ADP-ribose Polymerase Inhibitors Research Articles

Abstract 4473: DNMT and PARP inhibitor combination therapy induces an interferon-driven homologous recombination defect in triple-negative breast cancers and acute myeloid leukemia

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) are effective in a subset of triple negative breast cancers (TNBC) with BRCA gene mutations, which generates homologous recombination deficiencies (HRD), through synthetic lethality. However, PARPis fail for the majority of sporadic TNBC with intact BRCA1/2 genes and other BRCA-proficient cancers, including acute myeloid leukemia (AML). PARPi, Talazoparib (TAL), has potent PARP trapping ability, in both BRCA mutant and proficient TNBCs and AML. We reported that a combination of TAL with low doses of DNA methyl transferase inhibitor (DNMTi) azacytidine (AZA) or decitabine (DAC), increased PARP trapping even further, leading to increased levels of cytotoxic double strand breaks in vitro and decreased tumor burden in vivo. Low doses of DNMTis have been shown previously to reprogram genome wide cancer signaling, including immune and DNA damage/repair pathways. We now tie these two signaling pathways together for events through which DNMTis plus TAL drive HRD. BRCA proficient TNBC (N=2) and AML (cell lines (N=2) and primary cells (N=4)) treated with low doses of DNMTis (DAC 10nM and AZA 250-500nM), significantly downregulate expression of genes in HR and Fanconi Anemia (FA) pathways, decreasing HR activity, thus generating HRD. HR is further decreased when DNMTis are combined with TAL. We now link DNMTi/TAL induced HRD to a coupled interferon (IFN) response in both TNBC and AML. In these systems, we elucidate DNMTi/TAL potentiated cGAS-Sting and type 1 IFN signaling, culminating in the induction of IFN responsive genes and inflammatory cytokine production. Importantly, in DNMTi/TAL treated cells, increased expression of IFN signaling genes is strongly linked to downregulation of FA/HR genes through protein interaction network mapping. This inverse association between IFN and HR related genes, was further validated in TCGA data sets for AML and invasive BC, suggesting broad applicability of this observed transcriptional program. The acquisition of HRD from IFN signaling specifically, is verified by the observation of reduced HR gene expression and activity in the presence of exogenous double stranded RNA (Poly(I:C)) or IFNβ treatment in both TNBC and AML cell lines. Finally, knockdown of a key connectivity node between IFN and HR related genes, interferon sensitive gene 15 (ISG15), abrogates downregulation of FA gene expression when treated with IFNβ, suggesting mediation of HRD through ISG15. In conclusion, we have identified a novel mechanism through which IFN-sensitive genes can create an HRD phenotype in BRCA proficient TNBC and AML, suggesting that innate immunity and inflammatory processes may be intrinsically linked to DNA repair in cancer. These studies suggest that immune therapies and/or epigenetic therapy are likely to enhance responses to PARPis in BRCA proficient cancers. Citation Format: Lena J. McLaughlin, Aksinija A. Kogan, Eun Yong Choi, Rena S. Lapidus, Ying Zou, Huili Li, Stephen B. Baylin, Michael J. Topper, Feyruz V. Rassool. DNMT and PARP inhibitor combination therapy induces an interferon-driven homologous recombination defect in triple-negative breast cancers and acute myeloid leukemia [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 4473.

Abstract 2952: Predicting concentration of PARP inhibitors in human tumor tissue using PBPK modeling

