SMAD4 Regulates the Expression of LCK Affecting Chimeric Antigen Receptor‐T Cells Proliferation Through PI3K/Akt Signaling Pathway

ABSTRACT The proliferation of CAR‐T cells was hindered and cannot play its killing function well in solid tumors. And yet the regulatory mechanism of CAR‐T cell proliferation is not fully understood. Here, we showed that recombinant expression of CD19CAR in T cells significantly increased the basal activation level of CAR‐T cells and LCK activation. Both LCK and SMAD4 were essential for CAR‐T cells proliferation since over‐express LCK or SMAD4 significantly promotes CAR‐T cells proliferation, while knock‐down LCK or SMAD4 expression inhibited the proliferation of CAR‐T cells seriously. More cells go into apoptosis when knock‐down LCK or SMAD4 expression, and the cell cycle was arrested in G2/M or S phase, respectively. Over‐express LCK or SMAD4 significantly promotes phosphorylation of PI3K and Akt, while it was inhibited when cells were treated with PI3K and Akt inhibitors (LY294002 or MK2206). Further mechanism exploration experiments showed that SMAD4 bound on the promoter region of LCK regulating its expression. Taken together, we reported that the transcription factor SMAD4 regulated the expression of LCK and further involved in the PI3K/Akt signaling pathway to affect the proliferation of CAR‐T cells.