Abstract 4258: In vivo genome-scale enhancer screen decodes T cell fate decisions in the tumor

Abstract During chronic infection and cancer progression, T cells enter a dysfunctional state called exhaustion, posing a significant challenge to disease control and immunotherapy outcomes. Previously, we and others have shown that T cell exhaustion is a differentiation process regulated at the epigenetic level. Exhausted T cells share a conserved program of chromatin accessibility landscapes across chronic infections and tumors (core exhaustion program) that is imprinted in early T cell differentiation. However, it remains unclear whether and which open chromatin regions within the core exhaustion program play a causal role in driving the exhausted state. We hypothesize that the core exhaustion program contains transcriptional enhancers that regulate T cell persistence and differentiation in exhaustion, preventing reinvigoration of function. To test it, we leveraged our novel enhancer editing platform called Systematic Non-coding element Interrogation by Paired sgRNAs (SNIP-R), a pooled CRISPR-based enhancer deletion platform optimized for kilobase-scale perturbation in primary T cells. We performed the first in vivo genome-scale enhancer deletion screen on ova-specific CD8+ T cells (OT-1) in OVA-expressing tumors. We identified networks of regulatory elements in the core exhaustion program that regulate T cell persistence and subset formation in the tumor. Validation studies from the screen showed that deleting one such regulatory element 100kb upstream of the Klf6 gene (Klf6-100kb) in T cells improved tumor control. Klf6-100kb perturbed T cells strongly outcompeted control populations and had increased generation of effector-like T cells in the tumor compared to control perturbations. Together, we showed that the core exhaustion program contains causal regulatory elements underlying T cell exhaustion and pinpointed new targets for developing next-generation adoptive T cell therapies. Citation Format: Keely Y. Ji, Alex Chang-Yu Chen, Laura Hinojosa, Bolutito Babatunde, Daniela Martinez, Thomas J. LaSalle, Maria Zschummel, Marc A. Schwartz, Ferhat Ay, Debattama Sen. In vivo genome-scale enhancer screen decodes T cell fate decisions in the tumor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4258.