Rachel J. West

A cell stress signaling model of fetal hemoglobin induction: what doesn't kill red blood cells may make them stronger.

A major goal of hemoglobinopathy research is to develop treatments that correct the underlying molecular defects responsible for sickle cell disease and beta-thalassemia. One approach to achieving this goal is the pharmacologic induction of fetal hemoglobin (HbF). This strategy is capable of inhibiting the polymerization of sickle hemoglobin and correcting the globin chain imbalance of beta-thalassemia. Despite this promise, none of the currently available HbF-inducing agents exhibit the combination of efficacy, safety, and convenience of use that would make them applicable to most patients. The recent success of targeted drug therapies for malignant diseases suggests that this approach could be effective for developing optimal HbF-inducing agents. A first step in applying this approach is the identification of specific molecular targets. However, while >70 HbF-inducing agents have been described, neither molecular mechanisms nor target molecules have been definitively verified for any of these compounds. To help focus investigation in this area, we have reviewed known HbF-inducing agents and their proposed mechanisms of action. We find that in many cases, current models inadequately explain key experimental results. By integrating features of the erythropoietic stress model of HbF induction with data from recent intracellular signaling experiments, we have developed a new model that has the potential to explain several findings that are inconsistent with previous models and to unify most HbF-inducing agents under a common mechanism: cell stress signaling. If correct, this or related models could lead to new opportunities for development of targeted therapies for the beta-hemoglobinopathies.

The C. elegans gon-2 gene encodes a putative TRP cation channel protein required for mitotic cell cycle progression

The C. elegans gon-2 gene is required for the post-embryonic mitotic cell divisions of the gonadal precursor cells. A single major transcript of approximately 6.7 kb is derived from the gon-2 locus. This mRNA encodes a protein related to the TRP family of cation channels and has a high degree of similarity to several vertebrate genes, including melastatin. Mutant alleles of gon-2 affect evolutionarily conserved amino acid residues. Northern analyses suggest that gon-2 expression is not limited to gonadal tissues.

Simvastatin and t-butylhydroquinone suppress KLF1 and BCL11A gene expression and additively increase fetal hemoglobin in primary human erythroid cells.

UNLABELLED Although increased fetal hemoglobin (HbF) levels have proven benefit for people with β-hemoglobinopathies, all current HbF-inducing agents have limitations. We previously reported that drugs that activate the NRF2 antioxidant response signaling pathway increase HbF in primary human erythroid cells. In an attempt to increase HbF levels achieved with NRF2 activators, in the present study, we investigated potential complementary activity between these agents and HMG-CoA reductase inhibitors (statins) based on their ability to induce KLF2 protein levels. Experiments in K562 cells showed that simvastatin increased KLF2 mRNA and protein and KLF2 binding to HS2 of the β-globin locus control region and enhanced -globin mRNA production by the NRF2 activator Tert-butylhydroquinone (tBHQ). When tested in differentiating primary human erythroid cells, simvastatin induced HbF alone and additively with tBHQ, but it did not increase KLF2 mRNA or locus control region binding above levels seen with normal differentiation. Investigating alternative mechanisms of action, we found that both simvastatin and tBHQ suppress β-globin mRNA and KLF1 and BCL11A mRNA and protein, similar to what is seen in people with an HPFH phenotype because of KLF1 haploinsufficiency. These findings identify statins as a potential class of HbF-inducing agents and suggest a novel mechanism of action based on pharmacologic suppression of KLF1 and BCL11A gene expression. KEY POINTS Simvastatin and tBHQ suppress KLF1 and BCL11 gene expression and additively increase fetal hemoglobin in primary human erythroid cells. Because both drugs are FDA-approved, these findings could lead to clinical trials in the relatively near future.

Multiple physical stresses induce γ-globin gene expression and fetal hemoglobin production in erythroid cells

Increased fetal hemoglobin (HbF) expression is beneficial for β-hemoglobinopathy patients; however, current inducing agents do not possess the ideal combination of efficacy, safety and ease of use. Better understanding the mechanisms involved in γ-globin gene induction is critical for designing improved therapies, as no complete mechanism for any inducing agent has been identified. Given the cytotoxic nature of most known inducing drugs, we hypothesized that γ-globin is a cell stress response gene, and that induction occurs via activation of cell stress signaling pathways. We tested this hypothesis by investigating the ability of physical stresses including heat-shock (HS), UV- and X-irradiation and osmotic shock to increase γ-globin gene expression in erythroid cells. Experiments in K562 and KU812 cells showed that each of these stresses increased steady-state γ-globin mRNA levels, but only after 3-5days of treatments. HS and UV also increased γ-globin mRNA and HbF levels in differentiating primary human erythroid cells. Mechanistic studies showed that HS affects γ-globin mRNA at multiple levels, including nascent transcription and transcript stability, and that induction is dependent on neither the master regulator of the canonical HS response, HSF1, nor p38 MAPK. Inhibitor panel testing identified PI3K inhibitor LY294002 as a novel inducing agent and revealed potential roles for NFκB and VEGFR/PDGFR/Raf kinases in HS-mediated γ-globin gene induction. These findings suggest that cell stress signaling pathways play an important role in γ-globin gene induction and may provide novel targets for the pharmacologic induction of fetal hemoglobin.