DNA Damage Repair Deficiency Research Articles

Abstract CT157: Phase IB study of nivolumab and veliparib in patients with advanced solid tumors and lymphoma with and without alterations in selected DNA repair genes

Abstract Background: Genomic alterations in DNA repair genes is associated with genomic instability and increase tumor mutation burden. PARP inhibitors including veliparib and PD-1 inhibitors including nivolumab are effective in tumors harboring DNA Damage Repair (DDR) deficiency and high TMB, respectively. Methods: We conducted a phase Ib study to evaluate the safety and efficacy of nivolumab and veliparib in patients with advanced solid tumors or aggressive lymphoma. Study population included patients with advanced malignancies who had progressed at least in one prior line of treatment and had adequate kidney and liver function. All patient had genomic profiling of the tumor prior to enrollment demonstrating at least one mutation in selected DNA repair genes. Nivolumab was administered 240 mg on D1 and D15 every 28 days for the first 4 cycles and continued 480 mg monthly thereafter. Veliparib was administrated orally in a two-stage dose escalation. Three patient received 300 mg BID with no evidence of dose limiting toxicities allowing to increase the dose to 400 mg BID. Primary endpoints were to assess tolerability of nivolumab and veliparib combination and also to determine maximum tolerated dose (MTD). Secondary endpoint was to evaluate objective response rate (ORR). Results: From May 2017 through August 2019, 15 patients with advanced malignancies meeting the eligibility criteria were enrolled in the study. The first three patients received the lower dose of veliparib, 300 mg BID. None of those had dose limited toxicity so the dose of veliparib increased to 400 mg BID. Three more patient received 400 mg BID with no unacceptable toxicity concluding that the MTD of veliparib to be 400 mg. All subsequent patient received the MTD of veliparib. The combination of veliparib 400 mg BID and nivolumab either 240 mg every two weeks or 480 mg monthly was reasonably well tolerated. Grade 3-4 adverse events occurred in 7 patients with the most common being anemia and fatigue. ORR and survival data were assessed for 14 patient, 1 patient denied treatment after screening and withdrew the informed consent. No response was observed. Of note, a patient with pancreatic cancer harboring BRIP1 mutation received 12 cycles of treatment (24 weeks) without disease progression. PFS and OS were 9.0 and 26.8 weeks, respectively. Discussion: Combination of nivolumab and veliparib is a very well tolerated regimen. Even though this study did not meet its primary endpoint in response evaluation, we observed a durable stabilization of disease in two patients with pancreatic cancer with and without mutations in DDR genes. Further large-scale clinical studies are warranted to evaluate the efficacy of combination treatment with immunotherapy and PARP inhibitor and to identify biomarkers that predict response to such treatment. Citation Format: Elena Vagia, Pedro Viveiros, Cyra-Yoonsun Kang, Aparna Kalyan, Valerie Nelson, Devalingam Mahalingam, Young Kwang Chae. Phase IB study of nivolumab and veliparib in patients with advanced solid tumors and lymphoma with and without alterations in selected DNA repair genes [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 CT157.

Abstract B61: The genotype of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immune checkpoint therapies

Abstract Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) DNA repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNASeq (scRNASeq) transcriptomic, and immunofluorescence imaging analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Tian Xia, Ferenc Reinhardt, Tony Chavarria, Esmee Hoefsmit, Vera Poort, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Paula T. Hammond, Benjamin G. Neel, Robert A. Weinberg. The genotype of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immune checkpoint therapies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B61.

Damage Incorporated: Discovery of the Potent, Highly Selective, Orally Available ATR Inhibitor BAY 1895344 with Favorable Pharmacokinetic Properties and Promising Efficacy in Monotherapy and in Combination Treatments in Preclinical Tumor Models.

The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).

Translational analysis of esophageal adenocarcinoma (EAC) patients treated with oxaliplatin and capecitabine (Xelox) +/- the dual ErbB inhibitor AZD8931 in the DEBIOC study.

