Antagonizing IAPs by SMAC Mimetic TL32711 Induces Apoptosis in AML Cells Including AML Stem/Progenitor Cells Alone and in Combination with Chemotherapy

Abstract 66

The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs), including cIAP1, cIAP2, and XIAP, is antagonized by SMAC (second mitochondrial-derived activator of caspases). XIAP directly binds and inhibits caspase-9 and caspase-3 and suppresses both mitochondrion-mediated intrinsic and death receptor-mediated extrinsic apoptosis pathways, while the cIAPs are components of the cytoplasmic signaling complex containing members of TNF receptor associated factors and suppress death receptor/caspase-8 mediated extrinsic pathway activation. SMAC mimetics are a new class of anti-cancer agents that induce rapid degradation of cIAP1, relieve XIAP-mediated caspase repression, and promote TRAIL or TNFa-dependent apoptosis in various malignant cell types.

To assess the therapeutic potential of SMAC mimetics in AML, we determined the protein levels of cIAP1 and XIAP, which are targets of SMAC mimetics, and caspase-8, the initiator caspase of the death receptor pathway by reverse phase protein array in blasts obtained from 511 newly diagnosed AML patients and in CD34⁺38⁻ stem/progenitor cells isolated from blasts of these patients. We found that all three proteins were expressed in AML blasts. Importantly, we observed that the protein levels of cIAP1 and caspase-8 in CD34⁺38⁻ AML stem/progenitor cells were significantly higher than those in bulk AML cells (P < 0.001).

TL32711 (TL) is a highly potent and well-tolerated SMAC mimetic in clinical development that promoted rapid degradation of cIAP1 at low nM concentrations and induced pronounced apoptosis in AML cell lines in the presence of TNFα. Cell death was enhanced in the presence of TRAIL, confirming activation of the death receptor pathway as a significant mechanism of apoptosis induction. Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to TL, further supporting the critical role of caspase-8 in TL-mediated cell death. TL synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy such as Ara-C, clofarabine, and demethylating agents decitabine and 5-azacytidine (5-AC). Mechanistic studies showed that decitabine and 5-AC increased and activated caspase-8, decreased cFLIP, and induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cell types. In addition, TL had single agent activity against blasts from primary AML samples with no toxicity in CD34⁺ cells from normal bone marrows at doses effective against AML cells. Importantly, TL not only induced apoptosis in bulk AML blast but also in CD34⁺38⁻ AML stem/progenitor cells.

Collectively, we showed that cIAP1 and caspase-8 are overexpressed in AML stem/progenitor cells and that inhibition of IAPs by the novel SMAC mimetic TL32711 synergistically enhances drug induced-death of AML cells and also has the potential to eliminate AML stem/progenitor cells.