BRCA1-deficient Cancer Cells Research Articles

Abstract 803: Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers

Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with an increased lifetime risk of breast and ovarian cancers. While the BRCA proteins play a well-established role in double-stranded DNA break repair, recent studies have revealed an emerging role of BRCA1/2 in replication stress response. While replication forks are extensively degraded by nucleases in BRCA-deficient cancer cells, activation of specialized fork recovery mechanisms enables resumption of DNA synthesis and promotes cell survival. My project aims to determine this fork recovery mechanism in BRCA1-deficient cells and to identify potential recovery factors that can be targeted to improve chemotherapeutic response in BRCA1-mutated breast and ovarian cancers. To monitor perturbations in replication fork dynamics on a genome-wide scale, we utilize a DNA fiber assay technique measuring rates of fork recovery and replication fork degradation. In parallel, electron microscopy analysis allows direct visualization of replication fork intermediates. Cell survival assays are employed to test how loss of fork recovery factors impacts cell proliferation and chemotherapeutic response in BRCA1-deficient cells. Our results reveal that RAD18 and UBC13, which catalyze ubiquitination of Proliferating Cellular Nuclear Antigen (PCNA), promote fork recovery in BRCA1-deficient, but not BRCA2-deficient, cancer cells. Previous work has also shown that PCNA polyubiquitination by UBC13 is important for reversed fork formation in BRCA-proficient cells. However, our findings show that extensive degradation of reversed fork substrates still occurs in BRCA1-deficient cells lacking RAD18 or UBC13, indicating that PCNA polyubiquitination is not essential for fork reversal in this genetic background. In addition, loss of RAD18 in BRCA1-deficient cells significantly slows cell proliferation, and UBC13 inhibition further sensitizes cells lacking BRCA1 to the replication stress inducer Hydroxyurea (HU). Based on our findings, we hypothesize that RAD18, UBC13, and PCNA ubiquitination may represent novel targets to improve chemoresponse in BRCA1-deficient cancers that rely on fork recovery mechanisms for survival. Citation Format: Emily Cybulla, Jessica Jackson, Stephanie Tirman, Annabel Quinet, Delphine Lemacon, Alessandro Vindigni. Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 803.

Abstract LB202: Discovery and preclinical characterization of novel small molecule inhibitors of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers

Abstract KIF18A is a mitotic kinesin that localizes to the plus-end tips of kinetochore microtubule (MT) spindle fibers during metaphase, where it regulates chromosome alignment, and promotes the viability of chromosomally unstable cancer cells. KIF18A is overexpressed in a subset of human cancers, and its elevated expression is associated with tumor aggressiveness. Chromosomal instability (CIN) is a hallmark of human cancers and is caused by persistent errors in chromosome segregation during mitosis. Aggressive types of human cancer such as high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have elevated levels of CIN and frequently harbor alterations in TP53 tumor suppressor gene. These two CIN+ cancer subtypes share molecular similarities but have limited treatment options at present. The rationale of pharmacological inhibition of KIF18A motor activity is to selectively target a tumor-specific mitotic spindle vulnerability in CIN+ cancer cells while largely sparing normal diploid dividing somatic cells. Here, we describe the identification of a novel series of potent and selective small molecule inhibitors of KIF18A MT-ATPase motor activity exemplified by AM-1882, that disrupt the mitotic spindle and selectively kill chromosomally unstable cancer cells. Our KIF18A inhibitors phenocopy genetic ablation of KIF18A and trigger spindle assembly checkpoint activation, multipolarity, and apoptosis in sensitive CIN+ cancer cell lines. The sensitivity profile of AM-1882 is focal-in-nature with cell potency in the low double-digit nanomolar range across a panel of breast and ovarian cancer cell lines, including lines that harbor genetic alterations (e.g., TP53, CCNE1, RB1, BRCA1, whole genome doubling) frequently enriched in CIN+ cancers and in HGSOC and TNBC tumor subtypes. Furthermore, the sensitivity profile of AM-1882 is distinct from comparator test agents ispinesib (Eg5, pan cytotoxic) and palbociclib (CDK4/6, focal cytostatic). The combination of AM-1882 with PARP inhibitor olaparib is synergistic in BRCA1-deficient cancer cell lines, with evidence of increased double-strand DNA breaks (p-H2AX) and apoptosis (cl-PARP). Importantly, KIF18A inhibitors have minimal toxicity on normal dividing somatic cell types in vitro, including proliferating human bone marrow mononuclear cells, distinct from paclitaxel and small molecule inhibitors of essential mitotic kinases and kinesins. In vivo, we demonstrate that administration of KIF18A inhibitors AM-1882 and AM-5308 induce a robust pharmacodynamic response (pH3, mitotic marker) and frank tumor regressions in two TP53 mutant human HGSOC xenograft models (OVCAR-3, OVCAR-8) at well-tolerated doses. Collectively, our preclinical data provides the first example of a therapeutic strategy to selectively target CIN+ cancers through inhibition of KIF18A motor protein. Citation Format: Jan Sun, Brain Belmontes, Jodi Moriguchi, Grace Chung, Kui Chen, John D. McCarter, Upendra P. Dahal, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Sheroy Minocherhomji, Matthew P. Bourbeau, Jennifer R. Allen, Angela Coxon, Paul E. Hughes, Nuria Tamayo, Marc N. Payton. Discovery and preclinical characterization of novel small molecule inhibitors of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB202.

