(841.10) Impact of loss or regain of Equilibrative Nucleoside Transporter (ENT) Subtype-1 on ENT Subtype-2 pharmacology in a Novel CRISPR-Cas9 HEK293-ENT1KO Cell Model

Poster Board Number: B124

Nayiar Shahid (University of Alberta), James Hammond (University of Alberta)

Background: Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside-analog drugs that are used to treat cancers and viral infections. Of the two major ENT subtypes (ENT1, ENT2), ENT1 has been studied the most due to its relative predominance in 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 because ENT2 is always expressed concomitantly at lower levels with ENT1, and there is no selective ENT2 inhibitor available. Given the emerging evidence suggesting that ENT2 has distinct physiological roles from ENT1, it is critical that its functional characteristics and modes of regulation are better defined in its native environment. To address these knowledge gaps, we removed ENT1 from Human Embryonic Kidney cells (HEK293) via CRISPR-Cas 9, creating a cell line that has ENT2 as the only functional ENT in the cells (HEK293-ENT1KO).

Objectives: To confirm that any changes observed in the HEK293-ENT1KO cells were due to the removal of ENT1, we have introduced recombinant ENT1 back into these cells and assessed the function and expression of the endogenous ENT2.

Methods: Stable transfection of MYC-ENT1 in pcDNA3.1 was conducted in HEK293-ENT1KO cell line using calcium phosphate precipitation method and stable clones selected with G418(Geneticin). Transporter function was assessed through measurement of the initial rates of [3H]2-chloroadenosine uptake (2.5–300µM). Ki values for inhibitors were defined using the IC50 derived from concentration-response curve analyses and the Km of [3H]2-chloroadenosine for ENT2 determined in this model. The presence or absence of ENT1 was determined based on the binding of [3H]NBMPR. Protein levels were assessed by immunoblotting using ENT-specific antibodies and changes in expression of metabolic genes involved in de novo synthesis of nucleosides were examined by qPCR. Data are expressed as mean±SEM.

Results: MYC-ENT1-HEK293-ENT1KO had a similar level of ENT2 uptake (Km-32µM±9.7, Vmax-0.6pmol/µl/s±0.1) as wild-type (WT) HEK293 (Km-19µM±1.2, Vmax-0.7pmol/µl/s±0.3) and HEK293-ENT1KO (Km-25µM±17, Vmax-0.6pmol/µl/s±0.1). In contrast, kinetic profile of the MYC-ENT1 transfected model showed significant higher ENT1-mediated uptake (Km-97µM±68, Vmax-11pmol/µl/s±9.1) than that observed in the WT-HEK293 (Km-90µM±27, Vmax-3.6pmol/µl/s±2.4). Also, the number of [3H]NBMPR binding sites were also found to be higher in the transfected model. The complete regain of ENT1 in MYC-ENT1-HEK293- ENT1KO cell model was further confirmed in protein and gene expression studies. However, the presence of recombinant ENT1 did not change the endogenous ENT2 protein expression. Loss of endogenous ENT1 showed no changes in gene expression levels of other transporters or metabolic enzymes except NT5E (5-Nucleotidase Ecto) which reduced significantly.

Conclusion: Our data suggest that neither removing endogenous ENT1 from HEK293 cells nor introducing recombinant ENT1 in the HEK293-ENT1KO model impacts the expression or function of ENT2.

lt;bgt;NSERC lt;/bgt;(Natural Sciences and Engineering Research Council)