RHOA Loss of Function Impairs the IFNγ Response and Promotes CD19 Antigen Escape to Drive CAR-T Resistance in Diffuse Large B-cell Lymphoma

Abstract

CD19-directed chimeric antigen receptor (CAR)-T cells are breakthrough therapies for aggressive B-cell lymphomas, but less than half of patients achieve durable responses. We previously showed through whole-genome sequencing of tumors from CAR-T-treated patients that deletions of RHOA (3p21.31) are enriched in cases progressing after treatment. RHOAs roles in resistance and pathogenesis are poorly defined, despite loss-of-function alterations that occur in [~]20% of newly diagnosed large B-cell lymphoma (LBCL) cases. We created RHOA-deficient LBCL systems and confirmed cell-intrinsic loss of response to CAR-19 in vitro and in vivo driven by CD19 downregulation. Impact on CD19, however, was variable and would not explain selection for RHOA deletion in newly diagnosed cases. We therefore created RHOA-deficient tumors in immunocompetent mice and found remarkable correlation with dysfunctional lymphoma microenvironment (LME) signatures in CAR-19 resistant patients. These LMEs are marked by a type 1-like immune infiltrate with terminally exhausted CD8 T cells, Th1-like CD4 cytotoxic lymphocytes (CTLs), and increased production of interferon gamma (IFN{gamma}). RHOA-deficient tumor cells themselves have significantly impaired IFN{gamma} responses, providing resistance to CD8 T cell clearance by way of diminished induction of major histocompatibility complex class I (MHC-I). These findings support a model that depletion of healthy effector populations by RHOA-deficient lymphoma is a key driver of immune dysfunction thwarting CAR-19 clinical responses. Overall, we describe for the first time how a single-gene alteration found recurrently in CAR-19-resistant LBCL contributes to treatment failures.