Erythroid Phenotype Research Articles

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Erythroid Krüppel-Like Factor (KLF1): A Surprisingly Versatile Regulator of Erythroid Differentiation.

Erythroid Krüppel-like factor (KLF1), first discovered in 1992, is an erythroid-restricted transcription factor (TF) that is essential for terminal differentiation of erythroid progenitors. At face value, KLF1 is a rather inconspicuous member of the 26-strong SP/KLF TF family. However, 30years of research have revealed that KLF1 is a jack of all trades in the molecular control of erythropoiesis. Initially described as a one-trick pony required for high-level transcription of the adult HBB gene, we now know that it orchestrates the entire erythroid differentiation program. It does so not only as an activator but also as a repressor. In addition, KLF1 was the first TF shown to be directly involved in enhancer/promoter loop formation. KLF1 variants underlie a wide range of erythroid phenotypes in the human population, varying from very mild conditions such as hereditary persistence of fetal hemoglobin and the In(Lu) blood type in the case of haploinsufficiency, to much more serious non-spherocytic hemolytic anemias in the case of compound heterozygosity, to dominant congenital dyserythropoietic anemia type IV invariably caused by a de novo variant in a highly conserved amino acid in the KLF1 DNA-binding domain. In this chapter, we present an overview of the past and present of KLF1 research and discuss the significance of human KLF1 variants.

Single-Cell Characterization of TP53-Mutated AML Patients Treated with Frontline Azacitidine, Venetoclax, and Magrolimab Reveals Mechanisms of Response and Resistance

Background: TP53 mutations, found in around 20% of AML cases, often lead to therapy resistance and unfavorable outcomes. Recent studies hint at the potential efficacy of CD47, a ‘don't eat me’ signal, in treating TP53-mutated AML. The ongoing clinical trial (NCT04435691) is assessing the efficacy of a triple therapy combo-magrolimab (anti-CD47 mAb), azacitidine, and venetoclax (AVM)-with promising initial results in TP53-mutated AML patients. However, understanding resistance and relapse remains crucial. Methods: We performed paired 5' scRNA and scTCR profiling on 3 healthy donors and 27 samples from 11 treatment-naive TP53-mutated AML patients (median age 63, range 59-73 years; 8 responders (CR/CRi); 3 non-responders (NR)), both pre-AVM (n=11) and post-AVM-point of remission (n=11), relapse (n=2), or primary resistance (n=3). Results: 42,910 AML cells and 137,169 bone marrow microenvironment cells passed quality checks. AML cell identification was confirmed via correlation with clinical flow cytometry, inferred copy number alterations, and immunohistochemistry markers. We leveraged Symphony to phenotypically infer AML cell types based on their transcriptional similarity to normal hematopoietic cells. Overall, samples displayed heterogeneous distribution of cell types at time of diagnosis. However, the 3 samples from the 3 primary resistant patients demonstrated a marked and significant expansion of AML cell proportion exhibiting an erythroid differentiation phenotype post-AVM (Fig. A). Also, the 2 patients who relapsed 4.8- and 7.1-months following AVM demonstrated expansion of erythroid differentiation proportion (Fig. A) and higher LSC17 at time of relapse. This suggests a selection favoring erythroid clones as a mechanism of primary and acquired resistance following AVM therapy. We conducted a pathway enrichment analysis using Hallmark gene sets to identify significantly altered AML-intrinsic biological pathways between responders and non-responders. CR/CRi patient-derived AML cells exhibited an enriched inflammatory response, specifically in TNFα and IFNγ signaling pathways-known CD47 expression regulators-and increased CD47 expression (Fig. B). In contrast, AML cells from non-responsive patients showed an enrichment of oxidative phosphorylation (Fig. B). Of note, phagocytic activity of myeloid cells was high in non-responders suggesting that despite the likely activity of magrolimab, other mechanisms of primary resistance were at play. As anti-CD47-mediated phagocytosis can stimulate antigen presentation and trigger T cell activation, we delved into the T cell subtypes and their corresponding cellular responses. There were not significant differences in the distribution of CD4 and CD8 pretreatment in CR/CRi versus NR patients. However, CD8 dysfunction score was significantly higher in non-responders at baseline. Following therapy, the CD8 dysfunction score was also significantly higher at time of relapse among responders. Further, effector CD8 T cells of NR patients had enrichment for cytotoxicity and TCR signaling pathways, and higher expression of exhaustion-related genes. Conclusions: Through single cell transcriptomic profiling of TP53-mutated AML patients treated with the triplet combination of AVM on protocol we identified erythroid differentiation and oxidative phosphorylation as possible intrinsic resistance mechanisms. Additionally, non-responders exhibited a dysfunctional T cell state prior to treatment, while relapses were associated with development of CD8 dysfunction. Overall, this data provides valuable insight into the mechanisms of response and resistance to magrolimab-based therapy. TCR repertoire and AML-immune cell interaction analyses are ongoing.

