BRCA-deficient Tumors Research Articles

Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

DYNLL1 is hypomethylated and upregulated in a tumor stage- and grade-dependent manner and associated with increased mortality in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and cellular mechanisms regulating HCC pathogenesis and progression are not completely understood. DYNLL1 is essential for the development and expansion of MYC-driven B cell lymphoma, and also regulates genomic stability and responses to DNA-damaging chemotherapy in BRCA1-deficient tumors. However, the role and regulation of DYNLL1 has not been previously studied in the context of HCC. Here we report that DYNLL1 gene is hypomethylated and its expression is upregulated in HCC patients compared to healthy controls. The expression of DYNLL1 changes in a tumor grade- and stage-dependent manner in HCC. In this study, we further show that high DYNLL1 expression results in shorter overall and progression-free survival in hepatocellular carcinoma patients. Similar to DYNLL1, one of its protein interactors, RACK1, also shows decreased CpG-aggregated methylation and increased expression in HCC. RACK1 expression increases from early to late stage and from low to high grade in HCC. We found that high RACK1 expression is significantly associated with increased mortality of HCC patients. The present study shows that the epigenetic regulation of DYNLL1 and its consequent upregulation might be contributing to cancer development and progression in HCC. Its higher expression in late stage or high grade HCC may favor more aggressive disease as pointed by the increased mortality in high expression cohort. A better mechanistic understanding of the role of DYNLL1 in HCC will be needed to develop targeted treatment strategies in the future.

PCAF-Mediated Histone Acetylation Promotes Replication Fork Degradation by MRE11 and EXO1 in BRCA-Deficient Cells

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.

The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

SummaryPARP inhibitors (PARPi) cause synthetic lethality in BRCA-deficient tumors. Whether specific vulnerabilities to PARPi exist beyond BRCA mutations and related defects in homology-directed repair (HDR) is not well understood. Here, we identify the ubiquitin E3 ligase TRIP12 as negative regulator of PARPi sensitivity. We show that TRIP12 controls steady-state PARP1 levels and limits PARPi-induced cytotoxic PARP1 trapping. Upon loss of TRIP12, elevated PARPi-induced PARP1 trapping causes increased DNA replication stress, DNA damage, cell cycle arrest, and cell death. Mechanistically, we demonstrate that TRIP12 binds PARP1 via a central PAR-binding WWE domain and, using its carboxy-terminal HECT domain, catalyzes polyubiquitylation of PARP1, triggering proteasomal degradation and preventing supra-physiological PARP1 accumulation. Further, in cohorts of breast and ovarian cancer patients, PARP1 abundance is negatively correlated with TRIP12 expression. We thus propose TRIP12 as regulator of PARP1 stability and PARPi-induced PARP trapping, with potential implications for PARPi sensitivity and resistance.

BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining

Failure to preserve the integrity of the genome is a hallmark of cancer. Recent studies have revealed that loss of the capacity to repair DNA breaks via homologous recombination (HR) results in a mutational profile termed BRCAness. The enzymatic activity that repairs HR substrates in BRCA-deficient conditions to produce this profile is currently unknown. We here show that the mutational landscape of BRCA1 deficiency in C. elegans closely resembles that of BRCA1-deficient tumours. We identify polymerase theta-mediated end-joining (TMEJ) to be responsible: knocking out polq-1 suppresses the accumulation of deletions and tandem duplications in brc-1 and brd-1 animals. We find no additional back-up repair in HR and TMEJ compromised animals; non-homologous end-joining does not affect BRCAness. The notion that TMEJ acts as an alternative to HR, promoting the genome alteration of HR-deficient cells, supports the idea that polymerase theta is a promising therapeutic target for HR-deficient tumours.

