Abstract / Summary
Abstract Background The therapeutic efficacy of chimeric antigen receptor T (CAR-T) cells targeting CD19 in chronic lymphocytic leukemia (CLL) treatment can be limited by target cell antigen loss. This study aims to elucidate how the intrinsic NOTCH1 signaling activity in CLL cells modulates their sensitivity to CD19-targeted CAR-T cell therapy. Methods In this study, we modulated NOTCH1 signaling in MEC-1 cells through gene knockout and pharmacological inhibition, and employed molecular and cellular biology techniques to elucidate the mechanism by which NOTCH1 regulates CD19 degradation. On this basis, we evaluated the effect of combining NOTCH1 inhibition on enhancing the efficacy of CD19 CAR-T therapy both in vitro and in vivo. Results NOTCH1 deficiency in MEC-1 cells profoundly enhanced potency of CD19 CAR-T cell-mediated killing. Mechanistically, upon CAR-T cell engagement, NOTCH1 signaling in target cells promoted the rapid lysosomal degradation of the CD19 antigen. This was achieved through its downstream effector HEY1, which transcriptionally represses RAB31. The downregulation of RAB31, a negative regulator of the late endosome/lysosome transporter RAB7, was essential for facilitating CD19 degradation. Both NOTCH1-deficient and pharmacologically inhibited CLL cells exhibited impaired CD19 degradation, higher surface antigen retention, and increased susceptibility to CAR-T cell killing, correlating with elevated CAR-T cell activation and effector molecule secretion. Conclusions This study identifies NOTCH1 signaling as a key negative regulator of CD19 stability in CLL, as this pathway promotes the lysosomal degradation of CD19 following its internalization. Therefore, targeting this pathway represents a potential strategy to enhance the efficacy of CD19 CAR-T therapy in the treatment of CLL.