A Phase I study of topotecan, carboplatin and the PARP inhibitor veliparib in acute leukemias, aggressive myeloproliferative neoplasms and chronic myelomonocytic leukemia

Abstract

Purpose

The poly(ADP-ribose) polymerase (PARP) inhibitor veliparib delays DNA repair and potentiates cytotoxicity of multiple classes of chemotherapy drugs, including topoisomerase I inhibitors and platinating agents. This study evaluated veliparib incorporation into leukemia induction therapy using a previously described topotecan/carboplatin backbone.

Experimental Design

Employing a 3+3 trial design, we administered escalating doses of veliparib combined with topotecan + carboplatin in relapsed or refractory acute leukemias, aggressive myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (CMML).

Results

A total of 99 patients received veliparib 10-100 mg orally twice daily on Days 1-8, 1-14 or 1-21 along with continuous infusion topotecan 1.0-1.2 mg/m²/d + carboplatin 120-150 mg/m²/d on Days 3-7. The maximum tolerated dose was veliparib 80 mg twice daily for up to 21 days with topotecan 1.2 mg/m²/d + carboplatin 150 mg/m²/d. Mucositis was dose limiting and correlated with high veliparib concentrations. The response rate was 33% overall (33/99: 14 CR, 11 CRi, 8 PR) but was 64% (14/22) for patients with antecedent or associated aggressive MPNs or CMML. Leukemias with baseline DNA repair defects, as evidenced by impaired DNA damage-induced FANCD2 monoubiquitination, had improved survival (hazard ratio .56 (95% CI .27-.92)). A single 80 mg dose of veliparib, as well as veliparib in combination with topotecan + carboplatin, induced DNA damage as manifested by histone H2AX phosphorylation in CD34⁺ leukemia cells, with greater phosphorylation in cells from responders.

Conclusions

The veliparib/topotecan/carboplatin combination warrants further investigation, particularly in patients with aggressive MPNs, CMML, and MPN- or CMML-related acute leukemias.

Introduction

Despite advances in the treatment of acute myeloid leukemia (AML) (1), certain groups of AML patients, including the elderly, those with therapy-associated disease, and patients with prior chronic myelomonocytic leukemia (CMML) or myeloproliferative neoplasms (MPNs) have a particularly poor response to conventional cytarabine/anthracycline-based therapy and have an unmet clinical need (2-4). For example, patients with AML that arises in the setting of prior myelofibrosis with myeloid metaplasia have a 41% response rate (all regressing to prior chronic phase MPN and not meeting conventional CR criteria for AML) with conventional therapy but only a 9% 1-year survival (5). More generally, median survivals in patients with leukemic trans-formation from MPN reportedly range from 2.7 to 6.6 months, with those receiving induction therapy showing little if any prolongation of survival (median survival 3.9 to 6.0 months) (2). Moreover, response rates in transformed MPN reportedly range from 0 to 47%, but no uniform response criteria were applied to this diverse group of patients (2). JAK2 inhibitors, which have shown activity in chronic phase MPNs, exhibit limited efficacy after transformation to AML (6).

The poly(ADP-ribose) polymerase (PARP) enzymes PARP1 and PARP2 are involved in a wide variety of nuclear processes, including DNA damage sensing and repair through the base excision repair, single strand break repair, and double strand break (DSB) repair pathways (7, 8). Further studies have shown that PARP inhibition can be synthetically 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 (9-12). This synthetic lethality has been validated clinically in BRCA1- and BRCA2-mutated breast, ovarian and prostate cancers using PARP inhibitors (13-16).

Studies in diverse myeloid leukemia cell lines and primary human CD34⁺ chronic myelogenous leukemia (CML) cells have documented baseline defects in DNA repair pathways (17, 18). Moreover, primary marrow cells from patients with CMML and aggressive MPNs exhibit functional defects in DNA repair and hypersensitivity to multiple PARP inhibitors ex vivo (19). Certain AML-associated gene rearrangements, including AML1-ETO and PML-RARα fusions, have also been shown to confer PARP inhibitor sensitivity through deficiencies in homologous recombination (20). Conversely, elevated HOXA9 activity in MLL-rearranged acute leukemias has been associated with PARP inhibitor resistance (20).

