BRAFV600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

BRAF(V600E)confers poor prognosis and is associated with a distinct subtype of colorectal cancer (CRC). Little is known, however, about the genetic events driving the initiation and progression of BRAF(V600E)mutant CRCs. Recent genetic analyses of CRCs indicate that BRAF(V600E)often coexists with alterations in the WNT- and p53 pathways, but their cooperation remains ill-defined. Therefore, we systematically compared small and large intestinal organoids from mice harboring conditionalBraf(floxV600E),Trp53(LSL-R172H), and/orApc(flox/flox)alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAF(V600E)expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAF(V600E)alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAF(V600E)induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAF(V600E)-driven CRC. Co-expression of p53(R172H)delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53(R172H)cooperates with BRAF(V600E)to modulate the abundance of transcripts linked to carcinogenesis, in particular within colonic organoids. Remarkably, WNT-pathway activation byApcdeletion fully protects organoids against BRAF(V600E)-induced disintegration and confers growth/niche factor independence. Still,Apc-deficient BRAF(V600E)-mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53(R172H)confers partial resistance against this clinically relevant compound. In summary, our systematic comparison of the response of small and large intestinal organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. In addition, our work on BRAF-, p53-, and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.