Abstract 4862: Targeting <i>fli1</i> improves antitumor response of CD8 T cells by promoting their activation and limiting exhaustion

Abstract Fli-1 (Friend leukemia integration 1) is a transcription factor critical to cell proliferation and differentiation, and its overexpression has been identified in various cancers, including leukemia, Ewing sarcoma, and certain breast and prostate cancers. Emerging evidence links Fli-1 to T cell activation and exhaustion. In our recent study, we demonstrated that Fli-1 plays a key role in regulating the gene transcription for CD8 T-cell activation and their differentiation into effector and memory subsets after allogeneic hematopoietic cell transplantation. We hypothesize that targeting fli1 in T cells will potentially improve the effectiveness of cellular immunotherapy by enhancing the persistence and function of CD8 T cells. To study how Fli-1 regulates CD8 T cell-mediated antigen-specific response, we utilized OT-I and Pmel TCR transgenic mice in which T cells are specific to ovalbumin (OVA) and melanoma-associated gp-100 antigen, specifically respectively. We generated fli1-deficient OT-I and Pmel mice by crossing them with fli1fl/fl CD4cre micestrain. Upon stimulation of OT-I T cells with OVA SIINFEKL peptide, fli1-/- OT-I T cells had increased cytolytic activity against peptide-pulsed tumor targets compared to wild-type (WT) OT-I T cells in vitro. Additionally, we observed enhanced cytokine secretion and reduced exhaustion in fli1-/- OT-I T cells after repetitive activation stimulation by OVA peptide in vitro. Following adoptive cell therapy in vivo, fli1-/- OT-I T cells had significantly improved ability to control MC-38OVA growth as compared with WT counterpart cells. Deficiency of Fli-1 also enhanced antitumor response of Pmel T cells against B16 melanoma in vivo. The fli1-/- Pmel or OT-I CD8 T cells had significantly increased effector activity, persistence, and central memory differentiation in spleen and tumor. ScRNA-seq analysis revealed that fli1 was mainly expressed in exhausted, but not in progenitor-like subsets of CD8 T cells in tumor. Increased Granzyme B and decreased CTLA-4 gene transcription were observed in fli1-/- OT-I T cells compared to WT cells. To investigate the role of fli1 in regulating human T cells, we used CRISPR/CAS9 nucleofection-based gene silencing approach to knock down fli1 in hPBMCs. Preliminary data suggest deficiency that knock-down of fli1 in human CD19 CAR-T cells, improve their killing ability against Raji cells human B cell lymphoma in vitro. In conclusion, Fli-1 plays key roles in regulating the differentiation and anti-tumor function of CD8 T cells and fli1 silence in T cells may improve cancer immunotherapy. Citation Format: Reza Alimohammadi, Yongxia Wu, Allison Pugel, Gustavo Leone, Xue-Zhong Yu. Targeting fli1 improves antitumor response of CD8 T cells by promoting their activation and limiting exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4862.