Homologous Recombination DNA Repair Pathway Research Articles

Emerging strategies: PARP inhibitors in combination with immune checkpoint blockade in BRCA1 and BRCA2 mutation-associated and triple-negative breast cancer.

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor monotherapy in germline BRCA1 and BRCA2 mutation-associated metastatic breast cancer is a well-tolerated and an effective therapeutic strategy, however, the durability of response can be limited. Checkpoint inhibitors targeting the PD-1/PD-L1 axis as monotherapy in metastatic triple-negative breast cancer (mTNBC) have a limited role due to low response rates, but are capable of long, durable responses. Combination PARP inhibition with checkpoint blockade is an emerging area of investigation with potential synergy to produce robust responses with durability. Mechanistically, PARP inhibition activates the stimulator of interferon gene (STING) pathway to promote dendritic cell and T lymphocyte recruitment, increases tumor neoantigens, and upregulates PD-L1 expression to increase the immunogenicity of the tumor and thereby potentially enhance responses to immunotherapy. Several clinical trials have reported early results on PARP inhibitor and PD-1/PD-L1 checkpoint inhibitor combinations. All studies have shown safety and tolerability of this combination regimen. In advanced breast cancer associated with a germline BRCA1 or BRCA2 mutation, response rates have been high and similar to what is observed with PARP inhibitor monotherapy. Additional follow-up is needed to see if combination with a checkpoint inhibitor can lead to a clinically meaningful extension of durability of response in patients with germline mutations in BRCA1 and BRCA2. In unselected mTNBC in the 1st-3rd line setting, response rates of combined PARP inhibitor and PD-1/PD-L1 inhibitors have ranged from 18-21%, with higher rates of response among those with alterations in homologous recombination DNA repair pathway genes. Multiple ongoing studies will report additional data on combinations of PARP inhibitors and checkpoint blockade in the future and this combination strategy remains an active area of investigation.

KRAS Mutation Variants and Co-occurring PI3K Pathway Alterations Impact Survival for Patients with Pancreatic Ductal Adenocarcinomas.

KRAS variant alleles may have differential biological properties which impact prognosis and therapeutic options in pancreatic ductal adenocarcinomas (PDA). We retrospectively identified patients with advanced PDA who received first-line therapy and underwent blood and/or tumor genomic sequencing at the University of Washington between 2013 and 2020. We examined the incidence of KRAS mutation variants with and without co-occurring PI3K or other genomic alterations and evaluated the association of these mutations with clinicopathological characteristics and survival using a Cox proportional hazards model. One hundred twenty-six patients had genomic sequencing data; KRAS mutations were identified in 111 PDA and included the following variants: G12D (43)/G12V (35)/G12R (23)/other (10). PI3K pathway mutations (26% vs. 8%) and homologous recombination DNA repair (HRR) defects (35% vs. 12.5%) were more common among KRAS G12R vs. non-G12R mutated cancers. Patients with KRAS G12R vs. non-G12R cancers had significantly longer overall survival (OS) (HR 0.55) and progression-free survival (PFS) (HR 0.58), adjusted for HRR pathway co-mutations among other covariates. Within the KRAS G12R group, co-occurring PI3K pathway mutations were associated with numerically shorter OS (HR 1.58), while no effect was observed on PFS. Patients with PDA harboring KRAS G12R vs. non-G12R mutations have longer survival, but this advantage was offset by co-occurring PI3K alterations. The KRAS/PI3K genomic profile could inform therapeutic vulnerabilities in patients with PDA.

Targeting Krebs-cycle-deficient renal cell carcinoma with Poly ADP-ribose polymerase inhibitors and low-dose alkylating chemotherapy.

Loss-of-function mutations in genes encoding the Krebs cycle enzymes Fumarate Hydratase (FH) and Succinate Dehydrogenase (SDH) induce accumulation of fumarate and succinate, respectively and predispose patients to hereditary cancer syndromes including the development of aggressive renal cell carcinoma (RCC). Fumarate and succinate competitively inhibit αKG-dependent dioxygenases, including Lysine-specific demethylase 4A/B (KDM4A/B), leading to suppression of the homologous recombination (HR) DNA repair pathway. In this study, we have developed new syngeneic Fh1- and Sdhb-deficient murine models of RCC, which demonstrate the expected accumulation of fumarate and succinate, alterations in the transcriptomic and methylation profile, and an increase in unresolved DNA double-strand breaks (DSBs). The efficacy of poly ADP-ribose polymerase inhibitors (PARPis) and temozolomide (TMZ), alone and in combination, was evaluated both in vitro and in vivo. Combination treatment with PARPi and TMZ results in marked in vitro cytotoxicity in Fh1- and Sdhb-deficient cells. In vivo, treatment with standard dosing of the PARP inhibitor BGB-290 and low-dose TMZ significantly inhibits tumor growth without a significant increase in toxicity. These findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDH-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy.