4539 Background: The Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC) trial reported an acceptable safety profile for neoadjuvant Xelox +/- AZD8931 but limited efficacy. We utilized EAC patient samples from DEBIOC to evaluate the impact of neoadjuvant Xelox +/-AZD8931 on biological pathways using a unique software driven solution. Methods: 24 pre-treatment FFPE EAC biopsies and 17 matched surgical resection specimens were transcriptionally profiled using the Almac Diagnostics Xcel Array. Gene expression data was analyzed using the Almac claraT total mRNA report V3.0.0, reporting on 92 gene expression signatures and 7337 single genes associated with 10 key biologies. Paired Wilcoxon tests (5% significance level) were used to evaluate changes in claraT scores pre- and post-treatment. EGFR and Her2 expression were assessed by IHC and FISH. Results: 15 patients received Xelox+AZD8931 and 9 Xelox alone. Hierarchical clustering of biopsies identified 4 major clusters: Inflammation active, Genomic Instability active, EGFR & MAPK active, and EMT & Angiogenesis active. Comparison of signature scores pre- and post- neoadjuvant treatment demonstrated a significant reduction in scores relating to DNA damage repair (DDR) deficiency (Almac DNA Damage assay, p< 0.0001; BRCAness Profile, p= 0.0025; HRD Gene Signature, p< 0.0001; BRCA1ness Signature, p= 0.0004) and a significant increase in angiogenesis signatures (Almac Angiogenesis Assay, p= 0.0002; Angio Predictive G model, p= 0.0228; Angiogenesis Signature A, p= 0.0034) and EMT signatures (EMT Signature, p= 0.0031, EMT Enrichment Score, p= 0.0013, Pan-Can EMT Signature B, p= 0.0001). Comparing pre- and post-treatment signature scores in patients treated with Xelox +/-AZD8931 revealed a significant reduction in EGFR Sensitivity Signature ( p= 0.0088), ERBB2-specific Gene Expression Signature ( p= 0.0127) and Hallmark PI3K-AKT-MTOR Signaling ( p= 0.0195) in those treated with Xelox + AZD8931 in keeping with the mechanism of action of AZD8931. Downregulation of AKT signaling was confirmed in AZD8931 treated and resistant cell lines. Conclusions: We report the use of a novel software tool to apply 92 gene expression signatures to EAC biopsy and resection specimens from the DEBIOC trial to provide insight into mechanisms of action. Neoadjuvant treatment was associated with a reduction in DDR deficiency and an increase in angiogenesis and EMT signatures whilst a reduction in EGFR, Her2 and AKT pathways was noted with AZD8931 treatment.

Germline and somatic DNA damage repair gene mutations potentially predict the efficacy of relevant treatment in Chinese patients with pancreatic ductal adenocarcinoma.

e16732 Background: PDAC is a fatal disease with molecular heterogeneity, inducing differences in biological behaviour and therapeutic strategy. We conducted a study to reveal the mutation landscape of Chinese PDAC patients, and investigate the predictive role of germline and somatic DNA damage repair (DDR) status in precise treatment. Methods: 195 PDAC patients were enrolled from multiple medical centers of China between Jan 2016 to Nov 2019. Baseline clinical or genetic characteristics, and survival status were collected. NGS were performed on paraffin-embedded resected tissues or peripheral blood using a panel of 417 genes, including 50 DDR-related genes. Survival analysis was conducted using Kaplan-Meier, and Cox proportional hazard regression model. Results: The main driver genes were KRAS, TP53, CDKN2A, and SMAD4. Patients with KRAS mutation showed worse OS than those without (p = 0.048). DDR deficiency were identified in 15.38% of overall patients, mainly occurred in BRCA2 (4.62%), ATM (4.10%), RAD50 (1.54%) and MLH1 genes (1.03%). No significant improvement of OS existed between patients with or without DDR mutations (p = 0.88). Treatment with olaparib (adjusted HR, 0.2550; P = 0.0720) or platinum-based chemotherapy (adjusted HR, 0.1308; P = 0.0185) respectively decreased hazard of death in patients with DDR mutation. Besides BRCA gene, ATM mutant patients treated with olaparib harbored prolonged median OS than those without olaparib treatment (22.25 vs 15.2 month). Despite a little higher PD-L1 expression rate were seen in DDR mutant patients (29.17% vs 20.51%), no statistical correlation between tumor mutation burden level and DDR mutation was identified. And in patients treated with PD-1 blockade, 2 patients of DDR wild-type group both had SD, whereas of the remaining 5 patients with DDR deficiency, 1 was evaluated as PR, 3 as SD, and 1 as PD (ORR, 0 wt vs 20% mut). Conclusions: In this multi-center retrospective study, we deciphered the intra-tumoral genetic heterogeneity in Chinese PDAC population, which differs from western patients cohort to some extent. We found the potential role of germline and somatic DDR mutation status in predicting the response to olaparib and platinum-based chemotherapy, especially with BRCA or ATM mutation. However, DDR alteration was limited in prediction of hypermutational status and sensitivity to PD-1 blockade. Our study may provide a valuable evidence for clinical application of DDR mutation as a potential biomarker for precise treatment.

