BRCA1 Function Research Articles

Modeling Therapy Resistance in BRCA1/2-Mutant Cancers.

Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or "revertant" mutations in BRCA1 or BRCA2 Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor-sensitive and PARP inhibitor-resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in "revertant" tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022-34. ©2017 AACR.

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P < 0.0001, two-tailed Student’s t-test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy (P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.

Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth

Tumor growth relies on efficient DNA repair to mitigate the detrimental impact of DNA damage associated with excessive cell division. Modulating repair factor function, thus, provides a promising strategy to manipulate malignant growth. Here, we identify the ubiquitin-specific protease USP21 as a positive regulator of BRCA2, a key mediator of DNA repair by homologous recombination. USP21 interacts with, deubiquitinates and stabilizes BRCA2 to promote efficient RAD51 loading at DNA double-strand breaks. As a result, depletion of USP21 decreases homologous recombination efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated survival defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma, where it promotes BRCA2 stability and inversely correlates with patient survival. Together, our findings identify deubiquitination as a means to regulate BRCA2 function and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.

A functional BRCA1 coding sequence genetic variant contributes to prognosis of triple-negative breast cancer, especially after radiotherapy.

As a subtype of breast cancer, triple-negative breast cancer (TNBC) shows poorprognosis and high heterogeneity. Precise identification of TNBC subgroups relevant to clinical prognosis is crucial in the design and administration of individualized treatments.This study aimed to evaluatethe prognostic value of the functionalBRCA1rs799917genetic variant in TNBC. Associations between the rs799917 polymorphism and progression risk were investigated after genotyping 370 TNBC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated by Cox regression. RESULTS: We found that the rs799917T allele was associated with a significantly increased risk of diseaseprogressionand shortened progression-free survival time (PFS) (P = 0.001 for log-rank test). Notably,TNBC patients with thers799917 CC genotype showed about 22 months prolonged PFS compared to the TT genotype after radiotherapy (HR 4.44, 95% CI 1.98-9.93;P = 2.9×10-4). Additionally, in overweight patients, the mean PFS of the rs799917TT genotype was 10 months shorter than that of the CC genotype (HR 3.57, 95% CI 1.46-8.73,P = 0.005). Our findings demonstrate that the functionalBRCA1genetic variant contributes to prognosis of TNBC. Our study also highlights the clinical potential of this polymorphism in the screening of high-risk TNBC patients for recurrence and the possibility of patient-tailored decisions especiallyduringradiotherapy.

Synergistic Effects of Arsenite on Radiosensitization of Glioblastoma Cells.

Glioblastoma is a frequent type of brain tumor and is radioresistant. Arsenite, which crosses the blood-brain barrier, shows synergistic effects with radiation in vitro and in vivo. The mechanism remains unclear. As synergistic radiosensitization has been reported in p53-deficient cancer cells, radiosensitization was evaluated using the glioblastoma cell line, U87MG-E6, which harbors inactivated p53, in comparison with the cell line, HCT116 p53 (-/-). Radiosensitivity was evaluated using clonogenic assays and detection of abnormal amplification of centrosomes (AAC). Synergistic effects of arsenite on radiosensitivity were observed in both cell lines. The radiosensitization induced by arsenite was abolished by N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger. Increased radiosensitivity by arsenite was also abolished following knock-down of BRCA2. In addition, the increased radiosensitization by arsenite was correlated with AAC, which was abolished by BRCA2 knock-down. We conclude that radiosensitization by arsenite is related to ROS and BRCA2 function.

Abstract 3074: Selective lethality of cisplatin in pancreatic cancer is dependent on mitotic functions of BRCA2