Improving PARP inhibitor efficacy in high-grade serous ovarian carcinoma: A focus on the immune system.

High-grade serous ovarian carcinoma (HGSOC) is a genomically unstable malignancy responsible for over 70% of all deaths due to ovarian cancer. With roughly 50% of all HGSOC harboring defects in the homologous recombination (HR) DNA repair pathway (e.g., BRCA1/2 mutations), the introduction of poly ADP-ribose polymerase inhibitors (PARPi) has dramatically improved outcomes for women with HR defective HGSOC. By blocking the repair of single-stranded DNA damage in cancer cells already lacking high-fidelity HR pathways, PARPi causes the accumulation of double-stranded DNA breaks, leading to cell death. Thus, this synthetic lethality results in PARPi selectively targeting cancer cells, resulting in impressive efficacy. Despite this, resistance to PARPi commonly develops through diverse mechanisms, such as the acquisition of secondary BRCA1/2 mutations. Perhaps less well documented is that PARPi can impact both the tumour microenvironment and the immune response, through upregulation of the stimulator of interferon genes (STING) pathway, upregulation of immune checkpoints such as PD-L1, and by stimulating the production of pro-inflammatory cytokines. Whilst targeted immunotherapies have not yet found their place in the clinic for HGSOC, the evidence above, as well as ongoing studies exploring the synergistic effects of PARPi with immune agents, including immune checkpoint inhibitors, suggests potential for targeting the immune response in HGSOC. Additionally, combining PARPi with epigenetic-modulating drugs may improve PARPi efficacy, by inducing a BRCA-defective phenotype to sensitise resistant cancer cells to PARPi. Finally, invigorating an immune response during PARPi therapy may engage anti-cancer immune responses that potentiate efficacy and mitigate the development of PARPi resistance. Here, we will review the emerging PARPi literature with a focus on PARPi effects on the immune response in HGSOC, as well as the potential of epigenetic combination therapies. We highlight the potential of transforming HGSOC from a lethal to a chronic disease and increasing the likelihood of cure.

Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

Opinion: PARP inhibitors in cancer-what do we still need to know?

PARP inhibitors (PARPi) have been demonstrated to exhibit profound anti-tumour activity in individuals whose cancers have a defect in the homologous recombination DNA repair pathway. Here, we describe the current consensus as to how PARPi work and how drug resistance to these agents emerges. We discuss the need to refine the current repertoire of clinical-grade companion biomarkers to be used with PARPi, so that patient stratification can be improved, the early emergence of drug resistance can be detected and dose-limiting toxicity can be predicted. We also highlight current thoughts about how PARPi resistance might be treated.

Abstract 3249: Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer

Abstract PARP inhibitors (PARPi) offer major clinical benefit for patients with ovarian or other cancers, especially those with deficiencies in the homologous recombination DNA repair pathway. However, most patients with advanced cancer eventually acquire resistance to PARPi, and the mechanisms by which tumors escape PARPi therapy are still not fully understood. Here, we report that treatment with the PARPi olaparib induces the formation of polyploid giant cancer cells (PGCCs) in ovarian and breast cancer cell lines (Hey, SKOV3, MCF-7), three human high-grade serous cancer (HGSC)-derived organoids, and HGSC patient-derived xenografts. Live-cell fluorescence time-lapse tracking of ovarian cancer cells labeled with FUCCI (fluorescent ubiquitination cell-cycle indicator) or histone H2B-mCherry/α-tubulin-EGFP revealed that PGCCs primarily developed from the endoreplication of diploid cancer cells after exposure to sublethal concentrations of olaparib. PGCCs exhibited markers of senescent cells; however, they were able to escape senescence to generate mitotically competent daughter cells via budding, multipolar mitosis, and acytokinetic mitosis following olaparib withdrawal. PGCCs and derived daughter cells conferred resistance to olaparib-induced cytotoxicity, which could be blocked by the anti-progesterone drug mifepristone. Whole transcriptome analysis by RNA sequencing of ovarian cancer cell lines and ovarian cancer-derived organoids showed activation of a senescence-associated secretory phenotype with upregulated cytokines and chemokines and downregulation of MYC signaling in PGCCs. Mifepristone/olaparib combination therapy significantly mitigated tumor growth in patient-derived xenograft models with acquired resistance to olaparib. Thus, targeting of PGCCs may represent a promising approach to overcome PARPi resistance in recurrent ovarian cancer. Citation Format: Xudong Zhang, Jun Yao, Xiaoran Li, Na Niu, Yan Liu, Anil K. Sood, Jinsong Liu. Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer [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 3249.

