SNPs in the Promoter of the Gene Encoding Transmembrane Transporter SLC22A4 (hOCTN1) Are Significantly Associated with an Alteration of Gene Expression and with Resistance to the Imatinib Treatment in Chronic Myeloid Leukemia

Introduction: Apart from the mutations in the kinase domain of BCR-ABL, other important mechanisms of resistance to the first line imatinib (IM) therapy in chronic myeloid leukemia (CML) are pharmacokinetics factors, especially an intracellular concentration of IM. ATP Binding Cassette (ABC, ensuring efflux) and Solute Carrier (SLC, ensuring uptake) super families are responsible for transportation several drugs including IM. Gene expression and activity of the SLC and ABC transporters may be affected by polymorphisms in their promoter regions and may notably contribute to the treatment failure.

Objectives: The aim of this study was to identify polymorphisms in the promoter regions of the selected SLC and ABC transporter encoding genes and evaluate their association with the response to the first line IM therapy in CML patients in chronic phase. 

Material and Methods: The patient cohort consists of 40 CML patients with optimal response and 40 resistant patients (without mutations in the BCR-ABL kinase domain) to the IM in the first line with 24 months follow-up from the therapy initiation. All 80 CML patients were taking a standard dose of IM and in none of them were evidence of non-compliance. The promoter regions (~1000bp) of selected ABC (n=4) and SLC (n=15) genes with the annotated function of drug transportation were amplified. The next generation sequencing was applied for ultra-wide sequencing of altogether 1419 amplicons (454 GS Junior, Roche AppliedScience; MiSeq Series, Illumina). The obtained sequences were analyzed and evaluated with the focus on the presence of single nucleotide polymorphisms (SNPs) using NextGENe software (Softgenetics). The Fisher´s exact binomial test of goodness of fit was used to evaluate haplotype frequency distribution among patient cohort. The expression analysis of a selected gene was analyzed using RT-qPCR (TaqMan® Assays) in total leukocytes of peripheral blood with GUS as a housekeeping gene.

Results: Among 1419 evaluated sequences we identified 96 SNPs (2-12 SNPs per one promoter) of which nine was not yet annotated. We identified 2 SNPs, rs460089 (C/G) and rs460271 (C/G), with uniform alleles co-segregations in the promoter of the gene SLC22A4 when GG haplotypes were significantly more frequent in resistant patients (P<.05). Of all resistant patients, 69% carried GG haplotypes, 17% CG and 14% CC. The frequency of haplotypes among responding patients was 31% of GG, 53% CG and 16% CC. As SLC22A4 is imatinib transporter (He L et al. Hum Genomics 2009), we measured the level of the gene expression associated with both co-segregated SNPs. Among all patients analyzed regardless of the response to IM, the GG haplotypes showed a significantly lower expression of SLC22A4 in comparison to CG and CC haplotypes (P<.05).  Even greater significant differences in the expression were found when comparing GG haplotypes within patients resistant to IM in comparison to CG haplotypes within patients with optimal response (P<.01).

Conclusion: In this work, we found a significantly decreased gene expression of SLC22A4 in total leukocytes of peripheral blood that was associated with GG haplotypes of 2 SNPs in the SLC22A4 promoter, where GG haplotypes were significantly more frequent in CML patients resistant to the first line imatinib treatment in comparison to the patients with an optimal response. We assume that GG haplotypes have altered activity of SLC22A4 promoter resulting in the reduced hOCTN1 expression and thus in lower intracellular IM concentration that may be insufficient for optimal response. The experiments on the SLC22A4 promoter activity alterations associated with GG haplotypes are ongoing. We believe that screening for rs460089 (C/G) and rs460271 (C/G) SNPs among patients with newly diagnosed CML could be an important prognostic genetic factor helping to select a tyrosine kinase inhibitor that is not dependent on the active transportation through the cell membrane.

This work was supported by the Ministry of Health of Czech Republic, grant IGA MZ CR NT/13899 and Charles University in Prague, project GA UK/177815.