McDevitt, Michael A.

Abstract Abstract 400FN2 Patients with primary and secondary myelofibrosis (MF) have a worse prognosis than those with essential thrombocytosis (ET) or polycythemia vera (PV). MF and chronic myelomonocytic leukemia (CMML) share chromosomal and genetic lesions such as ASXL1 and JAK2 mutations, but effective treatments are lacking. The aim of the study was to identify perturbed pathways in MPN and CMML that were amenable to targeted molecular therapies. In a large genomic analysis of MPN cases, we identified recurrent deletions of multiple DNA repair genes, including BRCA2 and Fanconi anemia (FANC) proteins, which have been associated with poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity. Neoplastic cells from these patients are hypersensitive to PARP inhibitors in vitro and may provide a therapeutic strategy to improve treatment response for patients with advanced MPNs. Genomic screens utilizing SNP-A karyotyping were performed on primary cells from 144 patients with either ET, PV, or MF (90 were previously reported, McDevitt et al., ASH 2009). We focused on the potential functional relevance of recurrent genomic deletions, including BRCA2, by performing a GO analysis using the EASE software application (Hosack et al. 2003). This revealed statistically significant (p≤0.05) enrichment of 13 biological pathways. Notably, genes involved in cell cycle regulation and DNA repair (DR) were enriched in MF loss regions (p=0.007) as compared to regions lost in ET and PV. Several genes directly involved in DR including BRCA2, XRCC2, and several members of the FANC pathway, also critical for DNA crosslink repair, were deleted or present in copy neutral LOH regions. DNA methyltransferase inhibitors are being tested clinically in MF patients, and both cell cycle and DNA repair genes have been identified as epigenetically silenced in hematological malignancies. We therefore screened for BRCA1, BRCA2, FANC-C, L, and N promoter CpG island methylation in a series of 48 MPN samples. No methylation was identified in BRCA2 and FANC members, suggesting a potential haploinsufficiency mechanism. Methylation of BRCA1 was identified in a small number of cases and is under further study in a larger cohort to investigate the effects on BRCA1 expression, DR and sensitivity to PARP inhibitors. Based on these genetic and epigenetic findings, we then focused on a functional analysis of the homologous recombination (HR) pathway. Leukocytes from normal controls and MPN patients were exposed to ionizing radiation (IR) and stained for Rad51 foci, a marker of HR activity. Six of 15 MPN/CMML samples (40%), but none of the controls failed to form IR-induced Rad51 foci, suggesting impaired HR. Of the samples that demonstrated defects in HR [MPN-U, MF, CMML (2), MDS/MPN-U, and CML], there was no significant correlation between the MPN subtype, the JAK2 mutation status, or the specific metaphase karyotype, but the most sensitive case in our series exhibited a deletion of Ch13q that spanned the BRCA2 gene. We then examined sensitivity of MPN vs. normal myeloid progenitors to the PARP inhibitors veliparib and/or olaparib in methylcellulose (MC)-based colony forming assays. Failure to form Rad51 foci with irradiation was statistically correlated with PARP inhibitor sensitivity (p = 0.021). When an expanded series of cases were then analyzed for PARP inhibitor sensitivity using the MC colony assay, lower IC50s for both veliparib and olaparib were observed in MPN cases (N= 25, IC50S=2.69uM vs. 11.2uM, N=15 IC50S=.64uM vs. 1.27uM, respectively) relative to normal controls (p =.0003, p =.05, via Wilconon rank-sum test respectively). Two cases in this series were treated on a clinical trial combining the ABT-888 PARP inhibitor with salvage chemotherapy consisting of carboplatin and topotecan (Karp et al., ASH 2010). Both patients, one with sAML evolving out of PPV-MF, and a second with accelerating MPN-U, had failed four prior therapies but achieved complete clearance of bone marrow blasts at day 14. Interestingly, cells from this MPN-U patient (13q-) were the most sensitive to PARP inhibition in vitro. Additional accrual and correlative evaluations are underway and will be updated at the meeting. Collectively, these results demonstrate that a subset of MPN and CMML patients have intrinsic genetic and epigenetic defects affecting the FANC and HR pathways, providing pre-clinical rationale for the use of PARP inhibitors in the treatment of aggressive MPN and CMML. Disclosures: No relevant conflicts of interest to declare.

Abstract Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009). Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals. Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs. Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03). Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment. Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. Figure 1. Figure 1. Disclosures Off Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

Abstract Purpose: The 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 (MPN), 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 MTD 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 [HR = 0.56 (95% confidence interval, 0.27–0.92)]. 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. Clin Cancer Res; 23(4); 899–907. ©2016 AACR.