Preliminary study on the function of the POLD1 (CDC2) EXON2 c.56G>A mutation.

BackgroundFanconi anemia (FA) is a rare recessive disease characterized by DNA damage repair deficiency, and DNA polymerase δ (whose catalytic subunit is encoded by POLD1, also known as CDC2) is closely related to DNA damage repair. Our previous study identified a novel POLD1 missense mutation c.56G>A (p. Arg19>His) in FA family members. However, the function of the POLD1 missense mutation is currently unknown. This study aimed to uncover the biological function of the POLD1 missense mutation.MethodsStable cell lines overexpressing wild‐type POLD1 or mutant POLD1 (c.56G>A, p.Arg19His) were constructed by lentivirus infection. Cell growth curve analysis, cell cycle analysis, and a comet assay were used to analyze the function of the POLD1 mutation.ResultsThe growth and proliferative ability of the cells with POLD1 mutation was decreased significantly compared with those of the wild‐type cells (Student's t test, p < .05). The percentage of cells in the G0/G1 phase increased, and the percentage of cells in the S phase decreased significantly when POLD1 was mutated (Student's t test, p < .05). Moreover, the Olive tail moment value of the cells with the POLD1 mutation was significantly higher than that of the cells with wild‐type POLD1 after H2O2 treatment.ConclusionsThe POLD1 mutation inhibited cell proliferation, slowed cell cycle progression, and reduced DNA damage repair.

Exceptional sustained responses to ipilimumab/nivolumab (ipi/nivo) in patients (pts) with advanced pancreaticobiliary cancers and germline DNA damage repair (DDR) mutations.

e16757 Background: Immune checkpoint inhibitors (ICIs) have not shown meaningful clinical activity in unselected pts with pancreaticobiliary cancer. DDR-deficient tumors have increased genomic instability, including increased tumor mutation burden (TMB), more tumor-infiltrating immune cells, and enrichment of a T cell-inflamed signature. We hypothesized that pts with DDR deficiency may be sensitive to ICIs. Methods: Utilizing the IRB-approved database at the University of Miami, we identified pts with advanced pancreaticobiliary cancer with germline DDR mutations who were treated with dual ICI. Results: Eleven pts were identified (3 BRCA1, 4 BRCA2, 1 RAD51C, 1 RAD51D, 1 ATM, and 1 PALB2). There were two complete responses (CR), one partial response (PR), three stable disease (SD), two progressive disease (PD), and three pending evaluation. Evaluable responses are sustained for 22 months (m), 19m, and 7m, respectively. All pts had microsatellite stable tumors and none had high TMB. Among pts with responses, TMB was 4, 8, and 8 mut/Mb, respectively. One pt with a CR had gBRCA1 with pancreatic adenocarcinoma (PDAC) s/p distal pancreatectomy/splenectomy, adjuvant gemcitabine (gem)/capecitabine, biopsy-proven recurrence in lung and retroperitoneum 12m later, initiated ipi/nivo, and achieved a CR ongoing for 22m on nivo maintenance. Second pt with a CR had gBRCA1 and unresectable cholangiocarcinoma (CC), with disease progression after gem/cisplatin (cis), therapy switched to ipi/nivo. After 4 doses, an incidental cerebellar lesion was found which was biopsy-proven CC, therefore taken off treatment. Pt has been in CR for 19m without further therapy. The pt with a PR had RAD51C and metastatic PDAC s/p FOLFIRINOX for 6m, followed by olaparib for 12m. At progression, disease rapidly progressed through 5-fluorouracil/nanoliposomal irinotecan, gem/nab-paclitaxel/cis, and FOLFIRINOX. Pt was initiated on ipi/nivo with immediate improvement in pain and tumor markers; PR ongoing for 7m with continued clinical and tumor marker improvement on nivo maintenance. Additional correlative studies (RNA sequencing, Hyperion Imaging, mutation allele fraction analysis) among responders vs. nonresponders will be reported at the meeting. Conclusions: In this series, 6 out of 8 evaluable pts with advanced pancreaticobiliary cancers had disease control with ipi/nivo. This series suggests that tumors with germline DDR mutations may be responsive to ICIs. Further evaluation is warranted.

