Crypt density and recruited enhancers underlie intestinal tumour initiation

Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence. Mutation of Adenomatous polyposis coli (Apc), the most common initiating event in conventional adenomas, activates Wnt signaling, hence conferring fitness on mutant intestinal stem cells (ISCs). Apc mutations may occur in ISCs that arose by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar, it is unclear if both generate tumours equally well in uninjured intestines. Also unknown is whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here, we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs not associated with adenomas. These cis-elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumourigenesis. To examine the effects of Apc loss (constitutive Wnt activation) on different cell types and in different contexts, we performed RNA-seq on primary mouse intestinal stem cells (ISCs) and intestinal organoids with and without Apc loss. Primary ISCs were isolated from the proximal (Duodenum) or distal (Ileum) one-third of the intestinal length and Apc loss was driven by either Lgr5-Cre (ISC-restricted) or Atoh1-Cre (intestinal secretory lineage) mouse strains. Whole organoids were examined 10 days after isolated crypts were plated.