BRCA1 Deficiency Research Articles

BRCA2 Promotes Spontaneous Homologous Recombination In Vivo.

Simple SummaryInherited mutation of either BRCA1 or BRCA2 is associated with familial breast and ovarian cancer. As a tumor suppressor, BRCA2 functions to maintain genome stability but we do not know the genomic impact of this deficiency on normal tissues in normal contexts, rather than cancer cells that carry other gene mutations. Numerous murine models of BRCA2 have been developed to investigate BRCA2 tumor suppressor function but have been met with difficulties due to the lethality of most of these models during embryogenesis. To circumvent embryonic lethality here, we employ the cre conditional system combined with an assay for measuring changes in stability of a large tandem DNA repeat by homology-directed recombination. The relevance of this assay is that it can measure DNA recombination events in a normal growing/developing tissue. Here, we show that BRCA2 is required for homologous recombination in a normal developing tissue long before cancer arises.Background: BRCA2 is known to be a tumor suppressor involved in homologous recombination repair and presumed to prevent genome instability in normal tissues prior to the development of tumors. Typical assessment of BRCA2 deficiency on the genome involves cell-based models using cancer cells with mixed genetic contexts, but the role in normal tissue in vivo has not been clearly demonstrated. Methods: Using conditional deletion of Brca2 exon 11, the region containing all eight BRC repeats, in the retinal pigment epithelium and the pink-eyed unstable mouse model, we evaluate the frequency of DNA deletion events. Results: In the current study, we show that conditional loss of Brca2 exon 11 results in a decreased frequency of spontaneous homologous recombination compared to wild-type mice. Of note, we observe no apparent concomitant increase in events that indicate single-strand annealing by the pink-eyed unstable mouse model. Conclusions: Therefore, our results demonstrate that BRCA2, as expected, is required for high-fidelity homologous recombination DNA repair in normal tissues, here in a tissue undergoing normal proliferation through normal development.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Abstract LB159: Overcoming immunosuppression and therapeutic resistance to PARP inhibition in BRCA1-deficient breast cancer with STING agonists

Abstract PARP inhibitors (PARPi) have drastically changed the treatment landscape of advanced ovarian tumors with BRCA mutations. However, the impact of this class of inhibitor is relatively modest in patients with advanced BRCA-mutant breast cancer, as shown by the final reports on overall survival described by the OlympiAD and EMBRACA clinical trials. We have recently generated in parallel two syngeneic genetically engineered mouse (GEM) models of BRCA1/p53-deficient high-grade serous ovarian cancer (HGSOC) and invasive basal-like breast cancer. Notably, our pre-clinical results of PARPi in these two models are in line with clinical outcomes showing different responses to PARPi between breast and ovarian tumors, which cannot be explained by synthetic lethality alone. Here, we uncovered a major mechanism by which BRCA1-deficient breast tumors typically progress through treatment with PARPi as monotherapy or in combination with PD-1 blockade, in contrast to the much higher rates of success observed for BRCA1-deficient ovarian cancer. Using our syngeneic GEM model of breast cancer driven by Brca1 deficiency, we show that tumor-associated macrophages (TAMs) abrogate PARPi efficacy both in vivo and in vitro. Mechanistically, BRCA1-deficient breast tumor cells induce protumor polarization of TAMs, which in turn suppress PARPi-elicited DNA damage in tumor cells, leading to reduced production of cytosolic double-stranded DNA (dsDNA) and synthetic lethality, hence impairing STING-dependent antitumor immunity. STING agonists reprogram M2-like TAMs into an M1-like anti-tumor state in a macrophage STING-dependent fashion. Systemic administration of STING agonists breaches multiple layers of tumor cell-mediated suppression on macrophages and dendritic cells, and synergizes with PARPi. Notably, while STING-deficient/BRCA1-deficient breast tumors fail to respond to the combination of PARPi and STING agonists administered intratumorally, they respond effectively to systemic delivery of STING agonists in combination with PARPi. Our data is thus different from the majority of other studies suggesting that STING agonists act in the tumor cells but is consistent with a rising tide of work pointing to the importance of STING activation in macrophages and DCs. This finding also suggests a novel therapeutic approach for treating a significant fraction of patients with cancers deficient in tumor cell-intrinsic STING. Together, our study provides an important conceptual shift in understanding the resistance to PARP inhibitors, which has mostly been described in terms of restoration of homologous recombination. Our mechanism-based, novel therapeutic approach to circumvent the refractory nature of BRCA1-deficient breast cancers should prove instrumental in guiding the design of clinical trials evaluating PARPi-based rational combination therapy in the near future. Citation Format: Qiwei Wang, Johann S. Bergholz, Liya Ding, Ziying Lin, Shaozhen Xie, Tao Jiang, Hye-Jung Kim, Thomas M. Roberts, Jean J. Zhao. Overcoming immunosuppression and therapeutic resistance to PARP inhibition in BRCA1-deficient breast cancer with STING agonists [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 LB159.