Prime Editing of the α-Thalassemia Hb Constant Spring Mutation

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. Although the α-globin is expressed from two copies of homologous genes ( HBA2 and HBA1), the constant spring (CS) mutation CD142 (UAA > CAA) occurs only in HBA2 gene that leads to the instability of both mRNA and protein in erythroid cells. The newly developed gene editing technology prime editors (PEs) can generate all types of substitutions, providing therapeutic potential in different types of diseases. However, the efficiency of primer editor in primary hematopoietic stem cells is still a big challenge and lack of study in the HbCS mutation. We first created the endogenous HbCS mutation in human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells, providing as a model for prime editing optimization. The protein of α cs chain (a larger protein with extra 31 amino acids) was detected in the model cells with HBA2 bi-allelic CS mutation and normal HBA1, which exhibited the delay of expansion, higher apoptosis rate and altered erythroid maturation markers after induced differentiation. To correct the CS mutation for converting HbCS to HbA, we performed ex vivo delivery of mRNA encoding the prime editor together with a pegRNA and a nick guide RNA into the Hb CS HUDEP2 model cells and achieved the successful in-situ correction with an efficiency of 39.883±9.720% of CS alleles without enrichment, while no measurable editing in the same loci of HBA1 as evaluated by both Sanger sequencing and NGS. More importantly, the correction resulted in a significant decrease of α cs chain, thereby alleviated erythroid phenotype after induced maturation with reducing the apoptosis rate. After that, we collected the primary Hb H-CS patients derived CD34 + hematopoietic stem/progenitor cells (HSPCs) and confirmed that the defect on erythroid expansion, differentiation in Hb H-CS patients derived CD34 + HSPCs compared to the normal donor, especially demonstrated by lower enucleation rate and higher apoptosis. Moreover, the protein of α cs chain was detected in the erythroblast from Hb H-CS CD34+ HSPCs. We further successfully corrected the CS mutation in Hb H-CS patient derived CD34 + HSPCs with an efficiency of 23.68% by mRNA electroporation. At the same time, the editing efficiency was achieved with minimal generation of insertions and deletions and a low level of Cas9-dependent DNA off-target editing. Importantly, we observed an ~23% decrease of α cs chain protein and significant restoration of the enucleation defect from the differentiated erythroblast after PE correction in CD34 + HSPCs. In summary, these results suggests that the delivery of PE mRNA together with synthesized pegRNA and nick sgRNA can successfully achieve the in-situ correction in Hb HbCS mutation site in the model cells and the primary Hb H-CS patient derived CD34 + HSPCs. Our results provide proof of principle for prime editing for its therapeutic potential in directly and precisely correction of the Hb-CS mutation.

Elucidation of the low-expressing erythroid CR1 phenotype by bioinformatic mining of the GATA1-driven blood-group regulome

Genetic determinants underlying most human blood groups are now clarified but variation in expression levels remains largely unexplored. By developing a bioinformatics pipeline analyzing GATA1/Chromatin immunoprecipitation followed by sequencing (ChIP-seq) datasets, we identify 193 potential regulatory sites in 33 blood-group genes. As proof-of-concept, we aimed to delineate the low-expressing complement receptor 1 (CR1) Helgeson phenotype on erythrocytes, which is correlated with several diseases and protects against severe malaria. We demonstrate that two candidate CR1 enhancer motifs in intron 4 bind GATA1 and drive transcription. Both are functionally abolished by naturally-occurring SNVs. Erythrocyte CR1-mRNA and CR1 levels correlate dose-dependently with genotype of one SNV (rs11117991) in two healthy donor cohorts. Haplotype analysis of rs11117991 with previously proposed markers for Helgeson shows high linkage disequilibrium in Europeans but explains the poor prediction reported for Africans. These data resolve the longstanding debate on the genetic basis of inherited low CR1 and form a systematic starting point to investigate the blood group regulome.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Iron homeostasis governs erythroid phenotype in polycythemia vera

AbstractPolycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients’ hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Blast crisis in chronic myeloid leukemia: An immunophenotypic analysis

