Impaired S-phase arrest in acute myeloid leukemia cells with a FLT3 internal tandem duplication treated with clofarabine.
OBJECTIVE
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.


METHODS
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 (gammaH2AX 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.


CONCLUSIONS
There is a reversal of phenotype in mutant FLT3 cells dependent on the length of exposure to clofarabine. Efficient DNA repair may render the cells resistant to a short pulse of the drug, but a failure of cell cycle checkpoint(s) in S phase renders the cells sensitive to prolonged exposure.
Seedhouse
Claire
C
University of Nottingham, Nottingham, United Kingdom. Claire.seedhouse@nottingham.ac.uk
Grundy
Martin
M
Shang
Shili
S
Ronan
John
J
Pimblett
Heather
H
Russell
Nigel
N
Pallis
Monica
M
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001087:Arabinonucleosides / Q000494:pharmacology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D000077866:Clofarabine; D004260:DNA Repair; D015973:Gene Expression Regulation, Leukemic; D006657:Histones / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D010641:Phenotype; D016196:S Phase / Q000187:drug effects*; D020080:Tandem Repeat Sequences; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; C468783:H2AX protein, human; D006657:Histones; D000077866:Clofarabine; D051941:fms-Like Tyrosine Kinase 3