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors exert their effect intracellularly within tumor, thus sufficient tumor penetration is essential for a pharmacological response. Preclinical mouse xenograft data show a 3.3-fold higher tumor versus plasma exposure of niraparib, while for olaparib tumor exposure was less than plasma. This study aimed to build a physiologically-based pharmacokinetic (PBPK) model extended with a tissue composition-based permeability-limited tumor model to: (a) gain a mechanistic understanding of the differences in tumor exposure of niraparib and olaparib; and (b) to predict clinical tumor exposure in ovarian cancer patients at clinically relevant dosing regimens. A permeability-limited tumor model was developed that integrates data on tumor composition and drug physicochemical properties analogous to the established permeability-limited organ model available for the liver in the Simcyp Simulator [1,2]. The model assumes that unbound unionized drug is in equilibrium between the vascular and interstitial compartments and movement of the drug between the interstitial and intracellular space is via passive permeability. Total tumor concentration is dependent on passive permeability of the drug, drug binding to PARP and nonspecific binding to neutral lipids, neutral phospholipids, and acidic phospholipids in the intracellular space, albumin in the interstitial and pH of the tumor interstitial and intracellular spaces. Clinical and preclinical tumor physiological parameters such as volume, blood flow, and tissue composition are defined using published data. The model was developed using the Simcyp Simulator V17.1 and R [3]. Consistent with preclinical data, the model predicts a 5-7-fold higher tumor exposure relative to plasma, as measured by the AUC tumor to plasma ratio of niraparib compared with olaparib. Significant binding to acidic phospholipids contributes to the increased tumor exposure to niraparib, a basic drug, a mechanism that is not relevant to the neutral drug olaparib. Ongoing work aims to extrapolate the model to predict the clinical tumor concentration of olaparib and niraparib in ovarian cancer patients and to investigate the sensitivity of the model to key tumor attributes, including blood flow and interstitial pH that may contribute to variability in tumor drug exposure. A similar modeling approach may be used to predict the tumor exposure of other small molecule anticancer drugs from their plasma concentration and physicochemical properties in different types of solid tumor. References Jamei M et al, Clin Pharmacokinet. 2014;53:73-87 Poulin P et al, J Pharm Sci. 2015;104:1508-21 R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. Citation Format: Rachel H. Rose, Kaiming Sun, Linzhong Li, Keyur Gada, Jing Yu Wang, Yongchang Qiu. Predicting concentration of PARP inhibitors in human tumor tissue using PBPK modeling [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 2952.

Abstract 2981: Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor

Abstract Poly (ADPribose) polymerase inhibitors (PARPi) have shown great promise in the treatment of cancer, however, their current clinical use has been largely limited to homologous recombination-deficient (HRD) tumors. While these drugs are efficacious as monotherapies, durable responses beyond 12 months are uncommon and their activity against HRD-negative tumors is limited. These findings have prompted great interest in combining PARPi’s with chemotherapy to increase the duration of response in HRD-positive tumors and expand the activity of these drugs against HRD-negative tumors which comprise a much greater fraction of the total number of cancer cases each year. While such combinations are highly active against tumors independent of HRD status, they are also extremely cytotoxic against bone marrow cells. As such, dose reductions ranging from 5- to 20-fold are required when PARPi’s are combined with chemotherapy which has resulted in favorable safety profiles but limited efficacy. Tumor-targeting strategies could overcome this efficacy barrier but the technologies developed thus far are limited by numerous issues, including: (a) lack of universal, non-saturating tumor-targeting mechanisms which limit their use to specific tumor types and their corresponding antigens, (b) inability to deliver therapeutically relevant levels of drug(s) directly into tumor cells, and (c) insufficient tumor penetration leading to sub-optimal tumor exposure. Herein, we report the development of alphalex, a novel tumor-targeted drug delivery platform which allows the efficient and selective delivery of PARPi’s into tumors with significant normal-tissue sparing. Tumor-targeting is achieved using a pH- sensitive peptide which forms an alpha-helix in cellular membrane under low pH conditions associated with the tumor microenvironment. Under these conditions, the peptide translocates across cancer cell membranes to deliver the PARPi directly into the cytoplasm. We demonstrate that alphalex peptide-based conjugates can be combined safely and effectively with both cytotoxic chemotherapies and radiation therapy (RT) and that this approach can be used to selectively kill both HRD-positive and –negative tumors with significant sparing of the bone marrow. The safety and efficacy of the approach was demonstrated in both engineered and patient-derived xenografts (PDXs) in vivo using two FDA-approved PARPi’s across two independent laboratories. These data highlight an entirely new approach to apply PARPi’s against solid tumors independent of HRD status. Furthermore, our approach can be applied to a diverse range of DNA repair inhibitors for potent and selective chemo/radio-sensitization in a tissue-agnostic manner. Citation Format: Vishwas Paralkar, Robert J. Aiello, Dan Marshall, Johanna Csengery, Patricia Bourassa, Qing Zhang, Brett S. Robinson, Lori Lopresti-Morrow, Jane Bechtold, Laurie Tylaska, Timothy Paradis, Gregory Slaybaugh, Hanna Visca, Anna Moshnikova, Dhammika Weerakkody, Oleg Andreev, Yana K. Reshetnyak, Donald Engelman, Ranjit Bindra, Peter Glazer, Per Hellsund. Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor [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 2981.

