PARP Inhibitor Sensitivity Research Articles

Abstract GMM-027: DYNLL1 INHIBITS DNA END RESECTION IN BRCA1-DEFICIENT CELLS AND REGULATES PARP INHIBITOR SENSITIVITY

Abstract High-grade serous ovarian carcinoma (HGSOC) patients with germline mutations in BRCA1/2 exhibit high sensitivity and improved outcome to double strand DNA break (DSB)-inducing agents [i.e. platinum and Poly(ADP-ribose) polymerase inhibitors (PARPi)] due to underlying defects in DNA repair via homologous recombination (HR). Due to their effectiveness, three PARP inhibitors (olaparib, rucaparib, niraparib) have recently gained FDA approval for the treatment of HGSOCs. However, de novo and acquired resistance to these agents is common even in the BRCA mutation carriers, and pose a significant, and unsolved, clinical challenge. Therefore, we adopted a systematic approach to comprehensibly identify unexplored factors/pathways that could be responsible for PARPi/platinum resistance in BRCA-defective HGSOC patients. Here we identify DYNLL1 as a negative regulator of DNA end resection through a loss-of-function CRISPR screen in BRCA1-mutant ovarian carcinoma cells. DNA end resection is a vital process that initiates homologous recombination (HR)-mediated repair of double-stranded DNA breaks (DSBs), and consequently influences genome stability. In BRCA-defective HGSOC patients, DNA end resection is greatly compromised and contribute to the loss of HR and PARP inhibitor sensitivity. Loss of DYNLL1 allows DNA end resection and restores HR in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and PARP inhibitors. In primary ovarian carcinomas low BRCA1 expression correlates with increased chromosomal aberrations, and the junction sequences of somatic structural variants indicate the loss of HR. Concurrent decrease in DYNLL1 expression in BRCA1 low ovarian cancers ‘rescued' this phenotype with reduced genomic alterations and increased homology at putative lesions. DYNLL1 limits nucleolytic degradation of DNA ends by interacting with the DNA end resection machinery (MRN complex, BLM helicase and DNA2) in cells. The impact of DYNLL1 on end resection can be re-capitulated in vitro and this is dependent on direct interaction with MRE11. In the absence of exogenous stress, depletion of DYNLL1 slows DNA replication fork progression due to ectopic activity of MRE11. Therefore, we infer that DYNLL1 is an important anti-resection factor that significantly influences genomic stability and response to DNA damaging chemotherapy. Citation Format: Yizhou Joseph He, Khyati Meghani, Marie-Christine Caron, Chunyu Yang, Daryl A. Ronato, Jie Bian, Anchal Sharma, Jessica Miller, Niraj Joshi, Alexandre Detappe, John G. Doench, Gaelle Legube, David E. Root, Alan D. D'Andrea, Pascal Drané, Subhojyoti De, Panagiotis Konstantinopoulos, Jean-Yves Masson, and Dipanjan Chowdhury. DYNLL1 INHIBITS DNA END RESECTION IN BRCA1-DEFICIENT CELLS AND REGULATES PARP INHIBITOR SENSITIVITY [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 GMM-027.

Abstract AP10: REAL-TIME ASSESSMENT OF HGSC DNA DAMAGE REPAIR DEFECTS AND DEFECT-INDUCED RESPONSE TO THERAPY IN OVARIAN CANCER ORGANOIDS

