Expression of the DNA Repair Gene RAD51 Is Associated with FLT3 ITD Transcript Level and Is Down-Regulated by PKC412 Resulting in Suppression of DNA Repair.

The presence of internal tandem duplication (ITD) mutations in the FLT3 receptor tyrosine kinase confer an adverse prognosis in AML due to an increased risk of disease relapse. However the mechanisms underlying this increased relapse risk are unclear. We have investigated whether AML cells with FLT3 ITD mutations have an enhanced capacity for DNA repair following cytotoxic drug exposure. RAD51 is a key protein in the high-fidelity homologous recombination double strand break repair pathway and is the limiting factor for this pathway in mammalian cells. Using quantitative real-time PCR we found that the level of RAD51 transcripts are significantly correlated with the level of FLT3 transcripts in FLT3 ITD cells (n=27; p=0.017) but not in FLT3 WT cells (n=57; p=0.58). Clinically FLT3 ITDs have the most significant prognostic impact in AML patients with normal cytogenetics and if this group is studied the association between FLT3 ITD and RAD51 transcript levels are particularly pronounced (n=12; p=0.003).

To establish whether increases in RAD51 expression correlate with enhanced DNA repair activity we have adopted the comet assay and studied the MV4-11 cell line (FLT3 ITD), HL60 cell line (FLT3 WT) and a number of AML patient cells. The background level of DNA damage in untreated FLT3 ITD AML patients and the MV4-11 cell line was significantly lower than in FLT3 WT patients and the HL60 cell line (n=10; p=0.02), suggesting a constitutive up-regulation of DNA repair in cells harbouring the FLT3 ITD mutation resulting in lower background levels of DNA damage.

To test whether the increase in RAD51 and DNA repair was a consequence of the FLT3 ITD, we treated cells with the FLT3 inhibitor PKC412 and then examined the response of the cells to sub-toxic doses of daunorubicin. The MV4-11 FLT3 ITD cells, but not the FLT3 WT HL60 cells, demonstrated a statistically significant suppression of early DNA repair when treated with PKC412 and daunorubicin (p<0.001). Similar results were obtained in primary AML cells, with a loss of early DNA repair in the FLT3 ITD cells and no effect on the WT FLT3 cells. Furthermore the reduction in daunorubicin-induced DNA repair seen in PKC412 treated FLT3 ITD cells was associated with down-regulation of RAD51 expression. There was a statistically significant decrease in RAD51 transcript level following PKC412 treatment in the FLT3 ITD patients and MV4-11 cell line but not in the FLT3 WT patients or HL60 cell line (p<0.05). The decrease in the expression of RAD51 is closely associated with the reduction in DNA repair function in the inhibitor treated FLT3 ITD cells.

This work suggests that high expression levels of FLT3 transcripts in AML cells with a FLT3 ITD up-regulate RAD51 resulting in more efficient DNA repair following chemotherapy treatment. This may lead to resistance to cytotoxic therapies and genomic instability, ultimately resulting in the manifestation of a more resistant disease and a greater likelihood of relapse. The use of FLT3 inhibitors concurrently with or following AML therapy may suppress enhanced DNA repair in FLT3 mutated cells.