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Purpose Our preclinical work identified depletion of ATR as a top candidate for topoisomerase 1 (TOP1) inhibitor synthetic lethality and showed that ATR inhibition sensitizes tumors to TOP1 inhibitors. We hypothesized that a combination of selective ATR inhibitor M6620 (previously VX-970) and topotecan, a selective TOP1 inhibitor, would be tolerable and active, particularly in tumors with high replicative stress. Patients and Methods This phase I study tested the combination of M6620 and topotecan in 3-week cycles using 3 + 3 dose escalation. The primary end point was the identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Results Between September 2016 and February 2017, 21 patients enrolled. The combination was well tolerated, which allowed for dose escalation to the highest planned dose level (topotecan 1.25 mg/m2, days 1 to 5; M6620 210 mg/m2, days 2 and 5). One of six patients at this dose level experienced grade 4 thrombocytopenia that required transfusion, a dose-limiting toxicity. Most common treatment-related grade 3 or 4 toxicities were anemia, leukopenia, and neutropenia (19% each); lymphopenia (14%); and thrombocytopenia (10%). Two partial responses (≥ 18 months, ≥ 7 months) and seven stable disease responses ≥ 3 months (median, 9 months; range, 3 to 12 months) were seen. Three of five patients with small-cell lung cancer, all of whom had platinum-refractory disease, had a partial response or prolonged stable disease (10, ≥ 6, and ≥ 7 months). Pharmacodynamic studies showed preliminary evidence of ATR inhibition and enhanced DNA double-stranded breaks in response to the combination. Conclusion To our knowledge, this report is the first of an ATR inhibitor-chemotherapy combination. The maximum dose of topotecan plus M6620 is tolerable. The combination seems particularly active in platinum-refractory small-cell lung cancer, which tends not to respond to topotecan alone. Phase II studies with biomarker evaluation are ongoing.

Purpose Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AX (γH2AX) levels in paired tumor biopsies. Patients and Methods AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. Results Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Confirmed partial responses were observed in two patients carrying BRCA mutations: one with head and neck cancer and one with ovarian cancer. Common toxicities were myelosuppression and diarrhea. Dose-limiting toxicities were supraventricular tachyarrhythmia and myelosuppression. Accumulation of drug (t1/2 approximately 11 hours) was observed. Reduction in pY15-Cdk levels (two of five paired biopsies) and increases in γH2AX levels (three of five paired biopsies) were demonstrated. Conclusion This is the first report of AZD1775 single-agent activity in patients carrying BRCA mutations. Proof-of-mechanism was demonstrated by target modulation and DNA damage response in paired tumor biopsies.

2520 Background: The PARPi O has single-agent activity against BRCA1/2 mutation carriers (Br/Ov mut+) Br/OvCa. We hypothesized addition of C to O to stress the DNA repair machinery would further im...

3000 Background: The nuclear enzyme PARP is essential in recognition and repair of DNA damage. In preclinical models, PARP inhibitors work as sensitizing agents for DNA-damaging agents such as Ir. V is an orally bioavailable PARP 1 and 2 inhibitor. This phase I study determined the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), PK and PD of V together with Ir. Methods: Eligibility included patients with performance status 0-2; ≥ age 18; adequate bone marrow, hepatic and renal function. Cycles were 21 days. Ir was given i.v. 100 mg/m2 over 90 min on Days 1 and 8. Twice daily (BID) oral dosing of V (10-50mg) occurred Days 3-14 (Cycle 1) and Days -1-14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Tumor biopsies and matched peripheral blood mononuclear cells (PBMCs) were collected for PD evaluation of PAR, γ-H2AX, PARP1, TOPO1, and ERCC1 after Ir alone and after the combination. Results: 32 pts were treated (2 lung, 14 breas...

2605 Background: Inhibition of poly (ADP-ribose) (PAR) polymerase (PARP) is expected to enhance the DNA-damaging effects of chemotherapy. PARP has also been implicated in endothelial cell migration. A is an orally available, potent inhibitor of PARP-1 and PARP-2. C is an alkylator that has effects on thrombospondin 1, endothelial cells and T reg cells. Methods: Objectives were to establish the safety, tolerability, and MTD of the combination of A with metronomic C; determine the PK of A; and evaluate PARP inhibition in PBMCs and tumor samples. Patients (pts) had histologically documented solid tumors and lymphoid malignancies that had progressed following standard therapy; ≥ 18 years old; KPS > 70%; adequate bone marrow, hepatic and renal function. Cycle length was 21 d. DLT was defined as drug-related gr ≥ 3 nonhematologic or gr 4 hematologic toxicities during cycle 1. PBMCs and optional tumor biopsies were collected, and PAR levels were determined using a validated ELISA. Results: Eighteen pts have been...

