Recombination DNA Repair Pathway Research Articles

Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors.

Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

CDK1 inhibitor controls G2/M phase transition and reverses DNA damage sensitivity

CDK1 plays key roles in cell cycle progression through the G2/M phase transition and activation of homologous recombination (HR) DNA repair pathway. Accordingly, various CDK1 inhibitors have been developed for cancer therapy that induce prolonged G2 arrest and/or sensitize cells to DNA damaging agents in tumor cells, resulting in cell death. However, CDK1 inhibition can induce resistance to DNA damage in certain conditions. The mechanism of different DNA damage sensitivity is not completely understood. We performed immunofluorescence and flow cytometry analysis to investigate DNA damage responses in human tumor cells during low and high dose treatments with RO-3306, a selective CDK1 inhibitor. This comparative investigation demonstrated that RO-3306-induced G2 arrest prevented cells with DNA double-strand breaks from transitioning into the M-phase and that the cells maintained their DNA repair capacity in G2-phase, even under RO-3306 dose-dependent DNA repair inhibition. These findings reveal that CDK1 inhibitor-induced DNA repair inhibition and cell cycle control, which regulate each other during the G2/M phase transition determine the cellular sensitivity to DNA damage, providing insight useful for developing clinical strategies targeting CDK1 inhibition in tumor cells.

Abstract OT-13-05: Dolaf- an international multicenter phase 2 trial of durvalumab (medi4736) plus olaparib plus fulvestrant in metastatic or locally advanced er-positive, her2-negative breast cancer patients selected using criteria that predict sensitivity to olaparib (UCBG308)

Abstract Background: Olaparib is a PARP inhibitor that has shown high response rates and clinical activity in patients with advanced HER2-negative breast cancer (BC) and a germline BRCA1 and BRCA2 mutation. There is also evidence of cellular sensitivity to PARP inhibitors associated to defects in other genes involved in homologous recombination DNA repair (HRR) or mismatch repair pathway (microsatellite instability MSI). Moreover, about 15% of patients with ER+/HER2- metastatic BC have at least 100 mutations in their tumor, leading to genomic instability and potential sensitivity to PARP inhibitors. Several preclinical and clinical studies have suggested the benefit of an association of immune checkpoint blockade, like durvalumab, with PARP inhibitor. Indeed, tumors with BRCA1/2 mutations or other deficiency in HRR have high mutagenic burden and produce a larger number of neoantigens. Most BRCA-associated BC are ER+/HER2-. Although loss of the BRCA gene function is a key driver of oncogenesis in these patients, ER-pathway appears to remain a key target for their therapy. Preclinical data indicate that olaparib may enhance endocrine therapy efficacy and circumvent resistance. Therefore, the combination of durvalumab, olaparib and endocrine therapy could be a therapeutic option for patients with BRCA1/2 mutation or for patients with selected ER+/HER2- advanced BC. Trial design: DOLAF is an open-label, international, multicentric, phase II trial assessing the combination of olaparib, fulvestrant, and durvalumab. Olaparib will be administered orally twice daily at 300 mg. Fulvestrant will be administered as two intramuscular injections of 250 mg (Cycle 1 Days 1 and 15, and Day 1 of each subsequent 28-day cycle). Durvalumab will be started 4 weeks after the first dose of olaparib at 1500 mg intravenous every 4 weeks. Eligibility criteria include: ER+/HER2- metastatic or locally advanced BC with documented germline alteration in BRCA1 or BRCA2 or deleterious germline or somatic alterations implicated in the HRR pathway (ATM, BARD1, BRCA1, BRCA2, 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 next generation DNA sequencing. Patients could have received 1 line of endocrine therapy and/or 1 line of chemotherapy in the metastatic setting.Specific aims: To evaluate the efficacy of the combination in terms of progression-free survival rate at 24 weeks using RECIST v1.1. Secondary endpoints include: safety (according to the NCI-CTCAE v5.0), overall survival, progression-free survival, objective response rate, duration of response in the overall study population and in the germline BRCA mutated population. Statistical methods: Given the lack of safety data from this association, a safety run-in of 6 patients was planned. With an optimum two-stage Simon design,α = 2.5%, β = 5%, p0 (the probability of inefficiency maximum) = 50%, p1 (the probability of minimum efficiency) = 65%, it would be necessary to include 149 evaluable patients. The strategy could be considered sufficiently effective if there are at least 87 successes. According to the established design (including a rate of 5% of patients lost to follow-up or non-evaluable), it will be necessary to include 158 patients. We hypothesized that about 20% of screened patients will have a molecular alteration to allow enrollment. Thus, we need to screen 790 patients. The study is recruiting. The safety run-in is over. By July 1, 2020, 62 patients have been screened and 8 have been treated (NCT04053322). Contact information: DOLAF@unicancer.fr Citation Format: Severine Guiu, Judith Balmana, Lise Roca, Anthony Goncalves, Audrey Mailliez, Frederic Bigot, Thomas Bachelot, Florence Dalenc, Nadine Dohollou, Dominique Genet, Isabelle Desmoulins, Camille Chakiba, Suzette Delaloge, Geraldine Martineau, Veronique Haddad, Philippe Follana. Dolaf- an international multicenter phase 2 trial of durvalumab (medi4736) plus olaparib plus fulvestrant in metastatic or locally advanced er-positive, her2-negative breast cancer patients selected using criteria that predict sensitivity to olaparib (UCBG308) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-13-05.

