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.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Raf inhibitors target ras spatiotemporal dynamics.

BACKGROUND: The lateral segregation of Ras proteins into transient plasma membrane nanoclusters is essential for high-fidelity signal transmission by the Ras mitogen-activated protein kinase (MAPK) cascade. In this spatially constrained signaling system, the dynamics of Ras nanocluster assembly and disassembly control MAPK signal output. RESULTS: We show here that BRaf inhibitors paradoxically activate CRaf and MAPK signaling in Ras transformed cells by profoundly dysregulating Ras nanocluster dynamics. Specifically, BRaf inhibitors selectively enhance the plasma membrane nanoclustering of oncogenic K-Ras and N-Ras but have no effect on H-Ras nanoclustering. Raf inhibitors are known to drive the formation of stable BRaf-CRaf and CRaf-CRaf dimers. Our results demonstrate that the presence of two Ras-binding domains in a single Raf dimer is sufficient and required to increase Ras nanoclustering, indicating that Raf dimers promote K- and N-Ras nanocluster formation by crosslinking constituent Ras proteins. Ras crosslinking increases the fraction of K-Ras and N-Ras in their cognate nanoclusters, leading to an increase in MAPK output from the plasma membrane. Intriguingly, increased MAPK signaling in BRaf inhibited cells is accompanied by significantly decreased Akt activation. We show that this signal pathway crosstalk results from a novel mechanism of competition between stabilized Raf dimers and p110α for recruitment to Ras nanoclusters. CONCLUSIONS: Our findings reveal that BRaf inhibitors disrupt Ras nanocluster dynamics with significant, yet divergent, consequences for MAPK and PI3K signaling.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia.

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.