Targeted disruption of BRAF V600E in colorectal cancer as a model system for the identification of novel approaches towards individualized tumor therapy

BRAF kinase is part of the Raf-Ras-Mek-Erk signaling pathway and is mutated in various tumor entities. In colorectal cancer (CRC), the BRAF V600E mutation is found in approximately 18% of tumors. It is a bona fide modulator of resistance against EGFR-antibody therapy.

We utilized AAV-mediated gene targeting in the CRC cell line RKO, which harbors two mutated (V600E) and one wild-type (WT) BRAF allele, to establish tumor cell clones with a single WT or mutated allele, respectively.

The knockout (KO) was structurally confirmed by direct sequencing and functionally validated by detection of decreased Erk phosphorylation in WT cells. Unexpectedly, neither differences in response to conventional chemotherapeutics nor to agents targeting EGFR or RAF were observed. However, cells lacking a mutated BRAF allele showed a decreased proliferation, an effect enhanced by serum deprivation. This phenotype was not ascribable to senescence, as shown by X-gal staining and qPCR, but to strongly increased apoptosis, as shown by FACS.

In summary, using a novel cellular KO model, we provide evidence that disruption of mutant BRAF results in the loss of self-sufficiency of growth signals. On the other hand, targeting of BRAF alone is not sufficient to treat BRAF-mutated cancer, further supporting a non-linear model of MAPK signaling. Current studies focus on the mechanism underlying the newly identified BRAF phenotype, which might facilitate novel therapeutic strategies in BRAF-mutated cancer subsets.