Abstract 153: Identification of a gastrointestinal (GI) toxicity gene signature that predicts GI-toxicity induction in small intestinal organoids

Abstract

The early identification of GI toxicity caused by novel therapeutics is crucial for drug development pipelines. GI toxicity is one of the most frequent adverse effects amongst chemotherapeutics. Impaired barrier function and epithelial ulceration can lead to symptoms including diarrhoea, dehydration and susceptibility to systemic infection. We have identified a gene signature that can be utilised in an in vitro organoid model to predict relevant GI toxicity. Methods: Both mice and small intestinal organoids derived from human and mouse were exposed to chemotherapeutic agents known to cause GI toxicity. For in vivo assays mice were treated with 50 or 100mg/kg CPT11 for 4 or 8 hours or 100, 200 or 400mg/kg Iressa for 8 or 24 hours before small intestinal (SI) tissue was removed for RNA-Seq analysis. SI tissue was also analysed by histological staining and damage measured to confirm induction of GI-toxicity. Human and mouse organoids were treated with CPT11 and Iressa for 8 hours at a range of concentrations. The organoids were lysed, RNA extracted and an mRNA-Seq library was generated. Samples were sequenced using 2 × 75bp PE reads on a Next-Seq550. Sequences were aligned to the relevant genomes using BWA and normalised using DESeq2. Normalised gene counts were analysed using Partek Genomics Suite and Ingenuity Pathway Analysis (IPA) was used for gene set enrichment (GSEA) and pathway analysis of differentially expressed genes. Results: For all treatments and time points the mechanism of action (MOA) of each agent was evident from the GSEA and pathway analysis. CPT11 induced DNA damage and cell cycle arrest pathways, whereas Iressa demonstrated downregulation of cell cycle and chromosomal replication and mitotic genes. Analysis of toxicity pathways in IPA identified a number of differentially expressed genes that were consistent with increased toxicity. A gene signature was identified and a multivariate scoring system used to demonstrate signature engagement. This signature was activated in all SI tissues treated with CPT11 or Iressa, irrespective of the MOA of both agents. Conclusions: Deployment of this signature on mouse and human organoids treated with chemotherapeutic agents demonstrated activation of the signature, which could be an alternative method for GI toxicity testing. Further analysis of this signature in gene expression studies from GI toxicity inducing agents submitted to the NCBI Gene expression omnibus (GEO) demonstrated that the signature was activated with a variety of agents with different MOA’s such as NSAIDs, gamma-secretase inhibitors, CDK8/19 inhibitors and CAR-T-cells. Citation Format: Gary S. Beale, Aude Marine-Bonavita, Valentina Ubertini, Greg Tudor, Francesca Philips, Catherine Booth. Identification of a gastrointestinal (GI) toxicity gene signature that predicts GI-toxicity induction in small intestinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 153.