title: Activity-Based Protein Profiling Shows Heterogeneous Signaling Adaptations to BRAF Inhibition.
abstract: Patients with BRAF V600E mutant melanoma are typically treated with targeted BRAF kinase inhibitors, such as vemurafenib and dabrafenib. Although these drugs are initially effective, they are not curative. Most of the focus to date has been upon genetic mechanisms of acquired resistance; therefore, we must better understand the global signaling adaptations that mediate escape from BRAF inhibition. In the current study, we have used activity-based protein profiling (ABPP) with ATP-analogue probes to enrich kinases and other enzyme classes that contribute to BRAF inhibitor (BRAFi) resistance in four paired isogenic BRAFi-naïve/resistant cell line models. Our analysis showed these cell line models, which also differ in their PTEN status, have considerable heterogeneity in their kinase ATP probe uptake in comparing both naïve cells and adaptations to chronic drug exposure. A number of kinases including FAK1, SLK, and TAOK2 had increased ATP probe uptake in BRAFi resistant cells, while KHS1 (M4K5) and BRAF had decreased ATP probe uptake in the BRAFi-resistant cells. Gene ontology (GO) enrichment analysis revealed BRAFi resistance is associated with a significant enhancement in ATP probe uptake in proteins implicated in cytoskeletal organization and adhesion, and decreases in ATP probe uptake in proteins associated with cell metabolic processes. The ABPP approach was able to identify key phenotypic mediators critical for each BRAFi resistant cell line. Together, these data show that common phenotypic adaptations to BRAF inhibition can be mediated through very different signaling networks, suggesting considerable redundancy within the signaling of BRAF mutant melanoma cells.
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: Ritin
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: Inna
authors.initials: I
authors.lastname: Fedorenko
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: Paige T
authors.initials: PT
authors.lastname: Spence
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: Vernon K
authors.initials: VK
authors.lastname: Sondak
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: Keiran S M
authors.initials: KS
authors.lastname: Smalley
authors.affiliation: Molecular Oncology, ‡Tumor Biology, §Cutaneous Oncology, and ∥The Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center & Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Koomen
mesh_terms: D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004798:Enzymes / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000503:physiopathology*; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D004798:Enzymes; D047428:Protein Kinase Inhibitors; D010770:Phosphotransferases; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; activity-based protein profiling; drug resistance; melanoma; proteomics