Erythroid Development Research Articles

Changes of hemoglobin resulted by distant hybridization contributed to increased hypoxia tolerance of allotriploid crucian carp

Hypoxia tolerance, one of the important goals of fish breeding, is beneficial for aquaculture and the transporting of fish. Based on the bisexual fertile allotetraploid crucian carp hybrid lineage obtained by distant hybridization, we successfully developed the allotriploid crucian carp via interploidy crossing with diploid red crucian carp. This allotriploid fish exhibited obvious hypoxia tolerance with lower values of dissolved oxygen at Pcrit (critical oxygen press) and 50 % ASR (aquatic surface respiration) than its original parents. Hemoglobin is the main vector of oxygen in vivo. This study examined the changes in amino acid sequences and natural protein conformation of hemoglobin, and found that the Hbα sequences of allotriploid crucian carp displayed obvious hybridity, including recombination and variation, when compared with its original parents. Moreover, the expression of hbα gene is higher in the liver and blood of allotriploid crucian carp than in its original parents. After acute anoxia treatment, the expression of gata1 gene, which served as the general switch for erythroid development, decreased in the blood of all the three kinds of fish. However, only in the allotriploid crucian carp hbα gene expression significantly decreased, coupled with the erythrocytes displaying an increased ratio of nuclei/cytoplasm. Taken together, this study suggested that changes in hemoglobin coupled with the erythrocyte morphological alterations under hypoxia increased the hypoxia tolerance ability of allotriploid crucian carp, which would provide a new perspective to guide fish breeding to obtain high-quality fish species with increased hypoxia tolerance.

Serine Hydroxymethyltransferase 2 Deficiency in the Hematopoietic System Disrupts Erythropoiesis and Induces Anemia in Murine Models.

Serine and folate metabolism play critical roles in erythroid development in both embryonic and adult mice; however, the precise roles of these metabolic pathways in erythropoiesis and the pathophysiology of anemia remain inadequately characterized in the literature. To delineate the contributions of serine and folate metabolism to erythroid differentiation, we focused on serine hydroxymethyltransferase 2 (SHMT2), a key regulatory enzyme within these metabolic pathways. Using gene-editing techniques, we created fetal and adult mouse models with targeted deletion of Shmt2 in the hematopoietic system. Our findings demonstrated that the deletion of Shmt2 within the hematopoietic system led to the distinctive anemia phenotype in both fetal and adult mice. Detailed progression analysis of anemia revealed that Shmt2 deletion exerts stage-specific effects on the development and maturation of erythroid cells. Specifically, Shmt2 deficiency promoted erythroid differentiation in the R2 (CD71+ Ter119-) cell population residing in the bone marrow while concurrently inhibiting the proliferation and erythroid differentiation of the R3 (CD71+ Ter119+) cell population. This disruption resulted in developmental arrest at the R3 stage, significantly contributing to the anemia phenotype observed in the models. This study elucidates the critical role of Shmt2 in erythroid development within the hematopoietic system, highlighting the underlying mechanisms of erythroid developmental arrest associated with Shmt2 loss.

TMEM56 deficiency impairs the haem metabolism and cell cycle progression during human erythropoiesis.

TMEM56, a gene coding a transmembrane protein, is abundantly expressed in erythroid cells. Despite this, its role in erythropoiesis has not been well characterized. In this study, we sought to clarify the function of TMEM56 in erythroid development, focusing specifically on its involvement in haem biosynthesis and cell cycle progression. To do this, we used CD34+ haematopoietic stem cells derived from umbilical cord blood and differentiated them into erythroid cells in an exvivo model. Our results indicate that the loss of TMEM56 disrupts haem biosynthesis and impairs erythroid differentiation. Furthermore, deletion of Tmem56 in the erythroid lineage in murine models using erythropoietin receptor (EpoR)-Cre revealed defects in erythroid progenitors within the bone marrow under both normal conditions and during haemolytic anaemia. These observations underscore the regulatory role of TMEM56 in maintaining erythroid lineage homeostasis. Taken together, our results unveil a previously unrecognized function of TMEM56 in erythroid differentiation and suggest its potential as an unfounded target for therapeutic strategies in the treatment of erythropoietic disorders.

Creg1 Regulates Erythroid Development via TGF-β/Smad2-Klf1 Axis in Zebrafish.

Understanding the regulation of normal erythroid development will help to develop new potential therapeutic strategies for disorders of the erythroid lineage. Cellular repressor of E1A-stimulated genes 1 (CREG1) is a glycoprotein that has been implicated in the regulation of tissue homeostasis. However, its role in erythropoiesis remains largely undefined. In this study, it is found that CREG1 expression increases progressively during erythroid differentiation. In zebrafish, creg1 mRNA is preferentially expressed within the intermediate cell mass (ICM)/peripheral blood island (PBI) region where primitive erythropoiesis occurs. Loss of creg1 leads to anemia caused by defective erythroid differentiation and excessive apoptosis of erythroid progenitors. Mechanistically, creg1 deficiency results in reduced activation of TGF-β/Smad2 signaling pathway. Treatment with an agonist of the Smad2 pathway (IDE2) could significantly restore the defective erythroid development in creg1-/- mutants. Further, Klf1, identified as a key target gene downstream of the TGF-β/Smad2 signaling pathway, is involved in creg1 deficiency-induced aberrant erythropoiesis. Thus, this study reveals a previously unrecognized role for Creg1 as a critical regulator of erythropoiesis, mediated at least in part by the TGF-β/Smad2-Klf1 axis. This finding may contribute to the understanding of normal erythropoiesis and the pathogenesis of erythroid disorders.

Updates on clinical and laboratory aspects of hereditary dyserythropoietic anemias.

