Abstract A019: CRISPR-based investigation of CHIP mutations reveals their role in CAR-T therapy-related inflammatory toxicities

Abstract Background: Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations (e.g. DNMT3A, TET2, and ASXL1I) in hematopoietic stem cells, and linked to age-related clonal expansions and inflammatory states. We previously showed that patients with CHIP receiving CAR-T cell therapy have a four-fold risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP (Transplant Cell Ther 2024). This prompted us to investigate the mechanisms through which CHIP mutations could contribute to CAR-T-related toxicities. Methods: We employed CRISPR-based gene editing to target CHIP-associated genes—DNMT3A, TET2, and ASXL1—in M1-like macrophages, which were differentiated from human monocytes in the presence of 20 ng/mL of GM-CSF. Gene perturbation was achieved with cell-penetrating phosphorothioated single-stranded DNA oligonucleotides to deliver the CRISPR-Cas9 and gRNA complex (PS-Cas9/gRNA). Editing was performed using multiple gRNAs for each gene: DNMT3A (2 gRNAs), TET2 (3 gRNAs), and ASXL1 (2 gRNAs), and decreased protein expression was confirmed post-editing. We co-cultured CS1 CAR-T cells targeting multiple myeloma (MM), MM.1S cells, and edited autologous macrophages at a 1:1:0.1 ratio for 24 hours to assess the inflammatory response. Cytokine secretion was quantified using the LEGENDplex™ Human Inflammation Panel 1 (13-plex). Results: Compared to non-edited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages consistently secreted significantly higher levels of IL-6 (642, 920, 1338 pg/ml vs. 325 pg/ml respectively, P<0.05-0.001), MCP-1(90, 122, 151 pg/ml vs. 55 pg/ml, respectively, P<0.01-0.001), TNFα (2015, 1722, 1874 pg/ml vs. 1043 pg/ml, respectively P:ns - < 0.001), IL-1β (75, 99, 146 pg/ml vs. 38 pg/ml, respectively, P<0.05-0.001), and IL-8 (21, 19, 22 ng/ml vs. 15 ng/ml, respectively, P:ns- <0.001). Conclusions: These findings suggest CHIP mutations in human macrophages may exacerbate the inflammatory response in CAR-T therapy, highlighting a potential biomarker for stratifying patients at higher risk for toxicities such as CRS and guiding more personalized prophylactic interventions. Citation Format: Xiuli Wang, Scott R. Goldsmith, June-Wha Rhee, Stephen J. Forman, Chunyan Zhang, Saro Armenian. CRISPR-based investigation of CHIP mutations reveals their role in CAR-T therapy-related inflammatory toxicities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A019.