A Phase I Study of Topotecan, Carboplatin and the PARP Inhibitor

Published OnlineFirst August 22, 2016; DOI: 10.1158/1078-0432.CCR-16-1274

Cancer Therapy: Clinical Clinical Cancer Research A Phase I Study of Topotecan, Carboplatin and the PARP Inhibitor Veliparib in Acute Leukemias, Aggressive Myeloproliferative Neoplasms, and Chronic Myelomonocytic Leukemia Keith W. Pratz1, Michelle A. Rudek1, Ivana Gojo1, Mark R. Litzow2, Michael A. McDevitt1, Jiuping Ji3, Larry M. Karnitz2, James G. Herman1, Robert J. Kinders4, B. Douglas Smith1, Steven D. Gore1, Hetty E. Carraway1, Margaret M. Showel1, Douglas E. Gladstone1, Mark J. Levis1, Hua-Ling Tsai1, Gary Rosner1, Alice Chen5, Scott H. Kaufmann2, and Judith E. Karp1

Abstract

Purpose: The PARP inhibitor veliparib delays DNA repair and 150 mg/m2/d. Mucositis was dose limiting and correlated with potentiates cytotoxicity of multiple classes of chemotherapy high veliparib concentrations. The response rate was 33% overall drugs, including topoisomerase I inhibitors and platinating (33/99: 14 CR, 11 CRi, 8 PR) but was 64% (14/22) for patients agents. This study evaluated veliparib incorporation into leuke- with antecedent or associated aggressive MPNs or CMML. Leu- mia induction therapy using a previously described topotecan/ kemias with baseline DNA repair defects, as evidenced by carboplatin backbone. impaired DNA damage–induced FANCD2 monoubiquitination, Experimental Design: Employing a 3þ3 trial design, we had improved survival [HR ¼ 0.56 (95% conﬁdence interval, administered escalating doses of veliparib combined with topo- 0.27–0.92)]. A single 80-mg dose of veliparib, as well as veliparib tecan þ carboplatin in relapsed or refractory acute leukemias, in combination with topotecan þ carboplatin, induced DNA aggressive myeloproliferative neoplasms (MPN), and chronic damage as manifested by histone H2AX phosphorylation in þ myelomonocytic leukemia (CMML). CD34 leukemia cells, with greater phosphorylation in cells from Results: A total of 99 patients received veliparib 10–100 mg responders. orally twice daily on days 1–8, 1–14, or 1–21 along with con- Conclusions: The veliparib/topotecan/carboplatin combina- tinuous infusion topotecan 1.0–1.2 mg/m2/d þ carboplatin 120– tion warrants further investigation, particularly in patients with 150 mg/m2/d on days 3–7. The MTD was veliparib 80 mg twice aggressive MPNs, CMML, and MPN- or CMML-related acute daily for up to 21 days with topotecan 1.2 mg/m2/d þ carboplatin leukemias. Clin Cancer Res; 1–9. 2016 AACR.

Introduction elderly, those with therapy-associated disease, and patients with prior chronic myelomonocytic leukemia (CMML) or myelopro- Despite advances in the treatment of acute myeloid leukemia liferative neoplasms (MPN) have a particularly poor response to (AML; ref. 1), certain groups of AML patients, including the conventional cytarabine/anthracycline–based therapy and have an unmet clinical need (2–4). For example, patients with AML 1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Balti- that arise in the setting of prior myeloﬁbrosis with myeloid more, Maryland. 2Mayo Clinic, Rochester, Minnesota. 3National Clinical Target metaplasia have a 41% response rate (all regressing to prior Validation Laboratory, Frederick National Laboratory for Cancer Research, chronic phase MPN and not meeting conventional CR criteria 4 Bethesda, Maryland. Frederick National Laboratory for Cancer Research, Freder- for AML) with conventional therapy but only a 9% 1-year survival ick, Maryland. 5IDB/CTEP/NCI, National Cancer Institute, Rockville, Maryland. (5). More generally, median survivals in patients with leukemic Note: Supplementary data for this article are available at Clinical Cancer transformation from MPN reportedly range from 2.7 to 6.6 Research Online (http://clincancerres.aacrjournals.org/). months, with those receiving induction therapy showing little if S.H. Kaufmann and J.E. Karp contributed equally as last authors. any prolongation of survival (median survival 3.9–6.0 months; Current address for J.G. Herman: University of Pittsburgh, Pittsburgh, PA; ref. 2). Moreover, response rates in transformed MPN reportedly current address for S.D. Gore: Yale Cancer Center, New Haven, CT; and current range from 0% to 47%, but no uniform response criteria were address for H.E. Carraway, Cleveland Clinic Foundation, Cleveland, OH. applied to this diverse group of patients (2). JAK2 inhibitors, Corresponding Author: Keith W. Pratz, Division of Hematologic Malignancies, The which have shown activity in chronic phase MPNs, exhibit limited Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Bunting efﬁcacy after transformation to AML (6). Blaustein Cancer Research Building, 1650 Orleans Street, Room 2M45, Baltimore, The PARP enzymes PARP1 and PARP2 are involved in a wide MD 21231. Phone: 410-502-7726; Fax: 410-614-1005; E-mail: [email protected] variety of nuclear processes, including DNA damage sensing and doi: 10.1158/1078-0432.CCR-16-1274 repair through the base excision repair, single-strand break repair, 2016 American Association for Cancer Research. and double strand break (DSB) repair pathways (7, 8). Further

