Control of the PI3K/AKT Pathway by Phosphodiesterase 4B Modulates Glucocorticoid Response in B-Cell Lymphomas.

Abstract 728

Cyclic-AMP (cAMP) is a ubiquitous second messenger with marked growth inhibitory effects in lymphocytes. The phosphodiesterase 4B (PDE4B) is the principal negative regulator cAMP levels in normal and malignant B-cells. We previously showed that PDE4B is expressed at abnormally high levels in fatal diffuse large B-cell lymphoma (DLBCL), rendering these tumors resistant to cAMP's inhibitory activities towards the PI3K/AKT pathway (Blood, 105:308-16, 2005). Glucocorticoids (GC) remain a critical component of treatment of lymphoid cancers, but the mechanism for GC resistant in these tumors remains poorly characterized. A recent study indicated that the AKT/mTOR pathway mediates GC effectiveness in acute lymphoid leukemia (ALL), and identified PDE4B as one of the genes overexpressed in GC-resistant ALLs (Cancer Cell, 10:331-42, 2006). These data, together with our findings that PDE4B regulates PI3K/AKT, suggested that PDE4B may play a role in GC sensitivity. If this concept is correct, PDE4B expression may impinge on the same genes and pathways that control GC response in malignant lymphocytes. To test this hypothesis, we used gene set enrichment analysis (GSEA) and found a robust association between the gene expression signature of DLBCLs expressing high levels of PDE4B and ALLs resistance to GC-induced apoptosis (p and FDR =.014). Consistent with the GSEA data, dexamethasone treatment (50nM), in the presence of intra-cellular cAMP, inhibited the proliferation (as determined by MTS assays) of multiple PDE4B-low (DHL6, DHL10, and WSU-NHL), but not PDE4B-high (Ly1, Ly3, Ly10, DHL7 and Ramos) lymphoma cell lines (53% vs. 4% mean growth inhibition for PDE4B-low and -high cell lines respectively, p<0.01, Student's t-test). To further our investigation, we stably expressed PDE4B (wild-type [WT] or phosphodiesterase inactive [PI] mutant) in the DHL6 cell line, and determined whether this specific genetic modulation could alter the response of these cells to GC. Indeed, DHL6 cells ectopically expressing PDE4B-WT became less sensitive to the growth inhibitory effects of dexamethasone than their PDE4B-PI isogenic counterparts (31% vs. 57% growth inhibition, respectively, p<0.01). To validate the potential therapeutic benefits of this functional interplay, we tested if pharmacological inhibition of PDE4B could reverse the GC resistance found in the Ramos cell line. In agreement with our hypothesis, exposure to the PDE4-specific inhibitor rolipram (20μM) rendered these cells sensitive to dexamethasone, as determined by MTS-based cell proliferation assays (10% growth inhibition for dexamethasone or rolipram alone vs. 60% dexamethasone + rolipram, p<0.01). These data established PDE4B activity as an important regulator of GC resistance in mature B-cell malignancies, possibly via modulation of PI3K/AKT activity. To mechanistically test this theory, we stably expressed a constitutively active AKT mutant (CA-AKT) in the PDE4B-null/GC-sensitive DHL6 cell line. Subsequently, we exposed the DHL6-CA-AKT cell line, and its isogenic counterparts expressing PDE4B-WT or PDE4B-PI to dexamethasone; expression of PDE4B-WT and CA-AKT induced similar degree of GC-resistance in this DLBCL model (31% and 29% growth inhibition respectively vs. 57% for PDE4B-PI, p<0.01). In agreement with an important role for PI3K/AKT/mTOR signaling in this process, the differential sensitivity to GC observed in PDE4B-WT or -PI-expressing lymphoma cells was abrogated by Wortmannin, a PI3K inhibitor. Importantly, the improved GC sensitivity that followed PDE4B modulation was accompanied by inhibition of the AKT/mTOR pathway, as defined by substantial decrease in the phosphorylation levels of AKT, mTOR, eIF4G, p70S6K, S6R, and 4EBP1, measured in western blots. Collectively, our findings indicate that in B-cell lymphomas, PDE4B overexpression, via the modulation of the PI3K/AKT/mTOR pathway, induces GC-resistance. Together with our recent demonstration that PDE4B targeting improves the efficacy of SYK inhibitors in DLBCL (Blood, 113:6153-60, 2009), the data presented here provide the rationale to clinically test PDE4 inhibitors in B-cell lymphomas, alone or in combination with novel or classical chemotherapeutic agents.