Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition

ObjectivesEpigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours.DesignAlternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes.ResultsAPBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using ‘humanised mice’ harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032).ConclusionThese findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

Epigenetic alternate promoter utilization and association with PD-L1 expression in Epstein–Barr virus positive gastric cancer.

e15509 Background: We recently elicited the role of epigenetic promoter alterations as a mechanism of immune-evasion and primary resistance to immune checkpoint inhibition in gastric cancer. High prevalence of epigenetic modifications are known to occur in Epstein-Barr virus associated gastric cancer (EBVaGC). EBVaGC has high response rates to anti-PD-1 immune checkpoint inhibitors and is associated with high levels of PD-L1 expression. However, not all EBVaGC express PD-L1 and mechanisms that mediate these phenomena are unknown. Methods: We performed NanoString profiling and PD-L1 immunohistochemistry (using Dako PD-L1 IHC 22C3) on tissue from gastric cancer patients undergoing primary tumor resections at Samsung Medical Centre, South Korea. NanoString panel was designed for 90 recurrent somatic alternate promoter-related genes, and immune-related genes including PD-L1. EBV status was determined using EBV-encoded RNA in situ hybridization and categorized as EBVaGC and EBV-negative. Samples in the top-quartile of alternate promoter utilization were classified as APhigh and the remaining APlow. Results: A total of 272 samples (EBVaGC n = 79; EBV-negative n = 193) were included in this study. EBVaGC had significantly higher PD-L1 expression (p < 0.001) compared to EBV-negative samples. APhigh group (n = 67) consisted of 61 EBV-negative and 6 EBVaGC samples. EBVaGC APhigh tumors had significantly lower PD-L1 transcript expression compared to EBVaGC APlow tumors (p = 0.011, Wilcoxon-rank sum). Similar correlation was also found with PD-L1 IHC combined positive score (CPS)(median CPS score 1 vs 8, p = 0.047). There was a trend towards poorer survival for EBVaGC APhigh tumors (vs EBVaGC APlow; HR 0.23, 95% CI: 0.046 – 1.23, p = 0.087). EBV-negative APhigh tumors also had lower PD-L1 expression (vs EBV-negative APlow; p = 0.046, Wilcoxon-rank sum). Conclusions: Increased utilization of epigenetic alternate promoter isoforms correlates with lower transcriptomic and protein expression of PD-L1 in EBVaGC. Here we describe a potential mechanism of immune-evasion to explain low immune-infiltration and PD-L1 expression that occurs in a group of EBVaGC that is traditionally considered highly immunogenic.

CAGExploreR: an R package for the analysis and visualization of promoter dynamics across multiple experiments

Summary Alternate promoter usage is an important molecular mechanism for generating RNA and protein diversity. Cap Analysis Gene Expression (CAGE) is a powerful approach for revealing the multiplicity of transcription start site (TSS) events across experiments and conditions. An understanding of the dynamics of TSS choice across these conditions requires both sensitive quantification and comparative visualization. We have developed CAGExploreR, an R package to detect and visualize changes in the use of specific TSS in wider promoter regions in the context of changes in overall gene expression when comparing different CAGE samples. These changes provide insight into the modification of transcript isoform generation and regulatory network alterations associated with cell types and conditions. CAGExploreR is based on the FANTOM5 and MPromDb promoter set definitions but can also work with user-supplied regions. The package compares multiple CAGE libraries simultaneously. Supplementary Materials describe methods in detail, and a vignette demonstrates a workflow with a real data example. Availability and implementation: The package is freely available under the MIT license from CRAN (http://cran.r-project.org/web/packages/CAGExploreR). Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.