Abstract 2049: Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition

Abstract DNA double-strand breaks (DSBs) are life-threatening lesions that must be repaired to preserve chromosomal integrity. These lesions are repaired by two primary pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The factors that mediate pathway choice are critical to maintain genomic integrity. While many of these factors, epigenetic changes and cell cycle stages are known, with HR predominating in S and G2, the underlying mechanisms that determine pathway choice are not well understood. Previously, we found that the transcription factor Sp1 is rapidly recruited to DSBs and is necessary for repair. Here, we demonstrate that Sp1 integrates repair pathway choice with cell cycle by promoting NHEJ in G1. Sp1 localizes to DSBs in G1 to recruit the NHEJ repair factors 53BP1 and Ku70. Sp1 is phosphorylated by Cyclin A/cdk2 upon entry into S phase, evicting both Sp1 and 53BP1 from the break site, thereby permitting BRCA1 binding and HR. Mutation of this phosphorylation site (Sp1-S59A) results in persistence of Sp1 and 53BP1 at DSBs in S phase cells, precluding BRCA1 binding and HR. We also demonstrate that Sp1 is sumoylated in S phase in response to damage. This sumoylation in recognized by the E3-ubiquitin ligase, RNF4, and therefore polyubiquitylates Sp1 and targets it for proteasomal degradation. Expression of a sumoylation deficient Sp1 (Sp1-K16R) results in Sp1 and 53BP1 occupancy at DSBs in S phase, inhibiting BRCA1 binding to DSBs and HR. HR proteins are commonly mutated in cancers, resulting in cellular “addiction” to alternative repair pathways. Exploiting this addiction is the basis of synthetic lethality and is applicable to the treatment of many types of malignancies. Similar to BRCA1 deficiency, expression of Sp1-S59A or Sp1-K16R also increases cell sensitivity to PARP inhibition in BRCA1+/+ cells because of the inability to recruit BRCA1. Alternatively, synthetic lethality in BRCA1-/- cells requires 53BP1, whereas loss of 53BP1 confers PARPi resistance in a BRCA1 deficient cell. Consistent with this, depletion of Sp1 also results in PARPi resistance in BRCA1-/- cells. Interestingly, Sp1 is overexpressed in many tumors and is associated with poor prognosis. These data corroborate our finding that Sp1 is required for NHEJ and provide preliminary evidence that Sp1 may be a biomarker for PARPi sensitivity. Citation Format: Michelle Swift, Jane Azizkhan-Clifford. Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition [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 2049.

