Homologous Recombination DNA Repair Pathway Research Articles

PARP inhibitors: Clinical utility and possibilities of overcoming resistance.

PARP inhibitors represent a major breakthrough in ovarian cancer care. Almost half of all ovarian cancers have deficiencies in the homologous recombination (HR) DNA repair pathway, namely BRCA1/2 mutations. Given the limited therapeutic options for recurrent ovarian cancer patients there has been a significant effort to develop novel therapies to exploit DNA repair deficiencies. In 2005 and 2006, inhibiting PARP enzymes was first observed to be highly effective against cancers with HR deficiencies. PARP inhibitors are being utilized in the clinic to manage recurrent ovarian cancers that display defects in the HR repair pathway. However, PARP inhibitors also show significant clinical benefit in patients without HR deficiencies. There are currently three FDA-approved PARP inhibitors for recurrent ovarian cancer and an additional two PARP inhibitors being evaluated in late stage clinical trials. Given the expanding clinical use of PARP inhibitors and the high likelihood of acquired resistance, there is a significant need for clinical strategies to manage PARP inhibitor resistant disease. This review will examine PARP inhibitors in the context of: indications and toxicities, novel biomarkers to predict response, targeted-therapy resistance, and potential approaches to manage resistant disease.

Abstract A15: Downregulation of c-myc is synthetic lethal with PARP inhibitors in high MYC cancers independent of BRCA status

Abstract PARP inhibitors represent a novel class of cancer therapeutics that exploit a synthetic lethal mechanism specific to cancer cells with a defect within the Homologous Recombination (HR) DNA repair pathway. Patients with a BRCA1/2 germ-line mutation have the highest response rate to PARP inhibitors. However, early clinical trial data has indicated that only ~30-40% BRCA mutant patients respond to PARP inhibitors. Coincidentally, the oncogene c-myc is the most frequently amplified gene in BRCA mutant tumors (~40-60%) which typically present as Triple Negative Breast Cancer (TNBC). The oncogene c-myc transcriptionally regulates several DNA repair genes, and correlates with poor survival among breast cancer patients. Therefore we hypothesized that dual combination therapy targeting c-myc and PARP is an effective therapeutic strategy in BRCA mutant tumors. C-myc siRNA induced increased sensitivity to PARP inhibitors in BRCA wild-type (MB231) & BRCA mutant (SUM149) TNBC cell lines and down regulated HR repair. Pan CDK inhibitor Dinaciclib demonstrated significant MYC inhibition across several TNBC cell lines. Additionally, Dinaciclib demonstrated synergistic growth inhibition in combination with PARP inhibitor Niraparib in both BRCA wild-type and mutant c-myc expressing TNBC cell lines. Combination therapy also leads to an increase in DNA damage (via γH2AX) with a decrease in HR repair (BRCA1/RAD51). TNBC cell line mouse xenografts demonstrated down regulation of c-myc and RAD51 in Dinaciclib + Niraparib treated tumors. Dual Dinaciclib + Niraparib therapy also induced synergistic growth inhibition in PARP inhibitor acquired resistant cell lines while demonstrating significant growth inhibition across a panel of c-myc overexpressing cancer cell lines (Ovarian, Prostate, Pancreatic, Colon & Lung). In conclusion, we demonstrated that dual CDK + PARP inhibition is synthetic lethal in both BRCA wild-type and mutant TNBC cell lines and is dependent upon down regulation of c-myc. This study supports c-myc as predictor of response to PARP inhibitor therapy and may also serve as a biomarker of response to Dinaciclib + PARPi therapy in high MYC expressing tumors. Citation Format: Jason PW Carey, Smruthi Vjayaraghavan, Kelly Hunt, Khandan Keyomarsi. Downregulation of c-myc is synthetic lethal with PARP inhibitors in high MYC cancers independent of BRCA status [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A15.