Veliparib (ABT-888) is an orally bioavailable, small molecule PARP inhibitor that enhances the cytotoxicity of diverse classes of DNA damaging agents, including ionizing radiation, alkylating agents, platinating agents and topoisomerase I (topo I) poisons in vitro and in vivo (21-23). Studies in AML cell lines and clinical samples have demonstrated that veliparib enhances the antiproliferative and proapoptotic effects of topotecan in vitro, whereas no synergy is observed with cytarabine or etoposide (24). Further analysis indicated that the synergy results from trapping of PARP1 on the damaged DNA, thereby inhibiting repair downstream of topo I-DNA covalent complex stabilization (24, 25).

In earlier studies, topotecan and carboplatin induced synergistic cytotoxicity in acute leukemia cell lines and primary AML cells in vitro (26). This observation was the basis for a phase I study of topotecan + carboplatin by five-day intravenous continuous infusion (IV CI) in adults with relapsed or refractory acute leukemia (26). Oral and gastrointestinal (GI) mucositis were the dose limiting toxicities (DLTs) of this combination. Complete remissions (CRs) were observed at multiple dose levels, including 3 of 6 patients at the maximum tolerated dose (MTD) of topotecan 1.6 mg/m²/day and carboplatin 150 mg/m²/day simultaneously for 5 days (26). A subsequent ECOG Phase II trial of topotecan/carboplatin at this MTD resulted in CRs in 5 of 35 (14%) patients with relapsed and refractory AML, a response rate similar to a mitoxantrone/etoposide/cytarabine- (MEC-) based arm in the same trial (27).

Building on recent demonstrations that veliparib enhances the antineoplastic effects of topotecan and carboplatin, we conducted a Phase I dose-escalation trial of veliparib given twice daily prior to and during the administration of topotecan and carboplatin by 120-hour IV CI on days 3-7. At the MTD, the patient population was streamlined to focus on patients with aggressive CMML and progressive MPNs as well as AMLs evolving from these diseases based on preclinical studies suggesting PARP inhibitor hypersensitivity of this disease subset (19). The duration of veliparib administration was 8 days during the dose escalation. Veliparib dosing was extended in two MPN/CMML cohorts to 14 and 21 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 determinants of response.

Methods

Patient selection

Patients over age 18 with pathologically confirmed relapsed or refractory AMLs, newly diagnosed aggressive MPN, or aggressive CMML were eligible. Consistent with our previous study (29), for aggressive MPNs (primary myelofibrosis, agnogenic myeloid metaplasia, polycythemia vera, essential thrombocythemia, Ph-negative atypical CML) or CMML, one or more of the following disease acceleration criteria had to be present (30): marrow blasts >5%, peripheral blood blasts + promonocytes >10%, new onset or increasing myelofibrosis, new onset or >25% increase in hepatomegaly or splenomegaly, or new onset constitutional symptoms (fever, weight loss, splenic pain, bone pain). Patients were required to have: an Eastern Cooperative Oncology Group performance status of 0-2; left ventricular ejection fraction of ≥ 45%; hepatic enzymes ≤ 5 × upper limit of normal; bilirubin ≤ 2.0 mg/dL unless due to Gilbert's syndrome; interval of >4 weeks since allogeneic bone marrow transplantation (BMT) if performed; and absence of active GVHD, active CNS leukemia, and active uncontrolled infection. All patients gave informed consent to participate in the IRB-approved protocol according to the Declaration of Helsinki.

Treatment Plan

Veliparib was administered orally on a twice-daily schedule with dose escalation according to Table 1. Topotecan and carboplatin were given together by IV CI over 120 hours on days 3-7 of each cycle. Topotecan was dose-reduced for decreases in creatinine clearance (CrCl) as follows: CrCl 20-39 ml/min: 50% reduction; CrCl <20 ml/min: topotecan not administered. Carboplatin was dose-reduced for decreases in CrCl as follows: CrCl 40-59 ml/min: 30% reduction; CrCl 16-39 ml/min: 45% reduction; CrCl ≤ 15 ml/min: Carboplatin not administered.

