All-Trans Retinoic Acid Enhances CS1-BCMA Dual-Target CAR-T Cells in Multiple Myeloma

Background:CS1-BCMA dual-target chimeric antigen receptor T cell (CAR-T) has exhibited superior efficacy in multiple myeloma (MM). However, there are still obstacles such as target down-regulation and barriers to immune infiltration, which results in refractory or relapse in certain patients. All-trans retinoic acid (ATRA) has been reported to upregulate the expression of CD38 and modulate the tumor microenvironment (TME), but its synergistic effect on CS1-BCMA CAR-T and potential mechanism remains unexplored. Methods:CS1-BCMA dual-target CAR-T cells were generated by lentiviral transduction of healthy donor T cells. BCMA/CS1 expression was measured by flow cytometry and quantitative real-time PCR after 24 hours coculture of MM cells and ATRA. Tumor lysis of ATRA combined with CS1-BCMA dual-target CAR-T cells was assessed in vitro and in NXG xenograft models. Cancer related fibroblast (CAF) was induced by bone marrow mesenchymal stem cell coculturing with MM cell lines in transwell inserts (0.4um) for 48 hours. Transcriptome sequencing was conducted to reveal the mechanism of ATRA sensibilizing MM cells to CAR-T therapy. Results: Experiments in vitro demonstrated thatcoculturing MM cell lines with ATRA at concentrations of 10nM-1000nM upregulated BCMA expression (MM.1S: 1.5-fold, P<0.01 at 1000nM by flow cytometry), which was verified in the NXG xenograft model. ARTA synergized CS1-BCMA dual-target CAR-T cells against MM cells both in vitro (MM.1S: P<0.01 at 1000nM for 6-hour coculturing) and in the NXG xenograft model. The addition of CAF to MM cells impaired the cytotoxicity of CAR-T by 27% (P<0.05) but its immunosuppression was reversed by ATRA (P<0.05). Transcriptome sequencing analysis of MM cells showed that ATRA upregulated the arachidonic acid metabolism pathway and down-regulated of the TGF-β pathway.. Conclusion: Our study expands on the hypothesized mechanism that ATRA promotes the cytotoxicity of CS1-BCMA dual-target CAR-T cells by upregulating the expression of BCMA and reversing the immunosuppressive effect of CAF. Sequencing demonstrates that tumor sensitivity is associated with metabolism and TGF-β pathway. Further underlying mechanisms and clinical translation need to be explored.