PROJECT SUMMARY Cancer cell genomic plasticity can enable resistance to cancer therapy for both solid tumors and hematologic malignancy. Escape from cytotoxic or molecularly targeted therapy through an inherent capacity to reprogram differentiation state or lineage has now been described following adoptive cell therapy or immune checkpoint blockade in adult epithelial tumors. Transfer of T cells genetically modified to express chimeric antigen receptors (CAR T cells) targeting the B cell surface antigen CD19 induces remission in 70-90% of patients with relapsed/refractory B cell acute lymphocytic leukemia (B-ALL) resulting in FDA-approval for this indication. However, a large fraction of those patients relapse within one year of treatment. This occurs with two main patterns, 1) early antigen-positive (CD19pos) relapse, attributed to poor CAR T expansion or lack of persistence, and 2) later antigen-negative relapse. Evasion of CD19-targeted immunotherapy can result from loss of all B lineage phenotypic markers with acquisition of stable, alternative phenotypes in MLL-rearranged (MLL-r), BCR-ABL driven, TCF3-ZNF384 and other subtypes of ALL. Remarkably, emergence of phenotypic switch can occur years after CD19-targeted immunotherapy. Understanding the mechanisms of immunotherapeutic resistance and identifying strategies to overcome these will be critical in improving remission depth and durability of response. Our proposal will address two major deficits in cancer models to identify factors contributing to relapse from immunotherapy: the lack of immune-intact model systems that recapitulate the lineage switching phenomenon observed using CD19-targeted immunotherapy and the lack of faithful mouse models recapitulating infant/childhood MLL-r B-ALL. This collaborative proposal brings together the extensive expertise of the Ernst group in the biology of MLL-r leukemia and hematopoiesis with the CAR T cell expertise of the Fry/Kohler groups to develop innovative new models systems to study evasion from CAR T cell therapy through lineage reprogramming. Our preliminary CAR T cell data employs immune-intact mouse models to illustrate that CD19neg relapse includes cells that exhibit gain of myeloid antigens and a myeloid transcriptional profile. On the pediatric B-ALL front, we develop a retroviral system to produce B-ALL that captures the inherent plasticity of MLL-r leukemias and switches to AML in vivo. Our proposal assesses the ability for both leukemia-intrinsic as well as extrinsic host- environmental components to influence escape from CAR T killing through lineage reprogramming. The findings of our studies, including the discovery of novel strategies to block lineage reprogramming have the potential to inform the development of similar approaches in other forms of cancer treated with cellular therapy and, potentially, immune checkpoint inhibitors. In addition, these studies may lead to a better understanding of lineage plasticity and the extent to ...