3027 Background: Poly (ADP-ribose) polymerase (PARP) group of NAD+-dependent enzymes participate in multiple DNA- related functions. Increased PARP activity can lead to drug resistance. Olaparib (O), a potent oral inhibitor of PARP-1 and PARP-2 may partially overcome resistance to C and G when used in combination. Methods: Eligibility: histologically confirmed metastatic or unresectable solid tumors, PS 0–2, normal organ functions. Treatment at dose level 1 (DL1): O 100 mg orally bid on days (D) −2 to 2, G 500 mg/m2/h on D1 and D8, C 60 mg/m2 on D1 after G; q21 days ×6. Doses (same schedule) at DL −1: O 100 mg once daily, G 400 mg/m2/h, C 50 mg/m2. Peripheral blood mononuclear cells (PBMCs) were collected and processed within 2 hours and poly (ADP-ribose) (PAR) concentration determined by immunoassay. Results: From 5/2008 to 11/2009, 23 patients (pts) enrolled; 14 males, median age 52 yrs (25- 88). Tumor types: 8 NSCLC; 3 ovarian; 3 pancreatic; 2 esophageal; 2 thymic ca; 1 mesothelioma; 1 adrenocortical c...

14024 Background: Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a molecular sensor of DNA breaks that facilitates DNA repair and controls genomic stability. Treatment with single doses of ABT-888, a novel potent PARP-1 inhibitor, reduced PAR levels in peripheral blood mononuclear cells (PBMCs) and tumor biopsies in an on-going Phase-0 trial at the NIH Clinical Center. As a corollary to this study, we investigated whether ABT-888 can act as a sensitizer for radiation therapy and chemotherapy in human cancer cell culture, xenograft tumors and PBMCs to support future combination clinical trials. Methods: Inhibition of PARP-1 by ABT-888 was determined by a quantitative PAR chemiluminescence immunoassay validated for the Phase 0 trial. Since gamma-H2AX (?-H2AX) is a marker of DNA damage, we also developed and validated ?H2AX assays to monitor the effects of PARP-1 inhibition during treatment with Topo I inhibitors and radiation. Human monocytic leukemia (THP-1) and breast carcinoma (MCF-7) cell lines were treated with Topo I inhibitors including indenoisoquinoline, camptothecin and topotecan or irradiated with 0.5 to 10 Gy in the presence of ABT 888. We further evaluated these effects in human blood ex vivo to confirm the observations made in cell culture. Results: We found that ABT-888 inhibited PAR, but did not significantly increase DNA damage. Combination of ABT-888 with a Topo I inhibitor produced over 275% increase of DNA damage in THP-1 leukemia cells compared to indenoisoquinoline alone. ?H2AX foci per cell were 9.5 ± 0.8 in MCF-7 treated with 0.5 Gy/50 nM ABT-888 in comparison to 4.0 ± 0.6 with radiation alone. When whole blood was treated in the presence of ABT-888, camptothecin- induced DNA damage in PBMCs was also increased 2–3 fold, with maximum ?H2AX expression at 2 hours post treatment. Conclusions: We conclude that ABT-888 is a highly potent PARP-1 inhibitor that can enhance the DNA damaging effects of chemotherapy and radiation therapy of human cancer. Funded by NCI Contract N01-CO-12400. No significant financial relationships to disclose.

10565 Background: DNA double-strand breaks (DSBs) caused by exposure to DNA damaging agents initiate phosphorylation of histone H2AX to form gamma-H2AX, which is considered a surrogate marker of DSBs. However, it is a challenge to quantitatively measure gamma-H2AX in clinical samples such as tumor biopsies. The aim of this study was to develop an immunohistochemical detection method for gamma-H2AX and to quantitatively evaluate the levels of gamma-H2AX in paraffin-embedded tumor samples. Methods: Human breast cancer MCF-7 cells were treated with the topoisomerase I inhibitor irinotecan at 1 μM or vehicle for 1 h, and fixed in 10% neutral buffered formalin and embedded in paraffin. Staining with gamma-H2AX antibody was performed on sections of treated MCF-7 cells, tumor specimens, and biopsies at baseline and after doxorubicin-containing chemotherapy from cancer patients. Numbers of foci and level of gamma-H2AX expression per tumor nucleus were determined by manual counting under a light microscope and an Automated Cellular Imaging System. Results: There was a rise in the mean numbers of nuclear foci and intensity of gamma-H2AX in MCF-7 cells treated with irinotecan versus vehicle (19.9 ± 2.7 vs. 9.95 ± 3.6; P < 0.0001 and 61.2 ± 8.5 vs. 16.2 ± 13.6; P < 0.0001 by Wilcoxon rank sum test). The level of gamma-H2AX foci in human tumor samples was 18.8 ± 13.1, 44.8 ± 14.5, 51.2 ± 20.8, or 69.7 ± 21.2 in carcinomas of the breast, colon, ovary, or prostate. In a patient with stable disease, levels of gamma-H2AX foci were 62.7 ± 26.9 at baseline and 67.2 ± 25.3 after doxorubicin-containing regimen chemotherapy. Conclusions: Our data suggest that the quantitative immunohistochemical detection of gamma-H2AX levels is facilitated by a digital imaging system, and is a reliable method to measure the effects of DNA damaging agents in cells and paraffin-embedded human tumor samples. Its application may help evaluate tumor response to various DNA damaging agents currently in the clinic and those presently undergoing clinical development. No significant financial relationships to disclose.