Abstract 1280: Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) are becoming the standard of care for ovarian cancer. The greatest benefit has been observed in patients who have mutations in BRCA1/2 or related genes in the homologous recombination (HR) DNA repair pathway. Nonetheless, responses to PARPi were not durable, indicating that strategies to prevent recurrence and overcome resistance, such as combinations with immune checkpoint inhibitors (ICIs), are of high priority. However, most studies on PARPi have focused on the tumor itself and synthetic lethality. The immunological effect, particularly the effect on adaptive immune response, has been overlooked. PARP is expressed in almost all cells, and it regulates the transcription of various genes through poly(ADP-ribosyl)ation. Therefore, we hypothesized that PARPi affect the composition and functions of T cells in the maintenance therapy setting, and consequently reconstitute the anti-tumor immune response in ovarian cancer. Here, we collected serial peripheral blood mononuclear cells (PBMCs) from PARPi-treated patients and analyzed their dynamic immunologic changes using muticolor flow cytometry. The PBMCs were collected during PARPi maintenance therapy, and the time points included pre-treatment as well as 1 month and 3 months after the initiation of treatment. Olaparib or niraparib was used as maintenance therapy. First, the percentages of CD4, CD8, and FoxP3+regulatory T cells (Treg cells) were compared at each time point and by PARPi type. Interestingly, the proportion of Treg cells increased at 1 and 3 months after initiation, especially in the niraparib group. However, there were no significant changes in the proportion of CD4 and CD8 T cells. Second, we analyzed immune checkpoints and properties in each T cell population. We found that the PD-1 expression on Treg cells decreased after 1 and 3 months. Although the percentage of Treg cells increased, proliferative Ki-67+Treg cells decreased after 1 and 3 months, especially in the niraparib group. Third, intracellular staining assay was performed to measure functional changes of T cells during PARPi treatment. IFN-γ and/or TNF-α producing CD8 and CD4 T cells decreased after 1 month compared to pre-treatment. Finally, proliferative Ki-67+CD8 T cells and PD-1+CD8 T cells decreased after 72 hours of in vitro olaparib treatment compared to the control group. In conclusion, long-term PAPRi treatment may affect Treg cells and CD8 T cells. However, strategies to overcome negative effects of PARPi and their detailed mechanisms need to be further investigated. Additional data in this regard will be presented at the meeting. Citation Format: Junsik Park, Miran Lee, Yup Kim, Yoo-Na Kim, Yong-Jae Lee, Jung-Yun Lee. Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients [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 1280.

Abstract 5103: Characterization of the genomic landscape and actionable mutations in Taiwanese prostate cancer patients

Abstract Background: Prostate Cancer (PCa) is the second most common male cancer worldwide and the most common cancer in males over 65 years old. PCa is the sixth most common cancer in Taiwan, and its incidence rate has increased gradually over the past 30 years. PAPR inhibitors are the only type of targeted drug approved by the FDA to treat advanced castration-resistant prostate cancer (CRPC) that has grown after the hormone therapy drugs. This study aimed to characterize the genomic profile of Taiwanese PCa and explore whether there are potential treatment guidances derived. Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 99 patients diagnosed with PCa and collected at Chi-Mei Medical Center (CMMC) were subject to next-generation sequencing using ACTOnco® CGP panel that cover coding regions of 440 cancer-related genes. Genomic alterations (GAs) and signatures, including single nucleotide variations (SNVs), short insertions and deletions (InDels), copy-number variations (CNVs), fusion genes, tumor mutations burden (TMB), and microsatellite instability (MSI) status were retrieved. The gene mutation prevalence was compared with the Western population using the TCGA prostate cancer dataset. Results: A total of 1558 variants was identified, including 215 SNVs, 22 InDels, 1178 CN gain and 135 homozygous deletions. Similar to the Western population, the most frequently mutated genes were SPOP (21.2%), KMT2C (10.1%), MUC16 (8.1%), TP53 (6.1%), ARID2 (5.1%), IDH1 (5.1%), PTGS2 (5.1%). Further analysis showed that 12 patients harbored deleterious/likely deleterious mutations in genes implicated in homologous recombination (HRR) DNA repair pathway, including three BRCA2, two CDK12, BRAD1 and ARID1A, respectively, and one ATR, ATM and PALB2, respectively. All BRCA2 mutations were predicted to be germline origin and only one-third of 12 HRR mutants had a biallelic gene loss. Median TMB calculated from NGS data is 0.6 muts/Mb (range 0 to 4.5) and all samples are microsatellite-stable (MSS). Therapeutically actionable gene and pathway analysis revealed that up to 24.2% of patients harbored at least one aberration against FDA-approved targeted drugs, such as MET copy number (CN) gain (11.1%), mTOR pathway aberration (6.1%), HRR gene biallelic loss (4%), BRAF activating mutation (4%), IDH1 activating mutations (4%), EGFR CN gain (3.0%), FGFR1 CN gain (2.0%), MET fusion (1.0%) and BRAF fusion (1%). Conclusion: This CGP study unraveled the genomic profile of Taiwanese prostate cancers, and the results showed that GAs identified could serve as a potential biomarker to guide personalized, targeted therapies in one-fourth of patients. Citation Format: Kien Thiam Tan, Chia-Ling Wu, Yen-Jung Lu, Ka-Po Tse, Chien-Feng L Li. Characterization of the genomic landscape and actionable mutations in Taiwanese prostate cancer patients [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 5103.

