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.

Purpose: Acute myeloid leukemia cells with an internal tandem duplication mutation of FLT3 (FLT3-ITD) have effective DNA repair mechanisms on exposure to drugs. Despite this, the phenotype is not associated with primary resistant disease. We show defects in the response of mutant FLT3 AML cells to the S-phase drug clofarabine that could account for the apparent contradiction. Experimental Design: We studied responses of AML cells to clofarabine in vitro. Results: When treated with a short pulse of clofarabine, FLT3-ITD-harboring MOLM-13 and MV4.11 cells undergo similar damage levels (γH2AX foci) to wild-type cells but have a better repair capability than wild-type cells. However, whereas the wild-type cells undergo rapid S-phase arrest, the S-phase checkpoint fails in mutant cells. Cell cycle arrest in response to DNA damage in S phase is effected via loss of the transcriptional regulator cdc25A. This loss is reduced or absent in clofarabine-treated FLT3 mutant cells. Furthermore, cdc25A message levels are maintained by the FLT3-ITD, such that message is reduced by 87.5% on exposure to FLT3 small interfering RNA. Primary FLT3-ITD samples from untreated patients also display impaired cell cycle arrest and show enhanced sensitivity on prolonged treatment with clofarabine compared with wild-type samples. Clofarabine is a second-generation purine nucleoside analogue with favorable pharmacologic properties (resistance to deaminase degradation and stability in gastric acid) and is in increasing clinical use for the treatment of AML (5-9). Because of the potential oral availability and its acceptable toxicity with respect to mucositis and alopecia, its use has recently been explored in older AML patients with promising results. 3Mechanisms supporting heterogeneity in sensitivity and resistance to clofarabine have been reported in the literature; particularly, a variability in the cellular accumulation of clofarabine triphosphate in circulating blasts has been recorded (6, 7). We have previously shown efficient DNA repair following anthracycline exposure in AML cells with a FLT3-ITD (10) and were thus initially interested to discover whether DNA damage and repair mechanisms are important in the response of FLT3-ITD cells to clofarabine. There is a well-characterized assay (the measurement of γH2AX) that can be used as a biomarker of the effectiveness of clofarabine at reaching its DNA target and inducing a damage response (7,11). The use of this assay has enabled us to focus on events subsequent to clofarabine-induced DNA damage and to dissect mechanisms by which cells proceed to apoptosis or recovery subsequent to the initial damage response signals.The damage response pathway is closely linked to cell cycle arrest mechanisms. In a well-documented but fast-evolving model of cell cycle arrest in S phase (the phase in which 3 A.K. Burnett et al

No abstract

SummaryFLT3-internal tandem duplication (ITD) mutations are heterogeneous with regards to length and proportion of DNA harbouring the mutation and the expression level of FLT3 also varies widely, however very little is known about the biological effects of these variables. We studied FLT3-associated biological parameters in 322 acute myeloid leukaemia samples to establish their importance. Expression of total FLT3 transcripts was shown to be significantly higher in the FLT3-ITD cohort (n = 121) compared to the wild-type cohort (P = 0AE004). Whilst phosphorylated signal transducer and activator of transcription 5 (phospho-STAT5) was not confined to FLT3-ITD samples, within the FLT3-ITD group phosphorylation correlated with adjusted FLT3-ITD levels assessed by determining the total transcripts and proportion of FLT3-ITD within a sample. Expression of the STAT5 downstream target Bcl-xl (an isoform of BCL2L1) was strongly correlated with FLT3 total and adjusted FLT3-ITD levels in FLT3-ITD samples (P < 0AE001), however there was no association between Bcl-xl and phospho-STAT5 levels suggesting that STAT5 is not the sole regulator of Bcl-xl in FLT3-ITD cells. We further stratified our cohort by the presence/ absence of a cytoplasmic nucleophosmin NPMc+ mutation. Samples co-expressing NPMc+ had longer FLT3-ITD mutations (P = 0AE01) and there was a high occurrence of NPMc+ in samples that had >1 FLT3-ITD mutation. Phospho-STAT5 levels were reduced in the FLT3-ITD/NPMc+ group (P = 0AE04) suggesting that NPMc+ may oppose the FLT3-ITDdependent activation of STAT5.

No abstract