Genome-Wide CRISPR Screen Reveals IRF1 and TFAP4 As Transcriptional Regulators of Galectin-9 in T-Cell Acute Lymphoblastic Leukemia

Galectin-9 plays a crucial role in immunosuppression and has been implicated in various cancers, including T-cell acute lymphoblastic leukemia (T-ALL). Galectin-9 regulates T cell exhaustion by interacting with TIM-3, making it a promising target for cancer immunotherapy. This research focuses on Galectin-9 and its regulatory epigenetic circuits as potential therapeutic targets. We investigated the transcriptional regulation of Galectin-9 in pediatric T-ALL using genome-wide CRISPR screens, ELISA, Western blot, and qPCR. Additionally, histone modification ChIP-seq and HiChIP-seq were used to map enhancer-promoter interactions involved in Galectin-9 regulation.

We first assessed Galectin-9 expression in pediatric T-ALL patient samples and cell lines. High Galectin-9 expression was linked to worse disease outcome in 933 pediatric T-ALL patients in Gabriella Miller Kids First Pediatric Research Program. In pediatric T-ALL, Galectin-9 expression varied across subtypes based on genetic rearrangements, with expression levels similar to normal thymocytes but higher than normal T cells. In T-ALL cell lines, Galectin-9 expression varied across genetic subtypes, with the highest in Jurkat and CCRF-CEM cells and the lowest in HPB-ALL. This variability was seen in both intracellular and secreted Galectin-9 levels, indicating the clinical relevance of Galectin-9 in pediatric T-ALL.

To elucidate the regulatory mechanisms that govern Galectin-9 expression in pediatric T-ALL, we conducted a genome-wide CRISPR screen in Jurkat T-ALL cells, a TAL1-rearranged, PTEN-mutated pediatric T-ALL cell line with high Galectin-9 expression, using the Brunello CRISPR library. Galectin-9 was the top hit with an FDR < 0.1. Additional minipool screens, using negative and positive hits from the two genome-wide screen, identified the transcription factors IRF1 and TFAP4 and the cytoskeleton protein ADD1 as top-scoring hits.

To translate these results to primary T-ALL, we analyzed bulk RNA-seq data from 265 pediatric T-ALL samples by using TARGET (Therapeutically Applicable Research to Generate Effective Treatments) cohort across low, mid, and high Galectin-9 expressing groups. IRF1 and TFAP4 exhibited higher expression in samples with the highest Galectin-9 expression. We next investigated whether the IRF1 and TFAP4 bind regulatory DNA elements within the Galectin-9 locus. ChIP-seq analysis revealed IRF1 binding at the Galectin-9 promoter and TFAP4 binding at close-by enhancers. H3K27ac-HiChIP-seq demonstrated that enhancer-promoter looping is important for Galectin-9 expression in T-ALL cell lines with high Galectin-9 levels. Knockout of IRF1 substantially reduced Galectin-9 expression in T-ALL cell lines, confirmed by Western blot, ELISA, and qPCR. Knockout of TFAP4 also significantly reduced Galectin-9 levels, though to a lesser extent than IRF1. Knockouts of the two most prominent enhancers that are bound by TFAP4 also decreased Galectin-9 levels, highlighting the epigenetic roles of IRF1 and TFAP4 in regulating Galectin-9 expression.

Our study highlights the transcriptional regulation of Galectin-9 by IRF1 and TFAP4. These results provide a foundation for developing therapeutic strategies targeting the Galectin-9-Tim-3 axis in T-ALL and provide an approach to investigate the epigenetic circuitry in T-cell leukemia. Our approach, using a FACS-based genome-wide screen complemented by various validation techniques, creates new opportunities for understanding and manipulating epigenetic transcriptional regulation in cancer.