Pathophysiology Of Diamond-Blackfan Anemia Research Articles

Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1–4]).Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.

Metabolomic Profiling Confirms the Key Role of Oxidative Stress in the Pathophysiology of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), a rare congenital bone marrow failure syndrome, characterized by severe anemia, erythroblastopenia, and congenital malformations is mostly associated with pathogenic variants in ribosomal protein (RP) genes, which cause their haploinsufficiency. The marked heterogeneity of the disease, related to different severity of hematological and non-hematological symptoms and diverse response to treatment, even in patients harboring the same RP mutation is not fully understood. Similarly, the mechanisms responsible for the predisposition of DBA patients to cancer await elucidation. We previously showed that the formation of reactive oxygen species (ROS) exceeds the anti-oxidant capacity of DBA erythrocytes leading to their higher susceptibility to destruction. Using DBA patients' samples and CRISPR/Cas9-created Rps19- and Rpl5-haploinsufficient MEL cellular models we also showed, that excessive oxidative stress, DNA damage, and activation of DNA damage response of erythroblasts is associated with an increased production of pro-/inflammatory cytokines and induction of apoptosis and senescence. In recent years, the use of metabolomic analysis appears as a promising tool that could contribute to a better understanding of the pathophysiology of the disease and discovery of novel biochemical markers with their potential use in diagnostics and prognostication. We therefore performed targeted metabolomic analysis of serum and erythrocyte samples from DBA patients (n=26) and healthy controls (n=10) using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) where 129 metabolites were detected. Significant changes in the metabolome of DBA patients were observed, with the key deregulated metabolites involved in the antioxidant defense and energy metabolism; the reduction of amine compounds (creatinine, guanidinoacetate) and hippurate in DBA patients compared to controls was also noted. In two DBA families analyzed, the altered erythrocyte metabolism and markers of oxidative stress distinguished the patients from their asymptomatic family members with the same RP mutation. The incubation of erythrocytes from symptomatic DBA patients with antioxidant N-acetylcysteine in vitro led to normalization of ROS levels in erythrocytes. Similarly, the treatment of Rpl5- and Rps19-haploinsufficienct MEL cells with a pro-inflammatory cytokine inhibitor pomalidomide, resulted in the amelioration of oxidative DNA damage (assessed by the presence of 8-oxoguanine, 8-oxoG) and reduction of p53 activation. In addition, targeted metabolomic analysis of cellular extracts showed clear separation of Rps19- and Rpl5-haploinsufficient MEL cells from control cells with significant changes affecting the level of many carbohydrates and acylcarnitines. Elevated levels of glutathione and pentose phosphate cycle intermediates in Rps19- and Rpl5-haploinsufficient MEL cells correspond with their need to cope with increased oxidative stress. Significantly increased levels of deoxynucleotides, mainly in Rpl5-haploinsufficient MEL cells, could be related to DNA damage response (DDR) process to promote repair of ROS-mediated damaged DNA. Nucleotide formation is an energy intensive process and therefore the observed decrease in acylcarnitines could be related to increased beta oxidation and increased formation of reduced coenzymes. This result from model MEL cells is consistent with 8-oxoG foci and activation of ATM (a DDR kinase) observed in erythroblasts of DBA patients expanded in vitro. Importantly, immunoreactivity for 8-oxoG and phospho-ATM S1981 was detected also in non-erythroid bone marrow cells of two DBA patients, who recently developed myelodysplastic neoplasia (MDS). Collectively, our data support the involvement of oxidative stress and pro-/inflammatory cytokines in the pathophysiology of DBA and suggest that the treatment aiming to reduce oxidative stress and/or suppress inflammation could alleviate the severity of the disease, though additional studies are needed. The potential link between oxidative damage, inflammatory cytokines, and cancer risk in DBA also deserves further research. Grant support: The Ministry of Education, Youth and Sports of the Czech Republic (8F20005), European Union - Next Generation EU, Program EXCELES (LX22NPO5102); Internal grant of Palacky University (IGA_LF_2023_002).