Abstract IA17: PARP inhibition modulates the tumor immune microenvironment in Brca1-deficient ovarian tumor

Abstract Poly ADP ribose polymerase (PARP) inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We recently reported that inhibition with a PARP inhibitor (olaparib) triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. However, all PBM-bearing mice treated with olaparib alone or in combination with PD-1 blockade eventually succumbed to the disease. These data indicate that although multiple PARP inhibitors have been approved for the treatment of BRCA-deficient ovarian cancers, eradication of this type of cancer remains a challenge. I will discuss our recent efforts on identifying resistance mechanisms to PARP inhibition and developing potential therapeutic strategy that overcomes the resistance to improve the treatment outcome. Citation Format: Liya Ding, Qiwei Wang, Michael Kearns, Tao Jiang, Panagiotis Konstantinopoulos, Ursula Matulonis, Jean Zhao. PARP inhibition modulates the tumor immune microenvironment in Brca1-deficient ovarian tumor [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA17.

Abstract IA18: Genetic dissection of breast cancer development and therapy resistance in mouse models

Abstract Genetically engineered mouse models (GEMMs) of human cancer not only permit us to gain a detailed insight into the specific genetic changes that drive tumor development and metastasis but also provide powerful tools to study the mechanisms underlying drug response and acquired resistance. Once these processes are understood in sufficient detail, it may be possible to design combination therapies that not only cause complete remissions but also eliminate remnant cells that elicit recurrent disease. We have developed GEMMs of E-cadherin mutated lobular breast cancer. These mice develop mammary tumors that closely resemble the lobular morphology and the metastatic spectrum of the cognate tumors in humans. Using Sleeping Beauty (SB) transposon-based insertional mutagenesis in conditional E-cadherin mutant mice, we have found that heterozygous loss of MYH9 or overexpression of hyperactive truncated forms of MYPT1/2 and ASPP2 reduces actomyosin contractility and thereby promotes malignant transformation of E-cadherin-deficient mammary epithelial cells, resulting in ILC formation in mice. This work highlights actomyosin hypercontractility induced by E-cadherin loss as a critical barrier to ILC development. We have also developed several GEMMs for somatic modeling of BRCA1-mutated triple-negative breast cancer using intraductal injection of lentiviral vectors for stable overexpression of exogenous genes and CAS9-mediated disruption or APOBEC-CAS9n-UGI (BE3)-mediated base editing of endogenous genes. We have used these GEMMs to validate RB, PTEN, PIK3CA, MYC, and MCL1 as bona fide driver genes in BRCA1-associated breast cancer. Moreover, MCL1 inhibition potentiated the in vivo efficacy of the PARP inhibitor (PARPi) olaparib, underscoring the therapeutic potential of this combination for treatment of BRCA1-associated cancer patients with poor response to PARPi monotherapy. The BRCA1-deficient mammary tumors from our mouse models are characterized by genomic instability and hypersensitivity to DNA-damaging agents, including platinum drugs and PARP inhibitors. Nevertheless, none of these drugs are curative: tumors grow back after drug treatment and eventually become resistant. We found that PARPi resistance of BRCA1-deficient GEMM tumors can be induced by several mechanisms, including activation of drug efflux transporters, type of BRCA1 mutation, and loss of components of the 53BP1-RIF1-shieldin and CST complexes that govern end-protection of DNA double-strand breaks. Citation Format: Jos Jonkers. Genetic dissection of breast cancer development and therapy resistance in mouse models [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA18.

A signal-seeking trial of olaparib and durvalumab in homologous repair-deficient tumors: A sub-study of the cancer molecular screening and therapeutics (MoST) program.

