Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of γ-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced γ-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell mass. (Blood. 2004;104:2073-2080)

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL‐6 cytokine and JAK‐STAT signaling pathway related genes in circulating CD34⁺ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK‐STAT signaling pathway related genes had augmented expression in CD34⁺ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL‐6 cytokine levels were increased in plasma, as well as IL‐6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3′,5′-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 μM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.

Hydroxyurea is a cell-cycle-specific drug that has been used to treat myeloproliferative diseases and sickle cell anemia. We have recently shown that hydroxyurea, like nitric oxide (NO)-donor compounds, increased cGMP levels in human erythroid cells. We show now that hydroxyurea increases endothelial-cell production of NO; this induction of NO in human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC) is blocked by competitive inhibitors of NO synthase (NOS), such as NG-nitro-L-arginine-methyl ester (L-NAME) and NG-nitro-L-arginine. It is dependent on cAMP-dependent protein kinase (PKA) and protein kinase B (PKB/Akt) activity. We found that hydroxyurea dose- and time-dependently induced rapid and transient phosphorylation of eNOS at Ser1177 in a PKA-dependent manner; inhibitors of PKB/Akt could partially abrogate this effect. In addition, hydroxyurea induced cAMP and cGMP levels in a dose-dependent manner, as well as levels of intracellular calcium in HUVECs. These studies established an additional mechanism by which rapid and sustained effects of hydroxyurea may affect cellular NO levels and perhaps enhance the effect of NO in myeloproliferative diseases.

This article reports metabolic consequences of JAK2-mutant myeloproliferative neoplasms (MPNs) with a therapeutic translational impact: expression of mutant JAK2 induces abnormal metabolic activity of MPN cells, resulting in hypoglycemia, adipose tissue atrophy, and early mortality.