Abstract

The bone marrow (BM) niche serves as a critical protective environment for leukemia cells, particularly chemo-resistant leukemia cells, and plays a central role in driving therapeutic resistance and disease relapse in acute myeloid leukemia (AML). This specialized microenvironment not only promotes leukemia cell survival, but also inhibits T cell infiltration, which serves as a major obstacle to the effectiveness of CAR-T therapy in myeloid malignancies. To overcome this limitation, we targeted Rac1 GTPase, a central regulator of cytoskeletal dynamics that controls membrane protrusion and migration, by engineering primary human T cells and CD33 CAR-T cells to express constitutively active Rac1 (Rac1V12). Our results demonstrated that active Rac1 enhanced the migration of T cells and CD33 CAR-T cells and promoted their residence in the BM in vivo. Furthermore, CD33 CAR-T cells expressing Rac1V12 displayed enhanced cytotoxicity against leukemia cells in vitro, as demonstrated by transwell migration-dependent killing assays. Crucially, these engineered CAR-T cells achieved superior robust suppression of leukemia in vivo and significantly prolonged survival in xenograft models. Mechanistically, Rac1V12 CD33 CAR-T cells in the BM demonstrated enhanced immunological memory phenotype and lower tonic signaling, a combination that promotes T cell persistence and enhances anti-tumor efficacy in vivo. Our data suggest that active Rac1-engineered CD33 CAR-T cells represent a novel strategy for targeting BM leukemia cells, with the potential to eradicate AML cells. Graphical Active Rac1 augments the migration and trafficking of T cells and CAR-T cells to the bone marrow. Rac1V12-engineered CAR-T cells displayed enhanced in vitro cytotoxicity against leukemia cells and mediated superior leukemia eradication in xenograft models. Mechanistically, Rac1V12-CAR-T cells localized to the BM exhibited an enhanced immunological memory phenotype.