A risk stratification framework based on ammonia-induced cell death signatures for prognostic assessment and therapeutic insights in colorectal cancer

Discover Oncology https://doi.org/10.1007/s12672-026-05257-w Article in Press A risk stratification framework based on ammonia-induced cell death signatures for prognostic assessment and therapeutic insights in colorectal cancer Shuyan Sun, Yinuo Zhao, Zhiqiang Liu & Xiangying Ran Received: 11 December 2025 Accepted: 18 May 2026 Cite this article as: Sun S., Zhao Y., Liu Z. et al. A risk stratification framework based on ammoniainduced cell death signatures for prognostic assessment and therapeutic insights in colorectal cancer. Discov Onc (2026). https://doi.org/10.1007/ s12672-026-05257-w A S S We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply. IN E R P If this paper is publishing under a Transparent Peer Review model then Peer Review reports will publish with the final article. I T R E L C © The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. ARTICLE IN PRESS A Risk Stratification Framework Based on AmmoniaInduced Cell Death Signatures for Prognostic Assessment and Therapeutic Insights in Colorectal Cancer Shuyan Sun¹, Yinuo Zhao¹, Zhiqiang Liu², Xiangying Ran¹# ¹ Department of Pathology, Rizhao People’s Hospital, Rizhao, Shandong Province, 276800, People’s Republic of China. ² School of Pharmacy, Jining Medical University, Rizhao, Shandong Province, 276826, People’s Republic of China. Correspondence should be addressed to Xiangying Ran () Abstract Background: S S E R P Ammonia, which was traditionally regarded as a metabolic by-product, has more recently emerged as a crucial regulator of tumor metabolism and immune dysfunction. E L C IN Nevertheless, the prognostic implications and therapeutic significance of ammoniarelated genes in colorectal cancer (CRC) are still largely uncharted territory. This lack I T AR of exploration means that there is a significant gap in our understanding of how these genes may impact the prognosis of CRC patients and potentially serve as targets for new therapeutic strategies. Methods: Transcriptomic and clinical data from the TCGA-COAD dataset were analyzed to pinpoint ammonia-related genes that showed differential expression. A prognostic risk signature was then developed by employing univariate Cox, LASSO, and multivariate Cox regression analyses. To assess the immune landscape characteristics and pathway alterations, techniques such as CIBERSORT, GSVA, TIDE, and TCIA were utilized. External validation of the gene expression patterns across different cell types was carried out using single-cell RNA-seq data from the GSE132465 dataset. The exploration of potential therapeutic targets was further advanced through artificial intelligence-based virtual screening on the DrugCLIP platform. Finally, the key genes were experimentally verified using RT-qPCR. This comprehensive approach aimed to understand the role of ammonia-related genes in CRC, from their identification to their potential as therapeutic targets, while validating findings at multiple levels. Results: ARTICLE IN PRESS A three-gene signature consisting of PMM2, CLK2, and UCHL1, which is associated with ammonia-induced cellular stress, was identified. This signature was then utilized to construct a prognostic risk model. This model successfully classified CRC patients into high- and low- risk groups, with these groups showing significantly disparate survival outcomes. Through functional analyses, it was discovered that the high-risk group had an enrichment of pathways related to mitochondrial metabolism, proteostasis regulation, and oxidative stress responses. Immune analyses indicated that in the high-risk group, there was an increased infiltration of regulatory T cells and enhanced interferon - related signaling. This suggests the presence of an “inflamed but immunosuppressed” tumor microenvironment. Single-cell RNA-seq analysis further confirmed the celltype-specific expression patterns of the three genes within the tumor microenvironment. Additionally, artificial intelligence (AI)-based virtual screening identified candidate small molecules that are predicted to bind to the catalytic pocket of CLK2. This finding indicates that CLK2 has the potential to be a druggable metabolic target. Overall, these results provide valuable insights into the S S E R P role of ammonia-related genes in CRC and offer potential avenues for prognostic assessment and therapeutic intervention Conclusions: This gene signature associated with ammonia offers a new paradigm for prognostic IN stratification in CRC. It mirrors the metabolic stress responses triggered by ammonia E L C accumulation within the tumor microenvironment. These discoveries emphasize that metabolic adaptation related to ammonia could serve as a rich source of potential I T AR biomarkers. Moreover, they propose CLK2 as a highly promising therapeutic target, opening up new possibilities for more effective treatment strategies in CRC management. Introduction Colorectal cancer (CRC) is one of the most common malignancies worldwide. In 2022, there were approximately 1.9 million new cases and 0.9 million deaths. The incidence and mortality rates are particularly high in European and American countries, and by 2023 CRC had even become one of the leading contributors to the global cancer burden. [1–3]. The prognosis of CRC largely depends on the stage at diagnosis: the five-year survival rate exceeds 90% for patients with localized tumors, whereas it drops to about 10% among the 20% of patients who present with distant metastases at initial diagnosis [4,5]. The tumor microenvironment (TME) is considered to play a critical role in tumor initiation, progression, and metastasis. Recent studies have shown that the TME in CRC is characterized by immunosuppression and the accumulation of metabolic waste products. Metabolite (...truncated)