Role of Caspase-10-P13tBID axis in erythropoiesis regulation.

Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E/pro-erythroblast differentiation, depending on the balance between erythropoietin (EPO) levels and activation of the Fas/FasL pathway. At this stage, activation of transient caspases through depolarization via mitochondrial outer membrane permeabilization (MOMP) is also required for terminal erythroid differentiation. Molecular mechanisms regulating the differential levels of MOMP during differentiation and apoptosis, however, remain poorly understood. Here we show a novel and essential role for the caspase-10-P13-tBID axis in erythroid terminal differentiation. Caspase-10 (but not caspase-8, which is activated during apoptosis) is activated at the early stages of erythroid terminal differentiation leading to the cleavage of P22-BID into P18-tBID, and later into P13-tBID. Erythropoietin (EPO) by inducing casein kinase I alpha (CKIα) expression, which in turn phosphorylates P18-tBID, prevents the generation of MYR-P15-tBID (leading to apoptosis) and allows the generation of P13-tBID by caspase-10. Unlike P15-tBID, P13-tBID is not myristoylated and as such, does not irreversibly anchor the mitochondrial membrane resulting in a transient MOMP. Likewise, transduction of a P13-tBID fragment induces rapid and strong erythroid terminal differentiation. Thus, EPO modulates the pattern of BID cleavage to control the level of MOMP and determines the fate of erythroblasts between apoptosis and differentiation. This pathway is impaired in 5q- myelodysplastic syndromes because of CK1α haplo-insufficiency and may contribute to erythroid differentiation arrest and high sensitivity of this disease to lenalidomide (LEN).

BLNK mutation associated with T-cell LGL leukemia and autoimmune diseases: Case report in hematology.

We present the case of a female patient with a heterozygous somatic BLNK mutation, a T-cell LGL (large granular lymphocyte) leukemia, and multiple autoimmune diseases. Although this mutation seems uncommon especially in this kind of clinical observation, it could represent a new mechanism for autoimmune diseases associated with LGL leukemia. The patient developed several autoimmune diseases: pure red blood cell apalsia, thyroiditis, oophoritis, and alopecia areata. She also presented a T-cell LGL leukemia which required treatment with corticosteroids and cyclophosphamide, with good efficacy. Interestingly, she had no notable infectious history. The erythroblastopenia also resolved, the alopecia evolves by flare-ups, and the patient is still under hormonal supplementation for thyroiditis and oophoritis. We wanted to try to understand the unusual clinical picture presented by this patient. We therefore performed whole-genome sequencing, identifying a heterozygous somatic BLNK mutation. Her total gamma globulin level was slightly decreased. Regarding the lymphocyte subpopulations, she presented a B-cell deficiency with increased autoreactive B-cells and a CD4+ and Treg deficiency. This B-cell deficiency persisted after complete remission of erythroblastopenia and LGL leukemia. We propose that the persistent B-cell deficiency linked to the BLNK mutation can explain her clinical phenotype.

Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1.

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8+ T cells activity in the context of the antitumor immune response. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1+CD8+ T cells, cooperates and enhances PD-1 activity. In mice, CD8+ T cells specific deletion of Nrp1 improves anti-PD1 antibody antitumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8+ T cells predicts poor outcome of patients treated with anti-PD1. NRP1 is a promising target to overcome resistance to anti-PD1 therapies.

XPO1 regulates erythroid differentiation and is a new target for the treatment of ß-thalassemia.

ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.

Type I interferon-mediated autoinflammation due to DNase II deficiency.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.

Protein-based therapeutic for anemia caused by dyserythropoiesis.

