Abstract B013: Targeting CDC42 increases TCF1-mediated “stemness” and rejuvenates human T cells to enhance CAR-T efficacy

Chimeric Antigen Receptor (CAR) T cell therapy is an FDA-approved cancer immunotherapy which currently relies on autologous T cells from patients. Cancer such as lymphoma can accelerate the donor T cell aging, and current ex vivo expansion protocol of CAR-T cells further exacerbates T cell dysfunction by inducing T cell exhaustion and reducing stem-like T cells. CDC42, a cell polarity regulator and cell fate determinant, is an intracellular signal transducer controlling cell cytoskeleton organization, chromatin symmetry, and gene expression. Targeting CDC42 by CASIN, a CDC42 activity-specific inhibitor identified by our lab, is able to functionally rejuvenate hematopoietic stem cells and other stem cells in which CDC42-GTP is elevated upon aging. Here we aim to repress CDC42 activity by CASIN in T cells to maintain T cell stemness and counter T cell aging. In this study, human T cells from healthy donors of varying ages were isolated from the peripheral blood and cultured ex vivo in an exhaustion protocol. CDC42-GTP was found upregulated during T cell culture. Flow cytometry and scRNA-seq analyses showed that CASIN treatment remodeled the T cell composite of aged donors to resemble that of young donors. Specifically, CASIN was able to dose-dependently suppress CDC42-GTP level and increased the TCF1+ stem-like T cells while reducing the PD1+TIM3+ exhausted T cells. CASIN also increased CD4+/CD8+ T cell ratio and decreased the proportion of age-associated T cells (Taa). At the cellular level, similarly to what we reported for aged HSCs, CASIN acted on aged T cells to repolarize the depolarized T cells dose-dependently and realign the mis-localized autophagy flux to the lysosome. Molecularly, CASIN reduced p-Ser45 β-catenin (i.e. increased stability) and increased nuclear β-catenin, and significantly increased TCF1 protein level. CASIN treatment also markedly changed several histone methylation and acetylation markers indictive of epigenetic alterations, and remodeled chromatin accessibility at key immune regulatory loci including TCF7, HAVCR2, IFNG, and IL2, providing strong evidence that CASIN regulates the epigenetic landscape the cultured T cells. RNA-seq analyses saw a drastic reduction by CASIN treatment of gene sets from a series of inflammaging related pathways including that of IL2-STAT5, IL6-JAK-STAT3, TGF-β, and TNF-α. Functionally, we have carried out in vitro and in vivo tumor cell killing assays by the CAR-T cells with or without CASIN treatment and found that CASIN treated anti-CD19 CAR-T cells demonstrated an enhanced killing ability of the B-ALL Nalm1 and Nalm6 cells as well as MV411-CD19 AML cells in culture and significantly increased the activity in suppressing leukemia progression in Nalm6 xenograft NSG mice. Our work identifies CASIN-mediated pharmacological intervention as a unique approach in preventing T cell exhaustion and reverting T cell aging through CDC42-cell polarity mediated transcriptional and epigenetic remodeling, providing a promising strategy to improve CAR-T cell therapy efficacy. Mingjun Cai, Xin Duan, Ryan Jorgensen, Mark Wunderlich, Fukun Guo, Yi Zheng. Targeting CDC42 increases TCF1-mediated “stemness” and rejuvenates human T cells to enhance CAR-T efficacy [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B013.