ADP-ribose Polymerase Inhibitors Research Articles

Effect of inducing HR deficiency using AVB500, a receptor tyrosine kinase AXL inhibitor, on response to olaparib in uterine serous cancer.

e18108 Background: Evidence suggests DNA repair is a therapeutic target in endometrial cancer (EC). Given this, we determined whether combination therapy with AVB500, an AXL inhibitor, could improve response in a uterine serous cancer (USC) model. Methods: Two USC cell lines (ARK1 & ARK4) were treated with AVB500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB500 alone, olaparib alone or combination treatment (olaparib + AVB500); colonies were stained and absorbance was obtained to calculate relative cell viability using Graph Pad Prism. Baseline homologous recombination (HR) status was determined after radiating cells with 10Gy and identifying RAD51 foci by immunofluorescence (IF). Cell lines were considered to be HR proficient if over 30% of the cells expressed RAD51 ( > 5 foci per cell). IF was conducted using a Leica confocal microscope and foci were quantified using FociCounter. In vivo studies were performed using NOD-SCID mice injected with 1 x 107 ARK1 cells intraperitoneally followed by treatment q3 days for a 14 and 21 day treatment period. Treatment groups were vehicle control, AVB500 alone, olaparib alone and olaparib with AVB500. Results: The absorbance for olaparib + AVB500 was significantly less than the olaparib only group in two assays involving ARK1s (0.417nm vs 0.756nm, p = 0.001; 0.320nm vs 0.620nm, p = 0.008) as well as in ARK4s (0.186nm vs 0.641nm, p = 0.003). The HR assay indicated both cell lines were HR proficient. After baseline HR proficiency was established, the cell lines were pretreated with AVB500 prior to radiation. When compared to cells without treatment with AVB500, IF showed a decrease in RAD51 foci per cell in ARK1 (2.7 vs 7.3, p = 0.0003) and ARK4 (6.3 vs 13.0, p = 0.0054). The proportion of ARK1 cells expressing RAD51 decreased to 21%, indicating HR deficiency. Lastly, NOD-SCID mice receiving olaparib + AVB500 had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p = 0.002) and AVB500 alone (0.008g vs 0.145g, p = 0.0006) in a 14 day and a 21 day treatment period (0.212g vs 0.586g, p = 0.027 and 0.212 vs 0.494g, p = 0.005, respectively). Conclusions: HR proficient USC cell lines treated in vitro and in vivo with the combination of AVB500 and olaparib demonstrate an improved response to olaparib or AVB500 alone with a greater decrease in tumor burden. AVB500 appears to induce HR deficiency. Additional therapeutic and mechanistic experiments are ongoing.

Comparison of poly(ADP-ribose) polymerase inhibitors (PARPi's) as maintenance therapy for platinum-sensitive ovarian cancer: Systematic review and network meta-analysis.

e18070 Background: Over the past decade, 3 PARPi's (olaparib, niraparib and rucaparib) have been approved by the FDA as maintenance therapy for recurrent ovarian cancer. However, thus far, no trial compared the 3 approved PARPi's in the overall population, in patients with BRCA mutations (BRCAm), or in those with wild-type BRCA (BRCAwt). Methods: We performed a systematic review and identified relevant literature from Embase, Pubmed and Cochrane Library. To be included in the analysis, studies had to be phase 2 or 3 randomized controlled trials (RCT) of platinum-sensitive recurrent or newly diagnosed ovarian cancer, randomized to placebo or PARPi as maintenance therapy and where complete or partial response was documented. A frequentist network meta-analysis was used for indirect comparisons between the different PARPi's to assess superiority in terms of progression free survival (PFS), overall survival (OS), and adverse events. Results: Overall, 6 RCTs involving 2,770 patients, were included in the network meta-analysis. Results from the indirect comparisons revealed no statistically significant differences between the 3 different PARPi's with respect to OS. PFS results demonstrated a nonsignificant trend supporting superiority for niraparib in all patients as well as in BRCAm patients (Table). Only 2 studies reported OS results for niraparib and none reported OS results for rucaparib. Niraparib showed a statistically significant increased risk for all grade thrombocytopenia and neutropenia. Conclusions: Our analysis demonstrates no statistically significant differences between the 3 PARPi’s in any of the analyzed groups, and a trend supporting PFS superiority for niraparib in all patients and in BRCAm patients, while having greater risk for thrombocytopenia and neutropenia. Additional OS results are pending from current studies, and would be useful for elucidating the relative roles of the different PARPi's in platinum-sensitive ovarian cancer. [Table: see text]

