Characterization of a Novel HEK293 Cell Line (HEK293‐ENT1KO) to Assess the Role of Equilibrative Nucleoside Transporter Subtype‐2

Background Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside/nucleobase‐analog drugs that are used to treat breast, endometrial and uterine cancers (gemcitabine), lymphoma (cytarabine), as well as viral infections in pregnant women (ribavirin, abacavir). Of the two major ENT subtypes (ENT1, ENT2), ENT1 has been studied the most due to its relative predominance in most tissues, and the availability of the highly selective ENT1 inhibitor, nitrobenzylthioinosine (NBMPR). In contrast, there is much less information on how drugs interact with ENT2 or factors that affect ENT2 expression. This is mainly due to the fact that ENT2 is expressed at relatively lower levels concomitantly with ENT1, and no selective ENT2 inhibitor is available. Objectives To develop a model where ENT2 can be studied in isolation, we created a novel Human Embryonic Kidney (HEK293) cell mutant that lacks ENT1 (using CRISPR‐Cas9), leaving ENT2 as the only functional ENT in this system. We now report on the characterization of this HEK293‐ENT1KO model in terms of ENT2 expression, functional activity and the affinity of ENT2 for a range of known nucleoside/nucleobase compounds (endogenous and therapeutic analogs) in the absence of ENT1 interference. Potential compensatory changes in other genes associated with nucleoside metabolism were also assessed. Methods Transporter function was assessed through measurement of the initial rates of [ 3 H]2‐chloroadenosine uptake (2.5 ‐ 300 µM). K i values for inhibitors were defined using the IC 50 derived from concentration‐response curve analyses and the K m of [ 3 H]2‐chloroadenosine for ENT2 determined in this model. Data are expressed as mean ± SEM. The presence or absence of ENT1 was determined based on the binding of [ 3 H]NBMPR. Protein levels were assessed by immunoblotting using ENT‐specific antibodies, and changes in gene expression were examined by qPCR. Results HEK293‐ENT1KO cells had a similar level of ENT2 uptake (K m 104.7 µM ± 51.9, V max 1.6 pmol/µl/s ±0.4) as wild‐type (K m 105.3 µM ± 37.2, V max 1.4 pmol/µl/s ± 0.2). Neither [ 3 H]NBMPR binding sites nor ENT1 protein were observed in HEK293‐ENT1KO cells indicating the complete loss of ENT1. The relative expression of the gene encoding ENT1, SLC29A1, was negligible in the ENT1KO cells compared to the WT HEK293. ENT2 endogenously expressed in HEK293‐ENT1KO cells showed the expected affinities for known substrates such as adenine (2300 ± 750 µM), hypoxanthine (340 ± 54 µM), inosine (74.5 ± 29.9 µM), uridine (184.5 ± 33.7 µM), thymidine (42.4 ± 7.9 µM), guanosine (1500 ± 30 µM), gemcitabine (820 ± 79 µM), ribavirin (500 ± 140 µM) and abacavir (170 ± 18 µM). It also had the expected affinities for inhibitors such as NBMPR (2.9 ± 0.3 µM), dipyridamole (0.5 ± 0.1 µM), dilazep (2.6 ± 0.9 µM), and ticagrelor (11 ± 2.4 µM). Conclusion Our data suggest that removing ENT1 from HEK293 cells does not impact the expression, function or affinity of ENT2. This model can be used to advance our knowledge of pathways that regulate ENT2 expression and activity. It will also be a useful model for screening novel ENT2 inhibitors and substrates of potential therapeutic value.