Synthetic Lethal Interaction between Apto-253 and Impaired BRCA1/2 Function

APTO-253 is a novel indolyl-phenanthroline-imidazole small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies including leukemia, lymphoma, and colon, non-small cell lung, renal and prostate carcinomas. Of particular significance, APTO-253 did not elicit myelosuppression in its initial Phase 1 trial in patients with solid tumors even at the maximum tolerated dose. The goal of this project was to provide fundamental information on the mechanism of action and resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination drug studies.The human Burkitt'slymphoma cell line Rajiwas exposed to progressively increasing concentrations of APTO-253 over a period of 6 months to generate a resistant subline Raji/253R with 16.7 ± 3.9-fold resistance (IC50: Raji IC50 91.9 ± 22.3 nM; Raji /253R IC50 1387.7 ± 98.5 nM). RNA-seq analysis of the parental and resistant cells demonstrated marked up-regulation of ATP-binding cassette sub-family G member 2 (ABCG2) in the Raji/253R cells that was confirmed by qRT-PCR and Western blot analysis. Ko143 is a specific inhibitor of ABCG2 that was not toxic to either Raji or Raji/253R when used as a single agent. When Raji/253R cells were concurrently treated with either 5 nM or 50 nM Ko143 and APTO-253, resistance to APTO-253 was reversed by 1.6- and 6.8-fold, respectively, confirming that overexpression of ABCG2 is one mechanism of resistance to APTO-253. Raji/253R was found to be cross-resistant to topotecan, a known ABCG2 substrate, and Ko143 also reversed topotecan resistance in Raji/253R. Intriguingly, Raji/253R was also significantly cross-resistant to carboplatin even though carboplatin is not thought to be an ABCG2 substrate; treatment with Ko143 did not reduce the carboplatin IC50 in the Raji/253R cells. The curious finding that Raji/243R cells were cross-resistant to carboplatin led to the discovery that APTO-253 causes DNA damage as documented by the neutral comet assay and gH2AX phosphorylation and foci formation assays. Surprisingly, Raji/253R cells were hypersensitive to etoposide, an ABCG2 substrate and a potent DNA double strand break inducer. GO and pathway analysis from the RNA-seq data revealed that DNA repair pathways were downregulated in Raji/253R, which partially explained the hypersensitivity to etoposide. We then asked whether cells deficient in homologous recombination repair pathways were hypersensitive to APTO-253 and if there was synthetic lethality between APTO-253 and BRCA1 deficiency (using isogenic pairs of BRCA1-proficient and -deficient human cell lines). BRCA1 deficiency was documented by treating the BRCA1-proficient and -deficient isogenic cell lines with the PARP inhibitor olaparib. BRCA1-deficient sublines of MCF10A, MCF7 and hTERT -IMEC were all hypersensitive to both olaparib and APTO-253. We also found that the BRCA2deficient ovarian cancer cell line PEO4 and two colorectal HCT116 BRCA2 -/- subclones were hypersensitive to both olaparib and APTO-253.We conclude that AP-253 causes DNA damage and that it exhibits a degree of synthetic lethality comparable to olaparib in cells deficient in BRCA1 and BRCA2-function. The ability of APTO-253 to exploit homologous recombination deficiency is of particular interest because, unlike all the other drugs for which loss of this repair function results in hypersensitivity, APTO-253 does not produce myelosuppression even at the maximum tolerated dose. The observations reported here also identify gH2AX as a potential biomarker of clinical drug effect and open the window more detailed studies of how APTO-253 causes DNA damage. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees.

A randomized phase II trial of oral vinorelbine as second-line therapy for patients with malignant pleural mesothelioma.

