Elevated Levels Of Fetal Hemoglobin Research Articles

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

Effect of the Oral LSD1 Inhibitor Ory-1001 on F-Retics in Baboons ( Papio anubis)

Elevated levels of Fetal Hemoglobin (HbF) reduce the severity of symptoms and increase life span in patients with sickle cell disease (SCD). Experiments performed in baboons ( P. anubis) have been instrumental in discovering the potent pharmocological agents that increase HbF in vivo. Experimental baboon studies have been directly translated to clinical trials in patients with sickle cell disease. Drugs such as DNMT1 and LSD1 inhibitors that attack the repressive epigenetic regulatory mechanisms that silence expression of the γ-globin gene in adult erythroid cells increase HbF in baboons to levels necessary to provide therapeutic relief in SCD patients. Dose-dependent adverse events associated with the use of these drugs are minimized by modifications of dose and schedule as shown by a clinical trial of oral THU-decitabine in 25 SCD patients (Molokie et al, Plos Med 2017;14(9):e10022382). Our recent results have shown that subcutaneous administration of the DNMT1 inhibitor decitabine and the LSD1 inhibitor RN-1 2d/week produced synergistic increases in F-retics, F cells and γ-globin mRNA in normal baboons and increased HbF to therapeutic levels in phlebotomized anemic baboons after administration of only four doses (Ibanez et al, Blood Adv 2023;7(15):3891-3902). The goal of this current study is to develop an oral combinatorial drug regimen using THU-decitabine and the LSD1 inhibitor ORY-1001 that could be directly translated to clinical trials. Our previous studies demonstrated the effectiveness of oral THU-decitabine to increase HbF levels in baboons (Lavelle et al, Blood 2012;119(5):1240-1247). In this study we have performed 16 dose-response experiments in baboons to test the effect of oral ORY-1001 at doses ranging between 8 and 24µg/kg/d. ORY-1001 was chosen because it exhibited a good safety profile with known pharmacokinetic and pharmacodynamic parameters in a recent study in patients with relapsed/refractory acute myeloid leukemia (Salamero et al, J Clin Oncol 2020; 38(36):4260-4273). Four doses were tested (24µg/kg, n=2; 16µg/kg n=4; 12µg/kg, n-=7; 8µg/kg, n=3). The drug was administered on a single day to facilitate further combinatorial studies with THU-decitabine. Preliminary data from a current clinical trial of THU-decitabine in SCD patients (NCT04055818) suggests that administering the drug on a 1d per week schedule effectively increased HbF levels in patients. ORY-1001 was administered by gavage on a single day (d1) and the effect on F-retics measured by flow cytometry in peripheral blood samples obtained daily between days 6 and 10. The peak F-retic response was observed on d8. A linear dose-response relationship on F-retics was observed (Figure 1, r=0.79). Complete blood counts (CBC) were also performed daily between d6 and d10. Adverse hematological effects were not observed with no incidences of neutropenia or thrombocytopenia (Table 1). No consistent effects on reticulocyte counts were observed (Table 1). These results show that a single oral dose of ORY-1001 is sufficient to increase HBF in baboons without any adverse hematological effects and provide the necessary dose-response data to pursue further combinatorial studies with oral THU-decitabine

Direct and Indirect Functions of TR4 in the Control of Fetal Hemoglobin Silencing