948P - Genomic instability is associated with increased immune infiltration and PDL1 expression in epithelial ovarian cancer

Background: High mutation load secondary to mutagenic exposures such as smoking or to mutations in mismatch DNA repair genes has been associated with increased tumor immune infiltration and response to immune therapies. Ovarian cancers (OC) demonstrate low mutation load, but high degree of genomic instability (GI) attributable to frequent defects in the homologous recombination DNA repair pathway. We sought to investigate whether GI predicted increased infiltration by tumor infiltrating lymphocytes (TILs) and PDL1 expression in OC. Methods: TILs were evaluated on FFPE OC samples and scored as percentage of stromal area. PDL1 expression was quantified as percent positive immune cells. GI was measured as the number of copy number alterations >15Mb by Oncoscan SNParray on DNA from the same FFPE samples and high GI score (GIS) defined as > median. Correlations were evaluated using a Spearman rank and differences by Mann-Whitney. Results: 66 tumor samples were evaluable for both GIS and immune infiltration. GIS and TILs showed significant variability ranging from 0 to 64 (median=28) and from 5 to 90% (median=20%) for GIS and TILs, respectively. GIS was significantly higher among high grade serous or endometrioid OC than among low grade tumors (GIS=29 vs 5; p < 0.0001) and non-significantly greater among patients with BRCA mutations and/or family history compared to wild-type OC with no family history (p = 0.12). GIS and TILs were highly correlated (R = 0.4; p = 0.0019) and median TILs were significantly increased in GIS-high vs -low tumors (29% vs 15%; p = 0.0028, Fig 1) as was immune cell PDL1 expression (p = 0.025). Fig 1 Increased TILs in tumors with high genomic instability Conclusions: High genomic instability correlated with increased tumor infiltrating lymphocytes and PDL1 expression. Whether GIS could provide a simple and predictive biomarker for immune therapies in OC should be investigated. Legal entity responsible for the study: LEARY Alexandra Funding: INCa (Institut national du cancer) Disclosure: All authors have declared no conflicts of interest.

Mechanisms of Chromium and Uranium Toxicity in Pseudomonas stutzeri RCH2 Grown under Anaerobic Nitrate-Reducing Conditions.

Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI]) or uranyl oxycation (U[VI]). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.

Abstract 1425: Targeting cell cycle dependencies in CCNE1 amplified tumors

Abstract Genomic instability is a hallmark of high grade serous ovarian carcinoma (HGSOC). Based on The Cancer Genome Atlas (TCGA), it is estimated that approximately 50% of HGSOCs harbor a defect in the homologous recombination (HR) pathway of DNA repair. In contrast, the 20% that harbor CCNE1 amplifications appear to have an intact HR pathway. These tumors are associated with shorter overall survival and resistance to chemotherapy. Cyclin E is the activating partner of cyclin-dependent kinase 2 (CDK2) which controls cell cycle progression from G1 to S phase. Our previous data showed that CCNE1 amplification and overexpression occurs early in serous tumorigenesis. Importantly, in immortalized human fallopian tube secretory epithelial cells (FTSEC), constitutive Cyclin E overexpression imparts malignant characteristics to these cells. This leads to an accumulation of DNA damage and altered gene expression of genes involved in DNA replication and fork protection. However, in the setting of hTERT expression and a p53 mutant, Cyclin E overexpression alone was not capable of fully transforming the FTSECs. Therefore, in order to identify cooperating genetic alterations, we performed an in vitro gain-of-function (GOF) screen. One of those identified hits was the RAD51 paralog XRCC2, which is known to be involved in the HR DNA repair pathway and in fork protection. We could show that XRCC2 expression is upregulated in response to Cyclin E overexpression in FTSECs and we found a strong correlation between RNAseq expression of XRCC2 and Cyclin E in the TCGA patient cohorts. We could further demonstrate that the knock down of XRCC2 is synthetic lethal in CCNE1 amplified ovarian cancer cell lines but not in cells that harbor no CCNE1 amplification, indicating that the upregulation of XRCC2 creates a dependency in CCNE1 amplified tumors. Since overexpression of Cyclin E leads to unscheduled S-phase entry and stress on the replication fork, we speculated that one of the roles of XRCC2 might be to stabilize the replication fork in Cyclin E overexpressing cells. We found that the knock down of XRCC2 in Cyclin E overexpressing cells leads to a strong reduction in fork speed and fork recovery. To further understand this mechanism we analyzed the binding partners of XRCC2 in CCNE1 amplified cells by mass spectrometry. Interestingly, we found that XRCC2 interacts with the minichromosome maintenance deficient 7 (MCM7) protein. MCM7 is part of the MCM complex that unwinds the DNA during replication. Surprisingly, the downregulation XRCC2 also led to a strong reduction in MCM7 protein expression indicating that XRCC2 may play an important role in stabilization the MCM complex. This is especially interesting since CCNE1 amplified cells are more dependent on active MCM complexes and are more sensitive to MCM complex reduction compared to normal cells. Further defining the factors that contribute to the XRCC2-MCM7 interaction at the replication fork may define novel vulnerabilities in CCNE1 amplified tumors. Citation Format: Kai Doberstein, Alison Karst, Paul Jones, Azra Ligon, Michelle Hirsch, Dariush Etemadmoghadam, William Hahn, David Bowtell, Ronny Drapkin. Targeting cell cycle dependencies in CCNE1 amplified tumors [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 1425. doi:10.1158/1538-7445.AM2017-1425