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sion (IV CI) in adults with relapsed or refractory acute leukemia Translational Relevance (26). Oral and gastrointestinal (GI) mucositis were the dose- PARP plays multiple roles in DNA damage response path- limiting toxicities (DLT) of this combination. Complete remis- ways. Neoplasias associated with defective homologous sions (CR) were observed at multiple dose levels, including 3 of 6 recombination (e.g., BRCA1, BRCA2, ATM, or Fanconi anemia patients at the MTD of topotecan 1.6 mg/m2/d and carboplatin pathway mutations) are especially sensitive to PARP inhibi- 150 mg/m2/d simultaneously for 5 days (26). A subsequent tors. Here we report phase I clinical trial results in patients with ECOG Phase II trial of topotecan/carboplatin at this MTD resulted relapsed acute myeloid leukemia, chronic myelomonocytic in CRs in 5 of 35 (14%) patients with relapsed and refractory AML, leukemia (CMML), or progressive myeloproliferative neo- a response rate similar to a mitoxantrone/ etoposide/cytarabine- plasms (MPN) treated with the PARP inhibitor veliparib (MEC-) based arm in the same trial (27). in combination topotecan and carboplatin. Consistent with Building on recent demonstrations that veliparib enhances the recently published preclinical findings showing PARP inhib- antineoplastic effects of topotecan and carboplatin, we conducted itor hypersensitivity of CMML and MPNs, these disorders and a phase I dose-escalation trial of veliparib given twice daily prior to leukemias arising from them appear to be particularly sensitive and during the administration of topotecan and carboplatin by to this combination. Predictors of response include pretreat- 120-hour IV CI on days 3–7. At the MTD, the patient population ment defects in crosslinking agent–induced FANCD2 mono- was streamlined to focus on patients with aggressive CMML and ubiquitination and higher treatment-induced histone H2AX progressive MPNs as well as AMLs evolving from these diseases phosphorylation. These observations support further study of based on preclinical studies suggesting PARP inhibitor hypersen- the veliparib/topotecan/carboplatin combination in aggres- sitivity of this disease subset (19). The duration of veliparib sive myeloid disorders with inherent DNA repair deficits such administration was 8 days during the dose escalation. Veliparib as aggressive MPNs, CMML, and AML arising in the setting of dosing was extended in two MPN/CMML cohorts to 14- and 21- these disorders. day durations after completion of the topotecan/carboplatin infusion to take advantage of the PARP inhibitor sensitivity and defective DNA repair inherent in some CMML and MPN cells (19, 28). Here we report the results of this clinical trial, veliparib pharmacokinetics, and exploratory studies investigating potential studies have shown that PARP inhibition can be synthetically determinants of response. lethal with defective DSB repair, as seen in neoplasms with defective breast cancer susceptibility gene (BRCA), ataxia telangiectasia mutated (ATM), or Fanconi anemia (FA) proteins Materials and Methods (9–12). This synthetic lethality has been validated clinically Patient selection in BRCA1- and BRCA2-mutated breast, ovarian, and prostate Patients over the age of 18 years with pathologically confirmed cancers using PARP inhibitors (13–16). relapsed or refractory AMLs, newly diagnosed aggressive MPN, or Studies in diverse myeloid leukemia cell lines and primary aggressive CMML were eligible. Consistent with our previous þ human CD34 chronic myelogenous leukemia (CML) cells have study (29), for aggressive MPNs (primary myelofibrosis, agno- documented baseline defects in DNA repair pathways (17, 18). genic myeloid metaplasia, polycythemia vera, essential thrombo- Moreover, primary marrow cells from patients with CMML and cythemia, Ph-negative atypical CML) or CMML, one or more of aggressive MPNs exhibit functional defects in DNA repair and the following disease acceleration criteria had to be present (30): hypersensitivity to multiple PARP inhibitors ex vivo (19). Certain marrow blasts >5%, peripheral blood blasts þ promonocytes AML-associated gene rearrangements, including AML1-ETO and >10%, new onset or increasing myelofibrosis, new onset or PML-RARa fusions, have also been shown to confer PARP inhib- >25% increase in hepatomegaly or splenomegaly, or new onset itor sensitivity through deficiencies in homologous recombina- constitutional symptoms (fever, weight loss, splenic pain, bone tion (20). Conversely, elevated HOXA9 activity in MLL-rear- pain). Patients were required to have: an Eastern Cooperative ranged acute leukemias has been associated with PARP inhibitor Oncology Group performance status of 0–2; left ventricular resistance (20). ejection fraction of 45%; hepatic enzymes 5x upper limit of Veliparib (ABT-888) is an orally bioavailable, small-mole- normal; bilirubin 2.0 mg/dL unless due to Gilbert syndrome; cule PARP inhibitor that enhances the cytotoxicity of diverse interval of >4 weeks since allogeneic bone marrow transplanta- classes of DNA-damaging agents, including ionizing radiation, tion (BMT) if performed; and absence of active GVHD, active CNS alkylating agents, platinating agents, and topoisomerase I leukemia, and active uncontrolled infection. All patients gave (topo I) poisons in vitro and in vivo (21–23). Studies in AML informed consent to participate in the IRB-approved protocol cell lines and clinical samples have demonstrated that veliparib according to the Declaration of Helsinki. enhances the antiproliferative and proapoptotic effects of topotecan in vitro, whereas no synergy is observed with cytar- Treatment plan abine or etoposide (24). Further analysis indicated that the Veliparib was administered orally on a twice-daily schedule synergy results from trapping of PARP1 on the damaged DNA, with dose escalation according to Table 1. Topotecan and carbo- thereby inhibiting repair downstream of topo I–DNA covalent platin were given together by IV CI over 120 hours on days 3–7of complex stabilization (24, 25). each cycle. Topotecan was dose-reduced for decreases in creati- In earlier studies, topotecan and carboplatin induced synergis- nine clearance (CrCl) as follows: CrCl 20–39 mL/minute: 50% tic cytotoxicity in acute leukemia cell lines and primary AML cells reduction; CrCl < 20 mL/minute: topotecan not administered. in vitro (26). This observation was the basis for a phase I study of Carboplatin was dose-reduced for decreases in CrCl as follows: topotecan þ carboplatin by 5-day intravenous continuous infu- CrCl 40–59 mL/minute: 30% reduction; CrCl 16–39 mL/minute:

