Erythroid Development Research Articles

CRISPR/Cas9 Genome Editing to Treat Sickle Cell Disease and B-Thalassemia: Re-Creating Genetic Variants to Upregulate Fetal Hemoglobin Appear Well-Tolerated, Effective and Durable.

Persistent expression of fetal hemoglobin (HbF) beyond the neonatal period is a rare, naturally-occurring condition, referred to as the Hereditary Persistence of Fetal Hemoglobin, which substantially ameliorates the pathology of Sickle Cell Disease (SCD) and β-thalassemia (β-thal). Extensive human genetic and epidemiologic studies have demonstrated that this condition is associated with one of several specific point mutations or deletions that lead to expression of γ-globin, resulting in upregulation of HbF. Our strategy is to use the CRISPR/Cas9 genome editing technology in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) to efficiently re-create specific genetic variants associated with elevated HbF and to evaluate their safety, effectiveness and durability as a potential therapeutic strategy to treat SCD and β-thal.We have optimized CRISPR/Cas9 editing of human primary HSPCs mobilized from healthy donors to routinely achieve editing efficiencies of greater than 80%. Improved viability with no reduction in editing efficiency was achieved with Cas9 delivered as a recombinant protein as compared to Cas9 delivered as mRNA. We have further optimized the process at clinical scale in our manufacturing facility to achieve similar viability and editing efficiency with a GMP-compatible process. The high rate of editing in human primary HSPCs mobilized from healthy donors was associated with robust HbF expression following erythroid differentiation. In patient samples, clinically relevant increases in γ-globin mRNA to 39% (as a ratio of γ/α) in one β-thal patient sample and to 38±5% (Mean±SD, as a ratio of γ/(γ+β)) in six SCD patient samples were observed. Similarly high rate of editing was observed in the CD34+CD38-CD90+CD45RA- long-term repopulating subset of human primary HSPCs, as in the overall HSPC population (88±6% vs 90±4% Mean±SD, n=4). We have demonstrated the long-term persistence of transplanted cells in a NOD-SCID-Gamma (NSG) xeno-transplant mouse model at 16 weeks (44±12% of cells in controls versus 44±14% and 41±13% for two target modifications, Mean±SD, n=44-47 for each), and that the proportion of the various hematopoietic lineages derived from the transplanted edited human primary HSPCs was not affected by the editing process. Finally, the high editing rate of 85-90% persisted in human bone marrow cells at 16 weeks, demonstrating durability of the edited cells. Because the NSG model does not appropriately replicate erythroid development, we have also demonstrated normal erythroid potential of the engrafted human CD34+ cells isolated from mouse bone marrow in ex vivo differentiation assays, and the editing level is maintained in the erythroid lineage. In-depth analysis confirmed no detectable off-target sequence disruption at over 5,000 candidate off-target sites that were identified bioinformatically through homology to the on-target site or experimentally through a double strand break identification method. In vivo safety and toxicology studies are ongoing.Taken together, these findings show that a CRISPR/Cas9-based therapy has the potential to upregulate HbF and treat diseases of b-globin. We have optimized editing conditions to achieve high efficiency and viability of editing in human primary HSPCs without detectable off-target sequence disruption, and in turn, demonstrate the durability of this approach in vivo. We believe that the results to date support the initiation of clinical studies for a CRISPR/Cas9 treatment for patients with SCD and β-thal. DisclosuresLin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Burkhardt:CRISPR Therapeutics: Employment. Kernytsky:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Chen:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Cortes:CRISPR Therapeutics: Employment. Tan:CRISPR Therapeutics: Employment. Borland:CRISPR Therapeutics: Employment. Klein:CRISPR Therapeutics: Employment. Yen:Vertex: Employment. Mahajan:Vertex: Employment. Chan:Vertex: Employment. Eustace:Vertex: Employment. Porteus:CRISPRTx: Equity Ownership. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment. Novak:CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment.

Lyn Kinase Activity Is Required for Akt Mediated Erythroleukemia Cell Differentiation

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation (Fig-1).

Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts

Oxygen is a critical noncellular component of the bone marrow microenvironment that plays an important role in the development of hematopoietic cell lineages. In this study, we investigated the impact of low oxygen (hypoxia) on ex vivo myeloerythroid differentiation of human cord blood-derived CD34+ hematopoietic stem and progenitor cells. We characterized the culture conditions to demonstrate that low oxygen inhibits cell proliferation and causes a metabolic shift in the stem and progenitor populations. We found that hypoxia promotes erythroid differentiation by supporting the development of progenitor populations. Hypoxia also increases the megakaryoerythroid potential of the common myeloid progenitors and the erythroid potential of megakaryoerythroid progenitors and significantly accelerates maturation of erythroid cells. Specifically, we determined that hypoxia promotes the loss of CD71 and the appearance of the erythroid markers CD235a and CD239. Further, evaluation of erythroid populations revealed a hypoxia-induced increase in proerythroblasts and in enucleation of CD235a+ cells. These results reveal the extensive role of hypoxia at multiple steps during erythroid development. Overall, our work establishes a valuable model for further investigations into the relationship between erythroid progenitors and/or erythroblast populations and their hypoxic microenvironment.

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance.

Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.

Tumor Immune Evasion Induced by Dysregulation of Erythroid Progenitor Cells Development.

Simple SummaryTumor immune evasion is one of the hallmarks of tumor progression that enables tumor growth despite the activity of the host immune system. It is mediated by various types of cells. Recently, immature red blood cells called erythroid progenitor cells (EPCs) were identified as regulators of the immune response in cancer. EPCs expand in cancer as a result of dysregulated erythropoiesis and potently suppress the immune response. Thus, targeting dysregulated EPC differentiation appears to be a promising therapeutic strategy.Cancer cells harness normal cells to facilitate tumor growth and metastasis. Within this complex network of interactions, the establishment and maintenance of immune evasion mechanisms are crucial for cancer progression. The escape from the immune surveillance results from multiple independent mechanisms. Recent studies revealed that besides well-described myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) or regulatory T-cells (Tregs), erythroid progenitor cells (EPCs) play an important role in the regulation of immune response and tumor progression. EPCs are immature erythroid cells that differentiate into oxygen-transporting red blood cells. They expand in the extramedullary sites, including the spleen, as well as infiltrate tumors. EPCs in cancer produce reactive oxygen species (ROS), transforming growth factor β (TGF-β), interleukin-10 (IL-10) and express programmed death-ligand 1 (PD-L1) and potently suppress T-cells. Thus, EPCs regulate antitumor, antiviral, and antimicrobial immunity, leading to immune suppression. Moreover, EPCs promote tumor growth by the secretion of growth factors, including artemin. The expansion of EPCs in cancer is an effect of the dysregulation of erythropoiesis, leading to the differentiation arrest and enrichment of early-stage EPCs. Therefore, anemia treatment, targeting ineffective erythropoiesis, and the promotion of EPC differentiation are promising strategies to reduce cancer-induced immunosuppression and the tumor-promoting effects of EPCs.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one‑carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.

Modeling and targeting of erythroleukemia by hematopoietic genome editing

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor–mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a–mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

Requirement for antiapoptotic MCL-1 during early erythropoiesis

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

CAP1, a target of miR-144/451, negatively regulates erythroid differentiation and enucleation.

The exact molecular mechanism underlying erythroblast enucleation has been a fundamental biological question for decades. In this study, we found that miR‐144/451 critically regulated erythroid differentiation and enucleation. We further identified CAP1, a G‐actin‐binding protein, as a direct target of miR‐144/451 in these processes. During terminal erythropoiesis, CAP1 expression declines along with gradually increased miR‐144/451 levels. Enforced CAP1 up‐regulation inhibits the formation of contractile actin rings in erythroblasts and prevents their terminal differentiation and enucleation. Our findings reveal a negative regulatory role of CAP1 in miR‐144/451‐mediated erythropoiesis and thus shed light on how microRNAs fine‐tune terminal erythroid development through regulating actin dynamics.

P53 activation during ribosome biogenesis regulates normal erythroid differentiation

AbstractThe role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis

Cytoprotective mechanisms of heme oxygenases function by derivatizing heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin using two non-overlapping biliverdin reductases that display biliverdin isomer-restricted redox activity. Although cytoprotective functions of heme oxygenases are widely recognized, concomitant effects of downstream biliverdin reductases remain incomplete. A computational model predicated on murine hematopoietic single-cell transcriptomic data identified Blvrb as a biological driver linked to the tumor necrosis factor stress pathway as a predominant source of variation defining hematopoietic cell heterogeneity. In vivo studies using Blvrb-deficient mice established the dispensable role of Blvrb in steady-state hematopoiesis, although model validation using aged Blvrb-deficient mice established an important cytoprotective function in stress hematopoiesis with dichotomous megakaryocyte-biased hematopoietic recovery. Defective stress erythropoiesis was evident in Blvrb−/− spleens and in bone marrow erythroid development, occurring in conjunction with defective lipid peroxidation as a marker of oxidant mishandling. Cell autonomous effects on megakaryocyte lineage bias were documented using multipotential progenitor assays. These data provide the first physiological function of murine Blvrb in a non-redundant pathway of stress cytoprotection. Divergent effects on erythroid/megakaryocyte lineage speciation impute a novel redox-regulated mechanism for lineage partitioning.

Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

AbstractKit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

Erythroid Development in the Mammalian Embryo

Erythropoiesis is the method by which begetters for ruddy blood cells are created and terminally separate. In all vertebrates, two morphologically particular erythroid heredities (primitive, embryonic, and authoritative, fetal/ adult) shape progressively inside the yolk sac, fetal liver, and marrow and are basic for typical improvement. Ruddy blood cells have advanced profoundly specialized capacities in oxygen transport, defense against oxidation, and vascular remodeling. Here we survey key highlights of the ontogeny of ruddy blood cell improvement in well evolved creatures, highlight similitudes and contrasts uncovered by hereditary and quality expression profiling thinks about, and talk about strategies for recognizing erythroid cells at distinctive stages of improvement and separation.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor−/− knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor−/−/Dnmt3a−/− knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Regulation of GATA-1-Controlled Genes By O-Glcnacylation in Erythroid Cells

The role of post-translational modification (PTM) of proteins in regulating developmental and differentiation processes is understudied, but recently we established that O-GlcNAcylation regulates erythropoiesis. O-GlcNAc regulates numerous cellular functions including stress response, transcription, and cell cycle progression. O-GlcNAc is a single O-linked β-N-acetyl-D-glucosamine moiety added to serine/threonine amino acids of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes the modification, are responsible for the dynamic processing of the PTM. Newly developed erythroid-specific OGT conditional knockout mice show that OGT is essential for fetal definitive erythropoiesis, although during primitive erythropoiesis, erythrocytes exhibit hallmarks of ineffective erythropoiesis. In addition, using G1E-ER cells, we have shown that GATA-1 interacts with OGT and OGA and delivers OGT and OGA to GATA-1 regulated genes. When we perturb this process, we observe erythroid defects, most strikingly, a shift in commitment towards other hematopoietic lineages. We hypothesize that at the onset of erythroid lineage commitment, GATA-1 functions as an adaptor protein to deliver these enzymes to erythroid-specific cis-regulatory DNA elements, where the O-GlcNAc status of bound protein complexes is modified to direct transcriptional networks necessary for normal erythroid development and terminal differentiation. Two key proteins, LRF/ZBTB7A and BCL11A, silence the γ-globin promoter during adult erythropoiesis by recruiting the penultimate NuRD repressor complex to these genes. Loss of either of these proteins leads to partial restoration of γ-globin expression. Our previous work demonstrated that O-GlcNAc plays a role in γ-globin gene transcription. GATA-1 recruits FOG-1 and the NuRD complex to silence γ-globin by binding GATA sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin transcription start sites, respectively. OGT and OGA interact with GATA-1, FOG-1 and CHD4, a key component of the NuRD repressor complex and these three proteins are O-GlcNAcylated. Specifically, O-GlcNAcylation modulates the formation of the multi-protein NuRD repressor complex. OGT adds O-GlcNAc to CHD4 stimulating the formation of this repressor complex, whereas removal of this PTM by OGA results in the activation of γ-globin gene expression. In addition, the GATA-1 binding protein, GATAD2A, interacts with CHD4 and organizes it within the NuRD complex. The domain of GATAD2A that interacts with CHD4 is O-GlcNAcylated. Finally, the NuRD complex proteins RBBP7, HDAC1, and MTA2 are also modified by O-GlcNAc. We hypothesize that O-GlcNAcylation controls NuRD repressor assembly by directing subunit interactions. To ascertain the function of OGT and OGA at GATA-1 regulated genes, we developed novel CRISPR/Cas9 targeting tools in K562 cells. We fused OGT and OGA to catalytically dead Cas9 endonuclease (dCas9). Negative controls were generated by mutating a catalytic residue of OGT (H558F) or OGA (D174A). The CBP/p300 acetyltransferase core was fused to dCas9 as a positive control. As a proof of principle, we targeted the γ-globin promoters. Using the UCSC Genome Browser, single guide RNAs (sgRNAs) that do not disrupt endogenous cis-regulatory elements were selected, synthesized, and subsequently introduced into K562 cells to establish stably expressing monoclonal cell lines. Interestingly, when OGA-dCas9 was targeted to the -1 to -223 bp region relative to the mRNA start site of the γ-globin promoters, we observed a significant increase in γ-globin expression. OGT-dCas9 had a significant repressive effect when targeted to regions around the transcriptional start site. All controls behaved as predicted. To validate these results, we are performing chromatin immunoprecipitation (ChIP) to demonstrate occupancy of the targeted regions by our constructs and the presence or absence of O-GlcNAc. Our findings highlight a new post-translational mechanism regulating gene transcription during erythroid development, which affects γ-globin expression and erythropoiesis. Our data will provide insight into how O-GlcNAc controls erythroid differentiation and reveal O-GlcNAcylated protein targets that may be manipulated for the treatment of hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Lyn Kinase Activity Is Required for Akt Mediated Erythroleukemia Cell Differentiation