Abstract 2483: Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma

Abstract Standard treatments for glioblastoma (GBM) include surgery, radiation and chemotherapy with temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%, largely due to chemoresistance. Evidence shows that cancer cells utilize DNA damage repair pathways to overcome cytotoxic effects of chemotherapy. GBM tumors expressing O6-methylguanine-DNA-methyltransferase (MGMT) display intrinsic chemoresistance to TMZ and nitrosoureas, while a deficient DNA mismatch repair system (MMR) confers chemoresistance to TMZ and platinum agents. Alterations in p53, particularly gain-of-function mutations, are correlated with increased MGMT-expression and poor prognoses in GBM. Second line anti-angiogenic treatment with bevacizumab has not improved overall survival and has been shown to induce intratumor hypoxia and increased chemoresistance. VAL-083 is a bi-functional alkylating agent that readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has demonstrated activity against GBM in prior NCI-sponsored clinical trials. VAL-083 induces interstrand cross-links at guanine-N7 causing DNA double-strand breaks and cancer cell death. VAL-083 is equiactive against GBM cancer stem cells (CSCs) and non-CSCs independent of MGMT and p53 status, in vitro. We recently showed that VAL-083 leads to irreversible S/G2-phase cell cycle arrest, proposing synergy with S-phase specific chemotherapeutics, including topoisomerase and PARP inhibitors. VAL-083 further showed persistent activation of the homologous recombination (HR) DNA repair pathway and its potency was increased when HR was impaired, demonstrating that VAL-083-induced lesions are repaired via HR suggesting increased VAL-083 potency in HR-impaired tumors. Further, hypoxic cancer cells are known to downregulate their HR pathway, proposing increased VAL-083 potency in hypoxic tumors. Bevacizumab treatment increases hypoxia in tumor cells, presumably impairing HR, proposing VAL-083 as a treatment option in HR-deficient or hypoxic cancers following, or as part of a combination treatment with, bevacizumab. Here, VAL-083 cytotoxicity and DNA damage response was evaluated by crystal violet assays, western blot and flow cytometry. VAL-083’s ability to overcome MMR-related chemoresistance was investigated using lentiviral MLH1 and MSH2 vectors in HCT116 and LoVo cancer cells. We report synergy between VAL-083 and etoposide or camptothecin in A549 and PC3 cancer cell lines. We also investigated the potency of VAL-083 in GBM under hypoxia either in vitro or in vivo as part of a combination treatment with bevacizumab. Our results demonstrate a distinct anti-cancer mechanism for VAL-083, resulting in the ability to overcome resistance to TMZ and nitrosoureas, increased activity in cancers with impaired HR and synergy with etoposide or camptothecin. Citation Format: Beibei Zhai, Anna Gobielewska, Anne Steino, Jeffrey A. Bacha, Dennis M. Brown, Simone Niclou, Mads Daugaard. Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma [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 2483. doi:10.1158/1538-7445.AM2017-2483

