MATE1 regulates cellular uptake and sensitivity to imatinib in CML patients

Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25–30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels.


INTRODUCTION
Although the tyrosine kinase inhibitor imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), some patients do not respond to imatinib or relapse after initial response. 1,2 In all, 25-30% of CML patients treated with imatinib as a first-line therapy experience treatment failure leading to an increased risk of progression to accelerated or blast phase. The response rate varies within different stages of the disease. 3 Besides other mechanisms of resistance such as mutations in the BCR-ABL1 fusion gene, 4 the transport of imatinib into its targeted cells has been proposed to be crucial for the clinical effectiveness. [5][6][7] As an organic cation, imatinib has to be actively translocated across the cell membrane. Several clinical trials as well as in vitro studies investigated the role of the organic cation transporter 1 (OCT1) in this process. Although some studies suggested a critical role of OCT1 in regulating imatinib efficacy, [5][6][7] other studies could not confirm these findings. 8,9 The role of OCT1 for imatinib uptake has mostly been inferred from inhibition studies with prazosin and amantadine. This approach has been criticized recently, as these substances do not specifically inhibit OCT1 but are potential inhibitors of other imatinib uptake pathways distinct from OCT1. 10 Although OCT1 is generally capable to transport imatinib, it might have minor relevance for a transport under clinical conditions. Indeed, in vitro studies have shown a moderate affinity of OCT1 to imatinib 11,12 as well as low expression levels in mononuclear and CD34 + cells. 8,13 OCT1 belongs to the SLC22A family of polyspecific organic cation transporters that also includes OCT2 and OCT3, which have an overlapping spectrum of substrates. 14 Experiments on model cells have shown that other members of this group also transport imatinib. 11,12,15 Others and our group recently demonstrated that the multidrug and toxin extrusion protein 1 (MATE1), also belonging to the group of organic cation transporters, accepts imatinib as a substrate. 11,12 Transporters other than OCT1, especially MATE1, have not been investigated regarding their role for imatinib accumulation in targeted cells for CML treatment yet. Here we investigate transporters that might be relevant for imatinib uptake, and regarding their role in mediating cellular imatinib uptake, regulating molecular response and their correlation to the clinical therapeutic response to imatinib of CML patients.
Affinity of imatinib to single OCT To elucidate whether a transport is relevant under clinically used concentrations, peak plasma levels of imatinib were compared with apparent affinities of single OCT to imatinib as recommended by the International Transporter Consortium. 17 Imatinib plasma levels and the unbound fraction were obtained from the literature. 18,19 The half-maximal inhibitory drug concentration (IC 50 ) was taken from previously published data, where IC 50 values were obtained by inhibition of the well-known model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP + ). 11 Similar IC 50 values were obtained when inhibiting the uptake of 1-methyl-4-phenylpyridinium iodide (MPP + ) by imatinib underlining the stability of this method. 12 Cellular imatinib uptake and specific OCT inhibition HEK293, PMBC or K562 cells were incubated with 5 μM imatinib (LC Laboratories, Woburn, MA, USA) for 10 min at 4°C, where mainly passive diffusion of imatinib through the cell membrane is possible, and at 37°C, where active transporter-mediated uptake occurs. The transport process of the investigated transporters was specifically inhibited by chemical substances at concentrations that are known to inhibit specifically the distinct transporters (Supplementary Table 1). MATE1mediated transport was inhibited by 200 nM pyrimethamine (Sigma-Aldrich, Steinheim, Germany), 20 whereas 80 μM MPP + (Sigma-Aldrich) was used for OCT1 and OCT2 coinhibition. 14,20-23 Cells were then lysed and intracellular imatinib accumulation was quantified with high-performance liquid chromatography with UV detector as described previously. 11 MATE1 knockdown and effects on BCR-ABL1 activation or cytokinesis MATE1 knockdown in K562 cells was performed with Hyperfect transfection reagent (Qiagen) according to the manufacturer's instructions using either 40 nM MATE1 small interfering RNA (siRNA) (Sigma-Aldrich; 5′-GACAAUUUACUGUGAGUUA-3′) or control siRNA (Invitrogen; nonsilencing mock siRNA). The transfection efficiency was verified by PCR and western blot analysis for MATE1 as described earlier. 11 To assess the influence of MATE1 expression on imatinib-dependent biologic effects, transfected K562 cells were incubated with imatinib at different concentrations (5 or 10 μM) for 10 min and the activity of BCR-ABL1 protein was then detected by western blot analysis using anti-phospho-ABL (Cell signaling, Frankfurt, Germany) and GAPDH antibody (Sigma-Aldrich). Further, we evaluated the clonal growth of K562 cells stably transduced with lentiviral vectors (plKO.1-GFP) delivering a short hairpin RNA against MATE1 (shMATE1, 5′-GACAAUUUACUGUGAGUUA-3′) or control mRNA (shscramble). Thereon, short hairpin-transduced K562 cells (3 × 10 3 cells) were plated in 1 ml of methylcellulose medium (M3234 MethoCult; Stem Cell Technologies, Cologne, Germany) without cytokines in quadruplicates, supplemented with imatinib (0-1000 nM) and the colonies were counted on day 7.

