Homologous Recombination DNA Repair Pathway Research Articles

Post-transcriptional Regulation of BRCA2 through Interactions with miR-19a and miR-19b

Breast cancer type 2, early onset susceptibility gene (BRCA2) is a major component of the homologous recombination DNA repair pathway. It acts as a tumor suppressor whose function is often lost in cancers. Patients with specific mutations in the BRCA2 gene often display discrete clinical, histopathological, and molecular features. However, a subset of sporadic cancers has wild type BRCA2 and display defects in the homology-directed repair pathway, which is the hallmark of ‘BRCAness.’ The mechanisms by which BRCAness arises are not well understood but post-transcriptional regulation of BRCA2 gene expression by microRNAs (miRNAs) may contribute to this phenotype. Here, we examine the post-transcriptional effects that some members of the six-miRNA cluster known as the miR-17/92 cluster have on the abundance of BRCA2’s messenger RNA (mRNA) and protein. We discuss two interactions involving the miR-19a and miR-19b members of the cluster and the 3′UTR of BRCA2’s mRNA. We investigated these miRNA:mRNA interactions in 15 cell lines derived from pancreatic, breast, colon, and kidney tissue. We show that over-expression of these two miRNAs results in a concomitant decrease of BRCA2’s mRNA and protein expression in a subset of the tested cell lines. Additionally, using luciferase reporter assays we identified direct interactions between miR-19a/miR-19b and a miRNA response element (MRE) in BRCA2’s 3′UTR. Our results suggest that BRCA2 is subject to a complex post-transcriptional regulatory program that has specific dependencies on the genetic and phenotypic background of cell types.

The biological effects and clinical implications of BRCA mutations: where do we go from here?

BRCA1 and BRCA2 are tumour-suppressor genes encoding proteins that are essential for the repair of DNA double-strand breaks by homologous recombination (HR). Cells that lack either BRCA1 or BRCA2 repair these lesions by alternative, more error-prone mechanisms. Individuals carrying germline pathogenic mutations in BRCA1 or BRCA2 are at highly elevated risk of developing breast and/or ovarian cancer. Genetic testing for germline pathogenic mutations in BRCA1 and BRCA2 has proved to be a valuable tool for determining eligibility for cancer screening and prevention programmes. In view of increasing evidence that the HR DNA repair pathway can also be disrupted by sequence variants in other genes, screening for other BRCA-like defects has potential implications for patient care. Additionally, there is a growing argument for directly testing tumours for pathogenic mutations in BRCA1, BRCA2 and other genes involved in HR-DNA repair as inactivation of these genes may be strictly somatic. Tumours in which HR-DNA repair is altered are most likely to respond to emerging targeted therapies, such as inhibitors of poly-ADP ribose polymerase. This review highlights the biological role of pathogenic BRCA mutations and other associated defects in DNA damage repair mechanisms in breast and ovarian cancer, with particular focus on implications for patient management strategies.

Abstract 4730: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP-ribose polymerase inhibitor olaparib

Abstract Objective: Ovarian cancer is the second most common gynecological cancer and the number one cause of cancer death in gynecological cancers. In the United States alone, there are about 21,290 expected new cases in 2015 and about 14,180 expected deaths. Approximately 95% of ovarian malignancies are epithelial ovarian cancer (EOC). Most (50%) of these EOC tumors have BRCAness features - contain molecular defect in homologous recombination (HR) DNA repair pathways. About 20% of the non-BRACness subtypes of EOC exhibit increased expression of cyclin E (CCNE1) which is associated with resistance to DNA damaging drugs and increased mortality. A first-in-class drug, a poly(ADP-ribose) polymerase inhibitor (PARPi) olaparib was approved by the FDA for the treatment of advanced ovarian cancer patients with BRCA mutations who have had three or more lines chemotherapy. PARPi efficacy is lower in non-BRCAness tumors but may be enhanced by other drug combinations. Our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs. Our goal is to expand olaparib therapy to patients with chemoresistant tumors by sensitizing CCNE1 amplified EOC with the potent HDACi, panobinostat. Method: Ovarian cancer cell lines used were OVCAR-3 (CCNE1-amplified) and SKOV3 (CCNE1-non-amplified). They were co-treated with 0.01% DMSO vehicle, olaparib, panobinostat or olaparib/panobinostat combination. Sulforhodamine B (SRB) assays assessed cytotoxicity and immunofluorescence (IF) assessed markers of apoptosis (cleaved caspase-3). Images were analyzed using the Adobe Photoshop counting tool. Data analysis was performed via the Microsoft Excel average and percentage functions. Figures and p-value analysis were created using GraphPad Prism. Results: Our previous studies have demonstrated through western blot analysis of cleaved PARP expression that panobinostat enhances the pro-apoptotic effect of olaparib in CCNE1 amplified OVCAR-3 ovarian cancer cells. In this current study, IF was used to assess the established marker of apoptosis, cleaved caspase-3, in OVCAR3 and SKOV3 cells. Compared to vehicle-treated cells, olaparib alone induced no difference in the number of cleaved caspase-3 expression in both SKOV3 and OVCAR3 cells. However, there was significant upregulation in the percentage of cells positive for cleaved caspase-3 expression in combination treatments compared to single drug treatments in both cell lines. Conclusion: The response of ovarian cancer cells to the PARPi olaparib is enhanced by co-treatment with HDACi panobinostat in vitro. This indicates that, responses to olaparib in HR-proficient EOC can be improved by combination therapy with panobinostat. Thus we have identified a novel way to expand the use of olaparib for the treatment of advanced ovarian cancer to patients with non-BRCA tumors. Citation Format: Kofi Sarfo-Kantanka, Andrew J. Wilson, Alexandra Steck, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP-ribose polymerase inhibitor olaparib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4730.