Frequency and prognostic value of mutations associated with the homologous recombination DNA repair pathway in a large pan cancer cohort

PARP inhibitors have shown remarkable efficacy in the clinical management of several BRCA-mutated tumors. This approach is based on the long-standing hypothesis that PARP inhibition will impair the repair of single stranded breaks, causing synthetic lethality in tumors with loss of high-fidelity double-strand break homologous recombination. While this is now well accepted and has been the basis of several successful clinical trials, emerging evidence strongly suggests that mutation to several additional genes involved in homologous recombination may also have predictive value for PARP inhibitors. While this notion is supported by early clinical evidence, the mutation frequencies of these and other functionally related genes are largely unknown, particularly in cancers not classically associated with homologous recombination deficiency. We therefore evaluated the mutation status of 22 genes associated with the homologous recombination DNA repair pathway or PARP inhibitor sensitivity, first in a pan-cancer cohort of 55,586 patients, followed by a more focused analysis in The Cancer Genome Atlas cohort of 12,153 patients. In both groups we observed high rates of mutations in a variety of HR-associated genes largely unexplored in the setting of PARP inhibition, many of which were associated also with poor clinical outcomes. We then extended our study to determine which mutations have a known oncogenic role, as well as similar to known oncogenic mutations that may have a similar phenotype. Finally, we explored the individual cancer histologies in which these genomic alterations are most frequent. We concluded that the rates of deleterious mutations affecting genes associated with the homologous recombination pathway may be underrepresented in a wide range of human cancers, and several of these genes warrant further and more focused investigation, particularly in the setting of PARP inhibition and HR deficiency.

PIN-like ductal carcinoma of the prostate has frequent activating RAS/RAF mutations.

Prostatic intraepithelial neoplasia-like (PIN-like) ductal carcinoma is a rare tumour characterised by often cystically dilated glands architecturally resembling high-grade PIN, but lacking basal cells. These tumours are frequently accompanied by grade group 1 acinar cancer and behave relatively indolently. In contrast, conventional ductal adenocarcinoma of the prostate is an aggressive variant comparable to grade group 4 acinar cancer. Here, we used targeted next-generation sequencing to molecularly profile PIN-like ductal carcinoma cases at radical prostatectomy. Five PIN-like ductal carcinoma samples at radical prostatectomy with sufficient tumour tissue available were analysed for genomic alterations by targeted next-generation sequencing using the Johns Hopkins University (JHU) solid tumour panel. DNA was captured using SureSelect for 640 genes and sequenced on the Illumina HiSeq platform. Three of five (60%) of the PIN-like ductal carcinomas showed activating mutations in the RAS/RAF pathways, which are extraordinarily rare in conventional primary prostate carcinoma (<3% of cases), including an activating hot-spot BRAF mutation (p.K601E), an activating hot-spot mutation in HRAS (p.Q61K) and an in-frame activating deletion in BRAF (p.T488_Q493delinsK). An additional two cases lacked BRAF or HRAS mutations, but harboured in-frame insertions of uncertain significance in MAP2K4 and MAP3K6. One case had sufficient acinar tumour for sequencing, and showed a similar molecular profile as the concurrent PIN-like ductal carcinoma, suggesting a clonal relationship between the two components. PIN-like ductal carcinoma represents a molecularly unique tumour, enriched for potentially targetable oncogenic driver mutations in the RAS/RAF/MAPK pathway. This molecular profile contrasts with that of conventional ductal adenocarcinoma, which is typically enriched for pathogenic mutations in the mismatch repair (MMR) and homologous recombination (HR) DNA repair pathways.

