LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells

Abstract Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells. We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD. Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients. Disclosures No relevant conflicts of interest to declare.

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Lineage-Specific Activation of p53 in Response to Ribosomal Haploinsufficiency in Human Bone Marrow Cells.

Blood ◽

10.1182/blood.v114.22.948.948 ◽

2009 ◽

Vol 114 (22) ◽

pp. 948-948

Author(s):

Shilpee Dutt ◽

Anupama Narla ◽

Jeffery Lorne Kutok ◽

Benjamin L. Ebert

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

Erythroid Cell ◽

Erythroid Progenitor ◽

Intense Staining ◽

Bone Marrow Biopsies ◽

Erythroid Progenitor Cells ◽

Cd34 Cells

Abstract Abstract 948 Haploinsufficiency for the ribosomal protein genes RPS14 and RPS19 have been implicated in the erythroid defect in the 5q- syndrome and Diamond Blackfan Anemia, respectively. However, the mechanism by which defective ribosome biogenesis causes erythroid failure is unknown. In this study, we found that shRNA mediated knockdown of RPS14 or RPS19 in primary human CD34+ cells stabilize TP53 by day 4 after infection with concomitant arrest of these cells at G1 stage of cell cycle. The levels of TP53 attained are comparable to the levels observed following gamma irradiation (5Gy) of the CD34+ cells. Using quantitative PCR, we confirmed that stabilized TP53 activates expression of downstream target genes MDM2, p21, Bax and Wig-1. Furthermore, treatment of the CD34+ cells with Nutlin-3 phenocopies RPS14 or RPS19 knockdown, suggesting that the mechanism of TP53 activation is mediated by MDM2 pathway. Conversely, treatment with pifithrin-alpha, which inhibits the transactivation activity of TP53, rescues the effects of RPS14 or RPS19 knockdown. The in vitro activation of TP53 in CD34+ cells was restricted to erythroid cell lineage, consistent with the clinical phenotype of RPS14 or RPS19 haploinsufficiency. Moreover, immunohistochemical analysis of bone marrow biopsies from patient with the 5q- syndrome demonstrated intense staining of TP53 that was restricted to erythroid progenitor cells. Taken together our study indicates that inhibition of ribosomal biogenesis causes TP53 activation selectively in erythroid progenitor cells. Clinically, TP53 staining of patient samples could be used as a diagnostic marker for some types of MDS. Disclosures: No relevant conflicts of interest to declare.

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Cyclin E Regulation by the Fbw7 Ubiquitin Ligase Pathway Is Required for Normal Mitophagy and Restraining Reactive Oxidative Species During Erythroid Cell Maturation In Vivo,

Blood ◽

10.1182/blood.v118.21.3393.3393 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3393-3393

Author(s):

Yanfei Xu ◽

Ka Tat Siu ◽

Amit Verma ◽

Alexander C. Minella

Keyword(s):

Ubiquitin Ligase ◽

Conflicts Of Interest ◽

Cyclin E ◽

Cell Maturation ◽

Erythroid Cell ◽

Reactive Oxidative Species ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Oxidative Species

Abstract Abstract 3393 We previously described a knock-in mouse model that permits study of the physiologic consequences of cyclin E deregulation, by selective ablation of its regulation via the SCFFbw7 ubiquitin ligase. We found that erythroid progenitor cells in our cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. Most prominent among the gene expression alterations we have identified in the cyclin E knock-in erythroid cells is induction of multiple p53 target genes, consistent with p53 pathway activation. In contrast to several recently described models of ribosomal protein gene mutations, in which p53 activation appears to induce dyserythropoiesis, we determined that p53 function actually maintains partially compensated erythroid cell maturation in vivo, in the context of impaired Fbw7-mediated cyclin E degradation. We next found that dysregulated cyclin E-CDK2 activity in cyclin ET74A T393A erythroid cells is associated with increased reactive oxidative species and increased mitochondrial mass and activity. These results coincide with findings of abnormal mitochondria retention in late-stage erythroid cells and significantly down-regulated expression of BNIP3L (NIX). BNIP3L encodes a critical regulator of erythroid cell mitophagy, and the transcriptional controls maintaining its expression in maturing erythroid cells likely account for why this lineage is acutely sensitive to deregulated cyclin E activity. Finally, we show evidence that reduced expression of BNIP3L may play a role in some cases of early-stage myelodysplastic syndromes. Disclosures: No relevant conflicts of interest to declare.

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The Effects Of The Histone Methyltransferase Inhibitor UNC0638 Upon Gamma Globin Gene and Protein Expression Are Erythroblast Differentiation Stage Specific

Blood ◽

10.1182/blood.v122.21.3453.3453 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3453-3453

Author(s):

Ki Soon Kim ◽

Colleen Byrnes ◽

Y. Terry Lee ◽

Jaira F. de Vasconcellos ◽

Megha Kaushal ◽

...