Abstract 2487: Functional characterization of the BRCA2 variant, K3326X

Abstract Individuals with germline mutations in the breast cancer susceptibility gene, BRCA2, have an approximate 70% risk of developing breast cancer, a 30% risk of developing ovarian cancer, a 20-fold increased risk of developing prostate cancer, and a 10-fold risk of developing pancreatic cancer during their lifetime. Loss of function germline mutations in BRCA2 affect its role in the homologous recombination (HR) DNA repair pathway leading to significant genomic instability. In addition to deleterious truncating mutations, several sequence variants, collectively called Variants of Unknown Significance (VUS), have been identified and are distributed along its length. One such variant is rs11571833, a nonsense mutation in the last exon (c.9976A&amp;gt;T, K3326X), resulting in the loss of the C-terminal 93 amino acid residues in BRCA2. This truncated variant has been previously described as a polymorphism that does not increase susceptibility to breast and ovarian cancers, and as a neutral unclassified variant non-deleterious to its function. However, recent studies identified K3326X to be enriched in breast cancer cases and to increase the risk for lung, pancreatic, ovarian, and upper aero-digestive tract cancers. Preliminary data, obtained from the Rutgers Cancer Institute of New Jersey breast cancer case-control study, identified K3326X enrichment in 1.25% of cases compared to 0.7% of controls. Several of the carriers had second primaries and displayed a trend toward increased number of family members diagnosed with colon cancer. Notably, K3326X was also identified in 1.38% (11 of 796) of our histologically-diverse cohort of genomically-profiled tumors that included cancers of the breast, ovarian/fallopian tube, lung, vulvar, cancer of unknown primary, and one breast cancer case having prolonged response to platinum-based therapy. Thus, K3326X may represent a functional loss of wild type BRCA2 function, as we observe concomitant loss of heterozygosity at this locus. In a preliminary study, we evaluated the K3326X variant, in vitro, in a functional DR-GFP-based reporter assay measuring HR. Our data reveal the BRCA2 K3326X variant to be impaired in the HR pathway indicating a loss of wild-type protein function. We will also evaluate cell viability of the K3326X variant in the presence of DNA damaging drugs like cisplatin, poly-ADP ribose polymerase inhibitors and mitomycin C. Future studies will also incorporate a retrospective evaluation of tumor specimens that have undergone comprehensive genomic profiling. These data would indicate that BRCA2 K3326X represents a functional hypomorphic variant that may have implications in therapeutic approaches and cancer risk evaluations across a spectrum of tumor types. Citation Format: Srilatha R. Simhadri, Sonia C. Dolfi, Atul Kulkarni, Bing Xia, Shridar Ganesan, Kim M. Hirshfield. Functional characterization of the BRCA2 variant, K3326X [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 2487. doi:10.1158/1538-7445.AM2017-2487

Abstract 2477: Genomic instability in BRCA1-deficient cells is a result of the anti-recombinogenic activity of BLM helicase

Abstract Mutations in BRCA1 are responsible for approximately 5% of cases of breast cancer, and there are few treatment options that substantially alter the probability of disease onset in individuals with BRCA1 mutations. BRCA1-deficient cells exhibit increased genomic instability following DNA damaging treatments due to a defect in the homologous recombination (HR) DNA repair pathway. Presently, Olaparib, a PARP inhibitor, is approved as a targeted monotherapy for germline BRCA1 mutated advanced ovarian cancers. As use of this treatment has expanded, data suggests that patients exhibit a further relapse and resistance to PARP inhibitors via mechanisms that include the development of secondary BRCA1 reversion mutations, enhanced drug efflux relating to P-glycoprotein, and mutations in other DNA repair proteins that restore HR. Here, we show that the RECQ helicase, BLM, mediates the genomic instability observed in BRCA1-deficient cells by a mechanism that depends in part on its anti-recombinogenic activity. We have generated conditional knockout mice with single and combined deficiencies in BRCA1, BLM, and 53BP1 in the B lymphocyte cell population. Ablation of BLM in BRCA1-deficient cells allows the HR repair pathway to be restored, leading to a partial rescue of the genomic instability that is present in BRCA1-deficient cells. The stable accumulation of RAD51, a marker for HR, at DNA double-strand break sites is inhibited by BLM in BRCA1-deficient B cells. However, DNA end resection is not impacted by single or co-deletion of BRCA1 and BLM. Furthermore, cells co-deficient in BRCA1 and BLM display limited sensitivity to PARP inhibition. The rescue in HR and PARPi sensitivity phenotypes following deletion of BLM is only present in hypomorphic BRCA1Δ11/Δ11 cells and not the RING domain deficient BRCA1Δ2/Δ2. These results demonstrate that the anti-recombinogenic activity of BLM is of potentially great significance for regulating the balance of HR versus other error-prone repair pathways. Mutation of BLM in BRCA1-deficient tumors is therefore a potential pathway leading to resistance to PARPi and other DNA damaging agents. Citation Format: Dharm S. Patel, Sarah M. Misenko, Samuel F. Bunting. Genomic instability in BRCA1-deficient cells is a result of the anti-recombinogenic activity of BLM helicase [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 2477. doi:10.1158/1538-7445.AM2017-2477

