Pathogenesis Of Diamond-Blackfan Anemia Research Articles

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.

Small Molecule Inhibitor Targeting Nemo-like Kinase Improves Erythropoiesis in Human and Mouse Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies, and an increased risk of developing cancer. Approximately 80% of patients with DBA have a mutation in one of 20 different genes that encode ribosomal proteins, resulting in abnormal protein translation. The current standard of care for patients with DBA includes steroids, chronic red cell transfusions, or stem cell transplantation, but these are all associated with significant side effects including infections, iron overload, and the risk of graft versus host disease. Thus, the development of more effective and less-toxic therapies is needed to treat the anemia seen in patients with DBA. Our previous work demonstrated that Nemo-Like Kinase (NLK) is overly active in red cell precursors in models of DBA as well as in patient samples (Wilkes, et al., 2020). We hypothesize that NLK plays an important role in the pathogenesis of DBA, and is a potential target for DBA therapy. Human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) were transduced with lentivirus co-expressing GFP and shRNA against RPS19/RPL11 or luciferase (control), and were differentiated in erythroid media in the presence of small molecule NLK inhibitors. After screening small molecule compounds that inhibit NLK as an off-target from previously approved or clinically advanced drugs, we found that a known MELK (maternal embryonic leucine zipper kinase) inhibitor, OTS167, improved erythropoiesis (CD71+ cell expansion) by 31% and 27% at a concentration of 50nM in RPS19- and RPL11-knockdown HSPCs, respectively. We also investigated the effect of OTS167 on the erythropoiesis in Rpl11-haploinsufficient mouse model of DBA ( Rpl11 wt/LoxpCre-ERT2 +, Morgado-Palacin, et al., 2015 and Rpl11 wt/LoxpMx-Cre + Liu,et al.,2023). Lineage-negative (Lin-) hematopoietic progenitors isolated from the mouse bone marrow were cultured in erythroid differentiation medium with OTS167. Our data showed that the erythroid progenitors (CD71+ cells) expanded from the cultured Lin- cells were increased by 20% (n=2, p=0.065) at an OTS167 concentration of 30nM. We confirmed that NLK activity was increased in erythroid progenitors from the Rpl11-haploinsufficient mice for in vitro phosphorylation of the substrate compared with tamoxifen-treated wild type mice. Moreover, we performed colony formation assay with OTS167 in the mouse bone marrow cells. OTS167 increased BFU-E colony numbers in a dose-dependent manner with the highest effect at a concentration of 50nM (n=3, p=0.003). OTS167 did not increased CFU-E colony number, suggesting that OTS167 affected the BFU-E stage and/or earlier stages of erythroid development. Also, OTS167 did not affected CFU-GM colony formation as myeloid lineage. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to minimize the need for chronic red cell transfusions or treatment with steroids. Our in vitro data suggest that pharmacologic inhibition of NLK with OTS167 may achieve this goal. We have initiated studies to test the efficacy of OTS167 in vivo using Rpl11-haploinsufficient mouse models. Given that DBA is associated with a number of mutations in the ribosome, using one drug that targets NLK as a common therapeutic target could be a more practical treatment option for patients in contrast to gene therapy or gene editing followed by autologous stem cell rescue. Even if the drug targeting NLK does not completely rescue the anemia in ribosome-insufficient cell models of DBA, the improvement could be enough to help patients especially in combination with other drugs that are currently being used or studied .

