Equilibrative Nucleoside Transporter-2 (ENT2) Protein Correlates with Ex-Vivo Sensitivity to Fludarabine in Chronic Lymphocytic Leukemia (CLL)-Cells.
Authors: Marce Silvia , Molina-Arcas Miriam , Villamor Neus , Huber Isabel , Casado F. Javier , Bellosillo Beatriz , Montserrat Emili , Pastor-Anglada Marcal , Colomer Dolors 
Published: October 2019

Abstract
Nucleoside derivatives are widely employed in the treatment of hematological malignancies. In particular, fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). Anticancer therapy using nucleoside-derived analogs is dependent on drug uptake and metabolic activation. Although some enzymes have been shown to be suitable biomarkers of drug metabolism and may determine response to therapy, the role of transporters in determining nucleoside-derived drug bioavailability is less known. The uptake of these drugs into cells is mediated by one or more of the different nucleoside transporters that have recently been cloned. These proteins belong to two non-related gene families, SLC28 and SLC29, encoding CNT (Concentrative Nucleoside Transporters) and ENT (Equilibrative Nucleoside Transporters) proteins, respectively. In primary CLL cells we have described a high variability in ENT1, ENT2, CNT2 and CNT3 mRNA expression, whereas no CNT1 mRNA is detected. We have also shown that, in CLL cells, the uptake of fludarabine relies mainly ENT-type carriers and that ex vivo sensitivity significantly correlates with this ENT mediated uptake. In order to elucidate the role of ENT1 and ENT2 in the uptake and cytotoxicity of fludarabine, we have analyzed ENT1 and ENT2 expression by real time RT-PCR and Western Blot in cells from 19 CLL patients. ENT1 and ENT2 mRNA expression showed a high variability. ENT2 mRNA levels showed a range of variability of nearly 4-fold, whereas ENT1 variability was of 19-fold. Polyclonal antibodies against ENT1 and ENT2 consistently identified a single band of 50–55 kDa in all samples. Semiquantitative analysis of ENT1 and ENT2 expression was achieved by calculating the densitometry ratios versus α-tubulin, in a range of protein concentrations in which densitometric signal has been previously shown to be linear. Contrary to the mRNA expression, Western Blot analysis of ENT1 showed much less variability among patients (three-fold) than ENT2 (11-fold). The amounts of ENT1 protein did not significantly correlate with ENT1 mRNA expression, transporter function, or ex vivo fludarabine cytotoxicity. In contrast, although ENT2 protein does not bear any significant relationship with ENT2 mRNA, its expression correlates with transport activity. Moreover, ex-vivo sensitivity to fludarabine, at a pharmacological concentration (7.5 μM), directly correlated with the amount of ENT2 protein. Similar correlations were observed when the drug was tested at 3 and 15 μM. In summary, this study strongly suggests that fludarabine action on CLL cells is determined by the equilibrative nucleoside transporter ENT2. This is the first time ENT2 has been implicated in the therapeutic response to a nucleoside analog and might be useful to predict fluradabine response in CLL patients.