Background and Aim of Study: There are two different phases of untreated chronic myeloid leukemia – chronic phase, and blast crisis – according to the World Health Organization classification of Hematolymphoid tumors. The blast cells in the chronic myeloid leukemia blast crisis can express myeloid, lymphoid, bi-phenotypic, monocytic, megakaryocytic, and erythroid phenotypes. The immunophenotype of blast population determines how chronic myeloid leukemia – blast crisis patients respond to treatment, hence flowcytometric examination is required. The aim of the study: to assess immunophenotyping outcomes of flowcytometry performed on the chronic myeloid leukemia – blast crisis. Material and Methods: A five-year retrospective descriptive analysis was carried out in Pathology Department at King George’s Medical University Lucknow, India (2017-2021). The patient’s peripheral blood and bone marrow aspirate samples were analyzed. Clinical, hematological, and immunophenotypic data were retrieved. The flow cytometry samples were prepared using the standardized “lyse-stain-wash” method. Results: A total of 43 cases of chronic myeloid leukemia – blast crisis were retrieved from the departmental archive in 5 years. The mean age of study population was 39.62±14.86 years. There were 24 males and 19 females. 27 patients were diagnosed with myeloid blast crisis, 14 cases of B-lymphoid blast crisis and 2 cases of mixed phenotypic acute leukemia. Conclusions: Identification of the blast lineage of patients with chronic myeloid leukemia – blast crisis is crucial since the existence of atypical blast phenotypes influences the disease treatment and prognosis.

A Putative Erythroid Synapse Linking Developing Erythroblasts and Macrophages in Erythroblastic Islands

An estimated 25% of the global population suffer from anemia, a major cause being deficient erythropoiesis. Mammalian erythropoiesis in the adult occurs primarily in the bone marrow (BM), with the spleen being an active site of extramedullary erythropoiesis during anemic stress. Here, we examine regulators of the anemic stress response using an unbiased transcriptomic approach to compare FACS-sorted splenic Ter-119+ erythroid precursors, from untreated mice or phenylhydrazine (PHZ)-treated mice exhibiting chemically-induced anemia. Transcriptomic analyses revealed unexpected expression of a broad range of immune markers in untreated mouse splenic erythroid cells, most which were conspicuously diminished in expression during PHZ-induced anemic stress. These surface antigens, previously unreported on erythroid cells, include markers of B lymphocytes (CD22, CD74), T lymphocytes (CD2, CD3ε) and leukocytes including CD11a/LFA-1, CD45, CD48, CD53, as well as the homing receptors L-selectin and PSGL-1. Immunophenotyping confirmed expression of these immune markers on a subpopulation of steady state BM and splenic erythroid precursors, with a significant reduction in immune marker-expressing erythroid cells seen in the spleen during anemic stress. Erythropoiesis is governed by a close relationship between erythroid precursors and central macrophages in multicellular clusters termed erythroblastic islands (EBIs). Confocal microscopy demonstrated localization of the immune markers CD2, CD3ε, CD11a and PSGL-1 on Ter-119+ mouse erythroid precursors with frequent clustering of markers seen at intercellular contact points where erythroid cells interact with the central EBI macrophage. PHZ-treated mice exhibited a significantly reduced frequency of EBIs in both the BM and spleen compared to control mice. PHZ-treated mice also demonstrated downregulated Interferon Regulatory Factor-8 (Irf8) transcripts on transcriptomic analysis of splenic erythroid precursors. Irf8-deficient mice have been reported to have a profoundly reduced erythroid compartment. There are also reports that Irf8 may be a regulator of macrophage morphology. We therefore studied Irf8-deficient mice to evaluate whether Irf8 has a role in EBI-regulation. BM analysis of Irf8-deficient mice showed a reduced frequency of EBI-like structures compared to wildtype (WT) mice. Scanning electron micrographs revealed that Irf8-/- macrophages are smaller with fewer erythroid precursors attached compared to WT macrophages. Irf8-/- EBIs harbored no identifiable reticulocytes in contrast to WT EBIs. EBI rosette reconstitution assays demonstrated that Irf8-/- erythroid precursors were unable to bind to WT or Irf8-/- macrophages, whereas Irf8-/- macrophages could interact with WT erythroid cells. Immunophenotyping of Irf8-/- erythroid precursors revealed that immune marker expression (CD2, CD38, CD43, CD45, PSGL-1) was significantly lower compared to WT counterparts. These data suggest that Irf8 may regulate both macrophage morphology and erythroid cell surface phenotype influencing erythropoiesis. The newly uncovered immune markers expressed by erythroid cells are also involved in formation of immunological synapses between lymphocytes and macrophages. This led us to question whether an analogous synapse-like structure may exist in EBIs. Serial block face scanning electron microscopy (SBF-SEM) was used to assess EBI morphology at the nanometer scale. SBF-SEM micrographs show erythroblasts extending pseudopods towards the macrophage for attachment. Three-dimensional volumetric reconstruction of SBF-SEM micrographs revealed en face spatial topologies present on the surface of EBI central macrophages similar to those seen in immunological synapses. Our findings suggest that immune markers, possibly mediated by Irf8, may function to form synaptic-like interactions between erythroid cells and macrophages that result in EBIs - an "erythroid synapse". Failure in forming these interactions may result in alterations in EBI formation with potential downstream impacts on erythropoiesis. Improved understanding of how EBIs form and function may aid in the development of therapeutic agents to combat anemia.