DOLAF: An international multicenter phase II trial of durvalumab (MEDI4736) plus olaparib plus fulvestrant in patients with metastatic or locally advanced ER-positive, HER2-negative breast cancer selected using criteria that predict sensitivity to olaparib.

TPS1116 Background: PARP inhibitors have documented clinical activity in patients with HER2 negative breast cancer (BC) and a germline pathogenic variant (PV) in BRCA1 or BRCA2. Defects in other genes involved in homologous recombination DNA repair (HRR) or mismatch repair pathway (microsatellite instability MSI) have been associated with preclinical cellular sensitivity to PARP inhibitors. Several preclinical and clinical studies have suggested synergy between immune checkpoint blockade and PARP inhibitors. Indeed, tumors with deficiency in HRR have higher mutagenic potential and produce a larger number of neoantigens. Around 60% of BC with a germline PV in BRCA1/ 2 are ER+/HER2- tumors, and the ER-pathway remains a key target of their therapy. The combination of PARP inhibitors with endocrine therapy has shown to be superior to monotherapy. Methods: DOLAF is an open-label, international, multicentric, phase II trial assessing the combination of olaparib, fulvestrant, and durvalumab in ER+/HER2- metastatic or locally advanced BC with somatic or germline PV in BRCA1, BRCA2 or other genes implicated in the HRR pathway (ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FAND2, FANCL, MRE11A, NBN, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L) or in MSI status or other actionable genes ( AKT1, ESR1, FGFR1, FGFR2, FGFR3, and PIK3CA) all based on central tumor NGS. Further an amendment in May 2021, patients with only alterations in these other actionable genes can no longer be included. Patients must have received 1 prior line of endocrine therapy for their metastatic BC, including CDK4/6 inhibitor and maximum of 1 line of chemotherapy in the metastatic setting. Patients receive olaparib (twice daily at 300 mg), fulvestrant (2 intramuscular injections of 250 mg every 28 days) and durvalumab (1500 mg intravenous every 4 weeks). The primary objective is to evaluate the progression-free survival rate at 24 weeks. Secondary endpoints include safety, overall survival, objective response rate, in the overall population and in the germline BRCA mutated population. With an optimum two-stage Simon design, α = 2.5%, β = 5%, p0 (probability of inefficiency maximum) = 50%, p1 (probability of minimum efficiency) = 65%, it is necessary to include 158 patients. The strategy could be considered sufficiently effective if there are at least 87 patients without progression at 24 weeks. Given the lack of safety data from this association, a safety run-in phase including 6 patients has been completed without DLT. As of December 31, 2021, 266 patients have been screened of whom 102 have been treated. The first interim analysis occured in November 2021 after the inclusion of 64 evaluable patients. IDMC suggested that the trial continue as planned. Clinical trial information: NCT04053322.

Synergistic activity of PARP inhibitors (PARPi) in combination with standard chemotherapy (CTx) in leiomyosarcoma.

11560 Background: Leiomyosarcomas (LMS) are genetically heterogeneous tumors that arise from smooth muscle. Currently, the mainstay of systemic treatment for patients with advanced/metastatic disease is doxorubicin (Dox) based CTx. Several genomic analyses of LMS reveal defects in homologous recombination (HR) DNA repair pathway in about half of patients, consistent with a druggable “BRCAness” phenotype. Thus, we sought to determine which combinations of standard CTx and PARPi might be synergistic promising therapeutic strategies for LMS. Methods: Dox, Docetaxel (Doc), Temozolomide (Tmz) were evaluated in combination with PARPi (Olaparib [Ola], Niraparib [Nira] and Talazoparib [Tala]) at 12 different drug concentrations. Four LMS cell lines of different origins (gynecological - GY, abdominal - A, extremity - E) were tested in a high throughput manner. All drug concentrations were chosen according to EC50. Cells were incubated with each combination for 7 days. Viability was assessed by ATPlite Luminescence Assay System.Evaluation of drug combination effect was performed using a Bliss synergy score. This system quantifies the degree of synergy as multiplicative effect of single drugs as if they acted independently. With a synergy score of -5 to 5, the interaction between two drugs is considered as additive; <-5 antagonistic and > 5 synergistic, and therefore a promising combination. Results: Anticancer activity, ranging from additive to synergistic was seen with all combinations. Results were consistent among all cell lines, independent of site of cell line origin (Table) Most synergistic combination in the majority of LMS cell lines were Dox or Tmz when combined with Tala, reaching up to 15 % and 27% above Bliss respectively. In contrast, Doc showed only additive effect with all analyzed PARPi. Conclusions: The data suggest that the combination of Dox or Tmz with PARPi may represent promising treatment options for LMS patients. Recent clinical studies support this notion in uterine LMS. Importantly these results suggest that such approach may be extended to all sites of LMS. Pre-clinical studies are underway to identify the most promising combinations for future clinical trial design. [Table: see text]

Pharmacologic Induction of BRCAness in BRCA-Proficient Cancers: Expanding PARP Inhibitor Use.