Deficiency of ribosomal protein S26, which is mutated in a subset of patients with Diamond Blackfan anemia, impairs erythroid differentiation.

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis. Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells. Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder.

Developmental Stage-Specific Hematopoietic Failure In Vivo in a Novel Model of Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized by failure of erythropoiesis at the progenitor stages, congenital anomalies, and predisposition to cancer. DBA is most frequently due to mutations in genes encoding a ribosomal protein, the most common one being mutations in RPS19. Despite the well-established role of RPS19 as well as other ribosomal proteins in the erythropoietic failure in in vitro culture systems, there is a paucity of in vivo murine models of DBA due to differences between humans and mice. Indeed, stress erythropoiesis occurs in the spleen of mouse, compensating for the erythropoietic defects observed in the bone marrow. Using CRISPR/Cas9 technologies, we generated a conditional knockout of Rps19 by flanking exon 2 with 2 loxP sites and studied hematopoiesis in this model. While Tie2-cre+;Rps19+/- animals die in utero, Vav-iCre+; Rps19+/- mice are born at expected Mendelian ratios with severe macrocytic anemia. Surprisingly, and unlike any other model generated to date, these mice present with reticulocytopenia, a critical characteristic of patients with DBA and die by post-natal day 10 (P10), due to bone marrow failure and decreased splenic stress erythropoiesis. To understand the role of ribosomal proteins during hematopoietic development, we analyzed fetal livers of Vav-iCre+; Rps19+/- embryos and control littermates at E13.5 and E17.5. Using flow cytometry, we observed defective erythropoiesis and gradual hematopoietic stem cell exhaustion at the fetal to neonatal transition. Mechanistically, and consistent with previous studies using in vitro systems, Rps19mutant cells had increased p53 protein expression and reduced mTORC1 activity. Unexpectedly, hematopoietic progenitors had enhanced global protein synthesis. Using BrDU and EdU stainings, we observed a decrease in the cell cycle duration of erythroblasts, consistent with the macrocytosis and reticulocytopenia seen in neonatal Vav-iCre+; Rps19+/- mice. Since p53 is reported to have a central role in the pathophysiology of DBA, we asked whether deletion of one allele of p53 could rescue the defects observed in Vav-iCre+; Rps19+/-. The Vav-iCre+; Rps19+/-, trp53+/-presented with improved blood counts, increased spleen size and cellularity but died by four weeks after birth. Analyses of the hematopoietic compartment in these animals at P21 revealed bone marrow failure and HSC depletion with skewed progenitor populations. The increase in global protein synthesis observed in Vav-iCre+; Rps19+/- remained unchanged in Vav-iCre+; Rps19+/-, trp53+/-, suggesting mechanisms independent of p53. Taken together, we have developed a novel in vivo model for the study of DBA. Our findings could potentially explain why DBA typically manifests after birth and highlight a critical role for Rps19 during hematopoietic differentiation both in utero and after birth.

GATA-1 Defects in Diamond-Blackfan Anemia: Phenotypic Characterization Points to a Specific Subset of Disease.

Diamond–Blackfan anemia (DBA) is one of the inherited bone marrow failure syndromes marked by erythroid hypoplasia. Underlying variants in ribosomal protein (RP) genes account for 80% of cases, thereby classifying DBA as a ribosomopathy. In addition to RP genes, extremely rare variants in non-RP genes, including GATA1, the master transcription factor in erythropoiesis, have been reported in recent years in patients with a DBA-like phenotype. Subsequently, a pivotal role for GATA-1 in DBA pathophysiology was established by studies showing the impaired translation of GATA1 mRNA downstream of the RP haploinsufficiency. Here, we report on a patient from the Dutch DBA registry, in which we found a novel hemizygous variant in GATA1 (c.220+2T>C), and an Iranian patient with a previously reported variant in the initiation codon of GATA1 (c.2T>C). Although clinical features were concordant with DBA, the bone marrow morphology in both patients was not typical for DBA, showing moderate erythropoietic activity with signs of dyserythropoiesis and dysmegakaryopoiesis. This motivated us to re-evaluate the clinical characteristics of previously reported cases, which resulted in the comprehensive characterization of 18 patients with an inherited GATA-1 defect in exon 2 that is presented in this case-series. In addition, we re-investigated the bone marrow aspirate of one of the previously published cases. Altogether, our observations suggest that DBA caused by GATA1 defects is characterized by distinct phenotypic characteristics, including dyserythropoiesis and dysmegakaryopoiesis, and therefore represents a distinct phenotype within the DBA disease spectrum, which might need specific clinical management.

