Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/ extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogenactivated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.

Insulin-like growth factor-I inhibits transforming growth factor-b (TGF-b) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3-kinase (PI3K)/Aktdependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF-b responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (Akt WT and Akt Myr ) suppress TGF-b-induced phospho-activation of S3 but not Smad2 (S2), whereas kinasedead Akt1 (Akt1 K179M ) or dominant-negative PI3K enhances TGF-b-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-b type I receptor (ALK5), we demonstrate that mammalian target of Rap (mTOR) mediates Akt1 suppression of phospho-activation of S3. These and further data on Akt1-S3 binding do not support a recently proposed model that Akt blocks S3 activation through physical interaction and sequestration of S3 from TGF-b receptors. We propose a novel model whereby Akt suppresses activation of S3 in an Akt kinase-dependent manner through mTOR, a likely route for loss of tumor suppression by TGF-b in cancers.