Abstract 2278: Smac mimetic primes FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis and overcomes apoptosis resistance

Abstract

Abstract Evasion of apoptosis contributes to treatment resistance, one of the major, yet unresolved obstacles in oncology. Searching for new strategies to bypass apoptosis resistance we investigated the potential of Smac mimetic in acute lymphoblastic leukemia (ALL) cells deficient in FADD or caspase-8. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant leukemia cells for TNFα-induced necroptosis as an alternative mode of cell death. The interaction of Smac mimetic and TNFα occurs is highly synergistic as calculated by combination index (CI <0.1). Kinetic analysis reveals that Smac mimetic and TNFα cause cell death rapidly within few hours. Interestingly, Smac mimetic- and TNFα-mediated cell death in FADD-deficient cells occurs without characteristic features of apoptosis, namely without caspase activation or DNA fragmentation, pointing to non-apoptotic cell death. In sharp contrast, Smac mimetic and TNFα trigger activation of caspase-8, -9 and -3 and DNA fragmentation in apoptosis-proficient wildtype cells that express FADD and caspase-8. Consistently, the caspase inhibitor zVAD.fmk protects wildtype cells against Smac mimetic- and TNFα-triggered cell death, whereas zVAD.fmk fails to block cell death in FADD-deficient cells. Furthermore, production of reactive oxygen species (ROS) precedes Smac mimetic- and TNFα-induced cell death in FADD-deficient cells. In line with the notion that ROS contributes to cell death induction, the addition of ROS scavengers (i.e. butylated hydroxyanisole (BHA) and trolox) or the addition of the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduce cell death upon combination treatment. Moreover, Smac mimetic- and TNFα-triggered cell death is accompanied by enhanced RIP1 kinase activity in FADD-deficient cells. Of note, necrostatin-1, a RIP1 kinase inhibitor, reduces Smac mimetic- and TNFα-induced ROS generation and completely abolishes cell death in FADD-deficient cells, in accordance with necroptotic cell death. Importantly, Smac mimetic also sensitizes apoptosis-resistant, primary leukemic blasts derived from patients with ALL to TNFα-induced necroptosis, underscoring the clinical relevance of these findings. In conclusion, Smac mimetic primes leukemia cells to TNFα-induced apoptosis or necroptosis in a context-dependent manner. In apoptosis-proficient cells which express both FADD and caspase-8, Smac mimetic enhances TNFα-induced, caspase-dependent apoptosis. However in apoptosis-resistant cells lacking either FADD or caspase-8, Smac mimetic sensitizes cells for TNFα-initiated necroptosis that critically depends on RIP1 and ROS production. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance of cancers. 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 2278. doi:1538-7445.AM2012-2278