3073 Background: Data on the utility of a PARP inhibitor in combination with a checkpoint inhibitor remain limited, particularly in the histology agnostic setting with homologous recombination deficiency (HRD). This study evaluated the clinical activity of the combination of olaparib and durvalumab with the primary study endpoint of progression-free survival (PFS) at 6 months (PFS6m). Methods: This was a phase II, single-arm, signal-seeking study of the MoST program. Patients were recruited into two cohorts based on HRD genes, agnostic to histology: (1) BRCA 1/2 deficient tumours, excluding breast, prostate, ovarian cancers, and (2) other HRD related genes. Molecular testing was performed using in-house and commercial panels on archival tumour tissue centrally adjudicated by a molecular tumour board. All patients were treated with olaparib 300mg bid for 1 month, followed by combination with durvalumab 1500mg q4 weekly for 13 cycles. Olaparib treatment was then continued until disease progression. Results: Between Nov 2017-Feb 2019, 48 patients were enrolled (16 to BRCA 1/2 cohort 1 and 32 to HRD related genes cohort 2). Most common tumour sites were bone/soft tissue (15%, N=7), pancreas (13%, N=6) and stomach (8%, N=4). Overall best response in cohort 1 was PR (25%, N=4) and SD 4 (25%, N=4), and cohort 2 was PR (6%, N=2) and SD (56%, N=18). Median PFS was 3.65m (Cohort 1) and 3.56m (Cohort 2) respectively. PFS6m was 35% (Cohort 1) and 38% (Cohort 2) respectively. PDL1 status was not predictive of olaparib and durvalumab benefit. The most common grade 3/4 adverse events were anemia (11%%, N=4), abdominal pain (9%, N=3), increased lipase (9%,N=3), increased amylase (9%, N=3), dyspnea (6%, N=2), Hyperglycemia dyspnea (6%, N=2), Pancreatitis dyspnea (6%, N=2) and hematuria (6%, N=2). Conclusions: Olaparib and durvalumab show promising activity in a histology agnostic setting, particularly in BRCA deficient tumours. Further research is needed to identify biomarkers that correlate with treatment benefit. Results from longer clinical follow-up and additional biomarker analyses will be presented. Clinical trial information: ACTRN12617001000392 .

The development of a homologous recombination deficiency (HRD) score to identify HR-deficient tumors.

e18085 Background: BRCA1/2 mutated cancers are homologous recombination repair (HRR) deficient and often sensitive to drugs targeting DNA repair pathway. Emerging evidences suggest tumors with mutations in other HRR genes might also respond favorably to DNA repair pathway targeted drugs; however, the results were inconclusive. Genomic instability is characterized by telomeric allelic imbalance (TAI), large-scale transition (LST) and loss of heterozygosity (LOH). Here, we developed an HRD score based on assessing the status of these patterns in tumors. Methods: 73 cancer patients, including 36 breast cancer (BC), 35 ovarian cancer (OC) and 2 prostate cancer (PC) patients were recruited. 28 were BRCA-deficient tumors (10 BCs, 17OCs and 1 PC) harboring pathogenic/likely pathogenic (P/LP) germline mutation in BRCA1/2 with LOH. 13 BRCA-mutated tumors (10 BCs, 2 OCs and 1 PC) carried P/LP BRCA1/2 mutation without LOH. The rest 33 were HRR wild type controls (16 BCs and 16 OCs). All samples were profiled using a panel consisting of 24 classic HRR genes and their adjacent SNPs. The scores for TAI, LST and LOH were developed separately and an incorporated HRD score based on the 3 patterns was established. Clinical data of 42 OC pts were collected to validate the algorithm. Results: All pairwise correlations of TAI, LST and LOH scores showed high correlations (TAI vs. LOH: r = 0.89; TAI vs. LST: r = 0.92; LST vs. LOH: r = 0.83). The univariate logistic regression was used to assess the correlation of the score with BRCA deficiency. In the cohort of BRCA-deficient vs. HRR-negative, the scores of TAI (P = 9.72 x 10−4), LST (P = 8.75 x 10−5), LOH (P = 0.01) and HRD (P = 3.35 x 10−4) all showed significant associations with BRCA-deficiency. In the cohort of BRCA-deficient vs. BRCA-mutated +HRR-negative, all the scores also highly correlated with BRCA-deficiency (TAI: P = 2.35 x 10−4, LST: P = 1.48 x 10−5; LOH: P = 2.36 x 10−4, HRD: P = 6.29 x 10−5). Using 42 as a cutoff, it achieved sensitivities of 94.1%, 60% and 82% for identifying BRCA-deficient tumors in the 35 OCs, 36 BCs and in all 73 tumors, respectively. We further validated the predictive value of HRD score in an additional 42 OC pts and found significantly higher HRD sore in pts sensitive to platinum therapy (P = 0.036). Patients having an HRD score > 42 had a significantly longer PFS than pts with a score < 42 (p = 0.04). Conclusions: Our study developed a HRD score base on assessing 3 patterns of genomic instability (TAI, LST and LOH). The HRD score revealed a promising value in detecting HR-deficient tumors (especially BRCA-deficient).

