Attenuation of the CpG Island Methylator Phenotype and lack of WNT signaling activation restrainsKrasmutant intestinal neoplasia
Authors: Fennell Lochlan , Tria Simon , Liu Cheng , Kane Alexandra , McKeone Diane , Borowsky Jennifer , Chai Lu , Randall-Demllo Sarron , Bond Catherine , Leggett Barbara , Whitehall Vicki 
Published: December 2023

AbstractBackgroundSerrated neoplasia accounts for ∼25% of colorectal cancer. These cancers arise from serrated precursor lesions. Hyperplastic polyps initiated by eitherBRAForKRASmutation activating MAPK signalling are common, but premalignant sessile serrated lesions withKRASmutation are rare. Here, we modelKrasandBrafmutant neoplasiain vivoto compare histological, gene expression and DNA methylation manifestations associated with activation of these oncogenes.MethodsWe employ cre-recombinase dependentBrafV637andKrasG12Dmurine models, and cross animals with those bearing the Villin-CreERT2transgene to direct temporospatial activation of these oncogenes to the murine intestine. We examine histology, and genome-scale DNA methylation and gene expression via reduced representation bisulphite sequencing and RNA-Seq, respectively. We performed differential gene expression, methylation and pathways analysis to identify oncogene specific alterations.ResultsProlonged exposure to oncogenicBrafis associated with a time-dependent accumulation of murine serrated precursors (P=3x10-10) and advanced murine serrated lesions and invasive cancer (8x10-8).Krasmutant animals acquire fewer precursor lesions (P=0.06) and have a significantly lower probability of developing advanced serrated lesions (P=0.004).BrafandKrasmutant animals develop pronounced hyperplasia, however the severity is significantly less inKrasmutant animals.Krasmutant advanced serrated lesions rarely develop aberrant WNT signaling activation (1/23). Gene expression profiling showed divergent transcriptomic profiles betweenBrafandKrasmutant intestines, with the former overexpressing genes associated with immune and inflammatory signaling. Deconvolution analysis revealed a comparably higher macrophage infiltrate (P=0.025) and upregulation of M1 macrophage gene sets in theBrafmutant intestine (P=0.0008), contributing to chronic inflammatory signalling. BothKrasandBrafmutations lead to accumulation of substantial temporal DNA methylation alterations, however a subset of CpG sites (1,306) show an attenuated rate of DNA methylation accumulation in theKrasmutant intestine compared withBrafmutant animals.ConclusionsIn this study, we show thatKrasmutation can induce serrated intestinal neoplasia, however the latency period and penetrance is significantly lower when compared withBrafmutation. Aberrant WNT signalling is common in lesions arising in the context ofBrafmutation, but rare inKrasmutant neoplasms. We show marked transcriptomic disparities between these models, with a tendency for theBrafmutant intestine to upregulate immunological processes. Our DNA methylation analysis reveals an attenuated CIMP-like phenotype that is specific to theKrasmutant intestine, consistent with our previous works in humans. These data have significant implications for our understanding of how MAPK-induced neoplasia develops within the intestine.SynopsisBRAFandKRASmutant hyperplastic polyps have disparate malignant potential and the reason for this is unclear given both oncogenes activate MAPK signalling. We show that the DNA methylation alterations that followKrasmutation are attenuated and that hyperactivation of WNT signaling is rare, providing a molecular mechanism that restrains malignant transformation.