Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies.,In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK.,The DRS is a conserved region that lies distal to the active site and mediates ERK-protein interactions.,We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo.,BI-78D3 does not covalently modify p38MAPK, JNK or ERK5.,BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation.,These studies provide the basis for designing modulators of protein-protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.,The ERK signalling pathway is activated in many cancers, however ERK1 and ERK2 are difficult to target pharmacologically.,Here, the authors identify a small molecule inhibitor that binds covalently to the D-recruitment site of ERK and induces cell death and reduces tumour growth in mice.
Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors.,However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment.,For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets.,Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism.,Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved.,Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested.,We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells.,Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation.,BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation.,Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs.,Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells.,In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH.,As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels.,We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells.,Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).,The online version of this article (doi:10.1186/s12943-017-0667-y) contains supplementary material, which is available to authorized users.