Attenuation of the CpG Island Methylator Phenotype and lack of WNT signaling activation restrains Kras mutant intestinal neoplasia

Background: Serrated neoplasia accounts for ~25% of colorectal cancer. These cancers arise from serrated precursor lesions. Hyperplastic polyps initiated by either BRAF or KRAS mutation activating MAPK signalling are common, but premalignant sessile serrated lesions with KRAS mutation are rare. Here, we model Kras and Braf mutant neoplasia in vivo to compare histological, gene expression and DNA methylation manifestations associated with activation of these oncogenes. Methods: We employ cre-recombinase dependent BrafV637 and KrasG12D murine models, and cross animals with those bearing the Villin-CreERT2 transgene 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. Results: Prolonged exposure to oncogenic Braf is associated with a time-dependent accumulation of murine serrated precursors (P=3x10-10) and advanced murine serrated lesions and invasive cancer (8x10- 8). Kras mutant animals acquire fewer precursor lesions (P=0.06) and have a significantly lower probability of developing advanced serrated lesions (P=0.004). Braf and Kras mutant animals develop pronounced hyperplasia, however the severity is significantly less in Kras mutant animals. Kras mutant advanced serrated lesions rarely develop aberrant WNT signaling activation (1/23). Gene expression profiling showed divergent transcriptomic profiles between Braf and Kras mutant 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 the Braf mutant intestine (P=0.0008), contributing to chronic inflammatory signalling. Both Kras and Braf mutations 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 the Kras mutant intestine compared with Braf mutant animals. Conclusions: In this study, we show that Kras mutation can induce serrated intestinal neoplasia, however the latency period and penetrance is significantly lower when compared with Braf mutation. Aberrant WNT signalling is common in lesions arising in the context of Braf mutation, but rare in Kras mutant neoplasms. We show marked transcriptomic disparities between these models, with a tendency for the Braf mutant intestine to upregulate immunological processes. Our DNA methylation analysis reveals an attenuated CIMP-like phenotype that is specific to the Kras mutant 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. ### Competing Interest Statement The authors have declared no competing interest.