Interplay of SMARCAD1 and BRCA1 at Replication Forks to Maintain Genome Integrity

Chemotherapeutic regimens that poison DNA replication are used for the treatment of homologous recombination (HR)-deficient cancers. We have discovered a novel mechanism by which the SWI/SNF chromatin remodeler SMARCAD1 stabilizes replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and resistance to replication poisons, this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naive-or PARPi-resistant tumors require SMARCAD1 mediated fork stabilization to maintain cellular proliferation. Our data reveal a critical interplay between SMARCAD1 mediated fork stabilization and BRCA1 mediated fork protection in maintenance of genome stability.

Replication Fork Remodeling and Therapy Escape in DNA Damage Response-Deficient Cancers.

Most cancers have lost a critical DNA damage response (DDR) pathway during tumor evolution. These alterations provide a useful explanation for the initial sensitivity of tumors to DNA-targeting chemotherapy. A striking example is dysfunctional homology-directed repair (HDR), e.g., due to inactivating mutations in BRCA1 and BRCA2 genes. Extensive efforts are being made to develop novel targeted therapies exploiting such an HDR defect. Inhibitors of poly(ADP-ribose) polymerase (PARP) are an instructive example of this approach. Despite the success of PARP inhibitors, the presence of primary or acquired therapy resistance remains a major challenge in clinical oncology. To move the field of precision medicine forward, we need to understand the precise mechanisms causing therapy resistance. Using preclinical models, various mechanisms underlying chemotherapy resistance have been identified. Restoration of HDR seems to be a prevalent mechanism but this does not explain resistance in all cases. Interestingly, some factors involved in DNA damage response (DDR) have independent functions in replication fork (RF) biology and their loss causes RF instability and therapy sensitivity. However, in BRCA-deficient tumors, loss of these factors leads to restored stability of RFs and acquired drug resistance. In this review we discuss the recent advances in the field of RF biology and its potential implications for chemotherapy response in DDR-defective cancers. Additionally, we review the role of DNA damage tolerance (DDT) pathways in maintenance of genome integrity and their alterations in cancer. Furthermore, we refer to novel tools that, combined with a better understanding of drug resistance mechanisms, may constitute a great advance in personalized diagnosis and therapeutic strategies for patients with HDR-deficient tumors.

Influence of DNA damage repair defects on tumor immunogenicity

Abstract In recent years, immune checkpoint inhibitors (ICI) have dramatically altered the treatment landscape for patients with advanced stage and metastatic cancers. Durable benefit, however, is limited to subsets of histologies and patients, highlighting the importance of identifying predictive biomarkers to select for patients who may benefit. DNA damage repair and response (DDR) deficiency including mismatch repair deficiency and resulting increased tumor neoantigens have been associated with improved response. We hypothesize that a similar concept may extend to other defects in DDR pathways, the most common being homologous recombination (HR). In ongoing work, we have implicated mutations in the DDR pathways including the HR pathway with improved survival after ICI in a large retrospective cohort. However, divergent responses were noted depending on the individual HR gene mutated. Syngeneic murine models were used to study individual DDR genes using CRISPR-Cas9 mutagenesis and their role in altering the tumor microenvironment and response to immune checkpoint blockade in isogenic model systems. We report differential effects of mutations in genes of the homologous recombination (HR) pathway on response after ICI administration in mouse and human tumors, and show that truncating mutations in BRCA2 are associated with superior response to ICI compared to BRCA1-deficient tumors. Single cell RNA-sequencing of CD45+ cells sorted from tumors reveals distinct immune landscapes. These data have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy in the setting of DDR deficiency and ultimately expand the therapeutic window of immunotherapy.

