BRCA1 Deficiency Research Articles

Exploring Molecular Drivers of PARPi Resistance in BRCA1-Deficient Ovarian Cancer: The Role of LY6E and Immunomodulation.

Approximately 50% of patients diagnosed with ovarian cancer harbor tumors with mutations in BRCA1, BRCA2, or other genes involved in homologous recombination repair (HR). The presence of homologous recombination deficiency (HRD) is an approved biomarker for poly-ADP-ribose polymerase inhibitors (PARPis) as a maintenance treatment following a positive response to initial platinum-based chemotherapy. Despite this treatment option, the development of resistance to PARPis is common among recurrent disease patients, leading to a poor prognosis. In this study, we conducted a comprehensive analysis using publicly available datasets to elucidate the molecular mechanisms driving PARPi resistance in BRCA1-deficient ovarian cancer. Our findings reveal a central role for the interferon (IFN) pathway in mediating resistance in the context of BRCA1 deficiency. Through integrative bioinformatics approaches, we identified LY6E, an interferon-stimulated gene, as a key mediator of PARPi resistance, with its expression linked to an immunosuppressive tumor microenvironment (TME) encouraging tumor progression and invasion. LY6E amplification correlates with poor prognosis and increased expression of immune-related gene signatures, which is predictive of immunotherapy response. Interestingly, LY6E expression upon PARPi treatment resistance was found to be dependent on BRCA1 status. Gene expression analysis in the Orien/cBioPortal database revealed an association between LY6E and genes involved in DNA repair, such as Rad21 and PUF60, emphasizing the interplay between DNA repair pathways and immune modulation. Moreover, PUF60, Rad21, and LY6E are located on chromosome 8q24, a locus often amplified and associated with the progression of ovarian cancer. Overall, our study provides novel insights into the molecular determinants of PARPi resistance and highlights LY6E as a promising prognostic biomarker in the management of HRD ovarian cancer. Future studies are needed to fully elucidate the molecular mechanisms underlying the role of LY6E in PARPi resistance.

Synthesis and In Silico Evaluation of Piperazine-Substituted 2,3-Dichloro-5,8-dihydroxy-1,4-naphthoquinone Derivatives as Potential PARP-1 Inhibitors.

PARP-1 (poly(ADP-ribose)-polymerase 1) inhibitors are vital in synthetic lethality, primarily due to their specificity for PARP-1 over PARP-2 (PARP-1 > PARP-2). This specificity is crucial as it allows precise inhibition of PARP-1 in tumor cells with Breast Cancer 1 protein (BRCA1) or BRCA2 deficiencies. The development of highly specific PARP-1 inhibitors not only meets the therapeutic needs of tumor treatment but also has the potential to minimize the adverse effects associated with nonselective PARP-2 inhibition. In this study, a series of novel 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDNO) derivatives were synthesized, characterized, and evaluated regarding their PARP-1 inhibitory and cytotoxic activity. Compound 3 exhibited the highest cytotoxic potential against all cell lines, except for MDA-MB-231 cells. The inhibitory potential of these molecules against PARP-1 was evaluated through in silico molecular docking and molecular dynamics studies. Notably, compounds 5, 9, and 13 exhibited significant inhibitory activity in silico results, interacting with critical amino acids known to be important for PARP-1 inhibition during simulations. These compounds exhibited target-specific and strong binding profiles, with docking scores of -7.17, -7.41, and -7.37 kcal/mol, respectively, and MM/GBSA scores of -52.51, -43.77, and -62.87 kcal/mol, respectively. These novel compounds (DDNO derivatives) hold promise as potential PARP-1 inhibitors for the development of targeted therapeutics against cancer.

Real-World First-Line Maintenance Niraparib Monotherapy Use Following Chemotherapy Plus Bevacizumab: The SW1TCH Study.