Table 1. Dose levels and accruals to veliparib in combination with topotecan and carboplatin.

Dose Level	Veliparib Dose (p.o. BID) Days 1-8,14 or 21	Topotecan Continuous IV Days 3-7	Carboplatin Continuous IV Days 3-7	Number of Patients Accrued
1	10 mg × 8 d	1.0 mg/m2/day	-	3
2	10 mg × 8 d	1.3 mg/m2/day	-	3
3	10 mg × 8 d	1.3 mg/m2/day	150 mg/m2/day	4
4	10 mg × 8 d	1.0 mg/m2/day	120 mg/m2/day	6
5	10 mg × 8 d	1.2 mg/m2/day	120 mg/m2/day	6
6	10 mg × 8 d	1.2 mg/m2/day	150 mg/m2/day	6
7	20 mg × 8 d	1.0 mg/m2/day	120 mg/m2/day	6
8	20 mg × 8 d	1.2 mg/m2/day	120 mg/m2/day	6
9	20 mg × 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
11§	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

^§= Expansion cohort

Determination of DLT, MTD, and stopping rules

We evaluated 3-6 patients per dose level utilizing a standard 3 + 3 design. Toxicity was measured according to NCI-CTCAE version 4.0. The MTD was determined as the highest dose level where 0/3 or 1/6 experienced DLT. DLT consisted of: 1) any grade 4 non-hematologic toxicity; 2) any grade 3 non-hematologic toxicity that did not resolve ≤ grade 2 within 48 hrs, with the following exceptions: a) Grade 3 bilirubin, transaminases or alkaline phosphatase was considered dose-limiting only if resolution to ≤ grade 2 required ≥7 days; b) Grade 3 mucositis, diarrhea, nausea or vomiting was considered dose limiting only if resolution to ≤ grade 2 (including use of supportive care) required ≥7 days; c) any Grade 3 neurotoxicity or nephrotoxicity was considered dose limiting. Myelosuppression was not considered in evaluating toxicity in patients with acute leukemias except where bone marrow (BM) hypoplasia occurred for ≥50 days with BM cellularity ≤5% and no evidence of leukemia. At the MTD, 20 patients were treated in an expansion cohort to further establish the tolerability of this combination.

Evaluation of response

A BM aspirate and/or biopsy was obtained to assess response to therapy at the time of hematologic recovery or whenever leukemia regrowth was suspected based on circulating blasts or persistent cytopenias at 6-7 weeks after initiation of therapy. Hematologic recovery was defined as an absolute neutrophil count (ANC) of ≥500/mm³ and a transfusion-independent platelet count of ≥20,000/mm³. Standard definitions for CR, CR with incomplete count recovery (CRi) and partial response (PR) were used for response assessment in acute leukemia (31, 32).

Response in aggressive MPNs or CMML still in chronic phase (pre-treatment blasts less than 20%) was defined according to Giles et al. (30). In brief, CR in these patients required freedom from all symptoms or signs related to MPN; WBC 1-10 × 10⁹/L with no peripheral blood blasts, promyelocytes or myelocytes; normalization of bone marrow (<5% blasts in normocellular or hypercellular marrow) for ≥4 weeks; resolution of pretreatment cytopenias, including hemoglobin >12.0 gm/dl for males or >11.0 gm/dl for females without erythropoietin or transfusion support as well as ANC >1.0 × 10⁹/L and platelet count >100 × 10⁹/L without support; and resolution of pretreatment hyperleukocytosis and/or thrombocytosis. PR required improvement in two or more of the following: 1) ANC increase of 100% up to >10⁹/L for neutropenia and WBC count 1-10 × 10⁹/L with persistence of immature cells (blasts, progranulocytes, myelocytes); 2) increase in hemoglobin of ≥2 gm/dl if baseline value was <10 gm/dl, and a decrease in transfusion frequency and/or volume by at least 50%; 3) persistent thrombocytosis >450 × 10⁹/L but <50% of pretreatment values; 4) reduction in bone marrow blasts to <5% if blasts were originally >10% in normocellular or hypercellular marrow; and 5) reduction in splenomegaly and/or hepatomegaly by ≥50% of pretreatment dimensions.

