AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed,
α
,
and
mice develop ...hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin A
), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin A
), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.
Myelodysplastic syndromes (MDS) are heterogeneous diseases of the hematopoietic stem cell in the elderly. Anemia is the main symptom that mostly correlates with dysplastic erythropoiesis in the bone ...marrow. We will review the recent advances in understanding the diverse mechanisms of dyserythropoiesis.
Dyserythropoiesis defined as 10% dysplastic erythroid cells in the bone marrow is found in more than 80% of early MDS. Immature erythroblasts accumulate at the expense of mature erythroblasts due to differentiation arrest and apoptosis. In early MDS with dyserythropoiesis, caspase-dependent cleavage of the erythroid transcription factor GATA-1 occurring in basophilic erythroblasts accounts for impairment of final maturation. Depending on initiating genetic alteration, specific mechanisms contribute to erythroid defect. In MDS with 5q deletion, the haploinsufficiency of ribosomal protein gene, RPS14, opposes the transition of immature to mature erythroblasts by inducing a p53-dependent ribosome stress, cell cycle arrest and apoptosis. Recent work identifies the activation of a p53-S100A8/9 innate immune pathway that both intrinsically and extrinsically contributes to defective erythropoiesis. In MDS with ring sideroblasts, a paradigm of dyserythropoiesis, a unique mutation in SF3B1 splicing factor gene induces a multiplicity of alterations at RNA level that deeply modifies the patterns of gene expression.
Insights in the pathophysiology of MDS with dyserythropoiesis may guide the choice of the appropriate therapy, for instance lenalidomide in MDS with del(5q). A better understanding of the mechanisms of dyserthropoiesis is required to treat anemia in non-del(5q) MDS, especially in case of resistance to first-line therapy by erythropoiesis-stimulating agents.
Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the
splicing factor gene. Patients with MDS with
mutations often ...accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative
transcript in patients with MDS with the
mutation. Induction of this
transcript in primary
-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an
gene mutation than in patients with
wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with
-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant
transcript that was restricted to
-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.
Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases ...of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.
Introduction
SF3B1 hotspot mutations are associated with various cancers like uveal melanoma, chronic lymphocytic leukemia and myelodysplastic syndrome with ring sideroblasts (MDS-RS). These ...mutations affect RNA splicing by the use of alternative branchpoints resulting in an aberrant 3' splice site (ss) selection. RNA-sequencing (RNA-seq) analyzed to quantify exon-exon junctions identified aberrantly spliced transcripts in target genes, and half of them are predicted to be degraded by non-sense mediated decay. For this reason, target genes in SF3B1-mutated MDS remain partially characterized. In the present study, we performed deep RNA-seq analysis of bone marrow mononuclear cells in low/int-1 MDS with SF3B1 mutations to identify aberrant/cryptic splicing events among up or down-regulated gene sets.
Methods
SF3B1MUT MDS (n=21) were compared to 6 SF3B1WT cases and 5 controls. Analysis of RNA-seq read count was performed using the Voom method associated with the Limma empirical Bayes analysis pipeline (https://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r29). Up or downregulated gene sets were identified using Gene Set Enrichment Analysis (GSEA, false discovery rate<0.1). Gene expression profiling data (Affymetrix Hu2.0) were also available for 26/27 patient samples. TopHat (v2.0.6) was used to align the reads against the human reference genome Hg19 RefSeq (RNA sequences, GRCh37) downloaded from the UCSC Genome Browser (http://genome.ucsc.edu). Read counts for splicing junctions from junctions.bed TopHat output were considered for a differential analysis using DESeq2. Only alternative acceptor splice sites (two or more 3′ss with junctions to the same 5′ss) and alternative donor splice sites (two or more 5′ss with junctions to the same 3′ss) with P-values ≤10−5 (Benjamini-Hochberg) and absolute Log2 (fold change) ≥1 were considered.
Results
Principal component analysis (PCA) nicely discriminated controls from patients, and patients according to the presence of a SF3B1 mutation. A set of 6971 genes was differently expressed (P- value<0.05) between SF3B1MUT and SF3B1WT cases and allows unsupervised clustering in two separated groups (Fig. 1). Distinct gene sets also discriminated SF3B1MUT or SF3B1WT from controls.
