Allele-dependent activity of promoters of genes SLC22A4 and SLC22A5 encoding imatinib transporters in chronic myeloid leukemia Free

In our previous studies, we identified an association between the regulatory SNP rs460089, located in the promoter of SLC22A4 gene (encoding a transporter involved in imatinib transport), and the response to imatinib (Jaruskova et al., JECCR 2017) as well as treatment-free remission (Machova Polakova et al., Leukemia 2024) in patients with chronic myeloid leukemia (CML). The “GG” genotype was significantly linked to higher frequency of imatinib failure and molecular recurrence after therapy discontinuation. However, underlying biological mechanism for this association remains unclear. The initial hypothesis suggesting that “GG” genotype leads to reduced expression of SLC22A4 and lower intracellular imatinib concentration was not confirmed (Burda et al., Mol Metab 2024). We propose a new hypothesis that the regulatory SNP of SLC22A4 and SLC22A5 affect the intracellular concentrations of their natural substrates, ergothioneine (a stable and effective antioxidant) and carnitine, respectively, thereby supporting the survival of leukemic cells.

The promoter regions of SLC22A4 and SLC22A5 genes were analyzed using chromatin immunoprecipitation assays, luciferase reporter assays and DNA methylation analysis. Gene expression was assessed via real-time qPCR (RT-qPCR), while intracellular concentrations of carnitine and ergothioneine were quantified using mass spectrometry. The experiments were conducted using the KCL-22 myeloblast cell line (“GG” genotype), two blast phase patient-derived cell lines (PDs, “GC”) and primary PBMC cell isolated from CML patients at chronic phase at the time of diagnosis (n=12, Covering all 3 genotypes, GG, GC and CC).

Luciferase assays showed higher promoter activity with the “G” allele at the rs460089 compared to “C”. This was supported by significant increase in intracellular ergothioneine intake in KCL-22 (“GG”) vs. PD (“GC”) cells after 24 and 48 hours of incubation. Regardless of the allele, promoter activity decreased after JQ1 treatment (MYC expression inhibitor), confirming the rs460089 as a biologically relevant MYC binding site as predicted by previous in silico analysis. Chromatin immunoprecipitation showed MYC occupation at the rs460089 locus only in “GG” and incubation with ergothioneine even enhanced MYC binding only in KCL-22 (“GG”). Despite active chromatin marks (especially H3K4Me3 and H3K27Ac) and complete DNA demethylation at CpG within promoter (independent on rs460089 genotype), SLC22A4 expression was very low in KCL-22 (“GG”) cells and fell below the detection limit of RT-qPCR in “GC” PD cell lines. In contrast, SLC22A5 expression was two logs higher in both “GG” and “GC” genotypes. Notably, two other regulatory SNPs in SLC22A5 promoter (exhibiting 99% Linkage Disequilibrium with rs460089) were identified. We show that SLC22A5 promoter, including these high-LD loci, exhibited transcriptionally active chromatin state, fully demethylated CpG in promoter and MYC occupancy was detected only in KCL-22 “GG”. Carnitine incubation induced MYC expression and MYC (and its cofactor MAX) occupancy, stimulated SLC22A5 expression and increased corresponding intracellular carnitine level only in “GG” KCL-22 cells. Importantly, both “GC” genotype PD cell lines showed reduced intracellular carnitine concentrations compared to “GG” KCL-22. In relation to GG and GC cell lines analyzed, any DNA methylation at the promoters, regardless of the rs460089 allelic configuration in all 12 CML patients.

Our results revealed that the rs460089 locus, which is in strong linkage disequilibrium with regulatory elements in the SLC22A5 promoter, exerts a significant allele-specific effect on promoter activity, leading to altered SLC22A5 transcription and accumulation of intracellular carnitine. Elevated carnitine concentrations (potentially supported by ergothioneine antioxidant activity) may enhance the survival of CML cells. Modulated carnitine-dependent metabolic pathways may potentially explain the previously observed association between rs460089 and both imatinib response and treatment-free remission (TFR) in CML. We are currently investigating whether the non-optimal response in patients with the “GG“ genotype (compare to “GC“) may be related to a more efficient carnitine-dependent metabolism, thereby promoting better survival of leukemic cells.

MH CZ - DRO (IHBT 00023736)