and overcoming resistance. Defective DNA repair via HR is a pivotal vulnerability of EOC, particularly of the high-grade serous histologic subtype. Targeting defective HR offers the unique opportunity of exploiting molecular differences between tumor and normal cells, thereby inducing cancer-specific synthetic lethality; the promise and challenges of these approaches in ovarian cancer are discussed in this review.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Abstract 3850: Understanding PARP inhibitor sensitivity: Analyses of the genetic interactions between specific PARP inhibitor targets and DNA double-strand repair pathways

Abstract Poly (ADP-ribosyl)ation (PARylation) of histones and DNA repair factors is necessary to maintain genomic integrity in mammalian cells. In response to DNA damage, PARylation is primarily catalyzed by two Poly (ADP-ribose) polymerases (PARPs), PARP1 and PARP2. Ablation of PARP1 in the mouse results in embryonic lethality when combined with deficiencies for the DNA repair factors that mediate homologous recombination (HR; such as ATM or H2AX) or Nonhomologous End-joining (NHEJ; such as Ku80). In contrast, the interaction between PARP2 and DNA double-strand break (DSB) repair pathways has not been investigated in detail. In this context, all PARP inhibitors (PARPi) in clinical use block the catalytic domain of both PARP1 and PARP2. Since the two factors clearly play unique, non-overlapping roles in DNA repair and other cellular processes, we sought to further define their specific contribution to the therapeutic effect of PARPi. We find that combined loss of either PARP1 or PARP2 and histone H2AX results in embryonic lethality in the mouse, similar to previous observations with mice deficient for PARP1 or PARP2 and the ATM kinase. Because H2AX and ATM promote double-strand break (DSB) repair throughout the cell cycle, we next investigated whether this synthetic lethal interaction reflected on their functions in NHEJ and/or HR. Treatment of H2AX or ATM-deficient fibroblasts or lymphocytes with PARPi resulted in a marked increase in the frequency of chromatid-type aberrations relative to wild-type controls, suggesting that PARPi hypersensitivity in H2AX or ATM-deficient cells results primarily from defective HR. To further assess an interaction between PARP2 and NHEJ, we generated mice lacking PARP2 and either Ku80, a core NHEJ factor, or DNA-PKcs, a kinase required for a subset of NHEJ reactions. We find that, unlike the early embryonic lethality previously observed in PARP1/Ku80-deficient mice, loss of PARP2 does not result in additional phenotypes in growth and development in Ku80-deficient mice. Likewise, loss of PARP2 has no major effect on growth, development or lymphoma latency in DNA-PKcs-deficient mice, in marked contrast to the severe segmental progeroid syndrome and early-onset tumorigenesis observed in PARP1/DNA-PKcs-deficient mice. Furthermore, PARP1/DNA-PKcs-deficient lymphomas, but not PARP2/DNA-PKcs-deficient lymphomas, harbor marked aneuploidy and oncogenic p53 mutations. Altogether, these genetic analyses suggest that inhibition of both PARP1 and PARP2 contributes to PARPi-induced killing of HR-deficient human tumor cells. This hypothesis is currently being tested in our lab using 11q23-deleted neuroblastoma cells with monoallelic expression of H2AX and breast cancer cells lacking BRCA1 or BRCA2, and may provide a rationale for the design of PARP-specific inhibitors in the future. Citation Format: Rajib Ghosh, Sanchita Roy, Francoise Dantzer, Sonia Franco. Understanding PARP inhibitor sensitivity: Analyses of the genetic interactions between specific PARP inhibitor targets and DNA double-strand repair pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2015-3850

Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins.

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions.

The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions.

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.

DNA PoLθ Suppresses HR and Is Required for HR-Deficient Cell Survival

Abstract DNA polymerase θ (Polθ) inhibits homologous recombination (HR) and promotes alternative NHEJ.

Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone.Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX).Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone.Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. DisclosuresNo relevant conflicts of interest to declare.