Poly(ADP-Ribose) Glycohydrolase (PARG) vs. Poly(ADP-Ribose) Polymerase (PARP) - Function in Genome Maintenance and Relevance of Inhibitors for Anti-cancer Therapy.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that catalyze the addition of poly(ADP-ribose) (PAR) subunits onto themselves and other acceptor proteins. PARPs are known to function in a large range of cellular processes including DNA repair, DNA replication, transcription and modulation of chromatin structure. Inhibition of PARP holds great potential for therapy, especially in cancer. Several PARP1/2/3 inhibitors (PARPi) have had success in treating ovarian, breast and prostate tumors harboring defects in the homologous recombination (HR) DNA repair pathway, especially BRCA1/2 mutated tumors. However, treatment is limited to specific sub-groups of patients and resistance can occur, limiting the use of PARPi. Poly(ADP-ribose) glycohydrolase (PARG) reverses the action of PARP enzymes, hydrolysing the ribose-ribose bonds present in poly(ADP-ribose). Like PARPs, PARG is involved in DNA replication and repair and PARG depleted/inhibited cells show increased sensitivity to DNA damaging agents. They also display an accumulation of perturbed replication intermediates which can lead to synthetic lethality in certain contexts. In addition, PARG is thought to play an important role in preventing the accumulation of cytoplasmic PAR and therefore parthanatos, a caspase-independent PAR-mediated type of cell death. In contrast to PARP, the therapeutic potential of PARG has been largely ignored. However, several recent papers have demonstrated the exciting possibilities that inhibitors of this enzyme may have for cancer treatment, both as single agents and in combination with cytotoxic drugs and radiotherapy. This article discusses what is known about the functions of PARP and PARG and the potential future implications of pharmacological inhibition in anti-cancer therapy.

Abstract 1059: Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors

Abstract Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of solid tumors including low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. We have shown that in an in vivo flank model bearing IDH1 R132H mutation, AZD6738 (ATRi) and Olaparib (PARPi) combination treatment reduces the tumor burden significantly relative to monotherapy and vehicle. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of IDH1/2 mutant cancers. Citation Format: Amrita D. Sule, Ranjini Sundaram, Ranjit Bindra. Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors [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 1059.

Abstract IA04: Drug resistance in end-stage high-grade serous ovarian cancer

Abstract Background: Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent or end-stage disease who have been extensively treated with chemotherapy or newer targeted agents, such as antiangiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Aim: To characterize genomic features and identify mechanisms of acquired drug resistance in end-stage HGSC patients. Methods: Since 2012, we have collected tumor samples from 19 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, which is performed a median 6 hours after death. SNP array, WGS, and RNA sequencing were performed to detect germline and somatic mutations, copy number alterations and gene expression patterns. Results: A total of 89 end-stage tumor samples from 15 patients with germline or somatic mutations in components of the homologous recombination (HR) DNA repair pathway have been analyzed by SNP array, and a subset of those have undergone WGS (n=44) and RNAseq (n=47). As expected, all samples had a somatic pathogenic TP53 mutation and have highly aberrant somatic copy number profiles. Prominent mutational signatures (single- and double-base substitutions) are associated with defective HR and prior treatment with platinum drugs. The total single-base substitution mutation burden is similar across tumor samples within patients; however, many mutations are unique to individual tumor samples. Currently, reversion mutations, which are secondary mutations in HR genes, are the most common resistance mechanism identified in our cohort of end-stage patients. Conclusion: Our data are allowing us to understand HGSC at end stage, by examining the catalogue of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. Citation Format: Elizabeth L. Christie, Kathryn Alsop, George Au-Yeung, Ahwan Pandey, Australian Ovarian Cancer Study, David D.L. Bowtell. Drug resistance in end-stage high-grade serous ovarian cancer [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 IA04.

Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition

Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.

Comparison of genomic instability test scores used for predicting PARP activity in ovarian cancer.

