Plasma biomarkers of hemoglobin loss in Plasmodium falciparum-infected children identified by quantitative proteomics

Anemia is common among young children infected with Plasmodium falciparum (Pf) and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during Pf infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P. falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) versus those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower IGF-1 levels in children with reduced hemoglobin. We confirmed these findings in independent ELISA-based validation studies of subsets of children from the same cohort (20S proteasome, N=71; IGF-1, N=78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, while decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia.

Hemogen/BRG1 Cooperativity Is Required for Erythrocyte Maturation and Hemoglobin Production during Mammalian Erythropoiesis

Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unclear. We found that depletion of hemogen in human CD34 + erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to occupy chromatin at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization. Disclosures No relevant conflicts of interest to declare.

HEXIM1 Is Essential for the Establishment of Appropriate Patterns of Gene Expression and Chromatin Architecture during Terminal Erythroid Maturation

Terminal erythroid maturation is associated with dramatic changes in gene expression in the setting of a cell that is undergoing rapid division and nuclear condensation. Disruption of this process is associated with inherited anemias and myelodysplastic syndromes. Recent work from our laboratory revealed that terminal erythroid maturation is associated with a dramatic decline in the level of total and elongation competent RNA polymerase II (Pol II), and that control of pol II activity is a critical step in the regulation of gene expression during terminal erythroid maturation. We further demonstrated that HEXIM1, which is highly expressed in early erythroid cells compared to most other cell types (biogps.org; bloodspot.eu), is essential for erythropoiesis (Murphy Blood 2021). The goal of our current study is to understand the mechanisms by which HEXIM1 regulates erythroid gene expression. HEXIM1 can impact gene expression though multiple mechanisms, most notably by associating with pTEFb, which is required for release of "paused" pol II into active transcription (reviewed in Michels, Transcription, 2018). HEXIM1 can inhibit transcription through sequestration of pTEFb in the 7SK ribonuclear complex, rendering it incapable of facilitating pause release. Alternatively, it can activate transcription by delivering pTEFb to target loci (McNamara Genome Data 2016). In erythroid cells, disruption of HEXIM1 impaired the expression of many erythroid specific genes, such as GYPA and many of the heme synthesis enzymes, while overexpression (OE) of HEXIM1 promoted their expression (Murphy, Blood, 2021). We therefore hypothesized that in maturing erythroblasts, HEXIM1 targets pTEFb to erythroid specific genes, promoting the establishment of appropriate patterns of gene expression and facilitating terminal erythroid maturation. To address this hypothesis, we generated novel HUDEP2 lines that OE HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb (Mbonye Proteomics 2015). Biotinylated 7SK pulldown confirmed that the Y271A mutation maintains the ability to bind the 7SK complex in erythroid cell extracts and RNA immunoprecipitation confirmed that the Y271A mutation increases the affinity of HEXIM1 for the 7SK complex in HUDEP2 cells. The Y271A mutation has significant functional consequences in erythroid cells. OE of wild type (WT) HEXIM1 in HUDEP2 cells resulted in enhanced proliferation in both expansion and maturation conditions, which was accompanied by increased cell and nuclear size, and a dramatic increase in the level of CD235a. Similar to our previously published HEXIM1 mutant with tyrosine to phenylalanine mutations at residues 271 and 274, the Y271A HEXIM1 mutation abrogated the enhanced proliferation seen with HEXIM1 OE in both expansion and maturation conditions. The Y271A mutation also rescued the larger cell and nuclear area associated with HEXIM1 OE, as well as the dramatic increase in the level of CD235a. Conversely, disruption of HEXIM1 via genome editing resulted in poor expansion and viability of HUDEP2 cells, which was rescued by expression of WT but not Y271A mutated HEXIM1, highlighting the importance of HEXIM1-pTEFb interactions for erythroid proliferation and survival. Further, OE of WT HEXIM1, but not the Y271A mutant, promoted erythroid gene expression while facilitating repression of genes that are normally silenced during terminal maturation, such as RPS19. In cells expressing WT HEXIM1 these gene expression changes were accompanied by increases in the global levels of ser2 and ser5 phosphorylated Pol II, as well as genome wide changes in their distribution. In contrast, the Y271A mutant decreased the global level of ser2 and ser5 pol II, consistent with its reduced ability to release pTEFb at target genes. Intriguingly, levels of H3K79me2, a histone mark reflective of active transcription through gene bodies, were decreased with OE of both WT and Y271A mutant HEXIM1, suggesting that the ability of HEXIM1 to promote transcriptional activation or repression is context dependent. Together, these data demonstrate a critical role for HEXIM1 and its interaction with pTEFb and the 7SK complex in the establishment of appropriate patterns of gene expression and chromatin architecture in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