In Silico Discovery of Novel Flavonoids as Poly ADP Ribose Polymerase (PARP) Inhibitors

The concept of synthetic lethality is an emerging field in the treatment of cancer and can be applied for new drug development of cancer as already been represented by Poly (ADP-ribose) polymerase (PARPs) inhibitors. In this study, we performed virtual screening of 329 flavonoids obtained from the Naturally Occurring Plant-based Anti-cancer Compound-Activity-Target (NPACT) database to identify novel PARP inhibitors. Virtual screening carried out using different in silico methods which include molecular docking studies, prediction of drug-likeness and in silico toxicity studies. Fifteen out of 329 flavonoids achieved better docking score as compared to rucaparib which is an FDA approved PARP inhibitor. These 15 hits were again rescored using accurate docking mode and drug-likeliness properties were evaluated. The accuracy of the docking method was checked using re-docking. Finally NPACT00183 and NPACT00280 were identified as potential PARP inhibitors with docking score of -139.237 and -129.36, respectively. These two flavonoids also showed no AMES toxicity and no carcinogenicity which was predicted using admetSAR. Our finding suggests that NPACT00183 and NPACT00280 have promising potential to be further explored as PARP inhibitors.

Abstract IA23: Marker-guided targeted therapy, PARP inhibitors, and novel post-translational modification of PD-L1

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. Three PARP inhibitors have been approved by the FDA to treat ovarian cancer with (olaparib and rucaparib) or without BRCA mutations (niraparib). BRCA1 and BRCA2 play essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. My group recently demonstrated that receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907. Phosphorylation of PARP1 Tyr907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. Combining c-Met and PARP1 inhibitors synergized to suppress growth of breast cancer cells in vitro and xenograft tumor models. Similar synergistic effects were observed in a lung cancer xenograft tumor model. These results suggest that PARP1 pTyr907 abundance may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients bearing tumors with high c-Met expression who do not respond to PARP inhibition alone (Nature Medicine 2016;22:194). We are working with clinicians at MD Anderson to initiate marker-guided clinical trials using combinational therapy of PARP and c-MET inhibitors. Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumor-associated immune escape. We further explored whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis and determined whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression. PARP inhibitor (PARPi) treatment upregulates tumor cell PD-L1 expression, which attenuates PARPi efficacy via cancer-associated immunosuppression. The blockade of PD-L1 can restore the attenuated antitumor immunity and potentiate PARPi in tumor suppression (Clinical Cancer Res 2017;23:3711). My group also conducted a highly translational study, which identified a link between the ubiquitination and glycosylation pathways that regulate the immunosuppressive activity of PD-L1 (Nature Communications 2016;7:12632). We showed that glycosylation of PD-L1 is required for its protein stability and interaction with PD-1. In addition, we demonstrated that metformin-activated AMPK kinase downregulates PD-L1 through phosphorylation of Ser-195, which alters glycosylation of PD-L1 and functionally mimics anti-PD-L1 to block PD-L1/PD-1 interaction. It exhibited a highly potent synergistic effect of combination therapy of metformin, a drug that has been used to treat patients with diabetes and anti-CTLA4. The therapeutic efficacy could reach to survival rate of 50-70% in different syngeneic mouse models, which were treated by this combination therapy (Molecular Cell 2018;71:606). We also demonstrated that epithelial-mesenchymal transition (EMT) enhances PD-L1 in cancer stem-like cells (CSCs) by the EMT/β-catenin/STT3-PD-L1 signaling axis. Etoposide, a commonly used anticancer chemotherapy drug, is able to suppress this signaling axis, resulting in downregulation of PD-L1 to sensitize cancer cells to anti-Tim 3 therapy (Nature Communications 2018;9:1908). These findings provide potential therapeutic strategies to enhance cancer immune therapy efficacy by targeting PD-L1 stabilization to combat multiple cancer types. We identified TNFα as a major factor triggering cancer cell immunosuppression against T-cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1) (Cancer Cell 2016;30:925). To this end, in collaboration with StCube Pharmaceuticals Inc., we have developed monoclonal antibodies against glycosylation-specific PD-L1. Impressive therapeutic effect was observed through antibody-drug-conjugate approach (Cancer Cell 2018;33:187). Most recently, we unraveled an active defense system of PD-L1, previously known to only associate with passive defense activity. We discovered that cancer cells that express PD-L1 can secrete exosomes containing PD-L1 (exosome-PD-L1). The exosome-PD-L1 is able to directly interact with PD-1 to inhibit T-cell activation as well as T-cell killing activity (Cell Res 2018;28:862). Our new discoveries provide a new means to enhance therapeutic efficacy of immune checkpoint therapy. Citation Format: Mien-Chie Hung. Marker-guided targeted therapy, PARP inhibitors, and novel post-translational modification of PD-L1 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA23.