Hereditary dyserythropoietic anemias, or congenital dyserythropoietic anemias (CDAs), are rare disorders disrupting normal erythroid lineage development, resulting in ineffective erythropoiesis and monolinear cytopenia. CDAs include three main types (I, II, III), transcription-factor-related forms, and syndromic forms. The widespread use of next-generation sequencing in the last decade has unveiled novel causative genes and unexpected genotype-phenotype correlations. The discovery of the genetic defects underlying the CDAs not only facilitates accurate diagnosis but also enhances understanding of CDA pathophysiology. Notable advancements include identifying a hepatic-specific role of the SEC23B loss-of-function in iron metabolism dysregulation in CDA II, deepening CDIN1 dysfunction during erythroid differentiation, and uncovering a recessive CDA III form associated with RACGAP1 variants. Current treatments primarily rely on supportive measures tailored to disease severity and clinical features. Comparative studies with pyruvate kinase deficiency have illuminated new therapeutic avenues by elucidating iron dyshomeostasis and dyserythropoiesis mechanisms. We herein discuss recent progress in diagnostic methodologies, novel gene discoveries, and enhanced comprehension of CDA pathogenesis and molecular genetics.

Roles of Nuclear Orphan Receptors TR2 and TR4 during Hematopoiesis.

TR2 and TR4 (NR2C1 and NR2C2, respectively) are evolutionarily conserved nuclear orphan receptors capable of binding direct repeat sequences in a stage-specific manner. Like other nuclear receptors, TR2 and TR4 possess important roles in transcriptional activation or repression with developmental stage and tissue specificity. TR2 and TR4 bind DNA and possess the ability to complex with available cofactors mediating developmental stage-specific actions in primitive and definitive erythrocytes. In erythropoiesis, TR2 and TR4 are required for erythroid development, maturation, and key erythroid transcription factor regulation. TR2 and TR4 recruit and interact with transcriptional corepressors or coactivators to elicit developmental stage-specific gene regulation during hematopoiesis.

Epigenetic regulatory differentiation of hematopoietic stem cells to the red lineage Running head: Epigenetics of erythropoiesis

The differentiation process from hematopoietic (HSCs) to mature red blood cells (RBCs) is characterized by remarkable precision and is tightly controlled from the initial lineage commitment to eventual terminal differentiation. Erythropoiesis is the dynamic journey of HSCs through various functional and phenotypic stages. The physiological course of erythrogenesis is intricately linked to significant changes in chromatin accessibility, necessitating precise coordination of transcription factors and epigenetic elements. This review presents a comprehensive overview of recent investigations into the molecular-level epigenetic regulatory factors that influence differentiation of the erythroid lineage. This encompasses the exploration of transcriptional, post-transcriptional, and post-translational regulatory processes. The intricate interplay of epigenetic and transcriptional regulatory networks in erythroid differentiation not only enhances our understanding of this fundamental biological process, but also provides valuable insights into the underlying mechanisms contributing to the pathogenesis of disorders associated with abnormal erythroid development. These findings hold significant promise for the development of novel therapeutic strategies to address these diseases and improve patient outcomes.

Vhl deletion in Dmp1-expressing cells alters MEP metabolism and promotes stress erythropoiesis

IntroductionErythropoiesis occurs in a specialized niche in the bone marrow and is regulated in part by oxygen-sensing signaling pathways. VHL is an E3 ubiquitin ligase that regulates the degradation of hypoxia-inducible factor (HIF) proteins under normoxic conditions. In hypoxia, VHL is inactivated resulting in enhancement of HIF signaling. Methods and resultsTo investigate the effects of HIF-activation in bone cells on erythropoiesis, we performed longitudinal analyses of conditional Vhl knockout mice (Dmp1-Cre;Vhlfl/fl; VhlcKO) mice, in which Vhl is deleted primarily in osteocytes but is retained in hematopoietic cells. VhlcKO mice display elevated red blood cell counts and erythropoietin levels starting at 10 weeks of age, resembling polycythemia. Using flow cytometry, we observed increased frequency of CD71loTER119hiFSClo orthochromatophilic erythroblasts and reticulocytes in 10- and 24-week-old VhlcKO bone marrow. Features of extramedullary hematopoiesis were observed in the spleen, including red pulp hyperplasia, the presence of megakaryocytes, and increased frequency of basophilic and polychromatophilic erythroblasts and of mature stress erythroid progenitors. To investigate the mechanisms that drive the accelerated erythropoiesis in the bone marrow, we enumerated myeloid progenitors and observed higher frequencies of megakaryocyte-erythroid progenitors (MEPs) at 6 and 10 weeks of age, but MEP frequency was similar to controls at 24 weeks. Despite this normalization in MEP frequencies, bulk RNA-Seq of MEPs at this age showed upregulation of Epas1 (Hif2a), consistent with a response to hypoxia, as well as genes involved in erythrocyte development. Surprisingly, genes involved in the response to glucose were also upregulated in VhlcKO MEPs. DiscussionOur studies implicate that HIF-driven alterations in skeletal homeostasis drive changes to the bone marrow microenvironment that influence erythroid cell development and MEP metabolism.