Phenotypic Variations Related to Hypoxic Responses Among Andean Highlanders Living at Different Altitudes (400m, 4000m, 5000m)

Introduction Adaptation to high altitude poses selective evolutionary changes involving multiple and challenging adaptive responses, and one of them deals with the low pressure of oxygen. Native Bolivian Andeans have lived at an average of 3000-5000 m for about 14,000 years, and have developed different erythroid phenotypes compared to other populations living at high altitude (PMID 30781443; PMID 25342802). Although Andeans have developed genetic adaptations related to erythropoiesis regulation (Blood, ASH 2316, 2018) they still undergone with polycythemic states, and those of clinical relevance are often Chronic Mountain Sickness erythrocytosis (CMS-E), secondary erythrocytosis (SE) and polycythemia vera (PV) (Rev Hematol Mex. 2016 Jan;17(1):8-20). Either adaptation or erythrocytosis condition entails important changes in hemoglobin, SpO2, P50 and lactate, so evaluating significant changes among Andeans living at different altitudes as well as the modifications between erythrocytosis patients and healthy highlanders became of interest. Material and method We collected venous blood samples from Bolivian native Andeans born at 4000 m (n=124) but living at 3 different altitudes (400 m, 4000 m, 5000 m), aside from Europeans (n=11) residing at 4000 m. Complete blood count, venous blood gas, and oxygen saturation studies were performed. P50 was measured by using a formula described by Lichtman. Likewise, a differential diagnosis regarding healthy inhabitants and polycythemia patients was performed. Results In healthy Andean inhabitants in different altitudes, the Hb levels increased at increasing altitude (p:0.001), meanwhile SpO2 (p:0.001) and P50 (p:0.001) decreased. No lactate variations among them were observed (Table 1). European subjects at 4000 m in relation to Andean inhabitants at the same altitude displayed higher Hb levels (p: 0.01), without variations in SpO2 and P50, but a significant increased lactate (p: 0.001) (Table 1). When comparing healthy subjects and patients with erythrocytosis (CMS-E, SE, PV) at 4000 m, the latter displayed higher Hb levels (p:0.001), decreased SpO2 (p:0.001), no variations in P50, and increased lactate in CMS-E and SE patients (p:0.01) (Table 2). Similarly, patients with erythrocytosis (CMS-E, SE) at 5000 m, related to healthy subjects at the same altitude, reflected increased Hb (p:0.001), decreased SpO2 (p:0.01), P50 without variations, and increased lactate (p:0.01) (Table 2). Conclusions Native Andeans from Bolivia born at 4000 m but living at different altitudes have variations regarding hematological phenotypes, decreased P50 shifts the hemoglobin dissociation curve to left. Patients with erythrocytosis have distinct phenotypes in relation to healthy subjects characterized by decreased saturation and increased lactate. Higher Hb levels and increased lactate in Europeans living at 4000 m probably due to the different residing time at high altitude, since native Andean inhabited high-altitude regions for many years. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MBD2a-NuRD Binds to the γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Bcl11a Mediated Silencing of Hb F Expression

