Genetic and Epigenetic Defects in DNA Repair Lead to Synthetic Lethality of Poly (ADP-Ribose) Polymerase (PARP) Inhibitors in Aggressive Myeloproliferative Disorders

Abstract 400^FN2

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, IC_50S=2.69uM vs. 11.2uM, N=15 IC_50S=.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.