Targeting elevated heme levels to treat a mouse model for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.

Oxidative DNA Damage, Inflammatory Signature, and Altered Erythrocytes Properties in Diamond-Blackfan Anemia.

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients’ samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

Targeting of Calbindin 1 (CALB1) Rescues Erythropoiesis in a Human Model of Diamond Blackfan Anemia: Implications for Novel Therapies

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. The erythroid defects of DBA include macrocytosis, reticulocytopenia, and a selective decrease or absence of erythroid precursors in the bone marrow. It was the first disease linked to ribosomal dysfunction and >70% of patients have since been shown to have a haploinsufficiency of a ribosomal protein (RP) gene. Among these RP mutations, RPS19 mutations were the first to be identified and are the most common, found in up to 25% of DBA patients. Current therapies for DBA include corticosteroids, chronic transfusions, and/or hematopoietic stem cell transplantation. However, each of these therapies have potential for significant side effects and as such there is an urgent need to develop new therapies for the management of these patients. In addition there is a need for improved understanding of the mechanistic basis for the wide range of phenotypic expression and responses to therapy between patients with the same mutation, which implies a key role for modifier genes in this disease. To identify potential modifiers in the phenotype of patients and for developing potential novel therapeutic targets, we performed a genome wide CRISPR screen using A549 cells with RPS19 haploinsufficiency as a cellular model for DBA, which enabled large-scale screening. As the p21 pathway is known to be central to the pathophysiology of DBA, we identified hits that resulted in higher and lower levels of p21 expression in the DBA model vs. control, respectively. Among these hits, we consistently identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily that includes calmodulin and buffers entry of calcium into cells upon stimulation of glutamate receptors. Given the recent findings that calmodulin inhibition can rescue anemia in DBA models by blocking the p90 ribosomal S6 kinase (RSK)-p70 (p70S6K) pathway and the role for glucose and glutamine metabolism in the regulation of human HSC lineage specification, we decided to pursue CALB1 as a potential target of interest. We used peripheral blood derived-CD34+ cells placed in erythroid stimulating media following knockdown of RPS19, and investigated the effects of CALB1 on cell cycle progression and erythroid differentiation by flow cytometry. We observed that knockdown of CALB1 significantly improved cell viability, increased G0/G1 population and promoted erythroid maturation with increased expression of CD36 and glycophorin A in the context of RPS19 deficiency. Together, these data suggest that the knockdown of CALB1 rescues human erythropoiesis in a model of DBA. We are investigating the effects of CALB1 knockdown on signaling pathways and genes involved in DBA due to RPS19 mutations notably GATA-1/HSP70 and TNF-alpha. We anticipate that the identification of a role for CALB1 in the pathophysiology of DBA will provide a new therapeutic target for DBA either alone or in combination with steroids or calmodulin inhibition. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Untargeted Metabolomic Fingerprinting As a Potential Tool in the Diagnostic Evaluation of Diamond Blackfan Anemia