Keyword(s):

Conflicts Of Interest ◽

Epigenetic Modification ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Adult Human ◽

Gene And Protein Expression ◽

Differentiation Stage

Abstract Epigenetic modification of chromatin in erythroid cells represents an active field of study aimed, in part, toward increased expression of fetal hemoglobin in patients with beta-thalassemia. The homologous methyltransferases G9a and GLP regulate globin gene transcription by catalyzing mono- and dimethylation at Lys 9 and dimethylation at Lys 27 of histone H3. Inhibition of these methyltransferases by the small molecule named UNC0638 was recently shown to increase gamma-globin gene expression in adult human hematopoietic precursor and stem cells. Here UNC0638 was explored further to include fetal hemoglobin expression among more mature erythroid cells cultured from CD34(+) cells of three healthy adult human donors in a serum-free culture medium. According to this culture model, the main erythroblast population on culture days 0-7 consists of CD36(+), CD45(+), CD71(moderate), CD235a(-) erythroid progenitor cell. On culture days 7-14, the progenitor cells differentiate in the presence of erythropoietin to become CD36(+), CD45(-), CD71(high), CD235a(+) precursor cells. During the final week in culture, the erythroblasts undergo nuclear condensation, enucleation, and loss of RNA combined with the loss of CD36 and CD71 on the plasma membrane to become mature erythrocytes. To investigate different stages of erythroblast maturation, the cells were cultured in medium containing 1µM UNC0638 for periods of seven days (culture days 0-7, 7-14, or 14-21) and compared to control cultures without UNC0638. The effects of UNC0638 were determined by flow cytometry, Q-RT-PCR and hemoglobin chromatography (HPLC). Unexpectedly, fetal hemoglobin expression was highly-dependent upon the differentiation stage of the cells in the presence of UNC0638. When cultured in UNC0638 supplemented medium on culture days 0-7 or 14-21, the cells underwent terminal maturation, but there was no significant increase in the fetal hemoglobin content of the mature cells (see abstract figure). In contrast, UNC0638 added on culture days 7-14, caused a significant increase in fetal hemoglobin (HbF; control: 3.9 ± 3.5% vs. day 7-14 UNC0638: 32.6 ± 0.95%, p=0.007). The increase in HbF was associated with a similar increase in gamma-globin mRNA (control: 1.5E+06 ± 1.7E+05 copies/ng vs. day 7-14 UNC0638: 7.5E+06 ± 1.4E+06 copies/ng, p=0.021). Additionally, terminal maturation and enucleation were partially inhibited when compared to the other conditions or controls. These data suggest that UNC0638 causes a robust increase in fetal hemoglobin as the cells undergo maturation. Fetal hemoglobin increases were more pronounced after exposure to UNC0638 during the erythropoietin-dependent transition from CD235a(-) to CD235a(+) erythroblasts. The results suggest that fetal hemoglobin regulation by G9a and GLP may be differentiation stage dependent. Disclosures: No relevant conflicts of interest to declare.

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SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Blood ◽

10.1182/blood-2021-146071 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 963-963

Author(s):

Yannis Hara ◽

Mark Stottlemyer ◽

Kim Alving ◽

Nis Halland ◽

Alexandra Hicks ◽

...

Keyword(s):

Western Blot ◽

Blood Donors ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Cells ◽

Target Engagement ◽

Western Blots ◽

Cd34 Cells ◽

Hbf Induction ◽

In Erythroid Cells

Abstract Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF. Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer. Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls. Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. Disclosures No relevant conflicts of interest to declare.

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Knockdown of HNF4A Gene Increases Fetal Hemoglobin Synthesis in Hudep-2

Blood ◽

10.1182/blood-2019-130920 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 968-968

Author(s):

Ingrid Grazielle Sousa ◽

Priscila Keiko Matsumoto Martin ◽

Dulcinéia Martins de Albuquerque ◽

Carolina Lanaro ◽

Ryo Kurita ◽

...

Keyword(s):

Flow Cytometry ◽

Transcription Factor ◽

Conflicts Of Interest ◽

Clonal Selection ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Inherited Disease ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Cd34 Cells

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p<0.0001). These results corroborate the increased expression measured in CD34+ cells. To knock out HNF4A in HUDEP-2 cells, we used the CRISPR/Cas9 system. We generated INDELs in heterozygosity for HNF4A. Briefly, HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1µM of gRNA HNF4A using a CD34+ human cell kit and the E-001 program in an AMAXA Nucleofector 4D-device (Lonza). Two days after nucleofection, edited HUDEP-2 cells were submitted to clonal selection and expanded for approximately 28 days. Genomic DNA from clones was analyzed by a Sanger Sequencer, and four edited HNF4A-HUDEP-2 clones were selected with INDELs in the fourth exon of HNF4A gene. These clones were expanded in culture with controls, and the HbF levels were quantified by flow cytometry. HbF levels in these four clones were 8.2 ± 2.4%, while in the wild type HUDEP-2, HbF was 0.9 ± 0.02% (p< 0.05). The edited clones expressed significantly more HbF than the controls, although this expression was not homogeneous. Western blotting of edited HNF4A-HUDEP-2 clones demonstrated decreased HNF4A at the protein level (0.35 ± 0.9 AU N=8), compared to controls (2.02± 1.1 AU N=3, p< 0.05). Results suggest that HNF4A may play a role in the gamma globin gene transcription in HUPED-2 cells. The mechanism of how HNF4A may regulate gamma globin gene expression remains to be clarified. Disclosures No relevant conflicts of interest to declare.