Elevated levels of fetal hemoglobin (HbF) compensate for reduced normal β-globin chains and ameliorate clinical symptoms for patients with beta-globinopathies, such as sickle cell disease (SCD). The embryonic and fetal globin genes, but not the adult β-globin gene, have direct repeat (DR) elements in their promoters, which have been implicated in γ-globin gene repression (Tanabe et al., 2002). We previously purified a DR element-binding protein complex, which we named DRED, and was the first identified HBG gene repressor (Tanabe et al., 2002). DRED activity is conferred by a complex including LSD1, recruited by the nuclear receptors TR2/TR4 (Cui et al., 2011). BCL11A is a well-characterized transcription factor that independently represses the γ-globin genes through binding to their promoters (Liu et al., 2018). We have shown by co-immunoprecipitation that TR4 interacts with BCL11A in HUDEP2 cells. To understand how TR4 acts to repress the γ-globin gene and the relationship of TR4 to BCL11A in γ-globin repression, we characterized the chromatin occupancy of TR4 and BCL11A within the β-globin locus by CUT&RUN (CNR) in HUDEP2 cells. We established HUDEP2 cells bearing TR4 tagged with 3FLAG+3Myc epitopes (FM-TR4) and in the same cells, BCL11A tagged with 3HA+3Myc (HM-BCL11A) epitopes by genome editing. In addition, another HUDEP2 cell line bearing BCL11A tagged with the 3HA+3Myc (HM-BCL11A) epitope as well a cell line bearing TR4 tagged with the 3FLAG+3Myc epitopes (FM-TR4) were generated. The tagged cell lines not only improved the efficiency of antibody targeting, but also facilitated direct comparison of TR4 vs BCL11A chromatin occupancy by binding of the common Myc epitope in FM-TR4 and HM-BCL11A cell lines. Unbiased motif discovery of TR4 CNR binding of these epitopes in all 3 cell lines yielded the motif of a direct repeat consisting of 2 AGGTCA repeat elements with a 1 nt spacer. The exact match of BCL11A motif with one of the direct repeat elements of TR4 motif suggested that BCL11A and TR4 may be compete for binding to the γ-globin promoter. Overall, TR4 and BCL11A showed a similar pattern of occupancy within the globin locus, although of lower intensity. TR4 presented strong interaction with the locus control regions, among which HS2 and HS3 were the highest, consistent with the model of long-distance interaction through chromosomal looping. However, TR4 was found to be significantly less occupied at the γ-globin promoters than BCL11A. To test whether TR4 and BCL11A can bind in vitro to the motifs found in the γ-globin promoters, we performed TR4 and BCL11A electrophoretic mobility shift assays (EMSAs). We showed that TR4 binds to the DR1 element within the γ-globin promoters, which contains the distal TGACCA motif that was bound by BCL11A. Notably, mutation of the TGACCA motif disrupted both BCL11A and TR4 binding, while the other direct motif is indispensable for TR4 binding, supporting overlapping binding of BCL11A and TR4. Interestingly, DNA-TR4 protein complexes were competitively depleted by high concentrations of BCL11A and vice versa, suggesting competitive binding between the two factors. Studies of this mechanism in the erythroid cells are underway. In addition, unbiased motif discovery of TR4 CNR also enriched for KLF1, LRF and NF-Y motifs. Co-IP assays demonstrated interaction between TR4 and KLF1 and LRF, suggesting the presence of a large repressor complex. We also explore genome-wide occupancy of TR4 and discovered strong TR4 interactions at promoters of multiple HbF repressing transcription factors and corepressors, including NFIA and EHMT1, suggesting that the role of TR4 in HbF repression may be partially mediated by regulating expression of various HbF repressor genes. In summary, these data suggest that the function of TR4 in HbF silencing is mediated in part by direct repression of HBG promoters in a competitive manner with BCL11A, and in part by regulating the expression of multiple HbF repressor genes.

CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease

BackgroundSickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9–edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.MethodsWe performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin–immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1–2 clinical study to assess the safety and adverse-effect profile of OTQ923.ResultsIn preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.ConclusionsCRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.)

Prolonged Combinatorial Pharmacological Therapy Targeting the Epigenome Increases Fetal Hemoglobin to Clinically Relevant Levels in Normal, Nonanemic Baboons

