Obatoclax effects on MCL1 addiction in mesothelioma and activity in vivo.

Abstract

e13539 Background: Resistance to apoptosis is a common characteristic of mesothelioma and contributes to multidrug resistance, a particular problem in the clinical setting upon relapse following conventional chemotherapy. Overexpression of the pro-survival BCL-2 family of proteins which include BCL-2, BCL-XL, and MCL-1 contributes to anti-apoptotic signalling, and are highly expressed in mesothelioma. MCL-1 is one of the most commonly amplified genes at 1q21.2 in human cancer and this locus is also amplified in mesothelioma. Methods: All molecular and cell biology techniques used were carried out as described in Crawford et al 2010 or McTavish et al 2007. Results: Across a panel of mesothelioma cell lines several were demonstrated to be primed for death as silencing of Mcl-1 induced apoptosis. This addiction to Mcl-1 was dependent on the intrinsic mitochondrial death pathway as cells that stably expressed shRNA Bax and Bak rescued the addiction due to Mcl-1. Depletion of Bak but not Bax was sufficient to rescue from MCL1 knockdown. In the two Mcl-1 addicted mesothelioma cell lines studied (Ren and MSTO-211H), knockdown of the Noxa also rescued cells from MCL1 silencing induced apoptosis, however depletion of activator BH3 only proteins Bim or Bid only rescued in Ren cells. Amplification of Mcl-1 locus was not essential for addiction. Obatoclax is an inhibitor of MCL1 that is currently in clinical development. On-target activity of obatoclax was demonstrated by BAK/MCL1 co-immunoprecipitation demonstrating dissociation of Bak from Mcl-1 at early timepoints (6-12 hours). Across the panel of mesothelioma cell lines obatoclax exhibited cytotoxicity associated with reduction in cell viability and reduced clonogenic survival in both Mcl-1 addicted and non addicted cell lines. Activity in vivo was evaluated using REN and MSTO-211 xenografts. Obatoclax treatment (8mg/kg) of tumours exhibits significantly reduced tumour growth compared to control tumours. Conclusions: Obatoclax exhibits on target inhibition indicating its activity as a prosurvival BCL-2 antagonist however its activity is not solely dependent on cells being addicted to Mcl-1 for their survival. It is active in both in vitro and in vivo and clinical evaluation mesothelioma is warranted.