title: RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma.
abstract: Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.
authors.affiliation: Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.
authors.forename: Kristina A
authors.initials: KA
authors.lastname: Cole
authors.forename: Jonathan
authors.initials: J
authors.lastname: Huggins
authors.forename: Michael
authors.initials: M
authors.lastname: Laquaglia
authors.forename: Chase E
authors.initials: CE
authors.lastname: Hulderman
authors.forename: Mike R
authors.initials: MR
authors.lastname: Russell
authors.forename: Kristopher
authors.initials: K
authors.lastname: Bosse
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Diskin
authors.forename: Edward F
authors.initials: EF
authors.lastname: Attiyeh
authors.forename: Rachel
authors.initials: R
authors.lastname: Sennett
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Norris
authors.forename: Marci
authors.initials: M
authors.lastname: Laudenslager
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Wood
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Mayes
authors.forename: Jayanti
authors.initials: J
authors.lastname: Jagannathan
authors.forename: Cynthia
authors.initials: C
authors.lastname: Winter
authors.forename: Yael P
authors.initials: YP
authors.lastname: Mosse
authors.forename: John M
authors.initials: JM
authors.lastname: Maris
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D016503:Drug Delivery Systems; D004353:Drug Evaluation, Preclinical; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000188:drug therapy* / Q000473:pathology; D009687:Nuclear Proteins / Q000032:analysis; D015513:Oncogene Proteins / Q000032:analysis; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects
chemical_list: C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1