title: Differential inhibition of ex-vivo tumor kinase activity by vemurafenib in BRAF(V600E) and BRAF wild-type metastatic malignant melanoma.
abstract: BACKGROUND
  Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.
  
  
  METHODS
  In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.
  
  
  RESULTS
  Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.
  
  
  CONCLUSIONS
  Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.
authors.affiliation: Department of Clinical Molecular Biology and Laboratory Sciences, Akershus University Hospital, Lørenskog, Norway ; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: Andliena
authors.initials: A
authors.lastname: Tahiri
authors.forename: Kathrine
authors.initials: K
authors.lastname: Røe
authors.forename: Anne H
authors.initials: AH
authors.lastname: Ree
authors.forename: Rik
authors.initials: R
authors.lastname: de Wijn
authors.forename: Karianne
authors.initials: K
authors.lastname: Risberg
authors.forename: Christian
authors.initials: C
authors.lastname: Busch
authors.forename: Per E
authors.initials: PE
authors.lastname: Lønning
authors.forename: Vessela
authors.initials: V
authors.lastname: Kristensen
authors.forename: Jürgen
authors.initials: J
authors.lastname: Geisler
mesh_terms: D000368:Aged; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology* / Q000627:therapeutic use*; D008297:Male; D008545:Melanoma / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D008875:Middle Aged; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009362:Neoplasm Metastasis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D011758:Pyrroles / Q000494:pharmacology / Q000627:therapeutic use; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D012867:Skin / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D013379:Substrate Specificity / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology* / Q000627:therapeutic use*; D000077210:Sunitinib; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D013449:Sulfonamides; D000077484:Vemurafenib; D066246:ErbB Receptors; D020797:Receptor, Platelet-Derived Growth Factor beta; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D000077210:Sunitinib
references.citation: J Exp Clin Cancer Res. 2000 Mar;19(1):21-34
references.pmid: 10840932
references.citation: Mol Cancer Ther. 2009 Jul;8(7):1846-55
references.pmid: 19584230
references.citation: Nature. 2011 Nov 23;480(7377):387-90
references.pmid: 22113612
references.citation: Expert Opin Investig Drugs. 2011 Feb;20(2):153-208
references.pmid: 21235428
references.citation: J Invest Dermatol. 2010 Oct;130(10):2514-6
references.pmid: 20505745
references.citation: Cancer Res. 2001 May 1;61(9):3595-8
references.pmid: 11325826
references.citation: Int J Radiat Oncol Biol Phys. 2010 Oct 1;78(2):555-62
references.pmid: 20675069
references.citation: Cancer Res. 2011 Apr 1;71(7):2750-60
references.pmid: 21317224
references.citation: Nature. 2010 Sep 30;467(7315):596-9
references.pmid: 20823850
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):27-30
references.pmid: 10592173
references.citation: Acta Oncol. 2012 Jan;51(1):130-3
references.pmid: 22150168
references.citation: Cancer Res. 2009 Jul 15;69(14):5987-95
references.pmid: 19567681
references.citation: N Engl J Med. 2010 Aug 26;363(9):809-19
references.pmid: 20818844
references.citation: Clin Cancer Res. 2012 Jan 1;18(1):33-9
references.pmid: 22215904
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14903-8
references.pmid: 20668238
references.citation: Clin Cancer Res. 2010 Jul 1;16(13):3356-67
references.pmid: 20460471
references.citation: Pigment Cell Melanoma Res. 2010 Apr;23(2):190-200
references.pmid: 20149136
references.citation: Eur J Cancer. 2010 Jul;46(11):2127-33
references.pmid: 20541396
references.citation: Nature. 2010 Dec 16;468(7326):973-7
references.pmid: 21107323
references.citation: Nature. 2012 Jan 26;483(7387):100-3
references.pmid: 22281684
references.citation: Cancer Res. 2006 Oct 1;66(19):9483-91
references.pmid: 17018604
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D109-14
references.pmid: 22080510
references.citation: Cancer Res. 2011 Dec 1;71(23):7137-40
references.pmid: 22131348
references.citation: Melanoma Res. 2012 Jun;22(3):244-51
references.pmid: 22516966
references.citation: J Transl Med. 2010 Apr 20;8:39
references.pmid: 20406486
references.citation: Genes Dev. 2007 Dec 15;21(24):3214-31
references.pmid: 18079171
references.citation: Nature. 2010 Mar 18;464(7287):427-30
references.pmid: 20179705
references.citation: Adv Pharmacol. 2012;65:27-43
references.pmid: 22959022
references.citation: Lancet. 2012 Feb 18;379(9816):633-40
references.pmid: 22257673
references.citation: Neoplasia. 2010 Aug;12(8):637-49
references.pmid: 20689758
references.citation: Cancer Discov. 2012 Sep;2(9):791-7
references.pmid: 22798288
references.citation: Cancer Cell. 2010 Dec 14;18(6):683-95
references.pmid: 21156289
references.citation: Mol Oncol. 2012 Aug;6(4):418-27
references.pmid: 22521242
references.citation: Lancet Oncol. 2012 Mar;13(3):239-46
references.pmid: 22285168
references.citation: Clin Med Insights Oncol. 2012;6:275-86
references.pmid: 22904648
references.citation: N Engl J Med. 2012 Nov;367(18):1694-703
references.pmid: 23020132
references.citation: Lancet. 2012 Jul 28;380(9839):358-65
references.pmid: 22735384
references.citation: Oncogene. 2006 Jun 8;25(24):3357-64
references.pmid: 16462768