Abstract 5056: The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib

Abstract Introduction: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. Aims: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. Methods: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). Results: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. Conclusions: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib [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 5056. doi:10.1158/1538-7445.AM2017-5056

Polymorphisms and Mutations in GSTP1, RAD51, XRCC1 and XRCC3 Genes in Breast Cancer Patients

Genotoxic factors, including ionizing radiation and oxidative stress, are associated with genomic instability and development of breast cancer (BC). The homologous recombination DNA repair (HRR) pathway, base excision repair (BER) mechanism, and antioxidative enzymes are required as defense mechanisms against these DNA damaging agents. GSTP1, XRCC1, XRCC3 and RAD51 proteins are essential components of antioxidation, BER and HRR of DNA, respectively. Deficiencies in BER, HRR and antioxidation pathways are involved in the progression of cancer. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and blood samples of BC patients of an Italian population. Genomic DNA was also extracted from blood specimens of a control group. DNA sequencing was performed for six single-nucleotide polymorphisms (SNPs) in the GSTP1, RAD51, XRCC1 and XRCC3 genes in BC patients and the control group. Two variants in the 5'-UTR of the XRCC3 (rs1799794 A/G) and RAD51 (rs1801321) genes showed a significant association with susceptibility to BC (OR = 4.125; 95% CI 1.057-16.102; p = 0.03 and OR = 2.04; 95% CI 0.4925-8.449; p = 0.007, respectively). Additionally, we reported 2 mutations in intron 7 of the XRCC3 gene, CTdel (rs543072564) and A/G (rs369703243). Our results underscored the existence of an association between XRCC3-5'-UTR-A/G (rs1799794) and RAD51-5'-UTR G172T (rs1801321) genotypes and BC risk in an Italian population. The presence of mutations in the intronic region of the XRCC3 gene highlights the importance of more sequence screening of DNA repair genes for possible genetic penetrance in BC.

Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in RAS mutant cancers.

Mutant RAS has remained recalcitrant to targeted therapy efforts. We demonstrate that combined treatment with poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors and mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors evokes unanticipated, synergistic cytotoxic effects in vitro and in vivo in multiple RAS mutant tumor models across tumor lineages where RAS mutations are prevalent. The effects of PARP and MEK inhibitor combinations are independent of BRCA1/2 and p53 mutation status, suggesting that the synergistic activity is likely to be generalizable. Synergistic activity of PARP and MEK inhibitor combinations in RAS mutant tumors is associated with (i) induction of BIM-mediated apoptosis, (ii) decrease in expression of components of the homologous recombination DNA repair pathway, (iii) decrease in homologous recombination DNA damage repair capacity, (iv) decrease in DNA damage checkpoint activity, (v) increase in PARP inhibitor-induced DNA damage, (vi) decrease in vascularity that could increase PARP inhibitor efficacy by inducing hypoxia, and (vii) elevated PARP1 protein, which increases trapping activity of PARP inhibitors. Mechanistically, enforced expression of FOXO3a, which is a target of the RAS/MAPK pathway, was sufficient to recapitulate the functional consequences of MEK inhibitors including synergy with PARP inhibitors. Thus, the ability of mutant RAS to suppress FOXO3a and its reversal by MEK inhibitors accounts, at least in part, for the synergy of PARP and MEK inhibitors in RAS mutant tumors. The rational combination of PARP and MEK inhibitors warrants clinical investigation in patients with RAS mutant tumors where there are few effective therapeutic options.