Simple SummaryBRCA1 and−2 are critical components of the homologous recombination pathway of DNA repair required to effectively repair DNA double strand breaks, leading to an increased cancer risk in patients with inherited BRCA mutations. An additional subset of cancers exhibit ‘BRCAness’, harboring repair defects stemming from mutations in non-BRCA DNA repair genes. Both BRCA-mutant cancers and cancers with a BRCAness phenotype are sensitive to PARP inhibitors, a class of cancer therapy drugs that inhibit the repair of DNA single strand breaks. To expand the use of PARP inhibitors to a larger group of patients, studies have focused on new combination strategies using agents that can induce BRCAness. This review focuses on the current status of drug-induced BRCAness in combination with PARP inhibitors to enhance cancer treatment.The poly(ADP-ribose) polymerase (PARP) family of proteins has been implicated in numerous cellular processes, including DNA repair, translation, transcription, telomere maintenance, and chromatin remodeling. Best characterized is PARP1, which plays a central role in the repair of single strand DNA damage, thus prompting the development of small molecule PARP inhibitors (PARPi) with the intent of potentiating the genotoxic effects of DNA damaging agents such as chemo- and radiotherapy. However, preclinical studies rapidly uncovered tumor-specific cytotoxicity of PARPi in a subset of cancers carrying mutations in the BReast CAncer 1 and 2 genes (BRCA1/2), which are defective in the homologous recombination (HR) DNA repair pathway, and several PARPi are now FDA-approved for single agent treatment in BRCA-mutated tumors. This phenomenon, termed synthetic lethality, has now been demonstrated in tumors harboring a number of repair gene mutations that produce a BRCA-like impairment of HR (also known as a ‘BRCAness’ phenotype). However, BRCA mutations or BRCAness is present in only a small subset of cancers, limiting PARPi therapeutic utility. Fortunately, it is now increasingly recognized that many small molecule agents, targeting a variety of molecular pathways, can induce therapeutic BRCAness as a downstream effect of activity. This review will discuss the potential for targeting a broad range of molecular pathways to therapeutically induce BRCAness and PARPi synthetic lethality.

Transient Receptor Potential Cation Channel Subfamily V Member 1 Expression Promotes Chemoresistance in Non-Small-Cell Lung Cancer.

Approximately 85% of lung cancer cases are non-small-cell lung cancer (NSCLC). Chemoresistance is a leading cause of chemotherapy failure in NSCLC treatment. Transient receptor potential cation channel subfamily V, member 1 (TRPV1), a non-selective cation channel, plays multiple roles in tumorigenesis and tumor development, including tumor cell proliferation, death, and metastasis as well as the response to therapy. In this study, we found TRPV1 expression was increased in NSCLC. TRPV1 overexpression induced cisplatin (DDP) and fluorouracil (5-FU) resistance in A549 cells independent of its channel function. TRPV1 expression was upregulated in A549-DDP/5-FU resistant cells, and DDP/5-FU sensitivity was restored by TRPV1 knockdown. TRPV1 overexpression mediated DDP and 5-FU resistance by upregulation of ABCA5 drug transporter gene expression, thereby increasing drug efflux, enhancing homologous recombination (HR) DNA repair pathway to alleviate apoptosis and activating IL-8 signaling to promote cell survival. These findings demonstrate an essential role of TRPV1 in chemoresistance in NSCLC and implicate TRPV1 as a potential chemotherapeutic target.