8507 Background: All patients with malignant pleural mesothelioma (MPM) eventually relapse following standard chemotherapy. However, there is no standard treatment option in this setting. Vinorelbine, exhibits useful clinical activity but has not been formally evaluated in a randomised clinical trial, despite its widespread off-label use worldwide. BRCA1 regulates spindle assembly checkpoint in MPM and predicts vinorelbine sensitivity in preclinical models [1,2], suggesting that BRCA1 negative patients may be chemoresistant. Methods: VIM, a Cancer Research UK funded, investigator-initiated randomised controlled phase 2 multi-centre UK trial, enrolled patients with MPM who had progressed after first-line chemotherapy. Pts were randomised 2:1 to either vinorelbine (60mg/m2 weekly Q21d escalating to 80mg/m2 from cycle 2) + active supportive care (ASC) versus ASC until disease progression, unacceptable toxicity or withdrawal of consent. The primary outcome was progression free survival (PFS) defined as the time from randomisation to any progression (based on Modified RECIST criteria for assessment of response in malignant pleural mesothelioma) or death. The trial had 90% power to detect a hazard ratio of 0.65 at the one-sided 20% significance level. Secondary endpoints were overall survival (OS), tolerability and safety. Results: Between May 2016 and Oct 2018, 154 patients were recruited from 10 UK sites and randomised to vinorelbine + ASC (n=98) or ASC alone (n=56). In the Intention-to-treat analysis, after 129 events, median PFS was 4.2 months (m) for vinorelbine + ASC compared to 2.8m for ASC alone (Hazard Ratio (HR) 0.59; 95% CI: 0.41 to 0.85; one-sided p = 0.0017). 108 deaths were reported. Median OS was 9.3m for vinorelbine + ASC compared to 9.1m for ASC alone (HR=0.79; 95% CI: 0.53 to 1.17; two-sided p = 0.24). Toxicity data and subgroup analyses including the impact of BRCA1 deficiency will be presented. Conclusion: The trial met its primary endpoint. Vinorelbine demonstrates useful clinical efficacy in relapsed MPM, supporting its off-label use, as a treatment option for patients with relapsed MPM.[1] Busacca et al, J Pathol 2012, 227(2), 200. [2] Busacca et al, Mol Cancer Res, 2021, 20(2) 379. Clinical trial information: NCT02139904.

MET Expression and Cancer Stem Cell Networks Impact Outcome in High-Grade Serous Ovarian Cancer.

Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several cancer types, and MET has been suggested to interact with stem cell networks. In vitro studies have further suggested a possible benefit of a combined treatment using PARP and MET inhibitors. We used a tissue microarray (TMA) with 130 samples of advanced-stage high-grade serous fallopian tube/ovarian cancer (HGSC) to investigate the prognostic value of MET protein expression alone and in combination with the stem cell factor SOX2. The possible synergistic effects of a PARP and MET inhibitor treatment were evaluated in two cell lines with BRCA1 or BRCA2 deficiency and in their BRCA1/2-proficient counterparts. Patients with tumors positive for MET had worse overall survival (log-rank test, p = 0.015) compared to patients with MET-negative tumors. The prognostic role of MET was even more prominent in the subgroup of patients with SOX2-negative tumors (p = 0.0081). No synergistic effects of the combined treatment with PARP and MET inhibitors were found in the cell lines examined. We conclude that MET expression could be used as a marker for OS in HGSC and that stemness should be taken into consideration when evaluating the mechanisms of this effect.

The emerging determinants of replication fork stability.

A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.

Replication Gaps Underlie BRCA Deficiency and Therapy Response.

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.

DSB repair pathway choice is regulated by recruitment of 53BP1 through cell cycle-dependent regulation of Sp1

Although many of the factors, epigenetic changes, and cell cycle stages that distinguish repair of double-strand breaks (DSBs) by homologous recombination (HR) from non-homologous end joining (NHEJ) are known, the underlying mechanisms that determine pathway choice are incompletely understood. Previously, we found that the transcription factor Sp1 is recruited to DSBs and is necessary for repair. Here, we demonstrate that Sp1 localizes to DSBs in G1 and is necessary for recruitment of the NHEJ repair factor, 53BP1. Phosphorylation of Sp1-S59 in early S phase evicts Sp1 and 53BP1 from the break site; inhibition of that phosphorylation results in 53BP1 and Sp1 remaining at DSBs in S phase cells, precluding BRCA1 binding and suppressing HR. Expression of Sp1-S59A increases sensitivity of BRCA1+/+ cells to poly (ADP-ribose) polymerase (PARP) inhibition similar to BRCA1 deficiency. These data demonstrate how Sp1 integrates the cell cycle and DSB repair pathway choice to favor NHEJ.