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with a 5-year survival rate of just 8%. Mutations in BRCA2 are among the most common known germline alterations that predispose to PDA. While the sensitivity of BRCA2-mutant breast and ovarian tumors to platinum compounds has long been recognized, the recent approval a PARP inhibitor in BRCA2-mutant ovarian cancer confers hope that such tumors might be targeted with greatly reduced toxicity. To test whether PDA exhibits a similar genetic susceptibility to these agents, we carried out preclinical intervention studies in Kras/p53-mutant genetically engineered mouse models (KPC mice) with or without the additional mutation of BRCA2 (KPCB2 mice). Mice were enrolled based on 3D ultrasound imaging criteria to assure similar initial tumor burden, and tumor growth was monitored longitudinally by ultrasound. To our surprise, loss of Brca2 failed to sensitize pancreatic tumors to Parp inhibition and did not provide additional benefit in combination with cisplatin treatment. By contrast, cisplatin induced substantial regressions in most KPCB2 tumors, leading to a &amp;gt;3-fold increase in overall survival, while having no effect on the growth or survival of mice with wild-type BRCA2. More surprisingly, the efficacy of cisplatin was not shared with oxaliplatin, even at higher doses. While BRCA2 is primarily known for its role in homology-directed repair, a network biology analysis of the function of BRCA2 in human pancreatic cancer led us to investigate the role of BRCA2 in mitosis. Wild type BRCA2 protein localizes to the midbody- a structure formed during the final stages of telophase- and plays and poorly understood role in the final abscission of cytoplasm between two dividing cells. Timelapse video microscopy of KPCB2 tumor cells treated once weekly for 2 hours with cisplatin revealed the frequent induction of endoreduplication and eventual formation of polyploid giant cells subsequent death. Similar cells were found throughout KPCB2 tumors during regression in response to cisplatin treatment, but were largely absent from tumors in other treatment arms. Mechanism studies found that BRCA2 deficient PDA cells are susceptible to replicative stress following cisplatin treatment that selectively leads to the formation of radial chromosomes that cannot be resolved during mitosis. These studies argue for the consideration of cisplatin-based (rather than oxaliplatin-based) regimens for the treatment of BRCA2-mutant pancreatic tumors. Citation Format: Barbara Orelli, Hans C. Maurer, Carmine Palermo, Steven A. Sastra, Kenneth P. Olive. Selective lethality of cisplatin in pancreatic cancer is dependent on mitotic functions of BRCA2 [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 3074. doi:10.1158/1538-7445.AM2017-3074

Abstract 831: TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM

Abstract Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p&amp;lt;0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy. Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [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 831. doi:10.1158/1538-7445.AM2017-831

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 347: The role of Pin1 in chemosensitivity of BRCA1-deficient breast cancers

Abstract BRCA1-deficient breast cancers are largely triple negative (ERα, PR and HER2 negative) and express markers representing the basal-like subgroup. Triple receptor negativity means that there are no targeted therapies available for this subtype, which consequently has the poorest overall survival rates. Given that BRCA1 dysfunction or ‘BRCAness’ is often observed in triple negative breast cancers (TNBCs), it is likely that loss of BRCA1 also plays a key role in their pathogenesis. Therefore to improve TNBC treatment responses, investigation into the underlying biology is required. It has been reported that levels of the prolyl isomerase Pin1 are upregulated in the absence of functional BRCA1, and that levels correlate with tumor grade. Pin1 alters the conformation of target proteins to affect their regulation and function. Many of these target proteins are implicated in key oncogenic signalling pathways, including BRCA1-associated pathways, such as cell cycle and growth regulation, DNA damage response, transcriptional regulation and NFκB signalling. We have observed that Pin1 levels are indeed upregulated in BRCA1-deficent cell lines compared to matched BRCA1 reconstituted cells, and that Pin1 plays a role in their growth rate and responsiveness to chemotherapy. BRCA1-deficient cells are sensitive to DNA damaging agents due to loss of DNA repair functions, notably homologous recombination (HR) repair of double strand breaks. We have identified a correlation between Pin1 levels and sensitivity to PARP inhibitors, which are toxic to HR deficient cells due to synthetic lethality. Ongoing work will establish whether Pin1 levels may be utilized as a biomarker of BRCAness and PARP inhibitor sensitivity. In contrast, during treatment of breast cancer cells with microtubule damaging agents, the presence of BRCA1 is required to promote apoptosis through MEKK3 and JNK, therefore BRCA1 deficient cells are less sensitive. Knockdown of Pin1 in BRCA1-deficient cells sensitizes them to the taxane Paclitaxel, due to repression of the anti-apoptotic protein Mcl-1, while Pin1 knockdown in BRCA1 proficient cells has no effect on sensitivity as Mcl-1 levels are repressed through the MEKK3 pathway. We have also identified that several Src family kinases (SFKs) are upregulated by Pin1 in a BRCA1-deficient background, and may play a role in taxane sensitivity. Pre-treatment with the Src/SFK inhibitor Dasatinib increases cytotoxicity of Paclitaxel, suggesting that combination treatment of Dasatinib and Paclitaxel may be an effective treatment option for BRCA1-deficient breast cancers. In conclusion, we have found that Pin1 may be an important oncogenic mediator downstream of dysfunctional BRCA1 facilitating the viability of TNBCs. We propose that Pin1 could serve as a predictive marker of PARP and Src inhibitor treatments, allowing clinicians to make better informed treatment decisions for this heterogeneous and aggressive subtype of breast cancer. Citation Format: Catherine Knowlson, Paul Mullan, Niamh Buckley. The role of Pin1 in chemosensitivity of BRCA1-deficient breast cancers [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 347. doi:10.1158/1538-7445.AM2017-347

