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.

Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.metabolite profiling | metabolic subtypes in PDAC | glycolysis | lipid synthesis | biomarkers for metabolic inhibitors M etabolic reprogramming during tumorigenesis is an essential process in nearly all cancer cells. Tumors share a common phenotype of uncontrolled cell proliferation and must efficiently generate the energy and macromolecules required for cellular growth. The first example of metabolic reprogramming was discovered more than 80 y ago by Otto Warburg: tumor cells can shift from oxidative to fermentative metabolism in the course of oncogenesis (1). More recently, there has been a resurgence of interest in targeting cancer metabolism (2-4) because it may not only be effective in inhibiting tumor growth, but may also provide a therapeutic window (5, 6). For example, inactivation of lactate dehydrogenase-A (LDHA), an enzyme that catalyzes the final step of aerobic glycolysis, thereby reducing pyruvate to lactate, decreases tumorigenesis and induces regression of established tumors in mouse models of lung cancer driven by oncogenic KRAS or epidermal growth factor receptor (EGFR) while minimally affecting normal cell function (7). The finding that cancers have altered metabolism has prompted substantial investigation, both preclinically and in clinical trials, of several metabolically targeted agents, including those that elevate reactive oxygen species (ROS) or block glycolysis, lipid synthesis, mitochondrial function, and glutamine synthesis pathways (8).The identification of distinct metabolic reprogramming events or metabolic subtypes in cancer may inform patient selection for investigational...

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.

KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.

The enzyme glutaminase (GLS1) is currently in clinical trials for oncology, yet there are no clear diagnostic criteria to identify responders. The evaluation of 25 basal breast lines expressing GLS1, predominantly through its splice isoform GAC, demonstrated that only GLS1-dependent basal B lines required it for maintaining de novo glutathione synthesis in addition to mitochondrial bioenergetics. Drug sensitivity profiling of 407 tumor lines with GLS1 and gamma-glutamylcysteine synthetase (GCS) inhibitors revealed a high degree of co-dependency on both enzymes across indications, suggesting that redox balance is a key function of GLS1 in tumors. To leverage these findings, we derived a pan-cancer metabolic signature predictive of GLS1/GCS co-dependency and validated it in vivo using four lung patient-derived xenograft models, revealing the additional requirement for expression of GAC above a threshold (logRPKM + 1 ≥ 4.5, where RPKM is reads per kilobase per million mapped reads). Analysis of the pan-TCGA dataset with our signature identified multiple indications, including mesenchymal tumors, as putative responders to GLS1 inhibitors.