title: USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer cells expressing mid-to-high MCL1.
abstract: OBJECTIVE
  Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT.
  
  
  METHODS
  Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday-Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting.
  
  
  RESULTS
  USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition.
  
  
  CONCLUSIONS
  These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.
authors.affiliation: a Department of Radiation Oncology ; Dana-Farber Cancer Institute ; Boston , MA USA.
authors.forename: Deepa
authors.initials: D
authors.lastname: Kushwaha
authors.forename: Colin
authors.initials: C
authors.lastname: O'Leary
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Cron
authors.forename: Peter
authors.initials: P
authors.lastname: Deraska
authors.forename: Kaya
authors.initials: K
authors.lastname: Zhu
authors.forename: Alan D
authors.initials: AD
authors.lastname: D'Andrea
authors.forename: David
authors.initials: D
authors.lastname: Kozono
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D014408:Biomarkers, Tumor / Q000096:biosynthesis* / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology / Q000532:radiotherapy; D045744:Cell Line, Tumor; D003487:Cyanoacrylates / Q000008:administration & dosage; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D006801:Humans; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000096:biosynthesis* / Q000235:genetics; D011725:Pyridines / Q000008:administration & dosage; D011827:Radiation; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D003487:Cyanoacrylates; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011725:Pyridines; C506234:USP9X protein, human; C519751:degrasyn; D043222:Ubiquitin Thiolesterase
keywords: DUB, deubiquitinase; IR, ionizing radiation; MCL1; NSCLC, non-small cell lung cancer; RNAi screen; RT, radiotherapy; USP9X; apoptosis; lung cancer; radiosensitizing agents; shRNA, short hairpin RNA; siRNA, small interfering RNA (siRNA)