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.

Prognostic impact of white blood cell count in intermediate risk acute myeloid leukemia: relevance of mutated NPM1 and FLT3-ITD

“…It is likely that higher levels of FLT3-ITD might trigger pathways involved in chemoresistance more intensively, for example by enhancing DNA repair and salvage of damaged cells. 36 Our observations confirm the importance of the FLT3-ITD/FLT3 ratio, representing the FLT3-ITD allelic burden, with regards to prognosis and demonstrate that WBC count is not a surrogate marker for the FLT3-ITD/FLT3 ratio, but indeed is an independent prognostic factor.…”

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

“…FLT3-ITD was demonstrated to activate alternative unfaithful DNA repair pathways that leads to increased levels of unrepaired DNA damage (22). Interestingly, it was also shown that increased efficiency of FLT3-ITDstimulated DNA repair contributes to drug resistance (23).…”

Internal tandem duplication and tyrosine kinase domain mutations in FLT3 alter the response to daunorubicin in Ba/F3 cells

“…In addition to the well-known role of p53 in apoptosis induction, it has also been shown to induce multiple prosurvival and DNA repair genes (26). Consistent with this, FLT3-ITD-expressing AML cell lines and primary patient samples have increased the levels of reactive oxygen species, double-strand DNA breaks and increased DNA repair capacity (17,27).…”

“…Additionally, DNA repair contributes to the FLT3-ITD drug-resistant phenotype of primary AML (17). Several studies demonstrate drug resistance conferred by FLT3-ITD (16,18,19); however, thus far, no studies have demonstrated the effect of mutations in the FLT3 TKD on anticancer drug resistance.…”