Prevalence and tissue concordance of BRCA2 copy number loss evaluated by single-cell, shallow whole genome sequencing of circulating tumor cells (CTCs) in castration-resistant prostate cancer (CRPC).

5531 Background: Genomic studies have shown that up to 25% of prostate cancer tissue specimens harbor alterations in DNA Damage Repair (DDR) genes, which may sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Trials evaluating PARPi in patients with DDR deficiencies have shown varied response rates and differences regarding which genomic alterations predict for sensitivity to these agents, with the majority of objective responses seen in BRCA2-altered tumors. These results highlight the need to develop biomarker assays which can predict benefit from PARPi therapy. Tissue and cell-free DNA (cfDNA) have been the most utilized sources of tumor material for analysis in this setting, but success rates of obtaining sufficient tumor for analysis from bone are low and detecting tumor-derived copy number variants (CNVs) in cfDNA is challenging. Circulating tumor cells (CTCs) represent an alternate source of genetic information, for which assays are available to isolate and sequence individual cells in a manner that eliminates background noise from stroma and healthy cells, while capturing inter-cellular heterogeneity. Methods: Blood samples, collected from 138 progressing metastatic CRPC patients within 30 days of a pre-treatment biopsy intended for sequencing using MSK-IMPACT, were sent to EPIC Sciences for CTC analysis. Detected CTCs underwent single cell, low pass whole genome sequencing. Prevalence and concordance of BRCA2 copy-loss, regardless of whether single copy or homozygous, was compared in matched tissue and CTC samples. Results: BRCA2 copy-loss was identified in 21% (23/108) and 50% (58/115) of successfully sequenced tissue and CTC samples, respectively. In the 58 patients with CTC-detected BRCA2 loss, BRCA2 loss was detected in 36% (220/565) of the sequenced CTCs, representing a median of 46% (range 4-100%) of CTCs found in each individual sample. When both sequencing assays were successful, BRCA2 loss was detected in CTCs in 84% (16/19) of the tissue-positive cases, whereas tissue sequencing detected BRCA2 loss in 35% (16/46) of CTC-positive cases. Conclusions: Data from this study supports the notion that single-cell CTC sequencing can detect BRCA2 copy-loss at a high frequency, including cases that were negative in tissue, while also characterizing inter-cellular heterogeneity. Further studies will investigate whether CTC BRCA2 copy-loss can predict the likelihood of response to PARPi.

Alterations of DNA damage repair genes in Chinese colorectal cancer patients.

e16121 Background: Colorectal cancer (CRC) is the second most common cancer in women and third in men. DNA damage repair (DDR) deficiency has emerged as a predictive biomarker for chemotherapy, PARP and immune checkpoint inhibitors. However, comprehensive molecular characteristics of DDR variants in Chinese CRC patients is lacking. Methods: Formalin fixed, paraffin embedded (FFPE) tumor tissues and matched blood samples were collected for targeted next-generation sequencing (NGS) assay. The testing was carried out in a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Alterations of six functional gene sets involved in DDR pathways: homologous recombination (HR), mismatch repair (MMR), base excision repair (BER), nonhomologous end-joining (NHEJ), checkpoint factors (CPF) and Fanconi anemia (FA) were analyzed. The association of DDR gene mutations with TMB/MSI was assessed. Results: In total, 319 CRC patients were recruited, 127 female and 192 male with a median age of 59 (range 23-92). Over one third (36%,115/319) patients had at least one mutation in DDR genes. Mutation rates varied in different DDR pathway: HR (20.7%), CPF (15.1%), FA (11.3%), MMR (10.3%), BER (8.8%), and NHEJ (5.3%). The most frequently mutated DDR genes were ARID1A (13.4%), ATM (10.2%), BRCA2 (6.8%), MLH1 (5.6%), MSH6 (5.6%), POLE (5.0%). Germline mutations in MLH1 (2.5%), BRCA2 (1.2%), MSH2 (0.9%), MSH6 (0.9%), FANCA (0.9%), ATM (0.6%), BRCA1 (0.3%), and CHEK2 (0.3%) were detected in 24 patients. DDR variations were enriched in the right-side CRC compared to the left side CRC (50% vs. 32.8%, p= 0.024). Early stage (I-II) harbored more DDR variations. 20.1% of patients had high TMB (≥10 muts/Mb) with a median of 51 muts/Mb (10-326.7 mus/Mb). Patients with DDR mutations had a significantly higher TMB than patients with wild type DDR (8.5 vs. 4.6 muts/Mb, p&lt; 0.001). All CRC tumors with high MSI harbored DDR mutations. Importantly, the mutations in “HM” (HR/MMR), but not BER/CPF/NHEJ/FA mutations, were significantly correlated with high MSI ( p&lt; 0.001). Conclusions: DDR gene alterations occurred in 36% of Chinese CRC patients and were enriched in right sided tumors. DDR pathway alterations are relatively frequent in CRC and consideration for biomarker-enriched clinical trials with PARP, immune checkpoint inhibitors, and novel combinations are warranted.

Phase II study of pembrolizumab plus olaparib in the treatment of patients with advanced cholangiocarcinoma.

TPS4659 Background: Lack of an effective second-line treatment of patients (pts) with advanced cholangiocarcinoma (CC) necessitates the development of new therapies. Preclinical studies suggest CC susceptibility to PARP inhibition (PARPi): ERCC1 is underexpressed in 74% of CCs, and olaparib is selective for ERCC1 deficiency, profoundly inhibiting DNA repair. Additionally, PARPi exploits IDH mutation-related DNA damage repair deficiency, which is found in about 25% of CCs. Unfortunately, PARPi also upregulates PD-1-PD-L1 receptor-ligand binding, which attenuates anticancer immunity and counteracts the efficacy of PARPi. However, this can be prevented by PD-1 inhibition—blockade of PD-1-PD-L1 acts to re-sensitize cancer cells to T-cell killing. Hence, we hypothesize that the combination of olaparib and pembrolizumab will produce a durable anti-tumor response against CC by synergistically inducing DNA damage and increasing immune response. Methods: Thirty-six pts with advanced CC, who either failed to respond to or progressed on first-line therapy, will be enrolled to receive olaparib (300 mg PO bid) daily plus pembrolizumab (200 mg IV Q3 weeks) for 12 months, unless unacceptable toxicities or cancer progression occur, in which cases therapy will cease. MRI or CT tumor assessment will occur just before therapy, every 6 weeks for the first 6 months of therapy, and then every 9 weeks for the next 6 months of therapy (total, 12 months). Three tumor biopsies will be collected: at baseline; at week 4; and at time of progression. In each biopsy, ERCC1, PD-1, and PD-L1 expression, IDH1/2 mutation status, and immune cell (CD3 and CD8) response will be assessed. The total study duration will be 20-36 months. The primary endpoint will be overall response rate; the secondary endpoints will be PFS, OS, duration of response, and safety and tolerability. It is hypothesized that in pts with advanced CC, second-line therapy with olaparib plus pembrolizumab will improve the response rate from 17.5% to 35%, as well as increase PFS and OS compared to cytotoxic chemotherapy. Study enrollment began in Q1 2020. NCI number pending at time of abstract submission. Clinical trial information: pending at time of submission .

PARP inhibitors as a new therapeutic option in metastatic prostate cancer: a systematic review.

A great number of DNA-damage repair (DDR) pathways have been recognized to be frequently dysregulated in advanced stages of prostate cancer. DNA-repair defects in prostate cancer represents a clinically relevant disease subset. Tumors whose ability to repair double-strand DNA breaks by homologous recombination is compromised, are highly sensitive to blockade of the repair of DNA single-strand breaks via the inhibition of the enzyme poly(ADP) ribose polymerase (PARP). A systematic review of the literature has been conducted in January 2020 using PubMed Medline database in line with the recommendations from the PRISMA guidelines. The following string terms were used for searching clinical trial articles: castration resistant OR castrate resistance OR castration refractory AND prostate cancer AND PARP OR poly(ADP-ribose) polymerase inhibitor OR DNA-repair OR homologous recombination repair. On-going clinical trials with olaparib, niraparib, talazoparib, veliparib, and rucaparib in mCRPC were searched on the clinicalTrials.gov website. From this research 176 articles were identified. After title screening and abstract reading, five papers and four abstract were considered for the systematic review. Thirty-two clinical trials were also identified: from these 16 trials which did not include mCRPC patients or only prostate cancer patients, trials not yet recruiting and trials including radio-metabolic treatments were excluded. Sixteen trials were included and discussed in the paper. Olaparib has been the first agent showing a benefit in terms of rPFS and ORR alone or in combination with abiraterone plus prednisone in patients with DDR deficiency prostate cancer. Also rucaparib showed a benefit in terms of PSA response rate and ORR in patients with BRCA2 and BRCA1 mutation in a phase-II study. Other phase-III clinical trials are evaluating niraparib and talazoparib, alone or in combination with AR signaling inhibitors.