A Mediator Switch Tunes Transcription Pausing to Drive Erythropoiesis

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development. To address whether composition of the Mediator complex undergoes dynamic changes during erythropoiesis, we detected its expression in primary human erythroblasts derived from CD34 + hematopoietic stem and progenitor cells. We found that while the protein levels of most Mediator subunits decreased substantially in the terminal erythroid differentiation, MED26 remained relatively abundant. A gain-of-function assay in the primary human erythroid culture system showed that MED26 overexpression promoted erythroid differentiation. Furthermore, Med26 conditional knockout (cKO) mice exhibited severe erythroid and hematological defects. Erythroid precursors (especially the Cd71+Ter119+ stage) were absent in the bone marrow of Med26-cKO mice, which phenocopies the cKO mice of Gata1. Prussian blue staining of the spleen section showed more iron deposition in the cKO mice. Moreover, the cKO mice were unable to recover after phenylhydrazine-induced hemolytic anemia. To dissect the molecular basis underlying MED26 function, we analyzed the chromatin occupancy signals of MED26 by CUT&Tag assay and compared them to those of MED1, which is often regarded as a representative subunit of Mediator, in human CD34 + erythroid culture cells on Day 4. Intriguingly, we found that ~60% of chromatin sites with MED1 or MED26 occupancy did not colocalize, suggesting these subunits exert context-dependent gene regulation. Notably, MED26 colocalizes with GATA1 and GATA2 better than MED1. PRO-seq analysis further revealed that MED26-enriched loci were associated with RNA polymerase II (Pol II) pausing. To examine how MED26 might mediate transcription pausing, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify interacting proteins of MED1 and MED26. While MED26 and MED1 similarly coimmunoprecipitated with the majority of other Mediator subunits and the elongation complex, MED26 exhibited a substantially higher interaction with transcription pausing related factors (NELF, DSIF, and PAF complexes) compared to MED1. Knocking out MED26 significantly impairs the recruitment of PAF1 to MED26 occupancy sites, indicating the presence of MED26 facilitates the recruitment of PAF1. Our results also revealed that MED26 enrichment is associated with increased transcription pausing during terminal erythroid stages, which can promote erythroid development. Moreover, we uncovered that MED26 exhibited pronounced biomolecular condensate-forming capability. The intrinsically disordered region (IDR) of MED26 was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors to the MED26-enriched condensates. Taken together, our findings demonstrate that the relative enrichment of MED26 in terminal erythropoiesis leads to a shift of transcription condensates towards a MED26-enriched form. This shift, in turn, facilitates the recruitment of pausing related factors, thereby achieving genome-wide transcription repression during erythropoiesis. This study not only illustrates the indispensable function of MED26 in erythropoiesis but also elucidates the functional coordination of MED1 and MED26 during different erythroid stages. The dynamic switch of Mediator stoichiometry might serve as an important example for understanding the mechanism of the versatile Mediator function during development.

Human DOCK11 Deficiency Causes Defective Erythropoiesis and Systemic Inflammation

Erythropoiesis involves significant changes in the cells, which are mediated by the plasma membrane and the actin cytoskeleton. The composition of the actin cytoskeleton and the interaction between its components are dynamically modified during erythropoiesis, however the precise role that different actin regulators play during erythroid differentiation is poorly understood. The dedicator of cytokinesis (DOCK) family member DOCK11 regulates actin cytoskeleton dynamics via its guanine nucleotide exchange factor (GEF) activity, resulting in activation of the small Rho GTPase CDC42. CDC42 has been implicated in regulating the early stages of erythroid development, as well as the terminal maturation steps involving enucleation. However, the role of human DOCK11 in hematopoietic cell function and human disease had not been defined. We analyzed a cohort of four patients from four unrelated families presenting with recurrent infections, early-onset severe immune dysregulation, systemic inflammation, as well as normocytic anemia and anisocytosis of unknown origin. Using whole-exome sequencing, we identified rare, hemizygous germline mutations in DOCK11 in these patients. Two mutations - an early stop-gain mutation and a To study the role of DOCK11 during erythropoiesis, we generated a dock11-knockout zebrafish model. We found that the dock11-knockout zebrafish embryos recapitulated the anemia and aberrant erythrocyte morphology observed in human DOCK11 deficiency. The anemia was amenable to rescue with constitutively active CDC42, suggesting that DOCK11 regulates erythrocyte numbers in a CDC42-dependent manner. As a next step we modeled human erythroid differentiation in vitro using an erythroid liquid culture system starting from purified CD34+ cells. ShRNA-mediated knockdown of DOCK11 in CD34 + cells revealed impairment of cell growth and differentiation during erythroid development. In line with moderate erythroid hypoplasia observed in the bone marrow of one of the patients, these data suggest an erythroid-intrinsic role of DOCK11 during erythropoiesis. As the patients with germline DOCK11 defects also presented with recurrent infections and systemic inflammation, we further assessed the role of DOCK11 in immune cells. In line with its role in actin dynamics, we found that human DOCK11 regulates T-cell morphology and migration, suggesting that defects in DOCK11 might impact the T cells' capability to fight infections. We further uncovered that the immune dysregulation observed in the patients involved aberrant T-cell activation and cytokine production. Mechanistically, using cells from DOCK11-deficient patients and Dock11-knockout mice, we were able to show that Dock11 regulates cytokine production at the transcriptional level by modulating the nuclear translocation of the T-cell transcription factor NFATc1, known to regulate the production of several cytokines. Collectively, we identified germline loss-of-function mutations affecting the actin regulator DOCK11 in a previously unknown disorder associating anemia and systematic inflammation. This work earmarks the DOCK11-CDC42 axis as an attractive future target for the treatment of a broader range of immune and hematological diseases

GATA-1 and BCL11A Collaboratively Regulate Erythroid Gene Expression through HDAC1 Dependent Manner

Transcription factor GATA-1 plays important roles in hematopoiesis through interaction with other key transcription factors and epigenetic modulators. Previously, we established that GATA-1 can physically interact with HDAC1 at two arginine residues of the linker region between two zinc fingers. Arginine to alanine mutation at these sites blocks the HDAC1 interaction and results in constitutive GATA-1 acetylation, and reduced GATA-1 chromatin binding at the gene regulatory regions. In addition, the loss of HDAC1 interaction causes the blockage of erythroid and megakaryocytic differentiation in mice. Having established the importance of GATA-1 and HDAC1 interaction in GATA-1 function, next we aim to further investigate whether HDAC1-associated proteins are involved in regulating the function of GATA-1. We performed LC-MS/MS analysis using GATA-1 wild type and mutant expressing cells and identified that BCL11A was one of the major proteins that associates with GATA-1 through HDAC1. BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.

Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p <0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p <0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p <0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p <0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

Ex Vivo Treatment By Mitapivat, an Allosteric Pyruvate Kinase Activator, Reduced Oxidative Stress and Promoted Terminal Erythropoiesis in a Severe Hemolytic Anemia Patient Due to Krϋppel-like Factor 1 Mutations

Introduction: Human erythroid Krüppel-like Factor 1 (EKLF or KLF1), a hematopoietic-specific transcription factor encoded from KLF1 plays roles as the master regulator of erythroid development, including globin switching and expression, pyruvate kinase ( PKLR) function, heme synthesis, structural membrane, minor blood group, and cell cycle regulation. Most patients carrying KLF1 mutations present with severe chronic hemolytic anemia with low pyruvate kinase-red blood cell (PKR) activity and require regular transfusion. Previously, our group described the effects of ex vivo mitapivat (AG-348), an allosteric activator of PKR, treatment on the peripheral RBCs of two non-regular transfused chronic hemolytic anemia patients due to KLF1 mutations by increased PKR activity and ATP production of treated RBCs (Suksangpleng et al, Blood 2019; 134 (Supplement_1): 2220). We are currently studying the erythropoietic characteristics and investigating the effects of treatment with mitapivat to improve the severe anemia observed in these KLF1 patients. Objective: To evaluate the effects of ex vivo mitapivat treatment on the terminal erythropoiesis characteristics, including cell viability, oxidative stress, necrosis, and erythroid differentiation, of one severe hemolytic anemia patient due to KLF1 mutations. Methods: The 20-mL EDTA whole blood sample was collected from one regular transfused severe hemolytic anemia patient who was identified with KLF1 alleles mutations. The CD34 +-peripheral blood mononuclear cells (PBMCs) were separated by the specific anti-CD34 antibody-labelled magnetic beads and cultured by 3 phases of primary erythroid culture system, including phase 1: erythroblast transformation, phase 2: erythroblast expansion, and phase 3: erythroid differentiation. Cell viability was monitored in all 3 phases. The erythroid differentiation was monitored by stage-specific erythroid surface marker antibodies using flow cytometry. The ex vivo treatment was performed in phase 3 by incubating the cultured erythroid cells with the completed tissue cultured medium containing 40 nM mitapivat (Agios Pharmaceuticals, Inc.). Intracellular reactive oxygen species (ROS) were measured by fluorescent detection and necrotic cells were stained with Annexin V/PI and numbered by flow cytometry. Results: One index patient with regular transfused chronic hemolytic anemia showed low level of Hb (< 11 g/dL), high reticulocyte count (> 2%), high Hb F level (> 2%), and no PKLR mutation identified. The allele genotypes of KLF1 were identified as compound heterozygous mutation including unusual amino acid (Q58X) and missense mutation (A298P) on allele 1 and 2, respectively (Table 1). Proerythroblasts were transformed from the separated CD34 +-PBMCs in the Phase 1 culture period (Day 1 - Day 6) and highly expanded in Phase 2 (Day 7 - Day 13). After being induced to erythroid differentiation in Phase 3, the viability of erythroblasts gradually decreased while increased intracellular ROS was found. These phenomena corresponded to the increased numbers of necrotic cells during erythroid stage differentiation. Interestingly, the ex vivo treatment with mitapivat decreased intracellular ROS and necrotic cell numbers of the stage-differentiated erythroblasts, with more viable cell counts compared with the untreated condition in terminal erythropoiesis (Figure 1A, 1B, 1C). Moreover, the ex vivo treatment with mitapivat supported the effective stage differentiation shown by higher FITC- and lower PE-mean fluorescent intensity (MFI) that represented two stage-specific erythroid markers, including CD235a (glycophorin A) and CD71 (transferrin receptor), respectively, than those of untreated differentiated erythroblasts (Figure 1D). Conclusion: This study showed the effects of ex vivo treatment with mitapivat which could reduce oxidative stress, promote cell viability and stage differentiation in terminal erythropoiesis of one regular transfused hemolytic anemia patient due to KLF1 mutations. Mitapivat is being studied in clinical trials in PK deficiency (NCT03853798, NCT05144256, and NCT05175105), thalassemia (NCT04770753 and NCT04770779) and sickle cell disease (NCT05031780). Further evaluation of mitapivat in patients with severe hemolytic anemia due to KLF1 mutations is warranted as a potential therapeutic option for patients with KLF1 disease.

Synthetic Introns Identify the Novel RNA Splicing Factor GPATCH8 As Required for Mis-Splicing Induced By SF3B1 Mutations