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Downregulation of Transcription Factor LRF/ZBTB7A Increases Fetal Hemoglobin Expression in β-Thalassemia/Hemoglobin E Erythroid Cells

Blood ◽

10.1182/blood-2019-126664 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3549-3549

Author(s):

Sukanya Chumchuen ◽

Tanapat Pornsukjantra ◽

Pinyaphat Khamphikham ◽

Usanarat Anurathapan ◽

Orapan Sripichai ◽

...

Keyword(s):

Gene Therapy ◽

Transcription Factor ◽

Conflicts Of Interest ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Hemoglobin E ◽

In Erythroid Cells

LRF/ZBTB7A is a transcription factor that has been recently identified as a new key regulator of fetal hemoglobin (HbF; α2γ2) production in erythroid cells. Reduction of LRF/ZBTB7A expression led to increases in levels of HbF in human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroblast and in human immortalized erythroid line (HUDEP-2). Since reactivation of γ-globin gene is associated with the improvement of clinical manifestations of β-hemoglobinopathy patients, decrement in LRF/ZBTB7A expression might be a substantial interest as a novel target for gene therapy in β-thalassemia. In this study, we investigated the effects of LRF/ZBTB7A downregulation in erythroid cells derived from β-thalassemia/HbE patients in order to evaluate its therapeutic potential. The hematopoietic CD34+ progenitor cells were collected from 3 patients and 3 healthy normal individuals' peripheral blood and subjected for in vitro erythroblast culture. The cells were transduced with lentivirus carrying LRF/ZBTB7A specific shRNA, and used untransduced cells and non-targeted control shRNA (shNTC) as experimental controls. The LRF/ZBTB7A shRNA reduced LRF/ZBTB7A transcript and protein to nearly undetectable levels. Interestingly, downregulation of LRF/ZBTB7A increased expression of γ-globin, ε-globin and ζ-globin in both adult normal and β-thalassemia/HbE derived cells, whereas α-globin, β-globin and δ-globin expression were decreased. As previously reported, we found that the LRF/ZBTB7A knockdown produced a robust increase in HbF levels in both normal (43.3±9.0% vs. 5.9±2.1% in shNTC) and β-thalassemia/HbE erythroblasts (78.1±3.5% vs. 26.3±3.9% in shNTC). Noteworthy, the delay of erythroid differentiation was observed in the LRF/ZBTB7A knockdown cells of both derived from β-thalassemia/HbE patients and normal control, suggesting an additional role of LRF/ZBTB7A in regulating erythroid maturation. These data support the manipulation of LRF/ZBTB7A as one of the most interesting gene therapy candidates for treating the β-thalassemia, but the effect on erythroid cell maturation is needed to be concerned and required further investigation. Disclosures No relevant conflicts of interest to declare.

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Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Blood ◽

10.1182/blood.v75.9.1862.1862 ◽

1990 ◽

Vol 75 (9) ◽

pp. 1862-1869 ◽

Cited By ~ 13

Author(s):

P Constantoulakis ◽

B Nakamoto ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Cell Cultures ◽

Fetal Calf Serum ◽

Calf Serum ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Fetal Sheep ◽

Gamma Globin

Abstract Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

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Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽

10.1182/blood-2019-122770 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2063-2063

Author(s):

Naoya Uchida ◽

Claire Drysdale ◽

Morgan Yapundich ◽

Jackson Gamer ◽

Tina Nassehi ◽

...

Keyword(s):

Rhesus Macaques ◽

Fetal Hemoglobin ◽

Selective Advantage ◽

Erythroid Cells ◽

Post Transplant ◽

Human Cd34 ◽

Cd34 Cells ◽

Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

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Possible Interactions between the NS-1 Protein and Tumor Necrosis Factor Alpha Pathways in Erythroid Cell Apoptosis Induced by Human Parvovirus B19

Journal of Virology ◽

10.1128/jvi.73.10.8762-8770.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8762-8770 ◽

Cited By ~ 104

Author(s):

N. Sol ◽

J. Le Junter ◽

I. Vassias ◽

J. M. Freyssinier ◽

A. Thomas ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Parvovirus B19 ◽

Erythroid Cell ◽

Erythroid Cells ◽

Necrosis Factor Alpha ◽

Erythroid Progenitor ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Necrosis Factor

ABSTRACT Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G2phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-α)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-α and NS-1 in human erythroid cells.

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Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽

10.1182/blood-2019-123837 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 814-814

Author(s):

Peng Huang ◽

Scott A. Peslak ◽

Xianjiang Lan ◽

Eugene Khandros ◽

Malini Sharma ◽

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Keyword(s):

Transcription Factor ◽

Cell Line ◽

Research Funding ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Erythroid Cells ◽

Major Pathway ◽

Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

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