PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is one of the most common hematologic malignancies, representing a diverse collection of complex diseases. After 30-40 years without change of treatment strategy, the past 2 years have seen several drug approvals, including the most recent approval for the BCL2 inhibitor, venetoclax, used in combination with hypomethylating agents. While response rates of 60-80% with this combination are encouraging, the 1-year survival rate is still only at 30-40%. For the past decade, we have executed a functional genomics platform applied directly to primary samples from patients with AML and other hematologic malignancies. Using this platform, we have collectively studied over 2,500 primary patient specimens, and we have included a series of venetoclax combinations on this platform for the past five years. From this dataset, we have identified several novel venetoclax combinations with better activity than venetoclax plus hypomethylating agents, and we have started a clinical trial for one of the most promising of these combinations, venetoclax with the JAK inhibitor ruxolitinib. For this project, our long-term goals are to optimize and translate the most effective venetoclax drug combinations into the clinic for patients with AML. Our immediate goals are to understand the specific biology driving venetoclax combination synergy and identify biomarkers and mechanisms of response. Based on the central hypothesis that venetoclax combinations exhibit patterns and mechanisms of sensitivity and resistance that are specific to cell differentiation states and transcriptional programs. To accomplish these goals, 3 Aims are proposed: 1) Cell differentiation state as a mechanism of drug combination sensitivity and resistance – We will perform analytics of cell differentiation state in our large patient sample dataset as well as laboratory models of forced differentiation in cell lines and patient samples. 2) Synthetic lethality as a guide to mechanisms of drug combination sensitivity and resistance – Our preliminary data from genome-wide CRISPR/Cas screens point to specific candidate genes that impact on mediators of venetoclax combination activity. These candidates will be explored with genetic and pharmacologic perturbations. 3) Clinical validation of biomarkers and mechanisms of sensitivity and resistance – We have opened a clinical trial testing venetoclax combined with ruxolitinib in AML. We will have access to longitudinal specimens from patients on this trial. We will perform ex vivo drug testing on these trial specimens coupled with transcriptional and proteomic studies to evaluate the ability of each data type to predict clinical responses. Cumulatively, we expect these innovative analyses to have a major impact on our understanding of AML biology, with successful clinical translation of new, more effective drug combination strategies.