Abstract 3631: Development of targeted nanoformulation of talazoparib for combined chemoradiation therapy in lung cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy exploits a synthetic lethality strategy in cancers with inherent damage in DNA repair or transcription pathways. Talazoparib is a potent PARPi that is currently indicated for oral inhibitor therapy in several cancer clinical trials. Oral administration of these inhibitors typically results in poor bioavailability and tumor accumulation. In contrast, nanoparticle formulation provides a safe vehicle for parenteral administration of therapeutic drugs with sustainable release reducing systemic toxicity and also protect from surveillance of immune cells, thereby increasing the bioavailability of the drugs in vivo. Moreover, targeting strategy for the nanoparticles will improve the accumulation of drugs in the tumors. Here, we developed a targeted formulation of Talazoparib (NanoTLZ) which was decorated with anti-EGFR antibody as a ligand to ameliorate the cellular uptake of NanoTLZ via EGFR-mediated endocytosis. In vitro tests showed that NanoTLZ is more effective in cell growth inhibition than free Talazoparib. When combined with radiation with different doses from 2-10Gy, NanoTLZ showed a strong radiosensitization effect as evidenced with almost no colonies formation at 6 Gy of radiation dose. The high therapeutic efficacy of combined chemoradiation therapy in Calu 6 cell line can be attributed to the higher accumulation of NanoTLZ in the cancer cells as compared to the free Talazoparib which is also marred with efflux of the drug from the cells. The slow and sustained release of Talazoparib from nanoparticle formulation inside the cells lead enhanced inhibition of DNA repair pathways. These studies provide very encouraging results to evaluate the efficacy of these nanoparticles in lung cancer animal models. Citation Format: Bijay Singh, Mostafa Abdelhalim, Stephanee Warrington, Srinivas Sridhar. Development of targeted nanoformulation of talazoparib for combined chemoradiation therapy in lung cancer [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 3631.

PO-1084 Poly ADP-ribose polymerase-1 inhibitors enhance soft tissue sarcoma radiosensitivity: in vivo study.

PO-1084 Poly ADP-ribose polymerase-1 inhibitors enhance soft tissue sarcoma radiosensitivity: in vivo study.

Sequential therapeutic targeting of ovarian Cancer harboring dysfunctional BRCA1

BackgroundPoly (ADP-ribose) polymerase inhibitors (PARPi) have become the first targeted therapies available in the treatment of patients with high-grade serous ovarian cancer (HGSOC). We recently described a significant reduction in PARP1 protein levels in vitro and in vivo in patients treated with standard carboplatinum-paclitaxel chemotherapy, raising the question whether the sequence of treatment used today with chemotherapy followed by PARPi is optimal. In this study, we aim to evaluate if the sequence of PARPi followed by chemotherapy could be more beneficial.MethodsBRCA1-mutated (UWB1.287, SNU-251), epigenetically-silenced (OVCAR8), and wild-type (SKOV3, A2780PAR & A2780CR) ovarian cancer cell lines were exposed to clinically relevant doses of PARPi followed by different doses of standard chemotherapy and compared to the inverse treatment. The therapeutic efficacy was assessed using colony formation assays. Flow cytometry was used to evaluate cell apoptosis rate and the changes in cell cycle. Finally, apoptotic and cell cycle protein expression was immunodetected using western blot.ResultsExposure to PARPi prior to standard chemotherapy sensitized BRCA1-mutated or epigenetically-silenced BRCA1 cell lines to lower doses of chemotherapy. Similar results were observed in BRCA1 wild-type and cell lines in which BRCA1 functionality was restored. Moreover, this treatment increased the apoptotic rate in these cell lines.ConclusionPre-treatment with PARPi followed by standard chemotherapy in vitro is more efficient in growth inhibition and induction of apoptosis compared to the administration of standard chemotherapy followed by PARPi.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor

ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications.The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1.ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam.The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3–58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95–24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075 min−1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively.Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

Is SLFN11 a Promising Predictive Biomarker of Poly (ADP-ribose) Polymerase Inhibitor Sensitivity in Small-Cell Lung Cancer? Not Yet!

Is SLFN11 a Promising Predictive Biomarker of Poly (ADP-ribose) Polymerase Inhibitor Sensitivity in Small-Cell Lung Cancer? Not Yet!

Somatic Reversion of Germline BRCA2 Mutation Confers Resistance to Poly(ADP-ribose) Polymerase Inhibitor Therapy.

Somatic Reversion of Germline BRCA2 Mutation Confers Resistance to Poly(ADP-ribose) Polymerase Inhibitor Therapy.

