DNA instability, including increased DNA double strand breaks, has recently been demonstrated in myeloproliferative neoplasms (MPN). Because PARP1 has a central role in DNA repair and maintaining genomic integrity, we tested whether DNA repair inhibition with the PARP inhibitor ABT-888 (Abbvie, Chicago, IL) could affect the growth of myeloid neoplasms in-vitro and in-vivo, and/or enhance the cytotoxic effect of a standard alkylating agent commonly used in preparative regimens for stem cell transplants.
Since the JAK2V617Fmutation is common in chronic MPNs or acute myeloid leukemia (AML) secondary to MPNs, we initially utilized JAK2 mutated AML cell lines (SET2 and HEL) in-vitro and in-vivo. We then tested the effect of PARP inhibition on CD34+ cells obtained from patients with primary myelofibrosis.
JAK2 mutated SET2 and HEL cells were treated with ABT-888 in liquid cultures. Both cell lines were relatively sensitive with IC50 of 11.3μM and 74.2μM respectively. When the cells were treated with a combination of increasing doses of busulfan and 8μM ABT-888, significant synergy was observed, with busulfan IC50 decreasing from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. Treatment of SET2 cells with 8μM ABT-888, or with 8μM ABT888 and 10μM busulfan, was also accompanied by marked increase in γH2AX foci compared to control at 24 hours. We subsequently showed that ABT-888, busulfan and combination treatments resulted in increasing numbers of cells in G2/M arrest (30% vs. 35% vs 53% respectively, p=0.002). Western blot analysis of cells in G/2M arrest after combination treatment demonstrated a strongly increased phosphorylation of checkpoint kinase 1 (Chk1). In an immunofluorescence assay on treated cells we then showed that activated Chk1 translocated to the nucleus, suggesting that the Chk1-ATR pathway may be responsible for cell cycle arrest. To demonstrate the in-vivo effect of PARP inhibition, we transplanted 5x106 SET2 cells into a NOD/SCID/ gamma null mice to create a xenograft model. Mice developed AML with splenomegaly and bone marrow engraftment resulting in a median survival of 36 days. In mice that were treated with daily intraperitoneal injections of low dose ABT-888 (1.5mg/kg), starting 14 days after leukemic cell injection, survival increased to a median of 40 days (p=0.002).
We subsequently tested the effect of PARP inhibition on primary myelofibrosis CD34+ cells obtained from the bone marrow or peripheral blood of 3 patients with JAK2V617Fmutation and 2 with CALR mutation. Control experiments were performed with normal CD34+ progenitors. In a standard clonogenic assay in methylcellulose, cells were plated with or without 4μM ABT-888, 5μM busulfan or a combination of both. Compared to untreated cells, ABT-888, busulfan and the combination of the two drugs reduced colony formation by 39%, 70% and 89%, respectively (p=0.01). Both CALR and JAK2 mutated cells were sensitive to ABT-888. On the contrary, treatment of normal CD34+ cells with ABT-888 did not affect colony growth.
Here we describe the potential for PARP inhibition to exploit the genomic instability of MPNs. Potential clinical applications include the addition of PARP inhibition to busulfan in pretransplant conditioning or novel drug combinations including PARP inhibitors.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.
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