title: A kinome screen identifies checkpoint kinase 1 (CHK1) as a sensitizer for RRM1-dependent gemcitabine efficacy.
abstract: Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.
authors.affiliation: Molecular Therapeutics Program and Molecular Imaging and Biomarkers Program, Karmanos Cancer Institute, Detroit, MI, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.forename: Zhengming
authors.initials: Z
authors.lastname: Chen
authors.forename: Agnes
authors.initials: A
authors.lastname: Malysa
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Paula
authors.initials: P
authors.lastname: Oliveira
authors.forename: Yingtao
authors.initials: Y
authors.lastname: Zhang
authors.forename: Gerold
authors.initials: G
authors.lastname: Bepler
mesh_terms: D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012262:Ribonucleoside Diphosphate Reductase; D054852:Small Molecule Libraries / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D016896:Treatment Outcome; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: C532363:3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D054852:Small Molecule Libraries; D013876:Thiophenes; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; D014508:Urea; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D000093542:Gemcitabine