Pharmacokinetic studies

Plasma samples obtained on day 1 of veliparib and on day 4 after the first dose of veliparib in combination with topotecan + carboplatin were assayed for veliparib using the validated LC/MS/MS method (33). PK variables were calculated by standard noncompartmental methods using Phoenix^® WinNonlin^® version 6.3 (Pharsight A Certara™ Company, Cary, NC) as previously described (34).

Exploratory pretreatment and pharmacodynamic correlates

Pretreatment FA pathway integrity

Pretreatment BM samples were treated with the DNA cross-linker melphalan (10 μM) for 6 h and FANCD2 ubiquitylation was assessed by immunoblotting (35). BRCA1 promoter methylation was assessed using previously published methods (36).

Pretreatment expression of PARP1 and topo I

Marrow mononuclear cells were isolated on Ficoll-Hypaque step gradients, washed with serum-free RPMI 1640 medium containing 10 mM HEPES (pH 7.4 at 21 °C), and prepared for electrophoresis as previously described (37). Aliquots containing protein from 5 × 10⁵ cells were subjected to SDS-PAGE and immunoblotting with enhanced chemiluminscent detection (38). Each blot contained a serial dilution of HL-60 cells. Signals were digitized and quantified relative to the HL-60 cell signal as previously described (37).

Assessment of poly(ADP-ribose) polymer (PAR) suppression and DNA damage

PAR assays were performed as described (39). Assessments of Ser139-phospho-H2AX (γH2AX) in peripheral blood and BM mononuclear cells were performed via flow cytometry according to previously described methods, using the CD34⁺ fraction when appropriate to distinguish the leukemic population from other cells (40).

Statistical considerations

Overall survival (OS) was defined from starting date of treatment to death, or censored at the last follow up date. Relapse free survival (RFS) was defined from starting date of treatment to the first occurrence of relapse date or date of death. OS, RFS, and probabilities of outcomes at 6 and 12 months were estimated with the Kaplan-Meier method, along with medians and 95% confidence intervals. Exploratory biomarker comparisons of survival were performed using Log-rank test.

Results

Patient characteristics

Ninety-nine patients, including 34 with primary refractory AML, 35 with secondary AML (myelodysplasia- or treatment-related), 22 with aggressive chronic myeloid neoplasms (CMML, non-CML MPN) or AML arising out of aggressive chronic myeloid neoplasms, and 4 with refractory ALL, were accrued to the trial. As indicated in Table 2, the median number of prior therapies was 2 (range 0-4), including prior allogeneic BMT in 16.

Table 2. Patient Characteristics and Outcomes.

	Number	Responses (%)
Median Age	56 (25-76)	33/99 (33%)
Refractory to prior regimen	78	23/78 (29%)
Primary refractory	34	9/34 (26%)
Prior regimens	3 (0-4)
0 Regimens	6	5/6 (83%)
1 Regimen	38	14/38 (37%)
2 Regimens	20	6/20 (30%)
3 Regimens	25	7/25 (28%)
4 Regimens	10	1/10 (10%)
Prior allogeneic bone marrow transplant	16	3/16 (19 %)
Antecedent hematologic disorder in patients with AML	35	17/35 (49%)
Myeloproliferative neoplasm-associated	22	14/22 (64%)
Treatment related	10	3/10 (30%)
MDS associated (non-MPN)	6	1/6 (17%)
JAK2 mutated MPN	7	5/7(71%)
Cytogenetics
Intermediate	48	12/48 (25%)
Adverse	45	14/45 (31%)
BRCA1 mutant	3	2/3 (67%)
BRCA2 mutant	2	2/2 (100%)
Azacitidine failures	15	6/15 (40%)
Acute Lymphoblastic Leukemia	4	1/4 (25%)