INTRODUCTION: Major advances have been recently made in understanding the molecular determinants of dyserythropoiesis, particularly due to recent works in ß-thalassemia. The purpose of this review is devoted to underline the role of some proteins recently evidenced in the field, that may be new alternative therapeutic targets in the near future to alleviate different types of anemia. Areas covered: This review covers the contemporary aspects of some proteins involved in various types of dyserythropoiesis, including the transcriptional factor GATA-1 and its protective chaperone HSP70, but also cytokines of the transforming growth factor beta (TFG-ß) family, TGF-ß1 and GDF-11, and hormones as erythroferrone. It will be not exhaustive, but based on major recent published works from the literature in the past three years. Expert commentary: Sotatercept and lustatercept, two activin receptor II ligand traps that block GDF-11, are candidate drugs providing therapeutic hope in different types of ineffective erythropoiesis, including myelodysplastic syndromes (MDS) and ß-thalassemia. Furthermore, a new concept emerges to consider erythroid lineage in the bone marrow as an endocrine gland.

Eighty percent of French sport winners in Olympic, World and Europeans competitions have mutations in the hemochromatosis HFE gene.

The HFE gene encodes a protein involved in iron homeostasis; individuals with mutations in both alleles develop hemochromatosis. 27% of the French population is heterozygous for mutations in this gene. We found that 80% of the French athletes who won international competitions in rowing, Nordic skiing and judo display mutations in one allele of HFE, thus demonstrating the existence of a favourable phenotype linked to this heterozygosity.

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in ß-thalassaemia.

ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.

Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity.

Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.

Molecular mechanisms of the defective hepcidin inhibition in TMPRSS6 mutations associated with iron-refractory iron deficiency anemia.

Matriptase-2 is a transmembrane serine protease that negatively regulates hepcidin expression by cleaving membrane-bound hemojuvelin. Matriptase-2 has a complex ectodomain, including a C-terminal serine protease domain and its activation requires an autocatalytic cleavage. Matriptase-2 mutations have been reported in several patients with iron-refractory iron deficiency anemia. Here we describe a patient with 2 missense mutations in the second class A low-density lipoprotein receptor (LDLRA) domain. Functional studies of these 2 mutations and of a previously reported mutation in the second C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain were performed. Transfection of mutant cDNAs showed that membrane targeting of the 2 LDLRA mutants was impaired, with Golgi retention of the variants. The activating cleavage was absent for the LDLRA mutants and reduced for the CUB mutant. All 3 mutated proteins were still able to physically interact with hemojuvelin but only partially repressed hepcidin expression compared with wild-type matriptase-2. Our results underline the importance of LDLRA and CUB domains of matriptase-2.

Analysis of the largest tandemly repeated DNA families in the human genome.

BACKGROUND: Tandemly Repeated DNA represents a large portion of the human genome, and accounts for a significant amount of copy number variation. Here we present a genome wide analysis of the largest tandem repeats found in the human genome sequence. RESULTS: Using Tandem Repeats Finder (TRF), tandem repeat arrays greater than 10 kb in total size were identified, and classified into simple sequence e.g. GAATG, classical satellites e.g. alpha satellite DNA, and locus specific VNTR arrays. Analysis of these large sequenced regions revealed that several "simple sequence" arrays actually showed complex domain and/or higher order repeat organization. Using additional methods, we further identified a total of 96 additional arrays with tandem repeat units greater than 2 kb (the detection limit of TRF), 53 of which contained genes or repeated exons. The overall size of an array of tandem 12 kb repeats which spanned a gap on chromosome 8 was found to be 600 kb to 1.7 Mbp in size, representing one of the largest non-centromeric arrays characterized. Several novel megasatellite tandem DNA families were observed that are characterized by repeating patterns of interspersed transposable elements that have expanded presumably by unequal crossing over. One of these families is found on 11 different chromosomes in >25 arrays, and represents one of the largest most widespread megasatellite DNA families. CONCLUSION: This study represents the most comprehensive genome wide analysis of large tandem repeats in the human genome, and will serve as an important resource towards understanding the organization and copy number variation of these complex DNA families.

Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency.

Genetic causes of hypochromic microcytic anemia include thalassemias and some rare inherited diseases such as DMT1 deficiency. Here, we show that iron deficiency anemia with poor intestinal absorption and defective iron utilization of IV iron is caused by inherited mutations in TMPRSS6, a liver-expressed gene that encodes a membrane-bound serine protease of previously unknown role that was recently reported to be a regulator of hepcidin expression.