Olaparib tablets for the treatment of germ line BRCA-mutated metastatic breast cancer

ABSTRACTIntroduction: Germ line BRCA mutations (gBRCAm) are diagnosed in approximately 5% of unselected breast cancer patients. Olaparib is a new treatment option for patients with a gBRCAm who have metastatic HER2-negative breast cancer.Areas covered: Olaparib is an oral poly (ADP-ribose) polymerase inhibitor that has been shown in phase I–III clinical trials to have single-agent efficacy in breast cancer patients with gBRCAm. The recent phase III OlympiAD study demonstrated a statistically significant progression-free survival benefit compared with the chemotherapy control arm, although an overall survival benefit has not been demonstrated. The most common adverse events seen with olaparib include nausea, anemia, and vomiting. The most common grade 3 adverse events are anemia and neutropenia.Expert commentary: The US FDA-approved olaparib tablets in January 2018 for the treatment of patients with a gBRCAm and metastatic HER2-negative breast cancer. This is a well-tolerated and effective treatment option for this patient population, particularly in patients with triple-negative breast cancer in which chemotherapy is the only alternative. More data are needed to understand the role of olaparib in combination with endocrine therapy, other targeted agents, and chemotherapy, as well as sequentially with platinum chemotherapy in the metastatic setting.

Abstract 139: DNA damage and immunoprofiling with highly multiplexed tissue immunofluorescence (t-CycIF) in high-grade serous ovarian cancer

Abstract Immune checkpoint blockade (ICB) has emerged as a new promising therapeutic approach in multiple cancers, however, the responses to single-agent ICBs have been modest in high-grade serous ovarian cancer (HGSOC). Preclinical- and early clinical data show promising efficacy of combining DNA damaging agents with immunotherapy. An improved understanding of the interplay between DNA damage in cancer cells and anti-tumor immune responses may therefore accelerate the development of rational drug combinations and identify predictive biomarkers. The majority of HGSOC are deficient in homologous recombination (HR) DNA repair, and use alternative, error-prone DNA repair pathways, that have been shown to be associated with increased immune recognition (Strickland et al, 2016). Compelling evidence has shown that DNA damaging agents increase the expression of immune-regulatory genes, such as MCH class I antigens, and interferons in HR deficient tumors. Further, HR deficient tumor exhibit an increased response to ICBs, and DNA damage-driven activation of interferon signalling has been shown to overcome resistance to ICBs (Wang et al, 2016). To reveal the relationship between intrinsic and treatment-induced DNA damage and the HGSOC immune microenvironment we are employing a novel, high-multiplex tissue cyclic immunofluorescence (t-CycIF) platform (Lin et al, 2016& 2017) allowing for the simultaneous detection of up to 60 different antigens at single cell resolution. Utilizing t-CycIF we are in the process of profiling the DNA damage and immune responses in three unique HGSOC clinical cohorts including i) tumors with inherent DNA repair deficiencies ii) pre- and post DNA damaging therapy iii) tumors collected in an innovative clinical trial combining Poly- ADP Ribose Polymerase inhibitor (PARPi) Niraparib and ICB Pembrolizumab. Using this high-dimensional, quantitative data we are mapping the abundance, spatial arrangement and functional state of cancer cells, immune cells, and stroma in the HGSOC microenvironment. The highly multiplexed t-CycIF data are processed with established computational algorithms and correlated with clinical outcomes. Our preliminary data shows that t-CycIF sensitively captures immune cell subpopulations, tumor heterogeneity and DNA damage in HGSOC. We anticipate that t-CycIF could accelerate the development of rational strategies for combining DNA damaging agents with immunotherapy to ultimately improve the treatment and outcomes of patients with ovarian cancer. Citation Format: Anniina Farkkila, Sameer S. Chopra, Jia-Ren Lin, Zoltan Maliga, Bose Koruchupakkal, Kyle C. Strickland, Brooke E. Howitt, Sandro Santagata, Ursula A. Matulonis, Kevin Elias, Elizabeth M. Swisher, Panagiotis A. Konstantinopoulos, Peter Sorger, Alan D. D'Andrea. DNA damage and immunoprofiling with highly multiplexed tissue immunofluorescence (t-CycIF) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 139.

Abstract 568: Recurrent drug sensitivity patterns in myelodysplastic syndrome patients are recapitulated by ex vivo drug response profiling

