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

Abstract

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.

Keywords: CAR-T cell; Clonal hematopoiesis of indeterminate potential (CHIP); Cytokine Release Syndrome (CRS).