Background: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) marked by erythroid hypoplasia, reticulocytopenia and macrocytosis, and associated with congenital malformations and an increased risk of developing malignancies. From a clinical and molecular perspective this disease is highly heterogeneous, and no clear genotype-phenotype correlations can be found. Since the majority of molecular defects have been found in ribosomal protein (RP) genes, DBA is regarded a "ribosomopathy". While the molecular basis has been studied intensively, the pathophysiology of DBA is still not fully understood. One of the major unresolved issues is how RP gene mutations result in the specific erythroid defect seen in DBA, with macrocytic erythrocytes and increased adenosine deaminase activity. In addition, the highly heterogeneous and variable clinical presentation and disease course, even in patients with similar molecular defects, remains enigmatic. Hence, in order to investigate the cellular defect, and increase our understanding of disease pathophysiology and clinical heterogeneity, novel tools are needed. In this study, we explore the potential of untargeted metabolomics on dried blood spots and report for the first time a metabolic fingerprint for DBA. Aims: Defining a metabolic signature for DBA in order to: 1. Increase our understanding of cellular determinants of impaired ribosome biogenesis, and 2. Extend the toolbox for diagnostic evaluation. Methods: Untargeted metabolic profiling was performed on DBS samples obtained from 18 DBA patients and 45 healthy controls using direct infusion high resolution mass spectrometry following a previously established approach1. Statistical analysis was performed in MetaboAnalyst and predictive modeling was executed within R-software. Results: In total, 1917 unique metabolite features were identified in DBS samples from patients and controls. Multivariate analysis yielded distinct metabolic profiles, reflected by natural separation detected by principal component analysis (Figure 1A), and emphasized by clear distinction with partial least square discriminant analysis (Figure 1B). This 'metabolic fingerprint' was incorporated into a machine learning algorithm, and subsequently a binary classification (or prediction) model was constructed by randomly dividing patient and controls into 'training' (32 HC, 13 DBA) or 'test' set (13 HC, 5 DBA). Accurate class assignment was achieved for all patients and controls in the training set (Figure 1C). Prominent metabolites in the fingerprint and classification algorithm were a.o. menadione (a vitamin K precursor), 4-hydroxyproline (collagen component) and methylmalonylcarnitine (an acylcarnitine). In addition a large number of interesting metabolites were identified that could provide novel starting points for studying/understanding downstream effects of RP defects in DBA and therapeutic mechanisms (Figure 1D). Conclusion: In this study we performed untargeted metabolomics on dried blood spots from a substantial cohort of DBA patients and report for the first time a metabolic fingerprint of this disease. By incorporating this fingerprint in a machine learning algorithm we underlined the diagnostic potential of this approach. Moreover, the metabolites identified in this fingerprint, provide promising starting points for further studies to increase our insights in disease pathophysiology, including the mechanism involved in elevated eADA activity, as well as the development of new therapeutic strategies.

Disease Modeling and Phenotype Rescue Using Inducible Pluripotent Stem Cells from Patients with Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

PS1113 OLIGOCLONALITY OF T-CELL RECEPTOR AND THE MAIN SUBPOPULATION COMPOSITION IN PATIENTS WITH APLASTIC ANEMIA AT DIFFERENT STAGES OF THE DISEASE

