Synergistic Targeting of AML Stem/Progenitor Cells With IAP Antagonist Birinapant and Demethylating Agents

Activation of the intrinsic apoptosis pathway by chemotherapeutic agents is the primary treatment strategy for patients with acute myeloid leukemia (AML). Nevertheless, most patients ultimately relapse because of the persistence of disease-driving AML stem/progenitor cells that are refractory to chemotherapy (1,2). Inhibitor of apoptosis (IAP) proteins are important for regulating cell survival. They are expressed in various malignant cells, and this corresponds with poor treatment outcomes (3,4). IAP proteins have only recently received attention as therapeutic targets. We have previously identified survivin and the X-linked inhibitor of apoptosis protein (XIAP) as potential targets for AML therapy (5–7). Unfortunately, only antisense oligonucleotide (ASO) for XIAP is available. Interestingly, although the initial results seemed promising (8), XIAP ASO trials demonstrated little or no impact on cancer progression (9). However, we demonstrated that XIAP ASO induced apoptosis preferentially in AML stem/progenitor cells (10). IAP proteins also modulate NFκB activity, which is constitutively active in AML cells (11), which can inhibit the extrinsic apoptosis pathway (12–14). IAP proteins are antagonized by second mitochondrial-derived activator of caspases (SMAC) proteins (15,16). SMAC mimetics developed previously induce degradation of IAP proteins, especially baculoviral IAP repeat-containing protein 2 (cIAP1), promote death receptor ligand-induced caspase-8–mediated apoptosis in malignant cells (13,17,18), and are less toxic to normal cells (19). Birinapant, a novel bivalent SMAC mimetic with high affinity for IAP proteins, has excellent pharmacokinetic/pharmacodynamics properties, and it is in clinical trials both as a single agent and in combination agent chemotherapy (20,21). However, the expression of cIAP1 (the main target of SMAC mimetics), caspase-8 (the target of cIAP1), SMAC (the cellular antagonist of IAPs), and the antileukemic effectiveness of birinapant against AML cells and AML stem/progenitors have not been investigated. Currently and historically, antileukemia drugs are tested without consideration of the microenvironment in which leukemic cells reside. The bone marrow (BM) microenvironment plays critical roles in chemoresistance (22–24). AML cells, in particular AML stem/progenitor cells, are in close contact with mesenchymal stromal cells (MSCs) in a hypoxic environment (25), which makes them resistant to chemotherapy not only because of their cell-intrinsic mechanisms but also because of microenvironmental factors associated with low oxygen tension (eg, those associated with chemotherapeutic agent–induced reactive oxygen species production). In this study, we first examined the expression of cIAP1, caspase-8, and SMAC in AML blasts, AML stem/progenitor cells, and normal CD34+ cells by reverse-phase protein array. We then evaluated the therapeutic potential of birinapant and its combinations with demethylating agents in AML cells under physiologically relevant conditions and in an in vivo AML xenograft mouse model. Here we report that birinapant is effective, alone and in combination with demethylating agents, against AML cells and AML stem/progenitor cells, which we believe are clinically relevant findings that will greatly impact the therapy of AML.