Abstract Patients with High Grade Serous Ovarian Cancer (HGSC) have limited therapeutic options. Immuno-oncologic (IO) agents have had limited effect. DNA damage repair gene mutations that may confer repair defects have been identified in up to 50% of HGSCs, making therapies that target repair defects, like PARP, CHK1, and ATR inhibitors, additional options. We have no means of predicting which patients will respond to any of these therapies. A model system that allows for functional assays to assess for DNA damage repair defects, prediction of response to therapies targeting such defects, and assessment of the functionality of the tumor immune infiltrate and its response to IO agents is needed. Organoids are three-dimensional structures derived from human normal or tumor tissue cells that anatomically and functionally mimic the developed human organ. Organoids mimicking the parent tumor from which they were derived have aided in the study of multiple tumor types. They are inexpensive and easily manipulated and may be an ideal model system for studying ovarian cancer. We have devised a functional assay platform to profile the DNA damage repair capacity and immune targetability of short-term patient-derived HGSC organoids. The organoids mimic the tumors from which they were derived morphologically, molecularly, and genetically. We have tested 33 organoid cultures derived from 21 HGSC patients for homologous recombination (HR) and replication fork protection capacity and compared the functional results to the tumor genomic profile. Regardless of repair gene mutational status, an HR functional defect in the organoids correlated with PARP inhibitor sensitivity. A fork protection functional defect correlated with carboplatin, and ATR and CHK1 inhibitor sensitivity. Importantly, this work has led to the discovery of potential therapeutic combinations, such as a CHK1 inhibitor plus carboplatin or gemcitabine that may be useful in treating tumors otherwise resistant to most therapies. Drugs such as carboplatin or gemcitabine can synergize with a CHK1 inhibitor by enhancing replication stress and fork deprotection. In parallel, we have immune phenotyped the parent tumors and organoid cultures from 15 patients, and shown that the organoid cultures retain lymphocytes expressing relevant IO receptors in the short term. Upon treatment with carboplatin, olaparib, and pembrolizumab as single agents or in combination, we detect changes in IO receptor expression and production of different cytokines in the cultures, suggesting an immune response induced by these agents. We have detected receptor and cytokine alterations that would create an immune suppressive environment with specific drug combinations in tumors with specific repair defects, suggesting that these may be inappropriate combinations for harnessing the immune system in tumors with specific repair capacities. Continued combined immune and DNA damage repair phenotyping analyses of the organoids will lead to a better understanding of which mechanistic defects are needed to confer sensitivity to DNA damage repair agents, what functional properties and immune milieu lead to sensitivity to IO agents, and how best to combine such therapies. In addition, through further correlation with patient responses over time, HGSC organoids may become a useful tool for rapidly predicting patient response to therapeutic agents. Citation Format: Sarah J. Hill, Brennan Decker, Emma A. Roberts, Chunyu Yang, Neil S. Horowitz, Michael G. Muto, Michael J. Worley Jr., Colleen M. Feltmate, Marisa R. Nucci, Elizabeth M. Swisher, Ryuji Morizane, Bose Kochupurakkal, Khanh T. Do, Panagiotis Konstantinopoulos, Joyce F. Liu, Joseph V. Bonventre, Ursula A. Matulonis, Geoffrey I. Shapiro, Ross S. Berkowitz, Christopher P. Crum, and Alan D. D'Andrea. REAL-TIME ASSESSMENT OF HGSC DNA DAMAGE REPAIR DEFECTS AND DEFECT-INDUCED RESPONSE TO THERAPY IN OVARIAN CANCER ORGANOIDS [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 AP10.

Abstract AP13: GENOME-SCALE CRISPR KNOCKOUT SCREEN IDENTIFIES TIGAR AS A MODIFIER OF PARP INHIBITOR SENSITIVITY

Abstract A key challenge is to understand the mechanisms that contribute to “BRCAness” and hence sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. Using the sensitivity to PARP inhibitor olaparib as a surrogate for “BRCAness”, we performed a CRISPR/Cas9-based genome-scale loss-of-function screen and identified TIGAR as a modifier of olaparib response. TIGAR knockdown increases the level of intracellular reactive oxygen species, induces more DNA damage after olaparib treatment, and causes “BRCAness” by downregulating BRCA1 and genes in the Fanconi anemia pathway. Further, TIGAR knockdown induces senescence, reduces spheroid tumor cell growth, and enhances the cytotoxic effect of olaparib. Finally, TIGAR is amplified in ovarian cancer and a higher expression of TIGAR is associated with poor overall survival in high-grade serous ovarian cancer. Our study shows that TIGAR knockdown causes extensive transcriptional reprogramming and enhances sensitivity to PARP inhibitor, demonstrating that TIGAR is a relevant therapeutic target for treating ovarian cancer. Citation Format: Pingping Fang, Kay Minn, Jeremy Chien. GENOME-SCALE CRISPR KNOCKOUT SCREEN IDENTIFIES TIGAR AS A MODIFIER OF PARP INHIBITOR SENSITIVITY [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 AP13.