Background: Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder, characterized by severe hypoplastic anemia, associated congenital anomalies, and an increased risk of developing cancer. Based on the underlying mutations in ribosomal protein (RP) genes, DBA is classified as a ribosomopathy. While disruptions in ribosomal biogenesis would be expected to have global effects, hematological in DBA are primarely of the erythroid lineage. Moreover, since cellular determinants following ribosomal stress are still not well understood, and most functional studies are performed in models representing only the most frequently affected RP-genes, the development of novel therapeutic strategies for all DBA patients has been been hampered. Here we propose, for the first time, a novel untargeted metabolomics approach to study anemia in DBA patient cells. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, and a powerful functional tool, directly reflecting the underlying biochemical activity and the state of cells. The DBA-metabolome can be used to study commonly deregulated cellular pathways during erythroid development, irrespective of the underlying molecular defect. Aims: Defining a metabolic signature of RP-deficiency in DBA, will 1. Increase our understanding of erythroid-specific defects in DBA, and 2. Contribute to the identification of potential novel therapeutic targets. Methods: Quantification of metabolites was performed on dried blood spots from 6 DBA patients (RPS26, RPL5, RPL9, unknown) by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargeted metabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with dried blood spots samples of 10 healthy controls. Results: Preliminary data from 6 DBA patients with different genotypes demonstrate a significant increase in intermediates of the pentose phosphate pathway (PPP) (Figure 1A). This indicates that the PPP is upregulated in red blood cells from DBA patients, regardless of the underlying molecular defect. In line with this we observed a significant increase in erythrocyte reduced glutathione (GSH) content in 9 DBA patients (RPS26, RPL5, RPL11, unknown), compared to 12 controls (Figure 1B). In red blood cells the PPP mainly serves to provide the cell with sufficient amounts of NADPH to maintain high intracellular levels of GSH, which is needed to protect the cell against oxidative damage. Our results therefore support a role for oxidative stress response in DBA pathophysiology irrespective of the underlying genotype. Figure 1. (A) Increase in metabolites involved in PPP in DBA patient cells. (B) Increased GSH concentration in DBA patient erythrocytes.Summary/Conclusion: DBS metabolomics can be used as a novel functional tool to investigate deregulated cellular pathways in DBA irrespective of the underlying molecular defect. This will contribute to increased insights in cell type specific phenomena and DBA pathophysiology in general.

PS1111 QUANTIFICATION OF METABOLITES IN DRIED BLOOD SPOTS OF DIAMOND BLACKFAN ANEMIA PATIENTS REVEALS NOVEL INSIGHTS IN DEREGULATED CELLULAR PATHWAYS INVOLVED IN HYPOPLASTIC ANEMIA

Background:Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder, characterized by severe hypoplastic anemia, associated congenital anomalies, and an increased risk of developing cancer. Based on the underlying mutations in ribosomal protein (RP) genes, DBA is classified as a ribosomopathy. While disruptions in ribosomal biogenesis would be expected to have global effects, hematological in DBA are primarely of the erythroid lineage. Moreover, since cellular determinants following ribosomal stress are still not well understood, and most functional studies are performed in models representing only the most frequently affected RP‐genes, the development of novel therapeutic strategies for all DBA patients has been been hampered. Here we propose, for the first time, a novel untargeted metabolomics approach to study anemia in DBA patient cells. Metabolomics is the large‐scale, unbiased study of metabolites and their interactions within a biological system, and a powerful functional tool, directly reflecting the underlying biochemical activity and the state of cells. The DBA‐metabolome can be used to study commonly deregulated cellular pathways during erythroid development, irrespective of the underlying molecular defect.Aims:Defining a metabolic signature of RP‐deficiency in DBA, will 1. Increase our understanding of erythroid‐specific defects in DBA, and 2. Contribute to the identification of potential novel therapeutic targets.Methods:Quantification of metabolites was performed on dried blood spots from 6 DBA patients (RPS26, RPL5, RPL9, unknown) by direct infusion high resolution mass spectrometry (DI‐HRMS) followed by an untargeted metabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with dried blood spots samples of 10 healthy controls.Results:Preliminary data from 6 DBA patients with different genotypes demonstrate a significant increase in intermediates of the pentose phosphate pathway (PPP) (Figure 1A). This indicates that the PPP is upregulated in red blood cells from DBA patients, regardless of the underlying molecular defect. In line with this we observed a significant increase in erythrocyte reduced glutathione (GSH) content in 9 DBA patients (RPS26, RPL5, RPL11, unknown), compared to 12 controls (Figure 1B). In red blood cells the PPP mainly serves to provide the cell with sufficient amounts of NADPH to maintain high intracellular levels of GSH, which is needed to protect the cell against oxidative damage. Our results therefore support a role for oxidative stress response in DBA pathophysiology irrespective of the underlying genotype.Figure 1. (A) Increase in metabolites involved in PPP in DBA patient cells. (B) Increased GSH concentration in DBA patient erythrocytes.Summary/Conclusion:DBS metabolomics can be used as a novel functional tool to investigate deregulated cellular pathways in DBA irrespective of the underlying molecular defect. This will contribute to increased insights in cell type specific phenomena and DBA pathophysiology in general.image