Abstract NTOC-092: THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB

Abstract INTRODUCTION: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. AIMS: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. METHODS: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). RESULTS: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. CONCLUSIONS: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-092.

Detection of HRD gene mutations and copy number changes in cfDNA from prostate cancer patients.

e16546 Background: Recent genomic surveys of prostate cancer have identified somatic mutations in metastatic castration-resistant prostate cancer (mCRPC). In this study, we examined mCRPC patients for AR aberrations and mutations in the HRD (homologous recombination DNA-repair) pathway which may confer platinum or PARP inhibitor sensitivity. Methods: A novel targeted-hybrid-capture NGS assay capable of identifying deleterious mutations, copy number amplification and gene copy loss, was applied to circulating, cell-free DNA (cfDNA) extracted from plasma samples from 20 mCRPC patients. Samples were collected between 3/2010 – 10/2015 and stored at -20C. The gene panel used in the assay included AR and several genes in the HRD pathway — ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA, HDAC2, NBN, PALB2, and RAD51. Sequencing libraries created with the cfDNA extracted from 2.8-4 mLs of plasma had an average unique read coverage depth of 2282 genome equivalents (range 445 – 5136, median 2181). Results: Somatic variations were observed in 17 of the 20 samples analyzed. Copy number variation (CNV) was observed in 11 samples. AR amplification, linked to resistance to abiraterone and enzalutamide, was observed in 30% (6) of samples. Canonical AR ligand binding domain mutations, such as T787A and L702H were also detected. ATM, BRCA1, BRCA2, HDAC2, and FANCA gene deletions were also detected, as well as frameshift, nonsense, and other deleterious mutations in HRD genes. Significant CNV in multiple genes was observed in at least 5 patient samples. Mutations were detected across the entire collection date range, speaking to the robustness of cfDNA. Conclusions: Non-invasive tumor mCRPC genotyping appears to be feasible. The ability to detect HRD gene alterations suggests cfDNA testing may be suitable for the detection of HRD pathway defects and overall genome instability.

Phase ιι (window) preoperative study of olaparib with cisplatin or with durvalumab or alone or no treatment in patients with histologically proven head and neck squamous cell carcinoma who are candidates for surgery (OPHELIA).

TPS6096 Background: Novel agents are often investigated in unselected end-stage cancer patients and their efficacy is evaluated by the classical RECIST criteria making unlikely to fully exploit the antitumor potential of these targeted agents. Olaparib (O) is a potent inhibitor of PARP especially active in tumors that have homologous recombination DNA repair pathway deficiencies. Durvalumab (D) is a selective, high-affinity human IgG1 monoclonal antibody that blocks PD-L1 binding to PD-1 and CD80, overcoming PD-L1-mediated inhibition of T-cell activation. There is substantial evidence that tumor cells use PARP to repair platinum-induced DNA damage and thus escape apoptosis. In addition, O may complement the antitumor activity of D by increasing DNA damage through repair inhibition. Methods: OPHELIA is an open-label randomized multicenter phase II (window) trial in patients (pts) with head and neck squamous cell carcinoma (HNSCC). Treatment-naive HNSCC pts selected for primary curative study are randomized 3:3:3:1 in 4 neoadjuvant treatment groups: D 1500 mg on day 1 followed by O 600mg daily for 21-28 days (12 pts), cisplatin 60 mg/m2 on day 1 followed by O 75mg daily for 5 days (12 pts), monotherapy with O 600mg daily for 21-28 days (12 pts) and no treatment (5 pts). Preoperative therapy is discontinued 24 to 36 hours before surgery. Tumor biopsies, CT scans, PET and blood specimens are obtained at diagnosis and at surgery. Primary endpoint is the change in the tumor Ki-67 before and after treatment. Secondary endpoints are objective response rate according to RECIST 1.1 criteria, pathologic complete response rate and metabolic response rate assessed by FDG-PET/CT scan. Exploratory endpoints will include tumor and blood biomarkers. Translational correlates will be tested in tumor tissue, plasma and germline DNA and will include mutations in genes associated with DNA repair assessed by next generation sequencing and circulating tumor cells (CTCs) evaluated for DNA repair biomarkers and PD-L1. Trial is open to enrollment. Clinical trial information: NCT02882308.