Pharmacological inhibition of poly (ADP-ribose) polymerase by olaparib, prevents acute lung injury associated cognitive deficits potentially through suppression of inflammatory response

Acute lung injury (ALI) has been reported to be associated with high mortality rate. Moreover, ALI survivors, frequently present chronic cognitive deterioration. We have previously shown that ‘two hit’ (hydrochloric acid + lipopolysaccharide) induced ALI resulted in cognitive dysfunction through the induction of systemic inflammation. The present study was designed to explore the potential anti-inflammatory effects of olaparib (Poly ADP-ribose polymerase-1 inhibitor), on ALI mediated cognitive impairment. Olaparib was administered at dose of 5 mg/kg body weight (i.p.) 30 min before each hit. Data show that olaparib pre-treatment markedly reduced the neutrophil infiltration, alveolar capillary damage, inflammatory cytokines level (TNF-α/IL-1β/IL-6) and oxidative stress in the lungs at 24 h after ALI induction. Also, olaparib pre-treatment ameliorated the ALI associated cognitive impairment as assessed by Morris water maze test on weekly basis for 2 consecutive weeks. Further, restoration of cognitive function was associated with normalization of serum levels of TNF-α/IL-1β and improved the blood brain barrier (BBB) function, as reflected by data on expression of occludin/claudin-5 and extravasation of Evans-blue/FITC dextran in hippocampus at 1 week post injury. Finally, increased mRNA expression of VCAM-1, TNF-α and IL-1β and NF-κB activation in hippocampus indicate induction of neuro-inflammation, which was downregulated upon olaparib administration. Further, olaparib treatment 1 week after ALI induction blunted the systemic inflammation which was associated with improved BBB and cognitive function. Altogether, our results showed that olaparib protects against ALI and associated cognitive deficits in mice, and thus may offer a new treatment avenue in the area.

Tumor burden is a potential marker of PARP inhibitor effects in ovarian cancer: a head-to-head observational series

BackgroundOlaparib, a poly ADP-ribose polymerase (PARP) inhibitor, has proven to be effective and safe as maintenance therapy and multiline therapy in ovarian cancer, especially in patients with BRCA mutations. This study intended to observe the influence of tumor load on the efficacy and safety of olaparib in recurrent ovarian cancer.Cases presentationThree patients harbored gBRCAwt with low tumor load (LTL), while two women harbored BRCAmt with high tumor load (HTL) were recruited. Two of the three LTL patients achieved partial response, and the other showed stable disease. Both HTL patients were assessed to have progressive disease in a short time. Olaparib appears to be effective and safe for LTL recurrent ovarian cancer patients even if it is gBRCAwt, while the response is poor in HTL patients.ConclusionsTumor load may be another potential marker to predict the effect of PARP inhibitors. The present head-to-head observational series provides new evidence on this issue for further research from bench to bedside in the future.

A Qualitative Evaluation of Clinical Burden of Poly (ADP-ribose) Polymerase Inhibitor Usage in a Community Oncology Practice

A Qualitative Evaluation of Clinical Burden of Poly (ADP-ribose) Polymerase Inhibitor Usage in a Community Oncology Practice

Phase Ib study of niraparib plus androgen receptor-targeted therapy (ART) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC).

