Dissecting the impact of genomic variants on hallmarks of T cell anti-tumor activity

PROJECT SUMMARY/ABSTRACT While a fraction of patients with melanomas and leukemias experience durable response to T cell- based therapies, including adoptive transfer of tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor T (CAR-T) cells, respectively, a majority do not. Emerging studies have found that sporadically occurring or purposefully engineered single-nucleotide variants (SNVs) in T cell signaling genes have the potential to powerfully modulate cellular therapies. To this end, our group established novel methods for highly efficient screening of single-nucleotide variants in primary human T cells using CRISPR base editing. Using these methods, we identified a series of SNVs in genes encoding PI-3-kinase subunits (PIK3CD and PIK3R1), which enhance multiple hallmarks of T cell activity, as well as several SNVs that promote long-term in vivo persistence in humanized mice, including in PPP3CA (encoding the calmodulin-binding catalytic subunit of calcineurin). In multiple T cell immunotherapy models, including tumor-specific TCR-engineered T cells against melanoma and CAR-T cells against leukemia, engineering T cells with PIK3CD SNVs enhanced antigen-dependent PI3K pathway signaling, proinflammatory cytokine production, and in vitro tumor cell killing. Moreover, these SNVs enhanced tumor cell killing in melanoma models of CD58 loss, a mechanism of immunotherapy resistance shared across several cancer types. This proposal seeks to perform in-depth mechanistic and in vivo functional studies of these SNVs in a panel of immunotherapy models. In Aim 1, we propose to define the mechanisms by which PIK3CD SNVs modulate T cell activity and evaluate the in vivo impact of these SNVs in several murine xenograft immunotherapy models, including NY-ESO-1 specific T cells targeting melanoma and CD19-CAR T cells targeting leukemia. In Aim 2, we propose to use these models to perform mechanistic and in vivo efficacy studies of PIK3CD SNVs in the setting of tumor CD58 loss, a shared mechanism of therapy resistance in melanoma and leukemia. In Aim 3, we will perform rational combinatorial base editing studies in these in vivo models, coupling variants in PIK3CD which promote anti-tumor activity with variants in PPP3CA which enhance in vivo persistence, with the goal of further enhancing the durability of tumor clearance. These aims offer a powerful breadth and depth of study of promising SNVs which may enhance immunotherapies, spanning from mechanistic T cell biology to translational xenograft models. With the guidance from exceptional mentors and collaborators and access to the unparalleled scientific research environment at Columbia University’s Irving Medical Center, this project will prepare me for a successful career as a physician-scientist in the field of cellular immunotherapy.