Abstract P055: Targeting Krebs-cycle-deficient renal cell carcinoma with PARP inhibitor and low-dose alkylating chemotherapy

Abstract Loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) lead to excess accumulation of fumarate and succinate, respectively. Germline mutations in FH lead to a genetic predisposition to hereditary leiomyomatosis and renal cell cancer (HLRCC)-associated RCC. Similarly, loss-of-function alterations of SDH, most commonly SDHB, are associated with SDH-deficient neoplasms, including RCC. FH and SDHB deficient RCC tends to be aggressive and metastasize early with very limited treatment options. The oncometablites produced by these mutations, fumarate and succinate, competitively inhibit αKG-dependent dioxygenases, resulting in dysregulated DNA methylation and histone modification. We have previously demonstrated that elevated levels of fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway through inhibition of the lysine demethylase KDM4B, resulting in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks and masking a local H3K9 trimethylation signal that is essential for the proper execution of HR. In this study, we sought to identify novel treatment approaches that exploit genomic instability in FH- and SDHB-deficient RCC. Temozolomide (TMZ), an alkylating agent, mediates its cytotoxic effect by attaching methyl groups to DNA (O6-guanine, N7-guanine and N3-adenine). 16 N3-MetA and N7-MetG repair are mediated by the base excision repair (BER) in a process involving PARP. While the combination of PARP inhibitors with TMZ has been shown to increase TMZ-induced cytotoxicity, clinical trials investigating combination therapy have been hampered by significant toxicity. Using CRISPR/Cas9, we developed new syngeneic FH- and SDHB-deficient murine models of RCC. We demonstrate that FH- and SDHB-deficient cells have accumulation of fumarate and succinate leading to an increase in unresolved DNA double-strand breaks (DSBs). Treatment with PARP inhibition and temozolomide results in marked in vitro cytotoxicity in FH- and SDHB-deficient cells, even at low concentrations of TMZ (50 µM). In vivo, the combination of standard dose BGB-290 and low-dose TMZ (3mg/kg/dose) results in significantly delayed tumor progression in an SDHB deficient RENCA model without any significant increase in toxicity. Notably, the TMZ dose of 3mg/kg/dose is significantly lower than the 50mg/kg/dose that is commonly used for in vivo studies. Taken together, these findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDHB-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy. Furthermore, the development of a new syngeneic mouse model provides a tool for the future study of immunotherapy in Krebs-cycle-deficient RCC. Citation Format: Daiki Ueno, Amrita Sule, Jiayu Liang, Jinny van Doorn, Ranjini Sundaram, Randy Caliliw, Huihui Ye, Rong Rong Huang, Jing Li, Karla Boyd, Ranjit S. Bindra, Juan C. Vasquez, Brian M. Shuch. Targeting Krebs-cycle-deficient renal cell carcinoma with PARP inhibitor and low-dose alkylating chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P055.

Selected Articles from This Issue

Glioblastomas (GBM) are highly invasive brain tumors that can spread rapidly throughout the brain. GBM are marked by a high rate of disease recurrence after therap. by virtue of the self-renewal capacity of glioma stem cells (GSCs), which are highly heterogeneous. Here, Thomas and colleagues identify a subset of GSCs bearing copy number alterations of the ASNS gene, leading to elevated expression of asparagine synthase. ASNS-high GSCs bore distinct metabolic features from other GSCs, including increased capacity for both glycolysis and oxidative phosphorylation and the ability to switch between them as needed in response to environmental cues. This metabolic plasticity allowed for rapid dissemination of ASNS-high GSCs throughout the brain tissue and a marked resistance to oxidative stress, rendering these cells de novo resistant to radiation. These findings nominate ASNS as a potential therapeutic target for GBM, which could restrict the pathogenicity of GSCs and enhance the efficacy of standard treatment modalities.Inhibitors of poly (ADP-ribose) polymerase (PARPi) are known to be effective in treating a range of tumor types, particularly as a synthetic-lethal approach for tumors with defects in the homologous recombination DNA repair pathway. However, dose-limiting toxicities, particularly hematological toxicities, can cause early cessation of treatment and loss of therapeutic benefit. Here, using a CRISPR/Cas9 screen, Xu and colleagues identify the CHK2-p53 pathway as a key mediator of hematological toxicity in response to the clinically approved PARPi agent, olaparib. CHK2 was required for efficient activation of p53 in response to PARPi in blood cells, and genetic approaches that blocked this connection allowed cells to better tolerate olaparib treatment. Concurrent inhibition of CHK2 alongside olaparib emulated this protective effect, but importantly did not affect PARPi-mediated killing of ovarian cancer cell lines. If this finding extends to people, then these data nominate CHK2 inhibition as a potential target to spare patients from hematological adverse effects in response to PARPi, which could broaden the window of therapeutic benefit for these agents.Analogous to epigenetic control of gene expression through histone methylation, epitranscriptomic modifications such as N6-methyladenosine (m6A) function to modulate RNA stability, processing, and translation. In this study, Cotter and colleagues profile the activity of METTL3, a key constituent of the methyltransferase complex that catalyzes m6A modifications, and its role in establishing the pathologic transcriptome observed in metastatic castration-resistant prostate cancer (mCRPC). The authors observe that ablation of METTL3—and by extension, m6A modifications—is associated with resistance to androgen receptor signaling inhibitors, a key therapeutic modality in the clinical management of prostate cancer. This effect was shown to be mediated independently of the androgen signaling pathway, and instead relied on activation of a gastrointestinal-specific transcriptional signature driven by the expression of the nuclear receptor NR5A2/LRH-1. Taken together, these observations elucidate a novel mechanism by which epitranscriptomic m6A modifications may play a role in establishing the mCRp. transcriptome and in promoting disease progression.Pancreatic ductal adenocarcinoma (PDAC) patients often present with metastatic disease and face a dismal prognosis. Because of the high rate of metastasis observed in PDAC tumors, systemic therapies are frequently the only viable option for clinical management; however, PDAC lacks an effective armamentarium of targeted interventions, and tumors quickly develop resistance to standard chemotherapeutic regimens. Using single-cell sequencing of mouse PDAC tumors, Du and colleagues identify the receptor tyrosine kinase AXL as a mediator of PDAC progression and metastatic spread. The authors demonstrate that AXL is overexpressed in PDAC tumor cells with a mesenchymal phenotype, and that its activity promotes and is required for epithelial-mesenchymal transition and metastasis in vivo. Concordantly, PDAC tumors that developed in AXL-deficient mice were less aggressive, better differentiated, and more sensitive to gemcitabine chemotherapy than PDAC in mice bearing wild-type AXL. These data suggest that targeting AXL expression and/or activity may present an opportunity to suppress or treat PDAC progression.

