Molecular Characterization of Small Intestinal Adenoma and Adenocarcinoma in a Genetically Engineered Murine Model and their Derived 3D Organoids Containing Oncogenic KrasG12D and/or Cdkn2a/p16 loss
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Small intestine adenocarcinomas (SIAC) in humans occur most frequently as extra-ampullary duodenal adenocarcinomas (EDA) and demonstrate intestinal-type or gastric-type differentiation. There is limited knowledge of pathways driving each differentiation subtype of pre-cancer adenomas and adenocarcinomas. While APC alterations are predominant (80%) in colorectal adenocarcinomas (CRC), they are less frequent in SIAC (7-26%). On the other hand, KRAS activating mutations occur at similar frequencies in lesions at both sites, 40-60% in CRC and 42-54% in SIACs. Frequent in many malignancies, inactivation of CDKN2A/p16 occurs in 14-30% of SIAC, suggesting a role in a subset of SIAC. The p16 tumor suppressor is integral in upholding a senescence barrier in cells, a mechanism for growth arrest and cell death expressed during cellular stress, leading to bypass of tumor cell senescence as a step in tumor development. Oncogenic Ras-induced cellular senescence is alleviated by additional inactivation of CDKN2A/p16 and/or p53 in pancreatic ductal adenocarcinoma (PDAC), and co-alterations in RAS and CDKN2A/p16 or TRP53 occur in 56% of SIAC lesions. However, the phenotypic effects of combined oncogenic KRAS and p16 loss have not been characterized in small intestine (SI) adenocarcinogenesis. We hypothesized that a) KrasG12D and loss of p16 synergistically promote adenocarcinogenesis in the small intestine via bypass mechanisms of oncogenic KRAS-mediated senescence / apoptosis in vivo, in genetically engineered murine models, and b) SI-duodenum organoids derived from these murine models recapitulate molecular and phenotypic features of SIAC carcinogenesis. We developed a novel mouse model with conditional knockout of Cdkn2a/p16 (p16KO), conditional expression of Kras12D, or both (p16KOKras12D), targeted to mouse LGR5+ gastrointestinal progenitor cells leveraging Cre-mediated recombination. We characterized histologic alterations and molecular driver pathways due to Cdkn2a/p16 deletion and/or oncogenic expression of Kras in the SI-duodenum of our murine model and derived 3-dimensional (3D) organoids. This dissertation presents the findings of co-altered p16KOKras12D mice with frequent development of adenomas in the duodenum starting at young age and locally invasive adenocarcinomas in older mice, demonstrating synergism of combined Kras and p16 alterations in SI adenocarcinogenesis. We characterized the spatial transcriptomic landscapes and cellular phenotypes of SIAC/EDA intestinal- and gastric-type differentiated adenoma and adenocarcinoma subtypes in our murine model lesions. We further established 3D organoids derived from the novel murine models to characterize the in vitro and in vivo tumorigenic growth behavior resulting from these well-defined p16KOKras12D genetic alterations by assessing organoid histology and morphology, size and cell viability. We further utilized single-cell and bulk RNA-sequencing to identify underlying pathways that drive initiation and progression of SIAC and confirmed alterations in protein expression of MAPK pathways. In conclusion, we report a novel mouse model that mimics gastric-type and intestinal-type adenomas and SIAC/EDA and derived organoid lineages. Spatial transcriptomics provided a detailed understanding of differentiation lineages, individual subtype cellular microenvironment, and associated driving molecular pathways that may be leveraged to determine potential targets for precision cancer therapies.
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