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Sleeping Beauty mutagenesis identifies BACH2 and other regulators promoting CD8+ T cell persistence and effector function under tumor-associated chronic antigen stimulation - esTAG

ABSTRACT: ● Background: Genes that enhance T cell function could represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes that enhance 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. ● Methods: We developed transgenic mouse models carrying Doxycycline (Dox)-inducible SB engineered system (DiSBey) in primary T cells. Using DiSBey, we conducted screens to identify genes that enhance 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. ● Results: 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, previously recognized 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 persistence in the B16-Ova tumor model. We further demonstrated that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2high 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 in vivo. ● Conclusions: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE302683 | GEO | 2026/07/22

REPOSITORIES: GEO

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Project description:● Background: Genes that enhance T cell function could represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes that enhance 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. ● Methods: We developed transgenic mouse models carrying Doxycycline (Dox)-inducible SB engineered system (DiSBey) in primary T cells. Using DiSBey, we conducted screens to identify genes that enhance 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. ● Results: 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, previously recognized 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 persistence in the B16-Ova tumor model. We further demonstrated that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2high 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 in vivo. ● Conclusions: 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.

Project description:Adoptive T cell therapies have been transformative in the treatment of a subset of hematological malignancies. However, many patients either fail to respond or relapse, and these therapies still have had much more limited success in solid tumors. A critical challenge for increasing patient response rates and achieving durable efficacy of adoptive T cell therapies is overcoming suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints. Targeted gene editing has the potential to enhance T cell therapeutic function and improve clinical responses. Here we perform multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to nominate genes that could be targeted to prevent T cell dysfunction. These CRISPR screens converged on RASA2 as a target to confer resistance to suppressive conditions found in tumor microenvironments. We identify RASA2 as a signaling checkpoint in human T cells that is downregulated upon acute TCR stimulation but increases gradually with chronic antigen exposure. Antigen titration showed that genetic ablation of RASA2 enhances mitogen activated protein kinase (MAPK) signaling and CAR-T cell cytolytic activity in response to low antigen levels. Repeated tumor antigen stimulation in vitro revealed that RASA2-deficient TCR-T and CAR-T cells have increased activation, cytokine production, and metabolic activity compared to control cells, and show a striking advantage in persistent cancer cell killing. RASA2 KO CAR-T cells showed a competitive fitness advantage over CAR-T control cells in the bone marrow of an in vivo leukemia model. Deletion of RASA2 in multiple preclinical models of TCR- and CAR-T cell therapies prolonged survival in animals xenografted with either liquid or solid tumors. Altogether, our findings from multiple genome-wide screens and preclinical studies highlight RASA2 as a promising new target to enhance both persistence and effector function in TCR-T and CAR-T cell therapies for cancer treatment.

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Sleeping Beauty mutagenesis identifies BACH2 and other regulators promoting CD8+ T cell persistence and effector function under tumor-associated chronic antigen stimulation - esTAG

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