Treatment, toxicities and determination of dose and schedule

Patients received oral veliparib beginning day 1 and a 5-day continuous infusion of topotecan + carboplatin beginning on day 3 after steady state veliparib exposures were achieved. Subjects were accrued over 15 dose levels (Table 1). At the lowest dose of veliparib, topotecan 1.3 mg/m² was associated with dose-limiting GI mucositis. The study was redesigned with fixed doses of topotecan and carboplatin at 1.2 mg/m²/d and 150 mg/m²/d, respectively, to allow for veliparib dose escalation. A DLT of oral mucositis was identified at 100 mg veliparib twice daily (Table 3). De-escalation of veliparib to 90 mg twice a day also was not tolerable due to mucositis in two of four patients. Therefore, the MTD was 80 mg veliparib twice daily in combination with CI IV topotecan 1.2 mg/m²/d + carboplatin 150 mg/m²/day on days 3-7. At the MTD of 80 mg Veliparib twice daily, dosing was lengthened from 8 days to 14 and 21 days without increased toxicity (Dose levels 14 and 15, Table 1). Accordingly, the recommended phase 2 dose of the combination is veliparib 80 mg twice daily for up to 21 days in combination with CI IV topotecan 1.2 mg/m²/d + carboplatin 150 mg/m²/day on days 3-7.

Table 3. Toxicity by Veliparib Dose Level.

Category	Grade	10 mg (28 pts)	20 mg (18 pts)	40 mg (6 pts)	80 mg (26 pts)	80 mg × 14 days (6 pts)	80 mg × 21 days (6 pts)	90 mg (5 pts)	100 mg (4pts)
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

Clinical responses

As summarized in Table 4, 33% of 99 patients achieved objective responses: 14 CR, 11 CRi, and 8 PR. These responses were observed across a wide range of dose levels (Table 4).

Table 4. Summary of clinical responses to veliparib in combination with topotecan and carboplatin.

Dose Level	Clinical Responses/# Pts	Clinical Responses in patients with MPN 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)
11§	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)
Total	33/99 (14CR, 11 Cri, 8 PR	14/22 (3 CR, 7 Cri, 4 PR

Aza failure = patients who have been treated and failed to respond to azacitidine based therapy.

Among patients with AML but no history of MPN or CMML, the response rate was 25% (19/77). In contrast, 64% (14/22) with aggressive MPN, CMML or related AML responded, 11 of whom proceeded to allogeneic BMT with engraftment of donor cells (Supplemental Table S1). 93/99 (94%) patients were fully followed and died before analyzing the data. The median follow-up was 3.1 years among 6 alive patients. OS and RFS analysis of all patients, patients treated in the MTD expansion cohorts, and patients with associated MPN or CMML treated on any dose level are depicted in Figure 1.

Figure 1.

A. Survival analysis plotted in Kaplan Meier method for patients treated on study (expansion cohort = 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 Dec 2015, to Jan 2016). *p-value denoted in the figures was based on log-rank test.

The response rate varied depending on the number of prior regimens. All 6 patients without prior therapy had aggressive chronic myeloid neoplasms (aggressive CMML or MPN) or AML evolving in the setting of these diseases, with 5 responding. This subset was treated at or near the MTD of 80 mg veliparib twice daily for ≥14 days. Patients who received 1-4 prior therapies fared less well, with a response rate of 30% (28/93). Nonetheless, 15 heavily pretreated responders were able to proceed to allogeneic BMT (22 total transplanted after response).

The median duration of the response for all patients who achieved a CR, CRi or PR was 7.5 (95% CI: 5.4-13.1) months. In the subset with aggressive CMML, MPN or AML associated with these disorders, the duration of response was 11.5 (95% CI 7.5-NA) months (Figure 1A). Patients who achieved CR, CRi or PR had a median OS of 15.3 (95% CI: 10.5-23.0) months (Figure 1B). In contrast, median OS for patients who did not achieve a response was 4.2 (95% CI: 3.1-5.3) months (Figure 1B). Importantly, the duration of response and OS of patients achieving a CR/CRi or PR closely paralleled each other, consistent with the hypothesis that response contributes to the improved survival in responding patients.