Phase II open-label randomized multi-centre study of neoadjuvant olaparib in patients (pts) with platinum sensitive (PS) relapsed high grade serous ovarian cancer (OC): The NEO trial.

TPS5608 Background: Olaparib is a PARP inhibitor with clinical benefit in relapsed OC, especially in pts with germline BRCA1/2 mutation ( gBRCA). Study 19 (NCT00753545) found a progression free survival (PFS) gain from maintenance olaparib, post chemotherapy for PS relapse. Olaparib treatment in gBRCA OC pts relapsing post ≥ 3 prior chemotherapy regimens gave a response rate (RR) of 46% in the PS subgroup (Study 42; NCT01078662). Efficacy of olaparib may extend to OC with homologous recombination DNA repair pathway deficiency (HRD); susceptible to synthetic lethality from PARP inhibition. NEO [NCT02489006] is a window of opportunity study to assess tumor heterogeneity and the pharmacodynamic effects of olaparib given prior to surgery in PS OC, analyse the tumor genomic landscape pre and post olaparib, and assess for predictive biomarkers beyond BRCA mutation. Methods: This phase 2 study enrols pts with high grade serous OC, primary peritoneal or fallopian tube cancer with a progression free interval of ≥6 months and sensitive to their last line of platinum therapy. Pts must be suitable for secondary debulking surgery and agree to pre-operative tumour biopsy. All pts receive olaparib tablets 300mg po bid for 6 ± 2 weeks pre-surgery. Post-operatively, pts are randomised 1:1 to olaparib or 6 cycles of platinum-based chemotherapy followed by maintenance olaparib. The primary endpoint is the degree of PAR and PARP-1 inhibition in the blood and tumor following pre-operative olaparib in PS relapsed OC. Clinical efficacy is assessed by RR (RECIST 1.1), CA125, PFS and PFS2. Translational studies include next generation sequencing HRD panel to assess for somatic and germline mutations including RAD51B/C/D, PPM1D, FANCM, BRIP1, PALB2 and BARD1; evaluation of gene expression change in tumor tissue pre and post olaparib; assessment for resistance mechanisms and impact of heterogeneity. Circulating tumor DNA measured weekly pre-surgery is assessed for its prognostic value, alone and compared with CA125. The study will enrol 50-70 pts with estimated accrual of 3 pts/month across multiple sites, and opened at Princess Margaret Cancer Centre in 7/2016. Clinical trial information: NCT02489006.

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.

Abstract A25: Investigating the co-occurrence of potentially pathogenic DNA repair pathways alleles in BRCA1 or BRCA2 mutation carrier women with ovarian cancer.