Abstract NT-109: VAL-083 (DIANHYDROGALACTITOL) SYNERGIZES WITH PARP INHIBITORS IN BRCA-PROFICIENT AND BRCA-DEFICIENT OVARIAN CANCER MODELS

Abstract BACKGROUND: Ovarian cancer is normally treated with platinum (Pt)-based chemotherapy, but patients frequently develop Pt-resistance. Dysfunctional p53 is implicated in Pt-resistance, comprising a therapeutic challenge in high grade serous ovarian carcinoma (HGSC), where p53 is universally mutated (96%). Attempts to overcome Pt-resistance in HGSC include agents blocking the DNA repair pathways, most notably the PARP inhibitors (PARPi), leading to the accumulation of DNA double strand breaks (DSBs) and cancer cell death. Sensitivity to PARPi is correlated with deficiencies in the homologous recombination (HR) DNA repair system, which is in charge of accurately repairing DSBs. BRCA1 and BRCA2 are key proteins in HR, and mutated BRCA1/2 (present in 20% of HGSC) are well-established biomarkers for PARPi sensitivity. PARPi treatment of Pt-sensitive gynecologic malignancies have improved progression free survival (PFS); however, improvements in overall survival have not been achieved and a 5-year survival rate of only 40% remains in ovarian cancer. Additionally, resistance to PARPi in the clinical setting is emerging as a significant medical challenge. VAL-083 is a first-in-class bifunctional DNA damaging agent with demonstrated clinical activity against a range of cancers, including ovarian. VAL-083 rapidly induces interstrand cross-links at guanine-N7 leading to DSBs, activation of the HR repair pathway and cancer cell death. Notably, VAL-083 induces cell death through two parallel pathways - one p53-independent and one p53-dependent. We have shown that VAL-083 is able to overcome cisplatin-resistance in a panel of ovarian cancer in vitro models, independent of p53 status. We have also shown that VAL-083 maintains activity independent of prominent DNA repair mechanisms such as O6-methylguanine DNA methyltransferase (MGMT), non-homologous end-joining (NHEJ) and mismatch repair (MMR) implicated in resistance to numerous chemotherapeutics, including cisplatin and PARPi. Cancer cells thus rely heavily on functional HR pathway for repair of VAL-083-induced DSBs. This suggests combination therapy with agents further inducing DSBs or blocking their repair, including PARPi. Taken together, these data propose VAL-083's potential for targeting Pt-resistant HGSC and for combination treatment with PARPi. METHODS: In this study, we examined the cytotoxicity of VAL-083 in combination with PARPi (olaparib, niraparib, rucaparib, veliparib or talazoparib) against HR-proficient and HR-impaired ovarian cancer cells. VAL-083 cytotoxicity alone and in combination with PARPi was investigated using the 5-day MTT assay in HR-proficient (control siRNA) and HR-impaired (BRCA1 siRNA knockdown) A2780 ovarian cancer cells. RESULTS: We report increased VAL-083 cytotoxicity against HR-impaired A2780 cancer cells. We further report synergy between VAL-083 and PARPi olaparib, talazoparib and niraparib in both HR-proficient and HR-deficient settings. VAL-083 combination with rucaparib produced synergistic cytotoxicity in the HR-deficient setting, while VAL-083 combination with veliparib was no more than additive. These data demonstrate that VAL-083 can synergize with some PARP inhibitors in both a HR-proficient and HR-deficient setting. CONCLUSION: Our results demonstrate a distinct DNA damaging mechanism for VAL-083, resulting in the ability to overcome cisplatin-resistance, target HR-impaired tumors and overcome MGMT, MMR and NHEJ-related chemoresistance. In addition, VAL-083 synergized with several PARPi, particularly olaparib, rucaparib and talazoparib, in both HR-proficient and HR-deficient ovarian tumor cells. Citation Format: Anne Steino, Guangan He, Jeffrey Bacha, Dennis M. Brown and Zahid H. Siddik. VAL-083 (DIANHYDROGALACTITOL) SYNERGIZES WITH PARP INHIBITORS IN BRCA-PROFICIENT AND BRCA-DEFICIENT OVARIAN CANCER MODELS [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-109.

