Homologous Recombination-proficient Cells Research Articles

Abstract B004: Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells

Abstract DNA polymerase theta (Pol θ), functions in theta-mediated end joining (TMEJ), an error-prone DNA double strand break repair pathway. Pol θ is synthetic lethal with homologous recombination (HR) factors, and this finding has led to the development of Pol θ inhibitors and clinical trials to determine their effectiveness in HR-deficient tumors. Despite this, the biological functions of Pol q that promote genome integrity in normal cells are poorly understood. Here, we elucidate an essential role for Pol θ during mammalian interstrand crosslink (ICL) repair. We show that Pol θ is recruited downstream of ICL nucleolytic incision, BRCA2-dependent Rad51 loading, and FBXO5-mediated Rad51 ubiquitylation at sites of unsuccessful HR. Genomic scar analyses reveal that TMEJ is specifically required for repair of clustered ICLs, which are not amenable to HR-mediated repair. Thus, Pol q may be the predominant repair pathway for clustered lesions that generate two-ended double strand breaks during DNA replication. These findings suggest that Pol θ has essential roles in DNA repair in HR proficient cells, suggesting that Pol θ inhibitors may also be beneficial in the treatment of HR proficient tumors. Citation Format: Chelsea M. Smith, Dennis Simpson, Wanjuan Feng, Gaorav Gupta. Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B004.

Abstract B005: Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer

Abstract Introduction. The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing. Materials and methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype. Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable of quickly and efficiently repairing colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin. Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer. Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Erika Durinikova, Annalisa Lorenzato, Marco Avolio, Eleonora Piumatti, Mariangela Russo, Sabrina Arena, Livio Trusolino, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B005.

Abstract 5900: Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer

Abstract Introduction: The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing. Materials and Methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype.} Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable to quickly and efficiently repair colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin. Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer. Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Mariangela Russo, Annalisa Lorenzato, Erika Durinikova, Livio Trusolino, Sabrina Arena, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5900.

Hyperthermia synergistically enhances antitumor efficacy of PARP inhibitor through impacting homologous recombination repair and oxidative stress in vitro

Tumors with homologous recombination (HR) deficiency are particularly responsive to PARP inhibitors, however strategies to improve the sensitivity of epithelial ovarian carcinoma (EOC) with sufficient HR abilities still need to be deeply explored. In the present study, we firstly validated that hyperthermia (HT) changed diverse genes and signal pathways related to HR and oxidative stress in HR proficient EOC cells. HT impaired HR efficiency through inhibiting Olaparib (Olap) induced RAD51 foci formation in EOC cells, which was independent of the expression level of RAD51. Combination therapy of HT and Olap synergistically induced oxidative stress and oxidative DNA damage of EOC cells. Furthermore, we revealed that HT and Olap synergistically aggravated double-strand breaks of DNA in EOC cells. Conclusively, our findings confirmed that HT could synergistically enhance HR proficient EOC cells’ sensitivity to PARP inhibitor through impairing HR efficiency and increasing oxidative stress.

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p< 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [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 795.

Resveratrol sensitizes breast cancer to PARP inhibitor, talazoparib through dual inhibition of AKT and autophagy flux