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the ‘BRCAness’ profile.

BRCA1/2 reversion mutations revealed in breast and gynecologic cancers sequenced during routine clinical care using tissue or liquid biopsy.

5551 Background: Tumors with genomic alteration (GA) of BRCA1 or BRCA2 ( BRCA) may be more sensitive to platinum (Pt) therapies and PARP inhibitors (PARPi). However, secondary reversion mutations (revGA) can arise that may restore BRCA function and underlie reduced sensitivity to Pt compounds or PARPi. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for patients with predominantly relapsed, refractory or metastatic breast cancer (10967) or ovarian/peritoneal cancer (8352) was analyzed by hybrid-capture, next-generation sequencing for all classes of GA: base substitutions, indels, rearrangements, and copy number changes. RevGA were any GA that could restore the reading frame if in cis with a nonsense or frameshift (fs) GA. Results: 1900/19329 (9.8% ± 0.4%) tumors had ≥1 deleterious BRCA GA. 38 samples harbored potential revGA in BRCA1 (16) or BRCA2 (22): breast carcinoma (Ca) (21), ovarian or peritoneal serous Ca (10), ovarian or peritoneal adenocarcinoma (3), and ovarian epithelial Ca NOS (4). 35/38 sequenced samples were metastases. All potential revGA were somatic and fell into 3 classes: overlapping indel (21), compensatory fs (6), and missense mutation (11). One case harbored both an overlapping indel and a missense mutation with potential to revert a nonsense alteration. For 6 patients, testing of multiple tissue samples reveals the acquisition of revGA over time. RevGA are generally observed at allele frequencies lower than the deleterious GA they may revert. Clinical histories for patients with reversion mutations will be presented. Conclusions: Genomic profiling of breast and gynecological carcinomas, using either tissue or liquid biopsies, reveals potential revGA that may restore some level of BRCA function. RevGA, although rare, can be acquired during the course of treatment. We identified potentially compensatory missense, fs and indel mutations with CGP. Comparison of allele frequencies suggests that revGA often arise as subclones. The acquisition of a revGA over time can be observed through testing of multiple samples, either tissue or liquid biopsy.

BRCA1/2 reversion mutations in prostate cancer identified from clinical tissue and liquid biopsy samples.

5024 Background: Prostate tumors with genomic alterations (GA) in BRCA1 or BRCA2 ( BRCA) may be sensitive to treatment with PARP inhibitors (PARPi). However, secondary reversion mutations (revGA) can arise that may restore BRCA function and underlie reduced sensitivity to PARPi or platinum (Pt)-based therapy. Comprehensive genomic profiling (CGP), using either tissue or liquid biopsies, can detect the variety of clinically relevant revGA that can arise. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for 1911 patients with predominantly relapsed, refractory or metastatic prostate carcinoma was analyzed by hybrid-capture, next-generation sequencing for all classes of GA: base substitutions, indels, rearrangements, and copy number changes. RevGA were any GA that could restore the reading frame if in cis with a nonsense or frameshift (fs) GA. Results: 216/1911 (11.3% ± 1.4%) tumors had ≥1 deleterious BRCA GA. Of these, 7 samples harbored potential revGA in BRCA1 (1) or BRCA2 (6): prostate acinar adenocarcinoma (5), neuroendocrine carcinoma (1), or undifferentiated carcinoma (1). Of these, 2 samples were liquid biopsies (blood), 4 were FFPE tissue samples (liver), and 1 sample was a bone marrow core biopsy. All samples with revGA were metastases. Potential revGA were of 3 types: overlapping indel (4), compensatory fs (2), and missense (1). One case harbored 2 revGA, an overlapping indel and a compensatory fs. Alteration frequencies for TMPRSS2 (fusions), PTEN, and AR were similar with or without BRCA mutations; revGA-positive samples had a modest increase in PTEN alterations (42.9% vs 33.8%, NS). The frequency of CDK12 alterations was significantly reduced in BRCA-mutated tumors (0.9% vs 6.9%, p = 0.00002). Clinical histories for patients with reversion mutations will be presented. Conclusions: CGP of 1911 prostate carcinomas reveals ≥1 deleterious BRCA1/2 in 11.3% of samples. From these, a series of 7 cases, all metastases, with co-occurring potential revGA were identified. Although rare, revGA can be acquired during treatment and may underlie resistance to PARPi or Pt-based therapy. BRCA revGA can be detected from both tissue and liquid biopsies.