Abstract NT-093: THE N6-METHYLATION OF ADENOSINE (M6A) IN FZD10 MRNA CONTRIBUTES TO RESISTANCE TO PARP INHIBITOR

Abstract Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of messenger RNA. The m6A modification affects RNA fate and functions such as RNA stability. Despite the high initial clinical response rates to PARP inhibitors in BRCA1/2-mutated epithelial ovarian cancers, PARP inhibitor (PARPi) resistance remains a major challenge. The role of m6A modification in PARPi resistance has not previously been explored. Here we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/β-catenin pathway in BRCA-mutated EOC cells. Global m6A profile reveals a significant increase in m6A modification in FZD10 mRNA. This correlates with an increase in FZD10 mRNA stability and an upregulation of the Wnt/β-catenin pathway in PARPi resistant cells. FZD10 knockdown or inhibition of the Wnt/β-catenin sensitizes the resistant cells to PAPRi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 is sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity. Moreover, PAPRi and Wnt/β-catenin inhibitor showed synergistic suppression of growth of PAPRi resistant cancer both in vitro and in vivo in a xenograft ovarian cancer mouse model. Our results show that m6A contributes to PAPRi resistance in BRCA-deficient EOC cells by upregulating the Wnt/β-catenin pathway through stabilizing FZD10. They also suggest that inhibition of Wnt/β-catenin pathway represents a potential strategy to overcome PARPi resistance. Citation Format: Takeshi Fukumoto, Hengrui Zhu, Sergey Karakashev, Timothy Nacarelli, Nail Fatkhutdinov, Shuai Wu, Andrew V. Kossenkov, Louise Showe, Stephanie Jean, Lin Zhang, Rugang Zhang. THE N6-METHYLATION OF ADENOSINE (M6A) IN FZD10 MRNA CONTRIBUTES TO RESISTANCE TO PARP INHIBITOR [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-093.

Correlation of homologous recombination deficiency induced mutational signatures with sensitivity to PARP inhibitors and cytotoxic agents

BackgroundHomologous recombination (HR) repair deficiency arising from defects in BRCA1 or BRCA2 is associated with characteristic patterns of somatic mutations. In this genetic study, we ask whether inactivating mutations in further genes of the HR pathway or the DNA damage checkpoint also give rise to somatic mutation patterns that can be used for treatment prediction.ResultsUsing whole genome sequencing of an isogenic knockout cell line panel, we find a universal HR deficiency-specific base substitution signature that is similar to COSMIC signature 3. In contrast, we detect different deletion phenotypes corresponding to specific HR mutants. The inactivation of BRCA2 or PALB2 leads to larger deletions, typically with microhomology, when compared to the disruption of BRCA1, RAD51 paralogs, or RAD54. Comparison with the deletion spectrum of Cas9 cut sites suggests that most spontaneously arising genomic deletions are not the consequence of double-strand breaks. Surprisingly, the inactivation of checkpoint kinases ATM and CHK2 has no mutagenic consequences. Analysis of tumor exomes with biallelic inactivating mutations in the investigated genes confirms the validity of the cell line models. We present a comprehensive analysis of sensitivity of the investigated mutants to 13 therapeutic agents for the purpose of correlating genomic mutagenic phenotypes with drug sensitivity.ConclusionOur results suggest that no single genomic mutational class shows perfect correlation with sensitivity to common treatments, but the contribution of COSMIC signature 3 to base substitutions, or a combined measure of different features, may be reasonably good at predicting platinum and PARP inhibitor sensitivity.

DNA Methyltransferase Inhibitors Promote Homologous Recombination Deficiency through Induction of Immune Signaling, Sensitizing Acute Myeloid Leukemia Cells to PARP Inhibitors

