Abstract
ObjectivesClinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.MethodsMOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.Results and ConclusionsWe found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.
Highlights
A constitutively activated, mutated version of the class III receptor tyrosine kinase, FLT3 (Fms-Like Tyrosine kinase-3; STK-1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal Liver Kinase-2), is expressed in approximately 30% of acute myeloid leukemia (AML) patients and a subset of ALL patients [1]
Cells made resistant to PKC412 (MOLM13-R-PKC412) were several fold less sensitive to PKC412 than drug-sensitive cells (MOLM13-S) (Figure 1A)
The sensitivity of cells made resistant to HG-7-85-01 (MOLM13-R-PKC412) was several fold lower than that of MOLM13-S cells (Figure 1B)
Summary
A constitutively activated, mutated version of the class III receptor tyrosine kinase, FLT3 (Fms-Like Tyrosine kinase-3; STK-1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal Liver Kinase-2), is expressed in approximately 30% of AML patients and a subset of ALL patients [1]. Gain-offunction point mutants in FLT3, typically in the kinase activation loop (often at position 835) [7] are detected in about 7% of patients with AML. Additional FLT3 mutations that have been identified include N841I [8], Y842C [9], and novel, weakly activating point mutations in a 16 amino acid stretch of the FLT3 juxtamembrane [10]. The observed suboptimal clinical responses, coupled with detection of drug-resistant leukemic blast cells in FLT3 inhibitor-treated AML patients, have made understanding clinical resistance to FLT3 inhibitors a priority. The cause for clinical resistance has not yet been identified and there is little evidence for FLT3 mutations that are analogous to the BCR-ABL gate-keeper mutations in CML, observed in response to imatinib and related compounds
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