BRCA1 Research Articles

Genetic Aberrations of DNA Repair Pathways in Prostate Cancer: Translation to the Clinic.

Prostate cancer (PC) is the second most common cancer in men worldwide. Due to the large-scale sequencing efforts, there is currently a better understanding of the genomic landscape of PC. The identification of defects in DNA repair genes has led to clinical studies that provide a strong rationale for developing poly (ADP-ribose) polymerase (PARP) inhibitors and DNA-damaging agents in this molecularly defined subset of patients. The identification of molecularly defined subgroups of patients has also other clinical implications; for example, we now know that carriers of breast cancer 2 (BRCA2) pathogenic sequence variants (PSVs) have increased levels of serum prostate specific antigen (PSA) at diagnosis, increased proportion of high Gleason tumors, elevated rates of nodal and distant metastases, and high recurrence rate; BRCA2 PSVs confer lower overall survival (OS). Distinct tumor PSV, methylation, and expression patterns have been identified in BRCA2 compared with non-BRCA2 mutant prostate tumors. Several DNA damage response and repair (DDR)-targeting agents are currently being evaluated either as single agents or in combination in patients with PC. In this review article, we highlight the biology and clinical implications of deleterious inherited or acquired DNA repair pathway aberrations in PC and offer an overview of new agents being developed for the treatment of PC.

Synthesis, crystal structure, DFT, docking and biological activity studies of (NZ,N’Z)-3,3’-(piperazine-1,4-diyl)bis(N-(-4-methyl benzylidene)propane-1-amine)

The title compound (NZ,N’Z)-3,3’-(piperazine-1,4-diyl)bis(N-(-4-methyl benzylidene)propane-1-amine) (PMP) was synthesized by reacting N,N′-bis(3-aminopropyl)piperazine and 4- methyl benzaldehyde using microwave energy. The formed compound is characterized using FTIR, 1H and 13C NMR, UV–visible and elemental analysis techniques. Single crystal X-ray diffraction confirms that the title compound crystallizes in a monoclinic crystal system with space group of P21/c. The unusual C–H…π interaction facilitate the crystal packing proved by the Density Functional Theory (DFT) and Hirshfeld surface analysis. The PMP showed an excellent result against Enterococcus Faecalis with the value of 3.125 μg/ml. The antioxidant behavior of PMP showed strong antioxidant property. The in silico docking studies on breast cancer cell BRCA1 protein confirms that the PMP showed excellent binding affinity value of −6.80 kcal/mol. The lipophilicity of the molecule is high due to the non polar nature of the PMP. The in vitro anticancer studies on the breast cancer cell line MDA MB-231 showed promising results.

Olaparib effectively treats local recurrence of extrahepatic cholangiocarcinoma in a patient harboring a BRCA2-inactivating mutation: a case report

Cholangiocarcinoma (CCA) is a malignant tumor with poor prognosis and high recurrence rate. There is no standard treatment for advanced CCA beyond first-line chemotherapy, which provides only limited benefits. In this study, we report a case of a postoperative recurrence ECC patient harboring a breast cancer 2 (BRCA2)-inactivating rearrangement mutation that had an obvious reaction to olaparib therapy. The patient was a 68-year-old man with postoperative recurrence of extrahepatic CCA (ECC) who declined systemic chemotherapy. In August 2015, abdominal computed tomography (CT) of the patient revealed intrahepatic bile duct dilatation, obstruction at the hepatic hilar region proximal to the common hepatic duct, and splenomegaly, and radical surgical resection was performed. Postoperative histopathology diagnosis was ECC without metastases. In February 2017, abdominal CT revealed local recurrence, and the patient refused chemotherapy. BRCA2 rearrangement were detected by next-generation sequencing. Oral administration of olaparib was initiated. The patient achieved stable disease 1 month later, progression-free survival for >10 months without any significant adverse reactions, and an overall survival (OS) of 27 months. This is the first report demonstrating the clinical benefits of olaparib in a BRCA2 rearrangement-harboring patient with ECC. This observation would help determine the best treatment option for advanced ECC patients.

