PROJECT SUMMARY This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA-21-034. Acute myeloid leukemia (AML) is among the deadliest blood cancers with over 10,000 patients dying annually in the U.S. AML displays notorious genetic heterogeneity with thousands of mutations described across AML patient tumors to date. AML has benefited from the rise of targeted therapies although clinical impact of individual targeted agents has been modest. In AML, impressive initial response rates of 60- 80% have been observed in elderly patients using the combination of venetoclax, a BCL2 inhibitor, and hypomethylating agents. Yet, survival at one year is only 30-40%, suggesting more to learn about the efficacy of this combination. Novel ex vivo drug screening platforms have identified additional venetoclax combinations. In particular, venetoclax with ruxolitinib, a JAK tyrosine kinase inhibitor, is a promising combination therapy. Based on this preclinical data, a novel clinical trial has been initiated for patients with relapsed or refractory AML. Cancer drug combination decisions are primarily made on the basis of mutational heterogeneity, yet evidence of non-genetic drug resistance among isogenic cells is mounting, particularly with single cell analysis. Despite advances in single cell technologies, there are currently no strategies for making drug combination decisions that utilize single cell platforms to explicitly address intratumoral heterogeneity. Leveraging the strengths of CyTOF and addressing the need for analysis approaches, we developed a novel algorithm (DRUG-NEM) that analyzes single-cell, single-drug perturbation responses on individual leukemia cells to identify optimized drug combination strategies for the individual patient. Using mass cytometry analysis with focus on AML phenotype, signaling and metabolism, we will determine ex vivo response to single agent venetoclax or ruxolitinib or the combination. Using the single agent treatment data, we will use DRUG-NEM to predict response to the combination and compare to patient samples obtained on trial after staring combination therapy. If predictions are accurate, it provides proof of concept for using single-agent drug data to inform combination therapy, thus a more efficient and practical approach to study future combination treatments and will inform sensitivity or resistance to venetoclax in combination with ruxolitinib in patients with AML.