1586 Background: Clinical trials have explored the utility of various genomic instability (GI) scores or gene panels to assess deficiencies in the homologous recombination (HR) DNA repair pathway and support PARP inhibitor use in ovarian cancer; however, these methods of assessing homologous recombination deficiency (HRD) may not be equivalent. The myChoice HRD test is the only analytically and clinically validated, FDA-approved HRD test that includes BRCA1/2 mutation status and three measures of GI. We compared the proportion of patients identified as candidates for PARP inhibitor use by two measures of HRD [percent loss of heterozygosity (%LOH), 11-gene panel] to myChoice HRD. Methods: Whole-genome SNP analysis was used to reconstruct ovarian tumor genomic profiles to calculate the myChoice HRD score and %LOH in 2 cohorts (clinical laboratory cohort, N = 3,278; SCOTROC4 trial, N = 248). Mutation screening for a set of 11 genes in the HR pathway ( ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, MRE11A, NBN, PALB2, RAD51C, RAD51D) was performed for a subset of tumors from the SCOTROC trial (n = 187). Samples were considered positive if the myChoice HRD score was above the threshold (threshold scores of 42 and 33 were assessed), %LOH above the threshold (16%), or a pathogenic variant in one of the 11 HR genes. The correlation between positive results from %LOH and the 11-gene panel were compared to myChoice HRD. Percent positive agreement (PPA) was the proportion of positive test results from myChoice HRD that were also positive by %LOH or the 11-gene panel. Results: The table shows the correlation and PPA between myChoice HRD, %LOH, and the 11-gene panel. Overall, 19%-61% of patients identified as positive by myChoice HRD would have been missed by %LOH or the 11-gene panel in these two cohorts. Conclusions: These data show that HRD tests used in published and ongoing clinical trials are not equivalent, and they should not be considered interchangeable in predicting PARP inhibitor response in clinical practice. [Table: see text]

NCI protocol 10250: A phase II study of temozolomide and olaparib for the treatment of advanced uterine leiomyosarcoma.

TPS11570 Background: Soft tissue sarcoma (STS) is a heterogenous malignancy of mesenchymal origin and includes more than 50 biologically distinct subtypes. Leiomyosarcoma (LMS), a neoplasm of smooth muscle origin, represents up to 20% of STS. The uterus is the most common site of origin in women. Advanced uterine LMS (uLMS) is initially treated with gemcitabine + docetaxel or anthracycline-based chemotherapy but overall survival remains &lt; 24 mos. Besides recurrent alterations in RB1, TP53 and ATRX, insight into cancer biology of uLMS remains limited. Recently, whole exome and transcriptomic sequencing studies suggest uLMS harbors characteristic defects in the homologous recombination (HR) DNA repair pathway and thus features of BRCAness. HR-deficient cancers are unable to efficiently repair double-stranded DNA breaks and appear sensitive to treatment with poly ADP-ribose polymerase (PARP) inhibitors. In preclinical studies, the combination of temozolomide (T), an alkylating agent, and olaparib (O), a PARP inhibitor, was synergistic and markedly suppressed proliferation of uLMS models. A recent phase II study in small cell lung cancer defined the RP2D for T + O where the chief toxicity was myelosuppression. Methods: NCI Protocol #10250 is a single-arm, open-label, multi-center phase II clinical trial of T + O in patients with advanced uLMS. Eligible pts have ECOG PS ≤ 2, progression on ≥ 1 prior line of therapy and disease measurable by RECIST v1.1 and amenable to image-guided biopsy. Pts receive T 75 mg/m2 PO daily + O 200 mg PO BID on days 1-7 in 21-day cycles. The 1° endpoint is objective response rate (ORR). A one-stage binomial design is used to evaluate for an ORR ≤ 10% (null hypothesis) versus ≥ 35% (alternative hypothesis). The design calls for 22 patients. If 5/22 respond, the treatment is promising. This design yields 93% power and 1-sided type I error of 6%. 2° endpoints include progression free survival and safety. All pts undergo tumor biopsies pre-treatment and during cycle 2. Tissue is used for correlative analysis interrogating uLMS for features of BRCAness through (a) whole exome sequencing/RNAseq to evaluate for alterations in HR pathway component genes, (b) RAD51 foci formation by immunohistochemistry as a functional marker of HR pathway activity and (c) protein expression of Schlafen family member number 11 (SLFN11), an emerging biomarker for PARPi. Tumors are also evaluated for MGMT protein expression, a known determinant of sensitivity to T. The study opened to accrual 10/2019. Clinical trial information: NCT03880019.