BRCA1/2 reversion mutations in pancreatobiliary cancer identified from patient biopsies.

4130 Background: Germline genomic alterations (GA) in BRCA1 or BRCA2 ( BRCA) are associated with increased risk of pancreatic cancer. Tumors with BRCA GA may be more sensitive to platinum (Pt) therapies or PARP inhibitors (PARPi). However, secondary reversion mutations (revGA) that may restore BRCA function and underlie reduced sensitivity to Pt compounds or PARPi can arise. Methods: DNA extracted from FFPE tumor tissue obtained during routine clinical care for 7077 patients with predominantly relapsed, refractory or metastatic pancreatobiliary carcinoma was analyzed using comprehensive genomic profiling (CGP) for all classes of GA: base substitutions, indels, rearrangements, and copy number changes. RevGA were any GA that could restore the reading frame if in cis with a nonsense or frameshift (fs) GA. Tumor tissue only was used to predict germline (g BRCA) or somatic status by an algorithm with a call rate of 85% and &gt; 95% accuracy. Results: 417/7077 (5.9% ± 0.5%) tumors had ≥1 deleterious BRCA GA; 205 (2.9% ± 0.4%) had a predicted g BRCA GA. Significantly fewer women had deleterious BRCA GA (182/417; 43.6%)(p = 0.02); g BRCA GA frequency did not differ significantly (96/205; 46.8%)(p = NS). Overall, 49.1% of patients were women. 12 samples harbored potential revGA: pancreatic ductal adenocarcinoma (ACa) (7) or acinar cell carcinoma (Ca) (2), gallbladder ACa (2), and pancreatobiliary Ca NOS (1). 10/12 samples with revGA were metastases. Potential revGA were of 3 types: overlapping indel (7), compensatory fs (4), and missense (2). One case harbored 2 indels, both with potential to excise a fs GA. Apparent g BRCA were present in 7 cases, only somatic BRCA GA in 3 cases, and g BRCA/somatic status was ambiguous in 2 cases. For 2 patients, testing of multiple samples reveals the acquisition of revGA over time. KRAS mutation frequencies were similar for all samples (57.1%), BRCA-mutated samples (58.8%) and revGA-positive samples (58.3%). Clinical histories for patients with revGA will be presented. Conclusions: CGP of 7077 pancreatobiliary carcinomas reveals 5.9% had ≥1 deleterious BRCA GA, nearly 50% of which were g BRCA. Although rare, revGA can be acquired over time and potentially compensatory missense, fs and indel mutations were observed.

BRCA2 secondary mutation-mediated resistance to platinum and PARP inhibitor-based therapy in pancreatic cancer

Background:Pancreatic cancer has become the third leading cause of cancer death with minimal improvements in outcome for over 40 years. Recent trials of therapies that target-defective DNA maintenance using poly (ADP-ribose) polymerase (PARP) inhibitors are showing promising results, yet invariably patients recur and succumb to disease. Mechanisms of resistance to platinum-based and PARP inhibitor therapy in other cancer types include secondary mutations, which restore the integrity of DNA repair through an increasing number of different mechanisms.Methods:Here we present a case of a 63-year-old female patient with a germ line pathogenic BRCA2 mutation (6714 deletion) who developed pancreatic cancer and had an exceptional response to platinum and PARP inhibitor therapy. Through next-generation sequencing and clinical follow-up, we correlated tumour response and resistance to the BRCA2 mutational status in the tumour.Results:Initially, the patient had an exceptional response to platinum and PARP inhibitor therapy, most likely due to the BRCA2 mutation. However, the primary lesion recurred while on PARP inhibitor therapy and contained a secondary mutation in BRCA2, which mostly likely restored BRCA2 function in PARP inhibitor-resistant tumour cells.Conclusions:To our knowledge, this is the first report of a BRCA2 reversion mutation that conferred resistance to PARP inhibitor-based therapy in a pancreatic ductal adenocarcinoma patient. Future studies are needed to understand this important mechanism of resistance and how it may impact the choice of therapy for patients with pancreatic cancer.

