Abstract 4649: Nbk/Bik induced by flavopiridol switches GX15-070-induced autophagy to apoptosis in human multiple myeloma cells
Authors: Dai Yun , Chen Shuang , Wang Li , Pei Xin-Yan , Myers Jennifer , Schwartz Daniella , Thompson Nadine , Larsen Dustin , Richey Justin , Su Florence , Orlowski Robert Z. , Grant Steven 
Published: August 2012

Abstract
Combining the BH3 mimetic GX15-070 (GX, Obatoclax) with the CDK inhibitor flavopiridol (FP, Alvocidib) leads to striking in vitro and in vivo activity against human multiple myeloma (MM) cells, including those resistant to conventional or novel agents. Synergism is associated with both down-regulation of anti-apoptotic proteins (e.g., Mcl-1 and Bcl-xL) and induction of BH3-only proteins e.g., Bim and Bik (Bcl-2-interacting killer). While Mcl-1, Bcl-2, or Bcl-xL over-expression partially attenuates FP/GX lethality, knock-down of either Bim or Bik almost completely blocks apoptosis, suggesting a critical role for BH3-only proteins in this setting. Here, we demonstrate that in contrast to Bim, which directly triggers apoptosis, Bik promotes apoptosis by regulating autophagy. Prevention of Bik induction by shRNA failed to block release of Bak from Mcl-1, arguing against its direct action in apoptosis. Sub-toxic concentrations (500-750 nM) of GX released beclin 1 from Bcl-2 and induced autophagy (e.g., LC3 conversion and autophagosome formation), events occurring as early as 6 hr and sustained for 48 hr, but unaccompanied by marked apoptosis. Confocal microscopy revealed that in both drug-naïve and bortezomib-resistant MM cells, co-exposure to GX/FP strikingly increased autophagosomes extensively packed in the cytoplasm, accompanied by robust apoptosis. This phenomenon was associated with marked Bik up-regulation induced by FP +/− GX. Whereas Bim was up-regulated and predominantly localized to the mitochondria, Bik was induced and exclusively localized to the ER after FP/GX treatment. Autophagy includes initiation, elongation, autophagosome formation, and maturation to autolysosomes for cargo degradation. Interestingly, Bik expression occurred subsequent to autophagy induction and was diminished by beclin 1 or ATG5 shRNA, suggesting that Bik is induced during autophagy and negatively regulates later stages responsible for cargo clearance. Notably, whereas Bim shRNA diminished apoptosis but not autophagy induced by FP/GX, Bik shRNA blocked both processes. Moreover, beclin 1 or ATG5 shRNA significantly reduced apoptosis induced by FP/GX, whereas dominant-negative caspase-9 failed to prevent autophagy. Finally, overexpression of Bcl-xL, but not Bcl-2 or Mcl-1, largely attenuated Bik expression and autophagy induced by FP/GX. Co-immunoprecipitation showed that FP/GX-induced Bik bound to Bcl-xL, rather than to Bcl-2 or Mcl-1. Together, these findings suggest a model where in the presence of FP, Bik is expressed after autophagy induction by GX, and in turn interrupts later stages of autophagy, thus interfering with the ability of cells to remove cargo-loaded autophagosomes. Consequently, autophagosomes accumulate in cells co-exposed to FP/GX, leading to robust apoptosis. Thus, in this setting, Bik, acts as a switch that transforms autophagy into apoptosis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4649. doi:1538-7445.AM2012-4649