Abstract C79: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly (ADP-ribose) polymerase inhibitor olaparib

Abstract Objective: Ovarian cancer is the second most common gynecological cancer and the number one cause of cancer death in gynecological cancers. In the United States alone, there are about 21,290 expected new cases in 2015 and about 14,180 expected deaths. Approximately 95% of ovarian malignancies are epithelial ovarian cancer (EOC). Most (50%) of these EOC tumors have BRCAness features - contain molecular defect in homologous recombination (HR) DNA repair pathways. About 20% of the non-BRACness subtypes of EOC exhibit increased expression of cyclin E (CCNE1) which is associated with resistance to DNA damaging drugs and increased mortality. A first-in-class drug, a poly(ADP-ribose) polymerase inhibitor (PARPi) olaparib was approved by the FDA for the treatment of advanced ovarian cancer patients with BRCA mutations who have had three or more lines chemotherapy. PARPi efficacy is lower in non-BRCAness tumors but may be enhanced by other drug combinations. Our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs. Our goal is to expand olaparib therapy to patients with chemoresistant tumors by sensitizing CCNE1 amplified EOC with the potent HDACi, panobinostat. Method: Ovarian cancer cell lines used were OVCAR-3 (CCNE1-amplified) and SKOV3 (CCNE1-non-amplified). They were co-treated with 0.01% DMSO vehicle, olaparib, panobinostat or olaparib/panobinostat combination. Sulforhodamine B (SRB) assays assessed cytotoxicity and immunofluorescence (IF) assessed markers of apoptosis (cleaved caspase-3). Images were analyzed using the Adobe Photoshop counting tool. Data analysis was performed via the Microsoft Excel average and percentage functions. Figures and p-value analysis were created using GraphPad Prism. Results: Our previous studies have demonstrated through western blot analysis of cleaved PARP expression that panobinostat enhances the pro-apoptotic effect of olaparib in CCNE1 amplified OVCAR-3 ovarian cancer cells. In this current study, IF was used to assess the established marker of apoptosis, cleaved caspase-3, in OVCAR3 and SKOV3 cells. Compared to vehicle-treated cells, olaparib alone induced no difference in the number of cleaved caspase-3 expression in both SKOV3 and OVCAR3 cells. However, there was significant upregulation in the percentage of cells positive for cleaved caspase-3 expression in combination treatments compared to single drug treatments in both cell lines. These data were supported by SRB cytotoxicity assays, where the combination of panobinostat and olaparib synergized in both OVCAR-3 and SKOV3 cells. Conclusion: The response of ovarian cancer cells to the PARPi olaparib is enhanced by co-treatment with HDACi panobinostat in vitro. This indicates that, responses to olaparib in HR-proficient EOC can be improved by combination therapy with panobinostat. Thus we have identified a novel way to expand the use of olaparib for the treatment of advanced ovarian cancer to patients with non-BRCA tumors. Note: This abstract was not presented at the conference. Citation Format: Kofi Sarfo-Kantanka, Andrew J. Wilson, Alexandra Steck, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly (ADP-ribose) polymerase inhibitor olaparib. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C79.

Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation.

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Editorial: Maintenance of Genome Integrity: DNA Damage Sensing, Signaling, Repair, and Replication in Plants.

Editorial: Maintenance of Genome Integrity: DNA Damage Sensing, Signaling, Repair, and Replication in Plants.