Relationship between BRCA mutation status and adverse effects of PARP inhibitors.

e17566 Background: PARP inhibitors exert their anti-tumor effects by targeting BRCA-deficient cancer cells through a mechanism of synthetic lethality and PARP trapping. However, as a drug in the systemic route of administration, it has the same killing effect on BRCA-deficient normal cells in vivo, especially those with faster proliferation, such as those in the bone marrow hematopoietic cells and cells in the digestive tract. We, therefore, hypothesize that BRCA mutant patients are more likely than BRCA wild-type patients to have severe adverse effects that result in dose reduction, interruption, or discontinuation of the drug, thus affecting the patient's prognosis. Methods: Information was retrospectively collected on 46 patients diagnosed with ovarian cancer who had been treated with PARP inhibitors between August 2018 and October 2021. The patients were divided into BRCA mutation group (n = 24), BRCA wild-type group (n = 16), and BRCA unknown group (n = 6) according to the genetic test results. Results: The most common adverse effects in all three groups were gastrointestinal reactions, mostly grade 1 and 2. Hematologic reactions were the next common adverse effects, the most common of which was anemia. No extremely serious adverse effects such as myelodysplastic syndrome occurred in our patients. The most serious adverse effects in our patients were grade 3, 75% of which were anemia. In BRCA mutant patients, the incidence of gastrointestinal reactions and hematologic effects was 75% and 50%, respectively, while these two rates were 50% and 25% in BRCA wild-type patients. The incidence of adverse effects was significantly higher in BRCA mutant patients than in BRCA wild-type patients. The incidence of grade 3 adverse events was 37.5% in BRCA mutant patients and 12.5% in BRCA wild-type patients. The incidence of grade 3 adverse events was significantly higher in BRCA mutant patients than in BRCA wild-type patients. Overall, adverse events were higher and more severe in the BRCA mutant than in the BRCA wild type (P＜0.05). Conclusions: Patients with BRCA mutations are more likely than patients with BRCA wild-type to have serious adverse effects that result in dose reduction, interruption, or discontinuation of the drug. Therefore, we should pay more attention to the monitoring of BRCA mutant patients and take more measures to prevent or mitigate their adverse effects, so that they can always take PARP inhibitors according to the recommended dose as much as possible, which may lead to a better prognosis for more patients.[Table: see text]

The Apoptotic Resistance of BRCA1-Deficient Ovarian Cancer Cells is Mediated by cAMP.

Breast cancer type 1 susceptibility protein (BRCA1) is essential for homologous recombination repair of DNA double-strand breaks. Loss of BRCA1 is lethal to embryos due to extreme genomic instability and the activation of p53-dependent apoptosis. However, the apoptosis is resisted in BRCA1-deficient cancer cells even though their p53 is proficient. In this study, by analysis of transcriptome data of ovarian cancer patients bearing BRCA1 defects in TCGA database, we found that cAMP signaling pathway was significantly activated. Experimentally, we found that BRCA1 deficiency caused an increased expression of ADRB1, a transmembrane receptor that can promote the generation of cAMP. The elevated cAMP not only inhibited DNA damage-induced apoptosis through abrogating p53 accumulation, but also suppressed the proliferation of cytotoxic T lymphocytes by enhancing the expression of immunosuppressive factors DKK1. Inhibition of ADRB1 effectively killed cancer cells by abolishing the apoptotic resistance. These findings uncover a novel mechanism of apoptotic resistance in BRCA1-deficient ovarian cancer cells and point to a potentially new strategy for treating BRCA1-mutated tumors.

WRN helicase safeguards deprotected replication forks in BRCA2-mutated cancer cells

The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient.

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.

Identification of a RAD52 Inhibitor Inducing Synthetic Lethality in BRCA2-Deficient Cancer Cells.