In view of preclinical data suggesting PARP inhibitor hypersensitivity of a subset of aggressive chronic myeloid neoplasms (19), we were particularly interested in that group of patients. Patients with an antecedent aggressive MPN, CMML or AML in the setting of these disorders had a median OS of 13.3 (95% CI: 8.2-33.3) months (Figure 1C). One-year RFS was 41% (95% CI: 25-68%) in these patients vs 12% patients without prior MPN or CMML. Patients with antecedent aggressive MPN or CMML who had 0-1 prior therapy had a median survival of 14.4 months, whereas those with ≥2 prior therapies had a median survival of 9.3 months. Patients without an antecedent aggressive MPN or CMML had a median survival of 5.1 months. Since all but two responders in the MPN/CMML subset underwent allogeneic BMT, survival data were not censored for BMT.

Enrollment in the two extended dosing cohorts at the MTD (Dose levels 14 and 15, Table 1) was limited to patients with antecedent aggressive MPN or CMML (Figure 1D) based on preclinical data developed during the clinical study (19). Eight out of 12 patients had a clinical response (1 CR, 5CRi, and 2 PR). Median survival of patients treated in these two cohorts was 15.8 months vs 5.8 months for patients treated at dose levels 1-13.

Veliparib pharmacokinetics

Single and multiple dose veliparib plasma pharmacokinetics were analyzed in 86 patients after both single and multiple doses (Table 5). There was large interpatient variability in veliparib exposure. There was significant accumulation of veliparib (accumulation index) that corresponded with a decrease in both apparent clearance (Cl/F) and volume of distribution (V/F) (all P<0.0001). There were no significant differences in dose-normalized exposure (C_max or AUC) or other pharmacokinetic parameters (t_1/2, Cl/F, and V/F) across dose levels. Of note, there was a 1.8-fold increase in C_max and 2.0-fold increase in AUC after multiple doses between the MTD (80 mg) and the highest administered dose (100 mg) which is suggestive of non-linear pharmacokinetics. The development of mucositis correlated with single dose AUC (P=0.02) and multiple dose C_max (P = 0.04) suggesting a exposure-toxicity relationship.

Table 5. Plasma pharmacokinetic parameters¹ of veliparib when combined with topotecan and carboplatin.

Dose	Single or Multiple Dose	Cmax (ng/mL)	tmax (h)	AUCINF or Tau (µg*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)	1244 ± 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)	1474 ± 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)	1865 ± 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)	4057 ± 1278 (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)	5137 ± 1820 (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)	3042 ± 1319 (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)	3836 ± 1739 (3)	3.4, 3.6 (2)	15.8, 39.7 (2)	77.1, 206.8 (2)
100 mg	Single	1123.5 ± 718.5 (4)	1.0 (1.0 - 1.0; 4)	5044 ± 2655 (4)	5.9 ± 2.4 (4)	44.9 ± 50.6 (4)	504 ± 745 (4)
	Multiple	1515.8 ± 494.8 (4)	0.7 (0.5 - 1.0; 4)	10108 ± 4356 (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)

¹Data were obtained from patients receiving veliparib orally and are presented in the table as mean values ± SD (n). T _max is presented as median (range; n). If n<3, the actual values are reported.

Abbreviations: AUC_INF area under the plasma concentration-time curve to infinity; AUC_Tau area under the plasma concentration-time curve during the dosing interval at steady-state; Cl/F apparent systemic clearance; C_max, peak plasma concentration; t_max, time to peak concentration; t_1/2 half life; V/F apparent volume of distribution.

Exploratory Correlative Studies

Pretreatment assessment of FA pathway function

We examined functional integrity of the FA pathway, as assessed by FANCD2 monoubiquitination after melphalan exposure ex vivo (Supplemental Figure S1A), in pretreatment BM aspirates because i) FA pathway defects are reportedly common in AML and ii) loss of FA pathway function is associated with enhanced sensitivity to DNA crosslinking agents and PARP inhibitors (41). Impaired FANCD2 monoubiquitination was detected in 28/49 samples (57%) and was associated with modest prolongation of survival (median 6.1 vs 4.8 months, 1 year OS 39% vs 5%, P = 0.0342, Supplemental Figure S1B).