Abstract PR14: Phase I trial of first-in-class ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor VX-970 as monotherapy (mono) or in combination with carboplatin (CP) in advanced cancer patients (pts) with preliminary evidence of target modulation and antitumor activity

Abstract Background: ATR mediates the homologous recombination DNA repair pathway and cellular response to replication stress. VX-970 is a potent and selective inhibitor of ATR (Ki <0.2 nM) that showed enhanced synergy of ATR inhibition with cytotoxic chemotherapy and potential mono ATR inhibitor activity in tumor cell lines with high levels of replication stress, such as defects in the DNA damage repair (DDR) pathway (e.g. ATM loss). A phase I dose-escalation trial of VX-970 (sponsored by Vertex Pharmaceuticals Incorporated) was undertaken to assess the safety and tolerability of an ATR inhibitor as mono and with DNA-damaging chemotherapy, to show evidence of ATR inhibition in tumor tissue, and to explore antitumor activity. Methods: Pts with advanced solid tumors enrolled in 2 sequential parts. Part A: pts received IV VX-970 mono weekly in single-pt cohorts, with 3+3 cohorts initiated if grade (G) ≥2 VX-970-related adverse events (AEs) were observed. Part B: pts received CP on day 1 and VX-970 on days 2 and 9 of a 21-day cycle in a 3+3 dose-escalation design. Paired VX-970 tumor biopsies were obtained in selected CP treated pts pre- and post-VX-970, and pS345 Chk1 levels assessed by IHC. Results: 25 pts were treated; M/F 10/15; median age 67 yr (range 49-76 yr); ECOG PS 0/1: 11/14. In Part A, 11 pts (colorectal [CRC; n = 2]; mesothelioma [n = 2]; other [n = 7]; median prior lines of therapy = 3) received VX-970 at 60 mg/m2 (n = 1), 120 mg/m2 (n = 2), 240 mg/m2 (n = 1) and 480 mg/m2 (n = 7). In Part B, 14 pts (CRC [n = 6]; ovarian [n = 2]; other [n = 6]; median prior lines of therapy = 3) received VX-970 240 mg/m2 + CP AUC5 (n = 3; dose level 1 [DL1]), VX-970 120 mg/m2 + CP AUC5 (n = 3; DL2), VX-970 120 mg/m2 + CP AUC4 (n = 3; DL3) and VX-970 90 mg/m2 + CP AUC5 (n = 5; DL4). In Part A, no dose-limiting toxicities (DLT) or drug-related G3-4 AEs were seen. In Part B, 2 pts had DLT: G4 neutropenia and fever (n = 1; DL1) and G3 hypersensitivity (n = 1; DL2). Non-DLT G3-4 AEs were neutropenia (n = 4; DL1-2) and thrombocytopenia (n = 1; DL2) requiring dose delays. No G3-4 AEs were seen at DL3-4. RP2D cohort expansion is ongoing at DL4. VX-970 displayed linear AUC and Cmax at all DLs; median half-life was 16h with no accumulation. Based on preclinical models, efficacious exposures were achieved. When combined with CP, DL1 and DL2 showed similar VX-970 exposure, suggesting no apparent drug interactions. Decreased Chk1 phosphorylation was seen in 2/2 paired tumor biopsies (74% at DL4; 94% at DL2). An advanced CRC pt (serosal disease and abdominal lymphadenopathy; 3 prior lines of chemotherapy) with complete ATM loss by IHC achieved RECIST complete response to VX-970 mono at 60 mg/m2 and remains on trial at 59+ wks. RECIST stable disease (SD) was seen with VX-970 mono in 4 pts (median duration of SD = 11 wks [11-17.4 wks]) and VX-970 + CP in 7 pts, who were still ongoing (duration of SD = 5+ to 20+ wks), including several pts who had progressed on prior platinum therapy. Conclusion: VX-970 is well tolerated as monotherapy and in combination with CP, with preliminary evidence of target modulation and antitumor activity. VX-970 will be further explored in early phase II studies; in multiple tumor types, including triple-negative breast cancer and non-small cell lung cancer; and in patients with DDR aberrations. Citation Format: Timothy A. Yap, Maria J. de Miguel Luken, Brent O'Carrigan, Desam Roda, Dionysis Papadatos-Pastos, David Lorente, Nina Tunariu, Raquel Perez Lopez, Sasha Gayle, Ruth Riisnaes, Ines Figueiredo, Susana Miranda, Suzanne Carreira, Fang Yang, Sharon Karan, Marina Penney, John Pollard, L. Rhoda Molife, Udai Banerji, Mohammed Asmal, Scott Z. Fields, Johann S. de Bono. Phase I trial of first-in-class ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor VX-970 as monotherapy (mono) or in combination with carboplatin (CP) in advanced cancer patients (pts) with preliminary evidence of target modulation and antitumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR14.

Genetic variations in the homologous recombination repair pathway genes modify risk of glioma.

