The purine nucleoside phosphorylase inhibitor forodesine induces p53-independent apoptosis in chronic lymphocytic leukemia through caspase-8 activation, Mcl-1 downregulation, conformational changes of Bax and Bak and ROS generation.

A41

Chronic lymphocytic leukemia (CLL) is a disease derived from the monoclonal expansion of CD5+ B-lymphocytes. In spite of recent and important advances, treatment of CLL continues being challenging, particularly in those subjects with 17p- abnormalities (reflecting p53 alterations) since these patients do not respond to standard therapies. Finding novel agents capable of inhibiting survival genes or activating the mitochondria apoptotic pathway independent of p53 is therefore an important goal. Forodesine is a potent purine nucleoside phosphorylase (PNP) inhibitor that induces an increase of 2’-deoxyguanosine (dGuo) in plasma, the intracellular accumulation of deoxyguanosine triphosphate (dGTP), and the alteration of deoxynucleotide (dNTP) pools, all these leading to genetic instability, inhibition of DNA synthesis/repair, and cell death induction. In CLL cells, forodesine induces p53 stabilization and p21 activation, mitochondrial membrane depolarization and cell death. Unlike other purine nucleoside analogs, forodesine is not incorporated into DNA and represents a new class of selective anti-tumor agent with a novel and not fully understood mechanism of action. We analyzed the cytotoxic effect of forodesine in primary cells from 29 patients with CLL, 11 of them carrying p53 alterations (17p deletion). Pharmacologically achievable levels of forodesine (2 mM) and dGuo (10-20 mM) induced apoptosis at 24-48 hours in CLL cells (56.7±14.3 % of mean cytotoxicity) . As per the individual cytotoxic effect, this was higher than 60% in 17 cases (58.6% of all cases studied) and between 40-60% in 8 cases (27.5%). Next, we analyzed the effect of forodesine in cells from CLL patients with 17p deletion. Interestingly, these cases also showed good response to forodesine (11 cases, 58.5± 20 % of mean cytotoxicity vs. 18 CLL cases with no 17p deletion, 55.2±10.3% of mean cytotoxicity). To elucidate the mechanism of action of forodesine in CLL cells, several key elements of apoptotis were analyzed. Forodesine induced phosphatidylserine exposure, loss of mitochondrial membrane potential (ΔΨm) and generation of reactive-oxigen species (ROS). Conformational changes of the pro-apoptotic proteins Bax and Bak, caspase-8 activation and Bid cleavege were rapidly detected. Inhibition of caspases with z-VAD.fmk only partially blocked ΔΨm and ROS generation. It is known that CLL cells express high levels of the anti-apoptotic proteins Mcl-1 and Bcl-2 that play an important role in prolonging cells survival. Upon forodesine exposure, Bcl-2 levels were not affected, whereas Mcl-1 protein levels considerably decreased . No significative differences in these apoptotic markers were observed based on the p53 status, suggesting a common apoptotic pathway independent of p53-mediated cell death. These results support that forodesine might be highly effective in the treatment of patients with CLL regardless of the p53 status. Based on these data a phase II clinical trial of forodesine in CLL patients is being initiated at our institution.