A mechanistic understanding of γ-globin silencing is of clinical importance because reversal of silencing in adult erythroid cells results in high level expression Hb F which is therapeutic in sickle cell anemia and beta thalassemia. We describe here studies to elucidate the mechanism by which MBD2a-NuRD is an obligate partner of Bcl11a in silencing of HbF. We have previously shown that depletion of MBD2 or disruption of the MBD2-NuRD chromatin remodeling complex, but not MBD3-NuRD complex results in high levels of Hb F despite a concomitant 2-3 fold increase in expression of Bcl11a, a major silencing transcription factor (Yu etal. Haematologica, 2019). At the same time, Bcl11a can interact with both the MBD3 and MBD2 NuRD complexes. To elucidate the specific role of MBD2-NuRD, Chromatin Immunoprecipitation-qPCR(ChIP-qPCR) assays carried out in both adult erythroid phenotype HUDEP-2 and primary progenitor derived adult erythroid cells showed binding at the γ-globin gene promoter. To clarify the role of MBD2-NuRD in establishing closed chromatin configuration at the γ-globin gene loci, we carried out ATAC seq assays which showed that MBD2 knockout resulted in the same pattern of chromatin opening as reported for Bcl11a knockout in HUDEP-2 cells, consistent with our previous data showing ~40-50% Hb F in MBD2 knockout cells. In order to examine the effect of MBD2-NuRD on nucleosome positioning over the γ-globin gene promoter, NOME-SEQ assays were performed in HUDEP 2 cells which showed a nucleosome positioned at -110 to + 36 relative to the transcription start site (TSS) in MBD +/+ cells but not MBD2 KO HUDEP-2 cells, such that the NFY-A transcriptional activator binding site but not the Bcl11a repressor binding site is occluded. MBD2 exists in two isoforms in erythroid cells, resulting from alternative translation initiation sites. MBD2a contains an arginine rich (GR) domain which is absent in MBD2b and MBD3. Addback of MBD2a but not MBD2b in MBD2 KO HUDEP-2 cells resulted in silencing of γ-globin gene expression. Editing of the endogenous MBD2 gene such that predominantly MBD2b was expressed relieved silencing. To understand how the GR domain contributes to silencing we carried out unbiased proteomic analysis of MBD2a vs MBD2b NuRD complexes, which identified only 3 proteins uniquely associated with MBD2a. One of these, PRMT5, has been implicated in silencing of HbF. Knockdown of PRMT5 resulted in a`from <1% to ~15% γ-globin RNA in parental HUDEP-2 cells and a 4-5 fold increase in primary human progenitor derived erythroid cells, but caused no significant further increase in MBD2 KO HUDEP-2 cells. ChIP qPCR assays showed that PRMT5 and its obligate binding partner MEP50 occupy the γ- promoter in parental HUDEP-2 cells and primary erythroid cells but not in MBD2 KO HUDEP-2 cells. Furthermore, knockdown of PRMT5 resulted in frequent displacement of the nucleosome positioned at -110 to + 36 relative to the TSS. In order to determine the role that the GR domain might have in occupancy of MBD2a-NuRD at the γ-promoter, in vitro binding studies where done, which showed a requirement for both CpG methylation and the GR domain for high affinity binding of the methyl cytosine binding domain of MBD2 to the proximal γ-globin gene promoter sequences. We conclude that MBD2a-NuRD is required for silencing of Hb F due to its ability to localize at the methylated γ-globin gene promoter in conjunction with Bcl11a and recruit PRMT5, thereby stably positioning a nucleosome that blocks binding of the transcriptional activator NFY-A. These results reinforce the concept that targeted disruption of this specific complex has therapeutic potential to increase HbF in sickle cell anemia and β thalassemia.

HEXIM1 Overexpression Alters GATA1 Activity and Promotes Fetal Globin Expression

Erythropoiesis is a highly organized process whereby multipotent HSCs become committed to the erythroid linage and mature into enucleate erythrocytes. This process is associated with significant changes in gene expression as non-erythroid genes are switched off while expression of erythroid genes is maintained. HEXIM1 is a key regulator of RNA Polymerase II (RNPII) activity that is essential for erythropoiesis. Following transcription initiation, RNPII transcribes 30-50 base pairs before pausing. Phosphorylation of the c-terminal domain of RNPII by the dimer pTEFb (positive transcription elongation factor beta) is necessary for RNPII to resume active transcription elongation. HEXIM1 associates with pTEFb in the 7SK complex and renders it inactive. Phosphorylation of HEXIM1 releases pTEFb from the 7SK complex, allowing it to act on RNPII. As 7SK can be targeted to specific sequences, HEXIM1/7SK mediated pTEFb delivery is a crucial mechanism to facilitate on-site pTEFb activation and RNPII pause release. Recent work from our lab demonstrated that HEXIM1 is essential in the regulation of erythroid proliferation, viability and gene expression, and also promotes the expression of GATA1 target genes (Murphy Blood 2021). New analyses of HEXIM1 overexpression (OE) in HUDEP2 cells revealed a significant increase of gamma globin production, with a higher proportion of cells expressing fetal hemoglobin. These changes were accompanied by downregulation of beta globin and multiple key repressors of fetal hemoglobin, including BCL11A, MYB, and SOX6. These phenotypes were dependent on pTEFb activity, as the pTEFb (CDK9) inhibitor NVP-2 rescued the proliferative advantage granted by HEXIM1 OE and greatly decreased both gamma globin levels and the portion of fetal hemoglobin expressing cells. Further suggesting the effects of HEXIM1 OE are pTEFb-dependent, overexpression of HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb cannot enhance proliferation or gamma globin production. To gain insights into the mechanisms underlying the increased fetal globin expression, we performed CUT&RUN assays for Ser5 phosphorylated and total RNPII in HEXIM1 OE and control cells. Surprisingly, OE of HEXIM1 had only modest impacts on RNPII pausing, but instead lead to changes in the genes occupied by RNPII. Notably, the gene that lost the most RNPII was HBB and the gene that gained the most RNPII was HBG. Of note, GATA1 motifs were highly enriched in regions that gained RNPII occupancy. CUT&RUN identified peaks of GATA1 in the HEXIM1 OE cells at regions of increased RNPII occupancy that were not present in the EV or Y271A controls. These novel GATA1 sites occurred in regions of previously closed chromatin, suggesting that HEXIM1 OE enhanced the pioneer ability of GATA1. Similar to RNPII, GATA1 occupancy shifted from the beta globin gene in cells transduced with EV or HEXIM Y271A to the gamma globin genes in cells transduced with WT HEXIM1. Novel RNPII and GATA1 peaks were also identified in other genes associated with a fetal erythroid phenotype, most notably ARID3A and LIN28B. Together, these results support a model where elevated HEXIM1 levels promote the pioneer activity of GATA1, and facilitate the expression of a fetal transcriptional program in adult cells in a pTEFb-dependent manner.