Elevated levels of Fetal Hemoglobin (HbF; α2γ2) reduce the symptoms associated with Sickle Cell Disease (SCD) and increase the lifespan of patients. An effective pharmacological therapy that increases HbF would advance the treatment of SCD. HbF levels are developmentally regulated by transcriptional repression of the γ-globin gene in adult erythroid cells. This is achieved by a co-repressor complex containing multiple enzymes that catalyze repressive epigenetic modifications of chromatin targeted to the γ-globin promoter by site-specific DNA binding proteins. Pharmacological inhibitors of repressive epigenetic enzymes within the complex including DNMT1, LSD1 (KDM1A), G9A, and HDACs increase HbF in cell line and animal models and SCD patients. Many of these drugs have been studied in Phase 1 trials, but only the DNMT inhibitor decitabine (DAC) has advanced to Phase 3 trials. The successful development of a pharmacological therapy to increase HbF for SCD has thus far been hindered by dose-limiting side effects including neutropenia, thrombocytopenia, and/or thrombophilia. The goal of this current work is to develop a pharmacological therapy employing a combination of drugs that target different repressive epigenetic enzymes within the co-repressor complex based on the hypothesis that administration of these drugs in combination will reduce the dose and/or time of exposure to any single agent to minimize adverse effects while still stimulating clinically meaningful HbF elevations. To test this hypothesis, baboons, long considered the best animal model to test the effect of HbF-inducing agents due to conservation of the structure and developmental regulation of the β-globin gene locus between baboons and humans, were treated with a combination of two drugs, DAC and RN-1, that inhibit DNMT1 and LSD1 respectively. Analysis of seven baboons used in our studies showed that they comprised two phenotypic groups differentiated by baseline pre-treatment HbF expression (Figure 1). Two baboons of each phenotypic group were chosen to study the effect of DAC and RN-1, alone and in combination, on HbF. Either RN-1 (0.25mg/kg/d) or DAC (0.5mg/kg/d), were administered subcutaneously individually or in combination to normal, non-anemic animals for 2 days. The effect on γ-globin expression was measured by flow cytometry analysis of F-retics and RT-PCR of γ-globin mRNA in peripheral blood at least 3X per week post-drug treatments. Each drug induced a significant increase in F-retics and peripheral blood γ-globin mRNA in both phenotypic groups that peaked on d8 (Figure 1) while differences in F-retics and γ-globin mRNA between the two groups were maintained. Combined administration (RN-1 (0.25mg/kg/d; DAC (0.5mg/kg/d); 2d) increased levels of F-retics and γ-globin mRNA to greater than additive levels (Figure 1) while the difference in γ-globin mRNA (p<0.02), but not F-retics, between the two groups was still observed. To assess the effect of sustained administration of the two drug combination on HbF expression and evaluate possible hematological toxicity, two normal baboons, one of the low HbF group (PA 9254) and one of the high HbF group (PA 9194), were treated weekly with 12 total courses of the DAC-RN-1 combination (DAC, 0.5mg/kg/d; RN-1, 0.25mg/kg/d; 2d/wk). F cells increased approximately 6 fold to 60.5% in the high HbF animal and >10 fold to 42% in the low HbF baboon (Table 1). HPLC analysis of globin chain expression showed that HbF (γ/γ+β) increased tenfold in each animal (Table 1). The calculated % HbF/ F cell values were 33% for PA 9194 (High HbF group) and 23.5% for PA 9254 (Low HbF group). No instances of moderate or severe neutropenia, thrombocytopenia, or thrombophilia were observed during the treatment period (Table 1). Decreased Hct, Hb, and RBCs were apparent in the high HbF group baboon (PA 9194) but not the low HbF group baboon (PA 9254). In conclusion, combinatorial administration of DAC and RN-1 increased HbF and F cell numbers in a greater than additive manner. Prolonged treatment of two normal, nonanemic baboons increased F cells and HbF to levels predicted to be clinically significant in the absence of neutropenia, thrombocytopenia and/or thrombophilia throughout the treatment period although a reduction in Hct, RBCs, and Hb was observed in one animal. These results strongly suggest additional drug combinations, dosing, and schedules to increase HbF for the treatment of SCD should be pursued. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Examining the Role of Individual Baf Chromatin Remodeling Complex Components in Globin Gene Regulation

Regulation of the switch from fetal (HbF, α2γ2) to adult (HbA,α2β2) hemoglobin production is an important area of study as reversal of the switch is a key therapeutic approach for treatment of sickle cell disease and β-thalassemia. While multiple specific repressors of γ-globin gene transcription have been identified, there has not been a parallel finding of a γ-globin specific transcriptional activator. The mammalian BAF (BRG1/BRM-associated factor) complex is a multi-subunit, ATP-dependent, chromatin remodeling complex of the SWI/SNF family, with diverse composition and roles in regulation of gene expression. BRG1/SMARCA4 is a core component of the complex with ATPase catalytic activity. Prior studies showed that BRG1 is essential for proper β-globin expression and regulates long-range chromatin interactions between the locus control region (LCR) and the β-globin gene promoter. Studies in K562 cells, where γ-globin expression is predominant, have similarly shown the requirement for BRG1 for transcriptional activation. To date, the role of the BAF complex in regulation of γ-globin versus β-globin gene expression has not been well characterized. Moreover, the considerable diversity in the subtypes of this complex (such as polybromo-associated BAF or PBAF and non-canonical BAF), with subunit composition and functions that differ across cell types and developmental stages, suggests the hypothesis that specific subtypes or subunits of the BAF complex have specific roles in γ-globin gene regulation. We recently identified BRG1 as a potential activator of γ-globin production in a HUDEP2 CRISPR screen targeting ATPase and helicase domains. In validation experiments in HUDEP2 and primary human erythroid cultures, loss of BRG1 led to a marked decrease in γ-globin containing F-cells by flow cytometry and γ-globin protein by western blot, along with a smaller decrease in β-globin production. Because the BAF subunits have significant heterogeneity in expression and function, we asked whether any components may have specificity for γ-globin versus β-globin gene transcription in adult erythroid cells. Using CRISPR-Cas9 targeting in a HUDEP2 sub-clone with elevated fetal hemoglobin levels, we systematically ablated 12 individual BAF/PBAF complex components and assayed fetal hemoglobin by flow cytometry, HPLC, and quantitative western blots. We identified 5 components whose loss of function led to a reduction in γ-globin relative to β-globin: BAF53A, BAF155, BAF60A, BAF60B, and BAF57. We subsequently depleted these five components in primary human erythroid cultures from three independent donors using CRISPR-Cas9 targeting. While loss of BAF53A impaired cell proliferation, loss of remaining components was well tolerated. Loss of BAF155 and BAF60B had minimal effect on globin chain production. Loss of BAF60A had the most potent effect on γ-globin, with up to 80% reduction in γ-globin with no decrease in β-globin production. BAF57 knockdown led to a more modest decrease in γ-globin, but with some decrease in total globin production. Together these data suggest that the requirements for some specific components of the BAF complex differ for activation of fetal or adult globin gene transcription. Additional studies are under way to characterize chromatin accessibility changes at the β-globin locus in response to alterations in the BAF complex, as well as the response to loss of additional BAF complex components.

Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation

Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion.Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors.RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed.In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

Study of Xmn-I polymorphism in β-thalassemic children in Egypt

Background β-thalassemia (βT) has a wide spectrum of clinical severity that may be attributed to the wide variations in βT gene mutations. β-Globin mutations with the Xmn-I site might be associated with elevated fetal hemoglobin levels, which in turn may affect the severity of βT phenotype. Aim To investigate the frequency of Xmn-I polymorphism (−158 C&gt;T) among Egyptian children with βT, and to examine the relationship between Xmn-I polymorphism and βT phenotypes. Patients and methods This cross-sectional study was conducted on 112 βT patients (55 males and 57 females) with a mean age of 8.34±3.71 years (2–16 years). Laboratory investigations included complete blood count, hemoglobin electrophoresis, β-globin mutation, identification of Xmn-I polymorphism by two methods: PCR–restriction fragment length polymorphism and amplification refractory mutation system. Results All patients (76 with βT major and 36 with βT intermedia) were negative for the Xmn-I SNP whether in homozygous (+/+) or heterozygous (+/−) state. Conclusion This study demonstrated that Xmn-I polymorphism was not detected in the studied patients and this supports the low frequency of this polymorphism in other Egyptian studies.

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

High Adenosine Deaminase Level Among Healthy Probands of Diamond Blackfan Anemia (DBA) Cosegregates With the DBA Gene Region on Chromosome 19q13

AbstractPhenotypic characterization of Diamond Blackfan Anemia (DBA) patients and their relatives was performed in 54 families. Complete blood count, fetal hemoglobin level, erythrocyte i antigen expression, and erythrocyte adenosine deaminase (eADA) activities were quantitated in patients and relatives. eADA was elevated in 28 of 34 transfusion-independent DBA patients, whereas persistence of erythrocyte i antigen was noticed in only 10 of 20 DBA patients. High eADA activities were also found in 14 of 149 healthy family members, allowing us to identify an isolated high eADA phenotype in these families. In contrast, increase in erythrocyte i antigen expression, elevated fetal hemoglobin levels, and macrocytosis were much less frequently noted in nonaffected members of the DBA families studied. Importantly, isolated high eADA phenotype was found to be significantly associated with genetic markers on chromosome 19 that segregate with the DBA phenotype. Isolated high eADA phenotype thus seems to reflect a silent phenotype of DBA in affected families. These findings suggest that elevated eADA activity in unaffected individuals needs to be taken into account during genetic assessment of DBA families and could be used for accurate assessment of mode of inheritance.

