Acute myeloid leukemia (AML) is a devastating malignancy with dismal outcomes for patients, particularly in the elderly where most cases occur. The recent approval of the BCL2 inhibitor venetoclax (Ven) in combination with a hypomethylating agent (HMA; such as azacytidine/Aza) for the treatment of AML in elderly/unfit adults has substantially improved outcomes for these patients. Still, about a third of patients treated with Ven/HMA exhibit poor responses to therapy, and most patients who do respond will eventually relapse with Ven/HMA resistant disease. Moreover, patients whose AML contains mutations in the TP53 gene are refractory to Ven/HMA and all other forms of chemotherapy. The central premise of this project is that intra-patient heterogeneity of the leukemia initiating cell (LIC) population is a key driver of therapy resistance/failure. At least two distinct forms of LSCs can be found in AML patients, one with a more primitive phenotype (p-LIC) and one with a monocytic phenotype (m-LIC). While Ven-based therapies are an effective means to eradicate the p-LSCs in newly diagnosed AML patients, m-LIC are resistant to Ven/Aza and can mediate relapse. The goal of this work is to identify and characterize distinct LIC subtypes and their vulnerabilities to design more effective and more durable therapies. We have further shown that m-LIC exhibit upregulation of Inhibitor of Apoptosis Proteins (IAP) and that IAP inhibition using drugs called SMAC mimetics (SMACm) potently sensitizes AML cells to Ven/Aza- induced apoptosis in vitro and in vivo, particularly for the monocytic subpopulation highly resistant to Ven/Aza alone. Intriguingly, SMACm therapy is also highly effective at eradicating TP53 mutant/deleted AML cells, which are generally refractory to currently available therapies. Thus, varying subtypes of AML LIC are vulnerable to targeting by distinct strategies. Mechanistically, we show that SMACm in combination with Ven/Aza induces noncanonical NF-B signaling and high levels of TNF, which causes cell death by apoptosis. Here, we propose to characterize the molecular phenotypes and therapeutic vulnerabilities of different LIC subpopulations in order to design more effective therapies. In the first aim, we will characterize the pathways and molecular mechanisms that mediate differential vulnerabilities of resistant LIC subtypes, with a focus on monocytic and TP53 mutant subtypes. The second aim will leverage in vitro and in vivo models to gain a thorough understanding of the clonal heterogeneity of different primary AML specimens, the phenotypes of the various subclones, and how distinct LIC subclones differentially respond to Ven/Aza and SMACm therapy. Finally, in Aim 3, a clinical trial will assess the safety and efficacy of combining a SMACm drug, tolinapant, with Ven/Aza in the relapsed/refractory AML setting. Samples from this trial will be used to determine whether and how tolinapant sensitizes LICs to Ven/Aza therapy, thereby inducin...