1145P Reliable detection of BRCA1 and BRCA2 large genomic rearrangements in FFPE tissue: A new diagnostic benchmark for somatic BRCA testing

PARP inhibitors are used for treatment of tumors lacking function of the double-strand DNA break repair proteins BRCA1 or BRCA2 and are already approved for several cancer types. Thus, it is clinically crucial to determine germline as well as somatic BRCA1/2 mutations in those patients. The amplicon-based Oncomine BRCA1 and BRCA2 Assay is a test routinely used in diagnostics with FFPE specimens. The assay is validated for the detection of mutations, however, data on its performance in detecting large genomic rearrangements in FFPE tissue, is scarce.

Genetic epidemiology of BRCA1- and BRCA2-associated cancer across Latin America

The prevalence and contribution of BRCA1/2 (BRCA) pathogenic variants (PVs) to the cancer burden in Latin America are not well understood. This study aims to address this disparity. BRCA analyses were performed on prospectively enrolled Latin American Clinical Cancer Genomics Community Research Network participants via a combination of methods: a Hispanic Mutation Panel (HISPANEL) on MassARRAY; semiconductor sequencing; and copy number variant (CNV) detection. BRCA PV probability was calculated using BRCAPRO. Among 1,627 participants (95.2% with cancer), we detected 236 (14.5%) BRCA PVs; 160 BRCA1 (31% CNVs); 76 BRCA2 PV frequency varied by country: 26% Brazil, 9% Colombia, 13% Peru, and 17% Mexico. Recurrent PVs (seen ≥3 times), some region-specific, represented 42.8% (101/236) of PVs. There was no ClinVar entry for 14% (17/125) of unique PVs, and 57% (111/196) of unique VUS. The area under the ROC curve for BRCAPRO was 0.76. In summary, we implemented a low-cost BRCA testing strategy and documented a significant burden of non-ClinVar reported BRCA PVs among Latin Americans. There are recurrent, population-specific PVs and CNVs, and we note that the BRCAPRO mutation probability model performs adequately. This study helps address the gap in our understanding of BRCA-associated cancer in Latin America.

Revisiting the BRCA-pathway through the lens of replication gap suppression: “Gaps determine therapy response in BRCA mutant cancer”

The toxic lesion emanating from chemotherapy that targets the DNA was initially debated, but eventually the DNA double strand break (DSB) ultimately prevailed. The reasoning was in part based on the perception that repairing a fractured chromosome necessitated intricate processing or condemned the cell to death. Genetic evidence for the DSB model was also provided by the extreme sensitivity of cells that were deficient in DSB repair. In particular, sensitivity characterized cells harboring mutations in the hereditary breast/ovarian cancer genes, BRCA1 or BRCA2, that function in the repair of DSBs by homologous recombination (HR). Along with functions in HR, BRCA proteins were found to prevent DSBs by protecting stalled replication forks from nuclease degradation. Coming full-circle, BRCA mutant cancer cells that gained resistance to genotoxic chemotherapy often displayed restored DNA repair by HR and/or restored fork protection (FP) implicating that the therapy was tolerated when DSB repair was intact or DSBs were prevented. Despite this well-supported paradigm that has been the impetus for targeted cancer therapy, here we argue that the toxic DNA lesion conferring response is instead single stranded DNA (ssDNA) gaps. We discuss the evidence that persistent ssDNA gaps formed in the wake of DNA replication rather than DSBs are responsible for cell killing following treatment with genotoxic chemotherapeutic agents. We also highlight that proteins, such as BRCA1, BRCA2, and RAD51 known for canonical DSB repair also have critical roles in normal replication as well as replication gap suppression (RGS) and repair. We review the literature that supports the idea that widespread gap induction proximal to treatment triggers apoptosis in a process that does not need or stem from DSB induction. Lastly, we discuss the clinical evidence for gaps and how to exploit them to enhance genotoxic chemotherapy response.

Using an Anthropological Lens to Explore Motivators and Challenges for Follow-up Care Decision Making among Female BRCA1/2 Carriers at Risk for Inherited Cancer