Novel Genetic Loci That Influence Fetal Hemoglobin Expression in Children with Sickle Cell Anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p&amp;lt;0.05) in both datasets were then selected for two independent replication steps; these were designated either African-American (N=157) or African (N=269). Third, the SNVs that were significant in both dual discovery and at least one of the replication cohorts were then verified using an additional Caribbean cohort (N=149) with TaqMan techniques for genotyping specific variants. Through this multistep process, we searched for genomic loci with consistent HbF associations across multiple cohorts. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p&amp;lt;10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p&amp;lt;0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p&amp;lt;0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Expected HbF associations with BCL11A (rs1427407) and HBS1L-MYB (rs4895441) were identified. Among other 18 novel candidate variants, the rs77737207 variant (allele frequency ~0.10) near the RUNX1T1 locus was strongly associated with lower HbF levels, while coding variant rs2279587 (allele frequency ~0.03) in the ITGA1 gene approached statistical significance (p&amp;lt;0.08) in the final verification cohort and was associated with higher levels of HbF. Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

Combinatorial Administration of Pharmacological Inhibitors Targeting DNMT1 and KDM1A Produces Synergistic and Additive Increases in F Retiulocytes and γ-Globin Expression in Normal Baboons

Sickle cell disease (SCD) is an inherited blood disorder that severely impacts the quality of life and reduces the life expectancy of approximately 100,000 patients in the U.S. and millions worldwide. Elevated levels of Fetal Hemoglobin (HbF; α2γ2) reduce the severity of symptoms and lengthen the life span of patients with sickle cell disease by inhibiting deoxy HbS polymerization. Hydroxyurea (HU), the only FDA-approved drug shown to increase HbF, is not effective in a subset of patientsand importantly, in the refractory individuals the increased HbFis heterogeneously distributed within the erythrocyte population resulting in a large fraction of erythrocytes lacking protective levels. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates. Our previous studies in baboons have shown that the most powerful drugs for reactivation of HbF are DNMT1 and KDM1A inhibitors targeting enzymes located within the co-repressor complexes that act to establish and maintain repressive epigenetic modifications at the γ-globin promoter to repress γ-globin expression. Treatment of baboons with either decitabine, an inhibitor of DNMT1, or RN-1, an inhibitor of KDM1A, strongly stimulates HbF. Dose-limiting effects of these drugs include neutropenia, thrombocytopenia, and/or thrombophilia. In this investigation we have explored the hypothesis that a treatment regimen comprised of both decitabine and RN-1 could achieve additive or synergistic effects on HbF while minimizing adverse hematological side effects. Three baboons (PA8695, PA8697, PA8698) were treated with two courses of different doses of RN-1 (0.25 and 0.125mg/kg/d) administered subcutaneously for 3 days to select an optimal RN-1 dose for each animal. The response to RN-1 varied between individual animals and was associated with pretreatment levels of γ-globin expression. Higher pretreatment levels of γ-globin expression were observed in PA8697 (0.08 γ/γ+β) compared to PA8695 (0.014+0.005 γ/γ+β) and PA8698 (0.004+0.0007 γ/γ+β) and the effect on γ-globin expression in PA8697 (0.24+0.5 γ/γ+β) at the 0.125mg/kg/d dose was greater than the effect of 0.25 mg/kg/d dose in PA8695 (0.17+0.05 γ/γ+β) and PA8698 (0.121+0.006 γ/γ+β). For combinatorial studies with decitabine, an RN-1 dose of 0.125mg/kg/d was chosen for PA8697 and 0.25mg/kg/d for PA8695 and 8698. Decitabine (0.3mg/kg/d; 2d), administered as a single agent subcutaneously for two days increased F retics nearly 3 fold (23.6+12.5%; p&amp;lt;0.02) compared to pretreatment levels (8.26+9.90%) but changes in γ-globin expression (0.066+0.076 γ/γ+β) compared to the pretreatment levels (0.03+0.042 γ/γ+β) were not significant. For combinatorial experiments, decitabine (0.3mg/kg/d) was administered for 2 days followed by administration of RN-1 (0.25mg/kg/d PA8695 and 8698; 0.125mg/kg/d PA8697) for 3 days. This staggered dose schedule was used to avoid any possible effects of RN-1 on cell cycle kinetics that could potentially reduce incorporation of decitabine during S phase. The decitabine/RN-1 combination produced significantly higher increases in F retics (63.0+23.6%; p&amp;lt;0.002) than either RN-1 (29.9+27.1%) or decitabine (23.6+12.5%) alone. The effect of the decitabine/RN-1 combination on F retics was greater than additive (p&amp;lt;0.01; Figure 1A) in the two low HbF animals, PA8695 and 8698, and additive for the high HbF animal PA8697 (Figure 1B). Although increased levels of γ-globin expression produced by the decitabine/RN-1 combination (0.22+0.08 γ/γ+β) were not significantly different overall from those induced by RN-1 alone (0.178+0.06 γ/γ+β), individual analysis of PA8697 revealed an additive effect (Figure 1C). Plt and ANC remained with the normal range. In conclusion, targeting separate components of the γ-globin co-repressor complex by combinatorial administration of decitabine and RN-1 generated synergistic and additive increases in F retics and γ-globin expression in the absence of hematological toxicity. Future experiments will explore different doses, alternative schedules, and the effect of longer term treatment regimens. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

