Poh, Weijie

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: 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.