The prognostic value of HRD mutations in liver cancer.

e13546 Background: Liver cancer is the sixth most common cancer in the world and the fourth most common cause of cancer mortality. Genomic alterations including homologous recombination deficiency (HRD) mutations often lead to aberrant regulations of signaling pathways, which play an important role in tumorigenesis. The prognostic value of HRD mutation and its correlation with overall survival in liver cancer is unclear. Methods: We used cBioPortal platform to analyze a cohort of 978 liver cancer samples. Mutations in 13 genes (BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, ABRAXAS1, MRE11, NBN, PALB2, RAD51C, RAD51D) were screened due to the fact that they are the most common indication of deficiency in the homologous recombination (HR) DNA repair pathway. We define overall survival (OS) as the time between the sample collection (surgical) date and the date of death or the last follow-up visit. We conducted Kaplan-Meier survival analysis, and the statistical differences were compared by log-rank tests. Results: 228 of the cases harbored at least one mutation of the HRD-indicator genes. Based on the result, patients were divided into the HRD and non-HRD group. The median OS of the HRD group was 45.47, which is significantly shorter than the OS of the non-HRD group (log-rank test p = 1.6196e-4, Table). Conclusions: HRD mutations are significantly associated with a shorter OS in liver cancer patients. HRD mutation was a negative prognostic factor for overall survival in liver cancer, but the underlying mechanisms remain to be explored. [Table: see text]

BRIP1, RAD51C, and RAD51D mutations are associated with high susceptibility to ovarian cancer: mutation prevalence and precise risk estimates based on a pooled analysis of ~30,000 cases

BackgroundIt is estimated that more than 20% of ovarian cancer cases are associated with a genetic predisposition that is only partially explained by germline mutations in the BRCA1 and BRCA2 genes. Recently, several pieces of evidence showed that mutations in three genes involved in the homologous recombination DNA repair pathway, i.e., BRIP1, RAD51C, and RAD51D, are associated with a high risk of ovarian cancer. To more precisely estimate the ovarian cancer risk attributed to BRIP1, RAD51C, and RAD51D mutations, we performed a meta-analysis based on a comparison of a total of ~ 29,400 ovarian cancer patients from 63 studies and a total of ~ 116,000 controls from the gnomAD database.ResultsThe analysis allowed precise estimation of ovarian cancer risks attributed to mutations in BRIP1, RAD51C, and RAD51D, confirming that all three genes are ovarian cancer high-risk genes (odds ratio (OR) = 4.94, 95%CIs:4.07–6.00, p < 0.0001; OR = 5.59, 95%CIs:4.42–7.07, p < 0.0001; and OR = 6.94, 95%CIs:5.10–9.44, p < 0.0001, respectively). In the present report, we show, for the first time, a mutation-specific risk analysis associated with distinct, recurrent, mutations in the genes.ConclusionsThe meta-analysis provides evidence supporting the pathogenicity of BRIP1, RAD51C, and RAD51D mutations in relation to ovarian cancer. The level of ovarian cancer risk conferred by these mutations is relatively high, indicating that after BRCA1 and BRCA2, the BRIP1, RAD51C, and RAD51D genes are the most important ovarian cancer risk genes, cumulatively contributing to ~ 2% of ovarian cancer cases. The inclusion of the genes into routine diagnostic tests may influence both the prevention and the potential treatment of ovarian cancer.

Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway.

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.

ADP-Ribosylation Levels and Patterns Correlate with Gene Expression and Clinical Outcomes in Ovarian Cancers.

