Identification of genes that limit car t persistence in solid tumors

The National Cancer Institute awarded $164,190 to the University of Pennsylvania on January 15, 2026, under the Cancer Research Manpower program (CFDA 93.398) to support identification of genes that limit CAR T persistence in solid tumors. Work runs through July 31, 2029, and is performed in Philadelphia, Pennsylvania.

The project conducts genome-wide in vivo CRISPR screening of human CAR T cell persistence in solid tumor microenvironments. Prior work identified USP22 and ATXN7L3—components of the SAGA complex deubiquitylation module—as top candidates influencing CAR T function. Knockout of these genes increased T cell memory markers and enhanced CAR T expansion and persistence in vitro, yet paradoxically failed to enhance antitumor immunity or persistence in vivo and showed defects in IL-2 secretion. The research hypothesizes that knockout of USP22 or ATXN7L3 reprograms the CAR T transcriptome through histone ubiquitination changes and augments CAR T persistence in an IL-2 dependent manner. Aim 1 proposes to determine whether cell-extrinsic IL-2 is sufficient to rescue knockout CAR T persistence and enhance antitumor activity in solid tumors using pooled in vivo settings.

Abstract

Chimeric antigen receptor t (car t) cell therapy has been highly effective in a subset of hematological malignancies. However, this efficacy has not translated to solid tumors for a variety of reasons, including limited persistence in an immunosuppressive and metabolically challenging tumor microenvironment. Unbiased techniques such as genome-scale crispr screens have identified several pathways to enhance therapeutic efficacy. However, these screens have mainly utilized reductionist in vitro approaches, and do not recapture a solid tumor microenvironment. I have performed a genome-wide in vivo crispr screen of human car t persistence in solid tumors. The top two hits were ATXN7L3 and USP22, components of the SPT-ADA-GCN5 acetyltransferase (saga) complex deubiquitylation module (DUB). The saga DUB is primarily known for its role in histone H2B deubiquitination, but its role in t cell persistence and antitumor function remains to be described. I show knockout of USP22 or ATXN7L3 (uspko/atxko) increased t cell memory markers and enhanced car t expansion and persistence in vitro. Paradoxically, uspko/atxko car t exhibit a defect in IL- 2 secretion and fail to enhance antitumor immunity or persistence in vivo. The data indicate that USP22 and ATXN7L3 are fundamental regulators of car t cell function, yet the mechanisms through which they influence car t biology remain unclear. I hypothesize that knockout of USP22 or ATXN7L3 reprograms the car t transcriptome through changes in histone ubiquitination and augments car t persistence in an IL-2 dependent manner. In aim 1, i propose to determine the relationship between cell-extrinsic IL-2 and uspko/atxko car t persistence in solid tumors. I postulate that cell-extrinsic IL-2 is sufficient to rescue uspko/atxko persistence and enhance antitumor activity. As uspko/atxko car t persist in a pooled in vivo setting, i will pool wildtype car t cells with uspko/atxko car t at different ratios and infuse into tumor-bearing mice to assess persistence and antitumor activity. I will also determine the effects of exogenous IL-2 supplementation on in vivo uspko/atxko car t persistence through serial recombinant IL-2 infusions. In aim 2, i propose to investigate the impact of uspko/atxko and histone deubiquitination on car t transcriptional programs. I hypothesize that H2B deubiquitination through saga regulates gene expression programs that promote car t persistence. To evaluate this, i will perform bulk RNA-sequencing and histone ubiquitin cut & run-sequencing on uspko/atxko to determine the extent to which histone ubiquitin changes affect these programs. Together, the findings of this work will describe a novel role for the saga complex and histone ubiquitination in car t persistence and has potential for direct clinical translation for treatment of solid tumors.