BECN1 and BRCA1 Deficiency Sensitizes Ovarian Cancer to Platinum Therapy and Confers Better Prognosis.

Background: BRCA1, BECN1 and TP53 are three tumor suppressor genes located on chromosome 17 and frequently found deleted, silenced, or mutated in many cancers. These genes are involved in autophagy, apoptosis, and drug resistance in ovarian cancer. Haploinsufficiency or loss-of-function of either TP53, BRCA1 or BECN1 correlates with enhanced predisposition to cancer development and progression, and chemoresistance. Expectedly, the combined altered expression of these three tumor suppressor genes worsens the prognosis of ovarian cancer patients. However, whether such a genotypic pattern indeed affects the chemo-responsiveness to standard chemotherapy thus worsening patients’ survival has not been validated in a large cohort of ovarian cancer patients. Aim: We interrogated datasets from the TCGA database to analyze how the expression of these three tumor suppressor genes impacts on the clinical response to platinum-based chemotherapy thus affecting the survival of ovarian cancer patients. Results and conclusion: Compared to EOC with homozygous expression of BECN1 and BRCA1, tumors expressing low mRNA expression of these two tumor suppressor genes (either because of shallow (monoallelic) co-deletion or of promoter hypermethylation), showed higher sensitivity to platinum-based therapies and were associated with a better prognosis of ovarian cancer-bearing patients. This outcome was independent of TP53 status, though it was statistically more significant in the cohort of patients with mutated TP53. Thus, sensitivity to platinum therapy (and probably to other chemotherapeutics) correlates with low expression of a combination of critical tumor suppressor genes. Our study highlights the importance of thoroughly assessing the genetic lesions of the most frequently mutated genes to stratify the patients in view of a personalized therapy. More importantly, the present findings suggest that targeting the function of both BECN1 and BRCA1 could be a strategy to restore chemosensitivity in refractory tumors.

Abstract SP151: BRCA and homologous recombination deficiency mediated mutagenesis in breast cancer

Abstract The genes BRCA1, BRCA2 and PALB2 genes are the major high penetrance risk alleles in breast cancer have function in a DNA repair mechanism called homologous recombination (HR). These and a wider group of other genes including the RAD51 paralogues and kinase regulators of HR can also be subject to somatic mutation or epigenetic modification in cancer and, usually in the setting of bi-allelic inactivation, effect the use of HR. The dysregulation of BRCA1 by methylation of its regulatory region is frequent occurring, in approximately 30% of sporadic basal-like breast cancers. While there is evidence that BRCA1 silencing causes functional HR deficiency, there is also evidence suggesting this may be more “plastic” and subject to change to maintain cell fitness and DNA replication capacity under the evolutionary pressures that cancer cells are subjected to during progression and therapyexposure.HR deficient cancers change to use of other DNA break and DNA replication fork repair mechanisms that are mutagenic. A number of methods based on tumor genome sequencing have been developed to assesssomatic mutational signatures. These methods have revealed pathognomonic “scars” or “tattoos” indicating exposure to HR deficiency during breast cancer evolution and the variable performance of mutational signatures in the contexts of genetic and epigenetic loss of HR function. HR deficiency induced mutagenesis can also inactivate genes that negatively regulate HR, or re-activate (reversion mutation) the previously inactivated HR gene, driving a reversal of HR deficiency. As this changes a cell’s DNA repair phenotype and its ability to handle to therapeutic damage, these are increasingly found to be relevant to the development of resistance to DNA interacting drugs such as platinums and PARP inhibitors as well as to emerging DNA damage response (DDR) targeting therapeutics. In the setting of resistance both the residual HR deficiency induced genome landscape or the altered use of the DNA damage response in breast cancer cells may offer new vulnerabilities, enhanced in tumor cells, that are subject to targeted DDR directed or immunotherapy therapy and inform combination or sequential therapy approaches and associated patient selection biomarkers now being assessed in the clinic. Citation Format: A Tutt. BRCA and homologous recombination deficiency mediated mutagenesis in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP151.