Inhibitors of nuclear PARP enzymes (e.g., PARP-1) have improved clinical outcomes in ovarian cancer, especially in patients with BRCA1/2 gene mutations or additional homologous recombination (HR) DNA repair pathway deficiencies. These defects serve as biomarkers for response to PARP inhibitors (PARPi). We sought to identify an additional biomarker that could predict responses to both conventional chemotherapy and PARPi in ovarian cancers. We focused on cellular ADP-ribosylation (ADPRylation), which is catalyzed by PARP enzymes and detected by detection reagents we developed previously. We determined molecular phenotypes of 34 high-grade serous ovarian cancers and associated them with clinical outcomes. We used the levels and patterns of ADPRylation and PARP-1 to distribute ovarian cancers into distinct molecular phenotypes, which exhibit dramatically different gene expression profiles. In addition, the levels and patterns of ADPRylation, PARP-1 protein, and gene expression correlated with clinical outcomes in response to platinum-based chemotherapy, with cancers exhibiting the highest levels of ADPRylation having the best outcomes independent of BRCA1/2 status. Finally, in cell culture-based assays using patient-derived ovarian cancer cell lines, ADPRylation levels correlated with sensitivity to the PARPi, Olaparib, with cell lines exhibiting high levels of ADPRylation having greater sensitivity to Olaparib. Collectively, our study demonstrates that ovarian cancers exhibit a wide range of ADPRylation levels, which correlate with therapeutic responses and clinical outcomes. These results suggest ADPRylation may be a useful biomarker for PARPi sensitivity in ovarian cancers, independent of BRCA1/2 or homologous recombination deficiency status.

Abstract A104: Nanoformulations of PARP and CDK inhibitors for cancer therapy

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors have been majorly utilized in cancers with BRCA1/2 mutations that have deficiencies in the homologous recombination (HR) DNA repair pathway. Traditionally, PARP inhibitors have been administered through oral route in the clinic but they have resulted in poor bioavailability, low tumor accumulation and high systemic toxicity. Moreover, tumors develop resistance to PARP inhibitors necessitating the development of strategies to re-sensitize these resistant tumors. To overcome these limitations, nanoparticle drug formulation has demonstrated a great potential to increase the drug concentration and accumulation at tumor sites with low systemic toxicities. Here we report the development of several nanoformulations of PARP inhibitors and observe their efficacies by systemic administration to treat various cancer diseases. We used two FDA approved PARP inhibitors (Olaparib or Talazoparib) for nanoformulations of NanoOlaparib and NanoTalazoparib, decorated with EpCAM or EGFR antibody, and tested on breast, ovarian, prostate and lung cancer with or without irradiation. In HR deficient models, the nanoformulations showed significant inhibition of tumor cell growth in vitro and in vivo. Above all, combination of PARP inhibitor with irradiation resulted in greater efficacy in lung and prostate cancer models. To overcome PARP inhibitor resistance, we formulated nanoparticles, NanoDinaciclib of cyclin-dependent kinase 12 (CDK12) inhibitor (dinaciclib) and used in combination with nanoformulation of PARP inhibitor to arrest the tumor growth in animal model. Our current research reveals that the use of CDK12 inhibitor in PARP resistant tumors further arrests tumor growth compared with monotherapy alone. Further studies will help clarify to achieve clear understanding of the mechanism of action CDK12 inhibitor to determine the best way to use PARP inhibitors beyond HR deficiency. Citation Format: Bijay Singh, Shicheng Yang, Paige Baldwin, Anne van de Ven, Srinivas Sridhar. Nanoformulations of PARP and CDK inhibitors for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A104. doi:10.1158/1535-7163.TARG-19-A104

Fluzoparib increases radiation sensitivity of non-small cell lung cancer (NSCLC) cells without BRCA1/2 mutation, a novel PARP1 inhibitor undergoing clinical trials

Poly (ADP-ribose) polymerase 1 inhibitors were originally investigated as anti-cancer therapeutics with BRCA1/2 genes mutation. Here, we investigate the effectiveness of a novel PARP1 inhibitor fluzoparib, for enhancing the radiation sensitivity of NSCLC cells lacking BRCA1/2 mutation. We used MTS assays, western blotting, colony formation assays, immunofluorescence staining, and flow cytometry to evaluate the radiosensitization of NSCLC cells to fluzoparib and explore the underlying mechanisms in vitro. Through BRCA1 and RAD50 genes knockdown, we established dysfunctional homologous recombination (HR) DNA repair pathway models in NSCLC cells. We next investigated the radiosensitization effect of fluzoparib in vivo using human NSCLC xenograft models in mice. The expression of PARP1 and BRCA1 in human NSCLC tumor samples was measured by immunohistochemistry. Furthermore, we sequenced HR-related gene mutations and analyzed their frequencies in advanced NSCLC. In vitro experiments in NSCLC cell lines along with in vivo experiments using an NSCLC xenograft mouse model demonstrated the radiosensitization effect of fluzoparib. The underlying mechanisms involved increased apoptosis, cell-cycle arrest, enhanced irradiation-induced DNA damage, and delayed DNA-damage repair. Immunohistochemical staining showed no correlation between the expression of PARP1 and BRCA1. Moreover, our sequencing results revealed high mutation frequencies for the BRCA1/2, CHEK2, ATR, and RAD50 genes. The potential therapeutic value of fluzoparib for increasing the radiation sensitivity of NSCLC is well confirmed. Moreover, our findings of high mutation frequencies among HR genes suggest that PARP1 inhibition may be an effective treatment strategy for advanced non-small cell lung cancer patients.

