In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of β-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce HbF expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells, GSK3482364 decreased overall DNA methylation resulting in derepression of the -globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.     

Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation

Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS‐275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients. Am. J. Hematol. 90:624–628, 2015. © 2015 Wiley Periodicals, Inc.     

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.     

Accumulation of γ-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of γ‐globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well‐known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two‐phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and γ‐globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of γ‐globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically‐mediated regulation of the expression of human γ‐globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including β‐thalassemia and sickle cell anemia.     

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.     

Fetal haemoglobin induction in sickle cell disease

Fetal haemoglobin (HbF, α2γ2) induction has long been an area of investigation, as it is known to ameliorate the clinical complications of sickle cell disease (SCD). Progress in identifying novel HbF‐inducing strategies has been stymied by limited understanding of gamma (γ)–globin regulation. Genome‐wide association studies (GWAS) have identified variants in BCL11A and HBS1L‐MYB that are associated with HbF levels. Functional studies have established the roles of BCL11A, MYB, and KLF1 in γ–globin regulation, but this information has not yielded new pharmacological agents. Several drugs are under investigation in clinical trials as HbF‐inducing agents, but hydroxycarbamide remains the only widely used pharmacologic therapy for SCD. Autologous transplant of edited haematopoietic stem cells holds promise as a cure for SCD, either through HbF induction or correction of the causative mutation, but several technical and safety hurdles must be overcome before this therapy can be offered widely, and pharmacological therapies are still needed.     

A candidate transacting modulator of fetal hemoglobin gene expression in the Arab—Indian haplotype of sickle cell anemia

Fetal hemoglobin (HbF) levels are higher in the Arab–Indian (AI) β‐globin gene haplotype of sickle cell anemia compared with African‐origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes—seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34⁺ cells. As CD34⁺ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118–1122, 2016. © 2016 Wiley Periodicals, Inc.     

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

Background

The human hemoglobin switch (HbF→HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design and Methods

We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34⁺ cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results

In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80–90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions

Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221–222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.     

Hematology Morphology Forum Case 1

Hydroxyurea (HU), a chemotherapeutic agent, used increasingly in the treatment of sickle cell disease (SCD) stimulates the release of a tumor necrosis factor (TNF-) from human macrophages in vitro and the concentration of TNF- is greater than normal in subjects affected by SCD. It is widely accepted that HU may inhibit vaso-occlusive crisis (VOC) by stimulating the production of fetal hemoglobin (HbF) and nitric oxide (NO) in SCD; however, the beneficial effects of HU in vivo may be counteracted by the release of TNF- and, in turn, the expression of a vascular cell adhesion molecule (VCAM-1) on leukocytes. Previous studies have shown that the severity of SCD increases with the leukocyte count. Therefore, we examined the relationship between plasma levels of TNF- and HbF in SCD patients during steady-state (StSt) conditions (in the absence of VOC) and during VOC conditions after the acute administration of HU. Venous blood was collected in SCD patients over 6 h after administering a single dose of HU. Plasma TNF- was found to be greater in SCD subjects than in reported normal adult controls (p<0.05). TNF- in the StSt group was not significantly different than in the VOC group; however, the plasma TNF- tended to greater in the VOC group (p>0.1). An increase in the HbF concentration after acute administration of HU (p<0.01) was not associated with a significant change in plasma TNF- (p>0.1). Contrary to the results of in vitro studies, HU did not increase the plasma concentration of TNF-. These findings suggest that a HU-induced increase in TNF- does not contribute to VOC and sickle cell patients can be counseled that the HU-induced increase in TNF- does not counteract the beneficial effects of HU in SCD.     

Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells

The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.     

Serum-free culture of enriched hematopoietic progenitors reflects physiologic levels of fetal hemoglobin biosynthesis.

