Activity of the BH3 Mimetic ABT-737 on Polycythemia Vera (PV) Erythroid Precursor Cells

Abstract

Malignant hemopoietic cells are often characterized by ineffective death pathways, resulting in enhanced resistance to apoptosis and ultimately in the survival/expansion of the abnormal clone. Mechanistic studies have been undertaken to identify the aberrant signal transduction pathway and to develop small-molecule inhibitors targeting deregulated modules. Regulators of apoptotic pathways play a key role in the control of erythroid cell expansion; in particularl Bcl-XL is essential for erythroid cell development and, together with Bcl-2, it protects erythroblast survival from cytotoxic stimuli. Previous studies on erythroid cells derived from polycythemia vera (PV) patients have revealed an increased expression of Bcl-XL associated with cell survival in the absence of erythropoietin. The discovery of the JAK2V617F mutation in the vast majority of PV patients and its association with an increased resistance to apoptosis induced by death receptors prompted us to investigate the correlation between JAK2V617F and the expression of anti-apoptotic Bcl-2 family members and to explore the activity of a Bcl-2 inhibitor on PV erythroid cells. ABT-737 is a synthetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-XL, poorly to Mcl-1, promoting apoptosis as single agent in malignant hemopoietic cells. In this study we analyzed pure populations of primary erythroid precursors obtained from CD34+ cells of healthy donors and PV patients to investigate the expression of Bcl-2, Bcl-XL and Mcl-1. We found that the expression of Bcl-XL and Bcl-2 was increased in PV patients compared to controls, while Mcl-1 levels did not significantly differ. Then we analyzed Bcl-2 and Bcl-XL expression in erythroblasts derived from PV patients clustered on the basis of a different JAK2V617F allele burden. We found that on average the expression of both Bcl-2 and Bcl-XL was comparable to controls in PV erythroblasts with low/null mutation rates while both proteins were significantly (P< 0.001 and P<0.05, respectively) overexpressed in those with a high percentage of mutated JAK2. Subsequently, we investigated whether ABT-737 was effective in inducing apoptosis in normal and PV erythroblasts. A dose-response induction of apoptosis was elicited by ABT- 737 in PV erythroblasts with elevated JAK267F allele burden with higher effects than in normal cells starting from 500nM (P<0.05). The preferential effects of ABT-737 on PV erythroblasts were further documented by differences in ABT-737-induced mitochondrial depolarization and caspase cleavage. Treatment with ABT-737 did not induce significant alterations in cell cycle distribution on both PV and normal erythroid cells. The activity of ABT-737 was then studied in semisolid cultures. BFU-E colony formation was inhibited in PV-derived CD34+ cells but not in normal controls. To investigate the mechanisms underlying ABT-737 inhibition of erythroblast expansion we analyzed GATA-1 levels that resulted significantly (P<0.05) decreased in PV erytroblasts but not in normal cells, indicating a differential extent of caspase-mediated degradation of GATA-1. Taken together these results indicate that ABT-737 promotes apoptosis of mutated JAK2 PV erythroid cells with a preferential activity compared to normal erythroid cells, supporting a potential therapeutic role of this molecule in patients with PV.