Global transcriptomic analysis of Lactobacillus plantarum CAUH2 in response to hydrogen peroxide stress

To overcome the deleterious effects of hydrogen peroxide, Lactobacillus plantarum elicits an adaptive response to oxidative stress. In this study, global transcriptomic analysis revealed that L. plantarum CAUH2 expanded its carbon source utilizing profile and enhanced glycolysis to produce more ATP to confront with H2O2 stress. Some antioxidant enzymes including NADH peroxidase, thioredoxin reductase and glutathione peroxidase were 6.11, 36.76 and 6.23-fold up-regulated at transcription level for H2O2 scavenging. Meanwhile, free ferrous iron (Fe2+) was maintained at low concentrations in the cytoplasm, which could limit Fenton reaction and reduce the production of hydroxyl radicals. To repair DNA lesion caused by H2O2, both base excision repair system and recombinational DNA repair pathway were employed by L. plantarum CAUH2. In addition, the expression of methionine sulfoxide reductases and thioredoxin were up-regulated to repair oxidized proteins. It is noteworthy that some transcriptional regulators (Spx, CcpA and MarR1) were predicted to participate in the adaptive response to H2O2 stress, suggesting that L. plantarum CAUH2 utilized a wide array of sensors to monitor oxidative stress and modulated the transcriptional regulation network under H2O2 stress. These findings provide novel insight into the protective mechanisms developed by L. plantarum to cope with oxidative stress.

Abstract NT-098: WINDOW OF OPPORTUNITY TRIAL: ASSESSING THE ADAPTIVE RESPONSE OF HGSOC TO PARPI FOR INFORMED COMBINATION THERAPIES

Abstract At least 50% of high grade serous ovarian cancer (HGSOC) patients display defects in the components of the homologous recombination (HR) DNA repair pathway, which is the only high fidelity DNA repair mechanism for double strand breaks (DSB). This provides an exciting opportunity for targeted therapies using drugs such as poly (ADP-ribose) polymerase inhibitors (PARPi). PARP is an important component of the base excision repair and alternative non-homologous recombination DNA repair pathways. Therefore, inhibition of PARP enzymatic activity induces DNA damage and leads to synthetic lethality in HR defective tumors. Several clinical trials with PARPi have shown an improvement of progression-free survival in patients but have only shown a limited effect on overall survival. As for many other targeted therapies, the cancer cells rapidly adapt to the stress induced by the drug and develop resistance. Resistance can either be genomic or adaptive. In adaptive resistance the cellular networks are rewired in the absence of genomic changes to mediate resistance. To overcome this resistance, several groups, including ours, suggested the use of PARPi-based combination therapies. Several clinical trials are currently testing PARPi combinations with drugs targeting the immune checkpoint, the DNA damage checkpoint, the PI3K pathway, and the MAPK pathway. Although several of these combinations are promising, the main challenge remains in finding the right drug combination for the right patient since each tumor has the potential to adapt differently to PARPi. We thus implemented a window of opportunity trial to determine whether it is possible to identify different adaptive responses to PARPi by comparing pre and post-treatment tumor samples from multiple sites in the peritoneal cavity. We used reversed phase protein array (RPPA) analysis to measure the expression of over 300 proteins in each tumor samples and compared pathways activity in pre and post treatment samples from individual patients. We also compared the adaptive response detected in cancer patients with the one observed in cell lines. Base on pathway scores, we developed an approach to predict which combination with PARPi would be most likely to benefit a specific patient. Overall, our results indicate that the adaptive response to PARPi treatment can be detected early during treatment and that individual patients utilize different pathways to adapt to the stress induced by the drug. This study reinforces the need and opportunity for informed combination therapies to improve outcomes in this devastating disease. Citation Format: Marilyne Labrie, Tae-Beom Kim, Zhenlin Ju, Sanghoon Lee, Yiling Lu, Ken Chen, Gordon B. Mills and Shannon N. Westin. WINDOW OF OPPORTUNITY TRIAL: ASSESSING THE ADAPTIVE RESPONSE OF HGSOC TO PARPI FOR INFORMED COMBINATION THERAPIES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-098.