Exceptional responses to ipilimumab/nivolumab (ipi/nivo) in patients (pts) with refractory pancreatic ductal adenocarcinoma (PDAC) and germline BRCA or RAD51 mutations.

754 Background: Immune checkpoint inhibitors (ICI’s) have not shown meaningful clinical activity in unselected pts with PDAC. BRCA-deficient tumors have increased genomic instability, including increased tumor mutation burden (TMB), more tumor-infiltrating immune cells, and enrichment of a T cell-inflamed signature. We hypothesized that pts with mutations in BRCA or other homologous recombination repair genes may be sensitive to ICI’s. Methods: Utilizing the IRB-approved PDAC database at the University of Miami, we identified pts with relapsed/refractory PDAC with pathogenic germline mutations who were treated with combination ICI’s (ipi 1mg/kg and nivo 3mg/kg every 21 days followed by nivo 240mg every 2 weeks). Results: Five pts were identified (1 BRCA1, 2 BRCA2, 1 RAD51C and 1 RAD51D). Among the 3 evaluable pts, there was one complete response (CR), one partial response (PR) and one had progressive disease (PD). The pt with a CR had BRCA1; he had resection followed by adjuvant gem/cape and had a biopsy-proven recurrence in the lung and retroperitoneum 1y after the end of adjuvant therapy. He received ipi/nivo at recurrence and achieved a CR, ongoing for 17m on nivo maintenance. The patient with a PR had RAD51C; he was diagnosed with mPDAC and received FOLFIRINOX for 6m, followed by olaparib on a trial for 12m. Upon PD, the disease quickly progressed on 5FU/liposomal irinotecan, gemcitabine/nab-paclitaxel/cisplatin and FOLFIRINOX. He then started ipi/nivo with immediate improvement in pain and tumor markers. A radiological PR was seen after 2 doses and is ongoing for 3m with continued clinical and tumor marker improvement. The 3rd evaluable pt had BRCA2 and had PD with an exponential rise in tumor markers accompanied by clinical deterioration. Response data on the final two pts were pending at the time of submission and will be presented at the meeting. Conclusions: In this biomarker selected cohort, 2 out of 3 evaluable pts with PDAC had impressive responses to ipi/nivo. PDAC has generally been refractory to ICI therapy but this series suggests that this subgroup may be responsive to ICI’s. Further evaluation is warranted.

Murine models for familial pancreatic cancer: Histopathology, latency and drug sensitivity among cancers of Palb2, Brca1 and Brca2 mutant mouse strains.

