title: Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.
abstract: In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA; Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria; Leukemia Biology Group, Children's Cancer Research Institute, Vienna, Austria; School of Medicine, Sigmund Freud University, Vienna, Austria. Electronic address: cposch81@gmail.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Posch
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA; Department of Medical Sciences, Section of Dermatology, University of Turin, Turin, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Sanlorenzo
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA; Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria; School of Medicine, Sigmund Freud University, Vienna, Austria.
authors.forename: Igor
authors.initials: I
authors.lastname: Vujic
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, California, USA.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Oses-Prieto
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cholewa
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Sarasa T
authors.initials: ST
authors.lastname: Kim
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Lai
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Mitchell
authors.initials: M
authors.lastname: Zekhtser
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Rosaura
authors.initials: R
authors.lastname: Esteve-Puig
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Green
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, California, USA.
authors.forename: Shreya
authors.initials: S
authors.lastname: Chand
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, California, USA.
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.affiliation: Leukemia Biology Group, Children's Cancer Research Institute, Vienna, Austria.
authors.forename: Renate
authors.initials: R
authors.lastname: Panzer-Grümayer
authors.affiliation: Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria.
authors.forename: Klemens
authors.initials: K
authors.lastname: Rappersberger
authors.affiliation: Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA.
authors.forename: Susana
authors.initials: S
authors.lastname: Ortiz-Urda
mesh_terms: D047390:Casein Kinase II / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D008565:Membrane Proteins / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology*
chemical_list: D008565:Membrane Proteins; D010748:Phosphopeptides; D012333:RNA, Messenger; D058539:Phosphatidylinositol 3-Kinase; C570325:CSNK2A1 protein, human; D047390:Casein Kinase II; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human