title: Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status.
abstract: BACKGROUND
  Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.
  
  
  RESULTS
  The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.
  
  
  CONCLUSIONS
  Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.
authors.affiliation: Cancer and Cell Biology Division, Translational Genomics Research Institute, Phoenix, AZ, USA.
authors.forename: Sara A
authors.initials: SA
authors.lastname: Byron
authors.forename: David C
authors.initials: DC
authors.lastname: Loch
authors.forename: Candice L
authors.initials: CL
authors.lastname: Wellens
authors.forename: Andreas
authors.initials: A
authors.lastname: Wortmann
authors.forename: Jiayi
authors.initials: J
authors.lastname: Wu
authors.forename: John
authors.initials: J
authors.lastname: Wang
authors.forename: Kenichi
authors.initials: K
authors.lastname: Nomoto
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Pollock
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D007783:Lactones / Q000494:pharmacology*; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: C545120:14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione; D000970:Antineoplastic Agents; D002867:Chromones; D007783:Lactones; D009025:Morpholines; D047428:Protein Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human