Dual-Specificity Phosphatases (DUSPs) Are Potential Targets for Pharmacologic Induction of Fetal Hemoglobin

Abstract 2075

Morbidities of sickle cell anemia and β-thalassemia can be alleviated by increased fetal hemoglobin (HbF) production. However, currently available HbF inducing agents, such as HU, butyrate, and DNA methyltransferase inhibitors are not ideal due to suppression of hematopoiesis and other short and long-term side effects. Our goal is to identify novel HbF induction strategies that have the safety, efficacy and ease of use to make them applicable to most hemoglobinopathy patients. Recently, we proposed that most inducing agents increase γ-globin gene expression through activation of cell stress signaling pathways, including the p38 MAPK pathway (Mabaera et al, Exp Hematol. 2008). Evidence supporting the involvement of p38 signaling in HbF induction includes publications from multiple groups showing that γ-globin mRNA induction by several agents activates p38 signaling and can be suppressed by inhibitors of p38. Our lab has shown that physical stresses such as hyperosmotic shock, UV and X-ray irradiation, and heat shock are capable of activating p38 and inducing γ-globin mRNA expression in erythroid cells that is blocked by SB203580, a p38 inhibitor. If our model is correct, strategies that increase p38 signaling without cellular stress could be therapeutically beneficial. One possible approach is inhibition of the dual-specificity phosphatases (DUSPs) that are induced by MAPK signaling and then feedback to dephosphorylate activated MAPKs including p38. If this model is correct, suppression of DUSP activity could increase basal levels of p38 signaling without stress. Regulation of specific MAPK pathways by different DUSPs is cell-type and stress-type dependent. To determine which DUSPs are likely to be involved in p38 signaling in erythroid cells, we first used RT-PCR to determine which of ten DUSPs that are known to modulate MAPK signaling (DUSPs 1, 2, 4, 5, 6, 7, 8, 9, 10, and 16) are expressed in human erythroid progenitors and K562 cells. All ten DUSPs were expressed in both cell types. Next, we determined the effects of hemin (10 and 20μM) and Na butyrate (1 and 2mM), two known inducers of HbF and p38 MAPK signaling, on mRNA levels of the ten DUSP genes in K562 cells. γ-globin mRNA was induced by both drugs at these doses. DUSP gene expression was assessed during a short time course ranging from 5 minutes to 5 hours and a longer time course from 24 to 72 hours. At early time points, DUSP5 expression was induced up to 4-fold with both drugs when compared to untreated controls. DUSP5 continued to have the greatest level of induction at later time points (up to 8-fold) while DUSP4 and DUSP6 reached maximum induction (up to 4-fold) at 72 hours. Treatment of K562 cells with a small molecule inhibitor of DUSP1 and DUSP6, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-iden-1-one (BCI, gift of Dr. John Lazo) at 1μM increased γ-globin mRNA levels 5-fold, similar to levels seen with hemin and butyrate, and increased p38 phosphorylation by western blotting. Together, these data suggest that DUSPs may be a molecular target for the development of novel HbF inducing agents.