Abstract 904: Mapping the T cell-cold phenotype to the colorectal cancer genome identifies POFUT1 as a driver of tumor T cell inhibition

Abstract Colorectal cancer (CRC) patients with T cell-cold tumors experience rapid cancer progression and poor survival. While T cell-enhancing immunotherapies have improved outcomes for a subset of CRC patients, most remain nonresponsive. Therefore, better understanding the mechanisms that drive T cell dysfunction in CRC is needed. We used matched gene expression and copy number profiles from TCGA to evaluate CRC tumors (N=373) based on measures of T cell infiltrate, and developed a method to identify genes with potential roles in T cell exclusion. Leveraging the intersect between copy number amplification and gene expression in the T cell-cold state, we pinpointed a region of chromosome 20q11.21 where DNA- and RNA-levels were more strongly correlated with T cell exclusion than anywhere else in the CRC genome. This locus also contained the most frequently amplified and overexpressed genes in the CRC genome. Within this region, Protein O-Fucosyltransferase 1 (POFUT1) was the most distinguished gene, and correlated with CTL inhibition across 12/24 solid tumor types annotated in TCGA (SKCM, THCA, COAD, READ, LUSC, CESC, KIRC, STAD, HNSC, KIRP, LIHC, UCEC; p<0.0001). Stable overexpression of POFUT1 (POFUT1-OE) in MC38 mouse colon cancer cells enhanced proliferation relative to MC38-Vector (RTCA p<0.0001, CellTiterGlo p=0.0011); and in co-culture with c57BL/6 spleen-derived PBMCs, CD8+ T cell populations from MC38-POFUT1-OE co-cultures were significantly reduced over the course of 7 days. This effect persisted when PBMCs were stimulated with IL-2 and anti-CD2/CD3/CD28 in 50% conditioned medium (CM) from MC38-POFUT1-OE, prompting us to profile the CM for differences in secretomes. Of 110 profiled cytokines, two were at significantly higher levels in MC38-POFUT1-OE CM: IL-6 and IL-23 (p=0.0003, p=0.0055). These results were validated via ELISA and transient knockdown (KD) of POFUT1, where POFUT1-KD resulted in reduced cancer cell proliferation, diminished secretion of IL-6, and increased CD8+ T cell abundance in our CM models. Furthermore, siRNA-mediated KD of POFUT1 in MDST8 and HCT15 human CRC cell lines resulted in significant reductions in secreted IL-6 (p=0.0003, p<0.0001). Treatment of murine PBMCs with IL-6 (5.0 pg/mL - 50 ng/mL) decreased the proportion of CD8+ T cells to an analogous level as culture with POFUT1-OE CM. The decrease in CD8+ T cell proportion was also associated with less proliferation (CFSE, p=0.0049), and reduced surface expression of activating co-receptors CD25 and CD69 (p=0.1008, p=0.0015). Dosing with anti-IL-6 IgG (10 ng/mL) reversed the decrease in CD8+ T cell proliferation induced by CM from MC38-POFUT1-OE, establishing IL-6 as the downstream mechanism of POFUT1-dependent inhibition of CD8+ T cell activity. Together, these findings link POFUT1 amplification in CRC with dysregulation of CD8+ T cells via upregulation of cancer-intrinsic IL-6 secretion. Citation Format: Calvin Jordan Wagner. Mapping the T cell-cold phenotype to the colorectal cancer genome identifies POFUT1 as a driver of tumor T cell inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 904.