plx-4720 and Neoplasms

Oncogenic BRAF kinase deregulates the ERK signaling pathway in a large number of human tumors. FDA-approved BRAF inhibitors for BRAFV600E/K tumors have provided impressive clinical responses extending survival of melanoma patients. However, these drugs display paradoxical activation in normal tissue with BRAFWT due to RAF transactivation and priming, acquired drug resistance, and limited clinical effectiveness in non-V600 BRAF-dependent tumors, underscoring the urgent need to develop improved BRAF inhibitors. This review provides an overview of recent structural and biochemical insights into the mechanisms of BRAF regulation by BRAF inhibitors that are linked to their clinical activity, clinical liabilities, and medicinal chemistry properties. The effectiveness and challenges of structurally diverse next generation RAF inhibitors currently in preclinical and clinical development are discussed, along with mechanistic insights for developing more effective RAF inhibitors targeting different oncogenic BRAF conformations.

Topics: Animals; Drug Discovery; Humans; Mutation; Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bridged Bicyclo Compounds, Heterocyclic; Drug Screening Assays, Antitumor; Humans; Indoles; MAP Kinase Signaling System; Mitochondrial Dynamics; Molecular Targeted Therapy; Neoplasm Proteins; Neoplasms; Oncogene Addiction; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides

Data from several large high-throughput drug response screens have become available to the scientific community recently. Although many efforts have been made to use this information to predict drug sensitivity, our ability to accurately predict drug response based on genetic data remains limited. In order to systematically examine how different aspects of modelling affect the resulting prediction accuracy, we built a range of models for seven drugs (erlotinib, pacliatxel, lapatinib, PLX4720, sorafenib, nutlin-3 and nilotinib) using data from the largest available cell line and xenograft drug sensitivity screens. We found that the drug response metric, the choice of the molecular data type and the number of training samples have a substantial impact on prediction accuracy. We also compared the tasks of drug response prediction with tissue type prediction and found that, unlike for drug response, tissue type can be predicted with high accuracy. Furthermore, we assessed our ability to predict drug response in four xenograft cohorts (treated either with erlotinib, gemcitabine or paclitaxel) using models trained on cell line data. We could predict response in an erlotinib-treated cohort with a moderate accuracy (correlation ≈ 0.5), but were unable to correctly predict responses in cohorts treated with gemcitabine or paclitaxel.

Topics: Animals; Biomarkers, Pharmacological; Cell Line, Tumor; Erlotinib Hydrochloride; Humans; Imidazoles; Indoles; Lapatinib; Machine Learning; Mice; Neoplasms; Organ Specificity; Paclitaxel; Piperazines; Prognosis; Pyrimidines; Sorafenib; Sulfonamides; Xenograft Model Antitumor Assays

The development of resistance remains a major obstacle to long-term disease control in cancer patients treated with targeted therapies. In BRAF-mutant mouse models, we demonstrate that although targeted inhibition of either BRAF or VEGF initially suppresses the growth of BRAF-mutant tumors, combined inhibition of both pathways results in apoptosis, long-lasting tumor responses, reduction in lung colonization, and delayed onset of acquired resistance to the BRAF inhibitor PLX4720. As well as inducing tumor vascular normalization and ameliorating hypoxia, this approach induces remodeling of the extracellular matrix, infiltration of macrophages with an M1-like phenotype, and reduction in cancer-associated fibroblasts. At the molecular level, this therapeutic regimen results in a de novo transcriptional signature, which sustains and explains the observed efficacy with regard to cancer progression. Collectively, our findings offer new biological rationales for the management of clinical resistance to BRAF inhibitors based on the combination between BRAF

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Indoles; Mice; Mutant Proteins; Mutation, Missense; Neoplasms; Proto-Oncogene Proteins B-raf; Sulfonamides; Treatment Outcome; Vascular Endothelial Growth Factor A

Activation of the ERK1/2 mitogen-activated protein kinases (MAPK) confers resistance to the RAF inhibitors vemurafenib and dabrafenib in mutant BRAF-driven melanomas. Methods to understand how resistance develops are important to optimize the clinical use of RAF inhibitors in patients. Here, we report the development of a novel ERK1/2 reporter system that provides a noninvasive, quantitative, and temporal analysis of RAF inhibitor efficacy in vivo. Use of this system revealed heterogeneity in the level of ERK1/2 reactivation associated with acquired resistance to RAF inhibition. We identified several distinct novel and known molecular changes in resistant tumors emerging from treatment-naïve cell populations including BRAF V600E variants and HRAS mutation, both of which were required and sufficient for ERK1/2 reactivation and drug resistance. Our work offers an advance in understanding RAF inhibitor resistance and the heterogeneity in resistance mechanisms, which emerge from a malignant cell population.