Abstract Purpose: In preclinical studies, the PARP inhibitor veliparib enhanced the antileukemic action of temozolomide through potentiation of DNA damage. Accordingly, we conducted a phase 1 study of temozolomide with escalating doses of veliparib in patients with relapsed, refractory acute myeloid leukemia (AML) or AML arising from aggressive myeloid malignancies. Experimental Design: Patients received veliparib [20–200 mg once a day on day 1 and twice daily on days 4–12 in cycle 1 (days 1–8 in cycle ≥2)] and temozolomide [150–200 mg/m2 daily on days 3–9 in cycle 1 (days 1–5 in cycle ≥2)] every 28 to 56 days. Veliparib pharmacokinetics and pharmacodynamics [ability to inhibit poly(ADP-ribose) polymer (PAR) formation and induce H2AX phosphorylation] were assessed. Pretreatment levels of MGMT and PARP1 protein, methylation of the MGMT promoter, and integrity of the Fanconi anemia pathway were also examined. Results: Forty-eight patients were treated at seven dose levels. Dose-limiting toxicities were oral mucositis and esophagitis lasting >7 days. The MTD was veliparib 150 mg twice daily with temozolomide 200 mg/m2 daily. The complete response (CR) rate was 17% (8/48 patients). Veliparib exposure as well as inhibition of PAR polymer formation increased dose proportionately. A veliparib-induced increase in H2AX phosphorylation in CD34+ cells was observed in responders. Three of 4 patients with MGMT promoter methylation achieved CR. Conclusions: Veliparib plus temozolomide is well tolerated, with activity in advanced AML. Further evaluation of this regimen and of treatment-induced phosphorylation of H2AX and MGMT methylation as potential response predictors appears warranted. Clin Cancer Res; 23(3); 697–706. ©2016 AACR.

Abstract Purpose: DNA repair defects have been previously reported in myeloproliferative neoplasms (MPN). Inhibitors of PARP have shown activity in solid tumors with defects in homologous recombination (HR). This study was performed to assess MPN sensitivity to PARP inhibitors ex vivo. Experimental Design: HR pathway integrity in circulating myeloid cells was evaluated by assessing the formation of RAD51 foci after treatment with ionizing radiation or PARP inhibitors. Sensitivity of MPN erythroid and myeloid progenitors to PARP inhibitors was evaluated using colony formation assays. Results: Six of 14 MPN primary samples had reduced formation of RAD51 foci after exposure to ionizing radiation, suggesting impaired HR. This phenotype was not associated with a specific MPN subtype, JAK2 mutation status, or karyotype. MPN samples showed increased sensitivity to the PARP inhibitors veliparib and olaparib compared with normal myeloid progenitors. This hypersensitivity, which was most pronounced in samples deficient in DNA damage–induced RAD51 foci, was observed predominantly in samples from patients with diagnoses of chronic myelogenous leukemia, chronic myelomonocytic leukemia, or unspecified myelodysplastic/MPN overlap syndromes. Conclusions: Like other neoplasms with HR defects, MPNs exhibit PARP inhibitor hypersensitivity compared with normal marrow. These results suggest that further preclinical and possibly clinical study of PARP inhibitors in MPNs is warranted. Clin Cancer Res; 22(15); 3894–902. ©2016 AACR.

Abstract Purpose: Incorporation of cytarabine into DNA activates checkpoint kinase 1 (Chk1), which stabilizes stalled replication forks, induces S-phase slowing, and diminishes cytarabine cytotoxicity. The selective Chk1 inhibitor SCH 900776 abrogates cytarabine-induced S-phase arrest and enhances cytarabine cytotoxicity in acute leukemia cell lines and leukemic blasts in vitro. To extend these findings to the clinical setting, we have conducted a phase I study of cytarabine and SCH 900776. Experimental Design: Twenty-four adults with relapsed and refractory acute leukemias received timed sequential, continuous infusion cytarabine 2 g/m2 over 72 hours (667 mg/m2/24 hours) beginning on day 1 and again on day 10. SCH 900776 was administered as a 15- to 30-minute infusion on days 2, 3, 11, and 12. The starting dose of SCH 900776 was 10 mg/m2/dose. Results: Dose-limiting toxicities consisting of corrected QT interval prolongation and grade 3 palmar-plantar erythrodysesthesia occurred at 140 mg flat dosing (dose level 5, equivalent to 80 mg/m2). Complete remissions occurred in 8 of 24 (33%) patients, with 7 of 8 at 40 mg/m2 or higher. SCH 900776 did not accumulate at any dose level. Marrow blasts obtained pretreatment and during therapy showed increased phosphorylation of H2Ax after SCH 900776 beginning at 40 mg/m2, consistent with unrepaired DNA damage. Conclusions: These data support a randomized phase II trial of cytarabine +/− SCH 900776 at a recommended flat dose of 100 mg (equivalent to 56 mg/m2) for adults with poor-risk leukemias. The trial (SP P05247) was registered at www.clinicaltrials.gov as NCT00907517. Clin Cancer Res; 18(24); 6723–31. ©2012 AACR.

Abstract Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations. Cancer Res; 73(3); 1076–85. ©2012 AACR.

Abstract Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.