BRCA1, MGMT and MLH1 promoter methylation was examined in pretreatment samples from 11 of 12 patients in the prolonged dosing cohorts (Dose level 14 and 15, Table 1). One patient exhibited BRCA1 promoter hypermethylation and achieved a CRi.

Pretreatment PARP1 and topo I expression

Preclinical studies have demonstrated that PARP inhibitor-induced sensitization to topotecan involves conversion of each enzyme into an agent that perpetuates DNA damage upon treatment with the respective inhibitors (24). This model predicts that samples with higher levels of PARP1 or topo I will be more sensitive to the topotecan/veliparib combination. Accordingly, we examined expression of PARP1 and topo I by immunoblotting of pretreatment marrow samples from 21 patients treated at or above the veliparib MTD of 80 mg twice daily. As shown in Supplemental Figure S2, PARP1 and topo I levels varied without clear-cut relationship with each other. Although 5 of these 21 patients achieved response, there also was no clear-cut correlation with topo I or PARP1 expression.

Veliparib inhibits PAR formation in circulating and marrow leukemia

PAR assays were performed on samples obtained from 75 patients pretreatment and at various time points on days 1 and 4 of therapy. At 40 mg veliparib twice daily and above, inhibition of PAR formation by >75% was documented in 20 of 21 samples with baseline PAR >100 pg/10⁷ cells (Supplemental Fig. S3A). Greater suppression of PAR occurred at the higher dose levels in both peripheral blood and marrow samples (Supplemental Figs. S3A and S3C). PAR suppression at 2 hr post dosing time point on day 1 did not correlate with clinical response (Supplemental Fig. S3B).

Veliparib induces H2AX phosphorylation in circulating CD34⁺ blasts

Activation of the DNA damage response, as measured by H2AX phosphorylation via flow cytometry, was assessed in viable CD34⁺ BM and peripheral blood cells on day 1 at 4 hours after an initial veliparib dose of 80 mg and again on day 4 at 24 hours after the initiation of the topotecan/carboplatin infusion. As depicted in Supplemental Table S2, H2AX phosphorylation increased in CD34⁺ BM cells by median 2.6-fold (range 0.6-9.7) and peripheral blood by median 1.2-fold (median 0.1-19.2) following a single dose of veliparib on day 1 relative to pretreatment levels. Similarly, increases in H2AX phosphorylation were noted on day 4 in peripheral blood CD34⁺ cells during the topotecan/carboplatin infusion (median 2.2-fold, range 0.5-38). Peripheral blood CD34⁺ cohorts from patients who achieved clinical responses exhibited greater relative increases than CD34⁺ cohorts from non-responding patients on Day 4 (median 2.6-fold vs. 1.5-fold, P = 0.021 by unpaired t test, Supplemental Table S2).

Discussion

This study is the first to investigate the combination of PARP inhibitors with cytotoxic chemotherapy in acute leukemias in the clinical setting. The present trial demonstrates clinical activity of the topotecan/carboplatin/veliparib combination in patients with traditionally drug-resistant myeloid malignancies, in particular aggressive MPNs, CMML, and their associated acute leukemic phases. The MTD and recommended phase 2 dose of the regimen is veliparib 80 mg twice daily in combination with topotecan 1.2 mg/m²/d and carboplatin 150 mg/m²/d on days 3-7. Further escalation of veliparib was not tolerated due to mucositis, possibly due to non-linear pharmacokinetics at doses greater than 80 mg (Table 5).

Because activity was observed in MPN-associated leukemias during dose escalation and we also observed antiproliferative effects upon prolonged exposure of primary MPN and CMML cells to PARP inhibitors ex vivo (19), we added two cohorts (Dose Levels 14 and 15) for patients with antecedent MPN or CMML to allow extended duration PARP inhibition. Dosing beyond 8 days was well tolerated without impact on adverse events (Table 3). Combined cohorts of extended veliparib duration (14 and 21 days) had a response rate of 67% (8/12) and a favorable median survival of 15.8 months. Accordingly, we recommend examining an extended duration of PARP inhibitor treatment in any further trials of this combination due to the promising clinical activity and lack of added toxicity.