Adult erythroid progenitors produce significantly higher fetal hemoglobin (HbF) levels in cultures containing fetal calf serum (FCS) and erythropoietin (Ep) than in vivo. The precise mechanisms for this increased HbF production in culture have not been elucidated. We examined HbF biosynthesis by enriched human progenitors in serum-free (SF) culture. We measured globin chain biosynthesis by combination of isoelectric focusing and autoradiography and examined percent nucleated erythrocytes containing HbF (%FNRBC) using microscopic immunodiffusion. CD34 (My10)-positive marrow cells from a normal subject yielded an almost negligible amount of gamma-globin in SF culture stimulated by 100 U/mL interleukin-3 (IL-3) and 2 U/mL Ep, while corresponding FCS culture revealed significant gamma-globin biosynthesis. The %FNRBC of the erythroid bursts in SF cultures derived from nine normal adults (2.0% +/- 0.9% F cells) was 3.0% +/- 3.4%, while in FCS culture, it was 25% +/- 12% (mean +/- SD). Dosages of IL-3 between 10 and 10,000 U/mL did not increase %FNRBC in FCS of SF conditions. Mean Hb contents of nucleated erythrocytes (NRBC) assayed by microdensitometry of pericellular immunoprecipitate were similar in FCS and SF cultures. The number of erythroid bursts per 2 x 10(3) CD34-positive marrow cells was 48 +/- 20 in FCS and 36 +/- 12 in SF cultures. In two experiments, progenitors grown for 7 days under SF conditions were isolated and recultured in either SF or FCS conditions for 7 days, and the resulting erythroid bursts were analyzed for FNRBC. The bursts that had been returned to FCS cultures yielded values of %FNRBC intermediate between those obtained from progenitors grown entirely in SF or FCS cultures, indicating that serum effect is not solely due to growth selection for certain subpopulations of erythroid burst-forming units. This experiment also demonstrated that the factors present in serum responsible for HbF augmentation act at both early and late stages during erythroid burst development. SF culture of peripheral blood progenitors of one subject with heterocellular hereditary persistence of fetal hemoglobin (HPFH) yielded elevated levels of FNRBC (19% +/- 5%) that accurately reflected the F cell (18%) of the circulating blood. Similarly, FNRBC in cultures of progenitors from one umbilical cord blood sample (86% F reticulocytes) was 87 +/- 3% FNRBC. The SF culture for enriched human progenitors, which nearly reflects the physiologic HbF programs of the donor, should facilitate studies of the exact mechanisms of postnatal reactivation of HbF production.     

DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620–1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of ^Gγ-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 × 10⁻⁴²). Together, common SNPs at the BCL11A, HBS1L-MYB, and β-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.     

Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine

In vivo observations on the kinetics of F cells and of fetal hemoglobin (HbF) synthesis and in vitro studies of erythroid progenitors, their number, and the gamma-gene expression in their progeny were carried out in baboons (Papio cynocephalus) treated with 5-azacytidine. Maximum effect on the increase of HbF production in vivo was observed only when an expanded erythroid marrow population was present. In these animals, as well as in normal animals, treatment resulted in a significant reduction of the late erythroid progenitor cell pools (erythroid clusters and erythroid colony-forming units, CFU-E) in the marrow. This reduction was more pronounced among those progenitors grown in the absence of added erythropoietin, and it was followed by a rebound a few days after treatment cessation, reflecting the accumulation of regenerating progenitors. An early increase in the in vitro synthesis of HbF in erythroid clusters and CFU-E colonies was observed. This increase was further documented at the cellular level, with immunofluorescent labeling of colonies with monoclonal anti-gamma- globin chain antibodies. In contrast to the findings in late progenitors, the number of erythroid burst-forming unit (BFU-E) colonies and the synthesis of HbF in these colonies was not influenced significantly by 5-azacytidine treatment. It is proposed that the toxic effects of 5-azacytidine on late progenitors, leading to faster mobilization of earlier progenitors to the next more mature compartment, play a role in the in vivo augmentation of HbF synthesis by this drug. This perturbation in the progenitor cell population kinetics and the presumed hypomethylation of the surviving differentiating cells may act synergistically to produce a maximum HbF response after 5-azacytidine treatment.     

Fetal hemoglobin synthesis in vivo: direct evidence for control at the level of erythroid progenitors

To test directly whether the control of fetal hemoglobin (HbF) in the adult takes place at the level of erythroid progenitors or at the level of erythroblasts, we treated animals with high doses of erythropoietin and examined the effects of this manipulation on the globin gene programs of erythroid progenitors. We found that administration of erythropoietin produced a rapid expansion of all classes of erythroid progenitors. Almost all the expansion of colony-forming units-erythroid and 46-56% of erythroid clusters was due to the increase of HbF-programmed erythroid progenitors. The expansion of HbF-programmed erythroid progenitors was followed, 2-3 days later, by a wave of reticulocytes containing HbF in the peripheral blood. These results provide direct in vivo evidence that fetal-globin expression in the adult is controlled at the level of erythroid progenitors.