Consistent with increased amount of erythroblasts in MDS-RS bone marrows, a set of erythroid genes including several genes involved in hemebiosynthesis pathway (ALAD, UROS, ALAS2, UROD) was significantly enriched in SF3B1MUT samples. Genes selected for their involvement in the core iron-sulfur cluster mitochondrial machinery (FXN, BOLA3, FDXR, GLRX5, ISCA2, NFS1, ISCU), the iron binding and trafficking (SLC25A38, ABCB10, TFR2, SLC25A37, ABCB6, FAM132B, SLC25A39, FTH1) and the cellular iron homeostasis (ACO1, ACO2, GLRX3) were also significantly enriched (FDR<10% and nominal P-value<0.05) when input in GSEA. Moreover, other enriched gene sets were G2M checkpoint, MYC targets, oxidative phosphorylation and E2F targets. All of these observations were similarly obtained when analyzing Affymetrix data. Furthermore SF3B1MUT samples with a K700E substitution harbored a specific pattern of deregulated genes, which allowed the ordering of SF3B1MUT samples according to the type of substitution.
As previously reported by AlsafadiS et al (2016), analysis of splice junctions using DESeq2 revealed an overall high level of differences between SF3B1MUT and SF3B1WTsamples. Among more than 540 differentially spliced junctions, more than 80% involved an aberrant acceptor (3'ss) site. As determined by PCA, the top 50 genes associated with relevant aberrant junctions were linked to iron metabolism or erythropoiesis and differentially expressed between SF3B1MUT and SF3B1WTsamples.
Conclusion
In this study, we combined robust analyses of gene expression and aberrantly spliced transcript expression in MDS with SF3B1 mutation. By comparing SF3B1MUTversus SF3B1WT samples, we identified a set of deregulated genes in which both normally and aberrantly spliced transcripts were detected that could contribute to the physiopathology of MDS-RS.
Display omitted
No relevant conflicts of interest to declare.
Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders in which recurrent mutations define clonal hematopoiesis. The origin of the phenotypic diversity of non-del(5q) MDS remains ...unclear. Here, we investigated the clonal architecture of the CD34+CD38− hematopoietic stem/progenitor cell (HSPC) compartment and interrogated dominant clones for MDS-initiating cells. We found that clones mainly accumulate mutations in a linear succession with retention of a dominant subclone. The clone detected in the long-term culture-initiating cell compartment that reconstitutes short-term human hematopoiesis in xenotransplantation models is usually the dominant clone, which gives rise to the myeloid and to a lesser extent to the lymphoid lineage. The pattern of mutations may differ between common myeloid progenitors (CMPs), granulomonocytic progenitors (GMPs), and megakaryocytic-erythroid progenitors (MEPs). Rare STAG2 mutations can amplify at the level of GMPs, from which it may drive the transformation to acute myeloid leukemia. We report that major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knock-down (KD) in normal CD34+ progenitors modifies their granulocytic and erythroid differentiation. Clonal architecture of the HSPC compartment and mutations selected during differentiation contribute to the phenotypic heterogeneity of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation and may guide the search for novel therapeutic strategies.
•Genetic heterogeneity in non-del(5q) MDS arises within the HSPC and in committed progenitors.•Clonal selection in lineage-committed progenitors may drive the transformation to acute myeloid leukemia.
Background
Busulfan (Bu) is the cornerstone of conditioning regimens prior to hematopoietic stem cell transplantation, widely used in both adults and children for the treatment of malignant and ...nonmalignant diseases. Despite an intravenous formulation, interindividual variability (IIV) remains high and optimal exposure difficult to achieve, especially in neonates and infants.
Procedure
To ensure both efficacy and safety, we set up in 2005 an observational study designed for children not fully assessed during the drug registration procedure. From a large cohort of 540 patients, we developed a Bu population pharmacokinetic model based on body weight (BW) and maturation concepts to reduce IIV and optimize exposure. A new dosing nomogram was evaluated to better fit the population pharmacokinetic model.
Results
Bu clearance IIV was significantly decreased from 61.3% (covariate‐free model) to 28.6% when combining BW and maturation function. Median Bu area under the curve (AUC) was 1179 µmol/L × min compared to 1025 with the EMA dosing nomogram for children <9 kg. The target AUC was reached for each BW strata, significantly increasing the percentages of patients achieving reaching the targeted AUC as compared to FDA schedule.
Conclusion
This new model made it possible to propose a novel dosing nomogram that better considered children below 16 kg of BW and allowed better initial exposure as compared to existing dosing schedules. This nomogram, which would be easy to use to determine an optimal dosing schedule in daily practice, will need to be validated in clinical routine. Therapeutic drug monitoring remains strongly advisable for small children and those with specific diseases.
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