- Pediatric Hematology-Oncology
Chief Fellow, Children's Hospital Boston (2008 - 2009)
Director of Hematology Education, Children's Hospital Boston (2010 - 2012)
Director of Fellow Education Series, Children's Hospital Boston (2011 - 2012)
Boards, Advisory Committees, Professional Organizations
Member, American Society of Pediatric Hematology/Oncology (2006 - Present)
Member, American Society of Hematology (2006 - Present)
Member, North American Pediatric Aplastic Anemia Consortium (2013 - Present)
Scientific Committee Member, American Society of Hematology (2013 - Present)
Elected Member, Society of Pediatric Research (2014 - Present)
Liaison, ASH Foundation and Development Committee, American Society of Hematology (2016 - Present)
Board Certification: Pediatrics, American Board of Pediatrics (2006)
Board Certification: Pediatric Hematology-Oncology, American Board of Pediatrics (2011)
Fellowship:Children's Hospital Boston (2009) MA
Residency:UCSF-Graduate Medical Education (2006) CA
Internship:UCSF-Graduate Medical Education (2004) CA
Medical Education:University of Pennsylvania Registrar's Office (2003) PA
Current Research and Scholarly Interests
More than a decade ago, researchers discovered that a rare congenital bone marrow failure syndrome, Diamond Blackfan anemia (DBA), is caused by mutations in a ribosomal protein RPS19. Subsequently, my mentor Dr. Benjamin Ebert identified RPS14 as the gene responsible for the profound macrocytic anemia in the 5q- syndrome, a subtype of myelodysplastic syndrome. This reinforced the connection between ribosomal abnormalities and defects in erythropoiesis. Moreover, mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes including Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, Cartilage Hair Hypoplasia and Treacher Collins syndrome. Each of these disorders is associated with specific defects in ribosome biogenesis, which cause distinct clinical phenotypes, most often involving bone marrow failure, and have become collectively known as ribosomopathies.
I have studied the molecular mechanisms by which ribosomal dysfunction leads to bone marrow failure by further characterizing the signaling pathways that are triggered and the subsequent effects on hematopoiesis. I published work on ribosomal haploinsufficiency causing selective activation of p53 in human erythroid progenitor cells and on the effects of a microRNA cooperating in the pathogenesis of the 5q- syndrome. I will continue to focus on understanding the effects of specific drugs on these disorders which may uncover further clues about pathophysiology and as importantly, will directly benefit patients. I have published work on the effects of dexamethasone and lenalidomide, the first line therapies for DBA and 5q- MDS respectively, on erythropoiesis and am an author on a manuscript examining the effects of leucine, a stimulator of the mTOR pathway, in these disorders.
The goal of my lab is to make meaningful contributions to the elucidation of the pathophysiology of ribosomopathies, the development of novel therapies and the care of patients in the field.
A Study of AG-348 in Adult Patients With Pyruvate Kinase Deficiency
Study AG348-C-003 is a multicenter study designed to evaluate the safety and efficacy of different dose levels of AG-348 in patients with PK deficiency.
Stanford is currently not accepting patients for this trial. For more information, please contact Heather Hilmoe, 650-725-1662.
Pilot Phase I/II Study of Amino Acid Leucine in Treatment of Patients With Transfusion-Dependent Diamond Blackfan Anemia
This study will determine the safety and possibility of giving the amino acid, leucine, in patients with Diamond Blackfan anemia(DBA)who are on dependent on red blood cell transfusions. The leucine is expected to produce a response in patients with DBA to the point where red blood cell production is increased. Red cell transfusions can then be less frequent or possibly discontinued. The investigators will study the side effects, if any, of giving leucine to DBA patients. Leucine levels of leucine will be obtained at baseline and during the study. The drug leucine will be provided in capsule form and taken 3 times a day for a total of 9 months.
Stanford is currently not accepting patients for this trial. For more information, please contact Peds Hem/Onc CRA's, 650-497-8953.
MMP9 inhibition increases erythropoiesis in RPS14-deficient del(5q) MDS models through suppression of TGF-beta pathways.
2019; 3 (18): 2751–63
The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number ofcolony forming unit-erythroid colonies and the CD71+erythroid population from RPS14 knockdown human BMCD34+cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factorbeta(TGF-beta) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-betainhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-betasignaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.
View details for DOI 10.1182/bloodadvances.2019000537
View details for PubMedID 31540902
INHIBITION OF NEMO-LIKE KINASE IMPROVES ERYTHROPOIESIS IN MODELS OF DIAMOND BLACKFAN ANEMIA
View details for Web of Science ID 000490282100027
A case series of pediatric patients with direct antiglobulin test negative autoimmune hemolytic anemia.
BACKGROUND: The diagnosis of autoimmune hemolytic anemia (AIHA) can be challenging since the direct antiglobulin test (DAT) has been reported to be falsely negative in 3%-11% of cases. In children with anemia, laboratory and/or clinical evidence of hemolysis and a negative DAT, clinicians should consider further specialized testing to confirm AIHA to accurately diagnose and treat this uncommon pediatric entity.STUDY DESIGN AND METHODS: A retrospective chart review was undertaken at a large tertiary care academic pediatric hematology practice to describe our experience with DAT-negative AIHA.RESULTS: From January 1, 2010 through August 1, 2016, 10 children were described who had clinical and laboratory evidence of AIHA, a negative DAT, and further specialized serologic testing confirming this diagnosis.CONCLUSION: This case series highlights the need for further serologic workup when a child's clinical presentation is highly consistent with AIHA despite a negative DAT.
View details for DOI 10.1111/trf.15350
View details for PubMedID 31111963
- A fork in the road. Blood 2019; 134 (18): 1484–85
- The Genetic Landscape of Diamond-Blackfan Anemia AMERICAN JOURNAL OF HUMAN GENETICS 2018; 103 (6): 930–47
- Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation AMER SOC HEMATOLOGY. 2018
- Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia AMER SOC HEMATOLOGY. 2018
- MMP9 Inhibition Rescues the Erythroid Defect in RPS14-Deficient Del(5q) MDS Models AMER SOC HEMATOLOGY. 2018
- Leucine for the Treatment of Transfusion Dependence in Patients with Diamond Blackfan Anemia AMER SOC HEMATOLOGY. 2018
- Pharmacological Inhibition of Nlk (Nemo-like Kinase) Rescues Erythropoietic Defects in Pre-Clinical Models of Diamond Blackfan Anemia AMER SOC HEMATOLOGY. 2018
The Genetic Landscape of Diamond-Blackfan Anemia.
American journal of human genetics
Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.
View details for PubMedID 30503522
Developmental differences between neonatal and adult human erythropoiesis
AMERICAN JOURNAL OF HEMATOLOGY
2018; 93 (4): 494–503
Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)- and peripheral blood (PB)-HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB-derived cells proliferated 4-fold more than PB-derived cells. However, CB-derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony-forming unit (CFU-E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R- GPA- CD34+ CD36+ cells generated from PB- than CB-HSPCs. This subset was found to generate both burst-forming unit (BFU-E) and CFU-E colonies in colony-forming assays. To further understand the differences between CB- and PB-HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle- and autophagy-related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.
View details for PubMedID 29274096
View details for PubMedCentralID PMC5842122
- Erythropoiesis: insights into pathophysiology and treatments in 2017 MOLECULAR MEDICINE 2018; 24
An update on the pathogenesis and diagnosis of Diamond-Blackfan anemia.
Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia characterized by a block in erythropoiesis at the progenitor stage, although the exact stage at which this occurs remains to be fully defined. DBA presents primarily during infancy with macrocytic anemia and reticulocytopenia with 50% of cases associated with a variety of congenital malformations. DBA is most frequently due to a sporadic mutation (55%) in genes encoding several different ribosomal proteins, although there are many cases where there is a family history of the disease with varying phenotypes. The erythroid tropism of the disease is still a matter of debate for a disease related to a defect in global ribosome biogenesis. Assessment of biological features in conjunction with genetic testing has increased the accuracy of the diagnosis of DBA. However, in certain cases, it continues to be difficult to firmly establish a diagnosis. This review will focus on the diagnosis of DBA along with a description of new advances in our understanding of the pathophysiology and treatment recommendations for DBA.
View details for PubMedID 30228860
View details for PubMedCentralID PMC6117846
Erythropoiesis: insights into pathophysiology and treatments in 2017.
Molecular medicine (Cambridge, Mass.)
