Erythroid Differentiation Arrest Research Articles

1011 – INVESTIGATION OF HUMAN BONE MARROW PROGENITORS ELUCIDATES CELLULAR AND MOLECULAR MECHANISMS OF ERYTHROID FAILURE IN DIAMOND-BLACKFAN ANEMIA

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease. Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease.

Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing erythropoiesis in Diamond-Blackfan anemia.

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Here, we investigate how mutations in either ribosomal protein large (RPL) or ribosomal protein small (RPS) subunit genes selectively affect erythroid progenitor development and clinical phenotypes in Diamond-Blackfan anemia (DBA), a rare ribosomopathy with limited therapeutic options. Using single-cell assays of patient-derived bone marrow, we delineated two distinct cellular trajectories segregating with ribosomal protein genotypes. Almost complete loss of erythroid specification was observed in RPS-DBA. In contrast, we observed relative preservation of qualitatively abnormal erythroid progenitors and precursors in RPL-DBA. Although both DBA genotypes exhibited a proinflammatory bone marrow milieu, RPS-DBA was characterized by erythroid differentiation arrest, whereas RPL-DBA was characterized by preserved GATA1 expression and activity. Compensatory stress erythropoiesis in RPL-DBA exhibited disordered differentiation underpinned by an altered glucocorticoid molecular signature, including reduced ZFP36L2 expression, leading to milder anemia and improved corticosteroid response. This integrative analysis approach identified distinct pathways of erythroid failure and defined genotype-phenotype correlations in DBA. These findings may help facilitate therapeutic target discovery.

Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics.

Erythropoiesis is the most robust cellular differentiation and proliferation system, with a production of ∼2 × 1011 cells per day. In this fine-tuned process, the hematopoietic stem cells (HSCs) generate erythroid progenitors, which proliferate and mature into erythrocytes. During erythropoiesis, mitochondria are reprogrammed to drive the differentiation process before finally being eliminated by mitophagy. In erythropoiesis, mitochondrial dynamics (MtDy) are expected to be a key regulatory point that has not been described previously. We described that a specific MtDy pattern occurs in human erythropoiesis from EPO-induced human CD34+ cells, characterized predominantly by mitochondrial fusion at early stages followed by fission at late stages. The fusion protein MFN1 and the fission protein FIS1 are shown to play a key role in the progression of erythropoiesis. Fragmentation of the mitochondrial web by the overexpression of FIS1 (gain of fission) resulted in both the inhibition of hemoglobin biosynthesis and the arrest of erythroid differentiation, keeping cells in immature differentiation stages. These cells showed specific mitochondrial features as compared with control cells, such as an increase in round and large mitochondrial morphology, low mitochondrial membrane potential, a drop in the expression of the respiratory complexes II and IV and increased ROS. Interestingly, treatment with the mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporin A, rescued mitochondrial morphology, hemoglobin biosynthesis and erythropoiesis. Studies presented in this work reveal MtDy as a hot spot in the control of erythroid differentiation, which might signal downstream for metabolic reprogramming through regulation of the mPTP.

The effect of miR-223 on cellular behaviour in non-5q myelodysplastic syndromes through targeting RPS14

Myelodysplastic syndromes (MDS) are characterised by impaired haematopoiesis and a high risk of leukaemic transformation. A decrease in RPS14 expression in non-5q MDS patients was confirmed by immunohistochemical analyses of MDS bone marrow biopsies. To determine the cause of RPS14 reduction in non-5q MDS, we analysed the 3'-UTR of RPS14 and demonstrated that miR-223 binds to the 3'-UTR of RPS14 by bioinformatics-based approach combined with the luciferase reporter assay. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we observed a significantly increased expression of miR-223 in CD34+ cells and SKM-1 cells derived from non-5q MDS patients invitro and demonstrated a correlation between miR-223 levels and red blood cell counts. Exogenous miR-223 expression in SKM-1 cells could also inhibit RPS14 expression. In functional studies, overexpression of miR-223 was shown to promote cell proliferation and inhibit cell apoptosis in SKM-1 cells, and to impair erythroid differentiation in haemin-induced K562 cells. Taken together, our results revealed that the overexpression of miR-223 in MDS is closely associated with cell transformation and erythroid differentiation arrest, which is most likely mediated by targeting RPS14.

