Abstract #2786: Apoptosis induced by JAK2 inhibition requires Bim and is enhanced by a BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells

The activating mutation JAK2V617F has been shown to play a central role in the pathogenesis of polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. Inhibition of mutant JAK2 activity leads to growth inhibition and apoptosis in cells with mutated JAK2. However, the pro-apoptotic proteins involved in JAK2 inhibition-induced apoptosis remain unclear. Recently, it has been shown that the BH3-only protein Bim is one of the main effectors of tyrosine kinase inhibitor-mediated apoptosis in BCR-ABL positive chronic myelogenous leukemia cells, EGFR mutant lung cancer cells, and B-RAF mutant tumor cells. They show striking initial response to small molecule tyrosine kinase inhibitors and may use Bim as a common mediator during apoptosis. Therefore, we hypothesized that up-regulation of active Bim may be necessary for apoptosis induced by JAK2 inhibition in cells carrying constitutively activated JAK2 mutations. In this study, we examined two JAK2 mutant cell lines, HEL and CHRF-288-11 cells and observed that JAK2 inhibition-induced apoptosis led to inactivation of STAT5 and ERK1/2, up-regulation of de-phosphorylated Bim, as well as down-regulation of the anti-apoptotic protein Bcl-xL, suggesting that balance of Bim/BCL-xL may be critical for JAK2 inhibition-induced apoptosis. The BH3 mimetic agent ABT-737 induced apoptosis in a dose-dependent fashion in HEL cells. Knockdown of Bim in stably transfected HEL cells using a small hairpin RNA construct completely inhibited apoptosis induced by JAK2 inhibition. Knockdown of Bim led to lack of Bax activation and failure of inner mitochondrial membrane potential breakdown with JAK2 inhibition. Addition of ABT-737 fully restored induction of apoptosis with JAK2 inhibition in the Bim knockdown HEL cells. Thus, these data confirmed that the intrinsic apoptosis pathway was inactive in the absence of Bim. In addition, ABT-737 enhanced the apoptosis induced by JAK2 inhibition in wild type HEL cells. Moreover, the combination of JAK Inhibitor I and ABT-737 reduced the number of erythroid colonies derived from primary CD34 positive cells from PV patients more efficiently than either drug alone. Progenitor cells committed to the granulocytic or monocytic lineages were not significantly impaired by this combination of agents. Taken together, our data indicate that Bim is a key effector molecule in JAK2 inhibition-induced apoptosis and that manipulation of the apoptotic pathway could provide a novel therapeutic strategy for patients with myeloproliferative disorders carrying activating mutations of the tyrosine kinase JAK2.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2786.