JMML Fetal Identity Results Either from Retention of a Physiologic Signature or Aberrant Activation of Master Oncofetal Regulators

Objectives: Juvenile myelomonocytic leukemia (JMML) is a rare but aggressive myeloproliferative/myelodysplastic neoplasm affecting infants and young children. The narrow age-window of onset suggests that a prenatal environment is needed for JMML oncogenesis. In search of a transcriptional reminiscence of embryo-fetal characteristics that would confirm this hypothesis, we investigated how the gene expression profile of JMML hematopoietic progenitors compared to their healthy counterpart isolated at different stages of ontogeny. Methods: Hematopoietic stem cell and progenitor cell (HSPC) fractions of JMML (n=16), bone marrow (BM) of healthy children (n=7), fetal liver (FL; n=3) and fetal BM (FBM; n=2) were phenotyped and sorted using signatures validated in the fetal and adult BM (Notta et al, Science 2011). RNAseq was performed using the TruSeq® Stranded Total RNASample preparation kit. Unsupervised hierarchical clustering analysis was done with the Bioconductor edgeR package. Differentially expressed genes were identified with the Bioconductor limma package. Results: To eliminate the impact of variations in the HSPC distribution, the JMML transcriptome was assessed on FACS-sorted common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) cell fractions from 16 JMML and compared to healthy counterparts at different stages of ontogeny (FL, FBM, age-matched children BM). Unsupervised hierarchical clustering separated the samples into 4 groups (C1-4), primarily according to ontogeny, with 14/15 embryo-fetal fractions in C1 and all healthy post-natal progenitors in C2 (CMP, MEP) or C3 (GMP). Most JMML fractions clustered either with the prenatal fractions (C1; 17/47 fractions from 8/16 patients) or in a separate group containing no healthy sample (C4; 23/47 samples from 10/16 patients). Two groups were defined accordingly: one with JMML resembling embryo-fetal samples ('Fetal-JMML'; n=6/16), and a JMML-specific group ('Onco-JMML'; 8/16). Patients with Onco-JMML tended to be older, with a more severe presentation and elevated fetal hemoglobin levels. All PTPN11-mutated JMML were in this group whereas 5/6 Fetal-JMML had NRAS or KRAS mutations. Analysis of differential gene expression between Fetal and Onco-JMML highlighted 344 up-regulated genes versus 19 up-regulated genes in Onco-JMML. Surprisingly, LIN28B and WT1, both known to activate fetal pathways, were the most up-regulated genes in Onco-JMML. These key transcription factors were deregulated as early as the hematopoietic stem cell (HSC) compartment. Gene Set Enrichment Analysis (GSEA) confirmed enrichment in LIN28B and WT1-related signatures and showed enrichment in an AML signature in Onco-JMML. On the other hand, Fetal-JMML showed striking overexpression of monocytic /dendritic cell markers and inflammasome and innate immunity components. GSEA confirmed the strong monocyte identity of Fetal-JMML progenitors compared to onco-JMML or healthy postnatal progenitors. Part of the monocytic markers 'aberrantly' expressed in JMML progenitors was expressed in healthy fetal progenitors. Analysis of the HSC and multipotent progenitor (MPP) fractions showed that up regulation of monocytic markers was limited to the JMML progeny compartments. As we were able to confirm the transcriptional and functional identity of the sorted progenitors, these data suggest an early monocytic priming in these JMML progenitors, reminiscent of the monocyte-biased myelopoiesis characterizing physiologic fetal hematopoiesis. Conclusion: Our findings give a striking example of how ontogeny-related features are involved in childhood oncogenesis. They highlight a strong but complex link beween JMML and development, with a fetal identity resulting either from retention of a physiologic fetal monocytic signature or from aberrant (re)activation of master oncofetal regulators. Intriguingly, although LIN28B is thought to reprogram hematopoietic progenitors into a fetal-like state, its activation does not lead to an overall fetal profile in JMML, suggesting a regulatory mechanism distinct from that of physiological development. These two ontogeny-based signatures are likely to uncover the biology underlying previous classifiers based on AML-like profile or DNA methylation and suggest that a subset of JMML patient may benefit from immunomodulating therapies. Disclosures Dalle: Bellicum: Consultancy, Honoraria; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Orchard: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria; Astra Zeneca: Consultancy; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Shire: Research Funding; Bellicum: Consultancy.