The efficacy of poly (ADP-ribose) polymerase inhibitors (PARPi) is largely limited to the homologous recombination (HR) deficient cancers. Therefore, there is a necessity to explore novel drug combinations with PARPi to enhance its anti-cancer activity in HR-proficient cancers. By analysing the patient data in cBioPortal, we found copy number amplification of PARP1 in ∼ 22.8% of breast cancers. PARP1 upregulation has been correlated with unfavourable outcome with PARPi treatment. To overcome this adversity, we explored the effect of resveratrol, a natural molecule chemosensitizer, in enhancing the effects of the third generation PARPi, talazoparib (BMN673), against breast adenocarcinoma. Our results show that resveratrol effectively sensitized talazoparib induced cell death in HR proficient and BRCA wild-type breast cancer cells in vitro. Mechanistically, resveratrol caused dysregulation of cell cycle and enhanced talazoparib-induced double strand breaks (DSBs), leading to abnormal mitotic progression culminating in mitotic catastrophe. Intriguingly, our results showed potential of resveratrol in dual-inhibition of AKT-signalling and autophagy flux to impair HR-mediated DSB-repair in breast cancer cells. By using EGFP-LC3 and tf-LC3 (mRFP-EGFP-LC3) expressing breast cancer cells, we found that resveratrol attenuates fusion of autophagosome and lysosome though induction of lysosomal-membrane-permeabilization (LMP). The combination of resveratrol and talazoparib effectively reduced cell proliferation in the high-density cell proliferation assay and also led to tumour volume reduction in vivo pre-clinical SCID-mice model. The combination caused no or minimal cytotoxicity in three different normal cell lines in vitro. Taken together, our work proposes the usage of resveratrol as a chemosensitizer along with talazoparib for targeting HR-proficient breast cancers in clinical settings.

Increasing sensitivity to olaparib through inhibition of discoidin domain receptor 2 (DDR2) in homologous-recombination proficient ovarian cancer models

<h3>Objectives:</h3> Discoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. DDR2 expression is critical for invasion and migration of ovarian cancer tumor cells, making DDR2 a potential target for new treatments. This study aimed to determine whether genetic inactivation of DDR2 in ovarian cancer cells would increase sensitivity to treatment with PARP inhibitor. <h3>Methods:</h3> Two ovarian cancer cells lines, ES2 and COV362, were used for <i>in vitro</i> assays. Stable hairpin control and DDR2 knockdowns (shSCRM and shDDR2) were used for ES2 and transient small interfering RNA knockdown of DDR2 (siControl and siDDR2) were used for COV362. Homologous recombination (HR) status was determined by quantification of RAD-51 foci relative to controls using immunofluorescence after irradiation. Cell viability and survival after treatment with olaparib was quantified using MTS assays and clonogenics. The DNA damage response after treatment with olaparib was analyzed using immunofluorescence. <i>In vivo</i> studies to evaluate the sensitivity to olaparib in the presence or absence of DDR2 expression were performed in nude mice after intraperitoneal injection with ES2shSCRM or shDDR2 cells. <h3>Results:</h3> ES2 and COV362 cells were found to have a 2-fold increase in RAD51 foci after irradiation indicating HR proficiency. DDR2 genetically inactivated cells (ES2shDDR2) were found to be more HR deficient than DDR2 expressing cells (ES2shSCRM) which remained HR proficient (0.80 vs 4.78 relative RAD51 foci per cell). We found that genetic inactivation of DDR2 in the ES2s and COV362s led to increased sensitivity to olaparib with an 80% (15uM vs 73uM) and 68% (62uM vs 195uM) reduction in IC50s, respectively, compared to DDR2 expressing cells. Additionally, ES2shDDR2 cells had decreased cell survival by clonogenic assay after treatment with olaparib compared to ES2shSCRM cells as measured by absorbance (0.64 vs 0.80, p=0.005). We found that DNA damage and the repair response was altered in DDR2 genetically inactivated cells treated with olaparib with an increase in gamma-H2AX (8 vs 6 foci per cell, p<0.0001) and 53BP1 foci (3 vs 1 foci per cell, p<0.0001) when compared to DDR2 expressing cells. In an intraperitoneal metastatic mouse model with the HR-proficient cell line ES2, we found that genetic inactivation of DDR2 led to decreased tumor burden when treated with olaparib compared to DDR2-expressing cells treated with olaparib (1499mm³ vs 3236mm³, p=0.04). <h3>Conclusions:</h3> DDR2 knockdown sensitizes HR proficient ovarian cancer cells to treatment with PARP inhibitor in both <i>in vitro</i> and <i>in vivo</i> models through a mechanism of induced HR deficiency and increased DNA damage. Future experiments will explore whether a DDR2 inhibitor can improve sensitivity to PARP inhibitor in HR proficient ovarian cancer models.