Patient analysis
In total, 30 patients with BCR-ABL1 major transcript (p210)-positive CML were included in this study after written informed consent was obtained (Ethik-Kommission der Bayerischen Landesärztekammer; Ethik-Kommission No. 05117). BCR-ABL1 diagnostic procedures were performed in the Munich Leukemia Laboratory (MLL, München, Germany) and data were kindly provided by S Jeromin and T Haferlach. Upon diagnosis, MATE1-mRNA expression was quantified by qRT-PCR in total bone marrow (BM) cells as described above. All patients were treated with imatinib and MATE1 expression was compared between imatinib-responding and -refractory patients defined by major molecular response. BCR-ABL1/ ABL1 ⩽ 10% (International Scale) after 3 months of imatinib therapy was defined as optimal response, whereas poor response was defined by a BCR-ABL1/ABL1 41% after 6 months of therapy (warning and failure according to the ELN 2013 guidelines 24 ). Detailed patient characteristics are given in Table 1.

Statistical analysis
Data were analyzed with GraphPad Prism, V4.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance with Tukey's post hoc test or unpaired Student's t-test were used for statistical analysis as indicated.
To elucidate whether this transport is relevant under clinically used concentrations, we compared imatinib plasma levels to apparent affinities of the transporters according to the recommendations of the International Transporter Consortium. 17 Along with these guidelines, transporters with a quotient of unbound plasma drug concentration (C unbound ) divided by IC 50 40.1 are defined as clinically relevant. Imatinib plasma levels for therapy regimens of 400 and 600 mg daily, the unbound fraction and the half-maximal inhibitory imatinib concentration to the respective OCTs (IC 50 ) were obtained from the literature (Supplementary  Table 2). 11,18,19 For OCT1, the quotient of the unbound imatinib plasma concentration for therapy regimens of 400 or 600 mg daily divided by the apparent affinity to imatinib was 0.03 and 0.1, respectively. A relevant interaction of OCT1 with imatinib under clinically used concentration is thus unlikely. However, we found remarkably high quotients for MATE1 with 1.7 (for 400 mg imatinib daily) and 5.8 (for 600 mg imatinib daily), suggesting a possible interaction at clinically used concentrations. Quotients for OCT2 were manifold lower compared with that for MATE1, but in contrast to those of OCT1 still in the range of a potential contribution under clinically relevant concentrations (0.2 for 400 mg and 0.6 for 600 mg imatinib daily).
Besides the affinities of an OCT to imatinib, their expression level in targeted cells is important for a relevant cellular uptake of imatinib in patients. In a first step, we thus measured mRNA expression levels of the potential imatinib transporters in PBMCs of healthy volunteers as well as in the immortalized CML cell line K562, derived from a BCR-ABL-positive CML patient in blast crisis. 25 In both cell types mRNA for OCT1 (1/ΔC t = 0.07 and 0.07, NS), OCT2 (0.08 and 0.07, NS), and MATE1 (0.09 and 0.07, NS) in PBMC and K562 cells, respectively, were expressed at similar levels ( Figure 1c).
