Abstract 4674: Restoration of Smac-mediated apoptosis in chronic lymphocytic leukemia

Abstract

Abstract Defective apoptosis is a fundamental hallmark feature of CLL biology and is a major target of cancer therapy development. IAPs (Inhibitors of apoptosis proteins) expressed at high levels inhibit apoptosis by neutralizing activities of caspases, a family of proteases that act in concert to execute apoptosis signals. Smac/DIABLO is a pro-apoptotic mitochondrial protein released into cytosol in response to apoptotic stimuli. It functions as an endogenous antagonist of IAPs and promotes apoptosis. One attractive approach to antagonize IAPs is to exploit smac-mimetics that mimic pro-apoptotic N-terminal AVPI (Ala-Val-Pro-Ile) sequence as anti-cancer agents. Smac066 is a monomer that has greater affinity for IAPs and exhibits low nanomolar potency in leukemias and solid tumor cell lines. Because CLL lymphocytes express high levels of IAPs, we hypothesized that smac066 could sensitize CLL primary cells, neutralize anti-apoptotic functions of IAPs, activate caspases and restore apoptosis. When fresh CLL lymphocytes obtained from patients with different subsets of prognostic markers were incubated with smac066 (1, 3, 10, 30, 100 µM), the IC50 was in a range of 6-10 µM at 24 hr, 3-15 µM at 48 hr and 4-9 µM at 72 hr, respectively (n=5). Though there was heterogeneity in IC50 values for individual patients, overall there was restoration of cell-death in all samples tested (two tailed-paired t test; p<0.0001; n=59). Additional trait of CLL biology is the fatal attraction toward feeder-cells present in tissue microenvironments (ME) exerting drug-resistance signals. Using validated CLL-stromal model systems such as Nktert-line, representing bone marrow-ME, CD154-HeLa cell system and Nurse Like Cells both representing lymph node-ME, we investigated if smac066 can overcome the pro-survival signals delivered by diversified-MEs. Our data infer that the perseverance presented by representative-MEs is potentially strong for attenuation by 10 µM smac066 (two tailed-paired t test; p<0.0001 for NKtert; n=25 and p<0.0001 for NLC; n=20). One explanation for this observation is that there could be mechanisms through which stromal cells concurrently succumb higher levels of IAPs that eventually could not be encountered by exogenous smac. As a proof of this notion, when CLL lymphocytes were incubated with NKTert-stromal cells there was marked increase in XIAP levels measured by immunoblotting (2-3 fold; n=4). Because smac is a positive regulator of caspases, we tested if pan-caspase inhibitor Zvad-fmk can abrogate smac-induced cell death. Consistently, 30 µM Zvad-fmk inhibited 10 µM smac-induced apoptosis in CLL lymphoyctes (two tailed paired t test; p=0.0017; n=13). Further mechanisms of action of smac-066 are under investigation. Reactivating apoptosis by neutralizing IAPs is a novel strategy to target CLL cells. The results obtained will provide pertinent information on fine-tuning the balance between the pro-survival and pro-death pathways. 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 4674. doi:1538-7445.AM2012-4674