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Topotecan Carboplatin and PARPi Veliparib in Leukemia

Table 1. Dose levels and accruals to veliparib in combination with topotecan and carboplatin Dose Veliparib dose (p.o., BID) Topotecan continuous Carboplatin continuous Number of level days 1–8, 14 or 21 IV days 3–7 IV days 3–7 patients accrued 110mg 8 d 1.0 mg/m2/day — 3 210mg 8 d 1.3 mg/m2/day — 3 310mg 8 d 1.3 mg/m2/day 150 mg/m2/day 4 410mg 8 d 1.0 mg/m2/day 120 mg/m2/day 6 510mg 8 d 1.2 mg/m2/day 120 mg/m2/day 6 610mg 8 d 1.2 mg/m2/day 150 mg/m2/day 6 720mg 8 d 1.0 mg/m2/day 120 mg/m2/day 6 820mg 8 d 1.2 mg/m2/day 120 mg/m2/day 6 920mg 8 d 1.2 mg/m2/day 150 mg/m2/day 6 10 40 mg 8 d 1.2 mg/m2/day 150 mg/m2/day 6 11a 80 mg 8 d 1.2 mg/m2/day 150 mg/m2/day 26 12 100 mg 8 d 1.2 mg/m2/day 150 mg/m2/day 4 13 90 mg 8 d 1.2 mg/m2/day 150 mg/m2/day 5 14 80 mg 14 d 1.2 mg/m2/day 150 mg/m2/day 6 15 80 mg 21 d 1.2 mg/m2/day 150 mg/m2/day 6 Abbreviations: BID, twice daily; p.o., orally. aExpansion cohort.

45% reduction; CrCl 15 mL/minute: carboplatin not treatment cytopenias, including hemoglobin >12.0 gm/dL for administered. males or >11.0 gm/dL for females without erythropoietin or transfusion support as well as ANC >1.0 109/L and platelet Determination of DLT, MTD, and stopping rules count >100 109/L without support; and resolution of pretreat- We evaluated 3–6 patients per dose level utilizing a standard ment hyperleukocytosis and/or thrombocytosis. PR required 3 þ 3 design. Toxicity was measured according to NCI-CTCAE improvement in two or more of the following: (i) ANC increase version 4.0. The MTD was determined as the highest dose level of 100% up to >109/L for neutropenia and WBC count 1–10 109 where 0 of 3 or 1 of 6 experienced DLT. DLT consisted of: (i) any /L with persistence of immature cells (blasts, progranulocytes, grade 4 nonhematologic toxicity; (ii) any grade 3 nonhematologic myelocytes); (ii) increase in hemoglobin of 2 gm/dL if baseline toxicity that did not resolve grade 2 within 48 hours, with the value was <10 gm/dL, and a decrease in transfusion frequency following exceptions: (i) grade 3 bilirubin, transaminases or and/or volume by at least 50%; (iii) persistent thrombocytosis alkaline phosphatase was considered dose-limiting only if reso- >450 109/L but <50% of pretreatment values; (iv) reduction in lution to grade 2 required 7 days; (ii) grade 3 mucositis, bone marrow blasts to <5% if blasts were originally >10% in diarrhea, nausea, or vomiting was considered dose limiting only if normocellular or hypercellular marrow; and (v) reduction in resolution to grade 2 (including use of supportive care) required splenomegaly and/or hepatomegaly by 50% of pretreatment 7 days; (iii) any grade 3 neurotoxicity or nephrotoxicity was dimensions. considered dose limiting. Myelosuppression was not considered in evaluating toxicity in patients with acute leukemias except Pharmacokinetic studies where bone marrow hypoplasia occurred for 50 days with Plasma samples obtained on day 1 of veliparib and on bone marrow cellularity 5% and no evidence of leukemia. At the day 4 after the first dose of veliparib in combination with MTD, 20 patients were treated in an expansion cohort to further topotecan þ carboplatin were assayed for veliparib using the establish the tolerability of this combination. validated LC/MS-MS method (33). Pharmacokinetic variables were calculated by standard noncompartmental methods using Evaluation of response Phoenix WinNonlin version 6.3 (Pharsight A Certara Company) A bone marrow aspirate and/or biopsy was obtained to assess as described previously (34). response to therapy at the time of hematologic recovery or whenever leukemia regrowth was suspected on the basis of circulating blasts or persistent cytopenias at 6–7 weeks after Exploratory pretreatment and pharmacodynamic correlates initiation of therapy. Hematologic recovery was defined as an Pretreatment FA pathway integrity. Pretreatment bone marrow absolute neutrophil count (ANC) of 500/mm3 and a transfu- samples were treated with the DNA cross-linker melphalan sion-independent platelet count of 20,000/mm3. Standard (10 mmol/L) for 6 hours and FANCD2 ubiquitylation was definitions for CR, CR with incomplete count recovery (CRi), assessed by immunoblotting (35). BRCA1 promoter methylation and partial response (PR) were used for response assessment in was assessed using previously published methods (36). acute leukemia (31, 32). Response in aggressive MPNs or CMML still in chronic phase Pretreatment expression of PARP1 and topo I. Marrow mononu- (pretreatment blasts less than 20%) was defined according to clear cells were isolated on Ficoll-Hypaque step gradients, washed Giles and colleagues. (30). In brief, CR in these patients required with serum-free RPMI1640 medium containing 10 mmol/L freedom from all symptoms or signs related to MPN; WBC 1–10 HEPES (pH 7.4 at 21C), and prepared for electrophoresis 109/L with no peripheral blood blasts, promyelocytes or myelo- as described previously (37). Aliquots containing protein from cytes; normalization of bone marrow (<5% blasts in normocel- 5 105 cells were subjected to SDS-PAGE and immunoblotting lular or hypercellular marrow) for 4 weeks; resolution of pre- with enhanced chemiluminscent detection (38). Each blot