To elucidate whether the imatinib uptake in these cells is mediated by transporters in general and not only due to passive diffusion through the cell membrane, we measured the uptake at 37°C and 4°C, where transporter-mediated uptake is strongly reduced. For PBMC and K562 cells, the uptake was significantly higher at 37°C (PBMC: 1.56 ± 0.22 nmol/mg protein, P = 0.0027; K562: 1.83 ± 0.14 nmol/mg protein, P = 0.0001) than at 4°C (PBMC: 0.62 ± 0.10 nmol/mg protein; K562: 0.64 ± 0.01 nmol/mg protein). This suggests a transporter-mediated process as the main mechanism of imatinib uptake (Figure 1d). In an attempt to identify the responsible transporters, we performed uptake experiments in the presence of specific transporter inhibitors and confirmed our finding using a gene-knockdown approach. Specific inhibitors were chosen from the literature (Supplementary  Table 1). 14,[20][21][22][23]26,27 The selective inhibition of MATE1 with pyrimethamine significantly reduced the imatinib uptake in PBMCs by 34 ± 8% (P = 0.0262), whereas coinhibition of OCT1 and OCT2 with MPP + had no significant effect (P = 0.6051) (Figure 1e). Most of the studies showing that OCT1 is important for imatinib uptake and molecular response in CML patients used prazosin and amantadine as OCT1 inhibitors. 5,28 However, in accordance with recently published data, 10 prazosin and amantadine are not specific inhibitors for OCT1 but have similar or even higher affinities to MATE1 than to OCT1 (Supplementary Table 1). The experiments with pyrimethamine show that this substance at the concentration used did not inhibit the imatinib uptake completely. We did not increase the pyrimethamine concentration to reach a complete suppression of imatinib cellular accumulation, to avoid additional inhibition of other OCT. To unequivocally confirm the role of MATE1 in the process of imatinib uptake, we performed a gene knockdown of MATE1 in K562 cells using specific siRNA. The knockdown of MATE1, as confirmed by western blot analysis, led to an almost complete inhibition of the imatinib uptake compared with mock siRNAtransfected cells (−80 ± 12%, P = 0.0005) (Figure 2a). Consequently, we found that imatinib-induced inhibition of c-ABL phosphorylation was remarkably reduced in siMATE1-transfected cells compared with mock-transfected cells (Figure 2b). These results clearly show that MATE1 is the major transporter for imatinib uptake in K562 cells and is probably crucial for its intracellular therapeutic effects.
To further prove that MATE1 has an important role in regulating imatinib sensitivity in CML patients, we analyzed the transporter profile of total BM samples obtained from CML patients by qRT-PCR and correlated the expression levels with the major molecular response to imatinib treatment (Figures 2c and d). As some patients fail to respond to imatinib therapy because of BCR-ABL1 mutations, 4 patients with these mutations were excluded from the analysis. OCT2 mRNA was not expressed at detectable levels in the BM of all patients. The expression of mRNA for OCT1 was low (1/ΔC t = 0.07 ± 0.002 and 1/ΔC t = 0.08 ± 0.003 for imatinib responders and non-responders, respectively) and did not differ significantly between responders and non-responders.
However, MATE1-mRNA was expressed at higher levels and furthermore significantly decreased in imatinib non-responders (1/ΔC t = 0.07 ± 0.001) compared with imatinib responders (1/ΔC t = 0.11 ± 0.04, P o0.0001) (Figure 2c). We found a correlation (r 2 = 0.60 and r 2 = 0.57 for responder and non-responder, respectively) between MATE1 transporter mRNA expression and clinical response to imatinib treatment, suggesting that MATE1 levels might enable to predict whether CML patients are likely to respond to imatinib therapy (Figure 2d). In contrast, we did not find a correlation (r 2 = 0.01 and r 2 = 0.15 for responder and nonresponder, respectively) between OCT1 transporter expression and major molecular response (Figure 2d).
To underline the hypothesis that MATE1 expression is crucial for the response to imatinib, we examined the role of MATE1 for the Abbreviations: BM, bone marrow; IS, International Scale; PLT, platelets; WBC, white blood cell count in peripheral blood (cells/μl). a Response to imatinib therapy: good = 3 months after the start of imatinib therapy BCR-ABL1/ABL1 ⩽ 10%; poor = 6 months after the start of imatinib therapy BCR-ABL1/ABL1 41%. b WBC in peripheral blood (cells per μl). c PLT per μl peripheral blood. d BCR-ABL1/ABL1-ratio according to IS in %. e 1 = female; 2 = male.
