RPS19 Mutations Research Articles

Generation of iPSC lines and isogenic gene-corrected lines from two individuals with RPS19-mutated Diamond-Blackfan anemia syndrome

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients. CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells. To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

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.

Mutations in RPS19 may affect ribosome function and biogenesis in Diamond Blackfan anemia.

Ribosomes, the cellular organelles translating the genetic code to proteins, are assemblies of RNA chains and many proteins (RPs) arranged in precise fine‐tuned interwoven structures. Mutated ribosomal genes cause ribosomopathies, including Diamond Blackfan anemia (DBA, a rare heterogeneous red‐cell aplasia connected to ribosome malfunction) or failed biogenesis. Combined bioinformatical, structural, and predictive analyses of potential consequences of possibly expressed mutations in eS19, the protein product of the highly mutated RPS19, suggest that mutations in its exposed surface could alter its positioning during assembly and consequently prevent biogenesis, implying a natural selective strategy to avoid malfunctions in ribosome assembly. A search for RPS19 pseudogenes indicated > 90% sequence identity with the wild‐type, hinting at its expression in cases of absent or truncated gene products.

Large Deletions and Novel Mutations in Genes Coding for Ribosomal Proteins (RPL11, RPL35A, RPS19) in the Czech National Registry of Diamond-Blackfan Anemia (DBA)

Background: 50-70% cases of Diamond-Blackfan anemia (DBA) are underlied by mutations in genes coding for ribosomal proteins (RP), however, the etiology remains unexplained in the remaining patients. Recently, rare aberrations involving extraribosomal genes (e.g., GATA1, TSR2, MCM2, FLNB) have been reported as causative for DBA. Up to 10% of all DBA patients harbor large deletions involving some of the RP genes, which were not previously identified by sequencing.Patients and Methods: Czech National Registry, containing data of 56 patients with DBA from Czechia and Slovakia (both children and adults), has a high detection rate of RP gene mutations (currently 82%). Using exome sequencing (Agilent SureSelect v6 Kit, HiSeq 2500 sequencer, Illumina) and array comparative genomic hybridization (aCGH, SurePrint G3 CGH+SNP microarray, 4x180K, Agilent), we examined periferal blood samples of patients without any proven mutation in RP genes. Identified mutations were verified by Sanger sequencing at the gDNA and cDNA level and their functional impact was studied in a cell culture (RPS11-deficient MRC-5 fibroblasts).Results: Novel mutations, previously unpublished and unrecorded in gene databases, or large deletions involving RP genes were found in 5 patients with DBA:1)In a 10-year-old female with growth and developlmental delay, severe skeletal anomalies (hypertelorism, high-arched palate, low-set ears, triphalangeal thumbs) and with a markedly elevated activity of erythrocytic adenosine-deaminase (eADA), a large deletion in 1p36 chromosomal region - arr[hg19] 1p36.12p36.11(22308520_24074348)x1 - involving the RPL11 gene was found. Depletion of RPL11 in a cellular model resulted in attenuation of proteosynthesis and cell cycle and in the disruption of nucleolar morphology.2) In a 4-year-old female with transfusion-dependent DBA, large deletion of the 3q29 region - arr[hg38] 3q29(194309533_197837049)x1 - including the RPL35A gene was detected. The patient's height is at the 10th percentile, but her weight is below the 3rd percentile. Mild intellectual disability and both atrial and ventricular septal defect are present.3) A 4,5-year-old male with transfusion dependency, iron overload and hepatopathy harbors mutation in RPS19 - hg38[19:41869031_C>T], RPS19 p.A58V. The patient's father with an identical mutation required repeated blood transfusions in childhood (until 6 years of age) due to hypoplastic erythropoiesis. To date, all parameters of his blood count are normal without any intervention, only his eADA level is slightly elevated.All above mentioned patients are treated with leucine.4) A 3-year-old male with normal phenotype harbors mutation hg38[19:41860841_A>T] in RPS19 leading to premature stop codon K23Stop.5) Mutation in RPS19 : c.[3G>A], p.M1I in an 1 year old boy whose DBA is transfusion dependent and resistant to steroids.Conclusion: Together with next generation sequencing, aCGH examination is essential for detection of novel genetic defects in DBA due to its capacity to detect larger chromosomal aberrations. Reports on 3q29 deletions involving RPL35A are scarce (only 7 cases published to date). Suggested connection between deletions, leading to haploinsufficiency of RP genes, and growth retardation (Kuramitsu et al., Blood 2012) was confirmed only for RPL11 but not for RPL35A in our study. Phenotypic differences between family members harboring the novel identical RPS19 aberration are probably influenced by epigenetic regulation and we have already observed this phenomenon in individuals with RPS7 and RPS19 mutation (Maceckova et al., ASH 2016).Supported by: AZV 16-32105A, NPU LO 1304, GA15-13732S DisclosuresNo relevant conflicts of interest to declare.

Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71–CD34+ CD38mo/lo], megakaryocyte–erythroid progenitor cells (MEP) [CD71–CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.

Computational Studies of the Structural Basis of Human RPS19 Mutations Associated With Diamond-Blackfan Anemia.

Diamond-Blackfan Anemia (DBA) is an inherited rare disease characterized with severe pure red cell aplasia, and it is caused by the defective ribosome biogenesis stemming from the impairment of ribosomal proteins. Among all DBA-associated ribosomal proteins, RPS19 affects most patients and carries most DBA mutations. Revealing how these mutations lead to the impairment of RPS19 is highly demanded for understanding the pathogenesis of DBA, but a systematic study is currently lacking. In this work, based on the complex structure of human ribosome, we comprehensively studied the structural basis of DBA mutations of RPS19 by using computational methods. Main structure elements and five conserved surface patches involved in RPS19-18S rRNA interaction were identified. We further revealed that DBA mutations would destabilize RPS19 through disrupting the hydrophobic core or breaking the helix, or perturb the RPS19-18S rRNA interaction through destroying hydrogen bonds, introducing steric hindrance effect, or altering surface electrostatic property at the interface. Moreover, we trained a machine-learning model to predict the pathogenicity of all possible RPS19 mutations. Our work has laid a foundation for revealing the pathogenesis of DBA from the structural perspective.

Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

Single-Cell Transcriptional Landscapes of Human Bone Marrow Reveal Distinct Erythroid Phenotypes Underpinned By Genotype in Diamond-Blackfan Anemia

Background: Diamond- Blackfan Anemia (DBA) is a rare, heritable ribosomopathy caused by mutations in ribosomal protein large (RPL) and small (RPS) subunit genes. The diagnostic criteria of DBA include presentation in infancy with virtually no mature erythroblasts (EB) on bone marrow (BM) examination, however atypical presentations in later life with milder haematological manifestations are increasingly reported. The cellular and molecular mechanisms underpinning variable clinical phenotypes, and how they relate to genotype, are yet to be elucidated. Furthermore, as many DBA patients do not respond to corticosteroids, understanding the pathological processes contributing to erythroid failure is a prerequisite for developing novel, precision-medicine therapies. Aim: To elucidate phenotypic and functional differences in erythropoiesis in RPS- and RPL-DBA, using primary BM samples from patients. Methods: We performed single-cell transcriptome profiling (scRNAseq), using the 10X Genomics chromium platform, of 45,488 CD34+ Lineage negative (Lin-) BM hematopoietic stem and progenitor cells (HSPC), purified by fluorescence-activated cell sorting. We included six patients with red cell transfusion-dependent DBA (aged 2-19y) with mutations in RPS19 (n=3), RPL11 (n=1) and RPL5 (n=2) and three healthy donors (aged 3-17y). We validated our findings using bulk RNAseq, functional assays, and deep immunophenotyping-based dissection of the haematopoietic architecture of DBA BM ex vivo. Results: High quality sequencing data was obtained for all nine donors; after quality control, 41,415 single cells were carried forward for analysis. Unsupervised clustering analysis and lineage identification revealed two divergent cellular patterns in DBA compared with age-matched control BM that segregated with genotype: a selective loss of erythromegakaryocyte (E/MK) progenitors in RPS-DBA, but relative preservation of the erythroid developmental trajectory in RPL-DBA, at the expense of megakaryopoiesis (Fig 1a). Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between control and DBA HSPC clusters revealed p53-mediated apoptosis, TNFa-, IFNa- and IFNg-mediated inflammatory pathways in DBA EP. Although these pathways were enriched across all HSPC populations irrespective of genotype, they were detected at an earlier stage of the stem cell hierarchy, and more potently, in RPS- versus RPL-EP. Expression of transcriptional targets of the master E/MK transcription factor, GATA1, was significantly upregulated in RPL- versus RPS-DBA EP (Fig 1b), supporting the maintained erythroid program in RPL-DBA HSPC. However, expression of genes denoting erythroid differentiation, including adult haemoglobin (Hb) genes, was significantly elevated in RPL-DBA, suggesting aberrant accelerated differentiation to EB. These findings were confirmed by immunophenotypic examination of patient BM and bulk RNAseq of RPL-EB. Additionally, single-cell clonogenic assays of RPL-DBA EP showed severe qualitative defects. Thus, although erythroid commitment occurs in RPL-DBA, it is coupled with accelerated maturation beyond EP to functionally impaired mature EB, enriched in inflammatory and p53 pathways. Finally, we analysed the clinical characteristics of the 161 patients comprising the U.K DBA registry. In line with the milder erythroid specification defect, patients with RPL-DBA (n=44) presented with anaemia later (P=0.004), and with a higher average Hb concentration (P=0.04), than those with RPS-DBA (n=63). Furthermore, we identified higher corticosteroid responses in RPL-DBA, assessed at 6 months post initiation (P=0.006), consistent with our findings in RPL-DBA of preservation of the cellular EP populations that are targeted by corticosteroids. Impact: In conclusion, a preserved but distinct erythroid developmental trajectory, characterised by accelerated differentiation, underpins a milder haematological phenotype in RPL-DBA. Furthermore, we reveal the first single-cell transcriptomic dataset from haematopoietic cells in a ribosomopathy, uncovering novel cell intrinsic and extrinsic pathogenetic insights into failing erythropoiesis in DBA. Integration of these data with clinical genomics and phenomics provides a paradigm by which single cell approaches can be used to decipher genotype-phenotype relationships in Mendelian genetic disorders. Disclosures Mead: Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy; CTI: Consultancy; Gilead: Consultancy.