Abstract Recurrent mutations in BRCA1 and BRCA2, which are genes that play a major role in the homologous recombination (HR) DNA repair pathway, account for a significant proportion of hereditary breast cancer (HBC) and or breast-ovarian cancer (HBOC) families of French Canadian (FC) descent due to common founders. This has facilitated genetic testing for establishing germline mutation carrier status in hereditary cancer clinics in Quebec for offering cancer surveillance and prevention options for women at risk for hereditary breast and other cancers. Recent evidence suggests that rare germline mutations in other members of HR pathway also confer an increased risk to these cancers, and thus could account in part for some of the HBC and HBOC families from the general population found negative for germline BRCA1 and BRCA2 mutations. Interestingly, our group has identified rare potentially pathogenic germline alleles in other DNA repair pathway genes in BRCA1 or BRCA2 mutation carrier FC women with breast cancer from HBC families. This prompted our investigation of the co-occurrence of germline mutations in selected DNA repair genes in BRCA1 or BRCA2 mutation carrier FC women with ovarian cancer. Whole Exome Sequencing (WES) analysis was performed on 15 BRCA1 or BRCA2 mutation carriers from HBOC families with at least two cases of ovarian cancer. Data was analyzed for potentially damaging rare alleles occurring in 178 genes known to be involved in different DNA repair pathways, including BRCA1 and BRCA2. Minor allele frequencies (MAF) were inferred by surveying public databases for genotypes of the general European population: NHLBI GO Exome Sequencing Project (ESP), 1000 Genomes Project (1KG), and The Exome Aggregation Consortium (ExAC). In addition to confirming the BRCA1 and BRCA2 mutations, a total of 23 rare variants with MAF &amp;lt;1% were identified in 21 out of the 178 investigated DNA repair genes; none of these variants were found in the healthy FC cohort (N~200). Eight out of the 23 variants were very rare, each with MAF&amp;lt;0.002%, which is comparable to allele frequencies for the known founder pathogenic BRCA1 and BRCA2 mutations observed in FC carriers. Five variants had no reported MAFs in the public databases. Ten out of the 15 BRCA1 or BRCA2 mutation carriers were each found to carry one to three variants. Five out of the 13 variants were found in genes (POLG, PARP1, FEN1, NUDT1, and OGG1) involved in the Base Excision Repair (BER) pathway: one nonsense, one frameshift, and three missense mutations. One missense and an alternative splicing variant were found in Nucleotide Excision Repair (NER) pathway genes (RAD23A and ERCC2), and two missense variants were found in HR pathway genes (CHEK2 and BRCA1); the variant found in BRCA1 was different from the known pathogenic mutations found in the BRCA1 mutation carriers investigated). The remaining four variants, all conferring missense mutations, were found in genes (POLD1, POLE, and TOPBP1) involved in multiple DNA repair pathways. Interestingly, the identified BRCA1 missense mutation was found in each of two BRCA2 pathogenic mutation carriers, which was predicted to be damaging using prediction software tools for the functional implication of amino acid substitutions (SIFT). This BRCA1 missense mutation was also found in a BRCA1 pathogenic mutation carrier with breast cancer from one of 58 pathogenic BRCA1 or BRCA2 mutation carrier HBC families of FC descent. Although the sample size was limited, our results suggest that BRCA1 or BRCA2 mutation carrier FC women with ovarian cancer also may carry potentially damaging mutations in other DNA repair genes, warranting further research to investigate their biological implications. Citation Format: Wejdan M. Alenezi, Timothee Revil, Dunarel Badescu, Suzanna L. Arcand, Guy Rouleau, Ioannis Ragoussis, Patricia N. Tonin. Investigating the co-occurrence of potentially pathogenic DNA repair pathways alleles in BRCA1 or BRCA2 mutation carrier women with ovarian cancer. [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 A25.

Abstract A24: A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer

Abstract Cancer is characterized by deregulated cell growth and proliferation, both of which are associated with hyperactivation of ribosome biogenesis. Inhibition of ribosome biogenesis using CX-5461, a specific inhibitor of RNA polymerase I-dependent transcription, has shown therapeutic efficacy in a MYC driven B-cell lymphoma mouse model, which is enhanced when used in combination with the mTORC1 inhibitor Everolimus. However, the therapeutic potential of CX-5461 in solid cancers is yet to be determined. Our preliminary data utilizing a panel of 36 ovarian cancer (OVCA) cell lines suggest that acute CX-5461 treatment results in cell cycle arrest and does not induce apoptosis. We hypothesize that the identification of genes that can be targeted to cooperate with CX-5461 will define novel drug combinations for the improved treatment of OVCA. Therefore, we performed a genome-wide RNAi screen to identify synthetic lethal genes with CX-5461 in the high-grade serous ovarian cancer (HGSOC) cell line OVCAR4. Pathway enrichment analysis of the candidate hits showed significant enrichment in the homologous recombination DNA repair (HR) pathway. Synergy with CX-5461 was validated in multiple HGSOC cell lines by both genetic and pharmacological inhibition of HR pathway components. We are currently investigating the mechanism of this synergy and will further assess efficacy in vivo. As HR deficiency is observed in 20% of OVCA patients, we suggest that future application of our studies will lead to new therapeutic options to improve the survival of this cohort of patients. Citation Format: Shunfei Yan, Keefe T. Chan, Kaylene J. Simpson, Elaine Sanij, Karen E. Sheppard, Katherine M. Hannan, Ross D. Hannan, Richard B. Pearson. A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer [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 A24.

Rapid customised operon assembly by yeast recombinational cloning.

We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

ABSTRACTUntargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.