Accumulative epidemiological evidence suggests that single nucleotide polymorphisms (SNPs) in genes involved in homologous recombination (HR) DNA repair pathway play an important role in glioma susceptibility. However, the effects of such SNPs on glioma risk remain unclear. We used a used a candidate pathway-based approach to elucidate the relationship between glioma risk and 12 putative functional SNPs in genes involved in the HR pathway. Genotyping was conducted on 771 histologically-confirmed glioma patients and 752 cancer-free controls from the Chinese Han population. Odds ratios (OR) were calculated both for each SNP individually and for grouped analyses, examining the effects of the numbers of adverse alleles on glioma risk, and evaluated their potential gene-gene interactions using the multifactor dimensionality reduction (MDR). In the single-locus analysis, two variants, the NBS1 rs1805794 (OR 1.42, 95% CI 1.15-1.76, P = 0.001), and RAD54L rs1048771 (OR 1.61, 95% CI 1.17-2.22, P = 0.002) were significantly associated with glioma risk. When we examined the joint effects of the risk-conferring alleles of these three SNPs, we found a significant trend indicating that the risk increases as the number of adverse alleles increase (P = 0.005). Moreover, the MDR analysis suggested a significant three-locus interaction model involving NBS1 rs1805794, MRE11 rs10831234, and ATM rs227062. These results suggested that these variants of the genes involved in the HR pathway may contribute to glioma susceptibility.

Abstract POSTER-BIOL-1331: Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer

Abstract The marked platinum sensitivity of epithelial ovarian cancer (EOC) is thought to be related to underlying defects in the homologous recombination (HR) DNA repair pathway, which are present in approximately 50% of tumors with high-grade serous histology. However, repair of platinum-induced DNA damage does not always involve the HR pathway. More than 90% of platinum-DNA lesions are intrastrand cross-links which are repaired by the nucleotide excision repair (NER) pathway. We curated the TCGA ovarian cancer dataset and identified 22 tumors (7%) with nonsynonymous or splice site NER gene mutations among the 316 sequenced high grade serous ovarian tumors. All NER mutations were mutually exclusive and 8 (36%) were accompanied by heterozygous loss of the respective NER gene. In 12 (55%) of the 22 cases, there were no concomitant HR alterations identified. In vitro functional evaluation of two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in patients with advanced stage tumors and extreme response to first-line platinum therapy, showed that both mutations conferred platinum, but not PARPi, or doxorubicin sensitivity, and did not affect HR activity. Interestingly, ERCC6-Q524* was not associated with heterozygous loss but sensitized cells to platinum in a dominant negative fashion. Our findings suggest a novel mechanism of platinum sensitivity in EOC that involves NER gene mutations, which may occur independently of HR alterations. Unlike tumors with defective HR, tumors harboring only NER mutations may be sensitive to platinum but not PARP inhibition. Dissociation of platinum and PARPi sensitivity in this setting may have important implications for PARPi trials for both HR-proficient and HR-deficient EOCs. Citation Format: Raphael Ceccaldi, Kevin O’Connor, Kent W. Mouw, Adam Y. Li, Ursula A. Matulonis, Alan D. D’Andrea, Panagiotis A. Konstantinopoulos. Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1331.

Abstract 4251: Paclitaxel is necessary for improved survival in epithelial ovarian cancers with somatic homologous recombination gene mutations

