Cyclic nucleotide phosphodiesterases as targets for treatment of haematological malignancies

Abstract

The cAMP signalling pathway has emerged as a key regulator of haematopoietic cell proliferation, differentiation and apoptosis. In parallel, general understanding of the biology of cyclic nucleotide PDEs (phosphodiesterases) has advanced considerably, revealing the remarkable complexity of this enzyme system that regulates the amplitude, kinetics and location of intracellular cAMP-mediated signalling. The development of therapeutic inhibitors of specific PDE gene families has resulted in a growing appreciation of the potential therapeutic application of PDE inhibitors to the treatment of immune-mediated illnesses and haematopoietic malignancies. This review summarizes the expression and function of PDEs in normal haematopoietic cells and the evidence that family-specific inhibitors will be therapeutically useful in myeloid and lymphoid malignancies.

Figure 1. Role of PDEs in regulation of signal transduction

In the model of the second-messenger concept originally put forth by Sutherland and colleagues [295], first messengers, such as hormones, neurotransmitters, cytokines and growth factors, upon interacting with receptors on the cell surface, generate the production of a ‘second messenger’ such as cAMP, which then redirects the machinery of the cell, affecting many physiological processes. Currently, three different types of effector proteins to which cAMP can bind and carry out its actions are known: PKA [296], EPAC [251,252] and CNGCs (cyclic nucleotide-gated channels) [297]. In addition to these three known effector pathways for cAMP action, it remains possible that other currently undescribed cAMP effectors exist as well. Investigators examining cAMP inhibition of IL-5 production by T-cells, suppression of neutrophil apoptosis and inhibition of PKB/Akt leading to apoptosis in DLBCL cell lines have reported that these cAMP-mediated phenomena occur independently of currently described cAMP effector proteins [125,213,298]. cAMP effector molecules can also affect cellular processes directly through tethering mechanisms, as has been shown for the PKA catalytic subunit, which forms complexes with the GR, IκB and NF-κB [258,299]. Tethering of the PKA catalytic subunit in a complex with IκB and NF-κB provides a novel cAMP-independent means of regulating PKA, whereby degradation of IκB disinhibits PKA, allowing it to phosphorylate and activate NF-κB [299]. PKA is uniformly expressed and EPAC is expressed in specific subsets of haematopoietic cells, and these two effectors are probably responsible for mediating almost all of the known cAMP signalling events in these cells. Although CNGCs have not been explored in haematopoeitic cells, they are expressed in spleen and thymus [300], and future investigations may uncover a role(s) for these effectors in these cells as well. Activation of PKA by cAMP leads to changes both in cytosolic proteins and in gene transcription through phosphorylation of cAMP-responsive nuclear factors such as CREB, CREM (CRE modulator) and ATF-1 (activating transcription factor 1) [301]. Additionally, a powerful repressor, ICER (inducible cAMP early repressor), can be formed from the CREM gene, following stimulation of cAMP signalling, probably as a feedback mechanism to terminate cAMP-induced gene expression [301]. ICER has been shown to function in T-cells as a transcriptional repressor and to be induced by the PDE4 inhibitor, rolipram, in osteoblasts [302,303]. PDEs, by regulating cAMP levels, play a central role in modulating all of these cAMP signalling pathways and consequent physiological responses. AC, adenylate cyclase; R, receptor (seven-membrane-spanning G-protein-coupled metabotropic receptor).

Figure 2. PDEs as targets for inducing maturation of myeloid cells

It is proposed that PDE inhibitors through their elevation of cAMP can drive the maturation of myeloid cells through the PKA-mediated regulation of transcription. In cell lines and in animal models, PDE inhibitors can synergize with either RAR agonists or arsenic trioxide to drive differentiation of AML blast cells. Of note, PrKX, a variant catalytic subunit of PKA that binds to RI (regulatory subunit of PKAI) but not to RII (regulatory subunit of PKAII), is expressed in myeloid cells and has been implicated in myeloid differentiation [304,305].

Figure 3. PDE4 as a target for inducing apoptosis in B-CLL cells

PDE4-inhibitor-induced apoptosis in B-CLL cells appears to occur by a PKA-mediated pathway, as the percentage of cells that undergo apoptosis following rolipram treatment is reduced by co-treatment with the PKA antagonist Rp-8-Br-cAMPS. In contrast, activation of the cAMP effector EPAC1 by the EPAC-specific agonist 8-CPT-2Me-cAMP is anti-apoptotic in B-CLL cells. As the net effect of PDE4 inhibitor treatment is to induce apoptosis in B-CLL cells, the pro-apoptotic PKA-mediated pathway appears to predominate over the anti-apoptotic effects of PDE4-inhibitor-mediated EPAC activation. One study reported that PKA signalling induces apoptosis in B-CLL cells by activating PP2A, which in turn dephosphorylates BAD, releasing it from 14-3-3 proteins, and allowing BAD to initiate apoptosis through a mitochondrial pathway. Cyt C, cytochrome c.

Figure 4. PDE4 inhibitors augment glucocorticoid-induced apoptosis of B-CLL cells

In B-CLL cells that are relatively resistant to apoptosis induced by either hydrocortisone or dexamethasone, co-treatment with rolipram can increase the percentage of apoptotic cells in a supra-additive manner. Experiments with GRE-containing luciferase reporter constructs demonstrate that rolipram treatment also augments glucocorticoid-mediated apoptosis and glucocorticoid-induced GRE activation in B-CLL cells, suggesting that levels of PKA activity may serve as a ‘rheostat’ that determines the apoptotic outcome of glucocorticoid therapy in this B-cell malignancy. Although the precise mechanism by which PKA activity modulates glucocorticoid-induced apoptosis remains unknown, the catalytic subunit of PKA has been reported to associate with the GR and to phosphorylate co-activators or co-receptors that are associated with this transcriptional complex. Notably, the mechanism by which GR signalling induces apoptosis in lymphoid cells remains controversial. GR-mediated apoptosis could involve either transactivation of GRE-containing promoters or GR-mediated trans-repression of other transcription factors such as NF-κB or AP-1 (activator protein 1) through a tethering mechanism that does not require a functional GR DNA-binding domain. GC, glucocorticoid; GPCR, G-protein-coupled receptor.

Figure 5. PDE4 inhibitors induce apoptosis in DLBCL cell lines

Elevated levels of PDE4B2 protect DLBCL cell lines from forskolin-induced apoptosis, and PDE4B-selective inhibitors overcome this, allowing forskolin to elevate cAMP levels and induce apoptosis. In this case, cAMP was reported to act neither through EPAC nor through PKA, but instead through an as yet undefined effector to inhibit PI3K, thereby reducing PKB/Akt activity and consequent phosphorylation of BAD. Dephosphorylated BAD can then induce apoptosis through the mitochondrial pathway. Alteration of the phosphorylation state of other PKB/Akt phosphorylation targets such as FKHR (forkhead in rhabdomyosarcoma) [306] and GSK3β (glycogen synthase kinase 3β) [307] may also contribute. Cyt C, cytochrome c; PIP3, PtdIns(3,4,5)P₃.