The NOTCH-FOXM1 Axis Plays a Key Role in Mitochondrial Biogenesis in the Induction of Human Stem Cell Memory-like CAR-T Cells.

Recent studies have shown that stem cell memory T (T-SCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CART) cells. We previously reported that both human and murineactivated T cells are converted into stem cell memory-like T (iT(SCM)) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iT(SCM) reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, "CAR-iT(SCM)" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iT(SCM) formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iT(SCM) differentiation. Like NOTCH-induced CAR-iT(SCM) cells, FOXM1-induced CAR-iT(SCM) cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCHor FOXM1-driven CAR-iT(SCM) formation is an effective strategy for improving cancer immunotherapy. Significance: Manipulation of signaling and metabolic pathways important for directing production of stem cellmemory-like T cells may enable development of improved CAR-T cells.