Females with a BRCA1/2 (BRCA) pathogenic variant have high lifetime risks for cancer. Regularly updated guidelines are in place that recommend screening and/or surgery to monitor and/or significantly reduce breast or ovarian cancer risk. Follow-up care decision making among this population is important to explore to understand the multi-faceted influences guiding cancer screening behaviors and risk-reducing surgery decisions. Using lived-experience theory and cognitive anthropology, this study explored emotional and social influences on decision making and behavioral adherence to guideline recommendations among twenty-seven female BRCA carriers. Ethnographic data from in-depth interviews were analyzed in parallel with self-reported survey data on perceived threat, response efficacy, and self-efficacy constructs. Survey results demonstrated high rates of adherence to guideline recommendations and high levels of perceived threat and response efficacy with lower levels of self-efficacy, while interview data revealed multi-faceted motivators and challenges associated with behavioral adherence. These findings unearth complex lived-experiences within the context of perceived threat, response efficacy, and self-efficacy and explore the cognitive relationship between motivators and challenges to inherited cancer follow-up care decision making and behavioral adherence. This study demonstrates how anthropological practice can aid in inherited cancer research and be used to support the development of interventions that consider cognitive influences on behavior.

BRCAness as a Biomarker of Susceptibility to PARP Inhibitors in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common primary brain cancer. GBMs commonly acquire resistance to standard-of-care therapies. Among the novel means to sensitize GBM to DNA-damaging therapies, a promising strategy is to combine them with inhibitors of the DNA damage repair (DDR) machinery, such as inhibitors for poly(ADP-ribose) polymerase (PARP). PARP inhibitors (PARPis) have already shown efficacy and have received regulatory approval for breast, ovarian, prostate, and pancreatic cancer treatment. In these cancer types, after PARPi administration, patients carrying specific mutations in the breast cancer 1 (BRCA1) and 2 (BRCA2) suppressor genes have shown better response when compared to wild-type carriers. Mutated BRCA genes are infrequent in GBM tumors, but their cells can carry other genetic alterations that lead to the same phenotype collectively referred to as ‘BRCAness’. The most promising biomarkers of BRCAness in GBM are related to isocitrate dehydrogenases 1 and 2 (IDH1/2), epidermal growth factor receptor (EGFR), phosphatase and tensin homolog (PTEN), MYC proto-oncogene, and estrogen receptors beta (ERβ). BRCAness status identified by accurate biomarkers can ultimately predict responsiveness to PARPi therapy, thereby allowing patient selection for personalized treatment. This review discusses potential biomarkers of BRCAness for a ‘precision medicine’ of GBM patients.

1Rucaparib versus chemotherapy in patients with advanced, relapsed ovarian cancer and a deleterious BRCA mutation: efficacy and safety from ARIEL4, a randomized phase III study

Objectives: Prospective studies comparing poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors with standard-of-care (SOC) chemotherapy (CT) in patients (pts) with relapsed ovarian cancer (OC) are currently limited. ARIEL4 (NCT02855944) is a phase III, randomized, open-label, international, multicenter study of the efficacy and safety of rucaparib vs SOC CT as treatment for PARP-inhibitor naïve pts with relapsed high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer, who had a deleterious BRCA1/2 (BRCA) mutation and had received ≥2 prior CT regimens. Methods: Pts were randomized 2:1 to oral rucaparib 600 mg twice daily or SOC CT and stratified based on progression-free interval (≥1 to <6 months = platinum resistant; ≥6 to <12 months = partially platinum sensitive; ≥12 months = fully platinum sensitive). Pts in the CT arm with platinum-resistant or partially platinum-sensitive disease received weekly paclitaxel 60-80 mg/m2; pts with fully platinum-sensitive disease received investigator's choice of platinum-based CT (single-agent carboplatin or cisplatin, or platinum doublet [carboplatin + paclitaxel, carboplatin + gemcitabine, or cisplatin + gemcitabine]). Pre-study-treatment plasma samples were assessed for BRCA reversion mutations. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included objective response rate (ORR) per RECIST and safety. Each efficacy endpoint was first evaluated in the efficacy population (randomized pts with deleterious BRCA mutations excluding those with BRCA reversion mutations), stepping down to the intent-to-treat (ITT) population (all randomized pts). Results: Conclusions: Patients with BRCA-mutated advanced, relapsed OC who received rucaparib had a significant improvement in PFS vs SOC CT. No new safety signals were identified. This is the first prospective report from a randomized trial demonstrating that the presence of a BRCA reversion mutation predicts for primary resistance to rucaparib. Prospective studies comparing poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors with standard-of-care (SOC) chemotherapy (CT) in patients (pts) with relapsed ovarian cancer (OC) are currently limited. ARIEL4 (NCT02855944) is a phase III, randomized, open-label, international, multicenter study of the efficacy and safety of rucaparib vs SOC CT as treatment for PARP-inhibitor naïve pts with relapsed high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer, who had a deleterious BRCA1/2 (BRCA) mutation and had received ≥2 prior CT regimens. Pts were randomized 2:1 to oral rucaparib 600 mg twice daily or SOC CT and stratified based on progression-free interval (≥1 to <6 months = platinum resistant; ≥6 to <12 months = partially platinum sensitive; ≥12 months = fully platinum sensitive). Pts in the CT arm with platinum-resistant or partially platinum-sensitive disease received weekly paclitaxel 60-80 mg/m2; pts with fully platinum-sensitive disease received investigator's choice of platinum-based CT (single-agent carboplatin or cisplatin, or platinum doublet [carboplatin + paclitaxel, carboplatin + gemcitabine, or cisplatin + gemcitabine]). Pre-study-treatment plasma samples were assessed for BRCA reversion mutations. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included objective response rate (ORR) per RECIST and safety. Each efficacy endpoint was first evaluated in the efficacy population (randomized pts with deleterious BRCA mutations excluding those with BRCA reversion mutations), stepping down to the intent-to-treat (ITT) population (all randomized pts). Patients with BRCA-mutated advanced, relapsed OC who received rucaparib had a significant improvement in PFS vs SOC CT. No new safety signals were identified. This is the first prospective report from a randomized trial demonstrating that the presence of a BRCA reversion mutation predicts for primary resistance to rucaparib.

