title: DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412.
abstract: The presence of internal tandem duplications (ITD) mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor influences the risk of relapse in acute myeloid leukaemia (AML). We have investigated DNA repair in FLT3-ITD and wild-type (WT) cells. Using the comet assay, we have demonstrated that the FLT3 inhibitor PKC412 significantly inhibits repair of DNA damage in the MV4-11-FLT3-ITD cell line and FLT3-ITD patient samples but not in the HL-60-FLT3-WT cell line or FLT3-WT patient samples. Following the discovery that transcript levels of the DNA repair gene RAD51 are significantly correlated with FLT3 transcript levels in FLT3-ITD patients, we further investigated the role of RAD51 in FLT3-ITD-AML. The reduction in DNA repair in PKC412-treated FLT3-ITD cells was shown to be associated with downregulation of RAD51 mRNA and protein expression and correlates with the maintenance of phosphorylated H2AX levels, implying that PKC412 inhibits the homologous recombination double-strand break repair pathway in FLT3-ITD cells. Using FLT3-short interfering RNA (siRNA), we also demonstrated that genetic silencing of FLT3 results in RAD51 downregulation in FLT3-ITD cells but not in FLT3-WT cells. This work suggests that the use of FLT3 inhibitors such as PKC412 may reverse the drug-resistant phenotype of FLT3-ITD-AML cells by inhibiting repair of chemotherapy-induced genotoxic damage and thereby reduce the risk of disease relapse.
authors.affiliation: Department of Academic Haematology, School of Molecular Medical Sciences, Nottingham University Hospitals and University of Nottingham, Nottingham, UK. claire.seedhouse@nottingham.ac.uk
authors.forename: C H
authors.initials: CH
authors.lastname: Seedhouse
authors.forename: H M
authors.initials: HM
authors.lastname: Hunter
authors.forename: B
authors.initials: B
authors.lastname: Lloyd-Lewis
authors.forename: A-M
authors.initials: AM
authors.lastname: Massip
authors.forename: M
authors.initials: M
authors.lastname: Pallis
authors.forename: G I
authors.initials: GI
authors.lastname: Carter
authors.forename: M
authors.initials: M
authors.lastname: Grundy
authors.forename: S
authors.initials: S
authors.lastname: Shang
authors.forename: N H
authors.initials: NH
authors.lastname: Russell
mesh_terms: D004260:DNA Repair; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020080:Tandem Repeat Sequences; D051941:fms-Like Tyrosine Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics*
chemical_list: D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D050799:STAT5 Transcription Factor; D005047:Etoposide; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D051135:Rad51 Recombinase; D019311:Staurosporine; C059539:midostaurin