Topics: Animals; Drug Resistance, Neoplasm; Enzyme Activation; Female; Genes, Reporter; Genetic Heterogeneity; Green Fluorescent Proteins; Humans; Indoles; MAP Kinase Signaling System; Mice; Mice, Nude; Mice, Transgenic; Neoplasms; Proto-Oncogene Proteins B-raf; Selection, Genetic; Sulfonamides; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Carcinomas are comprised of transformed epithelial cells that are supported in their growth by a dedicated neovasculature. How the genetic milieu of the epithelial compartment influences tumor angiogenesis is largely unexplored. Drugs targeted to mutant cancer genes may act not only on tumor cells but also, directly or indirectly, on the surrounding stroma. We investigated the role of the BRAF(V600E) oncogene in tumor/vessel crosstalk and analyzed the effect of the BRAF inhibitor PLX4720 on tumor angiogenesis. Knock-in of the BRAF(V600E) allele into the genome of human epithelial cells triggered their angiogenic response. In cancer cells harboring oncogenic BRAF, the inhibitor PLX4720 switches off the ERK pathway and inhibits the expression of proangiogenic molecules. In tumor xenografts harboring the BRAF(V600E), PLX4720 extensively modifies the vascular network causing abrogation of hypoxia. Overall, our results provide a functional link between oncogenic BRAF and angiogenesis. Furthermore, they indicate how the tumor vasculature can be "indirectly" besieged through targeting of a genetic lesion to which the cancer cells are addicted.

Topics: Alleles; Angiogenesis Inducing Agents; Animals; Antibodies, Monoclonal, Humanized; Bevacizumab; Blood Vessels; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Chickens; Chorioallantoic Membrane; Cytostatic Agents; Down-Regulation; Gene Knock-In Techniques; Humans; Indoles; MAP Kinase Signaling System; Mice; Molecular Targeted Therapy; Mutation; Necrosis; Neoplasms; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Sulfonamides; Xenograft Model Antitumor Assays

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.

Topics: Adenosine Triphosphate; Animals; Benzamides; Cell Line; Cell Membrane; Cell Proliferation; Diphenylamine; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Indenes; Indoles; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Neoplasms; Protein Kinase Inhibitors; Protein Multimerization; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Pyrazoles; raf Kinases; ras Proteins; Sulfonamides; Xenograft Model Antitumor Assays

Tumours with mutant BRAF are dependent on the RAF-MEK-ERK signalling pathway for their growth. We found that ATP-competitive RAF inhibitors inhibit ERK signalling in cells with mutant BRAF, but unexpectedly enhance signalling in cells with wild-type BRAF. Here we demonstrate the mechanistic basis for these findings. We used chemical genetic methods to show that drug-mediated transactivation of RAF dimers is responsible for paradoxical activation of the enzyme by inhibitors. Induction of ERK signalling requires direct binding of the drug to the ATP-binding site of one kinase of the dimer and is dependent on RAS activity. Drug binding to one member of RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF) inhibits one protomer, but results in transactivation of the drug-free protomer. In BRAF(V600E) tumours, RAS is not activated, thus transactivation is minimal and ERK signalling is inhibited in cells exposed to RAF inhibitors. These results indicate that RAF inhibitors will be effective in tumours in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than mitogen-activated protein kinase (MEK) inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions have been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization by elevation of wild-type RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumours. In agreement with this prediction, RAF inhibitors do not inhibit ERK signalling in cells that coexpress BRAF(V600E) and mutant RAS.

Topics: Adenosine Triphosphate; Animals; Catalytic Domain; Cell Line; Cell Line, Tumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Indoles; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Neoplasms; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Protein Multimerization; Proto-Oncogene Proteins B-raf; raf Kinases; ras Proteins; Sulfonamides; Transcriptional Activation