Anticancer activity of RAPTA-EA1 in triple-negative BRCA1 proficient breast cancer cells: single and combined treatment with the PARP inhibitor olaparib

RAPTA-EA1 is a promising glutathione transferase (GSTP-1) inhibitor that has previously been shown to inhibit the growth of various breast cancer cells. We studied the anticancer activity of RAPTA-EA1 on triple-negative BRCA1 competent breast cancer MDA-MB-231 cells. MDA-MB-231 cells are significantly more sensitive to RAPTA-EA1 than MCF-7 cells. Treatment reveals a higher degree of cytotoxicity than cisplatin against both cell lines. Ruthenium accumulation in MDA-MB-231 cells is mainly in the nuclear fraction (43%), followed by the cytoplasm (30%), and the mitochondria (27%). RAPTA-EA1 blocks cell growth at the G2/M phase, leading to nuclear condensation and cell death. The compound slightly inhibits DNA replication of the 3,426-bp fragment of the BRCA1 exon 11 of the cells, with approximately 0.6 lesion per the BRCA1 fragment. The expression of BRCA1 mRNA and its protein in the Ru-treated cells is curtailed by 50–80% compared to the untreated controls. Growth inhibition of the triple-negative BRCA1 wild-type MDA-MB-231 and the sporadic BRCA1 wild-type MCF-7 cells by olaparib (a poly [ADP-ribose] polymerase (PARP) inhibitor) is dose-dependent, with MDA-MB-231 cells being two-fold less susceptible to the drug than MCF-7 cells. Combining olaparib with RAPTA-EA1 results in a combination index (CI) of 0.78 (almost additive) in MDA-MB-231 cells and 0.24 (potent synergy) in the MCF-7 cells. The PARP inhibitor alone differently regulates the expression of BRCA1 mRNA in both cell lines, whereas the olaparib-RAPTA-EA1 combination induces overexpression of BRCA1 mRNA in these cells. However, the expression level of the BRCA1 protein is dramatically reduced after treatment with the combined inhibitors, compared with the untreated controls. This observation highlights the cellular responses of triple-negative BRCA1 proficient breast cancer MDA-MB-231 cells to RAPTA-EA1 through BRCA1 inhibition and provides insights into alternative treatments for breast cancer.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events.

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.

BRCA-mutant pancreatic ductal adenocarcinoma.

Despite continued research, pancreatic ductal adenocarcinoma (PDAC) remains one of the main causes of cancer death. Interest is growing in the role of the tumour suppressors breast cancer 1 (BRCA1) and BRCA2-typically associated with breast and ovarian cancer-in the pathogenesis of PDAC. Indeed, both germline and sporadic mutations in BRCA1/2 have been found to play a role in the development of PDAC. However, data regarding BRCA1/2-mutant PDAC are lacking. In this review, we aim to outline the specific landscape of BRCA-mutant PDAC, focusing on heritability, clinical features, differences between BRCA1 and 2 mutations and between germline and sporadic alterations, as well as established therapeutic strategies and those that are still under evaluation.

