title: Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.
abstract: Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Penna
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Molla
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Grazia
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Cleris
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Nicolini
authors.affiliation: Laboratory of Molecular Pathology, Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Perrone
authors.affiliation: Laboratory of Molecular Pathology, Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Picciani
authors.affiliation: Medical Oncology Unit 1, Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Del Vecchio
authors.affiliation: Medical Oncology Unit 1, Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: de Braud
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Mortarini
authors.affiliation: Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.|Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Anichini
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005260:Female; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D009154:Mutation / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D048493:Proto-Oncogene Proteins B-raf; D012333:RNA, Messenger; D058570:TOR Serine-Threonine Kinases; C482128:MAP2K2 protein, human; C546842:MTOR protein, human; C482119:BRAF protein, human; C482125:MAP2K1 protein, human
keywords: BRAF; MEK; PI3K/mTOR; apoptosis; melanoma