MATE1 regulates imatinib uptake and sensitivity S Harrach et al reductive effect of imatinib on colony formation. Therapeutically used imatinib concentrations reduced colony growth in shscramble-transduced K562 cells concentration dependently. Imatinib (250 nM) led to a colony reduction of 58% (1280 colonies without and 532 colonies with 250 nM imatinib) and 1 μM nearly totally inhibited colony growth (74 colonies, reduction of 94%). In contrast, the effects of imatinib were reduced when MATE1 was knocked down. Thus, there was no significant reduction of colony growth by imatinib in shMATE1-transduced K562 cells until a concentration of 1 μM was used (1266 colonies without and 560 colonies with 1 μM imatinib, reduction of 56%; Figures 2e and f).

DISCUSSION
Besides other mechanisms of resistance, the transport of imatinib into target cells was proposed to regulate the effectiveness of imatinib treatment. The role of OCT1 in this process has been discussed controversially. 5,8,9,28 Most studies showing that OCT1 mediates the cellular uptake and regulates the response to imatinib rely on inhibition experiments with prazosin or amantadine. [5][6][7]28 This approach has been criticized recently and might explain the dichotomic results as both, the inhibition with prazosin and amantadine, are not specific for OCT1. 10 We here show that although OCT1 can potentially transport imatinib, the affinity to imatinib is too low to allow a significant interaction at therapeutically reached plasma levels. Indeed, inhibition of OCT1 and OCT2, which we also identified as a potential imatinib transporter, by MPP + had no significant effect of the uptake in PMBC. In contrast, MATE1 transports imatinib with a manifold higher affinity as shown in model experiments permitting an uptake at clinically observed plasma levels. Indeed, prazosin and amantadine have similar or even higher inhibitory affinities to MATE1 than to OCT1. 21,22,26,27 The observed inhibitory effects of prazosin and amantadine on imatinib uptake and its effectiveness might thus be rather because of MATE1 than OCT1 inhibition. Specific inhibition of single OCT is difficult as these transporters share various substrates. 14 To further prove the role of MATE1 for the cellular uptake and effectiveness of imatinib, we thus confirm the observed results of our model experiments with both specific inhibition of MATE1 by pyrimethamine and a MATE1 gene knockdown. Pyrimethamine was used at low concentrations, which significantly but not completely reduced imatinib uptake. However, we did not increase the pyrimethamine concentration to reach a total inhibition of MATE1 to guarantee specificity. This may explain the different degree of reduction in inhibition and knockdown experiment.
The importance of MATE1-mediated imatinib uptake for therapeutic effects is supported by our transduction experiments, where MATE1 knockdown markedly reduced the effects of imatinib on c-ABL phosphorylation and colony formation. Both effects are essential in the pathogenesis of CML, and c-ABL phosphorylation has been shown to be a good predictor for the molecular response of CML patients to imatinib. 29 Deep and early molecular response is suggested to be predictive of improved long-term outcomes in CML therapy. We therefore correlated MATE1-mRNA expression levels in CML patients with the molecular response to elucidate whether MATE1 can influence the response of CML patients to imatinib. There was a significant reduction of MATE1-mRNA expression in patients who did not respond to imatinib and a strong correlation of MATE1-mRNA levels in total BM cells with the molecular response of CML patients. Interestingly, this correlation was not observed for OCT1. Measuring MATE1-mRNA levels might therefore enable to predict whether CML patients are likely to respond to imatinib therapy. Clinical trials showed evidence that dasatinib and nilotinib generate faster and deeper short-term responses in first-line CML therapy compared with imatinib. 30,31 However, imatinib is the most cost-effective tyrosine kinase inhibitor therapy for CML and, in contrast to dasatinib and nilotinib, is associated with less severe side effects. Screening tools such as a MATE1 expression analysis or detection of MATE1 mutations, which have been shown to rigorously change the functionality of MATE1, 32 might thus help to individually choose the best, safest and economically reasonable therapy.
In conclusion, we demonstrate that MATE1 is the major transporter for the cellular uptake of imatinib and crucial for the therapeutic success in CML patients. We suggest that the detailed analysis of MATE1 expression levels and mutations could be a predictor for the response to imatinib therapy.

CONFLICT OF INTEREST
TH partly owns MLL Munich Leukemia Laboratory. SJ is employed by MLL Munich Leukemia Laboratory. The other authors declare no conflict of interest and nothing to disclose.