Impact of SLC43A3/ENBT1 Expression and Function on 6-Mercaptopurine Transport and Cytotoxicity in Human Acute Lymphoblastic Leukemia Cells.

6-Mercaptopurine (6-MP) is used extensively in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. Our laboratory determined previously, using a recombinant HEK293 cell model, that the SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) transports 6-MP into cells and significantly impacts the cytotoxicity of 6-MP in that model. To further investigate the clinical relevance of this finding, we now extend this work to an analysis of the impact of SLC43A3/ENBT1 expression and function on 6-MP uptake and cytotoxicity in leukemic lymphoblasts, the therapeutic target of 6-MP in ALL. A panel of ALL cell lines was assessed for SLC43A3/ENBT1 expression, ENBT1 function, and sensitivity to 6-MP. There was a significant difference in SLC43A3 expression among the cell lines that positively correlated with the rate of ENBT1-mediated 6-MP uptake. Cells with the lowest expression of SLC43A3 (SUP-B15: Vmax = 22± 5 pmol/µl per second) were also significantly less sensitive to 6-MP-induced cytotoxicity than were the highest expressing cells (ALL-1: Vmax = 69 ± 10 pmol/µl per second). Furthermore, knockdown of ENBT1 using short hairpin RNA interference (shRNAi) in RS4;11 cells caused a significant decrease in ENBT1-mediated 6-MP uptake (Vmax: RS4;11 = 40 ± 4 pmol/µl per second; RS4;11 shRNAi = 26 ± 3 pmol/µl per Second) and 6-MP cytotoxicity (EC50: RS4;11 = 0.58 ± 0.05 µM; RS4;11 shRNAi =1.44 ± 0.59 µM). This study showed that ENBT1 is a major contributor to 6-MP uptake in leukemia cell lines and may prove to be a biomarker for the therapeutic efficacy of 6-MP in patients with ALL. SIGNIFICANCE STATEMENT: This study shows that SLC43A3-encoded equilibrative nucleobase transporter 1 is responsible for the transport of 6-mercaptopurine (6-MP) into leukemia cells and that its level of expression can impact the cytotoxicity of 6-MP. Further studies are warranted to investigate the therapeutic implications in patient populations.

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter.

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 [equilibrative nucleobase transporter 1 (ENBT1)] has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. Human embryonic kidney 293 (HEK293) cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [3H]adenine and [14C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [14C]6-MP and [3H]adenine had K m values (±S.D.) of 163 ± 126 and 37 ± 26 µM, respectively, for this system. Decynium-22, 6-thioguanine, and 6-methylmercaptopurine inhibited 6-MP uptake with K i values of 1.0 ± 0.4, 67 ± 30, and 73 ± 20 µM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impact 6-MP cytotoxicity.