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

● 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.