title: Trametinib radiosensitises RAS- and BRAF-mutated melanoma by perturbing cell cycle and inducing senescence.
abstract: OBJECTIVE
  Radiotherapy (RT) is used frequently in patients with melanoma, but results are suboptimal because the disease is often radioresistant. This may be due to constitutive activation of MAPK pathway signalling through mutations involving RAS/RAF. Thus, we studied whether trametinib, a potent and selective allosteric inhibitor of MEK1/2 could improve the efficacy of RT.
  
  
  METHODS
  Clonogenic survival assays were performed in human BRAF-mutant (A375), NRAS-mutant (D04, WM1631), KRAS-mutant (WM1791c) and wild-type (PMWK) melanoma cell lines. The effects of trametinib with and without radiation on protein levels of MEK effectors were measured by immunoblot analyses. Cell cycle effects, DNA damage repair, mitotic catastrophe and senescence were measured using flow cytometry, γH2Ax staining, nuclear fragmentation and β-galactosidase staining, respectively. Additionally, athymic mice with D04 flank tumours were treated with fractionated RT after gavage with trametinib and monitored for tumour growth.
  
  
  RESULTS
  All cell lines, except PMWK, exhibited enhanced cytotoxicity when RT was combined with trametinib compared to either agent alone. Sensitiser enhancement ratios were 1.70, 1.32, 1.10, and 1.70 for A375, D04, WM1361 and WM1791c, respectively. Trametinib efficiently blocked RT-induced phosphorylation of ERK at nanomolar concentrations. Increased radiosensitivity correlated with prolonged G1 arrest and reduction in the radioresistant S phase up to 48 h following RT. A larger population of senescence-activated β-galactosidase-positive cells was seen in the trametinib pretreated group, and this correlated with activation of two of the major mediators of induced senescence, p53 and pRb. Mice receiving the combination treatment (trametinib 1mg/kg and RT over 3 days) showed a reduced mean tumour volume compared with mice receiving trametinib alone (p=0.016), or RT alone (p=0.047). No overt signs of drug toxicity were observed.
  
  
  CONCLUSIONS
  Trametinib radiosensitised RAS-/RAF-mutated melanoma cells by inducing prolonged G1 arrest and premature senescence. In this pre-clinical study we demonstrate that combining trametinib and RT is well tolerated, and reduces tumour growth in vivo.
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schick
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Joan
authors.initials: J
authors.lastname: Kyula
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Holly
authors.initials: H
authors.lastname: Barker
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Radhika
authors.initials: R
authors.lastname: Patel
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Shane
authors.initials: S
authors.lastname: Zaidi
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Gregory
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Hind
authors.initials: H
authors.lastname: Hafsi
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Victoria
authors.initials: V
authors.lastname: Roulstone
authors.affiliation: INSERM UMR 1030, Gustave Roussy, Villejuif, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Deutsch
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: McLaughlin
authors.affiliation: Targeted Therapy Team, The Institute of Cancer Research, London, United Kingdom. Electronic address: Kevin.Harrington@icr.ac.uk.
authors.forename: Kevin
authors.initials: K
authors.lastname: Harrington
mesh_terms: D000375:Aging / Q000187:drug effects*; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D005260:Female; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008546:Melanoma, Experimental / Q000188:drug therapy*; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000187:drug effects; D011728:Pyridones / Q000494:pharmacology*; D011744:Pyrimidinones / Q000494:pharmacology*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D018631:ras Proteins / Q000187:drug effects
chemical_list: D047428:Protein Kinase Inhibitors; D011728:Pyridones; D011744:Pyrimidinones; D011838:Radiation-Sensitizing Agents; C560077:trametinib; D048493:Proto-Oncogene Proteins B-raf; D018631:ras Proteins
keywords: GSK 1120212; MEK inhibitor; Radiosensitization; Senescence; Trametinib