Abstract 5369: Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition

Abstract

Abstract CD8+ T cells are a critical anti-cancer immune subset; however, most colorectal cancer (CRC) patients have inadequate levels of CD8+ T cells in their tumor microenvironment (TME), contributing to poor survival and increased CRC aggression. While current immune checkpoint inhibitors are able to elicit anti-cancer CD8+ T cell responses in some patients, a majority of patients are still nonresponsive, and have minimal levels of tumor-infiltrating CD8+ T cells. This effect is potentially due to still-unrecognized cancer-driven mechanisms of immunosuppression. To identify candidate genes with roles in CD8+ T cell exclusion, we used a T cell gene signature (Tsig) to quantify CD8+ T cell infiltration in tumors profiled by RNA-seq. Partial Spearman correlation adjusted for tumor purity was performed for all annotated genes (N=20501) in the TCGA CRC RNA-seq data set (N=373) to identify genes most inversely correlated with Tsig. A locus at chromosome 20q was identified as the region of the genome most inversely correlated with Tsig - both by copy number analysis and by RNA expression. Interestingly, this region is the most copy number-amplified region in CRC tumors, and the genes at this locus are among the most highly expressed genes in CRC tumors. Within this region, the amplified and overexpressed gene Protein O-Fucosyltransferase 1 (POFUT1) had the strongest inverse correlation to Tsig. POFUT1 function is essential to canonical NOTCH signaling, and CRC-expressed NOTCH is implicated in increased CRC secretion of immunosuppressive factors IL-6, TGF-β and VEGF, and reduced CD8+ T cell secretion of IFN-γ and IL-2. POFUT1 overexpressing (OE) CT26 and MC38 cell lines were generated, and confirmed by Western Blot to have elevated levels of both cleaved NOTCH1 (NICD1), and NOTCH pathway transcriptional target, HES1. Murine CD8+ T cells were stimulated in vitro with CD3/CD28 microbeads and IL-2 in conditioned medium (CM) from vector or POFUT1-OE lines. CD8+ T cells cultured in CM from POFUT1-OE lines had significantly lower levels of activation markers (CD45 (p=0.0022), CD8 (p=0.0095), CD25 (p=0.0069, CD69 (p=0.0099)), reduced proliferation (CFSE, p=0.1281), and generated less ATP (Cell-Titer Glo, p=0.0003) than CD8+ T cells cultured in vector CM. In pan-cancer correlational analyses, POFUT1 is in the top 5% of genes most inversely correlated with Tsig in 12/24 solid tumor types annotated in TCGA (SKCM, THCA, COAD, READ, LUSC, CESC, KIRC, STAD, HNSC, KIRP, LIHC, UCEC p<0.0001). POFUT1 is robustly negatively correlated with T cell infiltration in many cancer types. We observed a positive association in CRC between POFUT1 and NOTCH signaling, and observed that POFUT1 expression reduced CD8+ T cell activity, potentially through upregulation of extracellular factors (in progress). Our findings fit a model where POFUT1 amplification in CRC promotes CD8+ T cell dysfunction as a mechanism of immune escape. Citation Format: Calvin J. Wagner, Eric D. Routh, Raven Rutledge, Lance D. Miller. Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5369.