Novel Mouse Model That Recapitulates the Hematologic Phenotype of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that presents during early childhood and is characterized by macrocytic anemia, congenital malformations, and predisposition to cancer. More than 90% of DBA patients are diagnosed during their first year of life (median age 12 weeks). The pathogenesis of DBA is linked to loss-of-function mutations in genes encoding ribosomal proteins (RP), although mutations in 3 non-RP genes ( GATA1, TSR2, and HEATR3) have been detected. DBA patients present with a high degree of clinical heterogeneity with varying severity and responses to steroid therapy. DBA provides a unique disease model to study how RP deficiency impacts the phenotype and pathogenesis of ribosomopathies. However, one challenge is the lack of animal models that faithfully recapitulate the clinical features of DBA in terms of disease severity and onset age. Heterozygous loss-of-function mutations in RPL11 are found in 5-20% of DBA patients, but previous mouse models carrying Rpl11 haploinsufficiency have only a mild anemia. Here, we report a novel mouse model of DBA with inducible haploinsufficient expression of Rpl11 which results in a severe macrocytic anemia in juvenile mice. Mx1-CreRpl11+/flox experimental mice were generated by cross-breeding homozygous Rpl11 flox/flox and Mx1-cre breeders. Haploinsufficiency of Rpl11 ( Rpl11+/Δ) was induced by intraperitoneal injection of polyinosinic-polycytidylic acidto mice with Mx1-CreRpl11 +/flox on postnatal day 8 and 10. Diseased mice with Rpl11+/Δ showed macrocytic anemia at 2 weeks (wks) post-induction with hemoglobin (Hb) < 10 g/dL, eventually dying of severe anemia (Hb< 2 g/mL) by 30 wks (median survival 27 wks, p<0.001, n=7 )( Figure 1). We observed a 50% decreased expression of Rpl11 in blood neucleated cells from Rpl11+/Δ mice and several characteristic clinical features of DBA. Erythrocyte adenosine deaminase was significantly elevated in the blood of all Rpl11+/Δ mice when compared to wild type (WT) littermates (median 5 vs 1 EU/g Hg, p<0.001, n=6). Similarly, EPO concentrations were significantly elevated in Rpl11+/Δ mice compared to WT mice (median 38752 vs 23 pg/mL, p<0.01, n=6). On postmortem examination, the DBA mice displayed splenomegaly. FACS analysis data demonstrated that the differentiation of erythropoiesis is significantly blocked in peripheral blood, bone marrow (BM), and spleen of Rpl11+/Δ mice compared to WT mice (p<0.05, n = 5). In addition, methylcellulose colony assays revealed that CFU-E colonies were significantly decreased in BM from Rpl11+/Δ mice in comparison to WT mice (median 23.5 vs 40.5 per 0.2 million BM cells in the presence of EPO at a concentration of 1 unit/mL, p<0.05, n=4). To determine whether BM hematopoietic stem cells/progenitor cells from Rpl11+/Δ mice can transfer the DBA phenotype, we transplanted bone marrow cells of Rpl11+/ Δ mice (CD45.2+) into sublethally irradiated WT mice (CD45.1+). Our data demonstrate that the BM cells from Rpl11+/Δ donor mice can induce lethal macrocytic anemia in WT recipient mice. Studies on the molecular mechanisms underlying the dysregulated erythropoiesis in Rpl11+/Δ mice are ongoing. In conclusion, we have successfully generated the first DBA mouse model that recapitulates the hematologic features of DBA patients. This model provides an ideal tool to study the pathogenesis of DBA and other ribosomopathies.

Animal models of Diamond-Blackfan anemia: updates and challenges.

Diamond-Blackfan anemia (DBA) is a ribosomopathy that is characterized by macrocytic anemia, congenital malformations, and early onset during childhood. Genetic studies have demonstrated that most patients carry mutations in one of the 20 related genes, most of which encode ribosomal proteins (RP). Treatment of DBA includes corticosteroid therapy, chronic red blood cell transfusion, and other forms of immunosuppression. Currently, hematopoietic stem cell transplantation is the only cure for DBA. Interestingly, spontaneous remissions occur in 10-20% of transfusion-dependent DBA patients. However, there is no consistent association between specific mutations and clinical manifestations. In the past decades, researchers have made significant progress in understanding the pathogenesis of DBA, but it remains unclear how the ubiquitous RP haploinsufficiency causes the erythroid-specific defect in hematopoiesis in DBA patients, and why there is a difference in penetrance and spontaneous remission among individuals who carry identical mutations. In this paper, we provide a comprehensive review of the development of DBA animal models and discuss the future research directions for these important experimental systems.

