Treatment in Vitro with a Combination of Bcl-Xl Inhibitor-ABT-737 and a JAK2 Inhibitor Selectively Eliminates JAK2V617F MPN Progenitor Cells.

Abstract 752

The Ph^- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph⁻ MPNs.

We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (<5%). However, the combination of ABT-737 (0.25 uM) plus erlotinib (1.0 uM) was the least potent of inducing normal CD34⁺ cells to undergo apoptosis (∼5%) as compared to ABT-737 plus INCB018424 (∼20%). We next examined the effects of each of the JAK2 inhibitors alone or in combination with ABT-737 on CD34⁺ cells isolated from patients with PV or PMF. PV or PMF CD34⁺ cells were incubated in the absence or presence of agent(s) for 4 days; the percentage of apoptotic cells was then determined using Annexin V/PI staining by flow cytometry. A fraction of both untreated and treated CD34⁺ cells were assayed for hematopoietic colonies in the presence of SCF, IL-6, IL-3, G-CSF and EPO; individual colonies were then randomly plucked for JAK2V617F genotyping using nested allele-specific PCR. Similar to HEL cells, the percentage of MPD CD34⁺ cells undergoing apoptosis were similar when cells were treated with ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1.0 nM) (∼40%), but were significantly higher than that of MPN CD34⁺ cells treated with each of the agents alone (∼5%). Interestingly, when the percentages of JAK2V617F positive colonies within each of the treated and untreated cell populations were analyzed, MPN CD34⁺ cells treated with ABT-737 plus each of the JAK2 inhibitors contained a significantly lower percentage of JAK2V617F positive colonies than those untreated or treated with each of the single agents. These data indicate that the combination of ABT-737 plus a JAK2 inhibitor specifically targets on the JAK2V617F positive progenitor cells. Collectively, we identified an agent combination of ABT-737 (0.25 uM) plus a JAK2 inhibitor that is capable of selectively eliminating JAK2V617F positive MPN progenitor cells while sparing normal progenitor cells in vitro.