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.

Anemia of Blacfan Diamon in an infant at the Albert Royer National Children's Hospital in Dakar and Review of the Literature

Blackfan Diamon anemia is the only known congenital erythroblastopenia. Following the discovery of the genes involved, it has become the leader in ribosomal diseases and this has opened the way to a great deal of basic research into erythropoiesis. It is defined by severe erythroblastopenia with less than five percent of erythroid precursors in a single-cell marrow. Blackfan Diamon's anemia manifests early before the age of two and its incidence is low in Africa, we report a case with early onset in a 03-month-old infant with RPS19 mutation. The diagnosis often remains difficult due to the unavailability of genetic tests. Corticosteroid therapy was prescribed for two weeks without significant improvement. The patient is currently on a transfusion routine while awaiting a bone marrow transplant.

Diamond–Blackfan anemia with mutation in RPS19: A case report and an overview of published pieces of literature

ABSTRACTIntroduction:Diamond–Blackfan anemia (DBA), one of a rare group of inherited bone marrow failure syndromes, is characterized by red cell failure, the presence of congenital anomalies, and cancer predisposition. It can be caused by mutations in the RPS19 gene (25% of the cases).Methods:This case report describes a 10-month-old boy who presented with 2 months’ history of gradually increasing weakness and pallor.Results:The patient was diagnosed as a case of DBA based on peripheral blood finding, bone marrow aspiration with trephine biopsy reports, and genetic mutation analysis of the RPS19 gene. His father refused hematopoietic stem cell transplantation for financial constraints. Patient received prednisolone therapy with oral folic acid and iron supplements.Conclusion:Hemoglobin raised from 6.7 to 9.8g/dL after 1 month of therapeutic intervention.

Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

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.

Cells Deficient in the Shwachman-Diamond Syndrome Protein SBDS or the Diamond-Blackfan Anemia Protein RPS19 Have Impaired Homologous Recombination