122 Background: Poly ADP-ribose polymerase (PARP) inhibitor and abiraterone acetate + prednisone (AA+P) has activity in pts with mCRPC regardless of DNA repair gene defects (DRD). Methods: This phase 1b study (Bedivere; NCT02924766) began with a 3+3 dose escalation of niraparib (Part 1), followed by expansion (Part 2) of the recommended phase 2 dose (RP2D) of nira + AA+P. Pts with mCRPC received nira 200 or 300 mg/day with AA+P. Dose-limiting toxicities (DLTs) were evaluated the first 28 days. Serial PK for nira were collected on cycle 1 day 1 (C1D1) over 24 h; PK for nira and abiraterone were collected on C2D1 over 10 h. Results: Nira 200 mg + 1000 mg AA+P was selected as the RP2D dose. 4 pts in 200 mg cohort and 8 pts in 300 mg cohort were enrolled in Part 1. During Part 1, 1 patient in the 300 mg cohort had two DLTs: Gr 3 fatigue and Gr 4 increased gamma glutamyltransferase. Therefore, the 200 mg dose was evaluated in 15 pts in Part 2. In 19 total pts treated with 200 mg niraparib, 12 (63%) pts had Gr 3-4 AEs, 5 (26%) pts had an AE with outcome of drug discontinuation and 1 (5%) patient had an AE with outcome of death (general physical health deterioration; unrelated). AEs of special interest were: 6 pts (32%) thrombocytopenia, 6 pts (32%) hypertension, 5 pts (26%) anemia and 3 pts (16%) neutropenia of any grade. 21% pts in 200 mg cohort had treatment emergent serious AE (TESAE) while 50% treated with 300 mg had TESAE. The exposure (AUC24h) on Day 1 of Cycle 2 for 200 mg niraparib with 1000 mg of AA were 17745±9380 ng.h/mL and 712±140 ng.h/mL, respectively. These exposures are consistent with single agent PK. Summaries of all responses will be provided, including a patient with primarily bone disease and carrying an ATM mutation who had a circulating tumor cell conversion and was on study treatment for 22.1 mo. Conclusions: The safety and PK findings support the choice of nira 200 mg in combination with AA+P in pts with mCRPC. The efficacy of nira + AA+P is being evaluated in an ongoing phase 3 MAGNITUDE study (NCT03748641). Clinical trial information: NCT02924766.

Abstract P3-11-19: Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The BRCA-mutated and PTEN null TNBC cell line, SUM149PT, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an SUM149PT xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusion: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in BRCA-mutated and PTEN null TNBC. Citation Format: yuliang Sun, Xiaoqian Lin, Jennifer Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-19.

Platinum-based therapy of patients with gastrointestinal cancers that harbor homologous recombination DNA damage response (HR-DDR) mutations: A single institutional retrospective analysis.

836 Background: HR-DDR deficiencies have been described across cancer types and associated with response to specific therapies, including platinum (Pt)-based drugs and poly ADP-ribose polymerase (PARP) inhibitors. We sought to estimate the frequency of HR-DDR gene mutations in gastrointestinal (GI) cancers and determine if these patients (pts) benefit from Pt-based therapy. Methods: A retrospective chart review was performed on 421 pts with GI cancers treated at our institution. Tumors were categorized as colorectal (CRC), gastroesophageal (GE), pancreatic (PDA), hepatobiliary (HB), or other. All had next-generation sequencing with mutations in one or more of the following HR-DDR genes: BRCA1, BRCA2, PALB2, CHEK2, ATM, ARID1A, MRE11A, NBN, BARD1, and BL M. Overall survival (OS) of pts was analyzed using Log-rank test. Results: Of those analyzed, 127 pts (84 women and 43 men) met the inclusion criteria. Forty-eight percent had primary CRC tumors, and the remainder had primary GE (17%), PDA (14%), HB (10%), and other GI (11%) tumors. Fifty-four percent had stage IV disease. Sixty-five percent received Pt-based therapy at some point during their disease course. HR-DDR analysis revealed the following mutation rates: 34% ARID1A; 16% ATM; 19% BRCA2; and 9% BRCA1. OS analysis showed no significant survival advantage based on stage or mutation type for any of the disease-specific cohorts. There was no significant OS benefit in pts who received Pt-based therapy vs. those who did not, although pts with ARID1A-mutated tumors did show a trend towards improved OS (P = 0.09). Conclusions: Despite our limited cohort, our data suggest that pts with GI tumors harboring certain HR-DDR gene mutations might benefit from Pt-based therapy, supporting the development of mutation-specific Pt-based clinical trials.