A computational and structural analysis of germline and somatic variants affecting the DDR mechanism, and their impact on human diseases

DNA-Damage Response (DDR) proteins are crucial for maintaining the integrity of the genome by identifying and repairing errors in DNA. Variants affecting their function can have severe consequences since failure to repair damaged DNA can result in cells turning cancerous. Here, we compare germline and somatic variants in DDR genes, specifically looking at their locations in the corresponding three-dimensional (3D) structures, Pfam domains, and protein–protein interaction interfaces. We show that somatic variants in metastatic cases are more likely to be found in Pfam domains and protein interaction interfaces than are pathogenic germline variants or variants of unknown significance (VUS). We also show that there are hotspots in the structures of ATM and BRCA2 proteins where pathogenic germline, and recurrent somatic variants from primary and metastatic tumours, cluster together in 3D. Moreover, in the ATM, BRCA1 and BRCA2 genes from prostate cancer patients, the distributions of germline benign, pathogenic, VUS, and recurrent somatic variants differ across Pfam domains. Together, these results provide a better characterisation of the most recurrent affected regions in DDRs and could help in the understanding of individual susceptibility to tumour development.

Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.

BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.

Abstract 539: Utility of circulating tumor DNA in esophageal cancer: A potential prognostic biomarker in tumor staging, monitoring of treatment response and detection of recurrent disease

Abstract Background: Circulating tumor DNA (ctDNA) has clinical utility in monitoring treatment response and in the detection of disease recurrence in breast and colorectal cancer1,2. The aim of this study was to explore the role of ctDNA in the management of patients with esophageal cancer (EC). Methods:Blood samples and tumor biopsies were collected from 79 patients after diagnosis of EC. In patients planned for surgery, blood samples were taken before and after neoadjuvant treatment, and during the surveillance period. Blood and biopsy tissue samples were analysed for mutations using a custom targeted amplicon-based approach to cover mutational foci across 9 of the most commonly mutated genes in EC. Results:Somatic mutations in treatment-naïve EC tumor biopsies were detected in 71 out of 79 (90%) patients. Out of these 71 cases, 23 (32%) had detectable tumor-informed ctDNA in their plasma. The majority (90%) of patients who were ctDNA positive had either locally advanced or metastatic disease. For node negative locally advanced patients treated with curative intent, positive ctDNA status at diagnosis is a poor prognostic marker (HR 11.71; 1.16 - 118.80; p=0.037). In blood samples taken before and following neoadjuvant therapy (NAT), reversal of a ctDNA positive status after NAT was associated with an excellent response to treatment. In patients who had serial plasma samples taken during surveillance, detection of ctDNA was associated with inferior disease specific survival (HR 4.36; 1.07 - 17.88; p = 0.04). Discussion:This study demonstrates that ctDNA may have clinical utility in the management of patients with EC by providing additional prognostic information at pre-treatment staging. In the absence of a widely accepted paradigm for surveillance after curative-intent treatment, assessment of ctDNA in post treatment blood samples may lead to the detection of early recurrent disease. 1. Dawson, S.-J. et al. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. New Engl J Medicine 368, 1199-1209 (2013).2. Tie, J. et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med 8, 346ra92 (2016). Citation Format: Carlos Suhady Cabalag, Michael Yates, Mariana B. Corrales, Paul Yeh, Stephen Q. Wong, Bonnie Zhang, Kenji M. Fujihara, Lynn Chong, Michael Hii, Sarah-Jane Dawson, Wayne A. Phillips, Cuong P. Duong, Nicholas J. Clemons. Utility of circulating tumor DNA in esophageal cancer: A potential prognostic biomarker in tumor staging, monitoring of treatment response and detection of recurrent disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 539.

