Genome-Wide In Vivo CRISPR Screens in Human T Cells Identify GPCR Pathway Targets to Enhance CAR-T Therapies for Solid Tumors

Background: T cell therapies have been transformative for certain leukemias and lymphomas, however solid tumors have proven far more difficult due to challenges such as poor infiltration, harsh metabolic environments, and a myriad of suppressive factors in the tumor microenvironment (TME). Most large-scale genetic screens in human T cells have been performed in vitro, which fail to capture many solid tumor-specific barriers. However, performing large-scale screens in vivo has been impractical due to the low recovery of T cells from tumors, which significantly limits screening throughput. To overcome these limitations, we developed a novel screening platform that allows engagement of human CD4+ and CD8+ T cells with any polyclonal repertoire and achieves significantly higher T cell recovery from tumors, enabling genome-wide screens in relatively few mice. Preliminary Data: Using this system, we performed two genome-wide CRISPR loss-of-function screens for gene targets that enhance T cell abundance within tumors as well as effector function in tumors based on interferon- γ production. We uncovered many genes with known roles in T cell fitness, as well as many novel gene targets, including from two GPCR signaling pathways that regulate (1) T cell resistance to TME-mediated suppression and (2) T cell trafficking and infiltration into tumors. The PTGER4-GNAS (Gαs) signaling pathway was enriched in our screens and has been previously shown to induce dysfunction in mouse T cells in the TME in response to suppressive cues. Our preliminary data suggest knocking out GNAS in human T cell therapies can robustly enhance tumor control and survival. Our screens and preliminary validation data also identified P2RY8-Gα13 signaling, a pathway previously characterized in B cell migration, as a major regulator of human T cell trafficking into tumors. Research Strategy: In Aim 1, we will investigate the genetic disruption of GPCR-Gαs-PKA signaling to enhance T cell resistance to TME suppression. We will test the efficacy of GNAS KO in TCR- and CAR-T cells in multiple solid tumor models and we will dissect the dominant upstream GPCRs as well as key downstream PKA targets driving these effects. In Aim 2, we will establish the P2RY8-Gα13 GPCR signaling pathway as a novel regulator of T cell trafficking and immune exclusion. We will use a combination of in vivo and in vitro competition assays to assess how this pathway affects T cell migration into tumors, as well as functional genetics and microscopy approaches to evaluate its role in immune exclusion. We will test whether the P2RY8 ligand, S-geranylgeranyl-L-glutathione, is a repulsive cue used by tumors to block T cell infiltration. In both aims, in addition to our studies in healthy T cells, we will test the relevance of these pathways in tumor- infiltrating lymphocytes from solid tumor patients. Expected Results and Impact: These studies will rigorously test whether targeting key GPCR signaling pathways nominated by our screens can enhance T cell infiltration and durable effector function. Our findings will provide novel strategies for engineering more effective T cell therapies for solid tumors and establish a new standard for in vivo functional genomics screens in human T cells. Project Number: 1R01CA309039-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Julia Carnevale | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $680,597 | Activity Code: R01 | Study Section: Cellular Immunotherapy of Cancer Study Section[CIC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11279422