Abstract NT-088: PROGNOSTIC SIGNIFICANCE OF NR4A1/TR3 EXPRESSION IN OVARIAN CANCER

Abstract BACKGROUND: A key clinical challenge in ovarian cancer is identifying new strategies to treat patients who do not respond to poly ADP-ribose polymerase (PARP) inhibitor (PARPi) therapy. We previously linked the nuclear orphan receptor NR4A1/TR3 to pro-growth and pro-survival effects in ovarian cancer cells. However, it is unknown whether inhibiting NR4A1 function has therapeutic effects alone or in combination with PARPi in vitro. Moreover, the prognostic value of NR4A1 expression in patient tumors remains ill-defined, as two prior reports had contradictory findings for associations with ovarian cancer survival. OBJECTIVE: We undertook this study to test the therapeutic potential of inhibiting NR4A1 in ovarian cancer cells, and to clarify the prognostic value of NR4A1 expression in patient tumors. METHODS: In a panel of established ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3), we inhibited NR4A1 using the chemical antagonist, 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl) methane (C-DIM) and siRNA targeting NR4A1 (siNR4A1). Effects of C-DIM on cell growth, alone and in combination with the PARPi, olaparib and rucaparib, were assessed in sulforhodamine B (SRB) in vitro assays. Markers of apoptosis (cleaved PARP, cleaved caspase-3) and proliferation (Ki67, PCNA, p21) were measured by western blot or immunohistochemistry (IHC). In ovarian tumors, NR4A1 was measured by IHC in 203 clinically annotated formalin-fixed paraffin-embedded (FFPE) tissue samples from the Vanderbilt University Medical Center (VUMC) Tissue Repository for Ovarian Cancer (TROC). Staining intensity (1: weak; 2: moderate; 3: strong) and percent of positive nuclei (0-100) were multiplied to yield an H score for NR4A1 expression. Associations with progression-free survival (PFS) and overall survival (OS) were quantified by Hazards Ratios (HR) and 95% Confidence Intervals (CI) from proportional hazards regression. RESULTS: In ovarian cancer cell lines, C-DIM induced concentration-dependent decreases in cell growth and markers of proliferation, and stimulated apoptosis. These effects were mimicked in cells transfected with siNR4A1 compared to a non-targeting siRNA-transfected control. In combination with PARPi, C-DIM induced synergistic growth inhibition and apoptosis in vitro. In tumors, NR4A1 expression lower than the median (H score <153.6) was more common among later stage, higher grade, serous tumors with suboptimal cytoreduction or platinum resistant disease. Higher NR4A1 expression was associated with better OS (HR: 0.52, 95% CI: 0.37–0.74) in unadjusted analyses. However, after adjustment for important prognostic covariates, including age, stage, grade, and histologic subtype, higher NR4A1 was associated with significantly shorter PFS (HR: 2.35, 95% CI: 1.29–4.28). CONCLUSIONS: Our current results reconcile the discrepancy between prior NR4A1 reports, as associations differed due to confounding by clinical covariates. Shorter survival among cases with higher NR4A1 expression is supported by experimental evidence showing reduced ovarian cancer cell growth and increased apoptosis following NR4A1 inhibition, both alone and when combined with a PARPi. Together, our findings support further development of NR4A1 inhibition as a novel therapeutic approach that could improve response to PARPi therapy among ovarian cancer patients with chemoresistant disease. Citation Format: Alicia Beeghly-Fadiel, Johnathan Cooks, Dajah Chase, Marta Crispens, Dineo Khabele, and Andrew J Wilson. PROGNOSTIC SIGNIFICANCE OF NR4A1/TR3 EXPRESSION IN OVARIAN CANCER [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-088.

Abstract NT-094: TARGETED INHIBITION OF HSP90 IMPAIRS DNA-DAMAGE RESPONSE PROTEINS AND INCREASES THE SENSITIVITY OF OVARIAN CARCINOMA CELLS TO PARP INHIBITORS