The breast cancer susceptibility gene 1/2 (BRCA1/2) is frequently mutated in many malignant tumors, such as breast cancer and ovarian cancer. Studies have demonstrated that inhibition of RAD52 gene function in BRCA2-deficient cancer causes synthetic lethality, suggesting a potential application of RAD52 in cancer-targeted therapy. In this study, we have performed a virtual screening by targeting the self-association domain (residues 85–159) of RAD52 with a library of 66,608 compounds and found one compound, C791-0064, that specifically inhibited the proliferation of BRCA2-deficient cancer cells. Our biochemical and cell-based experimental data suggested that C791-0064 specifically bound to RAD52 and disrupted the single-strand annealing activity of RAD52. Taken together, C791-0064 is a promising leading compound worthy of further exploitation in the context of BRCA-deficient targeted cancer therapy.

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

Abstract IA-026: Gaps alone kill BRCA deficient cancer cells

Abstract Defects in DNA repair and/or 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, recent findings indicate that chemotherapies such as cisplatin used to treat BRCA-deficient tumors do not initially cause DNA double-strand-breaks (DSBs). Here, we show that single stranded DNA (ssDNA) replication gaps underlie the hypersensitivity of BRCA-deficient cancer, and we propose that defects in homologous recombination (HR) or fork protection (FP) do not. Specifically, in BRCA-deficient cells, ssDNA gaps develop because replication is not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling confers therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR can be uncoupled from therapy resistance when gaps are present. Moreover, we find that DSBs are not detected after therapy when apoptosis is inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather from cell death nucleases. Together, this data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapies. Citation Format: Sharon B. Cantor, Nicholas Panzarino. Gaps alone kill BRCA deficient cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-026.

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5′-phosphoryl and 3′-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown.Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells.Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids.Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

Synthetic Lethal Activity of Benzophenanthridine Alkaloids From Zanthoxylum coco Against BRCA1-Deficient Cancer Cells.

Several plants from South America show strong antitumoral properties based on anti-proliferative and/or pro-apoptotic activities. In this work we aimed to identify selective cytotoxic compounds that target BRCA1-deficient cancer cells by Synthetic Lethality (SL) induction. Using a high-throughput screening technology developed in our laboratory, we analyzed a collection of extracts from 46 native plant species from Argentina using a wide dose-response scheme. A highly selective SL-induction capacity was found in an alkaloidal extract from Zanthoxylum coco (Fam. Rutaceae). Bio-guided fractionation coupled to HPLC led to the identification of active benzophenanthridine alkaloids. The most potent SL activity was found with the compound oxynitidine, which showed a remarkably low relative abundance in the active fractions. Further validation experiments were performed using the commercially available and closely related analog nitidine, which showed SL-induction activity against various BRCA1-deficient cell lines with different genetic backgrounds, even in the nanomolar range. Exploration of the underlying mechanism of action using BRCA1-KO cells revealed AKT and topoisomerases as the potential targets responsible of nitidine-triggered SL-induction. Taken together, our findings expose an unforeseen therapeutic activity of alkaloids from Zanthoxylum-spp. that position them as novel lead molecules for drug discovery.

The PRIMPOL Protein Shields Replication Forks in BRCA-Deficient Cells

PRIMPOL shields replication forks after multiple rounds of cisplatin in BRCA-deficient cancer cells.

PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells.

SummaryAcute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.

Premature mitotic entry induced by ATR inhibition potentiates olaparib inhibition-mediated genomic instability, inflammatory signaling, and cytotoxicity in BRCA2-deficient cancer cells.

Poly(ADP‐ribose) polymerase (PARP) inhibitors are selectively cytotoxic in cancer cells with defects in homologous recombination (HR) (e.g., due to BRCA1/2 mutations). However, not all HR‐deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR‐deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2‐depleted and Brca2 knockout cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome‐wide single‐cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib‐treated BRCA2‐depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING‐associated inflammatory response in BRCA2‐deficient cells. ATR inhibition further increased the numbers of cGAS‐positive micronuclei and the extent of cytokine production in olaparib‐treated BRCA2‐deficient cancer cells. Altogether, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR‐deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease.

Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

BRCA1 represses DNA replication initiation through antagonizing estrogen signaling and maintains genome stability in parallel with WEE1-MCM2 signaling during pregnancy.