The inherited bone marrow failure syndromes (IBMFS) are rare genetic disorders caused by mutations in critical components of fundamental cellular processes such as ribosome biogenesis, DNA repair, and telomere maintenance. The IBMFS Shwachman-Diamond syndrome(SDS) and Diamond-Blackfan anemia (DBA) are classified as ribosomopathies due to etiologic mutations in genes encoding factors involved in ribosome biogenesis (SBDSin the majority of patients with SDS) or ribosomal proteins (RPS19most commonly in patients with DBA). Although these disorders can be distinguished clinically and from the other IBMFS, they share with each other and with other IBMFS increased predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas genomic instability due to defective DNA repair or telomere maintenance is thought to underlie cancer predisposition in the IBMFS Fanconi anemia and dyskeratosis congenita, respectively, the molecular mechanisms driving cancer in SDS and DBA are not fully understood. Our research has focused on DNA repair in SDS and DBA. A prior report suggested lymphoblastoid cell lines (LCLs) derived from patients with SDS arehypersensitive to ionizing radiation (IR). Consistent with this, we found SDS-LCLs had decreased survival following IR compared to control-LCLsin colony survival assays. To determine if this cellular phenotype was unique to SDS or present in the other IBMFS ribosomopathy, DBA, we examined LCLs derived from patients with DBA, including those with mutations in RPS19, RPS26, RPL5and RPL11. We found that the DBA-LCLs were similarly hypersensitive to IR as compared to control-LCLs. Further examination of γ-H2AX, a DNA damage response (DDR) factor and marker of DNA double strand breaks (DSBs), revealed that SDS- and DBA-LCLs had delayed resolution of γ-H2AX foci and increased protein levels at 24 hrs after IR as compared to control LCLs. p53, phospho-ATM, and DNA-PKcs protein levels were also higher in SDS-LCL compared to controls. The decreased survival and increased and sustained DDR following IR led us to hypothesize that SDS and DBA cells have a defect in DSB repair. There are two major pathways of DSB repair in mammals, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), and loss of either results in hypersensitivity to IR. To examine each pathway, we employed U2OS (human osteosarcoma) and HCT116 (human colon cancer) cells containing an integrated green fluorescent protein HDR or NHEJ reporter transgene. Interestingly, we found that knockdown of either SBDS or RPS19 proteins resulted in an approximately 50% reduction in HDR efficiency but no change in NHEJefficiency compared to the scrambled control in both cell lines. We next sought to determine the mechanism underlying the effect of SBDS and RPS19 deficiency on HDR. A survey of proteins required for HDR revealed a reduction in the recombinase RAD51 in SDS-LCLs and in SBDS-depleted HCT116 and U2OS cells, whereas, an initial survey in SDS-LCLs[e1] of factors involved in NHEJ did not reveal a specific NHEJ factor deficiency. Knockdown of eiF6 is known to rescue the defect in 40S and 60S ribosome subunit joining that manifests in SDS patient cells. However, we found eIF6 depletion failed to rescue the level of RAD51 protein and had no impact on HDR in SBDS-deficient cells. We conclude that decreased RAD51 levels in SBDS-deficient cells might contribute to impaired HDR, however, this decrease is independent of the ribosome subunit joining defect. Similarly, RPS19 knock down resulted in a reduction in RAD51 protein level, suggesting a potentially common pathway. We also asked whether SBDS or RPS19 might be more directly involved in the DDR or repair of DSBs. Consistent with this, we found SBDS and RPS19 recruited to chromatin surrounding an I-Sce1 site following DSB induction in chromatin immunoprecipitation assays. Collectively, these findings provide evidence that SBDS and RPS19 may be directly involved in the DDR or DSB repair and raise the possibility that loss of this function may contribute to MDS/AML predisposition in SDS and DBA patients. Disclosures No relevant conflicts of interest to declare.

Malignancies and MDS in Patients with Diamond-Blackfan Anemia (DBA) from the Czech National DBA Registry

Introduction: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by macrocytic anemia, reticulocytopenia, selective deficiency of erythroid precursors, presence of congenital anomalies and an increased risk of cancer. DBA is caused by germline mutations of genes coding for ribosomal proteins (RP). Interestingly, somatic RP mutations have also been found in several malignant diseases (T-ALL, CLL, Hodgkin lymphoma, myelodysplastic syndrome (MDS) and AML). The only representative overview of malignancies in DBA was published in 2012 based on the data from the North American DBA Registry. The malignancies developed in 3% of patients (MDS and AML in 4 patients and solid tumors in 15 patients). The observed-to-expected ratio for all cancers combined was 5.4 (p<0.05). Median age at diagnosis was 41 years and cumulative incidence of solid tumors/leukemias was approximately 20% at the age of 46 years. Five years later, additional solid tumors were reported, in particular gastrointestinal tumors, and an incidence of MDS was alarming. Our aim was to evaluate cancer incidence within Czech National DBA Registry and characterize underlying molecular pathology. Patients and methods: Czech National DBA Registry currently includes 62 patients. In cooperation with 8 national centers of pediatric and adult hematology, we collected data about patients followed with pre-malignant or malignant condition including a detailed analysis of the course of the disease, treatment and type of developed malignant disorder. In patients with an evolving MDS, a classical cytogenetic analysis, flow cytometry, mutational profile (TruSight Myeloid Sequencing Panel (Illumina) containing 54 genes) and commercial TUNEL assay for detection of apoptotic changes of erythroid cells in the bone marrow were performed. Results: Eight of 62 patients from the Registry (13%) had malignant or pre-malignant condition: four females (6.5%) had solid tumors, 3 males (4.8%) had MDS and one female (1.6%) had multiple myeloma. Age of the onset of these disorders ranged between 25-70 years. Three patients harbored RPL5 mutation, 3 patients RPL11 mutation and 2 patients RPS19 mutation. Cancer incidence was significantly higher within the RPL11 and RPL5 subgroups (p=0.0056, Fisher Exact Test). Two female patients were diagnosed with triple-negative breast carcinoma, both of them during pregnancy. The first patient died despite treatment at the age of 29 years, shortly after delivery of her second child. The second patient is currently undergoing neoadjuvant chemotherapy.One patient developed diffuse large B cell lymphoma (DLBCL), underwent chemotherapy and autologous BMT and is alive in second remission.One patient underwent hemicolectomy for colorectal adenocarcinoma at 53 years of age and is in remission 6 years after the surgery.One patient succumbed to multiple myeloma which evolved from monoclonal gammopathy of unknown significance (MGUS) after 14 years of follow-up.Three male patients developed suspected MDS at the age of 25, 28 and 29 years. Two of them had RPL5 mutation and one harbored RPS19 mutation. None of them had decrease of erythroid cells in the bone marrow, but apoptosis of erythroid progenitors was significantly increased in all cases. All 3 patients had bicytopenia in peripheral blood (anemia, leukopenia) and dysplasia in 2 or 3 hematopoietic lineages in the bone marrow, thus fulfilling criteria for MDS with multilineage dysplasia (MDS-MLD). They had no abnormalities detected by flow cytometry or cytogenetics. The patient with RPS19mut harbored ASXL1 mutation; in patient with RPL5 mut, mutational screening is ongoing. Conclusion: Our results confirmed increased incidence of cancer (13%) in patients with DBA at young age. Our cases of DLBCL and MGUS in DBA are the first published to date. In cases with suspected MDS, cytopenia and dysplastic changes in the bone marrow may either reflect specific features of ribosomopathy, or represent a severe disorder of regulatory mechanisms with propensity to clonal proliferation. International collaboration is required to refine the incidence of malignancies in DBA and to issue consensus guidelines for timely detection of solid tumors, leukemias and MDS. The best approach to DBA patients who developed MDS without harboring any mutation considered prognostically significant in MDS, is another subject of discussion. Support: NV16-32105A Disclosures No relevant conflicts of interest to declare.