Abstract Poly (ADP-ribose) Polymerase inhibitors (PARPi) are a promising class of inhibitors for the treatment of high grade serous ovarian carcinoma (HGSOC). The greatest activity of these agents is seen in patients with defects in DNA damage repair (DDR) mechanisms, including mutation or epigenetic inactivation of BRCA1 and BRCA2 genes and alterations in expression and/or function of DNA repair genes/proteins. PARPi are approved as second-line and maintenance therapies in recurrent HGSOCs. Notably, clinical trials have shown that single agent PARPi show activity in a significant number of HGSOC patients in the absence of alterations in BRCA genes, particularly in patients with platinum sensitive disease, possibly those with tumors exhibiting defects in homologous recombination (HR), or ‘BRCAness'. To extend the benefit of these agents beyond patients with inherent defects in HR, we wished to test the idea that combination of PARPi with agents that functionally abrogate HR could extend the benefit of PARPi's. An attractive molecular target for this purpose is heat shock protein 90 (HSP90) based on its essential role in mediating the maturation and stability of several key proteins required for the DDR. The goal of this study was to test the hypothesis that targeted inhibition of HSP90 with a small-molecule inhibitor ganetespib (STA-9090) would sensitize non-BRCA mutant OC cells to the PARPi talazoparib (BMN-673). To test this hypothesis, we used established OC cell lines (OVCAR3, UWB1.289), and novel OC cells lines (OC-38, OC-1) derived in our laboratory from de-identified tumors isolated from patients with HGSOC. Cells were treated with talazoparib and ganetespib alone and in combination, and the effects of drug treatment on expression of DDR proteins, ionizing radiation (IR)-induced RAD51+ and γH2AX+ foci, and cell viability was assessed. Ganetespib treatment led to dose- and time-dependent depletion of HSP90 client proteins participating in DDR including BRCA1 and 2, MRE11, CDK1, CHK1, RAD51. Treatment with ganetespib also led to a significant reduction in the percentage of RAD51+ nuclei following IR. The quantity of DNA double-strand break marker γH2Ax foci/nucleus decreased over time in vehicle treated, but not in ganetespib-treated cells. We next conducted a comprehensive analysis of cytotoxicity in cells treated with ganetespib and talazoparib alone, in combination and at differing molar ratios. Combination indexes (CI) were calculated to assess additive, synergistic and antagonistic effects using the methods of Greco plus the bootstrap to determine statistical significance. Ganetespib sensitized BRCA1-null UWB1.289 cells to the effects of talazoparib (CI=0.73, p<0.0005). Among the non-BRCA mutant cell lines analyzed, the combination of ganetespib and talazoparib were synergistic in some patient-derived cell lines, but antagonistic in others. Together, our data suggest that ganetespib effectively disrupts critical DDR pathway proteins in HGSOC cells and may sensitize non-BRCA-mutant OC cells to PARPi. From clinical perspective, this implicates the potential of sensitization of some HGSOC patients without HR pathway alterations to PARPi, and potentially other DNA-damage inducing agents. Citation Format: Rashid Gabbasov, I. Daniel Benrubi, Shane W. O'Brien, John J. Krais, Neil Johnson, Samuel Litwin, Denise C. Connolly. TARGETED INHIBITION OF HSP90 IMPAIRS DNA-DAMAGE RESPONSE PROTEINS AND INCREASES THE SENSITIVITY OF OVARIAN CARCINOMA CELLS TO PARP INHIBITORS [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-094.

Neurosurgical delivery of the poly ADP ribose polymerase-1 inhibitor olaparib from a thermo-responsive biodegradable paste potentiates radiotherapy and prolongs survival

Abstract There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste. Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study is ongoing where we already observe a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (50 days vs. 44 vs 30 respectively). A more pronounced survival benefit was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy. RNAseq data from 10 GBM patients show significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence–activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

P11.60 Neurosurgical delivery of the poly ADP ribose polymerase-1 inhibitor olaparib from a thermo-responsive biodegradable paste potentiates radiotherapy and prolongs survival

Abstract BACKGROUND There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste. MATERIAL AND METHODS Metabolic and clonogenic assays were used to assess effects on proliferation and clonal growth upon in vitro glioma exposure to olaparib. Flow cytometry and Annexin V/Propidium iodide were used to determine apoptosis. The 9L high-grade glioma orthotopic allograft model was utilised to assess survival upon intra-cavity olaparib delivery. RESULTS Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study revealed a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (95 vs. 44 vs. 30 days respectively). A more pronounced survival benefit, as measured by number of animals surviving long-term, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy, relative to standard-of-care (95 vs. 44 days). Clinical correlation was determined using RNAseq data from 10 GBM patients, showing significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence-activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. CONCLUSION Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

Targeting autophagy potentiates the anti-tumor effect of PARP inhibitor in pediatric chronic myeloid leukemia

Due to its potent cytotoxicity in BRCA-mutated tumors, synthetic lethality elicited by poly (ADP-ribose) polymerase (PARP) inhibitor gives renewed enthusiasm to researching and developing anti-cancer therapies. Chronic myeloid leukemia (CML) is a type of cancers that starts in certain blood-forming cells of the bone marrow. Here, we showed that poly (ADP-ribose) polymerase (PARP) inhibitor talazoparib could induce a concentration-dependent cytotoxicity in CML cells derived from pediatric patients. During talazoparib treatment, autophagy was markedly activated, which was confirmed by the accumulation of autophagosomes, decrease of SQSTM1 and up-regulation of LC3-II. Inhibition of autophagy by pharmaceutical inhibitor chloroquine or small-interfering RNA siATG5 significantly increased the cytotoxicity of talazoparib in pediatric CML cells and elicited synergistic anti-tumor effect in patient-derived xenograft model. Our data demonstrated that autophagy played a cyto-protective role in talazoparib-treated pediatric CML and co-treatment with talazoparib and autophagy inhibitor could induce synergetic anti-tumor effect, providing novel insights for pediatric CML treatment.

