The Purine Nucleoside Phosphorylase Inhibitor Forodesine (BCX-1777) Is a Potent Cytotoxic Agent and Has Synergistic Activity with Bendamustine in Chronic Lymphocytic Leukemia (CLL) Irrespective of ZAP-70 Levels and p53 Status.
Authors: Alonso Roberto , Villamor Neus , Upshaw Ramanda , Bantia Shanta , Mehrling Thomas , Campo Elias , Montserrat Emili , Colomer Dolors 
Published: October 2019

Abstract
Forodesine (BCX-1777) is a potent purine nucleoside phosphorylase (PNP) inhibitor. PNP inhibition results in elevation of plasma 2′-deoxyguanosine (dGuo) and intracellular accumulation of deoxyguanosine triphosphate (dGTP), which in turn affects deoxynucleotide-triphosphate pools and induces cell death. It has been reported that forodesine exerts a cytotoxic effect in cells from CLL probably due to high deoxycytidine kinase (dCK) activity in these cells. dCK is the primary enzyme for the conversion of dGuo to dGMP (dGuo monophosphate), which is then converted to dGTP. Several markers such as ZAP-70 and p53 status (17p deletions) identify CLL patients with a different biological and clinical behaviour. High ZAP-70 expression levels are associated with poorer overall survival and shorter time to disease progression, whereas p53 alterations convey drug resistance and short survival. We analyzed the in vitro cytotoxic effect of forodesine in primary cells from 29 patients with CLL, 11 of them carrying 17p deletions. Forodesine (2 μM) and dGuo (10–20 μM) induced apoptosis at 24–48 hours in CLL cells (56.7±14.3% of mean cytotoxicity in respect to control). As per the individual cytotoxic effect, this was higher than 60% in 17 cases (58.6%), 40–60% in 8 cases (27,5%), and lower than 40% in 4 cases (13.8%). No significant differences were observed between CLL cells with low levels of ZAP-70 (11 cases; 61.85±11.2% mean cytotoxicity) and CLL cells with high levels of ZAP-70 (16 cases; 55.7±16.8% mean cytotoxicity). Cases with 17p deletion showed good response to forodesine (11 cases; 58.5± 20% of mean cytoxicity vs. 18 CLL cases with no 17p deletion, 55.2±10.3 of mean cytotoxicity). Next, we analyzed the effect of combination of forodesine with fludarabine (3.75–7.5 μM) or bendamustine (10–25 μM). A significant synergistic effect (Chou Talalay Combination Index CI<1) was observed with forodesine/bendamustine combination (CI=0.56±0.26), without significant differences according to ZAP-70 levels (ZAP-70low CI=0.51±0.21 vs. ZAP-70high CI=0.61±0.31). On the contrary, an antagonistic effect was observed with the fludarabine/forodesine combination (CI=1.96) in all cases analyzed. Five patient samples with high levels of ZAP-70 (17% of all cases studied) that were resistant to bendamustine and fludarabine did respond to forodesine (65.7±7.3% of mean cytotoxicity). Three cases with 17p deletion showed low or very low response to bendamustine or fludarabine, but a high response to forodesine (71.5±5% of mean cytotoxicity). Cell death after forodesine and dGuo treatment correlated with an increase in intracellular dGTP levels. In addition, we detected mitochondrial depolarisation, ROS generation, Bax and Bak conformational changes and caspase activation after treatment of CLL cells with forodesine and dGuo. In conclusion, these results support that forodesine as single agent or in combination with bendamustine might be highly effective in the treatment of CLL independently of ZAP-70 expression levels and p53 status. Based on these data a phase II clinical trial of forodesine in patients with CLL will be planned in our institution.