P466: AMPLIFIED EPOR/JAK2 GENES DEFINE A UNIQUE SUBTYPE OF ACUTE ERYTHROID LEUKEMIA

Background: Acute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and erythroid/myeloid (EML) phenotypes based on the degree of erythroid hyperplasia. Despite an intensive mutational analysis, the mechanism of erythroid hyperplasia in AEL is still poorly understood and so are feasible therapeutic targets. Aims: To understand the mechanism of erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL. Methods: We analyzed a total of 124 adult AEL cases, where whole genome/exome sequencing of 35 cases were followed by targeted-capture sequencing in all cases. RNA sequencing was also performed in 23 cases. The mutational profile of AEL cases was compared to that of 409 cases with non-erythroid AML (non-AEL). Patient-derived xenograft (PDX) mouse models of AEL with JAK2 and/or EPOR amplification were established from 6 AEL patients with these abnormalities. These models were tested for their response to JAK1/2 inhibitor. Results: In accordance with a recent report, AEL cases were classified into 4 genomic groups (A-D), which are characterized by biallelic TP53 mutations and complex karyotype (Group-A), mutated NPM1 (Group-B) and STAG2 (Group-C), and other mutations in histone modifiers and transcription factors (Group-D). In particular, all but one PEL cases belonged to Group-A. Also found in non-AEL cases, these group-defining lesions in AEL were uniquely associated with focal gains/amplifications of EPOR, JAK2, and/or ERG/ETS2 loci (Group-A), PTPN11 mutations (Group-B), and KMT2A-PTD (Group-C), which might be responsible for the AEL phenotype. Highly enriched in PEL cases (7/13), EPOR/JAK2 focal gains/amplifications were implicated in their extreme erythroid hyperplasia and associated with particularly poor prognosis, even compared to other Group-A cases, who had shorter survival than those in other groups. As expected, JAK2/EPOR-amplified cases showed upregulated STAT5 signaling compared to non-AEL cases, which however, was also observed in other AEL cases, suggesting that upregulated STAT5 signaling is a hallmark of AEL. Based on these findings, we tested the effect of JAK2 inhibition on cell growth of EPOR/JAK2-amplified AEL cells in in vitro culture and xenograft model. Of interest, ruxolitinib-mediated JAK2 inhibition resulted in significantly suppressed in vitro cell growth of EPOR/JAK2-amplified AEL cells and prolonged overall survival in 4 PDX models with phospho-STAT5 downregulation, although other 2 models were resistant to JAK2 inhibition with persistent STAT5 activation. Summary/Conclusion: AEL is a heterogenous subgroups of AML characterized in common by upregulated JAK/STAT5 signaling. Gains/amplifications of JAK2/EPOR are frequent in TP53-mutated cases, particularly those with the PEL phenotype, and could be exploited as potential therapeutic targets using JAK2 inhibitors.

Translation defects in ribosomopathies.

Congenital or acquired ribosomopathies related to mutations or deletions in ribosomal proteins gene or ribosome-associated proteins exhibit defective ribosome biogenesis that expose the cell to translation defects. The mechanisms leading to low translation rate, loss-of-translation fidelity and translation selectivity are reviewed. New quantitative techniques to measure ribosome component stoichiometry reveal that the pool of ribosomes could be heterogeneous and/or decreased with a limited number of translationally competent ribosomes. During development or cell differentiation, the absence of specific ribosome components or their replacement by paralogs generate heterogeneous ribosomes that are specialized in the translation of specific mRNAs. Decreased ribosome content by defective biosynthesis of a subunit results in translation selectivity at the expense of short structured transcripts with high codon adaptation index. Activation of p53, as a witness of nucleolar stress associated with the hematological phenotype of ribosomopathies participates in translational reprogramming of the cell by interfering with cap-dependent translation. Translation selectivity is a common feature of ribosomopathies. p53 is more selectively activated in ribosomopathies with erythroid phenotype. The discovery of its dual role in regulating transcriptional and translational program supports new therapeutic perspectives.