Abstract 339: Mechanisms of resistance to poly (ADP-ribose) polymerase inhibitors in prostate cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated promise in treating cancers with DNA damage repair (DDR) gene abnormalities. As a result, olaparib and more recently, rucaparib have been given Breakthrough Therapy designation by the FDA for use in metastatic castration resistant prostate cancer (mCRPC) with BRCA1/2 and ATM mutations. One of the problems encountered with PARPi is drug resistance which generally limits drug efficacy. Mechanistic studies on PARPi resistance have shown one of the main mechanisms of acquired resistance to be a reversion mutation of BRCA1/2. However, this is likely more applicable to cancers in which impaired BRCA1/2 function is due to a mutation rather than in prostate cancer (PC) where BRCA2 tends to be frequently deleted. Therefore, we hypothesize that mechanisms of resistance to PARPi in PC may involve alternative molecular mechanisms rather than a reversion mutation. Methods: We used human castration-resistant PC cell lines that harbor genomic deletions of BRCA2, PC-3 and LnCaP-Abl, and performed cell viability (MTT) assays to determine the inhibitory growth (IG) concentrations of these cell lines with talazoparib and olaparib. We cultured parental PC-3 cells in sublethal concentrations (IG 50% and IG 90%) of talazoparib-supplemented media to develop talazoparib-resistant cells. RNA sequencing followed by gene-set enrichment analysis (GSEA) of hallmark gene sets was performed on the talazoparib-resistant PC-3 cells to understand the underlying molecular mechanisms. Results: We observed that the talazoparib-resistant PC-3 cells exhibited significantly enhanced cell growth compared to parental cells when cultured in the IG 90% concentration of olaparib. However, interestingly, the talazoparib-resistant cells grew much slower in 2D compared to parental PC-3 cells when cultured in the PARPi-free media. Our transcriptomic analysis showed significant enrichment of various inflammatory response pathways, including TNF-α and IFNα/γ signaling pathways, in the talazoparib-resistant cells and even in the parental PC-3 cells transiently treated with talazoparib. Conclusion: We hypothesize that resistance to PARPi in PC may be related to upregulation of inflammatory signaling. Therefore, further exploration of TNF-α and IFNα/γ and their role in PARPi resistance mechanisms may lead to the identification of targets that allow for overcoming PARPi resistance in PC. Citation Format: Mohammad Atiq, Goutam Chakraborty, Subhiksha Nandakumar, Ying Z. Mazzu, Konrad Stopsack, Yuki Yoshikawa, Nabeela Khan, Gwo-Shu Mary Lee, Philip W. Kantoff. Mechanisms of resistance to poly (ADP-ribose) polymerase inhibitors in prostate 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 339.

Abstract 3448: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination (Rugo HS et al. The New England Journal of Medicine. 2016). The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors (Finn RS et al. Clinical Cancer Research. 2011). In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The KRAS/BRAF-mutated TNBC cell line, MDA-MB-231, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-231 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusions: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in patients with KRAS/BRAF-mutated TNBC. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer C. Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC [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 3448.

Abstract 3816: Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways

Abstract We have identified that Corilagin is a major anti-tumor active component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L. Our previous work found that Corilagin inhibited the growth of ovarian cancer cells via TGF-β/AKT/ERK signaling pathways. Corilagin also sensitizes Paclitaxel and Carboplatin by primarily inhibiting Snail-glycolysis pathways. Recently, we have investigated if Corilagin could give a help to overcome poly ADP ribose polymerase (PARP) inhibitors (PARPi) resistance in ovarian cancer cells. As we knew, PARP inhibitors are DNA repair drugs, have been approved for ovarian cancer therapy, but drug resistance is a faced problem, combination therapy may give helps. In this study, two pairs of PARPi sensitive/resistant cells: A2780CP/A2780CP_R and UMB1.289/UMB1.289_R (Sci Transl Med. 2017; 9:392) were used. Cells were cloned again and confirmed their sensitivities to PARPi. The IC50 of PARPi-BMN673 is 0.02μM to A2780CP, &gt 10μM to A2780CP_R, 0.12μM to UMB1.289 and 60μM to UMB1.289_R. The combination assays showed Corilagin had almost equally cytotoxic activities to these two pairs and had synergistic effects with BMN673 to A2780CP_R and UMB1.289_R . Using CulcuSyn, the combination indexes (CI) presented at IC50 are 0.17365 and 0.62787 in A2780_R and UMB1.289_R, respectively. Signaling analysis found that PARPi did down-regulate the expression levels of PARP and up-regulate of pH2AX, PARPi only inhibited pERK activity in sensitive cells, but not in resistant cells. In contrast, Corilagin did not have DNA repair function, but it inhibited pERK activity in both sensitive and resistant cells. In summary, we demonstrate that combined treatment with Corilagin and PARPi evokes synergistic cytotoxic effects in two pairs of ovarian cancer cell models. Corilagin is an herb medicine with lower toxic effects to normal cells, especially hepatoprotective, which could be an ideal complimentary medicine when combinating with PARPi in ovarian cancer therapy. Citation Format: Baoxin Luan, Hongbo Zhao, Robert C. Bast, Jr., Zhen Lu, Yinhua Yu. Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways [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 3816.

