A Role for ETO-2 In Megakaryocytic Inhibition by Cyclic AMP Agonists

Abstract 289

The regulation of megakaryopoiesis by cyclic AMP (cAMP) signaling forms the basis for treatment approaches to thrombocytosis in patients with myeloproliferative neoplasms. Anagrelide, the longstanding treatment of choice, is an inhibitor of the cAMP catabolizing enzyme phosphodiesterase III, and acts to lower platelet counts by inhibiting early stages of megakaryocytic differentiation. Essential for programming megakaryocytic differentiation is the transcriptionally activating pentameric complex consisting of GATA-1, SCL/TAL-1, an E-protein (either E2A or the similar HEB), and the bridging factors Lmo2 and Ldb1. This complex becomes repressive upon the recruitment of the co-repressor ETO-2 (also called MTG16 and CBFA2T3) by either SCL or E-protein components. ETO-2 has also been described as an A-kinase floating protein (AKAP). AKAPs bind the regulatory subunits of Protein kinase A (PKA) and tether the kinase within a specific compartment of the cell, ensuring proximity to appropriate substrates following kinase activation. The current study addresses the hypothesis that elevations in cAMP, such as those induced by anagrelide, inhibit megakaryocytic differentiation through the activation of a novel PKA signaling pathway involving ETO-2 and other components of the pentameric complex. Using peripheral blood mobilized human CD34+ progenitors cultured under conditions promoting megakaryopoiesis, we have confirmed that 500 nM anagrelide or 10 μ M forskolin, a direct adenylyl cyclase agonist, suppress upregulation of CD41 by two to three-fold as well as polyploidization, as determined by FACS on day 6 cultures. Pre-treatment of cultures with 10 μ M H89, a specific inhibitor of PKA, prevented the inhibitory effect of forskolin on megakaryopoiesis, as reflected by CD41 upregulation at day 6. Analysis of the kinetics of PKA activation, by immunoblotting for phospho-CREB and for phospho-PKA substrates (Cell Signaling), surprisingly showed only transient PKA induction by forskolin, with signals returning to baseline level after 4 hours. Additionally, washout experiments in which cells were treated with forskolin for only the first 4 hours of megakaryocytic culture showed severely blunted CD41 upregulation on day 6. These findings argue that elevations in cAMP inhibit megakaryocytic differentiation via activation of PKA and that a brief window of PKA activation during early megakaryopoiesis suffices for a stable blockade of differentiation. To determine a potential role for ETO-2, expression levels were analyzed at various times during megakaryocytic culture. In contrast to the rapid downregulation described in fetal murine megakaryocyte culture (Hamlett I. et al. Blood 112:2738-2749, 2008), human CD34+ progenitors showed no downregulation of ETO-2 mRNA or protein during megakaryocytic differentiation, nor were levels significantly affected by forskolin treatment. To study the functional role of ETO-2, we utilized shRNA knockdown in CD34+ hematopoietic progenitor cells undergoing megakaryocytic culture. In contrast to cells transduced with control vector, cells expressing shRNA targeting ETO-2 showed resistance to the inhibitory effects of elevated cAMP (n = 3). In examining potential targets within the pentameric complex, we found that elevations in cAMP reproducibly lowered the protein and mRNA levels of E2A by 50% while not affecting mRNA levels of another E protein, HEB. Altogether, these data suggest that cAMP mediates inhibition of megakaryopoiesis via activation of PKA, which utilizes ETO-2, possibly as an AKAP, to modulate the composition of an essential activating complex by downregulating E2A. Interestingly, E2A+/− mice are impaired in their ability to generate megakaryocytic progenitors from bipotent megakaryocyte-erythroid precursors (Semerad CL et al. PNAS 106:1930-1935, 2009). This novel signaling pathway contributes to an understanding of the mechanism of action of anagrelide and also further elucidates the basic regulatory circuitry governing determination of the megakaryocytic lineage.