Abstract PS19-19: Whole exome sequencing and BRCAness estimation in TNBCs and their correlation with response to platinum

Abstract Background: Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. The hallmark of TNBC is genomic instability with very high rates of TP53 mutation. BRCAness has been defined by Ashworth et al (2004 &2014), as the phenocopying of BRCA1&2 mutation by alternate genetic or Epigenetic mechanisms. The consequence of this is DNA damage repair (DDR) deficiency. Though PARP inhibitors have been of utility in treating this subset of TNBC, trials have in general not found an unequivocal support for the use of platinum. We have developed an assay for BRCAness, the BRCA1 deficiency score (BDS) (Korlimarla et al 2016). In this study, we have applied BDS assay in tandem with whole exome sequencing to a small retrospective series of TNBC patients more than half of whom were treated with Platinum. Methods: 40 TNBC primary specimens along with complete clinical data from a retrospective series at the SSCHRC were obtained under all IERB approvals and informed patient consent for BDS assay and mutational analysis. BDS assay is a multianalyte assay involving measurement of BRCA1 (Transcript and protein) as well as additional epigenetic regulators of BRCA1, mir182 and ID4. Whole Exome Sequenced (WES) using the Agilent Sureselect V6 kit on Illumina HiSeq platform. Variant calling analysis was performed using GATK Mutect2 with Hg38 reference genome following the best practice workflow. Variants annotated as protein affecting based on their functional impact prediction and mapped to known cancer related genes (from COSMIC), were selected. Results: Clinical details: Mean age of patient in the series was 49. 37/40 qualified for BDS assay. 21/37 (57%) patients were treated with Carboplatin in combination with Docetaxel in an adjuvant setting. Remaining patients were treated with Cyclophosphamide Adriamycin with or without Docetaxel. 15/37 (45%) were BRCA1 deficient and10/15 were treated with Platinum and 8 (80%) were responders. 39 passed QC and considered for analysis. Spectrum of variants were missense mutations (86.5%), followed by stop gained (5.91%) and frame shift (3.76%). Results were compared to TCGA TNBC set (n=123). The most frequently mutated gene was TP53 (62%) as reported in TCGA. We also report higher frequency of deleterious mutations on DNA damage repair (DDR) genes like ATM and BRCA2 (15%). Response to Platinum therapy in this subset, correlated with Mutations in DNA damage repair genes (p ≤ 0.003). 4/6 samples mutated were also In the BDS deficient group. BRCAness score for predicting Platinum response also correlated significantly (p ≤ 0.05). Other genes which showed significant alterations were KMT2A and KMT2D (15% and 13%) which encode the histone methyl transferase and are responsible in altering the chromatin structure and RECQL4 (10%) which is a helicase involved in DDR Conclusion: Both BRCAness and mutation profile identified a DDR deficient group within TNBC patients. In addition to TP53 which is the most frequently mutated gene, our small sample set has shown higher frequencies of mutations in DDR genes like ATM and BRCA2. This group of patients showed favourable response to platinum therapy. Since very little is known about the molecular heterogeneity of TNBCs in Indian patients, our analysis aids in identification of actionable mutations in TNBCs and may be of use in selection of patients for platinum therapy. CHARACTERSn%Platinum Treatment - ( n )Treatment response%BDS3721Deficient154513BRCA1 deficient response861Proficient19518BRCA1 Proficient response450Undet34 Citation Format: Aruna Korlimarla, Sabarinathan Radhakrishnan, Snijesh VP, Jyothi S Prabhu, Naveen Luke Demonte, Savitha Rajarajan, Yatish Patil, Ravi Diwakar, Sandhya Apachu, BS Srinath, Sridhar TS, Anguraj Sadanandam. Whole exome sequencing and BRCAness estimation in TNBCs and their correlation with response to platinum [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-19.