Abstract 3505: AZD7648: A potent and selective inhibitor of DNA-PK with pharmacodynamic and monotherapy anti-tumor activity

Abstract DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB) and is a critical component of the DNA Damage Response (DDR). DSB can be induced by a range of agents, including chemotherapy, radiation or Poly ADP Ribose Polymerase (PARP) inhibitors such as olaparib, and thus a DNA-PK inhibitor is likely to sensitize to these agents. DNA-PK inhibitors may also be effective as monotherapy in tumors with high endogenous levels of DNA damage resulting from defects in other DNA repair pathways. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648 (pDNA-PK IC50 in A549 cells = 92 nM). AZD7648 shows broad growth inhibitory activity across a panel of 244 cancer cell lines (GI50 1.3 - 30 µM). Consistent with known synthetic lethal interactions with DNA-PK, AZD7648 shows a 10-13-fold greater growth inhibitory effect in FaDu head and neck and A549 non-small cell lung cancer cell lines with ATM knocked out (KO) by zinc finger nuclease or CRISPR respectively compared to their isogenic wild-type counterparts (WT). This growth inhibition is associated with increased levels of DNA damage as measured by micronuclei formation detected using high-content immunofluorescence (2-fold increase vs DMSO at 2 µM AZD7648). Moreover, a 6-fold increase in chromosomal breaks are detected in the ATM KO cells using metaphase spread analysis (mean breaks/cell: FaDu ATM KO = 1.7, ATM WT = 0.28) In vivo, monotherapy treatment with AZD7648 (75-100 mg/kg bid) inhibited tumor growth in a panel of 14 PDX and 2 xenograft models, derived from breast, lung, ovarian and head and neck cancers. This included models with and without loss of ATM. Treatment with AZD7648 resulted in dose-dependent inhibition of the phosphorylation of DNA-PK (S2056), RPA32 (S4/8) and nuclear γH2AX in FaDu ATM KO xenografts, where AZD7648 75mg/kg inhibited γH2AX and phosphorylation of DNA-PK (S2056) and RPA32 (S4/8) by 71, 98 and 95% respectively at 2 h after dosing. These data confirm that DNAPK inhibition using AZD7648 has potent pharmacodynamic and monotherapy anti-tumor activity in a range of pre-clinical models. This includes, but is not restricted to, models with engineered and endogenous loss of ATM. Citation Format: Elaine B. Cadogan, Jacqueline H. Fok, Antonio Ramos-Montoya, Neil James, Valeria Follia, Mercedes Vazquez-Chantada, Paul Winjhoven, Lenka Oplustil O’Connor, Ankur Karmokar, Anna Staniszewska, Emma Dean, Simon J. Hollingsworth, Barry Davies. AZD7648: A potent and selective inhibitor of DNA-PK with pharmacodynamic and monotherapy anti-tumor activity [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 3505.

Poly (ADP-ribose) polymerase inhibitors combined with other small-molecular compounds for the treatment of ovarian cancer.

Ovarian cancer is a heterogeneous disease with complex molecular and genetic hallmarks. Benefitting from profound understanding of molecular mechanisms in ovarian cancer pathogenesis, novel targeted drugs have been actively explored in preclinical studies and clinical trials. Considered as one of the most potent and effective targeted therapies for the treatment of ovarian cancer, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) take advantages of synthetic lethality mechanisms to prevent DNA damage repair in cancer cells and cause their death, especially in cancers with BRCA mutations. Mounting evidence has indicated that the combination of PARPis with cytotoxic drugs or other targeted drugs has shown favorable synergistic effects. Excitingly, the antitumor activity of combination therapy of PARPis has been actively tested in multiple clinical trials and in-vitro or in-vivo experiments. In this review, we will briefly discuss the molecular mechanisms of PARPis combined with other therapeutic small-molecular compounds for the treatment of ovarian cancer.