Clinical trials have demonstrated prolonged survival associated with niraparib first-line maintenance (1LM) therapy, compared with placebo, for patients with ovarian cancer (OC). However, data are limited on real-world 1LM niraparib monotherapy use, particularly as switch 1LM, following first-line (1L) combination chemotherapy plus bevacizumab. This real-world study aimed to describe patient demographics, clinical characteristics, and clinical outcomes of patients with OC receiving 1LM niraparib monotherapy following 1L combination chemotherapy plus bevacizumab. This retrospective observational study used data from a US-based nationwide database of deidentified, electronic health record-derived data. Patients diagnosed with OC during the study period (1 January 2011-30 November 2022, inclusive) were eligible if they received 1L chemotherapy plus bevacizumab treatment followed by 1LM niraparib monotherapy, initiated between 1 January 2017 (inclusive) and 2 September 2022. Patients were followed from index date (initiation of niraparib 1LM) until the first occurrence of death, end of follow-up, or end of study. Clinical outcomes were time to treatment discontinuation (TTD) and time to next treatment (TTNT). Kaplan-Meier curves were used to estimate TTD, TTNT, and 95% confidence intervals (CIs). Among 93 patients selected, median age at index was 67years (interquartile range [IQR] 60-72 years). Most patients had BRCA wild-type/homologous recombination (HR)-proficient or BRCA wild-type/HR unknown disease (75.3%). In all, 18 (19.4%) patients had HR-deficient disease. Five (5.4%) patients had unknown test results for both BRCA and HR deficiency status. Median follow-up time was 16.3months (IQR 8.7-25.4months), and median time from end of 1L therapy to 1LM initiation was 35.0days (IQR 25.0-53.9days). Median TTD was 9.3months (95% CI 6.1-11.3months). Median TTNT was 12.9months (95% CI 11.5-19.0months). This real-world study provided insights into switch maintenance with 1LM niraparib monotherapy, which may be a viable treatment option for patients with advanced OC.

LBA64 Outcomes of BRCA and in-house homologous recombination deficiency (HRD) testing in women with ovarian and breast cancer: A multicentre registry study

LBA64 Outcomes of BRCA and in-house homologous recombination deficiency (HRD) testing in women with ovarian and breast cancer: A multicentre registry study

DNA polymerase α-primase facilitates PARP inhibitor-induced fork acceleration and protects BRCA1-deficient cells against ssDNA gaps

PARP inhibitors (PARPi), known for their ability to induce replication gaps and accelerate replication forks, have become potent agents in anticancer therapy. However, the molecular mechanism underlying PARPi-induced fork acceleration has remained elusive. Here, we show that the first PARPi-induced effect on DNA replication is an increased replication fork rate, followed by a secondary reduction in origin activity. Through the systematic knockdown of human DNA polymerases, we identify POLA1 as mediator of PARPi-induced fork acceleration. This acceleration depends on both DNA polymerase α and primase activities. Additionally, the depletion of POLA1 increases the accumulation of replication gaps induced by PARP inhibition, sensitizing cells to PARPi. BRCA1-depleted cells are especially susceptible to the formation of replication gaps under POLA1 inhibition. Accordingly, BRCA1 deficiency sensitizes cells to POLA1 inhibition. Thus, our findings establish the POLA complex as important player in PARPi-induced fork acceleration and provide evidence that lagging strand synthesis represents a targetable vulnerability in BRCA1-deficient cells.

Medulloblastomas Initiated by Homologous Recombination Defects in Mice

Germline mutations of homologous-recombination (HR) genes are among the top contributors to medulloblastomas. A significant portion of human medulloblastomas exhibited genomic signatures of HR defects. We asked whether ablation of Brca2, Palb2, and their related Brca1 and Bccip genes, in the mouse brain can differentially initiate medulloblastomas. We established conditional knockout mouse models of these HR-genes, and a conditional knockdown of Bccip (shBccip-KD). Deletion of any of these genes led to microcephaly and neurological defects, with Brca1- and Bccip- producing the worst. Trp53 co-deletion significantly rescued the microcephaly with Brca1, Palb2, and Brca2 deficiency, but it exhibited limited impact on Bccip- mice. For the first time, inactivation of either Brca1 or Palb2 with Trp53 was found to induce medulloblastomas. Bccip/Trp53 deletions failed, despite that shBccip-CKD was highly penetrative, to induce medulloblastomas. The tumors displayed diverse immunohistochemical features and chromosome copy number variation. While there were widespread upregulations of cell proliferative pathways, most of the tumors expressed biomarkers of the Sonic Hedgehog subgroup. The MBs developed from Brca1-, Palb2-, and Brca2- mice were highly sensitive to a PARP inhibitor, but not the ones from shBccip-CKD mice. Our models recapitulate the spontaneous medulloblastoma development with high penetrance and narrow time-window, providing ideal platforms to test therapeutic agents with the ability to differentiate HR defective and proficient tumors.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma.