Acute myeloid leukemia (AML) patients unfit for intensive chemotherapy are treated with DNA methyltransferase inhibitors (DNMTis). However, while many AML patients respond to DNMTis, responses are not durable. We previously reporteda novel treatment strategy for AML that combines DNMTis with poly (ADP-ribose) polymerase inhibitors (PARPis), drugs classically used to treat breast and ovarian cancer patients with BRCA mutations and homologous recombination defects (HRD) (Faraoni and Graziani, 2018). We found that combining low doses of the potent PARP-trapping PARPi talazoparib with DNMTis increases PARP trapping and cytotoxicityin vitroand increases therapeutic efficacy in vivo (Muvarak et al, 2016). We have nowidentified a novel mechanism through which DNMTis may sensitize BRCA-proficient AML cells to PARPis. This mechanism is tied to the capacity of these drugs to reprogram cancer signaling networks, including altering DNA repair pathways (Tsai et al, 2012). In studies in AML cell lines (N=6) and peripheral blood mononuclear cells (PBMCs) from AML patients (N=4), we now show that treatment with the DNMTi decitabine (DAC) at a low concentration (10nM) can directly induce HRD, by significantly (p<0.01) down-regulating key genes central to HR activity, including multiple genes in the Fanconi anemia (FA) pathway, as a mechanism for enhanced PARPi sensitivity. How do DNMTis downregulate HR gene expression? We show for the first time that immune signaling is linked to induction of HRD. We have previously shown that DNMTis activate innate immune pathways involving interferon (IFN) □ and tumor necrosis factor (TNF) □, a phenomenon known as viral mimicry (Chiappinelli et al, 2015).First, The Cancer Genome Atlas (TCGA) AML data sets show an inverse correlation between type 1 interferon (IFN)/pro-inflammatory response and HR-related genes. Second, we verified in BRCA-proficient AML cell lines (N=6) that immune signaling by exogenous TNF□□or IFN□□treatment decreases HR gene expression and activity by more than two-fold for the majority of genes tested (p<0.0001). Third, treatment of AML cells with IFN□and the signal transducer and activator of transcription (STAT) 1/3 inhibitor ruxolitinib can rescue DAC-induced HRD. Importantly, we identified a common immune signaling pathway induced by both DNMTis and PARPis. PARPis have also been shown to activate type 1 IFN pathways via induction of cytoplasmic double-stranded DNA sensing through signaling of the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes(cGAS-STING) pathway. We now find that inhibition of STING with inhibitor H-151 (500nM) not only rescues immune signaling induced by PARPi, but also by DAC and PARPi combination treatment. Moreover, the STING inhibitor also rescues DAC- and/or PARPi-induced HRD. These data suggest that STING may be a central signaling hub linked to HRD and also suggest ways in which epigenetic therapy, inhibitors of DNA damage response proteins, and targeted immune therapy can synergize to treat AML. Disclosures Baer: Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

TET2 Loss Accelerates Leukemogenesis By Disrupting Mismatch Repair Proteins

TET2 mutations (TET2MT) occur around 40% of myeloid neoplasms (MN) and often constitute founder hits, as concluded from patient's clonal architecture and subclinical TET2MT clones in healthy individuals at risk for MN. The mechanisms of TET2MT in leukemogenesis involve at least two potential mechanisms: i) TET2 mutations could lead to stem cell expansion and altered differentiation and/or ii) they may convey a mutator phenotype with progression due to a higher rate of subsequent genetic hits. Both mechanisms are compatible with the weak driver function of TET2MT. Here, we studied whether TET2MT predispose to additional oncogenic mutations through faulty DNA repair,i.e., whether they produce a clonally acquired mutator phenotype. TET2MT lead to the inhibition of passive demethylation. Alternatively, they impair demethylation via excision repair of 5fC/5caC. Moreover, global 5mC accumulation may increase background C>T mutation rates via 5mC deamination linking TET2 to base excision repair machinery. We investigated this hypothesis because of the finding of increased mutability found in Tet2KO and Tet2kdin the mouse as determined by the numbers of coexisting subclonal hits in Tet2MTdisease (Pan F et.al., Nature Comm, 2017). Using WES analyses (n=435), we identified 95 cases with TET2MT stringently selected for high VAF and most damaging effects as confirmed by 2D-UPLC-MS/MS assay showing low levels of the TET2-dependent DNA oxidation products. To test whether these TET2MT indeed predisposed to additional mutations, we enumerated somatic SNV vs. TET2WT cases. Sequencing of MSH2, MSH3, MSH6, MLH1, CHEK2, and BRCA2 excluded the potential effects of alteration in these genes on our results. Similarly, the MMR gene expression were not significantly different among 20 TET2MT MDS, 71 TET2WT MDS pts, and 17 controls, indicating MMR downregulation was not the culprit. Consistent with our theory, TET2MT pts had a 1.5-fold increase in median WES SNV (p<.0001), particularly at 5hmC sites. Pts with a TET2MT VAF>50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect. HeLa TET2kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. In mice, 1.4-fold increased mutagenicity at Tet2-dependent active demethylation sites was found, in a manner also suggestive of a defective MMR. TET2MT cases (n=5) were shown to be microsatellite-stable at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites. MOLM-13 TET2kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2kd renders cells vulnerable to DNA damage. In murine cells, by overexpressing Flag/V5-tagged TET2 in MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. Using HPRT mutability assay, we measured mutational frequency in the HPRT1 gene in control, TET2kd, MSH6kd,and TET2kd/MSH6kd HeLa cells. Moreover, HPRT1 mutational frequency in TET2kd HeLa cells increased 24-fold compared to parental controls while MSH6kd HeLa cells had a further 10-fold increase in HPRT1, likely reflecting the presence of TET2-independent mechanisms for MSH6-mediated MMR. Intriguingly, the dual TET2kd/MSH6kd HeLa cells had equivalent MSI and mutational frequencies to the MSH6kd HeLa cells, supporting MSH6-dependence for the mutator phenotype induced by TET2 loss. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2kd altered MSH6 nuclear co-localization. TET2 immunoprecipitation from human CD34+ cells demonstrated abundant MSH2 binding to TET2, although we were unable to co-precipitate MSH6 in the same experiment. In sum, we uncovered novel connections among TET2, MMR proteins, epigenetic modifications, and genomic instability. Given that MSH2/6 is known to preferentially bind 5hmC, TET2 may target MSH2/6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of pts with TET2MT-driven MN. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

