Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.

CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation.

The CRAF protein kinase regulates proliferative, differentiation, and survival signals from activated RAS proteins to downstream effectors, most often by inducing MEK/ERK activation. A well-established model of CRAF regulation involves RAS-mediated translocation of CRAF to the plasma membrane, where it is activated by a series of events including phosphorylation. Here we have discovered a new mode of regulation that occurs prior to this step. By creating a kinase-defective version of CRAF in mice or by use of the RAF inhibitor sorafenib, we show that CRAF must first undergo autophosphorylation of serine 621 (S621). Autophosphorylation occurs in cis, does not involve MEK/ERK activation, and is essential to ensure the correct folding and stability of the protein. In the absence of S621 phosphorylation, CRAF is degraded by the proteasome by mechanisms that do not uniquely rely on the E3 ubiquitin ligase CHIP.

BRAF inactivation drives aneuploidy by deregulating CRAF.

Aspartate-594 is the third most common BRAF residue mutated in human cancer. Mutants of this residue are kinase inactive, and the mechanism(s) by which they contribute to cancer has remained perplexing. Using a conditional knock-in mouse model, we show that the (D594A)Braf mutant does not drive tumor development per se but is able to induce aneuploidy in murine splenocytes and mouse embryonic fibroblasts and contributes to immortalization through the propagation of aneuploid cells. (D594A)Braf lacks kinase activity but induces the related gene product Craf as well as the mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. Here, we show that the aneuploid phenotype is dependent on Craf. Treatment with the MEK inhibitor U0126 did not attenuate the emergence of aneuploidy but prevented the growth of aneuploid cells. These results provide a previously unidentified link between Craf and chromosomal stability, with important implications for our understanding of the development of cancers with driver mutations that hyperactivate Craf.