ABSTRACT
Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
Subject(s)
Drug Resistance, Neoplasm/genetics , Melanoma/drug therapy , Melanoma/genetics , Mutation , Proto-Oncogene Proteins A-raf/antagonists & inhibitors , Proto-Oncogene Proteins A-raf/genetics , raf Kinases/antagonists & inhibitors , Animals , Cell Line , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Female , Humans , Melanoma/pathology , Mice , Protein Multimerization/drug effects , Proto-Oncogene Proteins A-raf/chemistry , raf Kinases/chemistryABSTRACT
Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.
Subject(s)
Neoplasms/drug therapy , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Quinazolinones/therapeutic use , raf Kinases/antagonists & inhibitors , Animals , Azetidines/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dogs , Drug Combinations , Drug Synergism , Female , Humans , Madin Darby Canine Kidney Cells , Mice, Nude , Molecular Structure , Mutation , Phenylurea Compounds/chemistry , Phenylurea Compounds/metabolism , Piperidines/therapeutic use , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Quinazolinones/chemistry , Quinazolinones/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor Assays , raf Kinases/genetics , raf Kinases/metabolismABSTRACT
The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.