Abstract
Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor occur in approximately 25% of patients with acute myeloid leukaemia (AML) and are associated with poor prognosis. The ITDs can range from just three nucleotides to hundreds of nucleotides in length and the duplicated sequence is always in-frame. Recent data has suggested that the size of the ITD may be of importance prognostically and increasing ITD size has been shown to be associated with decreasing overall survival and relapse-free survival1. Very little is known about the biological effects of FLT3-ITDs of differing length and it is possible that such findings would help shed light upon the clinical findings. We have therefore studied various biological parameters in 102 primary AML blast cell samples including FLT3 mutation status, FLT3-ITD length, FLT3 and bcl-xl mRNA expression levels and STAT5 phosphorylation (activation).
38 AML samples had FLT3-ITDs and 64 samples were FLT3-wild type (WT); samples were also screened for D835 point mutations and any samples found to have such a mutation were not included in further analyses, therefore WT denotes neither an ITD nor a D835 mutation. FLT3 mRNA levels were determined by real time PCR and normalised to the housekeeper beta 2 microglobulin (b2M). Samples with a FLT3-ITD had a higher level of FLT3 mRNA than WT samples (p=0.02) and the transcript level of FLT3 was also correlated with phosphorylation of the downstream signalling target STAT5 (measured by flow cytometry; n=22; p=0.02), such an association was not seen in the WT samples (n=23; p=0.37). FLT3-ITD mutations have been shown to activate the anti-apoptotic gene bcl-xl via STAT5 signalling, therefore levels of bcl-xl were also assessed in these samples. A strong correlation was found between increasing levels of FLT3 and bcl-xl transcripts in the FLT3-ITD samples (n=30, p=0.002), this was not found in the FLT3-WT samples (n=50, p=0.42). Bcl-xl levels were found to be dependent upon the level of phosphorylated STAT5 (n=20, p=0.03) and the length of the ITD (n=17, p=0.03) in FLT3-ITD samples. However, as of yet no correlation has been found between STAT5 activation and FLT3-ITD length.
These findings suggest that increasing levels of FLT3 transcripts are associated with increasing activation of the STAT5 signalling molecule and its downstream target bcl-xl. Moreover, the length of the FLT3-ITD was also found to correlate with the level of bcl-xl, although whether this is via a STAT5-dependent mechanism remains to be established. Enhanced levels of bcl-xl may mediate chemoresistance and explain the adverse effect of FLT3-ITD length on outcome in AML.
Bcl-xl mRNA levels correlate with FLT3-ITD length
Disclosure: No relevant conflicts of interest to declare.
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