DRES-05. PREDICTORS OF SENSITIVITY TO COMBINED TEMOZOLOMIDE AND PARP INHIBITOR IN GLIOMA

Abstract Agents targeting the PARP enzyme family are under active development for the treatment of gliomas. PARP inhibitors (PARPi) can enhance the effect of temozolomide (TMZ) in IDH wild-type glioblastomas, and in addition recent studies have shown that PARP inhibitors can be selectively lethal in IDH1 mutant cancers. Here, we sought to identify predictors of sensitivity and resistance to PARP inhibition in gliomas. We treated 4 IDH1 mutant and 8 IDH wild-type glioma lines, as well as IDH1 mutant fibrosarcoma line HT1080, with TMZ, the PARPi olaparib, or the combination, and assessed cellular growth and survival. In dose response assessments, 7 out of 13 lines were sensitive to olaparib monotherapy (3 IDH1 mutant and 4 IDH wild-type). Combination with TMZ resulted in 6 of 13 lines responding to dual therapy, with an additive effect seen in 5 PARPi monotherapy sensitive gliomas. Notably, 5 of the 6 lines responsive to the combination harbored CDKN2A deletion, compared to none of the non-responsive lines (p=.0021). Treatment with CDK inhibitor palbociclib partially reversed sensitivity to TMZ+PARPi in HT1080, supporting a mechanistic basis for this association. PARPi sensitivity in glioma lines can be augmented by the addition of TMZ. CDKN2A deletion may serve as a potential biomarker identifying tumors sensitive to this combination therapy.

Depletion of DNA damage binding protein 2 sensitizes triple-negative breast cancer cells to poly ADP-ribose polymerase inhibition by destabilizing Rad51.

Poly ADP‐ribose polymerase inhibitors (PARPi) have shown promising therapeutic efficacy in triple‐negative breast cancer (TNBC) patients. However, resistance ultimately develops, preventing a curative effect from being attained. Extensive investigations have indicated the diversity in the mechanisms underlying the PARPi sensitivity of breast cancer. In this study, we found that DNA damage binding protein 2 (DDB2), a DNA damage‐recognition factor, could protect TNBC cells from PARPi by regulating DNA double‐strand break repair through the homologous recombination pathway, whereas the depletion of DDB2 sensitizes TNBC cells to PARPi. Furthermore, we found that DDB2 was able to stabilize Rad51 by physical association and disrupting its ubiquitination pathway‐induced proteasomal degradation. These findings highlight an essential role of DDB2 in modulating homologous recombination pathway activity and suggest a promising therapeutic target for TNBC.

A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor.