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Table 2. Patient characteristics and outcomes Results Number Responses (%) Median age 56 (25–76) 33/99 (33%) Patient characteristics Refractory to prior regimen 78 23/78 (29%) Ninety-nine patients, including 34 with primary refractory Primary refractory 34 9/34 (26%) AML, 35 with secondary AML (myelodysplasia- or treatment- Prior regimens 3 (0–4) related), 22 with aggressive chronic myeloid neoplasms (CMML, 0 Regimens 6 5/6 (83%) non-CML MPN) or AML arising out of aggressive chronic myeloid 1 Regimen 38 14/38 (37%) neoplasms, and 4 with refractory ALL, were accrued to the trial. As 2 Regimens 20 6/20 (30%) 3 Regimens 25 7/25 (28%) indicated in Table 2, the median number of prior therapies was 2 4 Regimens 10 1/10 (10%) (range 0–4), including prior allogeneic BMT in 16. Prior allogeneic bone marrow transplant 16 3/16 (19%) Antecedent hematologic disorder in patients 35 17/35 (49%) with AML Treatment, toxicities, and determination of dose and schedule Myeloproliferative neoplasm-associated 22 14/22 (64%) Patients received oral veliparib beginning day 1 and a 5-day Treatment related 10 3/10 (30%) continuous infusion of topotecan þ carboplatin beginning on day MDS associated (non-MPN) 6 1/6 (17%) 3 after steady-state veliparib exposures were achieved. Subjects JAK2 mutated MPN 7 5/7 (71%) were accrued over 15 dose levels (Table 1). At the lowest dose of Cytogenetics veliparib, topotecan 1.3 mg/m2 was associated with dose-limiting Intermediate 48 12/48 (25%) fi Adverse 45 14/45 (31%) GI mucositis. The study was redesigned with xed doses of 2 2 BRCA1 mutant 3 2/3 (67%) topotecan and carboplatin at 1.2 mg/m /d and 150 mg/m /d, BRCA2 mutant 2 2/2 (100%) respectively, to allow for veliparib dose escalation. A DLT of oral Azacitidine failures 15 6/15 (40%) mucositis was identified at 100 mg veliparib twice daily (Table 3). Acute lymphoblastic leukemia 4 1/4 (25%) De-escalation of veliparib to 90 mg twice a day also was not tolerable due to mucositis in 2 of 4 patients. Therefore, the MTD contained a serial dilution of HL-60 cells. Signals were digitized was 80 mg veliparib twice daily in combination with CI IV 2 2 and quantified relative to the HL-60 cell signal as described topotecan 1.2 mg/m /d þ carboplatin 150 mg/m /day on days previously (37). 3–7. At the MTD of 80 mg veliparib twice daily, dosing was lengthened from 8 days to 14 and 21 days without increased Assessment of PAR suppression and DNA damage. PAR assays were toxicity (dose levels 14 and 15, Table 1). Accordingly, the recom- performed as described previously (39). Assessments of Ser139- mended phase II dose of the combination is veliparib 80 mg phospho-H2AX (gH2AX) in peripheral blood and bone marrow twice daily for up to 21 days in combination with CI IV topotecan mononuclear cells were performed via flow cytometry according 1.2 mg/m2/d þ carboplatin 150 mg/m2/day on days 3–7. þ to previously described methods, using the CD34 fraction when appropriate to distinguish the leukemic population from other Clinical responses cells (40). As summarized in Table 4, 33% of 99 patients achieved objective responses: 14 CR, 11 CRi, and 8 PR. These responses Statistical analysis were observed across a wide range of dose levels (Table 4). Overall survival (OS) was defined from starting date of treat- Among patients with AML but no history of MPN or CMML, the ment to death, or censored at the last follow-up date. Relapse-free response rate was 25% (19/77). In contrast, 64% (14/22) with survival (RFS) was defined from starting date of treatment to the aggressive MPN, CMML, or related AML responded, 11 of whom first occurrence of relapse date or date of death. OS, RFS, and proceeded to allogeneic BMT with engraftment of donor cells probabilities of outcomes at 6 and 12 months were estimated with (Supplementary Table S1). Ninety-three of 99 (94%) patients the Kaplan–Meier method, along with medians and 95% confi- were fully followed and died before analyzing the data. The dence intervals. Exploratory biomarker comparisons of survival median follow-up was 3.1 years among 6 alive patients. OS and were performed using log-rank test. RFS analysis of all patients, patients treated in the MTD expansion

Table 3. Toxicity by veliparib dose level 10 mg 20 mg 40 mg 80 mg 80 mg x 14 80 mg x 21 90 mg 100 mg Category Grade (28 pts) (18 pts) (6 pts) (26 pts) days (6 pts) days (6 pts) (5 pts) (4 pts) Hepatic Bilirubin 3 3 3 1 0 0 0 0 1 ALT/AST 3 0 0 0 2 0 0 0 1 Infectious Febrile Neutropenia 3/4 9 9 3 15 3 3 3 1 Typhlitis/Colitis 3 0 1 1 2 0 0 0 0 Mucositis 3 3 2 0 5 1 1 2 2 Metabolic Hypophosphatemia 3 3 2 0 1 1 0 0 0 Hypokalemia 3 6 2 1 6 0 0 1 1 Hypocalcemia 3/4 0 2 0 1 1 0 0 1 Elevated Creatinine 3 0 1 1 2 0 0 0 0 Cardiac QTc Prolongation 3 0 1 0 1 0 0 0 0

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Table 4. Summary of clinical responses to veliparib in combination with topotecan and carboplatin Clinical responses in patients with MPN Dose level Clinical responses/# Pts or CMML (aggressive or transformed) 1 1/3 (CRi) 0 2 1/3 (CR) 0 3 0/4 0 4 1/6 (PR; 0/1 aza failure) 0 5 2/6 (CRi, PR) 0 6 2/6 (CR, CRi; 1/1 aza failure) 1/1 (CRi) 7 1/6 (PR) 0/1 8 2/6 (CR, PR) 0 9 2/6 (CR, CRi; 1/2 aza failure) 1/1 (1/1 aza failure) CRi 10 2/6 (2 CR) 0/1 (0/1 aza failure) 11a 8/26 (4 CR, 2 CRi, 2 PR; 1/4 aza failure) 3/5 (1/3 aza failure) CRi, 2 PR 12 3/4 (3 CR) 1/1 (CR) 13 0/5 (0/1 aza failure) 0 14 5/6 (CR, 3CRi, PR; 3/3 aza failure) 5/6 (3/3 aza failure; CR, 3CRi, PR) 15 3/6 (2CRi, PR' 0/3 aza failure) 3/6 (2CRi, PR' 0/3 aza failure) Total 33/99 (14CR, 11 Cri, 8 PR) 14/22 (3 CR, 7 Cri, 4 PR) NOTE: Aza failure, patients who have been treated and failed to respond to azacitidine-based therapy. aExpansion Cohort. cohorts, and patients with associated MPN or CMML treated on chronic myeloid neoplasms (aggressive CMML or MPN) or any dose level are depicted in Fig. 1. AML evolving in the setting of these diseases, with 5 respond- The response rate varied depending on the number of prior ing. This subset was treated at or near the MTD of 80 mg regimens. All 6 patients without prior therapy had aggressive veliparib twice daily for 14 days. Patients who received 1-4

Figure 1. A, Survival analysis plotted in Kaplan–Meier method for patients treated on study (expansion cohort ¼ dose level 11, MPN subgroups includes patients with MPN, CMML, or acute leukemias in the setting of prior MPN or CMML treated at any dose level). B, Survival analysis based on response to therapy. C, Survival analysis of patients with or without associated MPN or CMML irrespective of response. D, Survival analysis of patients treated with extended duration veliparib (days 1–14 and 1–21 for dose levels 14 and 15, respectively). Censored patients who are alive at their last follow-up dates (last follow-up dates were on December 2015 to Jan 2016). , P value denoted in the ﬁgures was based on log-rank test.

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Table 5. Plasma pharmacokinetic parametersa of veliparib when combined with topotecan and carboplatin

