Treatment with Pegylated Interferon Alpha 2a in Combination with the Bcl-Xl Inhibitor ABT-737 Specifically Targets JAK2V617F Positive Hematopoietic Progenitor Cells From Patients with Polycythemia Vera.

Abstract 3916

Poster Board III-852

Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34⁺ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34⁺ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34⁺ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34⁺ cells but not CB CD 34⁺ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a, <5%; ABT-737, 20%). PV CD34+ cells were incubated with Peg IFNa 2a (500 U) alone, ABT-737 (0.25 uM) alone or ABT-737 plus Peg IFNa 2a for 4 days and the numbers of cells were decreased by 35%, 40% and 65 %, respectively; and the corresponding percentage of apoptotic cells was 20%, 15% and 60%, respectively. Western blot analysis showed that the Bcl-xL protein level in PV but not CB mononuclear cells was reduced by treatments with ABT-737 alone or in combination with Peg IFNa 2a. Furthermore, treatment of PV CD34⁺ cells with ABT-737 plus Peg IFNa 2a (200U) lead to the appearance of a smaller proportion of JAK2 V617F-positive (46.7±26%) hematopoietic progenitor cells as compared to cells incubated with cytokines alone (81.7±17%) or cytokines plus Peg IFNa 2a (69±20%).

These data suggest that low doses of Peg IFNa 2a selectively and directly eliminate Jak2V617F hematopietic progenitor cells which likely accounts for the therapeutic responses that have been observed with the use of this agent in the clinic. The enhanced elimination of JAK2V617F hematopoietic progenitor cells observed with the combination of ABT-737 and Peg-IFNa-2a suggests that this strategy might be an even more optimal approach for the treatment of JAK2V617F positive MPN which merits further testing in the clinic.