Nemo-like kinase as a potential target for treatment of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, in which the major defect is ineffective erythropoiesis. Over 70% of patients with DBA have mutations in ribosomal protein subunits, although the precise molecular mechanisms contributing to the pathogenesis of DBA are not well understood

Decoding the pathogenesis of Diamond–Blackfan anemia using single-cell RNA-seq

Ribosomal protein dysfunction causes diverse human diseases, including Diamond–Blackfan anemia (DBA). Despite the universal need for ribosomes in all cell types, the mechanisms underlying ribosomopathies, which are characterized by tissue-specific defects, are still poorly understood. In the present study, we analyzed the transcriptomes of single purified erythroid progenitors isolated from the bone marrow of DBA patients. These patients were categorized into untreated, glucocorticoid (GC)-responsive and GC-non-responsive groups. We found that erythroid progenitors from untreated DBA patients entered S-phase of the cell cycle under considerable duress, resulting in replication stress and the activation of P53 signaling. In contrast, cell cycle progression was inhibited through induction of the type 1 interferon pathway in treated, GC-responsive patients, but not in GC-non-responsive patients. Notably, a low dose of interferon alpha treatment stimulated the production of erythrocytes derived from DBA patients. By linking the innately shorter cell cycle of erythroid progenitors to DBA pathogenesis, we demonstrated that interferon-mediated cell cycle control underlies the clinical efficacy of glucocorticoids. Our study suggests that interferon administration may constitute a new alternative therapeutic strategy for the treatment of DBA. The trial was registered at www.chictr.org.cn as ChiCTR2000038510.

Downregulation of SATB1 by miRNAs reduces megakaryocyte/erythroid progenitor expansion in preclinical models of Diamond–Blackfan anemia

Diamond−Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because more than 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is due predominantly to a block and delay in early committed erythropoiesis with reduced megakaryocyte/erythroid progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA sequencing on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19 insufficiency. However, SATB1 expression did not affect expansion of committed erythroid progenitors, indicating ribosomal insufficiency affects multiple stages during erythroid differentiation.

Research Progress on Pathogenesis of Congenital Pure Red Cell Aplasia---Review

Congenital pure red cell aplasia, also known as Diamond-Blackfan anemia (DBA), is a hereditary disease characterized by pure red cell aplasia and congenital malformation. Its main clinical features are anemia, dysplasia, and tumor susceptibility. Ribosomal protein (RP) gene mutation is the main pathogenesis of DBA. The most common type of gene mutation is RPS19 gene mutation. Heterozygous mutations in as many as 19 RP genes and other non-RP genes mutations have been identified in DBA. This review summarized briedfly the latest research advances in the pathogenesis of DBA.

Mutations in Ribosomal Protein Genes L9 and L15 Link to DBA with Specific Clinical Phenotypes

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein (RP) gene mutations. The European (EuroDBA) consortium reports here new mutations identified in RP genes L15 and L9 in patients with DBA. Although point mutations have not been previously reported for RPL15 in DBA, we identified 6 unrelated children of whom 4 carried truncating mutations c.242dupA; p.Tyr81X (N=3) and c.85C>T; p.Gln29X (N=1). Additional 2 patients carried RPL15 missense variants c.29T>C; p.Leu10Pro and c.458A>C; p.Lys153Thr. Notably, of the 4 patients with truncating mutations, 3 manifested prenatally with severe hydrops fetalis and required between 4-9 intrauterine transfusions. Even more remarkable is the observation that 3/4 patients with this mutation type rapidly became treatment-independent and maintained normal blood counts until the last follow-up (age 5-16 years). This finding suggests that a previously unknown correlation between genotype and hematological phenotype may underlie the tendency of some DBA patients to undergo treatment independence. Additionally we report a pathogenic splice donor mutation c. -2+1G>C in RPL9, a gene that has not been previously implicated in DBA pathogenesis. The patient presented with DBA and severe colitis at the age of 6 months, and showed dysmorphic features including microcephaly and bilateral thumb abnormality.Cells carrying either RPL15 or RPL9 mutations revealed pre-rRNA processing defects, reduced 60S ribosomal subunit formation, a reduction of de novo protein synthesis, and stabilization of the TP53 tumor suppressor. Zebrafish models reducing either RP recapitulated a loss of hemoglobin-expressing cells in early development. Red cell culture assays of RPL15 - or RPL9 -mutated primary erythroblast cells revealed reductions of cell proliferation and delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. Colon tissue from patient with RPL9 mutation revealed intense epithelial TP53 staining, CD3+ lymphocytic infiltrates, and insufficient crypt regenerationThis study identifies RPL9 as a new DBA-linked gene, identifies recurrent truncating hotspot mutation in RPL15, reaffirms a role for TP53 stabilization in disease pathophysiology, and suggests that certain DBA genotypes may be used for risk stratification. To our knowledge, this is the first report on multiple DBA patients presenting with hydrops fetalis or colitis due to haploinsufficiency of specific RP genes. DisclosuresNo relevant conflicts of interest to declare.