Mild dyserythropoiesis and \u03b2-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in &gt;70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Interrogating RNA Binding Proteins as Novel Regulators of Fetal Hemoglobin Expression

Elevated levels of fetal hemoglobin (HbF) can alleviate symptoms of hemoglobinopathies, such as sickle cell disease (SCD). Hydroxyurea is the only FDA approved drug that works through this mechanism of HbF induction; however, its efficacy is variable among patients and its mechanism of action is not well understood. Therefore, significant clinical benefit would arise from a more reliable treatment to upregulate HbF, such as developing inhibitors that target HbF repressors. The transcription factors, BCL11A and LRF, are two major independent repressors of HbF however, they have been challenging to control via pharmacologic means. While these transcription factors and their co-factors have been extensively studied, upstream regulation of these transcription factors, such as potential post-transcriptional regulators, are not as well studied. Exploration of these upstream regulators might yield new insights into basic mechanisms of transcriptional and post-transcriptional regulation of HbF, which has the potential to uncover novel therapeutic targets. For example, we have previously used a novel screening approach to successfully identify the protein kinase HRI as a regulator of HbF through BCL11A expression (Grevet and Lan et al., Science, 2018). Novel targets such as HRI may be more amenable to pharmacologic regulation. To uncover novel upstream regulators of HbF, we employed a CRISPR/Cas9 based screening approach to target a spectrum of RNA binding proteins (RBPs) potentially involved in post transcriptional regulation of HbF expression. Using a human erythroid progenitor cell line, termed HUDEP2, we interrogated 342 human RBPs using an sgRNA library that targets RBPs harboring RNA methyltransferase and RNA recognition motifs. This screen yielded four candidate RBPs, in which their disruption or depletion in human primary erythroid cultures and HUDEP2 cells raised HbF levels. Three of these are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which have not previously been implicated in HbF regulation. Of these candidates, polypyrimidine tract binding protein 1 (PTBP1) showed the greatest level of HbF induction following in vitro depletion. Significant depletion of PTBP1 protein (&amp;gt;60%) in HUDEP2 cells and human CD34+ derived erythroid progenitors via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by measuring % HbF positive cells, γ-globin (HBG, fetal β-like globin) mRNA, and HBG protein levels. Cell viability of PTBP1 perturbed samples are largely unaffected, however there is a delay in terminal differentiation as assessed by cell surface markers CD71 and CD235a (2-3 fold decrease in CD71-/CD235a+ cells at day 15 of differentiation). Unexpectedly, depletion of PTBP1 had minimal effect on BCL11A and LRF mRNA or protein levels. This suggests PTBP1 might impact the expression or activities of co-factors or upstream regulators of these transcription factors. Ongoing work is aimed at defining the mechanism of PTBP1 action by identifying its molecular targets. In sum, the identification of PTBP1 as a regulator of HbF production represents a previously undescribed layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this process which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.

Domain-Focused CRISPR-Cas9 Screen Identifies the E3 Ubiquitin Ligase Substrate Adaptor Protein SPOP as a Novel Repressor of Fetal Hemoglobin

Elevated levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease (SCD) and b-thalassemia. The identification of pharmacologically controllable pathways that regulate HbF levels remains an important goal in the treatment of these disorders. BCL11A and LRF (ZBTB7A) are the two major transcriptional repressors of g-globin (fetal beta-type globin). However, since transcription factors are challenging to inhibit with small molecules, we aimed to identify novel HbF regulators that might be amenable to control by pharmacologic means.Using an improved protein-domain based CRISPR-Cas9 screen recently developed in our laboratory (Grevet et al., Science 361:6399, 285-290), we screened for new HbF regulators using a gRNAs library targeting chromatin associated proteins including BTB domain containing factors. We identified SPOP, a substrate adaptor of the CUL3 ubiquitin ligase complex, as a novel inhibitor of g-globin expression. Depletion of SPOP strongly elevated the mRNA and protein levels of g-globin in both HUDEP-2 and primary human CD34+ derived erythroid cells. The effects of SPOP loss were remarkably selective as evidenced by RNA-seq and whole cell mass spectrometry. Importantly, SPOP depletion did not appear to impair cell growth or differentiation. Mechanistically, we demonstrated that the repressive role of SPOP on g-globin levels is dependent on the CUL3 ubiquitin ligase complex. Additionally, forced expression of a dominant negative form of SPOP (Y87N) unable to bind to its substrates strongly induced HbF production. We also found that SPOP depletion did not measurably alter LRF levels and modestly reduced BCL11A levels. Overexpression of BCL11A did not restore g-globin silencing in SPOP depleted cells, indicating that SPOP works largely independently of these two factors. Finally, we tested whether the effects of SPOP depletion can be augmented through the use of experimental pharmacologic HbF inducers, such as pomalidomide. In proof of principle experiments we found that SPOP depletion and pomalidomide treatment induced HbF production to levels higher than either treatment alone.In sum, our work uncovered SPOP as a new regulator of HbF and suggests targeting SPOP alone or in combination with other HbF inducers may provide a novel therapeutic strategy for the treatment of hemoglobinopathies such as sickle cell disease and b-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

