Abstract
An important treatment strategy for sickle cell anemia is increasing fetal hemoglobin (HbF) in circulating erythrocytes. OSI-2040 (Apicidin), a fungus-derived cyclic tetra-peptide, induces g-globin production in K562 cells. The effect of increasing doses of OSI-2040 (from 7.5 to 750 nM) was determined on cultures of primary human hematopoietic progenitor cells stimulated with erythropoietin (EPO). Cell proliferation and differentiation, globin production, and erythroid transcription factors expression were examined. At concentrations 7.5 nM - 75 nM there was minimal decrease in cell proliferation with little change in % benzidine positive cells after 12 days of culture with EPO. As OSI-2040 concentration increased above 75 nM, cell proliferation and % benzidine positive cells decreased, with concentrations of 300 and 750 nM being highly toxic, reducing cell number by 75% or more. Analysis of globin gene expression indicates that low to mid concentrations of OSI-2040 increase g-globin, with the peak increase occurring at 75 nM, while the highest concentrations (300 and 750 nM) suppress g-globin. OSI-2040 decreases b-globin expression with the highest concentrations resulting in the greatest decreases. The g/(g+b) ratio increases with increasing OSI-2040 concentration reaching a value of 4-fold and greater for concentrations of 75 nM or more, partially a consequence of the suppression of b-globin expression, particularly at higher concentrations. Although the g/(g+b) ratio is relatively high at the highest concentrations of OSI-2040 (300 and 750 nM), it is at a cost in overall globin production and cell toxicity. Hemoglobin expression is determined primarily at the transcription level. We found that OSI-2040 affects expression of select transcription factors, GATA-1, GATA-2, SCL/Tal-1 and EKLF, which are critical for erythroid differentiation. Peak EPO induction of GATA-1, a zinc-finger transcription factor essential for survival and differentiation of erythroid progenitor cells, is delayed with OSI-2040 treatment. OSI-2040 also delays expression of SCL/Tal-1, a basic-helix-loop-helix transcription factor that positively regulates erythroid differentiation and is required for the production of mature erythrocytes. In addition, there is a delay in the induction of EKLF, a zinc-finger transcription factor necessary for induction of b-globin in adult erythroid cells that acts by direct binding to the b-globin promoter. With increasing OSI-2040 concentrations, there is a dose-dependent decrease in overall levels of GATA-1, SCL/Tal-1 and EKLF. GATA-2, a member of the GATA-family that plays a critical role in the survival of early erythroid progenitor cells and is down-regulated with EPO stimulation, shows a slight delay in its reduction at 75 and 150 nM but overall is not greatly affected by OSI-2040. Thus, OSI-2040 concentration is crucial in optimizing the production of HbF. As we have also observed with hydroxyurea, the greatest increase in the g/g+b ratio is at high concentrations of OSI-2040 (300 and 750 nM), up to 25-fold, and is a consequence of reductions in both b- and g-globin. In contrast, a mid-level concentration (75 nM) yields a 2.5–4 fold increase in the g/(g+b) ratio with little or no cytotoxicity. These data suggest that like hydroxyurea, OSI-2040 may be effective in inducing HbF and may be a useful therapeutic alternative.
Phosphorylation status of transcription factor C/EBPα determines cell-surface poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis
