Abstract 4523: Inhibition of Bcl-2 antiapoptotic members by obatoclax dramatically potentiates sorafenib-induced apoptosis in human myeloid leukemia cells through a Bim-dependent process

Abstract

Previously, we demonstrated that the multikinase inhibitor sorafenib diminishes Mcl-1 protein expression in human leukemia cells by inhibiting translation through a MEK1/2/ERK1/2 signaling-independent mechanism, and that this phenomenon plays a key functional role in sorafenib lethality. However, recent evidence suggests that Bcl-2 and Bcl-xL may also limit the apoptotic activity of sorafenib. In efforts to potentiate the lethality of pharmacologically achievable concentrations of sorafenib (e.g., 5-7.5 μM) in human myeloid leukemia cells, we have examined the effects of the small molecule inhibitor obatoclax (GX15-070), which has been reported to disrupt the function of Bcl-2, Bcl-xL, and Mcl-1, on sorafenib anti-leukemic activity. Here we report that obatoclax strikingly enhances sorafenib-mediated mitochondrial dysfunction and cell death in U937 and other myeloid leukemia cell types. These events were manifested by cytochrome c and AIF release into the cytosol, caspase-3 and −9 activation, PARP cleavage, apoptosis, and a marked decrease in cell growth. Similar effects were observed in KG1 and HL-60 cells. These events were associated with the rapid and pronounced downregulation of Mcl-1. Notably, genetic studies using shRNA directed against Bim, a major Mcl-1 binding partner, revealed that knockdown of Bim strikingly attenuated obatoclax/sorafenib-induced apoptosis. Immunoprecipitation studies revealed that down-regulation of Mcl-1 in sorafenib-treated cells was associated with enhanced binding of Bim to Bcl-xL and Bcl-2. However, co-administration of obatoclax effectively prevented the increased association between Bim and Bcl-xL/Bcl-2. In addition, knockdown of Bak or Bax also markedly diminished obatoclax/sorafenib-mediated lethality. In contrast, knockdown of Bad or Noxa had no significant effect on obatoclax/sorafenib-mediated lethality. Together, these findings suggest that activation of Bim, but not Bad or Noxa, plays a key role in the pronounced antineoplastic activity of obatoclax/sorafenib toward human leukemia cells. They also raise the possibility that obatoclax may enhance sorafenib lethality by diminishing associations between Bcl-xL/Bcl-2 and Bim. Finally, in vivo studies in a murine xenograft U937 cell model revealed that co-treatment with sorafenib and obatoclax strikingly reduces in vivo tumor growth, whereas the agents administered individually had only modest effects. Combined treatment also reduced Mcl-1 expression in vivo. Collectively, these findings raise the possibility that combining sorafenib, which induces Mcl-1 down-regulation, with agents such as obatoclax that inhibit other Bcl-2 family antiapoptotic members (e.g., Bcl-2, Bcl-xL) may represent a novel and effective strategy against myeloid leukemia and possibly other hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4523.