Abstract 3325: Role of smac-mimetic in restoring apoptosis in chronic lymphocytic leukemia .

Abstract

Abstract Defective apoptosis is the fundamental hallmark feature of CLL biology. Inhibitors of apoptosis proteins (IAPs) inhibit apoptosis by neutralizing the activities of caspases, a family of proteases that act in concert to execute apoptosis signals. SMAC (Second Mitochondria Derived Activator of Caspases) released from mitochondria in response to apoptotic stimuli is an endogenous antagonist of IAP that promote apoptosis by neutralizing the anti-apoptotic properties of IAPs. The IAP-binding tetrapeptide motifs from SMAC (AVPI; Ala-Val-Pro-Ile) and from caspase-9 (ATPF; Ala-Thr-Pro-Phe) both bind to the same conserved surface groove on the BIR3 domain of XIAP, allowing SMAC to remove the XIAP mediated inhibition of caspase-9. This provided rationale to use SMAC mimetics to reverse the IAP-mediated inhibition of caspases. Monomeric smac mimetic (smac066) demonstrated low nM potency for MDA-MB231 cell line and low μM potency for HL-60 and PC-3 cells (Seneci et al., 2009). Using CLL primary cells, we show that IAPs (XIAP, cIAP1 and cIAP2) are expressed at high levels in CLL lymphocytes (n=9) compared to normal lymphocytes (n=7), while SMAC is present at similar levels suggesting that though SMAC is present, it is important that the caspases are unbound from IAPs to execute apoptosis. CLL lymphocytes treated with smac066 (10 μM; 24 hr) demonstrated a significant activation of apoptosis (n=37; p=0.0001; median 22%; range 1-74%) with IC50 values of 8 μM, 6 μM, and 6 μM at 24, 48 and 72 hr respectively. The correlative analysis between the sensitization and respective prognostic markers demonstrate that samples with poor prognostic markers such as 17p deletion (n=3) (tumor suppressor protein p53 locus), 11q deletion (n=3) (DNA damage response gene, ATM locus), unmutated IGVH gene status (n=7), trisomy12 (n=6) and number of prior treatments (n=4) required higher concentrations of drug compared to patients with no cytogenetic abnormalities. Alternatively, samples that had 13q deletion (n=6) (locus of miRs 15a and 16 that regulate the Mcl-1 and Bcl-2 expression) were significantly sensitive to smac-mediated apoptosis. The mechanism involved in restoring the apoptosis by smac mimetic in CLL cells was associated with decrease in IAPs (5 μM; n=16; 24 hr; p=0.003) such as XIAP and cIAP2, but not cIAP1. PARP cleavage (a substrate of caspases), cleavages of procaspase 8 and 9 (initiator caspases) and procaspase 3 (executioner caspase) suggested that both extrinsic and intrinsic pathways are activated by smac066. The immuno-precipitation studies showed that in untreated cells, XIAP and caspase-3 are in association with each other. However, upon treatment with smac mimetic caspase-3 dissociated significantly from the XIAP. The level of SMAC bound to XIAP increased with smac mimetic. In summary, smac066 disrupts the IAP-mediated inhibition of apoptosis in CLL primary cells; thereby should be combined and evaluated with other chemotherapeutic agents. Citation Format: Kumudha Balakrishnan, Min Fu, Francesco Onida, William G. Wierda, Michael Keating, Varsha Gandhi. Role of smac-mimetic in restoring apoptosis in chronic lymphocytic leukemia . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2013-3325