PROJECT SUMMARY Over 60% of T cell acute lymphoblastic leukemias (T-ALLs) harbor mutations in the NOTCH1 signaling pathway, making Notch activation the most common oncogenic pathway in T-ALL. However, pan-Notch inhibitors are associated with dose-limiting toxicities, primarily gastrointestinal. We previously showed that the transcription factor ETS1 is essential for Notch-activated T-ALL survival. ETS1 inhibition impairs T-ALL survival and downregulates major oncogenic Notch target genes. Further, ETS1 deletion has limited toxicities in murine models. These findings suggest targeting ETS1 might have strong anti-leukemic effects with more favorable toxicity profile than Notch inhibitors. While ETS1 and NOTCH1 activate many overlapping pathways, the cis- regulatory elements downstream of ETS1 are less well defined and may play a more essential role than NOTCH1 for activation of some T-ALL enhancers. I identified one such enhancer in which ETS1 may play a more essential role than NOTCH1. Here, ETS1 is required to activate expression of MYB, a critical T-ALL oncogene (tentatively named “E-Me” for ETS1-dependent MYB Enhancer). My preliminary data shows that ETS1 is required for E-Me activation and MYB expression, and E-Me repression in human T-ALL cell lines inhibits MYB expression and cell survival. Based on these preliminary studies, I hypothesize that ETS1 drives T-ALL survival by activating the E-Me and other essential regulatory elements for T-ALL oncogenes. I will investigate this hypothesis through two aims. In my first aim, I will determine the consequences of E-Me blockade in Notch-activated T-ALL survival in vivo using two murine models. In my second aim, I will further define the cis-regulatory elements and oncogenes downstream of ETS1 in T-ALL. The enhancers and transcription factor dependencies that regulate oncogene expression in T-ALL arecurrently poorly understood, raising barriers to targeted therapeutic strategies. My proposal will help close this gap in knowledge. Finally, the overall impact of this project will be to define the role of ETS1 as a driver of essential T-ALL oncogenic pathways and enable the discovery of other essential transcription factors in T-ALL.