Distribution of Bim Determines MCL-1 Dependence or Co-Dependence on BCL-x_L/BCL-2 In Multiple Myeloma

Abstract 4054

The inappropriate activation of oncogenes can result in the up-regulation of pro-apoptotic signals often in the form of BH3-only proteins such as Bim, Noxa or Puma. This renders cells dependent on anti-apoptotic proteins including BCL-2, BCL-x_L and MCL-1. Thus cancer cells would be predicted to be more susceptible to inhibition of BCL-2 family proteins, prompting the development and testing of several small molecule inhibitors of this class of proteins. Multiple myeloma is a plasma cell malignancy of the bone marrow and like normal plasma cells, myeloma plasma cells express MCL-1. A search of gene expression profile data from normal plasma cells (n=22), MGUS plasma cells (n=12), plasma cells from patients with asymptomatic (smoldering) myeloma (n=44) or newly diagnosed multiple myeloma (n=538) revealed no significant difference in MCL-1 mRNA expression associated with progression of disease. We then determined MCL-1 dependence through the introduction of siRNA in 4 MM cell lines and consistent with previous findings using anti-sense oligonucleotides in additional lines, we demonstrated that all cell lines tested were MCL-1 dependent. However using the BCL-2/BCL-x_L/BCL-w-selective inhibitor ABT-737 we found that 3 of the 6 MCL1-dependent cell lines tested were sensitive (IC50 for Annexin V-FITC positive at 24 hrs of 300 – 600 nM) and therefore also dependent on BCL-x_L/BCL-2. Taken together this is the first formal demonstration that cells can be co-dependent on multiple Bcl-2 family members. We have previously reported that ABT-737 sensitivity, and what we now refer to as co-dependence on MCL-1 and BCL-x_L/BCL-2, is determined by the distribution of BIM on the anti-apoptotic BCL-2 proteins in these cells. We have now expanded these findings to patient samples that displayed sensitivity to ABT-737 that is similar to what we have observed in the co-dependent cell line MM.1s. Consistent with these findings co-immunoprecipation revealed BIM binding predominantly to BCL-x_L. Additionally we have now demonstrated that BIM binding is not simply controlled by the expression levels of BCL-x_L or MCL-1 as enforced over-expression of each protein could alter the sensitivity of co-dependent cell lines to ABT-737 but did not alter the initial distribution of BIM amongst these proteins. These data suggest that additional factors regulate the association of BIM with anti-apoptotic BCL-2 proteins. These factors could include differences in cellular localization of these proteins as well as differences in post-translational modifications of either pro- or anti-apoptotic BCL-2 family proteins.