Objectives: Epigenomic 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.
Design: Alternate 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.
Results: APB 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).
Conclusion: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.
Competing Interests: Competing interests: The subject matter in this manuscript was submitted as a technology disclosure to the institutional Technology Transfer Office for potential intellectual property protection. PT had stock and other ownership interests in HealthSeq, research funding from Kyowa Hakko Kirin and Thermo Fisher Scientific, and patents/other intellectual property through the Agency for Science and Technology Research, Singapore (all outside the submitted work). RS received honoraria from Bristol-Myers Squibb, Lilly, Roche, Taiho, Astra Zeneca, DKSH and MSD; had advisory activity with Bristol-Myers Squibb, Eisai, Merck, Bayer, Taiho, Novartis, MSD and AstraZeneca; received research funding from Paxman Coolers and MSD; received travel grants from AstraZeneca, Roche, Eisai and Taiho Pharmaceutical (all outside the submitted work). FP received honoraria for speakers’ bureau/advisory activity from Amgen, Merck-Serono, Roche, Lilly, Sanofi, Bayer and Servier, and received research funding from BMS (all outside the submitted work).
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