Abstract Myelodysplastic syndromes (MDS) are a collection of clonal diseases of dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. Limited conventional treatment options exist for these patients, with hypomethylating agents remaining the standard of care for higher-risk MDS patients. Drug sensitivity and resistance testing on myelodysplastic syndromes (MDS) samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof-of-concept data by profiling the effects of 50 common oncology drugs on 14 myelodysplastic (MDS) samples from both treatment-naïve and refractory cases. Ex vivo high-throughput functional screening was performed on lysed peripheral blood and/or bone marrow aspirate patient samples. This set of samples was analyzed 72 hours post incubation and blasts were quantified by flow cytometry. Unsupervised hierarchical clustering was performed to determine similar responses between patient samples, and produced two distinct groups with differing sensitivity to tested drugs. We defined these groups as “sensitive” and “resistant” clusters based on the prevailing drug sensitivity patterns these groups displayed, the latter being strongly enriched for HMA refractory samples. Ex vivo testing recapitulated known patient drug sensitivity patterns, with 12/14 samples from known HMA refractory vs. sensitive patients showing similar ex vivo phenotypes. Interestingly, sensitive and resistant clusters strongly correlated with known features associated with therapy resistance and poor prognosis in MDS patients, including number of somatic driver mutations (average 2.38 vs. 4.5, P <0.05), absence vs. presence of RUNX1 or ASXL1 mutations (P 0.015 and 0.0256, respectively), and HMA sensitive vs. refractory status (P <0.05). Interestingly, percent bone marrow blasts, cytogenetic risk groups and other somatic mutations had no effect on sensitivity patterns. Furthermore, while the resistant cluster showed increased resistance to most drugs, some drugs seemed to have increased activity in this cluster, including calcitriol and poly ADP ribose polymerase (PARP) inhibitor rucaparib. This unique platform, applied to predict ex vivo therapeutic response of MDS patient samples to various classes of drugs, recapitulates known clinical and molecular predictors of therapeutic response, and possible new therapeutic targets. These data suggest the possible utility of using this methodology to aid decision making for therapeutic selection in the management of MDS patients. Citation Format: Alexey Aleshin, Bruno C. Medeiros, Savita Kamble, Diane Heiser, Marianne Santaguida, Sacha Prashad, Peter L. Greenberg. Recurrent drug sensitivity patterns in myelodysplastic syndrome patients are recapitulated by ex vivo drug response profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 568.

Abstract 1622: In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models

Abstract Background: Recently, poly ADP ribose polymerase (PARP) inhibitors have shown potential to increase the sensitivity of platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies in BRCA or DNA repair pathway genes altered or WT condition (Basourakos et al., 2017; Gampenrieder et al., 2017). Src, a non-receptor tyrosine kinase, is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib (D) is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which has displayed anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011). Our current study examines whether combinational inhibition of Src pathway (D) would exhibit greater antitumor activity of PARP inhibitor [veliparib (V)] plus carboplatin (C) in TNBC xenograft models with different genetic back grounds. Methodology: a) We utilized MDA-MB-231, MDA-MB-468 and SUM149PT cells for in vivo study. b) TNBC cells were implanted subcutaneously into immune-compromised mice (NCr Nu/Nu, Taconic) to generate tumor xenografts. c) We evaluated the effects on tumor growth inhibition of D (12.5 mg/kg, i.p., Qd for 4 weeks) / V (25 mg/kg, oral, Bid for 5 days) / C (40mg/kg, i.v., once) as single agent or in combinations (Chan et al., 2012; De et al., 2014). d) For IHC studies, tumor tissue sections were stained with respective antibodies for proliferation, cell survival, angiogenesis and apoptosis, including: phospho-Src, Ki-67, phospho-S6 RP, phospho-Akt, phospho-ERK, CD31, cyclin D1 and cleaved caspase3. e) Student's t test was used for data analyses. Results: We observed that 1) MDA-MB-231(KRAS/BRAF mutated) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, the best tumor inhibition result was induced by triple combination (p < 0.004); 2) MDA-MB-468 (PTEN null) xenograft model: D alone or combined with V or C or in triple combination significantly inhibited tumor growth, the best tumor inhibition result was observed by triple combination (p < 0.00006); 3) SUM149PT (BRCA1 mutated, PTEN null) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, and triple combination, as well as D plus C, showed greater (p < 0.0025) than other combinations / single agents; 4) Single agent of D / V / C was well tolerated with less than 10% mean body weight loss compared with control groups in all models. Conclusion: Our in vivo study suggests that D may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC xenograft tumors with different genetic backgrounds. Immunohistochemical studies of xenograft tumor samples are ongoing, the results of which will be presented at the meeting. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer Carlson, Pradip De, Nandini Dey, Casey Williams, Brian Leyland-Jones. In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1622.