TARGETING P53 AND NUCLEOLAR STRESS IN DIAMOND-BLACKFAN ANAEMIA

Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.

Ribosome biogenesis protein Urb2 regulates hematopoietic stem cells development via P53 pathway in zebrafish

Ribosome biogenesis is a significant process in cells. Dysfunction in this process will result in the defects of protein synthesis and consequently cause the development of specific diseases called ribosomopathies. Mutations in ribosome biogenesis protein Rps19, Rpl5, or Rpl11 can lead to hematopoietic defects in human, thus triggering the disease Diamond Blackfan anemia. However, the regulatory mechanisms of ribosome biogenesis in hematopoiesis remain incompletely understood. In this study, we describe a zebrafish mutant cq42, which carries a nonsense mutation in the gene that encodes ribosome biogenesis 2 homolog (Urb2). Urb2 is strongly expressed in the caudal hematopoietic tissue (CHT) during hematopoietic stem cells (HSCs) expanding. Molecular characterization of urb2cq42 larvae suggest that urb2 deficiency notably decrease the population of HSCs in CHT and early T cells in thymus. Further analysis shows that compromised cell proliferation and superfluous apoptosis are observed in the CHT of urb2cq42 mutant. P53 pathway is upregulated in the urb2cq42 larvae and loss-of-function of P53 can fully rescue the hematopoietic defects in urb2cq42 mutant. These data demonstrate that urb2 is essential for HSCs development through the regulation of P53 pathway.

Molecular analysis and genotype-phenotype correlation of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) features hypoplastic anemia and congenital malformations, largely caused by mutations in various ribosomal proteins. The aim of this study was to characterize the spectrum of genetic lesions causing DBA and identify genotypes that correlate with phenotypes of clinical significance. Seventy-four patients with DBA from across Canada were included. Nucleotide-level mutations or large deletions were identified in 10 ribosomal genes in 45 cases. The RPS19 mutation group was associated with higher requirement for chronic treatment for anemia than other DBA groups. Patients with RPS19 mutations, however, were more likely to maintain long-term corticosteroid response without requirement for further chronic transfusions. Conversely, patients with RPL11 mutations were less likely to need chronic treatment. Birth defects, including cardiac, skeletal, hand, cleft lip or palate and genitourinary malformations, also varied among the various genetic groups. Patients with RPS19 mutations had the fewest number of defects, while patients with RPL5 had the greatest number of birth defects. This is the first study to show differences between DBA genetic groups with regards to treatment. Previously unreported differences in the rate and types of birth defects were also identified. These data allow better patient counseling, a more personalized monitoring plan, and may also suggest differential functions of DBA genes on ribosome and extra-ribosomal functions.