Influence of DNA damage repair defects on tumor immunogenicity

Abstract In recent years, immune checkpoint inhibitors (ICI) have dramatically altered the treatment landscape for patients with advanced stage and metastatic cancers. Durable benefit, however, is limited to subsets of histologies and patients, highlighting the importance of identifying predictive biomarkers to select for patients who may benefit. DNA damage repair and response (DDR) deficiency including mismatch repair deficiency and resulting increased tumor neoantigens have been associated with improved response. We hypothesize that a similar concept may extend to other defects in DDR pathways, the most common being homologous recombination (HR). In ongoing work, we have implicated mutations in the DDR pathways including the HR pathway with improved survival after ICI in a large retrospective cohort. However, divergent responses were noted depending on the individual HR gene mutated. Syngeneic murine models were used to study individual DDR genes using CRISPR-Cas9 mutagenesis and their role in altering the tumor microenvironment and response to immune checkpoint blockade in isogenic model systems. We report differential effects of mutations in genes of the homologous recombination (HR) pathway on response after ICI administration in mouse and human tumors, and show that truncating mutations in BRCA2 are associated with superior response to ICI compared to BRCA1-deficient tumors. Single cell RNA-sequencing of CD45+ cells sorted from tumors reveals distinct immune landscapes. These data have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy in the setting of DDR deficiency and ultimately expand the therapeutic window of immunotherapy.

Abstract A29: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood, and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, Myc, Cyclin E1 (CCNE1), Akt2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNA sequencing, and transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and regulatory compartments between HR-proficient and -deficient tumors within the primary and metastatic tumors themselves and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean G. Smith, David Pepin, Raghav Mohan, Esmee Hoefsmit, Tian Xia, Ferenc Reinhardt, Tony E. Chavarria, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A29.

TALAPRO-2: A placebo-controlled phase III study of talazoparib (TALA) plus enzalutamide (ENZA) for patients with first-line metastatic castration-resistant prostate cancer (mCRPC).

TPS264 Background: TALA is a poly(ADP-ribose) polymerase (PARP) inhibitor that inhibits PARP1/2 and traps PARP on DNA, preventing DNA damage repair (DDR) and causing death of cells with DDR mutations (eg BRCA1/2). TALA is approved in multiple countries for germline BRCA1/2-mutated HER2- advanced breast cancer. ENZA is an established therapy for CRPC. PARP1 activity has been shown to support AR function, suggesting that co-blockade of PARP1 may synergize with AR-directed therapy, regardless of DDR deficiency. A combination of TALA with ENZA in mCRPC may improve clinical outcomes in patients with or without DDR-deficient tumors. TALAPRO-2 (NCT03395197) is a 2-part study to evaluate efficacy, safety, pharmacokinetics, and patient (pt)-reported outcomes (PROs) of the combination. Methods: Enrollment goal is 1037 pts (19 pts, part 1 dose-finding; 1,018 pts, part 2 placebo-controlled). Key eligibility criteria: age ≥18 y, asymptomatic/mildly symptomatic mCRPC, ECOG performance status ≤1, no brain metastases, and no prior life-prolonging systemic therapy in nonmetastatic CRPC or mCRPC state. Prior docetaxel or novel hormonal therapy (excluding novel AR inhibitors) in castration sensitive (CSPC) setting is allowed. The randomized double-blind portion will evaluate safety, efficacy, and PROs of TALA (0.5 mg QD) + ENZA (160 mg QD) vs placebo + ENZA (160 mg QD). Randomization is stratified by prior NHT or docetaxel for CSPC (yes/no) and DDR mutation status (deficient vs nondeficient/unknown). The primary endpoint is radiographic progression-free survival (rPFS), defined as time to progression in soft tissue per RECIST v.1.1 or in bone per PCWG3 criteria or death. The key secondary endpoint is overall survival (OS). Efficacy will be assessed by radiography every 8 weeks up to week 25 and every 8–12 weeks thereafter. rPFS will be compared between two arms by a 1-sided stratified log-rank test. Pt recruitment is ongoing in multiple regions including US, Europe/Eastern Europe, Israel, South America, South Africa, and Asia Pacific region. This study was sponsored by Pfizer Inc. Clinical trial information: NCT03395197.

