BCR-ABL activity and its response to drugs can be determined in CD34⁺ CML stem cells by CrkL phosphorylation status using flow cytometry

In chronic myeloid leukaemia, CD34<sup>+</sup> stem/progenitor cells appear resistant to imatinib mesylate (IM) <i>in vitro</i> and <i>in vivo</i>. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-AbI kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation(P-CrkL) in samples with < 10<sup>4</sup> cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL<sup>+</sup> as well as BCR-ABL<sup>-</sup> (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-AbI specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph<sup>+</sup> CD34<sup>+</sup> cells and was able to discriminate between Ph<sup>-</sup>, sensitive and resistant Ph<sup>+</sup> cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells