Abstract #5516: Mechanisms of marrow stromal cells (MSC) rescuing chronic lymphocytic leukemia (CLL) lymphocytes from forodesine-induced apoptosis
Authors: Kumudha Balakrishnan, Jan Burger, Maite Quiroga, William Wierda, Varsha Gandhi
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Previously we demonstrated that forodesine, a purine nucleoside phosphorylase inhibitor, in presence of deoxyguanosine (dGuo) showed efficacy during in vitro incubation of CLL lymphocytes by accumulating dGuo triphosphate (dGTP) without any effect on other deoxynucleotides. Intracellular increase in the level of dGTP in CLL-B cells was proportional to hall-mark features of apoptosis (Balakrishnan et al ; Blood 108:2392, 2006). Based on these encouraging data, a phase II clinical trial of forodesine is initiated for fludarabine-refractory CLL patients. Because microenvironments, such as the bone marrow,provide niches in which leukemia cells may survivestandard chemotherapy, we investigated the role MSC on forodesine-induced apoptosis, using 2 MSC lines (murine M2-10B4 and human NK-TERT). Both MSC lines decreased spontaneous apoptosis of CLL lymphocytes from 36% without stroma (48h, n=20) to 27% with M2-10B4 (n=15; p<0.05) and 9% with NK-TERT (n=7; p<0.05). Forodesine (2 µM) with 20 µM dGuo induced significant apoptosis in CLL lymphocytes with a median of 51% at 48h (n=20; p<0.05). Co-culture with both, M2-10B4 and NK-TERT significantly decreased forodesine-induced apoptosis to 29% (n=15; p<0.05) or 15% (n=7; p<0.05), respectively. Similar survival advantages were observed at 24h of treatment. Median endogenous dGTP levels in CLL lymphocytes were 5 µM, which increased with forodesine to a median of 21 µM (range: 10-31 µM). In co-cultures, both MSC lines slightly enhanced endogenous dGTP levels (median 7 µM with M2-10B4 and 12 µM with NK-TERT). With forodesine, there was no further increase in dGTP levels in CLL lymphocytes co-cultured with MSC. Other dNTPs were not affected either by stromal cells or in the presence of forodesine. Because dGTP perturbation was not fully associated with cell death, we tested additional mechanisms such as transcription inhibition. Although CLL lymphocytes are replicationally quiescent, they are transcriptionally active. Total RNA synthesis varied widely in CLL samples among patients but was inhibited by forodesine in 3 of 4 samples (median 33%, range 21-41%; 48h). When CLL lymphocytes were co-cultured on stromal cells, RNA synthesis increased dramatically (2-46 folds with M2-10B4 and 2-6 folds with NK-TERT). CLL lymphocytes co-cultured on stromal cells when incubated with forodesine, the RNA synthesis inhibition was partially recovered, but still not brought back to the levels in untreated cells. In conclusion, murine and human MSC rescue CLL lymphocytes from forodesine-induced apoptosis. The mechanisms for this resistance seem to involve decrease in the intracellular accumulation of dGTP and reversal in the inhibition of RNA synthesis in CLL lymphocytes co-cultured with MSC. Therefore, combination strategies of forodesine with agents that interfere with anti-apoptotic signals from the microenvironment might be beneficial.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5516.