Alterations of the PALB2 tumor suppressor gene have been identified in familial breast, ovarian and pancreatic cancer cases. PALB2 cooperates with BRCA1/2 proteins through physical interaction in initiation of homologous recombination, in maintenance of genome integrity following DNA double-strand breaks. To determine if the role of PALB2 as a linker between BRCA1 and BRCA2 is critical for BRCA1/2-mediated tumor suppression, we generated Palb2 mouse pancreatic cancer models and compared tumor latencies, phenotypes and drug responses with previously generated Brca1/2 pancreatic cancer models. For development of Palb2 pancreatic cancer, we crossed conditional Palb2 null mouse with mice carrying the KrasG12D; p53R270H; Pdx1-Cre (KPC) constructs, and these animals were observed for pancreatic tumor development. Individual deletion of Palb2, Brca1 or Brca2 genes in pancreas per se using Pdx1-Cre was insufficient to cause tumors, but it reduced pancreata size. Concurrent expression of mutant KrasG12D and p53R270H, with tumor suppressor inactivated strains in Palb2-KPC, Brca1-KPC or Brca2-KPC, accelerated pancreatic ductal adenocarcinoma (PDAC) development. Moreover, most Brca1-KPC and some Palb2-KPC animals developed mucinous cystic neoplasms with PDAC, while Brca2-KPC and KPC animals did not. 26% of Palb2-KPC mice developed MCNs in pancreata, which resemble closely the Brca1 deficient tumors. However, the remaining 74% of Palb2-KPC animals developed PDACs without any cysts like Brca2 deficient tumors. In addition, the number of ADM lesions and immune cells infiltrations (CD3+ and F/480+) were significantly increased in Brca1-KPC tumors, but not in Brca2-KPC tumors. Interestingly, the level of ADM lesions and infiltration of CD3+ or F/480+ cells in Palb2-KPC tumors were intermediate between Brca1-KPC and Brca2-KPC tumors. As expected, disruption of Palb2 and Brca1/2 sensitized tumor cells to DNA damaging agents in vitro and in vivo. Altogether, Palb2-KPC PDAC exhibited features observed in both Brca1-KPC and Brca2-KPC tumors, which could be due to its role, as a linker between Brca1 and Brca2.

Abstract B022: PARP1 complex composition as a predictor of response to PARP inhibitors in BRCA-linked ovarian carcinoma

Abstract Background: About 40-50% of epithelial ovarian cancers (EOC) show defects in DNA repair by homologous recombination (HR), which are mostly associated with BRCA1/2 loss-of-function mutations. The PARP inhibitors (PARPis) olaparib, niraparib and rucaparib were recently approved for treatment of ovarian cancer patients with platinum-sensitivity and recurrent ovarian cancer who carry inactivating BRCA1/2 mutations. These targeted drugs produce significant response rates ranging from 40-60% in patients with BRCA-linked advanced EOCs, but resistance is a continuing challenge. Whereas several studies have reported various mechanisms of acquired resistance to PARPis, the mechanisms of primary resistance are still poorly understood. Our goal is to develop predictors of PARPi response and to identify new targets for combination therapy to overcome primary resistance. We apply a novel integrated proteomics approach to develop mechanism-based biomarkers of response or primary resistance and to identify new therapeutic targets for rational combination approaches that can overcome resistance to single agent PARPi therapy. Methods: Primary patient-derived EOC cell lines and an isogenic EOC cell line pair with BRCA1 deletion and reconstituted with ectopic BRCA1 was used. Primary cell lines were genomically characterized by BROCA next generation sequencing. The effects of the FDA approved PARPis olaparib, rucaparib, and niraparib on BRCA1-null and BRCA1-reconstituted EOC cell lines regarding short- and long-term cell viability were determined by CellTiterGlo and crystal violet assays. Chemical and phosphoproteomics approach was used to identify the components of PARP1-based multiprotein complexes as well as protein post-translational modifications in the DNA damage signaling network in BRCA1/2-linked EOC cells. Specific PARP1-engaged protein complexes were further determined by immunoblotting. Frozen BRCA1-proficient and deficient ovarian cancer patient tumor samples collected at the time of debulking were also characterized by chemical proteomics. Results: Cell viability and clonogenic assays confirmed the expected correlation between PARPi response and BRCA1/2 status. Chemical proteomics with rucaparib and olaparib detected important elements of DNA damage repair, as components of PARP1 protein complexes, which are differentially represented in EOC cells that are sensitive to PARPi treatment compared to cells that show primary resistance to PARPi. In addition, phosphoproteomics analysis further identified significant and actionable changes in the DNA damage response signaling network in these cells. Most interestingly, these PARP1 protein complex differences are also observed in PARPi drug pulldowns (olaparib and rucaparib) from BRCA1-proficient compared to BRCA1-deficient tumor samples. Conclusion: Ovarian cancers that do not respond to PARPi displayed significant changes in PARPi-engaged protein complexes as well as protein modifications. We expect to identify new potential targets for combination therapy BRCA-linked EOC by further validation of our data. Citation Format: Ou Deng, Sweta Dash, Thales Nepomuceno, Ming D Han, Bin Fang, Doug Marchion, Alvaro N Monteiro, Uwe Rix. PARP1 complex composition as a predictor of response to PARP inhibitors in BRCA-linked ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B022. doi:10.1158/1535-7163.TARG-19-B022