RPL11 Haploinsufficient Mice Have a CFU-E/Proerythroblast Block, Elevated Erythroblast Heme, Reduced Gata1, and Increased Ribosomal Protein Gene Expression

Diamond-Blackfan anemia (DBA) is associated with hypomorphic mutations in at least 16 ribosomal proteins. Additionally, mutations in GATA1 that result in the preferential expression of variants lacking the transactivation domain cause a similar but not identical disease. Ribosomal haploinsufficiency can lead to imbalanced ribosome protein production resulting in the activation of p53 and additionally slows protein translation, including globin, resulting in excess heme during the early stages of erythropoiesis when heme synthesis (an enzymatic process) is brisk but globin levels are insufficient. Mice lacking the heme export protein FLVCR1 develop a severe macrocytic anemia similar to DBA resulting from a block in differentiation at the CFU-E/proerythroblast stage which is caused by excess heme and ROS (J Clin Invest 125:4681, 2015). This macrocytic anemia occurs independent of ribosomal haploinsufficiency or p53 activation, suggesting that elevated heme is a key factor in the pathophysiology of DBA. Indeed, erythroid cultures of marrow from individuals with DBA demonstrate delayed globin synthesis, excess heme, elevated ROS, and increased cell death of CFU-E/proerythroblasts (Sci Transl Med 8:338RA67, 2016). Rescue studies showed in vitro erythroid differentiation improved when heme synthesis was decreased. To further understand the role of heme and ribosomal haploinsufficiency in DBA, we are characterizing RPL11 haploinsufficient mice. RPL11 heterozygous mice develop a cell intrinsic macrocytic anemia with increased susceptibility to radiation-induced lymphomagenesis (Cell Reports 13:712, 2015). Our studies confirm RPL11 heterozygous mice have a chronic macrocytic anemia (HGB 12.0±1.7 vs 14.3±0.4 g/dL; MCV 58.0±2.2 vs 46.8±1.4 fL) concurrent with a block at the CFU-E/proerythroblasts stage (63% reduction in basophilic erythroblasts). Erythroblast heme content is 2-fold higher than control by the polychromatic erythroblast stage while ROS is elevated (15-75%) throughout terminal differentiation. To understand the pathophysiology leading to ineffective erythropoiesis in DBA we performed single cell RNA sequencing and cell surface protein quantification on erythroid precursors from control and Flvcr1 -deleted mice and are comparing these data to data from RPL11 haploinsufficient mice. Principal component analysis identified 4 transcriptionally unique clusters with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975) and increased as Ter119 expression increased. Gene set enrichment analysis comparing control cells to Flvcr1 -deleted cells revealed significant upregulation of the ribosome pathway genes and downregulation of the hallmark heme metabolism pathway genes including GATA1 and GATA1-target genes. Quantitative PCR analysis of RPL11 haploinsufficient erythroid cells show 2-fold increases in Rps19, Rps14, Rpl4, and Rpl35 transcript levels during terminal erythroid differentiation, however, Rpl11 transcript levels are reduced 50% in precursor cells and fail to increase to comparable levels with other ribosomal protein genes. Cdkn1a was increased, consistent with activation of the p53 pathway. Comparable studies with Flvcr1 -deleted mice do not show any activation of the p53 pathway, indicating that p53 pathway activation is unique to ribosomal haploinsufficiency and not a result of excess heme. Both Flvcr1 -deleted and RPL11 haploinsufficient erythroid cells have reduced Gata1 expression. To resolve the role of heme from other driving factors, we tested the effect of ALA and iron treatment to induce endogenous heme synthesis in sorted human marrow cells. Within 15 minutes of treatment the early erythroid cells (Lin-CD36+GlyA-) upregulated ribosomal gene transcript levels while later erythroid cells (Lin-CD36+GlyA+) did not. Additionally, GATA1 protein levels were rapidly decreased by ALA and iron, but not by exogenous heme or iron treatment alone. Thus poor translation of globin in ribosomal haploinsufficiencies leads to excess heme. It is this excess heme which leads to premature termination of erythroid differentiation by reducing GATA1 levels, additionally, it exacerbates ribosomal protein imbalance, increasing p53 activation and cell death. Thus the key pathologies in DBA are a direct result of excess heme, suggesting new approaches for treatment. DisclosuresNo relevant conflicts of interest to declare.

Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: Evidence from RPL11-deficient in vivo and in vitro models

Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of rpl11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, Rpl11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of Rpl11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency.

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis.These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA.

BMI1 Promotes Erythropoiesis Through Regulating Ribosome Biogenesis

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome of childhood manifested as macrocytic anemia with insufficient erythroid precursors in the bone marrow. Within the decade following the demonstration that mutations in the ribosomal protein gene RPS19 can lead to DBA, this disease has become a paradigm for an emerging group of pathologies (ribosomopathies) linked to defects in ribosome biogenesis. Mutations in ribosomal protein genes impair ribosome biogenesis, resulting in activation of the p53 tumor suppressor pathway, cell cycle arrest and defective erythropoiesis. While mutations in ribosomal protein genes have been found in 50-60% of DBA patients, genetic abnormalities in the remaining patients are largely unknown. Despite improvements in our understanding of the pathophysiology of DBA, the molecular basis for selective impairment of the erythroid lineage in this disorder is not understood. In particular, how ribosome biogenesis is regulated in erythroid precursors remains elusive. Our laboratory has been investigating the role of Polycomb group protein Bmi1 in regulating hematopoietic stem cell (HSC) self-renewal and lineage commitment. Recently, we found that Bmi1 is a critical downstream target of AKT signaling and AKT-mediated phosphorylation of Bmi1 inhibits HSC self-renewal (Liu et al., Science Signaling, 2012). Upon more detailed analysis of the hematopoietic phenotype of the Bmi1 knockout mice, we have observed that these mice develop macrocytic anemia and show delayed recovery following phenylhydrazine (PHZ)-induced hemolytic anemia. This phenotype suggests defective erythropoiesis and we identified that loss of Bmi1 expression results in a block in erythroid differentiation and decreased erythroid colony formation. Gene expression profiling indicated that multiple ribosomal protein genes were downregulated in Bmi1 null erythroid precursors. Moreover, we discovered that the p53 pathway is activated in Bmi1 null erythroid progenitor cells and genetic inhibition of p53 activity rescued the erythroid defects in the Bmi1 deficient mice. Thus, Bmi1 null mice recapitulate many critical features of human DBA. Furthermore, we demonstrated that BMI1 plays a critical role in human erythropoiesis as knockdown of BMI1 in human CD34+ cells decreases ribosomal protein gene expression, activates the p53 pathway, and blocks erythroid differentiation. Importantly, we observed that BMI1 expression is downregulated in bone marrow cells from some DBA patients. Thus, BMI1 plays a critical role in regulating ribosome biogenesis in erythroid precursors and BMI1 deficiency may contribute to the pathogenesis of DBA. Understanding how BMI1 regulates ribosome biogenesis and erythroid development will provide novel insight into the processes by which BMI1 and ribosomopathies contribute to the pathogenesis of DBA and potentially new targets for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Diamond Blackfan Anemia

Mutations affecting genes encoding ribosomal proteins cause Diamond Blackfan anemia (DBA), a rare congenital syndrome associated with physical anomalies, short stature, red cell aplasia, and an increased risk of malignancy. p53 activation has been identified as a key component in the pathophysiology of DBA after cellular and molecular studies of knockdown cellular and animal models of DBA and other disorders affecting ribosomal assembly or function. Other potential mechanisms that warrant further investigation include impaired translation as the result of ribosomal insufficiency, which may be ameliorated by leucine supplementation, and alternative splicing leading to reduced expression of a cytoplasmic heme exporter, the human homolog of the receptor for feline leukemia virus C (FVLCR). However, the molecular basis for the characteristic steroid responsiveness of the erythroid failure in DBA remains unknown. This review explores the clinical and therapeutic implications of the current state of knowledge and delineates important but as-yet-unanswered questions.