Abstract IA-003: Oxygen dependent resistance to PARP inhibitors

Abstract Severe hypoxia causes resistance to conventional chemotherapy and has been reported to synergize with PARP inhibitors (PARPi) through suppression of homologous recombination (HR). While this synergistic killing is true at oxygen levels less than 0.5%, our study shows that less severe hypoxia (e.g. 2% oxygen) is instead associated with resistance to PARPi in HR proficient cells. Interestingly, we demonstrate that HR deficient hypoxic tumors are significantly less responsive to PARPi, due to limited ROS-induced intrinsic DNA damage. To determine the contribution of hypoxic cells to PARPi, we used the hypoxic cytotoxin Tirapazamine to target hypoxic tumor cells. We found that the elimination of hypoxic tumor cells by Tirapazamine led to a substantial antitumor response with PARPi compared to PARPi treated tumors alone, without enhancing normal tissue toxicity. These studies indicate that tumor hypoxia reduces the efficacy of PARPi to tumor cells and that eliminating hypoxic tumor cells will enhance the efficacy of PARPi therapy. Citation Format: Manal Mehibel, Jimmy Xu, Grace Li, Jung Moon, Kaushik Thakkar, Anh Diep, Ryan Kim, Joshua Bloomstein, Siren Xiao, Julien Bacal, Joshua Saldivar, Quynh Le, Karlene Cimprich, Erinn Rankin, Amato Giaccia. Oxygen dependent resistance to PARP inhibitors [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-003.

Abstract 1379: Entinostat induces PARPi sensitivity across multiple ovarian cancer models

Abstract Introduction: Ovarian cancer is a rare, but fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising drugs, most effective in patients with tumors deficient in homologous recombination (HR) genes, mainly BRCA1/2. Our group has published that histone deacetylase inhibitors (HDACi) sensitized HR proficient BRCA wild-type ovarian cancer cells to PARPi, however the mechanism still remains elusive and the efficacy needed to be tested across multiple models. We recently tested Entinostat with and without Olaparib in three different ovarian cancer models, including human/mouse established cell lines, PDX derived primary cell lines and immune-competent/immune-compromised xenograft as well as PDX-derived preclinical mouse models. Objective: To delineate the efficacy of Entinostat to sensitize ovarian tumors to Olaparib across multiple models and determine the underlying mechanism. Methods: HR proficient BRCA wild-type ID8/SKOV3 and PDX derived primary cell lines were treated with Entinostat and Olaparib, alone or in combination and analyzed for cell proliferation. DNA damage was studied using Comet assay. Expression of cell proliferation, DNA damage and repair protein were studied using Western blot analysis and immunofluorescence. Further, ID8-treated immune-competent mouse models, SKOV3-treated immune-compromised mouse model and HGSOC patient derived PDX mouse model generated in our lab were treated with vehicle, Entinostat, Olaparib, or the combination. The mice were monitored for toxicity and body weight measured twice weekly, till tissue harvest or survival. Results: Cell proliferation was significantly decreased (p&amp;lt0.05) in Entinostat-Olaparib combination treated groups in all cell lines tested. Comet assay showed significantly longer comet tail length in combination treated cells compared to control (p&amp;lt0.05). Western blot and Immunofluorescence showed that combination treatment significantly decreased BRCA1, PCNA, RAD51 and increased cleaved PARP, ΓH2AX (p&amp;lt0.05). Further, SKOV3- xenograft mouse model showed decreased tumor burden ((p&amp;lt0.07), ID8-xenograft mouse tumors showed decreased Ki67 (p&amp;lt0.05) and HGSOC derived PDX model showed longer survival when treated with Entinostat-Olaparib combination therapy. Conclusion: Entinostat in combination with Olaparib decreased cell proliferation, increased DNA damage, induced HR deficiency in vitro, reduced tumor burden, decreased Ki67 in xenograft mouse model, and improved survival of HGSOC derived PDX mice, thereby potentially sensitizing homologous recombination proficient ovarian cancer to PARPi across multiple models. Citation Format: Vijayalaxmi G. Gupta, Shariska Petersen, Jeff Hirst, Katherine Roby, Harsh Pathak, Meghan Kusch, Andrew Wilson, Andrew Godwin, Dineo Khabele. Entinostat induces PARPi sensitivity across multiple ovarian cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1379.

Abstract 1716: The PARP inhibitor rucaparib activates the STING pathway and enhances antitumor responses of immune checkpoint inhibitors in BRCA deficient syngeneic models

Abstract Background: The poly(ADP ribose) polymerase (PARP) inhibitor rucaparib effectively kills homologous recombination (HR) deficient cells through impeding DNA repair that leads to DNA damage, apoptosis, and cell death. Detection of cytosolic DNA by the stimulator of interferon genes (STING) pathway mediates type I interferon (IFN) production and activates the immune system. Following rucaparib treatment, the accumulation of damaged DNA in HR impaired tumors may elicit an immune response through STING signaling, and enhance rucaparib activity as a single agent or in combination with immune checkpoint blockade. To test this hypothesis, rucaparib efficacy and mechanism of action were evaluated using BRCA deficient syngeneic ovarian tumor models. Results: Single agent rucaparib showed potent antitumor activity in the BRCAmut BrKras and ID8B3.15 models. In the BrKras model, rucaparib treatment resulted in complete regression and prevented tumor formation upon re-challenge. However, this efficacy was abolished with anti-CD8 but not anti-CD4 depletion. CD8 tumor infiltrating lymphocytes (TILs) but not CD4 TILs increased with rucaparib exposure, and expression profiling of rucaparib treated tumors showed activation of the type I IFN pathway. In vitro PCR assays identified several targets that were upregulated upon rucaparib treatment including Ccl5 and Cxcl10. Notably, increased Ccl5 and Cxcl10 levels were observed in BRCAmut cells but not in BRCAwt cells, and knockdown of the STING pathway genes MB21D1, IRF3, TBK1, and STING (TMEM173) abrogated rucaparib induction of Ccl5 and Cxcl10. Furthermore, rucaparib showed transcriptional activation of IFN type I signaling in BRCAmut reporter cells expressing the IFN stimulated response element consensus sequence driving luciferase expression, but required 6-fold higher concentrations in BRCAwt reporter cells. Similarly, a 10-fold higher rucaparib dose was needed to inhibit proliferation of BRCAwt cells compared to BRCAmut cells in a cell viability assay. Consistent with the in vitro results, rucaparib combined with anti-programmed death 1 or anti-programmed death ligand 1 therapy improved the survival and augmented antitumor responses in BRCA deficient syngeneic tumor models. Conclusions: Rucaparib treatment in HR deficient cells, and at a higher concentration in HR proficient cells, triggers type I IFN signaling through the STING pathway, which participates in the single agent efficacy of rucaparib and enhances the combination of rucaparib and checkpoint inhibitors in syngeneic models. These findings provide further evidence supporting the rationale for combining rucaparib with checkpoint therapy for the treatment of patients with HR defective cancers. Citation Format: Minh Nguyen, Liliane Robillard, Kevin K. Lin, Thomas C. Harding, Andrew D. Simmons. The PARP inhibitor rucaparib activates the STING pathway and enhances antitumor responses of immune checkpoint inhibitors in BRCA deficient syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1716.

Targeting BRCA1/2 deficient ovarian cancer with CNDAC-based drug combinations.

The mechanism of action of CNDAC (2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosyl-cytosine) is unique among deoxycytidine analogs because upon incorporation into DNA it causes a single strand break which is converted to a double strand break after DNA replication. This lesion requires homologous recombination (HR) for repair. CNDAC, as the parent nucleoside, DFP10917, and as an oral prodrug, sapacitabine, are undergoing clinical trials for hematological malignancies and solid tumors. The purpose of this study is to investigate the potential of CNDAC for the therapy of ovarian cancer (OC). Drug sensitivity was evaluated using a clonogenic survival assay. Drug combination effects were quantified by median effect analysis. OC cells lacking function of the key HR genes, BRCA1 or BRCA2, were more sensitive to CNDAC than corresponding HR proficient cells. The sensitization was associated with greater levels of DNA damage in response to CNDAC at clinically achievable concentrations, manifested as chromosomal aberrations. Three classes of CNDAC-based drug combinations were investigated. First, the PARP1 inhibitors, rucaparib and talazoparib, were selectively synergistic with CNDAC in BRCA1/2 deficient OC cells (combination index < 1) at a relatively low concentration range. Second, cisplatin and oxaliplatin had additive combination effects with CNDAC (combination index ~ 1). Finally, paclitaxel and docetaxel achieved additive cell-killing effects with CNDAC at concentration ranges of the taxanes similar for both BRCA1/2 deficient and proficient OC cells. This study provides mechanistic rationales for combining CNDAC with PARP inhibitors, platinum compounds and taxanes in ovarian cancer lacking BRCA1/2 function.

Suppression of Homologous Recombination by insulin-like growth factor-1 inhibition sensitizes cancer cells to PARP inhibitors.

BackgroundImpairment of homologous recombination (HR) is found in close to 50 % of ovarian and breast cancer. Tumors with BRCA1 mutations show increased expression of the Insulin-like growth factor type 1 receptor (IGF-1R). We previously have shown that inhibition of IGF-1R results in growth inhibition and apoptosis of ovarian tumor cells. In the current study, we aimed to investigate the correlation between HR and sensitivity to IGF-1R inhibition. Further, we hypothesized that IGF-1R inhibition might sensitize HR proficient cancers to Poly ADP ribose polymerase (PARP) inhibitors.MethodsUsing ovarian and breast cancer cellular models with known BRCA1 status, we evaluated their HR functionality by RAD51 foci formation assay. The 50 % lethal concentration (LC50) of Insulin-like growth factor type 1 receptor kinase inhibitor (IGF-1Rki) in these cells was assessed, and western immunoblotting was performed to determine the expression of proteins involved in the IGF-1R pathway. Moreover, IGF-1R inhibitors were added on HR proficient cell lines to assess mRNA and protein expression of RAD51 by qPCR and western blot. Also, we explored the interaction between RAD51 and Insulin receptor substance 1 (IRS-1) by immunoprecipitation. Next, combination effect of IGF-1R and PARP inhibitors was evaluated by clonogenic assay.ResultsCells with mutated/methylated BRCA1 showed an impaired HR function, and had an overactivation of the IGF-1R pathway. These cells were more sensitive to IGF-1R inhibition compared to HR proficient cells. In addition, the IGF-IR inhibitor reduced RAD51 expression at mRNA and protein levels in HR proficient cells, and sensitized these cells to PARP inhibitor.ConclusionTargeting IGF-1R might lead to improved personalized therapeutic approaches in cancer patients with HR deficiency. Targeting both PARP and IGF-1R might increase the clinical efficacy in HR deficient patients and increase the population of patients who may benefit from PARP inhibitors.

