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 APB_high, APB_int and APB_low. 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.ResultsAPB_high 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 APB_high tumours. Functional in vivo studies using ‘humanised mice’ harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APB_high tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APB_high tumours to immune checkpoint inhibition. APB_high gastric cancer exhibited significantly poorer progression-free survival compared with APB_low (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.