Abstract NT-110: EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER

Abstract Camptothecin derivatives topotecan and irinotecan are currently the only FDA-approved topoisomerase I (Top1) inhibitors. Despite their wide usage they have many drawbacks: chemical instability, short half-life, dose-limiting bone marrow and gastrointestinal toxicity, and they are drug efflux substrates. The novel non-camptothecin Top1 inhibitors, indenoisoquinolines (LMP400, LMP776 and LMP744), have been developed to overcome these limitations. We explored whether treatment with LMPs could induce “synthetic lethality” in cells with impaired homologous recombination (HR) repair mechanism and whether it could be enhanced by combining them with PARP inhibitor, olaparib. Using the DT40 cell line, we assessed in vitro the role of HR in the cellular responses to the LMPs. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the LMP compounds than wild type cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib. To confirm the differential response of Brca1 wild type or Brca1 deficient cells to LMP treatment we assessed compound potency in primary ovarian cancer cell lines derived from a genetically engineered mouse (GEM) model for serous epithelial ovarian cancer (SEOC). As in DT40 cells, we observed that murine Brca1 deficient cells were more sensitive to all 3 LMP compounds than Brca1 wild type cells. Our results suggest that Brca1 deficiency renders cells more sensitive to treatment with LMPs or combination of LMPs and olaparib, potentially directing selection of patients with impaired HR repair mechanism in the phase 2 clinical trials that are in preparation. To confirm this observation in vivo, we are performing efficacy studies using LMP compounds alone or in combination with olaparib in orthotopic allograft mouse models for ovarian cancer derived from the GEM model. In Brca1 deficient tumors, treatment with each of the 3 LMP compounds led to significant increase of survival compared to vehicle treated group. LMP400 had the greatest beneficial impact on survival and tumor regression. To confirm synergy between LMPs and olaparib we selected LMP400 to represent the LMPs and repeated the efficacy study using Brca1 deficient tumor model. We observed that suboptimal doses of LMP400 or olaparib used as single treatments resulted in some increase in survival, however, when combined they significantly increased survival due to tumor regression. Our data demonstrate that the LMPs synergize with olaparib and provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient ovarian cancers. Citation Format: Ludmila Szabova, Melanie Gordon, Laetitia Matzi, Yves Pommier, Zoe Weaver Ohler. EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-110.