Inhibition of S-Adenosylmethionine Synthesis Promotes Erythropoiesis Via Epigenetic Modifications

Erythroid differentiation involves global gene expression repression, chromatin condensation and enucleation, mitochondria removal and other marked cellular changes. Given the necessity for these dynamic alterations, it is hardly surprising that epigenetic modifications possess important roles for erythropoiesis. S-adenosylmethionine (SAM), a principle methyl donor for DNA and histone methylations, would be involved in this process. Yet little is known about the specific roles for SAM synthesis in erythropoiesis. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a and we evaluated the in vivo role of SAM synthesis by treating wild type mice (C57BL/6) with a selective Mat2a inhibitor (cycloleucine). As expected, the Mat2a inhibitor administration (henceforth Mat2ai) reduced SAM in bone marrow (BM) cells (SAM; 3.17±0.43 and 0.93±0.10 area ratio for ctrl and Mat2ai, p < 0.01, n = 4 mice). Interestingly, Mat2ai increased erythropoiesis in BM (Ter119 + cell; 46.3±3.1 and 116.4±14.2×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice) and in blood (hemoglobin concentrations in peripheral blood; 13.7±0.18 and 16.3±0.26 g/dl for ctrl and Mat2ai, p < 0.01, n = 8 mice). However, serum erythropoietin concentration decreased (erythropoietin; 254.2±34.1 and 42.7±5.70 pg/ml for ctrl and Mat2ai, p < 0.01, n = 10 mice). Therefore, Mat2ai promoted erythropoiesis in vivo without increasing erythropoietin. To reveal the point where the erythroid differentiation was affected, immature and mature erythroblast subsets in BM were assessed. Although immature erythroblasts were not changed by Mat2ai (24.1±2.80 and 23.8±3.86×10 6 cells for ctrl and Mat2ai, p = 0.95, n = 8 mice), mature erythroblasts in BM increased following Mat2ai (18.9±2.48 and 81.2±9.73×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice). Therefore, Mat2ai promoted erythroid maturation from immature erythroblast in BM. To reveal the mechanistic insight of this promotion of erythroid maturation by Mat2ai, we performed RNA sequencing of immature erythroblast in BM. This analysis revealed that most genes were down-regulated by Mat2ai (differentially expressed genes by Mat2ai; DOWN 2578 genes, UP 72 genes). In line with this notion, transposase-accessible chromatin sequencing (ATAC-seq) of immature erythroblasts revealed that chromatin accessibility was reduced. While DNA methylation analysis (whole genome bisulfite sequence) of immature erythroblasts revealed slightly reduced global DNA methylation (approximately 2%), there were no clear correlations between changes in promotor (or gene-body) DNA methylation and transcription. This result suggests that DNA methylation changes possess limited roles for the erythroid maturation promoted by Mat2ai. On the other hand, we found that an active histone methylation mark (H3K4me3) was selectively reduced by Mat2ai and that the changes of gene expression and H3K4me3 enrichment (revealed by chromatin immunoprecipitation followed by sequencing) correlated (r = 0.66). Therefore, the loss of H3K4me3, but not the DNA methylation, might contribute to the global gene expression repression for erythroid maturation induced by Mat2ai. Finally, in vitro human erythroid differentiation analysis using CD34 + cord blood cells further revealed that therapeutic and genetic inhibition of SAM synthesis induced erythroid maturation, which was cancelled by extracellular administration of SAM. Therefore, SAM synthesis inhibition is a non-erythropoietin trigger for erythroid maturation and this process occurs in human cells. Collectively, we found that SAM synthesis inhibition promoted erythroid maturation in both mouse and human. Histone methylation alteration induced by SAM synthesis inhibition might contribute to this phenomenon. These findings may pave the way to develop a new therapeutic strategy for anemia in erythropoietin independent manner. Disclosures Harigae: Kyowakirin: Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Ono pharma: Honoraria, Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Combination of a Luspatercept-like Drug (RAP-GRL) and Tmprss6-ASO Is Superior to Either Drug Alone for Correcting β-Thalassemia

