Impaired S Phase Arrest in AML Cells with a FLT3-ITD Treated with Clofarabine Allows Prolonged Drug exposure to Reverse the Chemoresistant Phenotype.

Abstract 1000

Poster Board I-22

We have previously reported that AML cells with a FLT3-ITD have enhanced DNA repair mechanisms following exposure to DNA-damaging drugs which may be a mechanism of chemoresistance. Clofarabine is a novel nucleoside analogue, active in S-phase, with efficacy in AML and is incorporated into DNA as clofarabine triphosphate. Here we show that in FLT3-ITD cells enhanced repair, and therefore resistance to clofarabine-induced DNA damage and toxicity, can be reversed by prolonged drug incubation. When treated with clofarabine, FLT3-ITD-harbouring MOLM13 and MV4.11 cells undergo similar levels of DNA damage (γH2A.X foci) to FLT3 wildtype (WT) cells (HL60 and KG1). After a short pulse of drug the FLT3-ITD cells have a superior repair capability than WT cells; following a 2 hour washout period γH2A.X positivity found immediately after treatment had almost completely disappeared in the FLT3-ITD cells (<10% γH2A.X remaining), whereas in the FLT3-WT cells significant damage (γH2A.X) remained (>40%). Furthermore, after a 1 hour pulse of clofarabine, whereas the FLT3-WT cells under go rapid S phase arrest the S-phase checkpoint fails in the FLT3-ITD cells: reduction in the proportion of cells synthesising DNA is >80% in FLT3-WT cells and <10% in FLT3-ITD cells. Cell cycle arrest in response to DNA damage in S phase is affected via loss of the transcriptional regulator cdc25A. This loss of expression of cdc25A fails to take place in clofarabine-treated FLT3 mutant cells compared to WT cells. In addition, cdc25A mRNA levels are maintained by the FLT3-ITD as demonstrated by siRNA to FLT3 which reduced cdc25A mRNA levels in MV4.11 cells by 87.5%. Primary FLT3 mutant samples from AML patients(n=3) also display impaired cell cycle arrest upon treatment with clofarabine and show enhanced sensitivity on prolonged treatment (24 hours) compared to wildtype samples (n=2). We conclude that there is a reversal of phenotype in mutant FLT3 cells dependant on the length of exposure to clofarabine. Efficient DNA repair renders the cells resistant to a short pulse of the drug, but a failure of cell cycle checkpoint(s) in S phase, mediated by cdc25A, renders the cells sensitive to prolonged exposure. These results may have implications for the scheduling of clofarabine in clinical studies.