Abstract TMIM-085: BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are urgently needed for patients with PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A recently recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) family of proteins. BET proteins such as BRD4 promote oncogenic transcription of genes promoting cell growth, survival and DNA repair. One example is the well-established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). In syngeneic mouse ovarian cancer models, M2-like pro-tumor macrophages are a prominent component of the ovarian cancer tumor microenvironment (TME). NF-κB inhibition can reduce the M2 macrophage population; however, more sustained treatment with a systemic NF-κB inhibitor leads to more ascites and reduced survival time. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in tumors and macrophages that may be beneficial or harmful depending on context. OBJECTIVE: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer cells and peritoneal macrophages. METHODS: Cultured ID8 mouse ovarian cancer cells, PMJ2-PC mouse peritoneal macrophages and immortalized bone marrow-derived macrophages were treated with vehicle, the PARPi olaparib, the BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth in vitro. C57BL/6 mice injected intra-peritoneally (IP) with ID8 cells were treated with JQ1 (30 days, 50mg/kg by IP injection) with volume of ascites fluid, weight of harvested tumor, and number of tumor implants assessed. Markers of apoptosis (cleaved PARP or cleaved caspase-3), and DNA damage (pH2AX) were measured by immunohistochemistry or western blot. NF-κB activity was measured by luciferase assays of a NF-κB reporter plasmid. Expression of M1 (CCL3) and M2 (CD206, arginase-1) macrophage markers was measured by quantitative real-time RT-PCR (QPCR). RESULTS: In culture, JQ1 treatment sensitized ID8 ovarian cancer cells to olaparib-induced growth inhibition, DNA damage and apoptosis. However, despite modest stimulatory effects on DNA damage and apoptosis of long-term JQ1 treatment in ID8 tumors in vivo, JQ1 unexpectedly increased ascites formation without reducing overall tumor burden. In macrophages, there were also contrasting effects between JQ1 treatment in vitro and in vivo. JQ1 alone or combined with olaparib reduced NF-κB activity in cultured macrophage cell lines, and increased expression of CCL3, and reduced CD206 and arginase-1 expression. In contrast, increased ascites due to JQ1 treatment in vivo was accompanied by a pronounced M2 macrophage shift. CONCLUSIONS: Sustained inhibition of NF-κB activity in macrophages and potentially other cells in the ovarian TME could have overall deleterious effects on tumor progression. Our results strongly suggest that the therapeutic effects of BETi in ovarian cancer, and any therapeutic agent with the potential to alter cell function in the tumor microenvironment, need to be more thoroughly tested in the context of functional immune system before being translated to patients. This is particularly relevant since BETi are currently being tested in human patients in early clinical trials. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona E. Yull. BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-085.

Emerging Roles of RAD52 in Genome Maintenance.

The maintenance of genome integrity is critical for cell survival. Homologous recombination (HR) is considered the major error-free repair pathway in combatting endogenously generated double-stranded lesions in DNA. Nevertheless, a number of alternative repair pathways have been described as protectors of genome stability, especially in HR-deficient cells. One of the factors that appears to have a role in many of these pathways is human RAD52, a DNA repair protein that was previously considered to be dispensable due to a lack of an observable phenotype in knock-out mice. In later studies, RAD52 deficiency has been shown to be synthetically lethal with defects in BRCA genes, making RAD52 an attractive therapeutic target, particularly in the context of BRCA-deficient tumors.

EZH2 Is Overexpressed in BRCA1-like Breast Tumors and Predictive for Sensitivity to High-Dose Platinum-Based Chemotherapy.

BRCA1-deficient breast cancers carry a specific DNA copy-number signature ("BRCA1-like") and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors. EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1-like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model. The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1-like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents. Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.

A Review of the Hereditary Component of Triple Negative Breast Cancer: High- and Moderate-Penetrance Breast Cancer Genes, Low-Penetrance Loci, and the Role of Nontraditional Genetic Elements.

Triple negative breast cancer (TNBC), representing 10-15% of breast tumors diagnosed each year, is a clinically defined subtype of breast cancer associated with poor prognosis. The higher incidence of TNBC in certain populations such as young women and/or women of African ancestry and a unique pathological phenotype shared between TNBC and BRCA1-deficient tumors suggest that TNBC may be inherited through germline mutations. In this article, we describe genes and genetic elements, beyond BRCA1 and BRCA2, which have been associated with increased risk of TNBC. Multigene panel testing has identified high- and moderate-penetrance cancer predisposition genes associated with increased risk for TNBC. Development of large-scale genome-wide SNP assays coupled with genome-wide association studies (GWAS) has led to the discovery of low-penetrance TNBC-associated loci. Next-generation sequencing has identified variants in noncoding RNAs, viral integration sites, and genes in underexplored regions of the human genome that may contribute to the genetic underpinnings of TNBC. Advances in our understanding of the genetics of TNBC are driving improvements in risk assessment and patient management.