BRCA1-Mutated Estrogen Receptor-Positive Breast Cancer Shows BRCAness, Suggesting Sensitivity to Drugs Targeting Homologous Recombination Deficiency.

Purpose: As estrogen receptor-positive (ER+) breast cancer in BRCA1 mutation carriers arises at an older age with less aggressive tumor characteristics than ER-negative (ER-) BRCA1-mutated breast cancer, it has been suggested that these tumors are "sporadic" and not BRCA1 driven. With the introduction of targeted treatments specific for tumors with a nonfunctioning BRCA1 or BRCA2 gene, the question whether the BRCA genes are impaired in the tumor is highly relevant. Therefore, we performed genomic profiling of BRCA1-mutated ER+ tumors.Experimental Design: Genomic profiling, BRCA1 promoter methylation assessment, and loss of heterozygosity analysis were done on 16 BRCA1-mutated ER+ tumors. Results were compared with 57 BRCA1-mutated ER- tumors, 36 BRCA2-mutated ER+-associated tumors, and 182 sporadic ER+ tumors.Results: The genomic profile of BRCA1-mutated ER+ tumors was different from BRCA1-mutated ER- breast tumors, but highly similar to BRCA2-mutated ER+ tumors. In 83% of the BRCA1-mutated ER+ tumors, loss of the wild-type BRCA1 allele was observed. In addition, clinicopathologic variables in BRCA1-mutated ER+ cancer were also more similar to BRCA2-mutated ER+ and sporadic ER+ breast cancer than to BRCA1-mutated ER- cancers.Conclusions: As BRCA1-mutated ER+ tumors show a BRCAness copy number profile and LOH, it is likely that the loss of a functional BRCA1 protein plays a role in tumorigenesis in BRCA1-mutated ER+ tumors. Therefore, we hypothesize that these tumors are sensitive to drugs targeting the BRCA1 gene defect, providing new targeted treatment modalities for advanced BRCA-deficient, ER+ breast cancer. Clin Cancer Res; 23(5); 1236-41. ©2016 AACR.

Abstract P5-06-01: Talazoparib antitumor effects in BRCA-deficient breast cancer models

