Impact of imatinib mesylate on SLC22A1 gene expression in chronic myeloid leukaemia cell line, K562

[摘要] English: Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder characterised by the BCR-ABL fusion gene. The BCR-ABL fusion gene encodes a constitutively active BCR-ABL tyrosine kinase, which is the driving force of the malignancy. Otherwise fatal, the use of imatinib mesylate has proved highly effective in the treatment of this disease in up to 85% of CML patients. However, approximately 25% of CML patients appear to respond suboptimally or experience treatment failure with imatinib. Suboptimal response in CML patients has been attributed to inadequate BCR-ABL kinase inhibition as a result of reduced intracellular accumulation of imatinib in target leukemic cells. The cellular influx of imatinib is mediated by the influx transport protein, SLC22A1. Therefore, its activity is considered a clinical determinant of imatinib uptake, and hence patients response to therapy. A number of studies use levels of SLC22A1 mRNA as a measure of SLC22A1 activity. It has been reported that cells over expressing levels of SLC22A1 mRNA showed significantly increased uptake of imatinib, thus, suggesting that levels of SLC22A1 mRNA can be used as a measure of SLC22A1 activity. However, there is a concern that imatinib may affect SLC22A1 expression. This consideration, however, is based on two studies involving a limited patient cohort and although widely accepted, has not been proven conclusively. Should it be proven that imatinib does influence SLC22A1 expression, levels of SLC22A1 mRNA may not be a reliable indicator of SLC22A1 activity. It is therefore important to understand the effect of treatment with imatinib on SLC22A1 gene expression. The data from this study demonstrated that imatinib induces expression of SLC22A1 mRNA in a non-linear dose dependent manner. It was also observed that expression of SLC22A1 was not dependent on time of exposure to imatinib. These results explain the differential expression of SLC22A1 mRNA reported in CML patients on a standard dose of 400 mg/day of imatinib. The trough plasma levels of imatinib achieved between patients after 24 hours of exposure to the same dose of imatinib may vary owing to inter individual differences. Since SLC22A1 expression is dependent on plasma levels of imatinib, therefore, patients administered the same dose of imatinib may show differential expression of SLC22A1. These findings suggest that imatinib does affect SLC22A1 mRNA expression and that the change in SLC22A1 expression observed at any particular time is dependent on the intracellular levels of imatinib achieved in CML patients within 24 hours of exposure to the drug. One of the challenges in this study was the availability of suitably qualified SLC22A1 antibodies for use in the Taqman protein assay to quantify SLC22A1 protein. Antibodies used in the Taqman assay have to fulfil specific criteria and out of 55 commercially available antibodies, only three SLC22A1 antibodies met the minimum requirements for use in the assay. However, despite various efforts focused at optimising the assay, the range of the assay was very limited and hence it was not possible to quantify SLC22A1 protein. We hypothesize that one of the reasons for assay failure could be as a result of antibodies not binding to the target protein at the required spatial distance to facilitate amplification by real-time PCR. Since the antibodies used in the assay have not been epitope mapped, it is uncertain whether they fulfil this requirement. Future research will be aimed at antibody production for manufacturing SLC22A1 antibodies suitable for use in the Taqman protein assay to enable successful quantification of SLC22A1 protein. In conclusion, this is the first study which specifically aimed to investigate the influence of imatinib on SLC22A1 expression. This is also the first study to demonstrate that expression of SLC22A1 is not time dependent, but follows a nonlinear correlation to imatinib concentration. Although it would have been useful to investigate the effect of increasing levels of SLC22A1 mRNA on intracellular uptake of imatinib in K562 cells, unfortunately, the latter technique requires the use of radio-labelled imatinib and specialized equipment which made it a limiting factor for use in this study. While this study does not invalidate the use of levels of SLC22A1 mRNA as a prognostic marker for treatment outcome, these findings suggest that levels of SLC22A1 mRNA as a measure of SLC22A1 activity is only applicable to newly diagnosed imatinib naive or previously untreated CML patients.

[发布机构] University of the Free State