Single or AUCINF or Tau Dose multiple dose Cmax (ng/mL) tmax (h) (mg/h/mL) t1/2 (h) Cl/F (L/h) V/F (L) 10 mg Single 74.6 33.0 (27) 2.0 (0.3–8.1; 27) 525 204 (23) 5.5 3.5 (23) 27.7 9.3 (23) 225 191 (23) Multiple 107.0 33.2 (25) 1.0 (0.5–4.0; 25) 696 210 (25) 5.8 1.9 (22) 16.1 4.2 (22) 132.3 55.1 (22) 20 mg Single 102.2 31.6 (12) 2.0 (1.0–4.1; 12) 849 243 (12) 4.7 1.1 (12) 31.7 7.7 (12) 216 70 (12) Multiple 182.2 45.5 (13) 1.0 (0.5–4.1; 13) 1,244 413 (13) 5.6 2.8 (11) 17.7 4.3 (11) 133 41 (11) 40 mg Single 231.2 128.2 (5) 1.0 (0.5–2.0; 5) 1,474 526 (5) 4.1 0.9 (5) 36.4 15.4 (5) 221 103 (5) Multiple 363.6 211.6 (5) 1.1 (0.5–4.0; 5) 1,865 570.2 (5) 4.5 0.7 (4) 20.5 5.5 (4) 131 27 (4) 80 mg Single 608.7 260.3 (35) 1.1 (0.3–6.0; 35) 4,057 1,278 (30) 4.7 1.4 (30) 26.2 7.1 (30) 173 53 (30) Multiple 856.8 317.6 (36) 1.0 (0.5–4.0; 36) 5,137 1,820 (36) 4.7 1.5 (26) 20.2 11.2 (26) 131 71 (26) 90 mg Single 509.0 100.6 (3) 1.0 (1.0–4.0; 3) 3,042 1,319 (3) 3.2 1.2 (3) 35.9 10.2 (3) 154 16 (3) Multiple 685.0 375.8 (3) 1.0 (0.5–4.1; 3) 3,836 1,739 (3) 3.4, 3.6 (2) 15.8, 39.7 (2) 77.1, 206.8 (2) 100 mg Single 1,123.5 718.5 (4) 1.0 (1.0–1.0; 4) 5,044 2,655 (4) 5.9 2.4 (4) 44.9 50.6 (4) 504 745 (4) Multiple 1,515.8 494.8 (4) 0.7 (0.5–1.0; 4) 10,108 4,356 (3) 3.6 1.0 (3) 12.9 6.3 (3) 72 47 (3) Accumulation index 1.88 1.83 (83) 1.47 1.07 (75)

Abbreviations: AUCINF, area under the plasma concentration–time curve to inﬁnity; AUCTau, area under the plasma concentration–time curve during the dosing interval at steady-state; Cl/F, apparent systemic clearance; Cmax, peak plasma concentration; tmax, time to peak concentration; t1/2 half life; V/F, apparent volume of distribution. a Data were obtained from patients receiving veliparib orally and are presented in the table as mean values SD (n). Tmax is presented as median (range; n). If n < 3, the actual values are reported.

prior therapies fared less well, with a response rate of 30% Veliparib pharmacokinetics (28/93). Nonetheless, 15 heavily pretreated responders were Single and multiple dose veliparib plasma pharmacokinetics able to proceed to allogeneic BMT (22 total transplanted after were analyzed in 86 patients after both single and multiple response). doses (Table 5). There was large interpatient variability in The median duration of the response for all patients who veliparib exposure. There was signiﬁcant accumulation of achieved a CR, CRi, or PR was 7.5 (95% CI, 5.4–13.1) months. veliparib (accumulation index) that corresponded with a In the subset with aggressive CMML, MPN or AML associated with decrease in both apparent clearance (Cl/F) and volume of these disorders, the duration of response was 11.5 (95% CI, 7.5– distribution (V/F; all P < 0.0001). There were no signiﬁcant NA) months (Fig. 1A). Patients who achieved CR, CRi, or PR had a differences in dose-normalized exposure (Cmax or AUC) or median OS of 15.3 (95% CI, 10.5–23.0) months (Fig. 1B). In other pharmacokinetic parameters (t1/2,Cl/F,andV/F)across contrast, median OS for patients who did not achieve a response dose levels. Of note, there was a 1.8-fold increase in Cmax and was 4.2 (95% CI, 3.1–5.3) months (Fig. 1B). Importantly, the 2.0-fold increase in AUC after multiple doses between the duration of response and OS of patients achieving a CR/CRi or PR MTD (80 mg) and the highest administered dose (100 mg) closely paralleled each other, consistent with the hypothesis that which is suggestive of nonlinear pharmacokinetics. The devel- response contributes to the improved survival in responding opment of mucositis correlated with single-dose AUC (P ¼ patients. 0.02) and multiple-dose Cmax (P ¼ 0.04) suggesting a expo- In view of preclinical data suggesting PARP inhibitor hyper- sure–toxicity relationship. sensitivity of a subset of aggressive chronic myeloid neoplasms (19), we were particularly interested in that group of patients. Exploratory correlative studies Patients with an antecedent aggressive MPN, CMML, or AML Pretreatment assessment of FA pathway function. We examined in the setting of these disorders had a median OS of 13.3 (95% functional integrity of the FA pathway, as assessed by FANCD2 CI, 8.2–33.3) months (Fig. 1C). One-year RFS was 41% (95% monoubiquitination after melphalan exposure ex vivo (Supple- CI, 25%–68%) in these patients versus 12% patients without mentary Fig. S1A), in pretreatment bone marrow aspirates prior MPN or CMML. Patients with antecedent aggressive MPN because (i) FA pathway defects are reportedly common in AML or CMML who had 0–1 prior therapy had a median survival of and (ii) loss of FA pathway function is associated with enhanced 14.4 months, whereas those with 2 prior therapies had a sensitivity to DNA crosslinking agents and PARP inhibitors (41). median survival of 9.3 months. Patients without an antecedent Impaired FANCD2 monoubiquitination was detected in 28 of 49 aggressive MPN or CMML had a median survival of 5.1 samples (57%) and was associated with modest prolongation of months. As all but two responders in the MPN/CMML subset survival (median 6.1 vs. 4.8 months, 1 year OS 39% vs. 5%, P ¼ underwent allogeneic BMT, survival data were not censored for 0.0342, Supplementary Fig. S1B). BMT. BRCA1, MGMT, and MLH1 promoter methylation was exam- Enrollment in the two extended dosing cohorts at the MTD ined in pretreatment samples from 11 of 12 patients in the (dose levels 14 and 15, Table 1) was limited to patients with prolonged dosing cohorts (dose level 14 and 15, Table 1). One antecedent aggressive MPN or CMML (Fig. 1D) based on preclin- patient exhibited BRCA1 promoter hypermethylation and ical data developed during the clinical study (19). Eight of 12 achieved a CRi. patients had a clinical response (1 CR, 5CRi, and 2 PR). Median survival of patients treated in these two cohorts was 15.8 months Pretreatment PARP1 and topo I expression. Preclinical studies have versus 5.8 months for patients treated at dose levels 1–13. demonstrated that PARP inhibitor–induced sensitization to