Abstract The impact of somatic mutations and genomic alterations in determining the clinical outcome of sporadic epithelial ovarian cancer (EOC) remains uncertain. Our goal was to identify molecular markers of optimal cytoreduction, chemotherapeutic response, and survival. We focused on genes in the homologous recombination (HR) DNA repair pathway, as germline mutations in these genes confer improved survival, and data suggest that somatic mutations may similarly predict improved survival and chemosensitivity. One hundred fifty-eight patients were included with diagnoses of serous, endometrioid, or clear cell EOC and underwent primary cytoreduction followed by platinum-based chemotherapy. We performed targeted massively parallel sequencing of 53 genes in known genes from pathways such as HR and checkpoint inhibition in primary surgical samples. Patients were treated with platinum and cyclophosphamide (n = 24) or platinum and paclitaxel (n = 67). We detected 226 deleterious variants in 35 distinct genes. TP53 mutations were found in tumors from 90 subjects (56%) and deleterious mutations in HR genes in 45 (28%). Twenty-three patient tumors (15%) were found to have homozygous copy number loss of genes in the HR pathway. We did not detect an overall survival (OS) benefit in advanced stage patients treated with platinum whose tumors had HR mutations/loss as compared to those without HR defects. However, OS was significantly different between patients (p = 0.0028) whose tumors had HR mutations/loss if they received cyclophosphamide (median 24.1 months) as compared to paclitaxel (median 50.4 months). Furthermore, in patients with both TP53 and HR gene mutations or deletions, median OS improved to 76.6 months in the platinum and paclitaxel treated cohort (p = 0.0001). In this analysis by chemotherapy regimen and mutation status, the patients with TP53 and HR mutation/loss demonstrated worse median OS than those without such defects when treated with platinum and cyclophosphamide (24.1 months vs. 32.7 months respectively), but improved survival over patients without TP53 and HR defects when treated with platinum and paclitaxel (76.6 months vs. 44.4 months respectively, p = 0.0100). We also found that a somatic HR mutation was significantly associated with suboptimal debulking status (p = 0.012). Chances for optimal debulking and early stage were significantly increased in patients with PTEN mutations (p = 0.042), leading to OS benefit. The survival benefit attributed to platinum agents in HR deficient ovarian cancers may depend upon the use of paclitaxel in addition to platinum. This merits elucidation of the mechanism, and may be due to the mitosis cell cycle specificity of paclitaxel, and ensuing inhibition of HR deficient genomically unstable tumor subclones. Citation Format: Stephanie Jean, Bradley Wubbenhorst, Jiaqi Li, Kara N. Maxwell, Lauren Fishbein, Michael W. McLane, Nandita Mitra, Lin Zhang, Katherine L. Nathanson, Janos L. Tanyi. Paclitaxel is necessary for improved survival in epithelial ovarian cancers with somatic homologous recombination gene mutations. [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 4251. doi:10.1158/1538-7445.AM2015-4251

B cell Rad52 deficiency enhances immunoglobulin class switching through increased recruitment of Ku70/Ku86 to S region double-strand DNA breaks (IRM10P.618)

Abstract Class switch DNA recombination (CSR) diversifies the biological effector functions of antibodies and is critical for the maturation of the immune response. It is initiated by AID-mediated generation of DSBs in the switch (S) regions that will undergo recombination, mainly through Ku70/Ku86-mediated nonhomologous end-joining. It has been shown, however, that Ku-deficient B cells can undergo CSR, suggesting that other DNA repair mechanisms are at work. Herein, we show that deficiency of Rad52, an important component of the homologous recombination DNA repair pathway, resulted in significantly increased CSR and is associated with increased recruitment of Ku70/Ku86 to the S region DSB ends. In Rad52-/- B cells, the increased role of Ku70/Ku86 in S region DNA recombination was further emphasized by decreased occurrence of microhomologies in S-S junctions and inter-chromosomal c-Myc/IgH translocations. Conversely, enforced expression of Rad52 in B cells impaired CSR and decreased Ku70/Ku86 recruitment to S region DNA, as revealed by ChIP assays. Accordingly, EMSAs using Sμ probes with blunt-ends or short overhangs, revealed that in CSR, Ku70/Ku86 and Rad52 bind to different types of DSB ends. Our findings show that Rad52 modulates CSR by binding to S region DSB ends. This may facilitate a Ku-independent repair pathway, which would resolve AID-mediated S region DSBs, thereby explaining the residual CSR that has been shown to occur in a conditional Ku70/Ku86 B cell knockout.

Abstract P5-06-01: Homologous recombination deficiency (HRD) score predicts response to standard neoadjuvant chemotherapy in patients with triple negative or BRCA1/2 mutation-associated breast cancer