While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

MA12.03 PARP Inhibitor Sensitivity Does Not Depend on BAP1 but Is Enhanced by Temozolomide in MGMT Deficient Human Mesothelioma Cells

BRCA1 associated protein 1 (BAP1), a nuclear deubiquitinase involved in DNA double-strand (DSB) break repair by homologous recombination (HR), is frequently mutated in mesotheliomas. Because poly (ADP-ribose) polymerase inhibitors (PARPIs) target PARP1 and PARP2 and induce synthetic lethality in BRCA1/2 mutant cancers deficient in HR, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesotheliomas. Ten patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression and function. Cellular sensitivity to two clinical PARPIs, olaparib and talazoparib were tested as single agents, and in combination with temozolomide. BAP1-deleted mesothelioma cellular models were generated by CRISPR/Cas9 and assessed for sensitivity to PARPIs. Because Schlafen 11 (SLFN11) and O6-methylguanine methyltransferase (MGMT) also drive response to temozolomide and PARPIs, we tested their expression and relationship with drug response. BAP1 inactivating mutations were present in eight of ten cell lines, with two harboring homozygous deletion. Cell lines exhibiting BAP1 expression also showed deubiquitinase activity (DUB). IC50 of olaparib and talazoparib plot classified them into sensitive or resistant population irrespective of BAP1 status (Figure 1). Although BAP1 knockout led to the loss of DUB activity, it did not increase the sensitivity of the cell ines to PARPI. Interestingly, cellular sensitivity to PARPI was increased by temozolomide in MGMT-negative and SLFN11-positive cell lines (Table 1).Table 1Summary reflecting combination study between talazoparib and temozolomide in different cell lines having varying MGMT and SLFN11 expression status.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BAP1 status does not determine cellular sensitivity to PARPIs in patient-derived mesothelioma cell lines. In MGMT-deficient and SLFN11-positive cells, combination of PARPI and temozolomide is synergistic.

P1.14-22 SASH1, a Novel Prognostic and Predictive Factor for PARP Inhibitors in Lung Cancer

Genomic instability is a universal hallmark of all cancers. Many of the most commonly used chemotherapeutic agents target this genomic instability by directly damaging the DNA, which results in tumour cell death. Our previous work has revealed that loss of SASH1 is associated with impaired apoptosis and increased cellular proliferation. A new generation of drugs have been developed that target the DNA repair enzyme PARP to induce DNA damage and cell death. SASH1 (SAM and SH3 domain containing protein 1) has been described as a tumour suppressor and in support of this SASH1 mRNA levels are decreased in lung, breast, thyroid and colorectal cancers. Our data demonstrates that SASH1 functions in the repair of DNA damage and loss of SASH1 protein expression could be used as a companion diagnostic for PARP inhibitors. SASH1 IHC staining of lung cancer was correlated with patient survival. DNA damage repair was assessed following the depleted of SASH1 (siRNA). SASH1 protein levels in cell lines were correlated to PARP inhibitor sensitivity. A lung cancer tissue microarray (TMA) of 225 patients was assessed for SASH1 protein level. Low SASH1 levels were associated with an improved patient prognosis in adenocarcinoma on univariate analysis (p = 0.03). Analysis of DNA repair pathways demonstrated that SASH1 plays a role in homologous recombination (HR). Based on this observation, the impact of SASH1 expression on sensitivity to PARP inhibitors was explored. An inverse correlation between SASH1 levels and sensitivity to Olaparib was identified in lung cancer cell lines Figure 1 (R2 = 0.882). We subsequently analysed Olaparib sensitivity in a panel of SASH1 depleted lung cancer cells that demonstrated increased Olaparib sensitivity. Our results indicate that SASH1 protein expression is a prognostic factor in lung cancer, high levels being associated with a worse prognosis in adenocarcinoma. Low SASH1 expression is associated with loss of HR and has the potential to be a predictive biomarker for sensitivity to PARP inhibitors in this disease.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

Genome-scale CRISPR knockout screen identifies TIGAR as a modifier of PARP inhibitor sensitivity

Treatment of cancer with poly (ADP-ribose) polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination (HR) pathway. Identification of genetic targets that induce or mimic HR deficiencies will extend the clinical utility of PARP inhibitors. Here we perform a CRISPR/Cas9-based genome-scale loss-of-function screen, using the sensitivity of PARP inhibitor olaparib as a surrogate. We identify C12orf5, encoding TP53 induced glycolysis and apoptosis regulator (TIGAR), as a modifier of PARP inhibitor response. We show that TIGAR is amplified in several cancer types, and higher expression of TIGAR associates with poor overall survival in ovarian cancer. TIGAR knockdown enhances sensitivity to olaparib in cancer cells via downregulation of BRCA1 and the Fanconi anemia pathway and increases senescence of these cells by affecting metabolic pathways and increasing the cytotoxic effects of olaparib. Our results indicate TIGAR should be explored as a therapeutic target for treating cancer and extending the use of PARP inhibitors.