Abstract 5721: Neoantigen load and HLA-class I expression characterize a subset of HR-proficient high-grade serous ovarian carcinomas with favorable prognosis and T cell-inflamed phenotype

Abstract Aim: Around 50% of high-grade serous ovarian carcinomas (HGSC) have deficiency in homologous recombination (HR) pathways. There has been increased evidence of benefit for the use of poly ADP ribose polymerase (PARP) inhibitors in a group of HGSC patients who especially have HR-deficient tumors. Therefore, it is needed to develop a new treatment strategy for the rest, an HR-proficient tumor. The aim of this study is to investigate immunological background of HR-proficient HGSC by integrated molecular analysis to explore the subset that would become candidates for immunotherapy such as immune checkpoint inhibitors. Methods: In total, 80 cases of HGSC were analyzed in this study. Exome and RNA sequencing were performed to identify germline and somatic mutations. Methylation arrays were also carried out to evaluate BRCA1 and RAD51C promoter methylation status. Predicted neoantigens derived from mutations were identified based on the MHC class I binding prediction algorithm NetMHCpan 2.8. Immune profile in the tumor was assessed by differential gene expression analysis and gene set enrichment analysis (GSEA) using RNA sequencing data. Neoantigen load, antigen presentation, immune profile and their relevance to clinical outcomes were also investigated. Results: Either BRCA1/2, RAD51C/D mutations, or BRCA1, RAD51C promoter methylation was defined as HR-deficient. A total of 34 (42.5%) and 46 (57.5%) patients were classified as having HR-deficient and HR-proficient tumors, respectively. As expected, the numbers of predicted neoantigens were lower in HR-proficient than HR-deficient tumors (p<0.01). However, 40% of the patients with HR-proficient tumors still had high numbers of neoantigens and displayed better survival trend than those who had lower numbers of neoantigens. Incorporation of HLA-class I expression status in the analysis revealed that the patients who had both high neoantigen number and high HLA-class I expression showed improved survival in HR-proficient HGSC (p=0.02). GSEA demonstrated that the gene sets for effector memory CD8, TH1, and type I and type II interferon (IFN) responses were enriched in those patients. Conclusions: The number of neoantigens and HLA-class I expression characterized a subset of HR-proficient HGSC with improved prognosis and immunologically hot phenotype. This subset might be a candidate target for immune checkpoint inhibitors rather than PARP inhibitors. Citation Format: Hirokazu Matsushita, Kosei Hasegawa, Katsutoshi Oda, Shogo Yamamoto, Kayo Asada, Akira Yabuno, Akira Nishijima, Takahiro Karasaki, Yuji Ikeda, Keiichi Fujiwara, Hiroyuki Aburatani, Kakimi Kazuhiro. Neoantigen load and HLA-class I expression characterize a subset of HR-proficient high-grade serous ovarian carcinomas with favorable prognosis and T cell-inflamed phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5721.

Abstract 4847: Evaluation of the combination of olaparib and cediranib in small-cell lung cancer cells

Abstract Metastatic Small cell lung cancer (SCLC) is exceptionally lethal, having a median survival of 9-10 months from the time of diagnosis and a 5 year survival rate of less than 2 percent. More effective treatment approaches to SCLC are desperately needed. Poly ADP-ribose polymerase (PARP) inhibitor such as olaparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. Preclinical data indicates that SCLC cell lines overexpress PARP and are sensitive to PARP inhibition, and PARP inhibitors show promise in the clinical setting of SCLC. The identification of combination regimens that potentiate the efficacy of PARP inhibition would be of great clinical value. Recent findings indicate that olaparib and the vascular endothelial growth factor receptor (VEGFR) inhibitor, cediranib, is an effective therapeutic combination in patients with BRCA wild-type ovarian cancer. Therefore, we evaluated the efficacy of the olaparib and cediranib combinations in SCLC models. Human SCLC cell lines, NCI-H841, H526, COR-L24 and murine SCLC cell line TKOmTmG were sensitive to olaparib or cediranib therapy in vitro as determined by Cell Titer Glo® assay. Moreover, a significantly greater effect on cell viability was observed when olaparib and cediranib were used in combination. Likewise, in H841 xenograft models, single agent treatment of olaparib or cediranib inhibited tumor growth, and combination treatment of olaparib plus cediranib induced tumor regression. Western blot analysis indicated that treatment with cediranib or the combination of olaparib and cediranib induced PD-L1 protein expression both in vivo and in vitro. In TKOmTmG xenograft model in syngeneic mice, programmed death-ligand 1 (PD-L1) inhibitor plus olaparib or olaparib/cediranib combination resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition. These data indicate that olaparib plus cediranib may be an effective therapeutic strategy for the treatment of SCLC. Further study is warranted to understand the mechanism of action and clinical activity. Citation Format: Naoto Morikawa, Monique B. Nilsson, Irene Guijarro, You-Hong Fan, Alissa Poteete, John V. Heymach. Evaluation of the combination of olaparib and cediranib in small-cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4847.

Preoperative olaparib in early-stage endometrial cancer (EC): A phase 0, window of opportunity trial to evaluate the PARP inhibition effect, targeting cell cycle-related proteins (POLEN study).