Mi2β-Mediated Silencing of the Fetal γ-Globin Gene in Adult Erythroid Cells

Abstract 825Epigenetics has emerged as a key regulator of the fetal to adult hemoglobin switch during development. Understanding the mechanisms of fetal γ-globin gene silencing offers the promise of effective targeted therapy of β- thalassemia and sickle cell anemia. Previous studies carried out by our group using adult erythroid cells from mice transgenic for a yeast artificial chromosome containing the entire human β-globin locus (β-YAC) and primary human erythroid cells have shown that methyl-CpG binding protein 2 (MBD2) is critical for full silencing of the fetal γ-globin gene. MBD2 binds to methylated CpG rich promoters and silences the associated target genes by recruiting the Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. Absent or ≥75% decreased expression of MBD2 has been shown to have a 10–50 fold stimulatory effect on the expression of the γ-globin gene in adult β-YAC transgenic mice and a 5–6 fold effect in CD34+ progenitor derived human erythroid cells in culture. Mi2, a major component of the NuRD complex, is an ATP-dependent chromatin remodeler consisting of two isoforms, Mi2a (also known as CHD3) and Mi2β (also known as CHD4). We have observed that 80% knockdown of Mi2β leads to a significantly higher expression of the γ-globin gene in βYAC containing murine adult hematopoietic CID cells (∼100 fold) when compared to equivalent knockdown of other components of the MBD2/NuRD complex (∼5-10 fold), including MBD2. Remarkably, in CD34+ progenitor derived adult human erythroid cells, as little as 40% knockdown of Mi2β resulted in a ∼30 fold increase in γ/γ + β mRNA levels. Moreover, simultaneous knockdown of MBD2 and Mi2β resulted in no greater γ-globin gene expression than Mi2β knockdown alone. This suggests that Mi2β is acting independently of as well as through its role in the MBD2-NuRD complex to exert its silencing effect on the γ-globin gene. Complete conditional knockout of Mi2β in transgenic mouse hematopoietic cells has been shown by others to result in arrest of erythroid differentiation. We observed that in human CD34+ progenitor cells ≥80% Mi2β knockdown also altered differentiation. In contrast, 40–50% knockdown of Mi2β does not affect erythroid differentiation compared to scramble shRNA controls and resulted in 25 – 40% γ/γ + β globin RNA expression versus 2–3% in scramble shRNA controls. To determine possible mechanisms of Mi2β-mediated silencing outside of its role in the MBD2-NuRD complex, we examined the effect of Mi2β knockdown on two key mediators of γ-globin gene silencing, Bcl11A and KLF-1 (EKLF). Our results showed that RNA and protein levels of these proteins are diminished by 40% and 70% respectively in cells with 40–50% Mi2β knockdown. Knockdown of MBD2 caused no decrease in either Bcl11A or KLF-1 levels. Thus at least part of the effect of Mi2β on γ-globin gene silencing is through its positive effects on KLF-1 and Bcl11A expression. In summary, we have shown that less than a 50% decrease in Mi2β expression in primary human adult erythroid cells resulted in high levels of γ/γ + β gene expression without altering erythroid differentiation. The silencing effects of Mi2β occur both through the MBD2-NuRD repressor complex and through positive regulation of the KLF-1 and Bcl11A genes. These results also suggest that partial inhibition of Mi2β function could be of therapeutic benefit in the treatment of β- thalassemia and sickle cell anemia. Disclosures:No relevant conflicts of interest to declare.