AbstractBRAF^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 conditional Braf^floxV600E, Trp53^LSL-R172H, and/or Apc^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 by Apc deletion 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.