Disclosures Mutations in the core RNA splicing factor SF3B1 are common across MDS, CLL, and clonal hematopoiesis. Prior studies have elucidated that mutations in SF3B1 result in neomorphic widespread changes in splicing due to usage of aberrant intronic branchpoint nucleotides. However, the molecular basis by which mutant SF3B1 induces mis-splicing is not established and therapeutic means to correct mis-splicing due to mutant SF3B1 do not exist. Recently, the aberrant gain-of-function splicing activity of mutant SF3B1 was harnessed to regulate expression of proteins in a mutant selective manner using synthetic version of endogenous mRNA sequences uniquely recognized by mutant SF3B1. Here, we utilized this technology to engineer a synthetic intron derived from MAP3K7 that interrupts the coding sequence of the fluorescent protein mEmerald such that mutant SF3B1 produces lower mEmerald expression than SF3B1 wild-type (WT) cells (Fig.A). This fluorescent splicing reporter was then used to perform positive enrichment whole genome CRISPR screens to identify genes whose deletion corrects SF3B1 mutant aberrant splicing. GPATCH8 was the single most robust hit. GPATCH8 knockout strongly corrected mis-splicing of both synthetic and endogenous MAP3K7 introns (Fig.A). A previously unexplored protein, GPATCH8 has domains characteristic of RNA splicing factors including a G-patch motif, thought to be important in activating RNA helicases. Interestingly, recent work suggests that mutations in SF3B1 result in mis-splicing by disrupting physical interaction of SF3B1 to the RNA helicase DHX15 via the G-patch domain containing protein SUGP1. We therefore compared SUGP1 to GPATCH8 activity, evaluating first their transcriptome-wide binding sites using TRIBE-seq. We found that both proteins predominantly target intronic sequences over other genomic regions suggesting roles in splicing. RNA-seq of SF3B1 mutant and WT cells with or without GPATCH8/SUGP1 deletion revealed that ~33% of SF3B1 mutant splicing alterations are corrected by GPATCH8 deletion, while ~60% of SF3B1 mutant splicing alterations are recapitulated upon SUGP1 deletion. GPATCH8 regulated splicing events have stronger branchpoints indicating a potential role for GPATCH8 in 3' splice site recognition. To further understand the connection between GPATCH8 and mutant SF3B1, we performed mass spectrometry studies of immunoprecipitated endogenous GPATCH8 in SF3B1 WT and mutant knockin cells. This revealed strong interaction between GPATCH8 and DHX15. GPATCH8 interacts with DHX15 at the same sites previously shown to be occupied by SUGP1 in crystal structures of the SUGP1/DHX15 interaction. These data and further biochemical studies elucidated that GPATCH8 competes with SUGP1 for interaction to DHX15. As such, deletion of GPATCH8 corrects SF3B1 mutant mis-splicing by enhancing the interaction of SUGP1 and DHX15 to the mutant SF3b complex. Given that GPATCH8 is required for a large proportion of the splicing alterations induced by mutant SF3B1, we next evaluated the phenotypic effect of correcting SF3B1 mutant mis-splicing. We first tested the impact of anti-Gpatch8 shRNAs in bone marrow from WT mice as well as animals with conditional knockin of Sf3b1 K700E, K666N, or R625H mutations. While each Sf3b1 mutation results in impaired colony formation of hematopoietic precursors in methylcellulose assays, silencing of Gpatch8 rescued colony formation from hematopoietic precursors of each of the different Sf3b1 mutant mice and was tolerated by normal hematopoietic precursors (Fig.B). Moreover, while CRISPR base-edited knockin of the SF3B1 K700E mutation in adult CD34 + cells impaired erythroid development, this erythroid differentiation defect was rescued by GPATCH8 deletion in the same cells (Fig.B). These data identify GPATCH8 as a novel RNA splicing factor involved in quality control of RNA branchpoint selection whose expression is required for mis-splicing by the different mutant forms of SF3B1. GPATCH8 antagonizes the activity of SUGP1 by competing for interaction with DHX15. These findings suggest that disrupting GPATCH8/DHX15 interaction could have important therapeutic benefit for the multitude of SF3B1 mutant hematopoietic diseases. This study also demonstrates the power of synthetic intronic splicing assays for discovery of trans factors and druggable proteins required by leukemia-associated mutant RNA splicing factors. Abdel-Wahab: AbbVie, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Loxo/Lilly: Consultancy; Nurix Therapeutics: Research Funding; Minovia Therapeutics: Research Funding; Amphista Therapeutics: Consultancy; AstraZeneca: Consultancy; Harmonic Discovery: Current holder of stock options in a privately-held company. Figure 1

Small Molecule Inhibitor Targeting Nemo-like Kinase Improves Erythropoiesis in Human and Mouse Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies, and an increased risk of developing cancer. Approximately 80% of patients with DBA have a mutation in one of 20 different genes that encode ribosomal proteins, resulting in abnormal protein translation. The current standard of care for patients with DBA includes steroids, chronic red cell transfusions, or stem cell transplantation, but these are all associated with significant side effects including infections, iron overload, and the risk of graft versus host disease. Thus, the development of more effective and less-toxic therapies is needed to treat the anemia seen in patients with DBA. Our previous work demonstrated that Nemo-Like Kinase (NLK) is overly active in red cell precursors in models of DBA as well as in patient samples (Wilkes, et al., 2020). We hypothesize that NLK plays an important role in the pathogenesis of DBA, and is a potential target for DBA therapy. Human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) were transduced with lentivirus co-expressing GFP and shRNA against RPS19/RPL11 or luciferase (control), and were differentiated in erythroid media in the presence of small molecule NLK inhibitors. After screening small molecule compounds that inhibit NLK as an off-target from previously approved or clinically advanced drugs, we found that a known MELK (maternal embryonic leucine zipper kinase) inhibitor, OTS167, improved erythropoiesis (CD71+ cell expansion) by 31% and 27% at a concentration of 50nM in RPS19- and RPL11-knockdown HSPCs, respectively. We also investigated the effect of OTS167 on the erythropoiesis in Rpl11-haploinsufficient mouse model of DBA ( Rpl11 wt/LoxpCre-ERT2 +, Morgado-Palacin, et al., 2015 and Rpl11 wt/LoxpMx-Cre + Liu,et al.,2023). Lineage-negative (Lin-) hematopoietic progenitors isolated from the mouse bone marrow were cultured in erythroid differentiation medium with OTS167. Our data showed that the erythroid progenitors (CD71+ cells) expanded from the cultured Lin- cells were increased by 20% (n=2, p=0.065) at an OTS167 concentration of 30nM. We confirmed that NLK activity was increased in erythroid progenitors from the Rpl11-haploinsufficient mice for in vitro phosphorylation of the substrate compared with tamoxifen-treated wild type mice. Moreover, we performed colony formation assay with OTS167 in the mouse bone marrow cells. OTS167 increased BFU-E colony numbers in a dose-dependent manner with the highest effect at a concentration of 50nM (n=3, p=0.003). OTS167 did not increased CFU-E colony number, suggesting that OTS167 affected the BFU-E stage and/or earlier stages of erythroid development. Also, OTS167 did not affected CFU-GM colony formation as myeloid lineage. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to minimize the need for chronic red cell transfusions or treatment with steroids. Our in vitro data suggest that pharmacologic inhibition of NLK with OTS167 may achieve this goal. We have initiated studies to test the efficacy of OTS167 in vivo using Rpl11-haploinsufficient mouse models. Given that DBA is associated with a number of mutations in the ribosome, using one drug that targets NLK as a common therapeutic target could be a more practical treatment option for patients in contrast to gene therapy or gene editing followed by autologous stem cell rescue. Even if the drug targeting NLK does not completely rescue the anemia in ribosome-insufficient cell models of DBA, the improvement could be enough to help patients especially in combination with other drugs that are currently being used or studied .

Whole-Genome CRISPR-Cas9 Screening Identifies Genes Required for Human and Mouse Erythroid Development

Red blood cell (RBC) development is regulated by a few external signaling molecules which act to influence the ongoing intracellular processes responsible for producing erythrocytes. While much is known about the signaling molecules important for blood production, comparatively little is known about the genes required for successfully generating erythrocytes. Several genes have already been identified as essential for RBC development through loss-of-function studies in the mouse or in human erythroid cells in vitro. However, these studies have been limited in scope to genes that are already suspected or known to influence RBC production. Since there are ~10,000 genes expressed in erythroid cells, it is unclear how many of these genes are functionally required for erythroid development. To define the repertoire of genes required for human erythroid development, we performed a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2, using the GeCKOv2 lentiviral library, which delivers Cas9 and one of 6 sgRNAs targeting virtually every gene in the genome. Following viral transduction and cell recovery for 9 days, we collected HUDEP-2 cells prior to the onset of differentiation as well as differentiated orthochromatic erythroblasts (right prior to enucleation) using flow sorting, on the basis of CD49d expression (CD49d is downregulated in the final stages of erythroid differentiation). As expected, sgRNAs targeting known erythroid essential genes (such as GATA1 and EPOR) were depleted in HUDEP-2 cells compared to the sgRNA library. Notably, we also identified genes for which sgRNAs were depleted in differentiated versus undifferentiated erythroid cells, representing genes that are likely required for terminal erythroid differentiation (such as ZFPM1, ALAS2, etc..). This supports the utility of the screen to identify novel regulators of erythropoiesis. Among highly ranked genes, NHLRC2, which was previously implicated in hemolytic anemia, was identified to be required in HUDEP-2 cells, suggesting that NHLRC2 is intrinsically required for human erythroid development. To validate the role of NHLRC2 in human erythropoiesis, we down-regulated NHLRC2 in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) undergoing erythroid differentiation, using one of 4 independent shRNAs. NHLRC2 down-regulation resulted in impaired proliferation and differentiation of human erythroid cells in vitro. To validate the results of the genome-scale screen, we generated a secondary library dedicated to examining genes that positively or negatively regulate the final stages of erythroid differentiation, while excluding common essential genes required for the survival of more than 90% of immortalized cell lines screened by the BROAD institute. The secondary screen yielded a list of over 500 genes found to influence erythroid differentiation (FDR<0.01). One of the novel genes, VAC14, was identified as a highly ranked gene in the secondary screen for erythroid differentiation. Validation experiments demonstrated that VAC14 down-regulation using one of 3 independent shRNAs result in erythroid cell proliferation defects, both in HUDEP2 cells and in erythroid cells derived from primary human HSPCs. Since germline loss of VAC14 is embryonic lethal in the mouse, to validate the role of VAC14 in erythropoiesis in vivo, we transplanted fetal livers from Vac14 null mice (or wild-type [WT] control mice) into WT recipients and examined the VAC14 deficient hematopoietic compartment. Mice transplanted with Vac14 null HSCs exhibited a profound reduction in absolute numbers of bone marrow erythroid cells with evidence of a block in erythroid maturation. Impaired bone marrow erythropoiesis was largely (but incompletely) compensated for by splenic extramedullary erythropoiesis. Vac14 null hematopoietic cells exhibited pronounced cytoplasmic vacuolation, also affecting numerous stages of erythroid cells. Altogether, we have performed an unbiased genome-scale CRISPR knock-out screen that identified (and validated) novel genes required in erythropoiesis.

Histone Lysine Methyltransferase MMSET : An Essential Regulator of Human Erythropoiesis.