The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). Recent studies have pointed to iron restriction (IR) to improve both anemia and IO in BT (Rivella, Blood). The decreased iron-uptake by early erythroid cells reduces hemichrome toxicity and prevents premature RBC hemolysis. One such IR therapy targets the matriptase-2 (Tmprss6) gene using antisense oligonucleotides (T-ASO). Our group has previously shown that treatment of Hbb th3/+(th3/+) mice (a mouse model for BT-intermedia) with T-ASO improved anemia, lengthened red blood cell (RBC) lifespan, reduced levels of erythroferrone (ERFE), hemichromes and reactive oxygen species, and ameliorated splenomegaly (Casu et al. Blood). Another novel therapeutic approach to improve anemia targets the Transforming Growth Factor (TGF)-β pathway to increase erythroid maturation. Luspatercept, a TGF-β trap-ligand, gained FDA approval in 2019 to treat transfusion dependent BT patients (Cappellini and Taher, Blood Adv). In mouse models of BT, its murine analog (RAP-536) was found to promote EPO-independent maturation of late-stage erythroid cells, and resulted in increased RBC parameters in a dose-dependent manner (Surgani, et al. Nat Med). In this work we treated th3/+ mice with an agent analogous to murine Luspatercept (RAP-GRL) in combination with the iron restriction (IR) drug T-ASO, (RAP-GRL+T-ASO) with the goal of targeting distinct morbidities associated with BT. To test our RAP-GRL construct, primary fibroblasts were transduced with an adenovirus containing the RAP-GRL sequence (FB Ad5RAP-GRL) and used to deliver RAP-GRL to mice. As a second strategy, RAP-GRL was expressed in a mammalian cell line and purified. Wild-type (WT) or th3/+ mice were subcutaneously (s.c.) implanted with 1x10 6 FB Ad5RAP-GRL or injected s.c. with 10mg/kg of RAP-GRL and monitored by complete blood counts. Implantation of FB Ad5RAP-GRL ortreatment with purified RAP-GRL increased RBC parameters in both WT and th3/+ mice (n=3-9, 2-4-month-old females and males). In the first combination therapy experiment we implanted FB Ad5RAP-GRL s.c. and delivered T-ASO via intraperitoneal (i.p.) injection in th3/+ mice. RBC parameters were increased in all treatment groups except controls after 6 weeks. The RAP-GRL+ T-ASO group displayed the most pronounced increase in RBC parameters with a mean increase in RBC of 3.067±0.73 10 6 cells/µL, Hb of 3.02±0.77 g/dL, and Hct of 5.88±2.36 % (Table 1). Additionally, we also treated th3/+ mice with two different doses of protein purified RAP-GRL in combination with T-ASO (Table 1). The best results using the protein purified RAP-GRL were achieved in the RAP-GRL+T-ASO group that was treated with two weekly 10mg/kg s.c. injections of RAP-GRL and two weekly 5mg/kg i.p. injections of T-ASO (Group 2) for 6 weeks. Flow cytometry analysis using CD71, TER119, and CD44 antibodies showed improvements in the bone marrow (BM) and spleen (SPL) of all treatment groups compared to controls. Additionally, ROS levels and splenomegaly were also greatly reduced in all T-ASO and RAP-GRL+T-ASO treated groups compared to controls. Serum assessment of T-ASO and RAP-GRL+T-ASO treated animals showed decreased levels of iron and transferrin saturations with a simultaneous increase in hepcidin levels. ERFE levels were decreased in all T-ASO and RAP-GRL+T-ASO groups, however, erythropoietin (EPO) levels were increased only in the RAP-GRL and RAP-GRL+T-ASO cohorts of Group 2. Additionally, although EPO was elevated in all RAP-GRL treated animals of Group 2, only the RAP-GRL+T-ASO group had reduced ERFE. This result is in agreements with our findings of decreased early (ERFE-producing) erythroid progenitors in the BM and SPL of RAP-GRL+T-ASO treated mice. This finding also suggests that higher doses of RAP-GRL may result in elevated EPO. Luspatercept, through heightened iron consumption, may increases EPO synthesis in the kidney via activation of the transcription factor HIF2-α, which can be stabilized not only by hypoxia, but also by iron deficiency. In conclusion our results provide pre-clinical support for combining IR and TFG-β trap-ligands in the treatment of BT. Our data shows that IR, in conjunction with the enhancing erythroid maturation action of Luspatercept (and potential activation of EPO), may offer an additive and more effective therapeutic strategy for BT patients. Figure 1 Figure 1. Disclosures Guo: Ionis Pharmaceuticals, Inc.: Current Employment. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Tpr Deficiency Disrupts Erythroid Maturation With Impaired Chromatin Condensation in Zebrafish Embryogenesis