Association between DNA damage repair gene somatic mutations and immune-related gene expression in ovarian cancer.

BackgroundDefects in DNA damage repair (DDR) system may lead to genomic instability and manifest as increased immunogenicity. DDR deficiency is prevalent in ovarian cancer (OvCa); however, the association of DDR mutation with immune profiles in OvCa remains largely unknown. This knowledge will provide an essential basis to the rational design of biomarker‐guided immune combination therapy of OvCa in the future.MethodsWhole‐exome sequencing data of 587 OvCa from The Cancer Genome Atlas (TCGA) were used to determine the expression profiles of 47 immune‐related genes and the abundance of tumor‐infiltrating immune cells. A Chinese OvCa cohort (n = 220) tested by next‐generation sequencing (NGS) was used to validate the association between DDR status and tumor mutation burden (TMB).ResultsA total of 19.3% in TCGA cohort and 25.9% in Chinese cohort harbored at least one DDR somatic mutation. DDR deficiency exhibited a distinct immune profile with significant higher expression levels of PTPRCAP, CCL5, IFI16, LAG3, IL15RA, and GBP1 in OvCa in the TCGA cohort. Different DDR pathway deficiency displayed various immune profiles. Increased levels of Th1 cells, TMB, and neoantigen were also observed in DDR‐deficient tumors.ConclusionsDDR deficiency was associated with specific immune signatures in OvCa. Our findings emphasize the urgent need for biomarker‐guided rational immune combination therapy to maximize the OvCa patients who could benefit from immunotherapy.

A pan-cancer transcriptome analysis identifies replication fork and innate immunity genes as modifiers of response to the CHK1 inhibitor prexasertib.

The combined influence of oncogenic drivers, genomic instability, and/or DNA damage repair deficiencies increases replication stress in cancer. Cells with high replication stress rely on the upregulation of checkpoints like those governed by CHK1 for survival. Previous studies of the CHK1 inhibitor prexasertib demonstrated activity across multiple cancer types. Therefore, we sought to (1) identify markers of prexasertib sensitivity and (2) define the molecular mechanism(s) of intrinsic and acquired resistance using preclinical models representing multiple tumor types. Our findings indicate that while cyclin E dysregulation is a driving mechanism of prexasertib response, biomarkers associated with this aberration lack sufficient predictive power to render them clinically actionable for patient selection. Transcriptome analysis of a pan-cancer cell line panel and in vivo models revealed an association between expression of E2F target genes and prexasertib sensitivity and identified innate immunity genes associated with prexasertib resistance. Functional RNAi studies supported a causal role of replication fork components as modulators of prexasertib response. Mechanisms that protect cells from oncogene-induced replication stress may safeguard tumors from such stress induced by a CHK1 inhibitor, resulting in acquired drug resistance. Furthermore, resistance to prexasertib may be shaped by innate immunity.

