CRISPR Knockdown of CHIP Genes in Macrophages Drives Increased Production of Inflammatory Cytokines in CAR-T Cell Therapy.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations in leukemogenic genes (e.g., DNMT3A, TET2, and ASXL1) in hematopoietic stem cells and is linked to age-related clonal expansion and inflammation. We previously showed that patients with CHIP receiving chimeric antigen receptor (CAR)-T cell therapy have a higher risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP. Here, we investigated the mechanisms through which CHIP mutations can contribute to CAR-T-related toxicities. To model the interaction of CAR-T cell-mediated inflammatory response and macrophages harboring CHIP gene mutations, we employed CRISPR-based gene editing to knock down three key CHIP-associated genes (DNMT3A, TET2, and ASXL1) in human macrophages, using multiple guide RNAs for each. We co-cultured gene-edited macrophages with autologous CS1 and BCMA CAR-T cells and multiple myeloma (MM) tumor cells. Compared to nonedited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages secreted significantly higher levels of pro-inflammatory cytokines characteristic of CRS, including IL-6, MCP-1, and IL-1β (P < .05 to .01). These results suggest that the presence of CHIP mutations in human macrophages may exacerbate the inflammatory response during CAR-T therapy. This study highlights CHIP mutations as potential biomarkers for identifying patients at high risk of developing CRS and for guiding personalized prophylactic interventions in MM CAR-T therapy.