ALBERT

Description: cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The PDE4 enzyme family regulates the catabolism of cAMP to AMP in a wide range of tissues. PDE4 inhibitors have recently been submitted for approval for use in asthma and COPD. In leukemic samples from 11 B-CLL patients, rolipram and RO20-1724, two structurally unrelated PDE4 inhibitors, synergized with either hydrocortisone or dexamethasone in inducing B-CLL but not T cell apoptosis. Dose titration studies demonstrated that addition of a PDE4 inhibitor augmented B-CLL apoptosis even when maximally effective doses of either glucocorticoid were utilized. In five patients so analyzed, 10 uM rolipram augmented the induction of apoptosis by 100 uM hydrocortisone by 40 +/− 18%. Using transient transfection of a GRE-luciferase construct with an Amaxa nucleofector technique, we determined that treatment with PDE4 inhibitors augmented glucocorticoid receptor (GR)-mediated GRE transactivation in primary B-CLL cells. Strikingly, inhibition of PKA with the cAMP antagonist Rp-8Br-cAMPS inhibited glucocorticoid-induced apoptosis by 86 +/− 14% in 6 patients so tested and GRE transactivation by 83% in 8 patients so tested. Similarly, treatment with Ht31 peptide, a 23 residue peptide derived from an AKAP that binds with 4.0 nM dissociation constant to PKA RII subunits, also reduced hydrocortisone-induced transactivation. These studies suggest that PKA activity is required for both the ability of glucocorticoids to induce apoptosis and GRE transactivation in B-CLL cells. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. We isolated both dexamethasone-sensitive and dexamethasone-resistant CCRF-CEM clones for these studies and demonstrated that forskolin induced glucocorticoid sensitivity even in the initially dexamethasone resistant clone. 1,9 dideoxyforskolin, a forskolin analogue that does not activate adenylyl cyclase, failed to augment glucocorticoid sensitivity in CCRF-CEM cells. Given the marked discrepancy in the sensitivity of B-CLL cells and CCRF-CEM cells to PDE4 inhibitor-induced augmentation of glucocorticoid apoptosis and GRE transactivation, we next examined the cAMP response and PDE4 isoforms in these two cell types. Inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram but not forskolin treatment up-regulated 63 and 68 kDa forms of PDE4B (most likely PDE4B2) in B-CLL, forskolin but not rolipram treatment up-regulated 67 and 72 kDa forms of PDE4D (most likely PDE4D1/D2) in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating GR signal transduction and that inhibition of PDE4 in the absence of exogenous adenylyl cyclase activation is a clinically tenable means by which to achieve such PKA activation. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL and other lymphoid malignancies are indicated.

Description: Chronic lymphocytic leukemia (CLL) cells, but not peripheral blood T cells, undergo apoptosis following treatment with inhibitors of type 4 cyclic nucleotide phosphodiesterase (PDE4), a process that correlates dose dependently with elevation of adenosine 3′,5′-cyclic monophosphate (cAMP) in leukemic cells. We show that treatment of CLL cells with rolipram, a prototypic PDE4 inhibitor, and forskolin, an adenylate cyclase activator, induces mitochondrial depolarization, release of cytochrome c into the cytosol, caspase-9 and -3 activation, and cleavage of poly(adenosine diphosphate [ADP]–ribose)polymerase. Inhibitors of caspase-9, but not caspase-8, block rolipram/forskolin-induced CLL apoptosis. In a subset of CLL patients, B-cell lymphoma 2 (Bcl-2)–associated death promoter homolog (Bad), a proapoptotic Bcl-2 family member that when phosphorylated on specific serine residues is sequestered in the cytosol by 14-3-3, was dephosphorylated at Ser112 following rolipram/forskolin treatment of leukemic cells. Rolipram/forskolin treatment also induced Bad to accumulate in CLL heavy-membrane fractions, consistent with Bad translocation to mitochondria. To determine the mechanism for rolipram/forskolin-induced Bad dephosphorylation, we examined CLL phosphatase activity. Rolipram/forskolin treatment augmented protein phosphatase 2A (PP2A) activity, as well as levels of immunoreactive PP2A catalytic subunit. Treatment of CLL cells with a concentration of okadaic acid (5 nM) that selectively inhibits PP2A, reduced both rolipram/forskolin-induced mitochondrial cytochrome c release and mitochondrial depolarization. Okadaic acid restored Bad Ser112 phosphorylation and Bad association with 14-3-3 in rolipram/forskolin-treated CLL cells. These results suggest that PDE4 inhibitors may induce CLL apoptosis by activating PP2A-induced dephosphorylation of proapoptotic BH3-only Bcl-2 family members such as Bad.

Description: Type 4 cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE4) inhibitors and other agents that raise intracellular cAMP levels induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) but not in T-CLL or peripheral blood T cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and EPAC (exchange protein directly activated by cAMP), a Rap guanosine 5′-diphosphate (GDP) exchange factor. We here examine whether varying expression of EPAC accounts for the discrepant sensitivity of B-CLL and T cells to PDE4 inhibitor-induced apoptosis. B-CLL and peripheral blood B cells express EPAC1 transcript, whereas T-CLL, peripheral blood T cells, monocytes, and neutrophils do not. Treatment with the PDE4 inhibitor rolipram induces Rap1 activation in B-CLL cells but not in peripheral blood B cells, T-CLL, or any of the normal hematopoietic lineages examined. The EPAC-specific cAMP analog 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP) activates Rap1 in B-CLL cells, but, unlike rolipram/forskolin or 8-Bromo-cAMP, it does not induce PKA activation, as judged by phosphorylation of the transcription factor cAMP-response element binding protein (CREB). Unexpectedly, whereas rolipram/forskolin and 8-Bromo-cAMP induce apoptosis in B-CLL cells, 8CPT-2Me-cAMP decreased basal apoptosis in B-CLL cells by an average of 25% (P 〈 .002). Our results demonstrate that B-CLL cells uniquely activate Rap1 in response to PDE4 inhibitors and suggest that physiologic stimuli that activate EPAC may transmit an antiapoptotic signal. (Blood. 2004;103:2661-2667)