Erythropoiesis is the process by which hematopoietic stem cells proliferate, differentiate, and eventually form mature erythrocytes. Steady-state erythropoiesis is the result of the coordinated efforts of transcription factors and epigenetic regulators during differentiation. Histone methyltransferases (HMTs) are critical regulators that drive activation and repression of histone marks during erythroid lineage progression. MMSET (multiple myeloma SET domain) protein is aH3K36 methyltransferase (HKMT) required for normal hematopoietic development and essential for B cell differentiation and mature B cell function. Knockout of MMSET induces Wolf-Hirschhorn syndrome, where lymphocytes show abnormal functions, including a deficiency in antibody production. Studies have highlighted that MMSET acts as an oncogenic driver, classically associated with solid tumours, with a special focus on Multiple myeloma. However, the biological role and function of MMSET in human erythropoiesis is untouched and needs to be explored to address its role in the erythroid cascade. To this end, we examined the role of MMSET in human erythropoiesis by studying the genotypic and phenotypic modulatory effects observed upon MMSET knockdown (KD) in erythroid cells. Erythroid cells were generated from human G-CSF mobilized peripheral blood, hematopoietic stem and progenitor cells (HSPCs) as described by (Palii et al.,2011). Cultured cells were transduced with a lentiviral vector (pLKO.1) expressing MMSET andKD was observed after 72 hours of induction. 70-80% KD of MMSET in HSPCs resulted in reduced BFU-Es and CFU-Es in the semi-solid assay (Methocult TM) as compared to the control (pLKO.1 carrying scrambled sequence) (Fig. A), which was co-validated by FACS on days 2 and 4 (Phase I). Taken together, these results indicate that MMSET is required for early erythroid differentiation. Further, to access the role of MMSET during terminal stages of erythroid differentiation, lentiviral-mediated KD of late erythroblasts (days 8-12) (phase II) was done. MMSET KD erythroblasts displayed a block in terminally differentiating (Polychromatic and Orthochromatic) erythroblasts with an increase in Proerythroblasts and Basophilic erythroblasts. Thus, our results indicate that MMSET KD at later stages is associated with disordered terminal erythropoiesis. H3K36 methylation marks are involved in the regulation of DNA replication, recombination, and repair. Specifically, these marks keep DNA replication in check; hence, we noted that loss of MMSET correlates with increased transition of G2M phase cells from S phase cells with respect to control. In lieu of the above context, we noted increased proliferation (via Click-It Edu) in both phases (I and II). Studies have shown that MMSET is associated with DNA damage and repair (Evans et al., 2016). DNA damage and fragmentation mitigates apoptosis as a result of the signalling cascade downstream of effector caspases. We, therefore, observed a significant increase in apoptotic cells (via Annexin/PI staining) in both early and late erythroblasts upon KD of MMSET. To further gain insight, we carried out whole transcriptome analysis upon KD of MMSET and observed differentially expressed erythroid genes at three time points (days 4, 8, and 12) which revealed that GATA1, ALAS2, TAL1, KLF1, and FECH were significantly altered at the specified days (Fig. B). We further observed that erythrocyte development and differentiation, followed by regulation of the G2M transition of the cell cycle, apoptosis (programmed cell death), and cell proliferation, were the most enriched processes. Thus, in the present study, we systematically characterised erythroid impairment developing upon MMSET KD, leading to global downregulation of erythroid-specific genes, which are key players in normal erythroid development. An in-depth assessment of the MMSET -mediated erythroid gene regulatory network needs elucidation for further validation of MMSET as a direct player or mediator in human erythroid development. Our findings uncovered a previously unknown role for MMSET in human erythropoiesis.

Cell-Type Specific Requirement of Glucose Catabolism in Erythropoiesis and Thymopoiesis

Glucose catabolism through glycolysis is central to cellular life. In glycolysis, pyruvate is converted to lactate by lactate dehydrogenase (LDH) or oxidized into the tricarboxylic acid (TCA) cycle via acetyl-CoA by pyruvate dehydrogenase (PDH). However, the two enzymes LDH and PDH required for completing glucose catabolism have never been ablated simultaneously in any tissue in vivo to test if glycolysis is required for cell survival and function. Ldha and Ldhb, the genes coding for LDH have not been ablated simultaneously in in the erythroid lineage to test if LDH plays a significant role in erythropoiesis. It is also unknown if erythoid progenitor cells, which unlike mature erythrocytes contain mitochondria, rely on LDH or are able to oxidize glucose via PDH to support erythroid development. To address these questions, we have generated conditional genetic knockouts (KO) of Ldha, Ldhb and PDH ( Pdha1) in the hematopoietic system to study the requirement of glucose catabolism in erythropoiesis. We observed that Ldha Δ/Δ and Ldha Δ/Δ;Ldhb Δ/Δ but not Ldhb Δ/Δmice had anemia, consistent with an essential role for LDHA in erythrocytes or erythropoiesis. Anemia was more severe in Ldha Δ/Δ;Pdha1 Δ mice as compared to Ldha Δ/Δ mice suggesting LDH and PDH are required in concert to maintain erythroid development in vivo. Ldha Δ/Δ mice accumulated CD71 +Ter119 + erythroid progenitors but Ldha Δ/Δ;Pdha1 Δ knockout mice had a significant decrease in this population. Moreover, there was a more pronounced expansion of upstream CD71 -Ter119 int and CD71 +Ter119 int cells in Ldha Δ/Δ;Pdha1 Δmice. This suggest that the accumulation of CD71 +Ter119 + cells in Ldha Δ/Δ mice is dependent on PDH and that the presence of either LDHA or PDH is required for the progression from CD71 +Ter119 int to CD71 +Ter119 + erythroid progenitors. The bone marrow of Ldha Δ/Δ;Pdha1 Δ showed an accumulation of pre-CFU-E and a depletion of CFU-E cells as compared to single Ldha Δ/Δ mice. Therefore, the presence of either LDHA or PDH is required to transit from pre-CFU-E to CFU-E stage during erythroid differentiation. Our previous results show that PDH is required for the double positive (DP) stage of T cell development. Analysis of thymus of Ldha Δ/Δ and Ldha Δ/Δ;Ldhb Δ/Δ mice did not show any changes in thymocytes. However, Ldha Δ/Δ;Pdha1 Δmice showed a significant decrease at the double negative (DN1) stage which persisted through all T cell development stages. Given that, PDH is required at the double positive (DP) stage and LDH deficiency alone does not affect T cell development, this suggest that LDH and PDH act redundantly at different stages to support T cell development. Altogether, these results suggest cell-type specific usage of glucose catabolic pathways in different differentiation stages. Our results also suggest that progenitor cells can switch between LDH and PDH for glucose catabolism while mature immune cells rely on either LDH or PDH for survival. To investigate this, we will use metabolomics and isotope tracing methods to analyze the route of glucose catabolism during erythroid and T cell development.