Functional profiling of nucleotide Excision repair in breast cancer

Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.

Analysis of CCDC6 as a novel biomarker for the clinical use of PARP1 inhibitors in malignant pleural mesothelioma

ObjectivesCCDC6 (coiled-coil domain containing 6) is a player of the HR response to DNA damage and has been predicted to interact with BAP1, another HR-DNA repair gene highly mutated in Malignant Pleural Mesothelioma (MPM), an aggressive cancer with poor prognosis. CCDC6 levels are modulated by the deubiquitinase USP7, and CCDC6 defects have been reported in several tumors determining PARP-inhibitors sensitivity. Our aim was to investigate the functional role of CCDC6 in MPM carcinogenesis and response to PARP-inhibitors. Materials and MethodsThe interaction between CCDC6 and BAP1 was confirmed in MPM cells, by co-immunoprecipitation. Upon USP7 inhibition, that induces CCDC6 degradation, the ability to repair the DSBs and the sensitivity to PARP inhibitors, was explored by HR reporter and by cells viability assays, respectively. A TMA including 34 MPM cores was immunostained for CCDC6, USP7 and BAP1 and the results correlated by statistical analysis. ResultsMPM cells depleted of CCDC6 showed defects in DSBs repair and sensitivity to PARP inhibitors. The silencing of CCDC6 when combined with the overexpression of BAP1-mutant (Δ221-238) enhanced the HR-DNA repair defects and the PARP inhibitors sensitivity.In the TMA of MPM primary samples, the staining of CCDC6 and of its de-ubiquitinase USP7 showed a significant correlation in the tested primary samples (p = 0.01). CCDC6 was barely detected in 30% of the tumors that also carried BAP1 defects. ConclusionThe combination of CCDC6 and BAP1 staining may indicate therapeutic options for DDR targeting, acting in synergism with cisplatinum.

Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.

Abstract 363: CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors

Abstract Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We have developed a novel RAD51 inhibitor, CYT01B, which we have previously shown to be preferentially active in Activation Induced Cytidine Deaminase (AID) expressing cells, and in other tumor derived cell lines with elevated levels of DNA damage and/or reduced DNA repair capabilities. CYT01B acts by reducing RAD51 focus formation, and depleting the nuclear pool of RAD51. We have observed anti-tumor effects with CYT01B in preclinical models, but have yet to determine if RAD51 could act as a sensitizer to current therapeutics. A main resistance mechanism to PARP inhibitors is the overexpression of RAD51, making it an attractive potential combination for our RAD51 inhibitor. To determine potential drug synergy, a matrix study was performed with CYT01B (concentration range of 20nM to 5µM) and 5 different PARP inhibitors including olaparib (78nM to 5µM), niraparib (78nM to 5µM), veliparib (780nM to 50µM), rucaparib (156nM to 10µM), and talazoparib (39nM to 2.5µM). The combination matrix was tested in cell lines of varying AID expression and PARP inhibitor sensitivity: ARPE19/HPV16 (HPV immortalized epithelial cell line), KYSE-70 (head and neck cancer cell line), Daudi (Burkitt’s Lymphoma cell line), HCC1143 (TNBC), and BT20 (TNBC). We used both the Loewe Additivity model and the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). In general, we observed synergy with all the combinations in the tumor derived cell lines. In the transformed epithelial cell line, synergy was observed only in the olaparib combination. The strength of the synergistic effect differed amongst the PARP inhibitors, with olaparib giving the strongest synergy scores. From this data we hypothesize that the synergistic activity with CYT01B is related to the PARP trapping efficiency of the PARP inhibitors; greater trapping efficiency the greater the synergy. These data suggest that CYT01B may be active as a combinatorial therapy with PARP inhibitors. Overall, we conclude that there is significant potential for combining RAD51 and PARP inhibition in future cancer treatment strategies, and warrants future exploration in vivo. Citation Format: Tyler Maclay, Melinda Day, Kevin Mills. CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 363.