2018; 24 (1): 11
Erythropoiesis is a tightly-regulated and complex process originating in the bone marrow from a multipotent stem cell and terminating in a mature, enucleated erythrocyte.Altered red cell production can result from the direct impairment of medullary erythropoiesis, as seen in the thalassemia syndromes, inherited bone marrow failure as well as in the anemia of chronic disease. Alternatively, in disorders such as sickle cell disease (SCD) as well as enzymopathies and membrane defects, medullary erythropoiesis is not, or only minimally, directly impaired. Despite these differences in pathophysiology, therapies have traditionally been non-specific, limited to symptomatic control of anemia via packed red blood cell (pRBC) transfusion, resulting in iron overload and the eventual need for iron chelation or splenectomy to reduce defective red cell destruction. Likewise, in polycythemia vera overproduction of red cells has historically been dealt with by non-specific myelosuppression or phlebotomy. With a deeper understanding of the molecular mechanisms underlying disease pathophysiology, new therapeutic targets have been identified including induction of fetal hemoglobin, interference with aberrant signaling pathways and gene therapy for definitive cure. This review, utilizing some representative disorders of erythropoiesis, will highlight novel therapeutic modalities currently in development for treatment of red cell disorders.
View details for PubMedID 30134792
View details for PubMedCentralID PMC6016880
The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70
2017; 1 (22): 1959–76
Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation-dependent DBA.
View details for PubMedID 29296843
View details for PubMedCentralID PMC5728147
Loss of FOXM1 promotes erythropoiesis through increased proliferation of erythroid progenitors.
Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34(+) cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.
View details for DOI 10.3324/haematol.2016.156257
View details for PubMedID 28154085
Characterization, regulation, and targeting of erythroid progenitors in normal and disordered human erythropoiesis.
Current opinion in hematology
The erythroid progenitors burst-forming unit-erythroid and colony-forming unit-erythroid have a critical role in erythropoiesis. These cells represent a heterogeneous and poorly characterized population with modifiable self-renewal, proliferation and differentiation capabilities. This review focuses on the current state of erythroid progenitor biology with regard to immunophenotypic identification and regulatory programs. In addition, we will discuss the therapeutic implications of using these erythroid progenitors as pharmacologic targets.Erythroid progenitors are classically characterized by the appearance of morphologically defined colonies in semisolid cultures. However, these prior systems preclude a more thorough understanding of the composite nature of progenitor populations. Recent studies employing novel flow cytometric and cell-based assays have helped to redefine hematopoiesis, and suggest that erythroid progenitors may arise from different levels of the hematopoietic tree. Moreover, the identification of cell surface marker patterns in human burst-forming unit-erythroid and colony-forming unit-erythroid enhance our ability to perform downstream functional and molecular analyses at the population and single cell level. Advances in these techniques have already revealed novel subpopulations with increased self-renewing capacity, roles for erythroid progenitors in globin gene expression, and insights into pharmacologic mechanisms of glucocorticoids and pomalidomide.Immunophenotypic and molecular characterization resolves the diversity of erythroid progenitors, and may ultimately lead to the ability to target these progenitors to ameliorate diseases of dyserythropoiesis.
View details for DOI 10.1097/MOH.0000000000000328
View details for PubMedID 28099275
Erythrocyte adenosine deaminase levels are elevated in Diamond Blackfan anemia but not in the 5q-syndrome
AMERICAN JOURNAL OF HEMATOLOGY
2016; 91 (12): E501–E502
View details for PubMedID 27556864
CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.
The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-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 β-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 β-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 β-haemoglobinopathies.
View details for DOI 10.1038/nature20134
View details for PubMedID 27820943
A novel pathogenic mutation in RPL11 identified in a patient diagnosed with diamond Blackfan anemia as a young adult
BLOOD CELLS MOLECULES AND DISEASES
2016; 61: 46–47
View details for PubMedID 27667165
- The road not taken? Blood 2016; 128 (7): 886-888
- Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes LEUKEMIA 2015; 29 (12): 2285-2295
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 PubMedID 26210353
View details for PubMedCentralID PMC4618471
- Jekyll and Hyde: the role of heme oxygenase-1 in erythroid biology. Haematologica 2015; 100 (5): 567–68
TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors.
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-α (TNF-α) 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-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, 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 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
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
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): 524–28
Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.
View details for PubMedID 25098371
View details for PubMedCentralID PMC4211992
Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q-syndrome
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
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
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
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
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
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
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
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