Abstract Background: Defects in the homologous recombination (HR) DNA repair pathway sensitize tumors to therapeutics that target this pathway. A significant proportion of triple negative breast cancers (TNBC) carry HR defects. Recently three DNA-based measures (LOH, Abkevich et al.; TAI, Birkbak et al; LST, Popova et al.) have been developed and shown to be highly associated with BRCA1/2 mutation status and sensitivity to platinum-based chemotherapy in TNBC. Standard chemotherapy consists of some combination of an anthracycline and cyclophosphamide, with or without a taxane. This study assesses the association of LOH, TAI, LST and the sum of these measures, the HRD Score, with response to standard neoadjuvant chemotherapy in patients with TNBC. Methods: Tumor samples were retrospectively obtained from 45 TNBC patients and 2 BRCA1/2 mutation positive hormone receptor-positive/HER2-negative breast cancer patients who received anthracycline-based neoadjuvant chemotherapy at Stanford University Medical Center or Cedars-Sinai Medical Center under IRB approved protocols. Measures of LOH, TAI, LST were obtained; tumor BRCA1/2 mutation analysis was conducted; and BRCA1 promoter methylation status was determined. Response was categorized by the residual cancer burden (RCB) score with responders defined as RCB 0 or 1, and non-responders as RCB 2 or 3. Data were also analyzed using the outcome of pathologic complete response (RCB 0). BRCA1/2 deficiency was defined as either BRCA1/2 germline or somatic mutant, or BRCA1 methylated with loss of the second allele in the tumor confirmed by LOH at the affected gene. Associations of LOH, TAI, LST, HRD Score, BRCA1/2 deficiency and mutation status with response to neoadjuvant therapy were evaluated with univariate logistic regression models. Results: All three metrics showed significant association with response (LOH: p=0.0018; TAI: p=0.0057; LST: p=0.0043) and pCR (LOH: p=0.0061; TAI: p=0.0044; LST: p=0.047). The sum of the scores, HRD Score, was also significantly associated with response (p=0.0021) and pCR (0.011). Neither BRCA1/2 mutation status (p=0.78 and p=0.31 respectively) nor BRCA1/2 deficiency (p=0.12 and p=0.34 respectively) were significantly associated with response or pCR in this cohort. When the analysis was restricted to BRCA1/2 intact samples (n=29) all scores were still significantly associated with pCR (LOH: p=0.019; TAI: p=0.0046; LST: p=0.013; HRD: p=0.0067), while only the sum was significant for response (p=0.042). Conclusions:All three measures of HR deficiency, LOH, TAI and LST and the sum of these three metrics, the HRD Score, are significantly associated with response to standard neoadjuvant chemotherapy in TNBC. This observation is consistent with the mechanisms of action of doxorubicin and cyclophosphamide as DNA damaging agents, though we have yet to examine cohorts of patients treated with non-anthracycline-containing standard chemotherapy regimens, such as docetaxel and cyclophosphamide. The HRD Score could be used clinically to identify patients with increased sensitivity to DNA damaging therapeutics. Citation Format: Melinda L Telli, William Audeh, Kirstin C Jensen, Shikha Bose, Kirsten Timms, Alexander Gutin, Victor Abkevich, Jerry Lanchbury, Chris Neff, Elisha Hughes, Zaina Sangale, Joshua Jones, Richard Wenstrup, Anne-Renee Hartman, Pei-Jen Chang, Shaveta Vinayak, James M Ford. Homologous recombination deficiency (HRD) score predicts response to standard neoadjuvant chemotherapy in patients with triple negative or BRCA1/2 mutation-associated breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-01.

Double PALB2 and BRCA1/BRCA2 mutation carriers are rare in breast cancer and breast-ovarian cancer syndrome families from the French Canadian founder population

French Canadian families with breast cancer and breast-ovarian cancer syndrome harbor specific BRCA1, BRCA2 and PALB2 germline mutations, which have been attributed to common founders. Mutations in these genes confer an increased risk to breast and ovarian cancers, and have been identified to play a role in and directly interact with the common homologous recombination DNA repair pathways. Our previous study described the case of a female diagnosed with breast cancer at 45 years old, who harbored the PALB2:c.2323C>T [p.Q775X] and BRCA2:c.9004G>A [p.E3002K] germline mutations, which have been found to recur in the French Canadian cancer families. As the frequency of double heterozygous carriers of breast-ovarian cancer susceptibility alleles is unknown, and due to the possibility that there may be implications for genetic counseling and management for these carriers, the present study investigated the co-occurrence of BRCA1/BRCA2 and PALB2 mutations in the French Canadian cancer families. The PALB2:c.2323C>T [p.Q775X] mutation, which is the only PALB2 mutation to have been identified in French Canadian cancer families, was screened in 214 breast cancer cases and 22 breast-ovarian cancer cases from 114 BRCA1/BRCA2 mutation-positive French Canadian breast cancer (n=61) and breast-ovarian cancer (n=53) families using a tailored polymerase chain reaction-based TaqMan® SNP Genotyping Assay. No additional PALB2:c.2323C>T [p.Q775X] mutation carriers were identified among the BRCA1/BRCA2 mutation carriers. The results suggest that carriers of the PALB2:c.2323C>T [p.Q775X] mutation rarely co-occur in French Canadian breast cancer and breast-ovarian cancer families harboring BRCA1 or BRCA2 mutations.

O6.4 - VX-970, selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) protein

ABSTRACT Background: ATR is a critical regulator of the homologous recombination DNA repair pathway and the cellular response to replication stress. Inhibition of ATR is predicted to selectively enhance the sensitivity of tumor cells, in which DNA repair is often compromised, to standard of care DNA damaging agents, while relatively sparing non-tumor cells which have DNA repair mechanisms to compensate for the loss of ATR. VX-970 is a potent and selective inhibitor of ATR with a Ki Results: VX-970 substantially increases the cytotoxic activity of multiple DNA damaging agents in a broad panel of tumor cell lines, with minimal increase in non-tumor cell cytotoxicity. VX-970 markedly synergizes with cytotoxic agents in mouse xenograft models of several tumor types. At concentrations of VX-970 which inhibit tumor growth, phosphorylation of the downstream biomarker, Chk1, is observed, as is up-modulation of markers of unrepaired double strand DNA breaks, including gamma-H2AX. VX-970 is currently in two phase 1 clinical trials which employ standard 3 + 3 dose escalations in subjects with advanced solid tumors to determine the tolerability of escalating doses of VX-970, administered intravenously, in combination with gemcitabine, cisplatin or carboplatin. Conclusions: VX-970 is an inhibitor of ATR that enhances the activity of DNA damaging agents in pre-clinical models, and is in phase 1 clinical development.

Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

A unique subset of epithelial ovarian cancers with platinum sensitivity and PARP inhibitor resistance.

Platinum and PARP inhibitor (PARPi) sensitivity commonly coexist in epithelial ovarian cancer (EOC) due to the high prevalence of alterations in the homologous recombination (HR) DNA repair pathway that confer sensitivity to both drugs. In this report, we describe a unique subset of EOC with alterations in another DNA repair pathway, the nucleotide excision repair (NER) pathway, which may exhibit a discordance in sensitivities to these drugs. Specifically, 8% of high-grade serous EOC from The Cancer Genome Atlas dataset exhibited NER alterations, including nonsynonymous or splice site mutations and homozygous deletions of NER genes. Tumors with NER alterations were associated with improved overall survival (OS) and progression-free survival (PFS), compared with patients without NER alterations or BRCA1/2 mutations. Furthermore, patients with tumors with NER alterations had similar OS and PFS as BRCA1/2-mutated patients, suggesting that NER pathway inactivation in EOC conferred enhanced platinum sensitivity, similar to BRCA1/2-mutated tumors. Moreover, two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in the two most platinum-sensitive tumors, were functionally associated with platinum sensitivity in vitro. Importantly, neither NER alteration affected HR or conferred sensitivity to PARPi or other double-strand break-inducing agents. Overall, our findings reveal a new mechanism of platinum sensitivity in EOC that, unlike defective HR, may lead to a discordance in sensitivity to platinum and PARPi, with potential implications for previously reported and ongoing PARPi trials in this disease.

Mutational landscape of gastric adenocarcinoma in Chinese: Implications for prognosis and therapy

Gastric cancer (GC) is a highly heterogeneous disease. To identify potential clinically actionable therapeutic targets that may inform individualized treatment strategies, we performed whole-exome sequencing on 78 GCs of differing histologies and anatomic locations, as well as whole-genome sequencing on two GC cases, each with three primary tumors and two matching lymph node metastases. The data showed two distinct GC subtypes with either high-clonality (HiC) or low-clonality (LoC). The HiC subtype of intratumoral heterogeneity was associated with older age, TP53 (tumor protein P53) mutation, enriched C > G transition, and significantly shorter survival, whereas the LoC subtype was associated with younger age, ARID1A (AT rich interactive domain 1A) mutation, and significantly longer survival. Phylogenetic tree analysis of whole-genome sequencing data from multiple samples of two patients supported the clonal evolution of GC metastasis and revealed the accumulation of genetic defects that necessitate combination therapeutics. The most recurrently mutated genes, which were validated in a separate cohort of 216 cases by targeted sequencing, were members of the homologous recombination DNA repair, Wnt, and PI3K-ERBB pathways. Notably, the drugable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair were mutated in 10% of GC cases. Mutations of the BRCA2 (breast cancer 2, early onset) gene, found in 8% of our cohort and validated in The Cancer Genome Atlas GC cohort, were associated with significantly longer survivals. These data define distinct clinicogenetic forms of GC in the Chinese population that are characterized by specific mutation sets that can be investigated for efficacy of single and combination therapies.

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. 56iron and 28silicon) by inhibiting Mre11 complex activity. Exposure of cells to 56iron or 28silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.