OF6 Clin Cancer Res; 2017 Clinical Cancer Research

Topotecan Carboplatin and PARPi Veliparib in Leukemia

topotecan involves conversion of each enzyme into an agent that levels 14 and 15) for patients with antecedent MPN or CMML to perpetuates DNA damage upon treatment with the respective allow extended duration PARP inhibition. Dosing beyond 8 days inhibitors (24). This model predicts that samples with higher was well tolerated without impact on adverse events (Table 3). levels of PARP1 or topo I will be more sensitive to the topotecan/ Combined cohorts of extended veliparib duration (14 and 21 veliparib combination. Accordingly, we examined expression of days) had a response rate of 67% (8/12) and a favorable median PARP1 and topo I by immunoblotting of pretreatment marrow survival of 15.8 months. Accordingly, we recommend examining samples from 21 patients treated at or above the veliparib MTD of an extended duration of PARP inhibitor treatment in any further 80 mg twice daily. As shown in Supplementary Fig. S2, PARP1 and trials of this combination due to the promising clinical activity topo I levels varied without clear-cut relationship with each other. and lack of added toxicity. Although 5 of these 21 patients achieved response, there also was The 80-mg twice daily MTD for veliparib in this trial is signif- no clear-cut correlation with topo I or PARP1 expression. icantly lower than the 400-mg twice daily dose administered in veliparib single-agent trials (42). Other studies combining veli- Veliparib inhibits PAR formation in circulating and marrow leuke- parib with chemotherapy in solid tumors have noted less than mia. PAR assays were performed on samples obtained from 75 proportional increases in veliparib exposure at doses above patients pretreatment and at various time points on days 1 and 4 120 mg twice daily (43, 44). In contrast, we observed greater of therapy. At 40-mg veliparib twice daily and above, inhibition of than proportionate increases in veliparib exposure above 80 mg PAR formation by >75% was documented in 20 of 21 samples (Table 5). A potential factor contributing to nonlinearity in our with baseline PAR >100 pg/107 cells (Supplementary Fig. S3A). study may be that renal elimination of veliparib was compro- Greater suppression of PAR occurred at the higher dose levels in mised in the presence of carboplatin and topotecan (44, 45). The both peripheral blood and marrow samples (Supplementary Figs. single-dose AUC and multiple-dose Cmax at the highest veliparib S3A and S3C). PAR suppression at 2 hours postdosing time point doses correlated with the DLT of mucositis (Table 5). on day 1 did not correlate with clinical response (Supplementary On the basis of levels of intracellular PAR, it appears that Fig. S3B). veliparib-induced PAR suppression may be necessary but not sufficient to determine clinical response. Thus, measurement of þ Veliparib induces H2AX phosphorylation in circulating CD34 PAR does not appear promising as a predictor of response. On the blasts. Activation of the DNA damage response, as measured by other hand, the ability of veliparib to increase DNA damage both H2AX phosphorylation via flow cytometry, was assessed in viable by itself and in combination with topotecan þ carboplatin could þ CD34 bone marrow and peripheral blood cells on day 1 at 4 serve as a potential biomarker of response. The increase in H2AX þ hours after an initial veliparib dose of 80 mg and again on day 4 at phosphorylation in circulating CD34 cells was roughly 3-fold 24 hours after the initiation of the topotecan/carboplatin infu- greater in responders than the modest increases seen in nonre- sion. As depicted in Supplementary Table S2, H2AX phosphor- sponders (median 160% increase vs. 50% increase, respectively). þ ylation increased in CD34 bone marrow cells by median 2.6-fold On the other hand, the increase in phospho-H2AX after treatment (range 0.6–9.7) and peripheral blood by median 1.2-fold (medi- was also greater in patients who had received fewer prior treat- an 0.1–19.2) following a single dose of veliparib on day 1 relative ments. Accordingly, further studies are needed to define the to pretreatment levels. Similarly, increases in H2AX phosphory- potential role of this parameter as an independent predictor of þ lation were noted on day 4 in peripheral blood CD34 cells response. during the topotecan/carboplatin infusion (median 2.2-fold, Why the therapy-induced damage is greater in responding þ range 0.5–38). Peripheral blood CD34 cohorts from patients leukemias also requires further investigation but may relate, at who achieved clinical responses exhibited greater relative least in part, to genetic features of individual leukemias that þ increases than CD34 cohorts from nonresponding patients on confer differences in DNA damage response and subsequent day 4 (median 2.6-fold vs. 1.5-fold, P ¼ 0.021 by unpaired t test, PARP inhibitor sensitivity (20). Preclinical studies demonstrat- Supplementary Table S2). ed that many primary human MPN cell populations are hyper- sensitive to the antiproliferative effects of PARP inhibitors (19). In the current study, FANCD2 ubiquitination was impaired in Discussion >50% of pretreatment leukemia samples and was associated This study is the first to investigate the combination of PARP with modest improvement in both median and overall survival inhibitors with cytotoxic chemotherapy in acute leukemias in the (Supplementary Fig. S1). Higher pretreatment PARP1 expres- clinical setting. The current trial demonstrates clinical activity of sion, which could result in greater potentiation of damage the topotecan/carboplatin/veliparib combination in patients through trapping of PARP1 on the DNA (24), may be another with traditionally drug-resistant myeloid malignancies, in partic- determinant of response (Supplementary Fig. S2) at doses ular aggressive MPNs, CMML, and their associated acute leukemic lower than those that inhibit catalytic acitivity (46) but requires phases. The MTD and recommended phase II dose of the regimen further study. is veliparib 80-mg twice daily in combination with topotecan In summary, the topotecan/carboplatin/veliparib combination 1.2 mg/m2/d and carboplatin 150 mg/m2/d on days 3–7. Further appears to have significant activity in myeloid malignancies that escalation of veliparib was not tolerated due to mucositis, pos- consist of or arise from aggressive MPNs and CMML. In this subset sibly due to nonlinear pharmacokinetics at doses greater than 80 of patients we observed a striking 64% overall response rate and mg (Table 5). prolonged overall survival compared with previous studies (2). Because activity was observed in MPN-associated leukemias While these results reflect observations from a heterogeneous during dose escalation and we also observed antiproliferative group of patients in a phase I study, they nonetheless suggest effects upon prolonged exposure of primary MPN and CMML that this regimen warrants further clinical investigation in these cells to PARP inhibitors ex vivo (19), we added two cohorts (dose disorders. In addition, several of the exploratory biomarkers

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Pratz et al.

examined here, including FANCD2 ubiquitylation and H2AX H.E. Carraway, D.E. Gladstone, M.J. Levis, H.-L. Tsai, G. Rosner, A. Chen, phosphorylation, warrant further investigation in future trials as S.H. Kaufmann, J. Karp well. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.W. Pratz, M.A. Rudek, M.R. Litzow, J. Ji, J. Karp Study supervision: K.W. Pratz, J. Ji, B.D. Smith, D.E. Gladstone, A. Chen, J. Karp Disclosure of Potential Conﬂicts of Interest M.A. Rudek is an employee of Novavax. M.A. McDevitt is an employee of Acknowledgments AstraZeneca. No potential conﬂicts of interest were disclosed by the other We thank the patients who participated in this study, the nurses and authors. physicians who cared for them, and Rebecca Rickliss, Karen Flatten, Kevin Peterson, Yiping Zhang, Paula Schneider, and Cathy Huntoon for technical Authors' Contributions assistance with the correlative studies. Conception and design: K.W. Pratz, M.A. Rudek, G. Rosner, A. Chen, S.H. Kaufmann, J. Karp Development of methodology: K.W. Pratz, M.A. Rudek, J. Ji, L.M. Karnitz, Grant Support R. Kinders, A. Chen, J. Karp This work is supported by NIH grants U01 CA070095, UM1 CA186691, U01 Acquisition of data (provided animals, acquired and managed patients, CA69912, and UM1 CA186686. The Analytical Pharmacology Core of the provided facilities, etc.): K.W. Pratz, M.A. Rudek, I. Gojo, M.R. Litzow, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins was funded M.A. McDevitt, J. Ji, L.M. Karnitz, B.D. Smith, S.D. Gore, H.E. Carraway, by NIH grants P30 CA006973 and UL1 TR 001079. The National Clinical Target M.M. Showel, D.E. Gladstone, M.J. Levis, S.H. Kaufmann, J. Karp validation lab was funded by NCI Contract no HHSN261200800001E. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment of advertisement computational analysis): K.W. Pratz, M.A. Rudek, I. Gojo, M.R. Litzow, page charges. This article must therefore be hereby marked in M.A. McDevitt, J. Ji, L.M. Karnitz, J.G. Herman, R.J. Kinders, H.-L. Tsai, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. G. Rosner, S.H. Kaufmann, J. Karp Writing, review, and/or revision of the manuscript: K.W. Pratz, M.A. Rudek, Received May 19, 2016; revised July 11, 2016; accepted August 3, 2016; I. Gojo, M.R. Litzow, M.A. McDevitt, J. Ji, J.G. Herman, B.D. Smith, S.D. Gore, published OnlineFirst August 22, 2016.

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www.aacrjournals.org Clin Cancer Res; 2017 OF9

A Phase I Study of Topotecan, Carboplatin and the PARP Inhibitor Veliparib in Acute Leukemias, Aggressive Myeloproliferative Neoplasms, and Chronic Myelomonocytic Leukemia

Keith W. Pratz, Michelle A. Rudek, Ivana Gojo, et al.

Clin Cancer Res Published OnlineFirst August 22, 2016.

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