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation. Disclosures No relevant conflicts of interest to declare.

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

3007 – POLYVINYL ALCOHOL AND THE EMERGING TRANSLATABILITY OF IPSC-DERIVED RED BLOOD CELLS FOR DISEASE MODELING AND CLINICAL USE

Human induced pluripotent stem cells (iPSC) are an invaluable resource in tissue and blood cell engineering due to their multi-lineage potential in culture systems. iPSC-derived hematopoietic progenitors that undergo successful erythropoiesis in vitro would allow for the modeling of inheritable red blood cell (RBC) diseases, such as Sickle Cell Disease (SCD), for novel therapeutic design and pre-clinical testing. Here, we describe an optimized method of generating induced RBCs (iRBCs) in vitro from healthy and sickle cell homozygous iPSCs, using a novel differentiation media formula containing Polyvinyl Alcohol (PVA). iPSC-derived iRBCs mature into GlyA+Band3+CD71lo cells which successfully enucleate at a high rate (65-80%). mRNA profiles demonstrate iRBCs undergo three distinct waves of globin activation and suppression throughout in vitro erythroid development, which closely mimics the in vivo globin-switching profile modeled from humans. Ultimately, iRBCs become robust adult β-globin expressing cells, which is confirmed at the protein level by HPLC. Sickling is easily observable in SCD iRBCs, regardless of oxygen environment, along with other morphological abnormalities characteristic of the human disease (microcytosis, hypochromasia, cell-cell adhesion, fragmentation etc). The use of PVA in this novel erythroid differentiation protocol successfully replaces BSA as a non-toxic, xeno-free media supplement that significantly boosts early erythroblast proliferation in culture, and ultimately primes the blood products for safe use in live human trials. Future blood products engineered using this method have the capacity to supplement donor-derived blood products for clinical use and can be used as a modeling platform to design and test novel drugs against human globinopathies.

Iron Nanoparticle Composite Hydrogels for Studying Effects of Iron Ion Release on Red Blood Cell In Vitro Production

Growing numbers of complex surgical interventions increase the need for blood transfusions, which cannot be fulfilled by the number of donors. Therefore, the interest in producing erythrocytes from their precursors-the hematopoietic stem and progenitor cells (HSPCs)-in laboratories is rising. To enable this, in vitro systems are needed, which allow analysis of the effects of essential factors such as iron on erythroid development. For this purpose, iron ion-releasing systems based on poly(ethylene glycol) (PEG)-iron nanocomposites are developed to assess if gradual iron release improves iron bioavailability during in vitro erythroid differentiation. The nanocomposites are synthesized using surfactant-free pulsed laser ablation of iron directly in the PEG solution. The iron concentrations released from the material are sufficient to influence in vitro erythropoiesis. In this way, the production of erythroid cells cultured on flat PEG-iron nanocomposite hydrogel pads can be enhanced. In contrast, erythroid differentiation is not enhanced in the biomimetic macroporous 3D composite scaffolds, possibly because of local iron overload within the pores of the system. In conclusion, the developed iron nanoparticle-PEG composite hydrogel allows constant iron ion release and thus paves the way (i) to understand the role of iron during erythropoiesis and (ii) toward the development of biomaterials with a controlled iron release for directing erythropoiesis in culture.