PDGFR\u03b2 is an essential therapeutic target for BRCA1-deficient mammary tumors

BackgroundBasal-like breast cancers (BLBCs) are a leading cause of cancer death due to their capacity to metastasize and lack of effective therapies. More than half of BLBCs have a dysfunctional BRCA1. Although most BRCA1-deficient cancers respond to DNA-damaging agents, resistance and tumor recurrence remain a challenge to survival outcomes for BLBC patients. Additional therapies targeting the pathways aberrantly activated by BRCA1 deficiency are urgently needed.MethodsMost BRCA1-deficient BLBCs carry a dysfunctional INK4-RB pathway. Thus, we created genetically engineered mice with Brca1 loss and deletion of p16INK4A, or separately p18INK4C, to model the deficient INK4-RB signaling in human BLBC. By using these mutant mice and human BRCA1-deficient and proficient breast cancer tissues and cells, we tested if there exists a druggable target in BRCA1-deficient breast cancers.ResultsHeterozygous germline or epithelium-specific deletion of Brca1 in p18INK4C- or p16INK4A-deficient mice activated Pdgfrβ signaling, induced epithelial-to-mesenchymal transition, and led to BLBCs. Confirming this role, targeted deletion of Pdgfrβ in Brca1-deficient tumor cells promoted cell death, induced mesenchymal-to-epithelial transition, and suppressed tumorigenesis. Importantly, we also found that pharmaceutical inhibition of Pdgfrβ and its downstream target Pkcα suppressed Brca1-deficient tumor initiation and progression and effectively killed BRCA1-deficient cancer cells.ConclusionsOur work offers the first genetic and biochemical evidence that PDGFRβ-PKCα signaling is repressed by BRCA1, which establishes PDGFRβ-PKCα signaling as a therapeutic target for BRCA1-deficient breast cancers.

PARP inhibitors in ovarian cancer: An overview of the practice-changing trials.

Poly (ADP-ribose) polymerase (PARP) inhibitors have transformed the management of recurrent ovarian cancer in patients with BRCA-mutations and beyond. Olaparib was the first PARP inhibitor to gain approval as maintenance therapy for patients with newly diagnosed, advanced BRCA-mutated ovarian cancer establishing a new standard of care. At the end of 2020, as a result of the SOLO1, PRIMA, and PAOLA-1 phase III trials, we are now in an era where three maintenance PARP inhibitor strategies have FDA and European Medicines Agency approval in the first-line setting. In this review, we provide an overview of the key PARP inhibitor trials that have changed clinical practice, discuss directing therapy according to biomarker status (BRCA and homologous recombination deficiency) and future strategies in ovarian cancer and other gynecological malignancies.

Dissecting the heterogeneity and tumorigenesis of BRCA1 deficient mammary tumors via single cell RNA sequencing.

Background: BRCA1 plays critical roles in mammary gland development and mammary tumorigenesis. And loss of BRCA1 induces mammary tumors in a stochastic manner. These tumors present great heterogeneity at both intertumor and intratumor levels.Methods: To comprehensively elucidate the heterogeneity of BRCA1 deficient mammary tumors and the underlying mechanisms for tumor initiation and progression, we conducted bulk and single cell RNA sequencing (scRNA-seq) on both mammary gland cells and mammary tumor cells isolated from Brca1 knockout mice.Results: We found the BRCA1 deficient tumors could be classified into four subtypes with distinct molecular features and different sensitivities to anti-cancer drugs at the intertumor level. Whereas within the tumors, heterogeneous subgroups were classified mainly due to the different activities of cell proliferation, DNA damage response/repair and epithelial-to-mesenchymal transition (EMT). Besides, we reconstructed the BRCA1 related mammary tumorigenesis to uncover the transcriptomes alterations during this process via pseudo-temporal analysis of the scRNA-seq data. Furthermore, from candidate markers for BRCA1 mutant tumors, we discovered and validated one oncogene Mrc2, whose loss could reduce mammary tumor growth in vitro and in vivo.Conclusion: Our study provides a useful resource for better understanding of mammary tumorigenesis induced by BRCA1 deficiency.