The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Schlafen 11 further sensitizes BRCA-deficient cells to PARP inhibitors through single-strand DNA gap accumulation behind replication forks

The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.

Single-Stranded DNA Gap Accumulation Is a Functional Biomarker for USP1 Inhibitor Sensitivity.

Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetically lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Herein, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with drug sensitivity. USP1 inhibition increased monoubiquitinated proliferating cell nuclear antigen at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials. Significance: USP1 inhibitors kill BRCA1-deficient cells and cause ssDNA gap accumulation, supporting the potential of using ssDNA gap detection as a functional biomarker for clinical trials on USP1 inhibitors.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1,DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Adaptive use of error-prone DNA polymerases provides flexibility in genome replication during tumorigenesis.

Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics.

Deficiency in DNA Damage Repair Proteins Promotes Prostate Cancer Cell Migration through Oxidative Stress

Introduction: DNA damage repair gene deficiency defines a subgroup of prostate cancer patients with early metastatic progression and unfavorable disease outcome. Whether deficiency in DNA damage repair genes directly promotes metastatic dissemination is not completely understood. Methods: The migratory behavior of prostate cancer cells was analyzed after siRNA-mediated knockdown of DNA damage repair and checkpoint proteins, including BRCA2, ATM, and others, using transwell migration assays, scratch assays and staining for F-actin to ascertain cell circularity. Cells deficient in BRCA2 or ATM were tested for oxidative stress by measuring reactive oxygen species (ROS). The effects of ROS inhibition on cell migration were analyzed using the antioxidant N-acetylcysteine (NAC). The correlation between BRCA2 deficiency and oxidative stress was ascertained via immunohistochemistry for methylglyoxal (MG)-modified proteins in 15 genetically defined primary prostate cancers. Results: Prostate cancer cells showed a significantly increased migratory activity after the knockdown of BRCA2 or ATM. There was a significant increase in ROS production in LNCaP cells after BRCA2 knockdown and in PC-3 cells after BRCA2 or ATM knockdown. Remarkably, the ROS scavenger NAC abolished the enhanced motility of prostate cancer cells after the knockdown of BRCA2 or ATM. Primary prostate cancers harboring genetic alterations in BRCA2 showed a significant increase in MG-modified proteins, indicating enhanced oxidative stress in vivo. Conclusions: Our results indicate that DNA damage repair gene deficiency may contribute to the metastatic dissemination of prostate cancer through enhanced tumor cell migration involving oxidative stress.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.

Abstract 4644: Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or a PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and a USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alexandre Andre B. A. Da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David Martigneti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Golbahar Sadatrezai, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey Shapiro, Kalindi Parmar, Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4644.

Abstract 3234: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3234.

Abstract 5801: Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody

Abstract The formulation of novel chemotherapeutics capable of precisely targeting and damaging tumor cells, while preserving the integrity of healthy tissues, represents a principal challenge and is recognized as one of the biggest achievements in cancer treatment in the last few decades. Antibody-drug conjugates (ADCs) hold significant potential as an effective cancer treatment, by targeting and delivering cytotoxic drugs exclusively to tumor cells that express distinct antigens associated with malignancy. Due to the necessity for specific surface antigens, therapies with ADCs are limited to specific tumor subtypes. In this study, we show the potential of a lupus-derived, cell-penetrating, anti-DNA antibody for delivering of cytotoxic payload to tumors in vivo. We utilized a fully humanized variant, named as V66, which was chemically linked to the Topoisomerase I inhibitor, Exatecan. The primary mechanism involved in V66 cellular penetration is mediated by the Equilibrative Nucleoside Transporter ENT2. The latter shows elevated expression in a substantial subset of cancer types in comparison to healthy tissues, broadening the range of applications for ADCs in tumor therapy. Using labeled V66, we demonstrated specific tumor delivery in vivo, with minimal impact on healthy tissues. In vitro experiments validated the nuclear localization of V66-Exatecan, where it triggers the activation of the DNA damage repair pathway (DDR). As a consequence of the profound DNA damage, exposure to V66-Exatecan leads to cell death, exhibiting low nanomolar EC50 values across a diverse range of cell lines. Furthermore, V66-Exatecan treatment presents synthetic lethality with BRCA2 deficiency, evidenced by a substantial increase in sensitivity. In vivo efficacy studies conducted in BRCA2 deficient tumors revealed a remarkable effectiveness of V66-Exatecan, leading to almost complete tumor regression and substantial improvement in the probability of survival. In addition to the lack of significant weight changes observed throughout the treatment, further toxicological studies conducted in serum and tissues demonstrated the absence of short- and long-term toxicity, with optimal liver and kidney function and the absence of damage to other tissues. Likewise, bone marrow cellularity remained unaffected. Collectively, these studies indicate that V66-Exatecan achieves targeted treatment of tumor cells, where it induces DNA damage leading to cell death. The in vivo application of V66-Exatecan leads to tumor regression without triggering general toxicity that is otherwise associated with Topoisomerase I inhibitors. This work provides the basis to advance a novel nuclear-penetrating ADC for delivering cytotoxic payloads. Citation Format: Zaira Ianniello, Elias Quijano, Peter M. Glazer. Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5801.