Abstract Background: BRCA1 and BRCA2 functions are essential for the DNA double-strand break repair process in living cells with DNA damages. Therefore, germline pathogenic mutations in BRCA1/2 increases the risk of developing cancer.1, 2Poly(ADP-ribose) polymerase (PARP) enzymes are proteins responsible for DNA single-strand break repair. Persistent inhibition of PARP-dependent DNA repair in BRCA-deficient breast cancer cells leads to increased DNA damages resulting in cancer cell death. Talazoparib is a novel and potent, orally bioavailable, small molecule PARP inhibitor. Talazoparib's dual mechanism of action inhibits PARP enzyme activity and effectively traps PARP on DNA, preventing DNA repair, resulting in cell death in BRCA1/2-mutated cells.3 In tissue culture studies, talazoparib is more potent at trapping PARP on DNA to induce cancer cell death compared to other PARP inhibitors.4 Previous studies have demonstrated talazoparib inhibited growth in tumors harboring BRCA1/2 gene mutations. In the MX-1 breast cancer model with BRCA1-deficiency, talazoparib inhibited cell growth in vitro and induced regression in solid mouse xenografts.5 Here, we demonstrate antitumor effects of talazoparib monotherapy in a panel of breast cancer cells and patient-derived breast cancer models with pathologic BRCA1/2 mutations. Method: A panel of human breast cancer cell lines was treated with talazoparib to determine its cytotoxic effects. BRCA1/2 mutations status was correlated to talazoparib cytotoxic effects. At the molecular level, BRCA1/2-mutant and wild type breast cancer cell lines were treated with dose-escalating talazoparib to also assess the relationship between PARP-DNA trapping complex formation and treatment response. Patient-derived breast cancer xenograft models were used to assess talazoparib monotherapy on tumorigenesis. Immunohistochemistry assays were performed to determine Ki-67, gH2AX and caspase 3 marker expression following talazoparib treatment. Results: Cytotoxicity was observed in 50% (7/14) cell lines at IC50 values that are achieved in the clinic. BRCA1/2 alterations were detected in 21.4% (3/14) of cell lines sensitive to talazoparib treatment. In 14 patient-derived breast cancer xenograft models selected for this study, 57.1% (8/14) responded to talazoparib monotherapy. Of these, 35.7% had mutations in the BRCA1 (28.6%) and BRCA2 (7.1%) genes. Stable disease was observed in 14.2% (2/14). One stable model had BRCA1 mutations. Tumor regression was observed in 42.8% (6/14) of the models treated with talazoparib monotherapy. Importantly, 66.7% (4/6) of patient-derived breast cancer models that regressed on talazoparib monotherapy had mutations in BRCA1/2 genes. Conclusions: Cytotoxicity was observed with talazoparib monotherapy in breast cancer cell lines and in patient-derived xenograft tumor models harboring BRCA1 or BRCA2 mutations. Our data demonstrate therapeutic potential of talazoparib monotherapy in breast cancer associated with pathologic BRCA1/2 mutations. 1.Couch FJ et al. J Clin Oncol. 2015;33:304-11. 2. Petrucelli N et al. Genet Med. 2010;12:245-59. 3. Wang B et al. J Med Chem. 2016;59:335-57. 4. Murai J et al. Mol Cancer Ther. 2014;13:433-43. 5. Shen Y et al. Clin Cancer Res. 2013;19:5003-15. Citation Format: Phan VT, Protter AA, Peterson A, Uppal H. Talazoparib antitumor effects in BRCA-deficient breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-01.

Abstract P5-06-04: Distinct BRCA1 and BRCA2 specific functions at stalled replication forks - Clinical implications for differences between BRCA1 and BRCA2 mutation driven cancer

Abstract This abstract was withdrawn by the authors.

Abstract P4-12-03: Estrogen receptor-positive breast cancer in BRCA1 mutation carriers shows a BRCAness profile, suggesting sensitivity to drugs targeting homologous recombination deficiency

Abstract Background As estrogen receptor (ER)-positive breast cancer in BRCA1 mutation carriers arises at an older age with less aggressive tumor characteristics than ER negative BRCA1 mutated breast cancer, it has been suggested that these tumors are 'sporadic' and not BRCA1-driven. With the introduction of targeted treatments specific for tumors with a non-functioning BRCA1 or BRCA2 gene, the question whether the BRCA genes are impaired in the tumor, is highly relevant. Therefore, we performed genomic profiling of BRCA1-mutated ER+ tumors. Methods Genomic profiling, BRCA1 promoter methylation assessment, and loss of heterozygosity (LOH) analysis were done on 16 BRCA1-mutated ER+ tumors. Results were compared with 57 BRCA1-mutated ER- tumors, 36 BRCA2-mutated ER+ associated tumors, and 182 sporadic ER+ tumors. Results The genomic profile of BRCA1-mutated ER+ tumors was different from BRCA1-mutated ER- breast tumors, but highly similar to BRCA2-mutated ER+ tumors. In 83% of the BRCA1-mutated ER+ tumors, loss of the wildtype BRCA1 allele was observed. Clinico-pathological variables in BRCA1-mutated ER+ cancer were more similar to BRCA2-mutated ER+ and sporadic ER+ breast cancer than to BRCA1 mutated ER- cancers. Conclusions As BRCA1-mutated ER+ tumors showed a BRCAness copy number profile and LOH, it is likely that the loss of a functional BRCA1 protein plays a role in tumorigenesis in BRCA1-mutated ER+ tumors. Therefore, we hypothesize that these tumors are sensitive to drugs targeting the BRCA1 gene defect. Citation Format: Lips EH, Debipersad R, Scheerman E, Mulder L, Sonke GS, van der Kolk LE, Wessling J, Hogervorst FBL, Nederlof PM. Estrogen receptor-positive breast cancer in BRCA1 mutation carriers shows a BRCAness profile, suggesting sensitivity to drugs targeting homologous recombination deficiency [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-03.

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells.

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.

ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.