Abstract 667: Mechanisms underlying synergistic interactions between the CDK inhibitor flavopiridol (Alvocidib) and the BH3 mimetic GX15-070 (Obatoclax) in human multiple myeloma cells

Abstract

Abstract To establish a mechanistic basis for a strategy combining CDK inhibitors and Bcl-2 antagonists, in vitro and in vivo interactions between flavopiridol (FP) and GX15-070 (GX) were investigated in human multiple myeloma (MM) cells. The activity of this regimen was also examined in primary CD138+ MM cells and in a murine MM xenograft model. Co-administration of sub-toxic concentrations of FP interacted synergistically with GX to induce apoptosis in U266, RPMI8226, H929, OPM-2, and MM.1S, as well as IL-6-dependent ANBL6 and KAS 6/1 MM cells. Combination treatment also effectively killed MM cells in the presence of stromal cells or growth factors. The regimen was active against multiple drug-resistant MM cell lines, including bortezomib/Velcade- (U266/VR and OPM-2/VR), Revlimid- (8226/R10R), dexamethasone- (MM.1R), melphalan- (8226/LR5), and doxorubicin-resistant (8226/Dox40) MM cells. FP also sharply promoted GX lethality in primary CD138+ MM cells, while sparing normal CD138- bone marrow cells. GX induced clear declines in Mcl-1 and Bcl-xL levels which were potentiated by FP, whereas Bcl-2 expression did not change. Significantly, FP/GX overcame cytoprotective effects mediated by ectopic Mcl-1, Bcl-xL, or Bcl-2 expression. Notably, BH3-only protein expression profiling revealed multiple changes: a) FP, in the presence or absence of GX, triggered a clear increase in Bim expression (both BimEL and BimL isoforms); b) FP/GX co-treatment sharply induced Bik (∼35kDa species) and Noxa (∼27 kDa species),; c) co-exposure (24hr) to FP and GX induced a clear reduction in Puma expression; and c) no changes in the expression of other BH3-only proteins occurred. These findings were confirmed in various MM cell lines, including multiple drug-resistant cells, and were associated with Bak/Bax activation, Bax translocation, cytochrome C release, and caspase activation. Importantly, Bim, Bik, or Noxa knock-down by stable transfection with shRNA dramatically diminished FP/GX lethality. Lastly, co-administration of FP and GX significantly suppressed tumor growth, reflected by diminished tumor size and bioluminescence intensity, in athymic nude mice inoculated with 8226 cells stably expressing luciferase. These events were associated with pronounced apoptosis, manifested by caspase 3 activation and PARP degradation. Western blot analysis also revealed down-regulation of Mcl-1, as well as up-regulation of Bik and Noxa in tumor tissues following FP/GX co-administration. Collectively, these findings provide a mechanistic framework for a strategy combining CDK inhibitors with Bcl-2 antagonists in which down-regulation of anti-apoptotic proteins and induction of BH3-only proteins cooperate to trigger synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents, both in vitro and in vivo. 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 667.