PARP Inhibitors Are Effective in IDH1/2 Mutant MDS and AML Resistant to Targeted IDH Inhibitors

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are heterogeneous clonal disorders. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations are detected in ~20% of AML and ~5% of MDS, in which they confer gain of a neomorphic function that leads to the production of (R)-2-hydroxyglutarate (2HG). Targeted inhibition of mutant IDH1/2 has resulted in significant responses in IDH1/2 mutant MDS and AML but is not curative and patients relapse (Stein et al. Blood 2016, DiNardo et al. N Engl J Med 2018). 2HG accumulation inhibits the function of histone demethylases (KDM4A and KDM4B) that are critical for the homologous recombination (HR) DNA repair pathway and consequently for the repair of DNA double strand breaks (DSBs) (Mallette et al. EMBO J 2012, Sulkowski et al. Sci Transl Med 2017). In HR deficient tumors, Poly-ADP-Ribose Polymerase (PARP) is essential for DNA single strand break (SSB) repair. In IDH mutant tumors PARP inhibitors induce synthetic lethality by suppressing the repair of SSBs, which eventually get converted into DSBs (Javle and Curtin Br J Cancer 2011). We previously demonstrated that in AML, IDH1/2 mutations impair DNA damage response by inducing a defect in HR, and that this renders leukemia cells susceptible to PARP inhibitors in vitro. We hypothesized that this vulnerability would also exist in IDH mutant MDS and more importantly, that this vulnerability would persist in MDS/AML resistant to IDH1/2 inhibitors. To determine whether PARP inhibition targets IDH mutant MDS/AML in vivo, we took advantage of 2 syngeneic mouse models of MDS and AML relying on co-mutation of SRSF2/IDH2 and FLT3/IDH2, respectively. Olaparib (PARP inhibitor) effectively targeted IDH2 mutant but not IDH2 wild type MDS/AML (Fig. 1A). We next sought to determine whether PARP inhibition mediated synthetic lethality persists in MDS/AML resistant to targeted IDH inhibition. We transduced IDH2 mutant murine cells with IDH2 WT or IDH2 MUT lentiviral vectors carrying one of two published resistance mutations. While these resistance mutations conferred resistance to the targeted IDH2 inhibitor Enasidenib, cells remained sensitive to Olaparib (Fig. 1B). Patient MDS/AML is heterogeneous and in general carries additional genetic mutations and epigenetic alterations. We therefore engrafted IDH1/2 WT and MUT MDS/AML patient samples in cytokine humanized immunodeficient mice and treated with vehicle of Olaparib. Engrafted mice were assigned to vehicle or Olaparib 8 weeks after transplantation based on equal engraftment levels determined by BM aspiration. Mice were treated with vehicle or Olaparib via IP injection for 21 days. Human engraftment levels and plasma 2-HG levels were significantly reduced in Olaparib treated animals when compared to pre-treatment and vehicle-treated mice (Fig. 1C). Of note, when equal numbers of huCD34+ cells from vehicle or Olaparib treated mice were transplanted into next generation mice, engraftment was significantly higher for recipients of human cells from vehicle than Olaparib treated mice, suggesting that Olaparib is toxic to leukemia initiating cells. In contrast, IDH WT MDS/AML was insensitive to Olaparib treatment (Fig. 1C). In conclusion, PARP inhibition is effective in vivo against IDH mutant MDS/AML and can overcome targeted IDH inhibitor resistance. Disclosures Flavell: Zai labs: Consultancy; SMOC: Equity Ownership; Troy: Equity Ownership; Artizan Biosciences: Equity Ownership; GSK: Consultancy; Rheos Biomedicines: Equity Ownership. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; novartis: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; pfizer: Honoraria. Bindra:Cybrexa: Consultancy, Equity Ownership.