Exchange protein activated by cAMP: Drug target and biomarker in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is associated with the accumulation of B-lymphocytes due to decreased apoptosis. The second messenger 3’5’-cyclic adenosine monophosphate (cAMP) promotes apoptosis by unknown mechanisms in peripheral blood mononuclear cells (PBMC) isolated from patients with CLL. The actions of cAMP are mediated by protein kinase A (PKA) and Exchange protein activated by cAMP-1 (Epac-1). Using real-time PCR we find that compared to PBMC from healthy subjects, CLL-cells have elevated Epac-1 and decreased PKA regulatory subunit RIIβ mRNA expression. Studies using traditional PCR reveal that this increase in Epac-1 mRNA expression does not result from single nucleotide polymorphisms (SNPs) in the promoter region of Epac-1. Use of fluorescent-activated cell sorting (FACS) (to assess annexin 5 staining, a marker for apoptosis) revealed that 48 hr treatment with an Epac-selective analog (8-pCPT-2Me-cAMP [8Me], 50 μM) inhibited apoptosis of CLL-cells but that a PKA-selective analog (N⁶-Phenyladenosine-cAMP [N6], 50 μM) induced apoptosis. Inhibition of Rap-1, the downstream mediator of Epac-1, with N-4-[2(R)-Amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine methyl ester [GGTI-298] (100 nM–10 μM, 48h) increased apoptosis of CLL-cells and prevented the anti-apoptotic effect of Epac activation. These results reveal that CLL is associated with increased Epac-1 expression and that unlike PKA activation, Epac-1 activation (in a Rap-1 dependent manner) enhances the survival of the malignant B-cells. Approaches that decrease the expression and function of Epac-1 and increase the expression and function of PKA thus may be beneficial in treating CLL by increasing the pro-apoptotic effects of cAMP.