Abstract 2057: BRCA1 D11q isoform expression impacts PARP inhibitor responses in high grade serous ovarian carcinoma patient derived xenografts

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in High Grade Serous Ovarian Carcinoma (HGSOC) commonly occurs via restored homologous recombination DNA repair due to secondary mutations in BRCA1/2. However, overexpression of certain BRCA1 splice isoforms can also contribute to PARPi resistance in HGSOC. This includes the D11q isoform of BRCA1, where deleterious mutations in the 11q region of BRCA1 (exon 10) can be spliced out resulting in a truncated but partially functional BRCA1 protein. Here we describe four HGSOC Patient Derived Xenografts (PDX) with primary BRCA1 11q mutations, where D11q isoform expression has been shown to correlate with platinum and PARPi responses in vivo. Methods: PDX were derived from chemo-naive and pre-treated patients (Table 1). PDX were treated with PARPi rucaparib (300mg/kg) and cisplatin (4mg/kg). D11q isoform expression was assessed using two-step RT-qPCR. DNA sequencing was performed using the BROCA Cancer Risk Panel. Results: PARPi resistant PDX #1032 and #1049 had high D11q expression relative to D11q-high cell line UWB1.289 and the PARPi-responsive models (Table 1). Interestingly, PDX #1049 harboured a second BRCA1 exon 11 donor splice site mutation, shown previously to enhance splicing and increase abundance of the D11q isoform. Indeed, other proximal splice site mutations have been found following relapse in patients with BRCA1 11q mutations post-PARPi. Conclusions: Our work in PDX provides additional functional evidence that BRCA1 D11q expression impacts on PARPi and platinum responses, and is a clinically relevant mechanism of PARPi resistance in a subset of patients with BRCA1 mutations. Whilst high D11q expression can be associated with secondary BRCA1 splice site mutations (PDX #1049), it can also occur in their absence (PDX #1032). Thus, BRCA1 D11q isoform expression should be considered a potential mechanism of PARP-resistance in patients with BRCA1 11q mutations. Table 1.Summary of results.PDX modelPrimary BRCA1 mutationBRCA1 D11q isoform expressionSecondary BRCA1 mutationsOther mutations (BROCA)PDX platinum responsePDX PARPi responsePatient treatments prior to PDX generation#1049Germline BRCA1: c.2475delCVery highBRCA1: c.4096+3A&amp;gt;C (33%) - splice site mutation known to cause increased D11q expressionTP53: c.428_429insCAStable diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#1032Somatic BRCA1: c.1251delTHighNone detectedTP53: c.97-1_97-6del; FANCA: c.3788_3790delResistant - progressive diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#56Germline BRCA1: c.894_895delTGModerateNone detectedNone detectedSensitive - complete responseMixed responsesNo prior treatment#206Germline BRCA1: c.3817C&amp;gt;TLowNone in PDX, one found in post-PARPi patient biopsy: BRCA1:c.3746_4081delTP53:c.451C&amp;gt;T; BLM rearrangementSensitive - complete responseResponsive - complete response1 line of platinum chemotherapy Citation Format: Ksenija Nesic, Cassandra J. Vandenberg, Olga Kondrashova, John J. Krais, Neil Johnson, Elizabeth M. Swisher, Matthew J. Wakefield, Clare L. Scott. BRCA1 D11q isoform expression impacts PARP inhibitor responses in high grade serous ovarian carcinoma patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2057.

Abstract 2110: Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer (OC). Platinum resistance is a common occurrence in HGSOC leading to tumor relapse and patient mortality. Multiple mechanisms contribute to platinum resistance in OC, including enrichment of ovarian cancer stem cells (OCSCs), which is observed in relapsed tumors. Platinum treatment activates the DNA damage response (DDR) and the DNA repair protein BRCA1 is an OC susceptibility factor. In our study, we demonstrate that platinum treatment induces a decrease in BRCA1 levels that leads to an increase in activity of ALDH, an OCSC marker. In a parallel pathway that is associated with G2/M arrest, platinum treatment also induces an increase in NAMPT expression and NAD+ levels, the cofactor required for ALDH activity. BRCA1 is repressed through platinum-induced promoter DNA hypermethylation. Importantly, combination of platinum with decitabine, a DNA methyltransferase (DNMT) inhibitor, prevents the platinum-induced increase in OCSCs. This study uniquely provides preclinical support for the use of DNMT inhibitors in the neoadjuvant setting in combination with platinum treatment for OC patients. Citation Format: Riddhi Sood, Sikai Xiao, Shruthi Sriramkumar, Christiane Hassel, Kenneth Nephew, Heather O'Hagan. Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2110.

