Extracellular Regulating Kinase (ERK) Inhibition by U0126: A Novel Mechanism for γ-Globin Gene Activation.

Abstract Fetal hemoglobin (HbF) effectively inhibits hemoglobin S polymerization in red blood cells and improves symptoms in sickle cell disease. Studies in K562 cells induced with sodium butyrate (NaB), apicidin and hydroxyurea suggest that ERK inhibition is required for induction of cell differentiation and HbF synthesis. Multiple K562 stable lines were established with the pcmvMEK1/2 expression vector which mediates constitutively active ERK signaling. We observed a 75% decrease in γ-globin expression at baseline which was completely reversed by the ERK pathway inhibitor, U0126. This data confirmed the ability of ERK to silence γ-gene expression. Western blot analysis was performed to determine whether ERK inhibition is a common mechanism for γ-globin activation by other HbF inducers as well. We tested the histone deacetylase inhibitors: trichostatin A (TSA), SAHA, and MS275, decitabine and hemin, and the ERK inhibitors U0126 and PD98059. ERK phosphorylation was decreased from 45% to 99% at 24 and 48 hrs in K562 cells treated with TSA, decitabine, U0126 and PD98059 supporting a common mechanism for HbF induction. Concomitantly, γ-globin mRNA synthesis increased 2 to 8-fold, measured by real-time PCR analysis. When U0126 was combined with NaB or TSA, γ-gene expression increased 9-fold and 13-fold respectively. To evaluate the therapeutic potential of U0126 in sickle cell disease, we tested its effects on cell viability at 5 to 40μg concentrations. K562 cell viability remained >60% at the highest concentration for 72 hrs. Another positive effect of U0126 is its ability to inhibit cell proliferation mediated by ERK thus promoting differentiation and hemoglobin induction. Subsequent studies were completed in ES10 stable lines established with an HS2γβ mini-construct in MEL cells to test γ-gene activation in the presence of the β-globin gene. We also determined whether ERK signaling is inhibited by HbF inducers in an adult intracellular environment. ERK phosphorylation was blocked by U0126, TSA and NaB in parallel with a 5 to 34-fold increase in γ-globin expression. Additional studies are in progress to test the ability of U0126 to induce HbF in primary erythroid progenitors. Investigations are underway to ascertain mechanisms for γ-globin regulation by U0126 using DNA microarray analysis. K562 cells were induced with 20μM U0126 for 48 hrs and total RNA with 5-fold γ-globin induction was analyzed using ArrayIt DNA microarray chips (TeleChem International, Inc., Sunnyvale, CA) containing 380 genes from several functional groups. Hierarchical cluster analysis confirmed a >2-fold increase in gene expression for heat shock 70 kD protein 6, insulin receptor substrate 2 and transcription factor Dp-1. Genes inhibited >2-fold by U0126 included the proto-oncogene fms-tyrosine kinase and glutathione-S-transferase. When NaB was combined with U0126 a synergistic 13-fold increase in Dp-1 was observed. The role of these and other transcription factors identified by microarray analysis in γ-globin regulation will be investigated. The ability of U0126 to act as a potent HbF inducer and the limited number of genes altered by this agent makes it an ideal lead compound for clinical development.