Molecular pathogenesis and therapeutic targets in acute erythroid leukemia

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by erythroid predominance and dysplasia. It is classified into two subtypes: pure erythroid (PEL) and erythroid/myeloid (EML) phenotypes. To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed 105 AEL and 214 non-AEL cases using whole-genome/exome and/or targeted-capture sequencing, with SNP probes for detecting copy number abnormalities. We also performed a transcriptome analysis of 12 AEL samples. Combining publicly available sequencing data, AEL was genetically clustered into four groups according to mutational status in TP53, STAG2, and NPM1 genes. Conspicuously, highly recurrent gains and amplifications affecting EPOR, JAK2, and/or ERG/ETS2 were recurrently detected in AEL cases, almost exclusively found in TP53-mutated cases. Among these, gains/amplifications of EPOR/JAK2 were more highly enriched in PEL than EML cases. Along with the activated STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism, and they showed high sensitivity to ruxolitinib in in vitro and in xenograft models, highlighting the potential role of JAK2 inhibition in AEL therapeutics.

EPOR/JAK/STAT Signaling Pathway As Therapeutic Target of Acute Erythroid Leukemia

EPOR/JAK/STAT Signaling Pathway As Therapeutic Target of Acute Erythroid Leukemia

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation

AbstractHistone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor–like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

Concurrent Ex Vivo Differentiation of a Vascular Niche and Hematopoietic Stem/Progenitor Cells from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) ex vivo. Recent reports have described the generation of engraftable HSPCs via forced expression of key transcription factors in either endothelial cells, or hemogenic endothelium (HE) derived from induced pluripotent stem cells (iPSCs). While protocols for the generation of transgene-free engraftable HSPCs suitable for therapy remain unavailable, these studies, along with observations in the developing embryo, have shown the close association of HE populations with arterial vascular endothelium is of critical importance to HSPC production and maturation. To facilitate the development of a vascular niche supporting the emergence of transgene-free, functional HSPCs from human iPSCs, we developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, a mesodermal lineage specification was induced with morphogens (BMP4, bFGF and VEGF). For the subsequent 18 days, cells were further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3-L and TPO). Starting at day 5, an adherent monolayer composed in part of CD34+CD43- endothelial cells rapidly formed, followed by the initiation of endothelial-to-hematopoietic transition (EHT), as demonstrated by the emergence of round cells directly budding off the endothelial monolayer in time-lapse imaging studies (Panel A). Importantly, these cells were released within the culture supernatant and could be easily harvested by simple pipetting. Flow cytometric analysis of the supernatant cells indicated that the emerging cells initially had a predominant CD45-CD235a+ erythroid phenotype (maximum 69.9±12.4% at day 7), consistent with a primitive yolk-sac wave of hematopoiesis (PanelB). From day 7, CD45+CD34+ HSPCs increased gradually, with a maximum number (average 1x105 cells/cm2, n=10) observed at day 12, and a peak percentage at day 12 (47.4±12.4%) and 14 (48.8±9.9%) (Panel B). These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to bona fide CD45+CD34+ cells. Strikingly, cells with a phenotype that enables the highest reported purity of human HSCs (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholo) were highly enriched in the culture supernatant at day 10 through 14 (Panel C). However, these cells did not result in efficient, long-term engraftment in immuno-deficient (NSG) mice. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we further characterized the endothelial monolayer between day 5 and 12 of differentiation. We focused on recently described endothelial subsets within the CD34hiCD43- population, including the in vitro equivalent of definitive HE (CD34hi CD43- CD73- CD184- DLL4-), arterial vascular endothelium (CD34hi CD43- CD73mid CD184+), and venous vascular endothelium (CD34hi CD43- CD73hi CD184-) (Ditadi et al., Nat. Cell Biol 2015). At each timepoint analyzed, definitive HE comprised <1% of the total cell population (Panel D). Arterial vascular endothelium was also scarce, rising to a maximum of 6.6% at day 7 before declining to <1% by day 9 of culture (Panel D). Venous vascular endothelium accounted for approximately 2% of total cells (Panel D). The remaining cells of the monolayer expressed varying levels of CD34 and CD43, consistent with the recently demonstrated heterogeneity of cells undergoing EHT (Guibentif et al., Cell Rep 2017). Thus, our data suggest that the absence of a definitive HE population within a developing arterial niche, as occurs in the dorsal aorta (DA) during development, may explain in part the lack of engraftment potential of iPSC-derived HSPCs. The unique adaptable and scalable nature of this monolayer-based differentiation protocol will facilitate precise temporal activation or downregulation of signaling pathways essential for the generation of engraftable human HSPCs. Quantitative and temporal modulations of Wnt/Nodal pathways for mesodermal specification as well as VEGF/Notch pathways for arterial vascular endothelium are underway. [Display omitted] DisclosuresChen:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Boehm:StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Larochelle:Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit; Novartis: Research Funding.