Whole transcriptome analysis of human erythropoietic cells during ontogenesis suggests a role of VEGFA gene as modulator of fetal hemoglobin and pharmacogenomic biomarker of treatment response to hydroxyurea in \u03b2-type hemoglobinopathy patients

BackgroundHuman erythropoiesis is characterized by distinct gene expression profiles at various developmental stages. Previous studies suggest that fetal-to-adult hemoglobin switch is regulated by a complex mechanism, in which many key players still remain unknown. Here, we report our findings from whole transcriptome analysis of erythroid cells, isolated from erythroid tissues at various developmental stages in an effort to identify distinct molecular signatures of each erythroid tissue.ResultsFrom our in-depth data analysis, pathway analysis, and text mining, we opted to focus on the VEGFA gene, given its gene expression characteristics. Selected VEGFA genomic variants, identified through linkage disequilibrium analysis, were explored further for their association with elevated fetal hemoglobin levels in β-type hemoglobinopathy patients. Our downstream analysis of non-transfusion-dependent β-thalassemia patients, β-thalassemia major patients, compound heterozygous sickle cell disease/β-thalassemia patients receiving hydroxyurea as fetal hemoglobin augmentation treatment, and non-thalassemic individuals indicated that VEGFA genomic variants were associated with disease severity in β-thalassemia patients and hydroxyurea treatment efficacy in SCD/β-thalassemia compound heterozygous patients.ConclusionsOur findings suggest that VEGFA may act as a modifier gene of human globin gene expression and, at the same time, serve as a genomic biomarker in β-type hemoglobinopathy disease severity and hydroxyurea treatment efficacy.

Correlation of SIN3A genomic variants with β-hemoglobinopathies disease severity and hydroxyurea treatment efficacy.

Hemoglobinopathies, particularly β-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways. Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production. We show that SIN3A genomic variants are associated both with β-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in β-hemoglobinopathies patients. Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.

Hemoglobin genetics: recent contributions of GWAS and gene editing.

The β-hemoglobinopathies are inherited disorders resulting from altered coding potential or expression of the adult β-globin gene. Impaired expression of β-globin reduces adult hemoglobin (α2β2) production, the hallmark of β-thalassemia. A single-base mutation at codon 6 leads to formation of HbS (α2βS2) and sickle cell disease. While the basis of these diseases is known, therapy remains largely supportive. Bone marrow transplantation is the only curative therapy. Patients with elevated levels of fetal hemoglobin (HbF, α2γ2) as adults exhibit reduced symptoms and enhanced survival. The β-globin gene locus is a paradigm of cell- and developmental stage-specific regulation. Although the principal erythroid cell transcription factors are known, mechanisms responsible for silencing of the γ-globin gene were obscure until application of genome-wide association studies (GWAS). Here, we review findings in the field. GWAS identified BCL11A as a candidate negative regulator of γ-globin expression. Subsequent studies have established BCL11A as a quantitative repressor. GWAS-related single-nucleotide polymorphisms lie within an essential erythroid enhancer of the BCL11A gene. Disruption of a discrete region within the enhancer reduces BCL11A expression and induces HbF expression, providing the basis for gene therapy using gene editing tools. A recently identified, second silencing factor, leukemia/lymphoma-related factor/Pokemon, shares features with BCL11A, including interaction with the nucleosome remodeling deacetylase repressive complex. These findings suggest involvement of a common pathway for HbF silencing. In addition, we discuss other factors that may be involved in γ-globin gene silencing and their potential manipulation for therapeutic benefit in treating the β-hemoglobinopathies.