Abstract 5374: Synergistic tumor cell killing with ganciclovir and vorinostat is due to inhibition of homologous recombination

Abstract The indiscriminate nature of traditional cancer chemotherapy, which targets all dividing cells, has initiated a search for more selective approaches such as suicide gene therapy. With this strategy, only the cells containing the suicide gene are capable of activating a prodrug to a toxic metabolite, thus conferring selectivity for tumor cells while sparing normally dividing tissues. One of the most commonly used suicide gene therapy strategies transfers the cDNA for herpes simplex virus thymidine kinase (HSV-TK) into tumor cells followed by treatment with the antiviral drug ganciclovir (GCV). GCV is phosphorylated initially by HSV-TK, and after further phosphorylation by cellular kinases the active triphosphate is incorporated into the DNA of dividing cells. Recently, we have demonstrated a role for homologous recombination (HR) repair in promoting survival after treatment of GCV in HSV-TK containing cells. Based on these findings, we hypothesize that pharmacologic inhibition of HR will result in synergistic cell kill when combined with GCV. Previous reports have demonstrated that inhibition of lysine deacetylases (KDACs) can inhibit HR. KDACs can also increase the expression of the cell receptor utilized by adenoviruses for infection, thus making this class of drugs a potentially beneficial addition to gene therapy with HSV-TK/GCV. Therefore we evaluated the effect of the KDAC inhibitor Vorinostat (SAHA) on HR and cytotoxicity with HSV-TK/GCV. The ability of SAHA to inhibit HR was evaluated in a cell based recombination reporter assay. SAHA (1 to 10µM) produced increasing inhibition (51 to 85%) of HR. Western blot analysis demonstrated that these concentrations of SAHA produced a dose-dependent decrease in Rad51, an essential protein for HR. HR can be visualized in intact cells by the formation of Rad51 foci at sites of DNA damage. In U251tk cells (stably expressing HSV-TK), GCV produced a time-dependent increase in Rad51 foci, demonstrating a role for HR in repair of GCV-induced DNA damage. At concentrations that inhibited HR and decreased Rad51 expression, SAHA completely blocked Rad51 foci formation with GCV treatment. Together, these data demonstrate that SAHA inhibits HR in response to GCV. We then evaluated cytotoxicity with the combination of GCV and SAHA and observed a synergistic increase in cell kill. To further assess the role of HR in the synergistic cytotoxicity with SAHA and GCV, we evaluated the drug combination in HR proficient and deficient CHO cells stably expressing HSV-TK. The results demonstrated that the combination of SAHA and GCV produced synergistic cell kill in HR proficient cells but only additive cell kill in HR deficient cells. Collectively, these data demonstrate that inhibition of HR by SAHA can account, at least in part, for the synergistic cytotoxicity with GCV. These data suggest that the combination of SAHA and HSV-TK/GCV merits further evaluation in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2011-5374

DNA repair in response to anthracycline–DNA adducts: A role for both homologous recombination and nucleotide excision repair

Doxorubicin, a widely used anthracycline anticancer agent, acts as a topoisomerase II poison but can also form formaldehyde-mediated DNA adducts. This has led to the development of doxorubicin derivatives such as doxoform, which can readily form adducts with DNA. This work aimed to determine which DNA repair pathways are involved in the recognition and possible repair of anthracycline–DNA adducts. Cell lines lacking functional proteins involved in each of the five main repair pathways, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) were examined for sensitivity to various anthracycline adduct-forming treatments. The treatments used were doxorubicin, barminomycin (a model adduct-forming anthracycline) and doxoform (a doxorubicin–formaldehyde conjugate). Cells with deficiencies in MMR, BER and NHEJ were equally sensitive to adduct-forming treatments compared to wild type cells and therefore these pathways are unlikely to play a role in the repair of these adducts. Some cells with deficiencies in the NER pathway (specifically, those lacking functional XPB, XPD and XPG), displayed tolerance to adducts induced by both barminomycin and doxoform and also exhibited a decreased level of apoptosis in response to adduct-forming treatments. Conversely, two HR deficient cell lines were shown to be more sensitive to barminomycin and doxoform than HR proficient cells, indicating that this pathway is also involved in the repair response to anthracycline–DNA adducts. These results suggest an unusual damage response pathway to anthracycline adducts involving both NER and HR that could be used to optimise cancer therapy for tumours with either high levels of NER or defective HR. Tumours with either of these characteristics would be predicted to respond particularly well to anthracycline–DNA adduct-forming treatments.