GATA3 functions downstream of BRCA1 to suppress EMT in breast cancer.

Purpose: Functional loss of BRCA1 is associated with poorly differentiated and metastatic breast cancers that are enriched with cancer stem cells (CSCs). CSCs can be generated from carcinoma cells through an epithelial-mesenchymal transition (EMT) program. We and others have previously demonstrated that BRCA1 suppresses EMT and regulates the expression of multiple EMT-related transcription factors. However, the downstream mediators of BRCA1 function in EMT suppression remain elusive.Methods: Depletion of BRCA1 or GATA3 activates p18INK4C, a cell cycle inhibitor which inhibits mammary epithelial cell proliferation. We have therefore created genetically engineered mice with Brca1 or Gata3 loss in addition to deletion of p18INK4C, to rescue proliferative defects caused by deficiency of Brca1 or Gata3. By using these mutant mice along with human BRCA1 deficient as well as proficient breast cancer tissues and cells, we investigated and compared the role of Brca1 and Gata3 loss in the activation of EMT in breast cancers.Results: We discovered that BRCA1 and GATA3 expressions were positively correlated in human breast cancer. Depletion of BRCA1 stimulated methylation of GATA3 promoter thereby repressing GATA3 transcription. We developed Brca1 and Gata3 deficient mouse system. We found that Gata3 deficiency in mice induced poorly-differentiated mammary tumors with the activation of EMT and promoted tumor initiating and metastatic potential. Gata3 deficient mammary tumors phenocopied Brca1 deficient tumors in the induction of EMT under the same genetic background. Reconstitution of Gata3 in Brca1-deficient tumor cells activated mesenchymal-epithelial transition, suppressing tumor initiation and metastasis.Conclusions: Our finding, for the first time, demonstrates that GATA3 functions downstream of BRCA1 to suppress EMT in controlling mammary tumorigenesis and metastasis.

The Oncogenic Helicase ALC1 Regulates PARP Inhibitor Potency by Trapping PARP2 at DNA Breaks

The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.

Loss of Nuclear DNA Ligase III Reverts PARP Inhibitor Resistance in BRCA1-Deficient Cells by Increasing DNA Replication Stress

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1, identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 is required for PARPi-induced fork acceleration in BRCA1-deficient cells and that LIG3 loss increases fork asymmetry. Furthermore, LIG3 depletion in BRCA1-deficient cells results in an increase in ssDNA gaps behind the replication forks, resulting in accumulation of chromosomal abnormalities. We also report that high expression of LIG3 in patients with invasive breast cancer correlates in with poorer overall survival, rendering LIG3 as a potential therapeutic target for enhancing PARPi sensitivity.

Brca2 deficiency drives gastrointestinal tumor formation and is selectively inhibited by mitomycin C

BRCA2 is crucial for repairing DNA double-strand breaks with high fidelity, and loss of BRCA2 increases the risks of developing breast and ovarian cancers. Herein, we show that BRCA2 is inactively mutated in 10% of gastric and 7% of colorectal adenocarcinomas, and that this inactivation is significantly correlated with microsatellite instability. Villin-driven Brca2 depletion promotes mouse gastrointestinal tumor formation when genome instability is increased. Whole-genome screening data showed that these BRCA2 monoallelic and biallelic mutant tumors were selectively inhibited by mitomycin C. Mechanistically, mitomycin C provoked double-strand breaks in cancer cells that often recruit wild-type BRCA2 for repair; the failure to repair double-strand breaks caused cell-cycle arrest at the S phase and p53-mediated cell apoptosis of BRCA2 monoallelic and biallelic mutant tumor cells. Our study unveils the role of BRCA2 loss in the development of gastrointestinal tumors and provides a potential therapeutic strategy to eliminate BRCA2 monoallelic and biallelic mutant tumors through mitomycin C.