The 80 mg twice daily MTD for veliparib in this trial is significantly lower than the 400 mg twice daily dose administered in veliparib single-agent trials (42). Other studies combining veliparib with chemotherapy in solid tumors have noted less than proportional increases in veliparib exposure at doses above 120 mg twice daily (43)(44). In contrast, we observed greater than proportionate increases in veliparib exposure above 80 mg (Table 5). A potential factor contributing to non-linearity in our study may be that renal elimination of veliparib was compromised in the presence of carboplatin and topotecan (44, 45). The single dose AUC and multiple dose C_max at the highest veliparib doses correlated with the DLT of mucositis (Table 5).

Based on levels of intracellular PAR, it appears that veliparib-induced PAR suppression may be necessary but not sufficient to determine clinical response. Thus, measurement of PAR does not appear promising as a predictor of response. On the other hand, the ability of veliparib to increase DNA damage both by itself and in combination with topotecan + carboplatin could serve as a potential biomarker of response: The increase in H2AX phosphorylation in circulating CD34+ cells was roughly 3-fold greater in responders than the modest increases seen in nonresponders (median 160% increase vs. 50% increase, respectively). On the other hand, the increase in phospho-H2AX after treatment was also greater in patients who had received fewer prior treatments. Accordingly, further studies are needed to define the potential role of this parameter as an independent predictor of response.

Why the therapy-induced damage is greater in responding leukemias also requires further investigation but may relate, at least in part, to genetic features of individual leukemias that confer differences in DNA damage response and subsequent PARP inhibitor sensitivity (20). Preclinical studies demonstrated that many primary human MPN cell populations are hypersensitive to the antiproliferative effects of PARP inhibitors (19). In the present study, FANCD2 ubiquitination was impaired in >50% of pretreatment leukemia samples and was associated with modest improvement in both median and overall survival (Fig. S1). Higher pretreatment PARP1 expression, which could result in greater potentiation of damage through trapping of PARP1 on the DNA (24), may be another determinant of response (Fig. S2) at doses lower than those that inhibit catalytic acitivity (46) but requires further study.

In summary, the topotecan/carboplatin/veliparib combination appears to have significant activity in myeloid malignancies that consist of or arise from aggressive MPNs and CMML. In this subset of patients we observed a striking 64% overall response rate and prolonged overall survival compared to previous studies (2). While these results reflect observations from a heterogeneous group of patients in a phase I study, they nonetheless suggest that this regimen warrants further clinical investigation in these disorders. In addition, several of the exploratory biomarkers examined here, including FANCD2 ubiquitylation and H2AX phosphorylation, warrant further investigation in future trials as well.

Translational Relevance.

Poly(ADP-ribose) polymerase (PARP) plays multiple roles in DNA damage response pathways. Neoplasias associated with defective homologous recombination (e.g., BRCA1, BRCA2, ATM, or Fanconi anemia pathway mutations) are especially sensitive to PARP inhibitors. Here we report phase I clinical trial results in patients with relapsed acute myeloid leukemia, chronic myelomonocytic leukemia (CMML) or progressive myeloproliferative neoplasms (MPNs) treated the PARP inhibitor veliparib in combination topotecan and carboplatin. Consistent with recently published preclinical findings showing PARP inhibitor hypersensitivity of CMML and MPNs, these disorders and leukemias arising from them appear to be particularly sensitive to this combination. Predictors of response include pretreatment defects in crosslinking agent-induced FANCD2 monoubiquitination and higher treatment-induced histone H2AX phosphorylation. These observations support further study of the veliparib/topotecan/carboplatin combination in aggressive myeloid disorders with inherent DNA repair deficits such as aggressive MPNs, CMML, and AML arising in the setting of these disorders.