CRISPR/Cas9 -globin gene targeting in human haematopoietic stem cells. Nature 2016
The -haemoglobinopathies, such as sickle cell disease and -thalassaemia, are caused by mutations in the -globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure -haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult -globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for -haemoglobinopathies.
View details for DOI 10.1038/nature20134
View details for PubMedID 27820943
The road not taken? Blood 2016; 128 (7): 886-888
Biology of the bone marrow microenvironment and myelodysplastic syndromes MOLECULAR GENETICS AND METABOLISM 2015; 116 (1-2): 24-28
Myelodysplastic syndromes (MDS) are characterized by cytopenias resulting from ineffective hematopoiesis with a predisposition to transform to acute myeloid leukemia (AML). Recent evidence suggests that the hematopoietic stem cell microenvironment contributes to the pathogenesis of MDS. Inflammation and hypoxia within the bone marrow are key regulators of hematopoietic stem and progenitor cells that can lead to several bone marrow failure syndromes, including MDS. In this brief review, we provide an overview of the clinical and molecular features of MDS, the bone marrow microenvironment, and specific pathways that lead to abnormal blood cell development in MDS. Characterization of key steps in the pathogenesis of MDS will lead to new approaches to treat patients with this disease.
View details for DOI 10.1016/j.ymgme.2015.07.004
View details for Web of Science ID 000361423600004
TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors BLOOD 2014; 124 (25): 3791-3798
Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor- (TNF-) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF- or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF- inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.
View details for DOI 10.1182/blood-2014-06-584656
View details for Web of Science ID 000347467400017
View details for PubMedID 25270909
L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way BRITISH JOURNAL OF HAEMATOLOGY 2014; 167 (4): 524-528
The emerging importance of ribosomal dysfunction in the pathogenesis of hematologic disorders. Leukemia & lymphoma 2014; 55 (3): 491-500
Abstract More than a decade has passed since the initial identification of ribosomal protein gene mutations in patients with Diamond-Blackfan anemia (DBA), a hematologic disorder that became the founding member of a class of diseases known as ribosomopathies. In these diseases, genetic abnormalities that result in defective ribosome biogenesis cause strikingly tissue-specific phenotypes in patients, specifically bone marrow failure, craniofacial abnormalities and skeletal defects. Several animal models and numerous in vitro studies have demonstrated that the p53 pathway is central to the ribosomopathy phenotype. Additionally, there is mounting evidence of a link between the dysregulation of components of the translational machinery and the pathology of various malignancies. The importance of the role of ribosomal dysfunction in the pathogenesis of hematologic disorders is becoming clearer, and elucidation of the underlying mechanisms could have broad implications for both basic cellular biology and clinical intervention strategies.
View details for DOI 10.3109/10428194.2013.812786
View details for PubMedID 23863123
Lenalidomide Causes Selective Degradation of IKZF1 and IKZF3 in Multiple Myeloma Cells SCIENCE 2014; 343 (6168): 301-305
Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.
View details for DOI 10.1126/science.1244851
View details for Web of Science ID 000329718600037
View details for PubMedID 24292625
Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q-syndrome BLOOD 2013; 122 (14): 2487-2490
Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.
View details for DOI 10.1182/blood-2013-06-509935
View details for Web of Science ID 000326078200032
View details for PubMedID 23943650
Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts NATURE 2012; 491 (7425): 608-612
Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.
View details for DOI 10.1038/nature11536
View details for Web of Science ID 000311339800055
View details for PubMedID 23135403
L-leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway BLOOD 2012; 120 (11): 2214-2224
Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34 cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.
View details for DOI 10.1182/blood-2011-10-382986
View details for Web of Science ID 000309044200013
View details for PubMedID 22734070
Fulminant thrombotic microangiopathy in Pediatrics: Where diagnostic and therapeutic dilemmas meet AMERICAN JOURNAL OF HEMATOLOGY 2012; 87 (8): 816-818
Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q(-) syndrome BLOOD 2011; 118 (17): 4666-4673
Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.
View details for DOI 10.1182/blood-2010-12-324715
View details for Web of Science ID 000296368700029
View details for PubMedID 21873545
Translational medicine: ribosomopathies BLOOD 2011; 118 (16): 4300-4301
Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis BLOOD 2011; 118 (8): 2296-2304
Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34(+) cells and from CD34(+) cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.