Abstract 2056: The genomic landscape of carriers of rare variants in FANCI, a new candidate ovarian cancer predisposing gene

Abstract The potentially pathogenic variant (PPV), FANCI c.1813C>T; p.L605F, in a new candidate ovarian cancer (OC) predisposing gene was discovered by whole exome sequencing (WES) of familial OC cases from the founder French Canadian (FC) population for discovering new OC predisposing genes. Modeling this variant in cellulo suggested this variant encodes an unstable protein. FANCI is an essential member upstream of the homologous recombination DNA repair pathway and intersects BRCA1 and BRCA2 function, proteins encoded by genes involved in hereditary OC. Investigating the FC founder population facilitates the discovery of PPVs as they are more likely to harbor recurrent variants due to common ancestors, increasing the likelihood of identifying candidate genes in cancer families. To further support the candidacy of FANCI as a new OC predisposing gene, we investigated the germline landscape of c.1813C>T carrier FC OC cases for co-occurring PPVs in known or proposed new (emerging) OC predisposing genes or other genes involved in similar pathways (DNA repair). Using WES of peripheral blood lymphocyte DNA, the genomic landscape of 10 FANCI carriers were investigated for heterozygous PPVs in 276 DNA repair pathways genes, which included known (BRCA1, BRCA2, MSH2, MLH1, MSH6, PMS2) and emerging (BRIP1, RAD51C, RAD51D) OC predisposing genes. Top ranking candidate variants (minor allele frequency <1%) were identified using 11 different in silico tools that assessed amino acid conversation or potential pathogenicity. Pathogenicity of known and emerging OC predisposing genes was assessed using BRCAExchange (www.brcaexchange.org) and ClinVar (www.ncbi.nlm.nih.gov/clinvar/). A similar analysis was done with WES data from 13 FC OC cases harboring pathogenic BRCA1/BRCA2 variants. We identified 31 variants in 27 genes in FANCI c.1813C>T carriers. A previously known carrier of a pathogenic BRCA1 variant (c.2836_2837del; p.Ile946GlnfsTer5) was also identified in a familial case. No carriers of other pathogenic variants in known or emerging OC predisposing genes were found. However, FANCI carriers were found to carry at least one other PPV in a DNA repair pathway gene (range 2-9; average=4.1). There were no other carriers of variants in common among all OC cases, though at least 2 cases carried a variant in the same gene. FC OC cases harboring BRCA1/BRCA2 variants carried at least one other PPV in a DNA repair pathway gene (range 2-7; average 4). It is possible that the identified variants influence or modify risk in conjunction with FANCI, though no PPV was identified in all carriers. As new cancer predisposing genes are identified it will become increasingly important to characterize the genetic context in which variants are identified. This will allow for further insight to clinical translatability once penetrance has been established. Citation Format: Caitlin Fierheller, Wejdan M Alenezi, Corinne Serruya, Timothée Revil, Javad Nadaf, Anne-Marie Mes-Masson, Diane Provencher, William D Foulkes, Zaki El Haffaf, Celia M T Greenwood, Jean-Yves Masson, Jiannis Ragoussis, Patricia N Tonin. The genomic landscape of carriers of rare variants in FANCI, a new candidate ovarian cancer predisposing gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2056.