Combined Targeting of Mutant p53 and Jumonji Family Histone Demethylase Augments Therapeutic Efficacy of Radiation in H3K27M DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with a 5-year survival of <1%. Up to 80% of the DIPG tumors contain a specific K27M mutation in one of the two genes encoding histone H3 (H3K27M). Furthermore, p53 mutations found in >70–80% of H3K27M DIPG, and mutant p53 status is associated with a decreased response to radiation treatment and worse overall prognosis. Recent evidence indicates that H3K27M mutation disrupts tri-methylation at H3K27 leading to aberrant gene expression. Jumonji family histone demethylases collaborates with H3K27 mutation in DIPG by erasing H3K27 trimethylation and thus contributing to derepression of genes involved in tumorigenesis. Since the first line of treatment for pediatric DIPG is fractionated radiation, we investigated the effects of Jumonji demethylase inhibition with GSK-J4, and mutant p53 targeting/oxidative stress induction with APR-246, on radio-sensitization of human H3K27M DIPG cells. Both APR-246 and GSK-J4 displayed growth inhibitory effects as single agents in H3K27M DIPG cells. Furthermore, both of these agents elicited mild radiosensitizing effects in human DIPG cells (sensitizer enhancement ratios (SERs) of 1.12 and 1.35, respectively; p < 0.05). Strikingly, a combination of APR-246 and GSK-J4 displayed a significant enhancement of radiosensitization, with SER of 1.50 (p < 0.05) at sub-micro-molar concentrations of the drugs (0.5 μM). The molecular mechanism of the observed radiosensitization appears to involve DNA damage repair deficiency triggered by APR-246/GSK-J4, leading to the induction of apoptotic cell death. Thus, a therapeutic approach of combined targeting of mutant p53, oxidative stress induction, and Jumonji demethylase inhibition with radiation in DIPG warrants further investigation.

The Novel ATR Inhibitor BAY 1895344 Is Efficacious as Monotherapy and Combined with DNA Damage-Inducing or Repair-Compromising Therapies in Preclinical Cancer Models.

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their efficacy.

EGFR exon 19-deletion aberrantly regulate ERCC1 expression that may partly impaired DNA damage repair ability in non-small cell lung cancer.

BackgroundEpidermal growth factor receptor (EGFR) activating mutations are usually associated with DNA damage repair (DDR) deficiency. However, the precise mechanism has remained elusive. In this study, we aimed to investigate whether EGFR exon 19 deletion mutation downstream signals contributed to DDR deficiency by downregulation of excision repair cross‐complementation group‐1 (ERCC1), a key factor in DDR, expression and function.MethodsWe first measured cell survival, DNA damage (γ‐H2AX foci formation) and damage repair (ERCC1 and RAD51 foci formation) ability in response to DNA cross‐linking drug in EGFR exon 19 deletion and EGFR wild‐type cells separately. We then investigated the involvement of EGFR downstream signals in regulating ERCC1 expression and function in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones.ResultsWe observed increased γ‐H2AX, but impaired ERCC1 and RAD51 nuclear foci formation in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones treated with DNA cross‐linker. In addition, we identified that inhibition of EGFR exon 19 deletion signals increased ERCC1 expression, whereas blocked wild‐type EGFR signals decreased ERCC1 expression, on both mRNA and protein levels. Furthermore, EGFR exon 19 deletion downstream signals not only inhibited ERCC1 expression but also influenced ERCC1 foci formation in response to DNA cross‐linker.ConclusionOur findings indicated that the aberrant EGFR exon 19 deletion signals were not only associated with decreased expression of ERCC1 but were also involved in impaired ERCC1 recruitment in response to DNA cross‐link damage, thereby providing us with more evidence for exploring the mechanism of DDR deficiency in EGFR mutant NSCLC.

HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling.

Base excision repair (BER) is the major cellular DNA repair pathway that recognises and excises damaged DNA bases to help maintain genome stability. Whilst the major enzymes and mechanisms co-ordinating BER are well known, the process of BER in chromatin where DNA is compacted with histones, remains unclear. Using reconstituted mononucleosomes containing a site-specific synthetic abasic site (tetrahydrofuran, THF), we demonstrate that the DNA damage is less efficiently incised by recombinant AP endonuclease 1 (APE1) when the DNA backbone is facing the histone core (THF-in) compared to that orientated away (THF-out). However, when utilizing HeLa whole cell extracts, the difference in incision of THF-in versus THF-out is less pronounced suggesting the presence of chromatin remodelling factors that stimulate THF accessibility to APE1. We subsequently purified an activity from HeLa cell extracts and identify this as the E3 ubiquitin ligase, HECTD1. We demonstrate that a recombinant truncated form of HECTD1 can stimulate incision of THF-in by APE1 in vitro by histone ubiquitylation, and that siRNA-mediated depletion of HECTD1 leads to deficiencies in DNA damage repair and decreased cell survival following x-ray irradiation, particularly in normal fibroblasts. Thus, we have now identified HECTD1 as an important factor in promoting BER in chromatin.