Vertebrate erythropoiesis involves nuclear and chromatin condensation at the early stages of terminal differentiation, which is a unique process to distinguish mature erythrocytes from erythroblasts. However, the underlying mechanisms of chromatin condensation during erythrocyte maturation remain elusive. Here, we reported a novel zebrafish mutantcas7 with erythroid maturation deficiency. Positional cloning showed that a single base mutation in tprb gene, which encodes nucleoporin translocated promoter region (Tpr), is responsible for the disrupted erythroid maturation and upregulation of erythroid genes, including ae1-globin and be1-globin. Further investigation revealed that deficient erythropoiesis in tprbcas7 mutant was independent on HIF signaling pathway. The proportion of euchromatin was significantly increased, whereas the percentage of heterochromatin was markedly decreased in tprbcas7 mutant. In addition, TPR knockdown in human K562 cells also disrupted erythroid differentiation and dramatically elevated the expression of globin genes, which suggests that the functions of TPR in erythropoiesis are highly conserved in vertebrates. Taken together, this study revealed that Tpr played vital roles in chromatin condensation and gene regulation during erythroid maturation in vertebrates.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of β0-Thalassemia/Hemoglobin E Patients

<b><i>Introduction:</i></b> β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. <b><i>Methods:</i></b> To elucidate the correlation between ineffective erythropoiesis and β⁰-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. <b><i>Results:</i></b> The β⁰-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β⁰-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. <b><i>Discussion/Conclusion:</i></b> The degree of erythroid expansion and maturation arrest contributes to the severity of β⁰-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

A new murine Rpl5(uL18) mutation provides a unique model of variably penetrant Diamond Blackfan Anemia

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins (RPs) are found in the majority of individuals with Diamond Blackfan anemia (DBA) while somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human DBA patients. Here we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations and increased mortality. Following genetic mapping and whole exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ mutant animals had a profound delay in erythroid maturation and increased mortality at embryonic day E12.5, which improved by E14.5. Surviving mutant animals had a macrocytic anemia at birth as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus mutant mouse will be useful to study the factors influencing the variable penetrance that is observed in DBA.