View details for DOI 10.1182/blood-2010-11-318543
View details for Web of Science ID 000294258000032
View details for PubMedID 21527522
Neonatal Enteroviral Sepsis/Meningoencephalitis and Hemophagocytic Lymphohistiocytosis: Diagnostic Challenges AMERICAN JOURNAL OF PERINATOLOGY 2011; 28 (5): 337-345
We present the clinical course of three neonates with proven enteroviral infection and an initial clinical picture suggestive of hemophagocytic lymphohistiocytosis (HLH). After a complete workup, only one was treated for HLH. Of particular interest, the first newborn presented with hemophagocytic cells in the cerebrospinal fluid (CSF) and proved to have enteroviral meningoencephalitis but was ultimately not diagnosed with HLH. A fourth infant, who fulfilled the diagnostic criteria for HLH but did not have enteroviral infection, is included for comparison. We suggest that severe neonatal enteroviral infection and HLH are difficult to distinguish. Careful assessment is recommended, as prognosis and treatment differ between these two entities. Literature regarding neonatal enteroviral infection and HLH is reviewed, to demonstrate the continuum between the inflammation triggered by enteroviral infection and the occurrence of HLH, as well as their comparable CSF findings.
View details for DOI 10.1055/s-0030-1268710
View details for Web of Science ID 000289666200001
View details for PubMedID 21089006
Diamond Blackfan Anemia Treatment: Past, Present, and Future SEMINARS IN HEMATOLOGY 2011; 48 (2): 117-123
Despite significant improvements in our understanding of the pathophysiology of Diamond Blackfan anemia (DBA), there have been few advances in therapy. The cornerstones of treatment remain corticosteroids, chronic red blood cell transfusions, and hematopoietic stem cell transplantation, each of which is fraught with complications. In this article, we will review the history of therapies that have been offered to patients with DBA, summarize the current standard of care, including management of side effects, and discuss novel therapeutics that are being developed in the context of the research into the roles of ribosomal haplo-insufficiency and p53 activation in Diamond Blackfan anemia.
View details for DOI 10.1053/j.seminhematol.2011.01.004
View details for Web of Science ID 000289037800007
View details for PubMedID 21435508
Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells BLOOD 2011; 117 (9): 2567-2576
Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q-syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q-syndrome, DBA, and perhaps other bone marrow failure syndromes.
View details for DOI 10.1182/blood-2010-07-295238
View details for Web of Science ID 000288207400008
View details for PubMedID 21068437
Ribosome defects in disorders of erythropoiesis INTERNATIONAL JOURNAL OF HEMATOLOGY 2011; 93 (2): 144-149
Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis.
View details for DOI 10.1007/s12185-011-0776-0
View details for Web of Science ID 000288027700002
View details for PubMedID 21279816
Difficulty Measuring Methotrexate in a Patient with High-Dose Methotrexate-Induced Nephrotoxicity CLINICAL CHEMISTRY 2010; 56 (12): 1792-1794
Ribosomopathies: human disorders of ribosome dysfunction BLOOD 2010; 115 (16): 3196-3205
Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases.
View details for DOI 10.1182/blood-2009-10-178129
View details for Web of Science ID 000276956500005
View details for PubMedID 20194897
Allogeneic hematopoietic stem cell transplantation for X-linked ectodermal dysplasia and immunodeficiency: case report and review of outcomes IMMUNOLOGIC RESEARCH 2009; 44 (1-3): 89-98
Hypomorphic mutations in nuclear factor kappa B essential modulator (NEMO) cause X-linked ectodermal dysplasia with immunodeficiency (X-ED-ID). Clinical manifestations in boys with X-ED-ID apart from ectodermal dysplasia and immunodeficiency include osteopetrosis, lymphedema, and colitis. Further description of atypical findings in this disorder is needed. Treatment with allogeneic hematopoietic stem cell transplantation (HSCT) is in its infancy, and how or whether non-immune manifestations of defective NEMO function are impacted by HSCT is poorly described. We report an interesting case of a boy with NEMO mutation who had symptoms reminiscent of Omenn's syndrome and small intestinal villous atrophy with features reminiscent of tufting enteropathy. We describe his treatment course as well as reconstitution of immune function and correction of osteopetrosis post-HSCT, and review the cases of allogeneic HSCT reported to date in the literature.
View details for DOI 10.1007/s12026-008-8085-2
View details for Web of Science ID 000266582800011
View details for PubMedID 19225723
Blood group antigens in health and disease CURRENT OPINION IN HEMATOLOGY 2005; 12 (2): 135-140
Blood group antigens are polymorphic, inherited structures located on the surface of the red blood cell. They have long played an important role in identifying matched blood products for transfusion. Recent studies have identified varied and important functions for some of these molecules in cell physiology and human pathology.Many novel functions associated with blood group antigens have recently been identified. These include contributing to erythrocyte membrane structural integrity, transport of molecules through the membrane, and complement regulation as well as acting as adhesion molecules, receptors for extracellular ligands, and enzymes. Importantly, deficiency of these membrane components is associated with certain red cell disorders. Furthermore, as the same components are expressed in a variety of non-erythroid cells, deficiency of these proteins can also result in various other pathologies.Novel functions for red cell membrane components carrying blood group antigens are being identified. These findings are providing new molecular insights into the pathophysiology of both red cell disorders as well as various related pathologies in other organ systems.
View details for Web of Science ID 000230658800006
View details for PubMedID 15725904