Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

We thank Sarah Haigh, Ada Kane, Nicole Reuter, David Carey, and Marilyn Perry Carey for dedicated and expert technical assistance and Cloret Carl for assistance with preparation of the manuscript.This work was supported by grants from the National Institutes of Health, R01 DK-52962, (SPP, Boston University), R41 HL-105816 (SPP, Phoenicia BioSciences), and R42 HL-110727 (Phoenicia BioSciences), 2 P40 ODO010988-16 (GLW, University of Oklahoma) and UL1-TR000157 (RFW, University of Oklahoma). SMN was supported by P50 HL-118006. The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.Yeshttp://www.plosone.org/static/editorial#pee

Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to<i>Pseudomonas aeruginosa</i>

The NLRC4 inflammasome is responsible for IL-1β processing by macrophages in response to Pseudomonas aeruginosa infection. We therefore hypothesized that mice that lack ASC, an NLRC4 inflammasome adaptor protein necessary for in vitro IL-1β production by macrophages, would be preferentially protected from a hyperinflammatory lethal challenge that is dependent on bacterial type three secretion system (T3SS) activity. We report herein that lack of ASC does not confer preferential protection in response to P. aeruginosa acute infection and that ASC−/−mice are capable of producing robust amounts of IL-1β comparable with C57BL/6 mice. We now identify that neutrophils represent the ASC-independent source of IL-1β production during the acute phases of infection both in models of acute pneumonia and peritonitis. Consequently, depletion of neutrophils in ASC−/−mice leads to a marked deficit in IL-1β production in vivo. The pulmonary neutrophil IL-1β response is predominantly dependent on caspase-1, which contrasts with data derived from ocular infection. These studies therefore identify a noncanonical mechanism of IL-1β production by neutrophils independent of ASC and demonstrate the first physiological contribution of neutrophils as an important source of IL-1β in response to acute P. aeruginosa infection during acute pneumonia and peritonitis.</jats:p

Developmental- and differentiation-specific patterns of human - and -globin promoter DNA methylation

The mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34(+) cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching

Neither DNA Hypomethylation or Changes in the Kinetics of Erythroid Differentiation Account for 5-Azacytidine’s Ability To Induce Human Fetal Hemoglobin.

Abstract 5-Azacytidine (5-Aza) is among the most potent inducers of fetal hemoglobin (HbF) in patients with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first model is based on the drug’s ability to inhibit DNA methyltransferase enzymes causing global DNA hypomethylation, including the promoters of the fetal globin genes, resulting in their expression during adult erythropoiesis. The second model is based on the drug’s other well-know property - cytotoxicity. In this model, a cohort of differentiating erythroblasts either dies or undergoes growth arrest during drug exposure. The marrow then responds with a rapid proliferation of erythroblasts which express high levels of fetal globin mRNA and HbF. To determine which model best explains HbF induction by 5-Aza, we used an in vitro differentiation system in which CD34+ cells from normal donors are cultured with SCF, IL-3 and Flt-3 ligand for 7 days and then switched to Epo for an additional 14 days. This results in &gt;90% erythroid cells, a 2,000-fold expansion in cell number and Hb expression similar to that seen in normal red cells (∼99% HbA, ∼1% HbF). To model common 5-Aza dosing schedules, we treated cultures daily with doses ranging from 0 to 1000 nM. Near-maximal induction of γ-globin mRNA (∼2-fold increase in γ/γ+β) and HbF (from 1% to 40%) occurred at the 300 nM dose level. While this dose was associated with hypomethylation of the 7 CpGs between −256 and +50 bp of the γ promoter (59% +/− 14% methylated CpGs vs. 92% +/− 8.1% in untreated controls, p &lt; 0.001), no changes were found in cell expansion rate, differentiation kinetics (as judged by glycophorin A expression) or cell cycle distribution. These results argue against the cytotoxicity model. We also observed that global DNA methylation was not significantly changed by the 300 nM dose of 5-Aza. Because 5-Aza is incorporated into both RNA and DNA, it affects many cellular processes beyond DNA methylation. To determine if DNA hypomethylation is sufficient for γ-globin mRNA and HbF induction, we treated differentiating cells with DNMT1 siRNA. This resulted in transiently decreased DNMT1 mRNA and protein and lowered global DNA methylation. γ-globin promoter methylation decreased to levels equivalent to those seen with 300 nM 5-Aza (61% +/− 7% of CpGs methylated vs. 88% +/− 8% in controls, p &lt; 0.001) but did not induce γ-globin mRNA or HbF. To further evaluate this unexpected finding we used a lentiviral vector expressing DNMT1 shRNA. This suppressed DNMT1 mRNA and protein throughout differentiation and decreased total γ-globin promoter methylation from 98% +/− 4% in control cells, to 20% +/− 11% (p&lt;0.001) in treated cells. This level of γ promoter methylation is similar to that seen in fetal erythroid cells (13% +/− 7%). Global CpG methylation was also decreased vs. controls (30% +/− 10% vs. 83% +/− 11%, p&lt;0.001). Again, despite significant γ promoter and global hypomethylation, no induction of γ-globin mRNA or HbF was observed. These results suggest that neither model explains 5-Aza’s ability to induce fetal Hb and that alternative mechanisms await discovery. If verified, these findings have important implications for the future development of clinically useful HbF inducing agents.</jats:p

Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway

Although hematopoietic stem cell transplantation and gene therapy have the potential to cure β-thalassemia and sickle cell disease, they are not currently available to most people with these diseases. In the near term, pharmacologic induction of fetal hemoglobin (HbF) may offer the best possibility for safe, effective, and widely available therapy. In an effort to define new pathways for targeted drug development for HbF induction, we evaluated the nuclear factor erythroid 2–related factor 2 (NRF2) antioxidant response element signaling pathway. We found that 3 well-known activators of this pathway increased γ-globin mRNA at nontoxic doses in K562 cells. Tert-butylhydroquinone (tBHQ), the most active of these compounds, increased cellular levels and nuclear translocation of NRF2 and binding of NRF2 to the γ-globin promoter. siRNA knockdown of NRF2 inhibited γ-globin induction by tBHQ. When tested in human primary erythroid cells, tBHQ induced NRF2 binding to the γ-globin promoter, increased γ-globin mRNA and HbF, and suppressed β-globin mRNA and HbA, resulting in a > 3-fold increase in the percentage of HbF. These results suggest that drugs that activate the NRF2/antioxidant response element signaling pathway have the potential to induce therapeutic levels of HbF in people with β-hemoglobinopathies

Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vitro erythroid differentiation system. We found that doses of 5-Aza that produce near maximal induction of γ-globin mRNA and HbF do not alter cell growth, differentiation kinetics, or cell cycle, but do cause a localized demethylation of the γ promoter. However, when we reduced γ promoter methylation to levels equivalent to those seen with 5-Aza or to the lower levels seen in primary fetal erythroid cells using DNMT1 siRNA and shRNA, we observed no induction of γ-globin mRNA or HbF. These results suggest that 5-Aza induction of HbF is not the result of global DNA demethylation or of changes in differentiation kinetics, but involves an alternative, previously unrecognized mechanism. Other results suggest that posttranscriptional regulation plays an important role in the 5-Aza response

In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia

Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4+CD25− T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4+CD25− T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3+ Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs