{"doi":"10.1101/2025.08.18.670666","title":"<i>Sleeping Beauty</i> mutagenesis identifies <i>BACH2</i> and other regulators of CD8 <sup>+</sup> T cell exhaustion, persistence <i>in vivo</i> , and CAR-T function under tumor-associated chronic antigen stimulation","abstract":"Abstract Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying D oxycycline (Dox)-inducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8⁺ T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8⁺ T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB -mutagenized CD8⁺ T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2 low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2 high restricted differentiation predominantly to the CD62L⁺ subset. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558889,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9561,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":417673,"name":"Alex T. Larsson","orcid":"0000-0002-1350-8271","position":1,"is_corresponding":false},{"id":995476,"name":"Tyler Jubenville","orcid":"0009-0003-9578-4847","position":2,"is_corresponding":false},{"id":418994,"name":"Wendy A. Hudson","orcid":null,"position":3,"is_corresponding":false},{"id":1177679,"name":"Carli M. Stewart","orcid":"0000-0002-2288-2230","position":4,"is_corresponding":false},{"id":1143623,"name":"Alexander K. Tsai","orcid":"0000-0002-4014-1946","position":5,"is_corresponding":false},{"id":445732,"name":"Adam L. Burrack","orcid":"0000-0002-4001-7849","position":6,"is_corresponding":false},{"id":1438295,"name":"Erin E. Nolan","orcid":null,"position":7,"is_corresponding":false},{"id":1460111,"name":"Zach J. Seeman","orcid":null,"position":8,"is_corresponding":false},{"id":1459673,"name":"Yuling Yang","orcid":"0000-0002-6387-611X","position":9,"is_corresponding":false},{"id":993153,"name":"Christopher M. Stehn","orcid":"0000-0003-2911-1954","position":10,"is_corresponding":false},{"id":582746,"name":"Daryl M. Gohl","orcid":"0000-0002-4434-2788","position":11,"is_corresponding":false},{"id":1169051,"name":"Margaret Donovan","orcid":"0009-0007-1779-4459","position":12,"is_corresponding":false},{"id":257752,"name":"Nuri A. Temiz","orcid":"0000-0003-1416-3639","position":13,"is_corresponding":false},{"id":1460112,"name":"Flavia E. Popescu","orcid":null,"position":14,"is_corresponding":false},{"id":227914,"name":"Søren Warming","orcid":"0000-0002-5721-0741","position":15,"is_corresponding":false},{"id":469024,"name":"Somasekar Seshagiri","orcid":"0000-0003-4272-6443","position":16,"is_corresponding":false},{"id":107245,"name":"Yun You","orcid":"0000-0002-0671-8123","position":17,"is_corresponding":false},{"id":513391,"name":"Ingunn M. Stromnes","orcid":"0000-0001-8120-4547","position":18,"is_corresponding":false},{"id":371975,"name":"Saad S. Kenderian","orcid":"0000-0003-2767-3830","position":19,"is_corresponding":false},{"id":257760,"name":"David A. Largaespada","orcid":"0000-0002-3183-0491","position":20,"is_corresponding":false},{"id":1459672,"name":"Chang-Jung Lee","orcid":"0000-0001-9052-3160","position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":null,"created_at":"2026-07-19T02:55:30.312295Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}