Reactivation of Smac-mediated apoptosis in chronic lymphocytic leukemia cells: mechanistic studies of Smac mimetic
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Kumudha Balakrishnan1,3, Min Fu3, Francesco Onida2, William G. Wierda3, Michael J. Keating3 and Varsha Gandhi1,3
1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
2 Department of Hematology Unit, Fondazione IRCCS Ospedale Maggiore Policlinico, University of Milan, Milan, Italy
3 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
Kumudha Balakrishnan, email:
Keywords: CLL, Smac mimetic, IAPs, apoptosis, XIAP
Received: December 17, 2015 Accepted: February 28, 2016 Published: March 29, 2016
Dysfunctional apoptotic machinery is a hallmark feature of chronic lymphocytic leukemia (CLL). Accordingly, targeting apoptosis regulators has been proven a rational approach for CLL treatment. We show that CLL lymphocytes express high levels of XIAP, cIAP1, and cIAP2 compared to normal lymphocytes. Smac mimetic, Smac066, designed to bind to BIR3-domain of IAPs, induce apoptosis in primary CLL cells (n=71; p<0.0001), irrespective of prognostic markers. Apoptosis was mediated by diminished levels of IAPs (XIAP-p=0.02; cIAP-p<0.0001) and increased activation of caspases-8,-9,-3. The caspase-cleavage was in direct association with the levels of apoptosis (r2=0.8 for caspases-8,-9,-3). Correlative analysis revealed a direct relationship between reduction in IAPs and degree of apoptosis (r2=0.6 (XIAP); 0.5 (cIAP2)). There was a strong association between apoptosis, IAP-degradation, and concurrent caspase-activation. Pan-caspase inhibitor Z-Vad-fmk reversed the degradation of Mcl-1, but not IAPs suggesting that smac066 is selective to IAPs, however, Mcl-1 degradation is through caspase-mediated cleavage. Immunoprecipitation experiments revealed physical interaction between caspase-3 and XIAP that was disrupted by smac066. Importantly, XIAP and cIAP2 were markedly induced in bone-marrow and lymph-node microenvironments, providing a basis for IAP antagonists as anti-tumor agents in CLL. Smac066 synergized with ABT-737, revealing a mechanistic rationale to jointly target BH3 and BIR3 domains.
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