The mammary gland undergoes fast cell proliferation during early pregnancy, yet the mechanism to ensure genome integrity during this highly proliferative stage is largely unknown. We show that pregnancy triggers replicative stresses leading to genetic instability in mice carrying a mammary specific disruption of breast cancer associated gene-1 (BRCA1). The fast cell proliferation was correlated with enhanced expression of most genes encoding replisomes, which are positively regulated by estrogen/ERα signaling but negatively regulated by BRCA1. Our further analysis revealed two parallel signaling pathways, which are mediated by ATR-CHK1 and WEE1-MCM2 and are responsible for regulating DNA replication checkpoint. Upon DNA damage, BRCA1 deficiency markedly enhances DNA replication initiation and preferably impairs DNA replication checkpoint mediated by ATR and CHK1. Meanwhile, DNA damage also activates WEE1-MCM2 signaling, which inhibits DNA replication initiation and enables BRCA1-deficient cells to avoid further genomic instability. Finally, we demonstrated that overriding this defense by WEE1 inhibition in combination with cisplatin, which causes DNA damage, serves as a promising therapeutic approach for killing BRCA1-deficient cancer cells.

TPX2/Aurora kinase A signaling as a potential therapeutic target in genomically unstable cancer cells

Genomic instability is a hallmark feature of cancer cells, and can be caused by defective DNA repair, for instance due to inactivation of BRCA2. Paradoxically, loss of Brca2 in mice results in embryonic lethality, whereas cancer cells can tolerate BRCA2 loss. This holds true for multiple DNA repair genes, and suggests that cancer cells are molecularly “rewired” to cope with defective DNA repair and the resulting high levels of genomic instability. In this study, we aim to identify genes that genomically unstable cancer cells rely on for their survival. Using functional genomic mRNA (FGmRNA) profiling, 16,172 cancer samples were previously ranked based on their degree of genomic instability. We analyzed the top 250 genes that showed a positive correlation between FGmRNA levels and the degree of genomic instability, in a co-functionality network. Within this co-functionality network, a strong cluster of 11 cell cycle-related genes was identified, including TPX2. We then assessed the dependency on these 11 genes in the context of survival of genomically unstable cancer cells, induced by BRCA2 inactivation. Depletion of TPX2 or its associated kinase Aurora-A preferentially reduced cell viability in a panel of BRCA2-deficient cancer cells. In line with these findings, BRCA2-depleted and BRCA2-mutant human cell lines, or tumor cell lines derived from Brca2–/–;p53–/– mice showed increased sensitivity to the Aurora-A kinase inhibitor alisertib, with delayed mitotic progression and frequent mitotic failure. Our findings reveal that BRCA2-deficient cancer cells show enhanced sensitivity to inactivation of TPX2 or its partner Aurora-A, which points at an actionable dependency of genomically unstable cancers.

Abstract IA17: PARP inhibitor resistance in prostate cancer

Abstract Large-scale genomic studies have demonstrated that approximately 15% of metastatic castration-resistant prostate cancer (mCRPC) harbors genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA2, BRCA1, and ATM, followed by other Fanconi anemia genes including FANCN/PALB2 and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues and PARP inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in BRCA2-deficient mCRPC where the PARPi olaparib has demonstrated improvements in progression-free survival. PARPi resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2-deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. These somatic reversions of mutant BRCA2 alleles can be monitored by sequencing of ctDNA. However, PARPi resistance may also develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism—namely, the restoration of RF stability—appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire may PARPi resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient ovarian and breast cancer cells can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. Whether or not the downregulation of the PRC2/MUS81 pathway is also a mechanism of PARPi resistance in mCRPC remains to be determined. A molecular understanding of PARPi resistance mechanisms may allow the generation of a new class of drugs, or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARPi to more tumor types. Citation Format: Alan D. D’Andrea. PARP inhibitor resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA17.

Compound F779-0434 causes synthetic lethality in BRCA2-deficient cancer cells by disrupting RAD52-ssDNA association.

Maintenance of genomic integrity is essential for the survival of all organisms. Homologous recombination (HR) is the major pathway for high-fidelity repair of DNA double-stranded breaks (DSBs). In addition to the classic BRCA–RAD51 pathway, another secondary HR sub-pathway dependent on RAD52 has been suggested to be functioning in mammalian cells. Importantly, RAD52 has been shown to be synthetically lethal to BRCA1/2-deficient cells, rendering RAD52 to be a desirable target in cancer therapy. In the current study, we performed a structure-based virtual screening of 47 737 drug-like compounds to identify RAD52-specific inhibitors. The top ranked virtual screening hits were further characterized using molecular dynamics simulation and biochemical and cell-based assays. We found that one compound, namely, F779-0434 specifically suppressed the growth of BRCA2-deficient cells and disrupted RAD52–ssDNA interaction in vitro. This RAD52-specific inhibitor identified in the current study is a promising compound for targeted cancer therapy, and it can also be used as a probe to study the mechanisms of DNA repair in human cells.