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

Nadine Reischmann and Tilman Brummer

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BRAFV600E is associated with a distinct subtype of colorectal cancer (CRC) and confers poor patient prognosis. However, the (epi)genetic alterations cooperating with BRAFV600E in driving the initiation and progression of this CRC subtype remain ill-defined. Recent genetic analyses of CRCs point towards the coexistence of BRAFV600E with mutations in the tumour suppressor TP53 and alterations in the Wnt signalling pathway. As reconstruction of interactions between different mutations is best conducted by a synthetic approach in which oncogene-naïve systems are transformed systematically by genetic manipulations, we made use of in vitro intestinal organoid cultures from transgenic mice [1]. Therefore, we generated and analysed small intestinal as well as colonic organoids harbouring conditional BraffloxV600E/+, Trp53LSL‑R172H/+ knock-in and Apcflox/flox knock-out alleles, either singly or in combination.

Using these isogenic models, we observed an initial burst of proliferation and disturbed epithelial organisation upon sudden BRAFV600E expression, which eventually led to organoid death. Our data on small as well as colonic organoids support the observations of other studies conducted on small intestinal organoids, which are described in detail in our publication. Thus, BRAFV600E alone is insufficient for malignant transformation and organoid disintegration might reflect a protective mechanism of the intestinal stem cell niche against tumourigenesis. RNA-sequencing of BRAFV600E-mutant organoids demonstrated induction of two simultaneous, but at first sight contrasting, processes: loss of Wnt-dependent stemness and loss of epithelial differentiation features. Interestingly, this gene expression pattern highly resembles the transcriptional profile of previously described fetal intestinal organoids, which is, as we show now in our publication, also observed in human BRAFV600E-driven CRC.

Co-expression of p53R172H delayed BRAFV600E triggered organoid disintegration, conferred anchorage independent growth and induced invasive properties. At the same time, BRAFV600E and p53R172H cooperated in modulating the abundance of transcripts associated with advanced metastatic CRC.

As RNA-sequencing revealed tissue-specific differences towards sudden oncogene expression in small intestinal and colonic organoids, the collaborating effects of BRAFV600E and p53R172H were particularly seen in the latter. The tissue-specificity could be attributed to different ERK pathway ground states that we observed in freshly isolated small and large intestinal crypts. These observations emphasize colonic organoids as the better tool to model the transformation requirements of human CRC and advises the rational choice of the organoid donor tissue in a context-dependent manner.

Remarkably, Wnt-pathway activation by Apc deletion rescued the BRAFV600E-induced loss of the intestinal stem cell niche and fully protected against organoid death. APC loss conferred complete growth/niche factor independence to BRAFV600E-mutant organoids, albeit organoid growth remained MEK-dependent. Of note, although the co-occurrence of BRAFV600E and APC mutations was thought to be less common in CRC patients so far, Fennel and colleagues recently identified an extremely aggressive BRAFV600E mutant CRC subset, which is characterized by the co-existence of APC truncations [2]. The underlying mechanisms and the behaviour of the double-mutant tumour cells were not investigated in this study, but our present publication on murine colonic organoids support their concept that BRAFV600E and APC deficiency cooperate in inducing an overt proliferative phenotype.

Interestingly, co-expression of p53R172H induced invasive properties in the BRAFV600E-mutant and APC-deficient organoids and, importantly, conferred resistance against the clinically relevant MEK inhibitor trametinib.

In summary, our systematic comparison of the response of small intestinal and colonic organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. Additionally, our work on BRAF-, p53- and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.

This image is taken from Reference [1].

References:

1          Reischmann N, Andrieux G, Griffin R, Reinheckel T, Boerries M, Brummer T. BRAFV600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation Oncogene 2020; https://doi.org/10.1038/s41388-020-01414-9.

2          Fennell LJ, Kane A, Liu C, McKeone D, Fernando W, Su C et al. APC Mutation Marks an Aggressive Subtype of BRAF Mutant Colorectal Cancers. Cancers (Basel) 2020; 12.

Nadine Reischmann

PhD candidate, Freiburg University

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