5598Background: Olaparib (AZD2281, KU-0059436) is a poly ADP ribose polymerase (PARP) inhibitor. Biomarkers that predict a response to olaparib in EC are not fully established. The aim of this stud...

EMBRACA: Efficacy outcomes in clinically relevant subgroups comparing talazoparib (TALA), an oral poly ADP ribose polymerase (PARP) inhibitor, to physician's choice of therapy (PCT) in patients with advanced breast cancer and a germline BRCA mutation.

1069Background: TALA is a dual-mechanism PARP inhibitor that traps PARP on DNA, preventing DNA damage repair and causing cell death in BRCA1/2-mutated cells. Methods: EMBRACA (NCT01945775) is an open-label, randomized, 2-arm, phase 3 trial comparing the efficacy and safety of TALA (1 mg/day) with standard single-agent PCT (capecitabine, eribulin, gemcitabine, or vinorelbine) in pts with advanced breast cancer (aBC) and a germline BRCA1/2 mutation (gBRCAmut). PFS by blinded independent central review (BICR) and ORR by investigator were investigated in clinically important subgroups. Results: Of 431 pts randomized, 287 were assigned to receive TALA and 144 to PCT. PFS and ORR are shown (Table). Conclusions: In pts with advanced gBRCAmut breast cancer, TALA demonstrated a statistically significant improvement in both ORR and PFS in clinically relevant subgroups compared with PCT, including pts with CNS metastases, poor performance status, younger age, and visceral disease. Clinical trial information: NCT0194...

Prognostic impact of DNA repair germline variants in hormone sensitive prostate cancer stage.

262 Background: Inherited and somatic aberrations in DNA repair genes in castrate resistant prostate cancer (CRPC) are associated with poor prognosis, but respond well to poly ADP ribose polymerase (PARP) inhibitors. We evaluated the prevalence and prognostic impact of harboring germline DNA repair variants in hormone sensitive prostate cancer (HSPC). Methods: Germline DNA from buffy coat was sequenced on HiSeq4000 with a median coverage of 200X for DNA repair variants in 20 genes in HSPC and CRPC patients (pts) enrolled in a hospital registry. Pts were divided into two groups; Group A: pts enrolled at the time of CRPC stage; Group B: treatment naïve HSPC stage pts. The primary endpoints were to determine any impact of harboring DNA repair variants on time to progression from HSPC to CRPC and, from CRPC to death. Group A pts were retrospectively analyzed for time to progression from HSPC to CRPC while Group B patients were followed prospectively for outcomes. Statistical analysis included Cox proportional hazard models and Wilcoxon Rank sum test with significance at p≤0.05. Results: In Group A, 51/562 CRPC pts (9.07%) had variants in the 20 genes (most frequently in BRCA2; n = 15). 44/51 pts with variants and 399/511 without variants had died. Median time of progression from HSPC to CRPC with/without variants was 22.1 vs. 25.1 months (mths); p-value = 0.679. Median time from CRPC to death with/without variants was 32.2 Vs. 27.7 mths (p = 0.6). In HSPC Group B, 14/100 pts were identified with germline variants in ATM (n = 5), CHEK2 (n = 3), BRCA1 (n = 2), BRCA2 (n = 2), RAD50 (n = 1), and MSH2 (n = 1). 31/100 have died and median time to progression from HSPC to CRPC with/without variants was 15.6 vs.11.8 mths, p-value = 0.76. Conclusions: Pts with germline DNA repair variants detected in HSPC stage were not associated with poor prognosis. Presence of additional somatic DNA repair gene aberrations in cell-free DNA, not investigated in this cohort may add to the prevalence of DNA repair gene variations in HSPC and together impact prognosis adversely so as to provide a rationale for PARP inhibitor therapy in select HSPC stage pts.

Abstract P6-06-02: Functional homologous recombination REpair CAPacity (RECAP) test in metastatic breast cancer biopsies