Erythroid and Cardiovascular High Altitude-Selected Haplotypes in Andean Aymaras and Tibetans

Humans migrating out of Africa encountered new conditions, including living at high altitude. Tibetans and Andean Aymaras have inhabited regions of 4,000 meters or more for ~44,000 and 14,000 years respectively (Hu et al, PLoS Genet 2017, Rademaker et al, Science 2014). There is a distinct difference in erythroid phenotypes: Aymaras are polycythemic at high altitude, while most Tibetans are not. Mutations providing an advantage in highlanders will improve fitness under hypoxic conditions,including modulation of erythropoiesis through the hypoxia-inducible factor (HIF) pathway. There are few shared, naturally-selected gene regions in Aymaras and Tibetans and these have different phenotypic associations (Bigham et al, Am J Hum Biol 2013). Aymara high-altitude selected haplotypes have not been published. Tibetan EPAS 1 (encoding HIF2a protein) haplotypes in part originated from ancient Denisovans and entered the Tibetan genome through introgression (Huerta-Sánchez et al, Nature 2014). Tibetan variant prolyl hydroxylase 2 (PHD2), a negative regulator of HIFs encoded by EGLN1 gene, encodes in cis both PHD2D4E and PHD2C127S which together have increased activity in hypoxia (Lorenzo et al, Nature Genetics 2014) and, together with a Tibetan EPAS 1 haplotype, protects from high altitude polycythemia (Tashi et al, JMM 2017). We and others previously identified other Tibetans haplotypes that are not unique but are enriched in Tibetans, including a “Tibetan” haplotype of PKLR encoding liver- and red cell-specific pyruvate kinase (PK) (Simonson et al, Science 2010, and Yi et al, Science 2010).We studied Tibetan-enriched haplotypes of EGLN1, EPAS1 and PKLR in 72 Bolivian Andean Aymaras, all residing at 4,000 m, and compared them with 347 Tibetans living at altitudes of 200 m and 4,300 m (Table). We genotyped PHD2D4E and PHD2C127S variants of EGLN1 and 10 Tibetan specific single nucleotide polymorphisms (SNPs) of EPAS1, 5 Denisovan and 5 non-Denisovan, each under different linkage disequilibrium (Hu et all, PLoS Genet 2017), 7 Tibetan enriched PKLR SNPs, and searched for Aymara selected variants by whole genome sequencing.The prevalence of the Tibetan-selected EGLN1 and EPAS1 haplotypes increased with increasing altitude of residence in Tibetans, suggesting a continuous evolutionary advantage (Tashi et al, JMM 2017). Aymaras did not have the PHD2D4E haplotype, and PHD2C127S was found at lower prevalence in heterozygote form. Aymaras shared two of five non-Denisovan EPAS1 SNPs selected in Tibetans; one, rs130005507 G allele, had a similar prevalence to Tibetans, but another, rs142764723 C allele, had a lower prevalence. We report that >90% of Tibetans and ~50% of Aymaras, but only ~10% of Europeans, have the “Tibetan ” PKLR haplotype. Aymara females with homozygous PKLR haplotypes have lower hemoglobins than heterozygotes (p=0.022). Further, the PKLR transcript in reticulocytes decreases with increasing altitude and this progressive decrease is even more pronounced in the “Tibetan” haplotype. We found Aymaras' selected haplotypes encoding BRINP3, NOS2, TBX5 ; these genes are associated with cardiovascular development and function but not hypoxia sensing. They are not enriched in Tibetans.We conclude that Aymara highlanders do not have the Tibetan PHD2D4E mutation or Denisovan-like EPAS1 variants. Furthermore, they share only two of five Tibetan non- Denisovan EPAS1 variants. The absence of these variants in Aymaras supports that Tibetan and Aymara high altitude inhabitants do not have the same ancestry, that they developed different evolutionary adaptations (Bigham et al, Am J Hum Biol 2013), and that Tibetans' EGLN1 and EPAS1 mutations are unique to that part of the world. We hypothesize that decreased PK enzyme activity would be expected to increase 2,3-diphosphoglycerate (2,3-DPG) (as is shown in people with PK enzyme deficiency) and thus be beneficial to high altitude adaptation by progressively augmenting tissue oxygen delivery with increasing altitude. Based on its association with lower hemoglobin in Aymara females, the selected “Tibetan” PKLR haplotype, present in about half of Aymaras, may contribute to their hypoxic adaptation. Additional evaluation of evolutionary selected genes including PKLR, BRINP3, NOS 2, and TBX5 and their functional consequences are in progress with Aymaras living at El Alto (4,150 m), Cochabamba (2,500 m) and Santa Cruz, (416m), Bolivia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.