PRMT5 Regulates DNA Repair By Controlling the Alternative Splicing of Epigenetic Factors

PRMT5 is the major type II arginine methyltransferase that symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. PRMT5 is overexpressed in many cancer types and PRMT5 inhibition has recently been shown to be a promising therapeutic approach for these diseases, including lymphoma and leukemia. PRMT5 plays an important role in adult mouse hematopoiesis, as its conditional deletion in the hematopoietic compartment (using an Mx1-Cre based system) leads to rapid, lethal, bone marrow aplasia (F. Liu et al, JCI 2015). To avoid excess toxicity, we have been investigating the myriad of cellular processes regulated by PRMT5, in order to identify combinatorial approaches, that can exploit potential synthetic vulnerabilities, similar to what has been shown for loss of MTAP and PRMT5 inhibition. Here we report that PRMT5 deletion or inhibition leads to the accumulation of DNA damage, at least in part due to defects in the homologous recombination (HR) DNA repair pathway, which ultimately activates the p53 pathway and leads to cell cycle arrest and cell death. Using a Vav1-Cre based PRMT5 knockout mouse model that we generated, we have found that PRMT5 knockout induces specific dysregulation of RNA splicing, including alternative splicing of the multifunctional epigenetic and DNA repair factor Tip60/KAT5. Aberrant splicing of Tip60 leads to selectively defective acetyl-transferase activity, including histone H4 acetylation, a key event in DNA repair pathway choice. Consistently, we found that ectopic expression of wild-type Tip60 in PRMT5-null cells rescues the homologous recombination phenotype. Thus, we have uncovered a novel interaction between PRMT5-dependent RNA splicing and DNA repair, that functions to regulate genomic stability and cellular homeostasis. Importantly, these findings can be exploited therapeutically as PARP inhibitors have been shown to be effective in HR defective cells, such as BRCA1 or BRCA2 mutant cells. We show here that PRMT5 inhibitors and PARP inhibitors have synergistic effects on leukemia cell lines and mouse models, thus demonstrating the advantages of combining targeted epigenetic and non-epigenetic inhibitors. DisclosuresNo relevant conflicts of interest to declare.

APOBEC3G rescues cells from the deleterious effects of DNA damage.

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and rescues transgenic mice from a lethal dose of ionizing radiation. Here, we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet irradiation and by combined bromodeoxyuridine and ultraviolet treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cell sensitivity to genotoxic treatments.

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

A phase II clinical trial of talazoparib monotherapy for PALB2 mutation-associated advanced breast cancer.

TPS1109 Background: PALB2 (Partner and Localizer of BRCA2) plays a critical role in the maintenance of genomic integrity through its role in the Fanconi anemia and homologous recombination DNA repair pathways. Our recently reported phase II clinical trial (Gruber, et al. JCO 2019) showed that talazoparib monotherapy was associated with single agent activity in germline PALB2 (gPALB2) mutation-associated advanced breast cancers. Of 5 patients with a germline PALB2 mutation, all 5 had reduction in target lesions > 20% with 3 of 5 achieving a RECIST 1.1 response. All patients had visceral metastases and were heavily pretreated; one response occurred in a patient with 8 prior lines of therapy for metastatic breast cancer. Clinical activity of the PARP inhibitor olaparib was also demonstrated in breast cancer patients with gPALB2 mutations in the Olaparib Expanded trial (TBCRC 048). Thus, we believe there is significant value in generating additional PARP inhibitor monotherapy data in subjects with advanced PALB2 mutation-associated breast cancer. We propose this phase II trial to better estimate the rate of RECIST 1.1 objective response in this patient population. Methods: This is a single-arm, two-stage, non-randomized study to assess the objective response rate (ORR) of talazoparib monotherapy in patients with PALB2 mutation-associated advanced breast cancer. Eligible subjects will be adults with inoperable locally advanced or metastatic breast cancer with a germline or somatic PALB2 mutation with 0-3 prior therapies for advanced disease. Eligible patients must have a deleterious or suspected deleterious mutation in PALB2 on a CLIA approved commercial germline or next generation sequencing tumor assay. Study treatment is talazoparib 1 mg PO daily. In stage 1, ORR will be assessed after 15 patients; if at least 2 responses are observed then an additional 15 patients will be enrolled; if less than 2 responses are observed the study will be terminated. The primary objective is to evaluate whether talazoparib monotherapy can induce a 30% ORR in subjects with advanced breast cancer associated with a PALB2 mutation. Response will be assessed by RECIST 1.1. Secondary objectives include safety, PFS, clinical benefit rate, and patient-reported quality-of-life. Exploratory endpoints will assess for the presence of PALB2 loss-of-heterozygosity, biallelic mutations, correlation of ctDNA profile with treatment response and the correlation of germline versus somatic mutations with response rate. This trial is currently activated and enrolling at Stanford Cancer Center and it is expected that 4 additional sites will be added. Conclusion: This trial will evaluate gPALB2 as a biomarker for PARP inhibitor monotherapy in advanced or metastatic HER2- breast cancer. Clinical trial information: pending .