Simultaneous Targeting of PARP1 and RAD52 Triggers Dual Synthetic Lethality in BRCA-Deficient Leukemia Cells

Cancer cells may be addicted to DNA repair, which is supported by more selective targeting of tumor cells by recently developed novel drugs/compounds against specific DNA repair mechanisms. The success of PARP1 inhibitor olaparib in BRCA1 and BRCA2-deficient breast and/or ovarian tumors has established a proof-of-concept for personalized cancer therapy utilizing synthetic lethality. Unfortunately, therapeutic effect is usually short-lived and tumor cells become unresponsive to PARP1 inhibitors. Recently using personalized medicine approach we discovered that cohorts of acute and chronic leukemias display deficiencies in BRCA1/2-RAD51 -mediated homologous recombination (HR) repair, but these leukemia cells could not be completely eradicated by PARP1 inhibitors (Nieborowska-Skorska et al., J.Clin.Invest., 2017). Therefore, more radical elimination of BRCA1/2-deficient leukemia cells is required to prevent time-dependent emergence of PARP1 inhibitor-resistant/refractory clones. Alternative mechanisms of DNA repair may emerge in tumor cells exhibiting genetic and/or epigenetic deficiencies in the BRCA protein network to protect cells from lethal effect of DNA double strand breaks (DSBs). It has been suggested that RAD52-RAD51 provides an alternative HR pathway in BRCA1/2-deficient cells. We detected that RAD52-RAD51 mediated HR repair of DSBs remained active in PARP1 inhibitor-treated BRCA1/2-deficient tumor cells. Therefore, we hypothesized that RAD52-RAD51 pathway may be a potential escape route from PARP1 inhibitor-mediated synthetic lethality in BRCA1/2-deficient cells and that targeting RAD52 should enhance synthetic lethal effect of PARP1 inhibitor. Here we show that simultaneous short-term targeting of PARP1 and RAD52 with dominant-negative mutants or small molecule inhibitors resulted in synergistic accumulation of DSBs and/or cell death of BRCA1/2-deficient solid tumor and leukemia cells, but not BRCA/1/2-proficient cells. Moreover, long-term simultaneous treatment with PARP1 inhibitor and RAD52 inhibitor completely eradicated BRCA1/2-deficient tumor cells in vitro. Combination of PARP1 inhibitor + RAD52 inhibitor was not toxic for normal cells and tissues. Moreover, Parp1-/-;Rad52-/- double-knockout mice are indistinguishable from their wild-type littermates. SImultaneous targeting of PARP1 and RAD52 also reduced leukemia burden in vivo. BCR-ABL1-induced BRCA1-deficient Parp1-/-;Rad52-/- leukemias displayed prolonged latency when compared to Parp1-/- or Rad52-/- counterparts. Finally, the combination of PARP1 inhibitor and RAD52 inhibitor exerted synergistic activity against BRCA1/1-deficient primary leukemia xenografts in immunodeficient mice. In conclusion, we postulate that addition of RAD52 inhibitor may dramatically improve therapeutic outcome of BRCA1/2-deficient leukemias treated with PARP1 inhibitor. DisclosuresValent:Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Whole-exome sequencing in eccrine porocarcinoma indicates promising therapeutic strategies

Malignant sweat gland tumours are rare, with the most common form being Eccrine porocarcinoma (EP). To investigate the mutational landscape of EP, we performed whole-exome sequencing (WES) on 14 formalin-fixed paraffin-embedded samples of matched primary EP and healthy surrounding tissue. Mutational profiling revealed a high overall median mutation rate. This was attributed to signatures of mutational processes related to ultraviolet (UV) exposure, APOBEC enzyme dysregulation, and defective homologous double-strand break repair. All of these processes cause genomic instability and are implicated in carcinogenesis. Recurrent driving somatic alterations were detected in the EP candidate drivers TP53, FAT2, CACNA1S, and KMT2D. The analyses also identified copy number alterations and recurrent gains and losses in several chromosomal regions including that containing BRCA2, as well as deleterious alterations in multiple HRR components. In accordance with this reduced or even a complete loss of BRCA2 protein expression was detected in 50% of the investigated EP tumours. Our results implicate crucial oncogenic driver pathways and suggest that defective homologous double-strand break repair and the p53 pathway are involved in EP aetiology. Targeting of the p53 axis and PARP inhibition, and/or immunotherapy may represent promising treatment strategies.