Project Summary T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. Although cure rates have improved with intensified multi-agent chemotherapy, relapsed or treatment refractory disease remains very difficult to treat. Therefore, there is a great medical need for identifying novel vulnerabilities and therapeutic approaches. Targeting of the immunosuppressive tumor microenvironment has led to promising results in many solid tumors. Yet, comprehensive studies of the T-ALL microenvironment, which are necessary to characterize leukemia/immune cell interactions and identify therapeutic vulnerabilities, have not been performed. Using single cell sequencing technologies of T-ALL blasts and their microenvironmental cells, we have generated preliminary data suggesting a novel role of immune evasion in T-ALL with the potential to incorporate immunotherapy approaches into current treatment regimens to overcome T-cell exhaustion. This proposal seeks to define novel mechanisms of immune evasion in T-ALL and develop means for their therapeutic targeting. We will define mechanisms of CD8+ T-cell dysfunction by combining RNA-Seq, immune repertoire and protein analysis in single leukemia and microenvironmental cells in primary T-ALL patient samples and determine the necessary and sufficient exhaustion receptor/ligands through functional perturbation in in vitro and in vivo models of T-ALL (aim 1); We will study epigenetic and transcriptional mechanisms that regulate immune evasion in T-ALL and develop novel strategies to target T-cell dysfunction in pediatric leukemias by perturbing the epigenetic machinery (aim 2). Successful completion of this proposal is expected to uncover novel mechanisms of immune evasion in T- ALL and will lead to development of innovative therapeutic strategies for targeting T-cell exhaustion in the immune microenvironment in patients with high-risk T-ALL. The principles described in T-ALL will have a significant impact on a wide variety of malignancies.