The Pyruvate Kinase (PK) Activator AG-946 Improves PK Properties and Red Blood Cell (RBC) Characteristics upon Ex Vivo treatment of RBCs from Patients with Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia, directly affecting quality of life. The limited therapeutic options often do not result in the desired clinical improvement. Interestingly, in MDS the activity of the red blood cell (RBC) enzyme pyruvate kinase (PK), a key regulatory enzyme of glycolysis, may be decreased. The activation of PK via small molecules is hypothesized to be beneficial in a wide range of anemias. Currently, a phase 2a, open-label, proof of concept trial is running on the use of the PK activator AG-946 in low-risk MDS patients (NCT05490446). In light of these developments, we investigated the effect of ex vivo treatment of MDS RBCs with AG-946. Objectives: To evaluate RBC PK and cellular properties of patients with MDS, and to determine the effect of ex vivo treatment with the PK activator AG-946. Methods: Eleven low-risk non-transfusion dependent MDS patients and six healthy controls (HCs) were studied. Baseline PK activity and PK thermostability were measured in RBC lysates of purified RBCs, under V max conditions (phosphoenolpyruvate (PEP) 5mM final concentration). Hexokinase (HK) activity was included to relate PK activity to mean RBC age. Ex vivo treatment was performed by incubating MDS RBCs for 16 hours at 37 °C in presence or absence of the PK activator AG-946 (10μM or 5μM) using dimethylsulfoxide (DMSO) as a blank. Effect was assessed by measuring PK activity (using a final concentration of PEP 0.5mM) and levels of adenosine triphosphate (ATP) (LC-MS/MS). Functional RBC analysis was performed by osmotic gradient ektacytometry (Lorrca MaxSis). To investigate the effect on PK thermostability, RBC lysates were incubated with AG-946 (2μM or 1μM) or DMSO, after which residual PK activity was measured (PEP 0.5mM). The effect of AG-946 on erythroid development was studied using peripheral blood mononuclear cells, cultured in MethoCult TM H4434 medium for 14 days in the presence or absence of AG-946 (10μM or 625nM, DMSO as blank). Differences between conditions were determined using Unpaired T-test, ANOVA with Dunnett's test or Kruskal-Wallis with Dunn's test via GraphPad Prism. Results: Mean PK/HK ratio was significantly lower in RBCs from MDS patients compared to HCs (6.1 (SD 1.8) versus 10.5 (SD 1.5), p<0.001). Upon ex vivo treatment, PK activity significantly increased (independent of dosage) when compared to DMSO (10μM AG-946, mean increase 30% ( p<0.001); 5μM, 28% ( p<0.0001)) (Figure 1A). MDS RBCs displayed decreased PK thermostability at baseline under V max conditions (HCs 79% residual activity versus 68% in MDS, p<0.01). This was restored by ex vivo treatment with AG-946 (DMSO, 33% residual activity, 2μM AG-946, 85% ( p<0.0001); 1μM, 87% ( p<0.0001)). The increase in PK activity was accompanied by significantly increased ATP levels (N=10, DMSO, mean ATP 14.9 μg/mL RBC; 10μM AG-946, 21.0 μg/mL RBC ( p<0.01); 5μM, 21.1 μg/mL RBC ( p<0.01)) (Figure 1B). Functionally, RBC properties improved upon ex vivo treatment with AG-946 as reflected by the modest yet significant increase in O hyper, indicating improved hydration status (10μM AG-946, mean increase 2.3% ( p<0.001); 5μM, 2.9% ( p<0.0001)). To date, culture assays were performed in 8 patients; 3/8 showed an increase in the number of burst forming units-erythroid (BFU-Es) upon treatment with AG-946 when compared to DMSO. The percentual increases differed per patient: increase of 25% with 10μM AG946 and 150% with 625nM, increase of 40% (10μM) and 39% (625nM), and for the third an increase of 22% (10μM) and 39% (625nM). Conclusion: Our findings show that RBCs from MDS patients have decreased PK activity and thermostability. We demonstrate that ex vivo treatment with AG-946 increases PK activity and ATP levels and restores PK thermostability. Moreover, ex vivo treatment with AG-946 improves RBC hydration, suggesting that improved energy status directly improves RBC functional properties. The preliminary results of the culture assay could indicate that in certain patients, dyserythropoiesis may be ameliorated upon PK activation. To better understand the effects on erythropoiesis additional experiments are required. In conclusion, our data might support a rationale for the use of PK activators as a novel therapeutic option for MDS.

Activating transcription factor 4 in erythroid development and -thalassemia: a powerful regulator with therapeutic potential.

Activating transcription factor 4 (ATF4) is a fundamental basic region/leucine zipper transcription factor, responds to various stress signals, and plays crucial roles in normal metabolic and stress response processes. Although its functions in human health and disease are not completely understood, compelling evidence underscores ATF4 is indispensable for multiple stages and lineages of erythroid development, including the regulation of fetal liver hematopoietic stem cells, induction of terminal erythroid differentiation, and maintenance of erythroid homeostasis. -Thalassemia is a blood disorder arising from mutations in the -globin gene. Reactivating the expression of the -globin gene in adult patients has emerged as a promising therapeutic strategy for ameliorating clinical symptoms associated with -thalassemia. Recent research has suggested that ATF4 contributes to decreased fetal hemoglobin (HbF) level through its binding to potent negative regulators of HbF, such as BCL11A and MYB. Notably, evidence also suggests a contrasting outcome where increased ATF4 protein levels are associated with enhanced HbF at the transcriptional level. Consequently, the identification of mechanisms that modulate ATF4-mediated -globin transcription and its effects on erythroid development may unveil novel targets for -thalassemia treatment.