Diamond Blackfan Anemia Treatment: Past, Present, and Future

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.

Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells

AbstractHaploinsufficiency 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.

Diamond Blackfan Anemia Defects In Rps19-Mutant Embryonic Stem Cells: Rescue by Gene Replacement but Not Glucocorticoid Treatment

AbstractAbstract 2237 Background:Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes (IBMFS), characterized by erythroid hypoplasia, congenital anomalies and a cancer predisposition. DBA is caused by ribosomal protein haploinsufficiency, which somehow triggers apoptosis of erythroid precursors, possibly through activation of p53. Some DBA patients show a response to steroid treatment, while others remain transfusion-dependent. While the mechanism of action of steroids in DBA is unclear, recent work has suggested that p53 may antagonize glucocorticoid-induced proliferation of normal erythroid progenitors1. Objective:Our goal was to create a murine embryonic stem (ES) cell model of DBA with a mutation in Rps19 to study the pathophysiology of DBA and to test glucocorticoid responsiveness. Methods:The Rps19-mutated murine embryonic stem cell line, S17-10H1 was created using a gene trap strategy. The ES cells were induced to undergo primary differentiation into embryoid bodies (EBs). Day 9–11 EBs, representing definitive hematopoiesis, were re-plated with hematopoietic cytokines (stem cell factor, interleukin (IL)-3, IL-6 and erythropoietin) in methylcellulose, and secondary differentiated colonies were scored on day 10. S17-10H1 cells were transfected by electroporation with a plasmid vector expressing either wild-type Rps19 cDNA or an empty vector control. Protein and mRNA levels of the tumor suppressor p53 were measured at the ES cell stage. Secondary differentiated hematopoietic colonies were grown with and without glucocorticoids (either dexamethasone at 1nM, 10nM, 100nM, 1μM or hydrocortisone at 50nM, 2.5μM concentration). Results:Western blot analyses confirmed S17-10H1 Rps19-haploinsufficiency. The mutant cell line had reduced EB formation (hematopoietic and non-hematopoietic) following primary differentiation. Significant defects in both erythroid (BFU-E) and myeloid (CFU-GM) formation were found following secondary hematopoietic differentiation of day 9–11 EBs. These defects were specific to Rps19 haploinsufficiency since all defects were rescued by stable transfection of the mutant cell lines with an Rps19-expressing correction vector. However, glucocorticoid treatment was unable to rescue defects in secondary differentiation (hematopoietic colony formation). Finally, we also examined baseline levels of the tumor suppressor p53, which may be induced following abortive ribosome assembly resulting from ribosomal protein haploinsufficiency. Although there was no difference in transcription levels of p53 mRNA between mutant and control cells as determined by quantitative PCR, baseline levels of p53 protein were significantly increased in the mutant ES cells as compared to controls. Conclusions:(1) In our model system, Rps19 mutation was associated with a relatively early defect in hematopoietic progenitors, since both erythroid and myeloid (or mixed) colony formation was diminished.(2) Hematopoietic defects in our murine ES cell model of DBA were rescued by Rps19 gene transfer but not by glucocorticoid therapy.(3) We found significant accumulation of baseline p53 protein (but not mRNA) levels in mutant ES cells, suggesting that Rps19 haploinsufficiency leads to inhibition of the p53-degradation pathway.(4) Taken together, these results suggest that p53 may antagonize glucocorticoid-induced proliferation in our Rps19-mutant ES cell model. Experiments to test this hypothesis are in progress. Reference:Ganguli G, Back J, Sengupta S, Wasylyk B. The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors. EMBO Rep. 2002;3:569-574. Disclosures:No relevant conflicts of interest to declare.