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.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

No abstract

The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D , displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit . Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition. INTRODUCTIONReceptor kinases (RKs) play a key role in important cellular processes in plants and animals (Fantl et al., 1993; Song et al., 1995;Becraft et al., 1996;Heldin and Ostman, 1996; Stein et al., 1996;Ten Dijke et al., 1996;Torii et al., 1996;Li and Chory, 1997). Three functional domains are commonly associated with RK proteins: an extracellular domain, a transmembrane domain, and an intracellular catalytic domain. Studies of animal RKs have revealed a common mechanism for RK-mediated cellular signaling (Hunter, 1995;Pawson, 1995;Heldin and Ostman, 1996). In this model, ligand binding to the extracellular receptor domain induces receptor dimerization and subsequent activation of the intracellular kinase domain. The specificity of the interaction with the ligand is controlled by amino acid residues in the extracellular domain (Heldin and Ostman, 1996).Plant RKs can be divided into six subclasses based on the protein motif in the presumed extracellular domains (Walker, 1994;Becraft et al., 1996). The largest subclass of plant RKs is the leucine-rich repeat (LRR) group, which encodes proteins with an extracellular domain containing 20 to 25 imperfect repeats of a 24-amino acid leucine-rich motif. The LRR subclass of plant RKs includes proteins that govern pollen development, plant elongation, regulation of meristem and flower development, disease resistance, and brassinosteroid signal transduction, as well as other functions that remain to be determined (Chang et al., 1992;Valon et al., 1993; Song et al., 1995;Torii et al., 1996;Clark et al., 1997;Li and Chory, 1997). Plant LRRs have also been found in secreted proteins (polygalacturonase inhibitor proteins or PGIPs) (De Lorenzo et al., 1994) and in membrane-bound resistance gene products (Dixon et al., 1996). LRR domains are present in a variety of proteins involved in peptide ligand recognition, cell adhesion, and various other functions and are thought to mediate protein-protein interactions (Braun et al., 1991;Kobe and Deisenhofer, 1994).The cloning and characterization of the rice Xa21 gene demonstrated that LRR-containing RKs function in plant disease resistance. Xa21 confers race-specific resistance to Xanthomonas oryzae pv ...