Abstract 1454: Expanding the therapeutic application of PARP inhibitor: AI evaluation of real-word clinico-genomic data from spontaneous canine tumors

Abstract The widespread application of Poly-ADP ribose polymerase (PARP) inhibitors in anti-cancer therapy is constantly increasing their role as a treatment strategy across various neoplasms, the majority of which are linked with BRCA deficiency. While the most significant advantages are observed in cancers with BRCA1/2 mutations, it is evident that the benefits extend beyond this specific group. Preclinical evidence encourages the exploration of PARP inhibitors in neoplasms demonstrating BRCAness or homologous recombination deficiency (HRD), both as monotherapy and in combination with chemotherapy. This study employs spontaneous canine tumors as a complementary model for cancer research, aiming to seamlessly bridge the transition from preclinical investigations to clinical application. Leveraging the FidoCure® Precision Medicine Platform, we identified biomarkers associated with prognosis and treatment prediction, specifically with the PARP inhibitor olaparib, facilitating a smoother translation from laboratory findings to practical clinical use. Analyzing real-world clinico-genomic data from 1278 dogs with cancer revealed intriguing insights of specific targeted treatments developed for humans showing a positive prognosis when applied to canine tumors with specific genomic alterations. Notably, olaparib exhibited efficacy in TP53 and BRCA1-mutated cases (OS HR 0.34, P &amp;lt; 0.001; HR 0.39, P = 0.004), while rapamycin (mTOR inhibitor) demonstrated promising outcomes in TP53 and RB1-mutated canine tumors (OS HR 0.73, P = 0.028; HR 0.32, P = 0.024). Further stratification by tumor types unveiled noteworthy correlations. TP53 mutant osteosarcomas exhibited improved prognosis with olaparib treatment (OS HR 0.11, P &amp;lt; 0.001), and soft tissue sarcomas with TP53 mutations demonstrated favorable responses to rapamycin and olaparib therapy (OS HR 0.11, P = 0.012; HR 0.07, P = 0.011). These findings underscore the potential of leveraging canine tumor models and clinico-genomic analyses to inform targeted treatment strategies, offering valuable insights for advancing precision medicine in both veterinary and human oncology. Further investigation into the association between TP53 mutation and olaparib response is warranted. The intriguing aspect lies in the role TP53 mutation has been linked in previous studies to heightened chromosomal instability and elevated HRD scores. Utilizing real-world evidence and data tools within the FidoCure dataset, we have effectively pinpointed correlations between gene mutations and survival, particularly in relation to responses to targeted therapy treatment. Canine models, mirroring human diseases with intact immune systems and comparable tumor genomic profiles, as verified by the FidoCure database, expedite clinical studies of novel treatments that face significant scalability challenges. Citation Format: Lucas Rodrigues, Kevin Wu, Garrett Harvey, Gerald Post, Abigail Hull, Aubrey Miller, Lindsay Lambert, Christina Lopes, James Zou. Expanding the therapeutic application of PARP inhibitor: AI evaluation of real-word clinico-genomic data from spontaneous canine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1454.

BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-β and vimentin) were significantly increased in BRCA1-deficient cells. Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.