Induction of DNA Damage Response and Repair Pathways in HSPCs Following Exposure to AML Exosomes

Exosomes are extracellular vesicles that function in cell-cell communication by trafficking protein and RNA species to bystander cells. While exosomes are produced by all cell types, those released by cancer cells have come to the forefront of investigation for their potential to modulate the tumorigenic niche. We recently reported that exosomes released from acute myelogenous leukemia (AML) cells impact the phenotype and function of stromal and hematopoietic stem and progenitor cells (HSPC) found in the bone marrow (Huan et al. Cancer Res. 2013). As part of these studies, we observed a decrease in clonogenic potential of murine HSPCs exposed to exosomes isolated from the Molm-14 AML cell line in vitro and in xenograft transplantation studies. To determine if this observation was more widely applicable, we next exposed murine c-kit-selected HSPCs to exosomes isolated from both primary patient samples and the HL-60 AML cell line in vitro. Strikingly, exposure to primary AML patient and HL-60 exosomes produced a significant reduction in colony formation; on average, only 7.3% as many colonies formed in exposed conditions compared to controls. We also previously showed that exosomes traffic a complex mixture of protein and RNA to bystander cells. A recent report demonstrated increased DNA damage in mammary epithelial cells due to elevated reactive oxygen species (ROS) following exposure to exosomes derived from multiple breast cancer cell lines (Dutta et al. PLOS One 2014). A similar mechanism has been shown to restrict the replicative capacity of human HSPCs. Here, we hypothesized that exosome transfer might elicit a DNA damage response in murine HSPCs, contributing to the decreased ability of exposed cells to form colonies. When we performed 48 hours of in vitro HL-60 exosome exposure of c-kit enriched progenitor cells, we found a statistically significant upregulation of genes involved in DNA damage sensing as well as homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways compared to unexposed controls. Immunofluorescence analysis on exosome-exposed cells also revealed an increase in the formation of γH2AX foci in cells exposed to HL-60 exosomes, indicating an increase in DNA damage burden. Next, we tested if increased ROS might account for DNA damage and the resulting progenitor frequency. Using a flow cytometric analysis of ROS (DCF-DA) revealed a clear upward shift in median fluorescence intensity of exosome-treated c-kit+ cells compared to untreated controls. To further confirm the involvement of ROS, we treated exosome-exposed cells with the antioxidant NAC. While this did not result in a substantial reduction in ROS levels as measured by flow cytometry, analysis of the transcriptional DNA damage response revealed a dose-response pharmacological rescue of HR and NHEJ pathway gene expression. Our work in aggregate suggests that AML exosomes have a direct suppressive effect on HSPCs that involves, at least in part, gains in ROS that promote DNA damage accumulation and genomic instability. We propose a model whereby the paracrine trafficking of exosomes plays an active role in the erosion of HSPC activity in the AML niche that leads to characteristic cytopenias even at low leukemic burden. DisclosuresNo relevant conflicts of interest to declare.

Association of BRCA1/2 defects with genomic scores predictive of DNA damage repair deficiency among breast cancer subtypes.

IntroductionHomologous recombination (HR) DNA repair is of clinical relevance in breast cancer. Three DNA-based homologous recombination deficiency (HRD) scores (HRD-loss of heterozygosity score (LOH), HRD-telomeric allelic imbalance score (TAI), and HRD-large-scale state transition score (LST)) have been developed that are highly correlated with defects in BRCA1/2, and are associated with response to platinum therapy in triple negative breast and ovarian cancer. This study examines the frequency of BRCA1/2 defects among different breast cancer subtypes, and the ability of the HRD scores to identify breast tumors with defects in the homologous recombination DNA repair pathway.Methods215 breast tumors representing all ER/HER2 subtypes were obtained from commercial vendors. Next-generation sequencing based assays were used to generate genome wide SNP profiles, BRCA1/2 mutation screening, and BRCA1 promoter methylation data.ResultsBRCA1/2 deleterious mutations were observed in all breast cancer subtypes. BRCA1 promoter methylation was observed almost exclusively in triple negative breast cancer. BRCA1/2 deficient tumors were identified with BRCA1/2 mutations, or BRCA1 promoter methylation, and loss of the second allele of the affected gene. All three HRD scores were highly associated with BRCA1/2 deficiency (HRD-LOH: P = 1.3 × 10-17; HRD-TAI: P = 1.5 × 10-19; HRD-LST: P = 3.5 × 10-18). A combined score (HRD-mean) was calculated using the arithmetic mean of the three scores. In multivariable analyses the HRD-mean score captured significant BRCA1/2 deficiency information not captured by the three individual scores, or by clinical variables (P values for HRD-Mean adjusted for HRD-LOH: P = 1.4 × 10-8; HRD-TAI: P = 2.9 × 10-7; HRD-LST: P = 2.8 × 10-8; clinical variables: P = 1.2 × 10-16).ConclusionsThe HRD scores showed strong correlation with BRCA1/2 deficiency regardless of breast cancer subtype. The frequency of elevated scores suggests that a significant proportion of all breast tumor subtypes may carry defects in the homologous recombination DNA repair pathway. The HRD scores can be combined to produce a more robust predictor of HRD. The combination of a robust score, and the FFPE compatible assay described in this study, may facilitate use of agents targeting homologous recombination DNA repair in the clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0475-x) contains supplementary material, which is available to authorized users.