Abstract Introduction Tumors of germline BRCA1/2 mutated carriers cannot repair DNA double strand breaks (DSB) due to homologous recombination deficiency (HRD), rendering them highly sensitive to Poly ADP Ribose Polymerase (PARP) inhibitors and DSB inducing chemotherapy. Although evidence is emerging that the use of these therapies could be extended beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. We have developed a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer (BrC) tissue: the RECAP test. Here, we investigated feasibility of this test on small biopsies from metastatic BrC lesions. Methods Patients with advanced or recurrent BrC with easily accessible metastases were eligible. Patients with pulmonary or bone metastases were excluded due to risk of pneumothorax and technical issues with calcifications. Biopsies were collected in customized DMEM/F12 medium, irradiated with 5Gy and cultured for 2 hours. Final test outcomes were available within 1 week after the biopsy procedure. The primary endpoint was the proportion of patients with a successful test result defined as either less than 20% (i.e. HRD), 20-50% (i.e. HR intermediate), or more than 50% (i.e. HR proficient) of S/G2 phase cells showing RAD51 foci. Using a Simon 2-stage design (p0=60%, p1=80%, α=0.1, β=0.1), 38 patients were required. When at least 27 had a successful test result, we decided to further develop this test. Results 51 patients enrolled in the RECAP trial. Core needle biopsies were collected from liver (n=16), breast (n=4), lymph nodes (n=13) and other sites (n=6), as well as punch biopsies from skin metastases (n=5). In 6 cases a biopsy could not be obtained; 1 patient was excluded as the biopsy appeared to be a metastasis of a neuroendocrine tumor. Of 44 obtained biopsies, 42 biopsies contained tumor cells. Successful test results were obtained in 40 of the 42 metastatic biopsies (95%, CI 89-100%). The reason for the two unsuccessful tests, both derived from ER+/PR+/HER2-liver metastases, was absence of tumor cells in S-phase. Success of the test in biopsies was independent of BrC subtype (p=0.12), histological grade (p=0.65), receptor status (p=0.74), biopsy type (p=0.82) and metastatic site (p=0.78). We found that 25 metastatic lesions were HR proficient, 13 were HR deficient and 2 were HR intermediate. Germline BRCA testing showed mutations in 6/13 HRD lesions. Somatic and epigenetic analyses are ongoing. Conclusion In this study, we enhance the diagnostic potential and clinical applicability of the functional RECAP test by demonstrating novel feasibility on metastatic biopsies from BrC patients. Robustness of the test is shown, as its applicability extends from core needle biopsies from different metastatic sites to punch biopsies from skin metastases. Comparison of HRD status of primary versus metastatic tumors and among BrC subtypes will be presented at the conference. Citation Format: Meijer TG, Verkaik NS, Naipal KAT, van Deurzen CHM, Kanaar R, van Gent DC, Jager A. Functional homologous recombination REpair CAPacity (RECAP) test in metastatic breast cancer biopsies [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-06-02.

Retrospective analysis of patients using olaparib (O) in pancreatic cancer (PC).

389 Background: Molecular analysis has revealed four subtypes of PC giving clinicians further insight into treating this deadly disease. One subtype that was elucidated termed “unstable” is significant for the presence of DNA damage repair deficiency and can be targeted therapeutically. One such therapy, O, from the drug class poly ADP ribose polymerase (PARP) inhibitors, has already been FDA approved for individuals with BRCA mutated ovarian cancers. We performed a retrospective analysis on patients with PC treated at a single institution who have DNA damage repair deficiency mutations and have been treated with O. Methods: A chart review identified pancreatic cancer patients with DNA repair pathway mutations who were treated with O. The primary objective examined ORR in patients with PC with DNA repair mutations receiving O. Secondary objectives included tolerability, overall survival (OS), CA 19-9 change, and changes in quantitative textural analysis (QTA) on CT. Results: 11 individuals were identified, 5 carriers of a pathogenic germline (g) BRCA2 mutation, 1 carrier of a pathogenic g ATM mutation, 1 carrier of a pathogenic g BRCA1 mutation. Variants of uncertain significance (VUS) included 1 g ATM mutation, 1 g PALB2 mutation, 1 somatic (s) C11orf30 mutation, and 1 s BRCA2 mutation. Median age at diagnosis was 59, with 4 M and 7 F. No patients met criteria for familial PC and 7 had a family history consistent for breast and ovarian cancer syndrome. All individuals had metastatic PC and had progressed on at least 1 line of systemic therapy. ORR was 18%. Median time of therapy on O was 5 months (mo) (Range 1.4 to 29.567 mo) with 5 of the individuals still undergoing treatment at the time of analysis. Mean OS was 12.35 mo, 9 of the 11 individuals still alive. QTA of baseline CTs from subjects with liver (8/11) and pancreatic tumors (7/11) revealed a strong association between lesion texture and OS (Pearson correlation coefficient (PCC): hepatic mets = 0.952, p = 0.0003) and time on O (PCC: panc lesions = 0.889, p = 0.006). Conclusions: In individuals with metastatic PC with mutations involved in DNA repair, O may provide clinical benefit. QTA of individual tumors may allow for additional information that predicts outcomes to PARP inhibitors in this population.