Inflammatory Signature, Oxidative Stress, and DNA Damage Response in DBA Pathogenesis

Diamond-Blackfan anemia (DBA) is an inherited pure red cell aplasia caused mostly by pathogenic variants in genes encoding for ribosomal proteins (RPs). It was previously published that haploinsufficiency of RP leads to increased apoptosis of erythroid precursors and p53 upregulation in CD34+cells from DBA patients and in animal models. However, the exact mechanisms of p53 activation are still not completely understood. In our study, we examined the link between activation of p53 pathway, inflammation-evoked oxidative stress and DNA damage in DBA patients and cellular model.To investigate the pathogenesis of DBA, we prepared the RPL5- and RPS19-deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. The particular clones were heterozygous for different nucleotide size deletions in RPL5 or RPS19 genes resulting in decreased levels of these proteins to half when compared to control cells thus mimicking the RP haploinsuficiency in DBA patients. We examined the pathological consequences of RPL5 and RPS19 deficiency in both undifferentiated and differentiated cells; the differentiation was induced by either dimethyl sulfoxide - DMSO or hexamethylene bisacetamide - HMBA for at least 48 hours. Significantly decreased proliferation capacity and significantly increased apoptosis rate was detected for all undifferentiated RPL5- and RPS19-deficient clones in comparison with controlcells. Upon induction of erythroid differentiation, RPL5- and RPS19-deficient clones showed abnormal GATA1 expression (2-times lower than in control cells) and higher percentage of TUNEL+ apoptotic cells (RPL5-deficient cells 2.04%; RPS19-deficient cells 1.57%;controlcells 0.50%).The data from cellular models were consistent with an increased rate of apoptosis detected in the bone marrow of RPL5 and RPS19 mutant DBA patients by TUNEL assay (15-times more TUNEL+ cells in RPL5 and RPS19 mutant patients than in normal controls). These patients showed also induction of p53 in their bone marrow (p53 positive cells: 8.4% in RPL5-mutant patient and 7.3% in RPS19-mutant patient vs. 0.4% in normal controls). The activation of p53 was also confirmed in RPL5- and RPS19-deficient clones by detection of increased level of p53 phosphorylation.Because oxidative stress is known to induce p53, we examined oxidative DNA damage marker 8-oxoguanine (8-oxoG) in MEL-DBA clones. Cytospin slides of differentiated RPL5- and RPS19-deficient clones revealed elevated positivity for 8-oxoG in contrast to control cells. It is known that oxidative damage is caused by exposure of cells to inflammatory cytokines, which activate DNA damage response (DDR) through reactive oxygen species (ROS)-mediated DNA damage. We found upregulation of pro-inflammatory cytokines TNF-α and IL6 in RPL5- and RPS19-deficient cells and IL1b in RPS19-deficient cells. The increased expression of Map3k8 kinase or increased phosphorylation of p38 was detected in RPL5-deficient or RPS19-deficient cells; both of these molecules are known to play essential roles in the production of pro-inflammatory cytokines. We then analyzed the levels of pro-inflammatory cytokines in our DBA patients and observed that besides TNF-α, IL1b, and IL6 also other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF-γ, and GM-CSF) were upregulated in their serum. We also detected higher levels of ROS in differentiated MEL-DBA clones in comparison with controlcells. Increased ROS levels were also present in the serum of all DBA patients with elevated inflammatory cytokines in contrast to healthy controls. As a result of ROS production, DNA double-strand breaks can occur responding by phosphorylation of Ser-139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RPL5- and RPS19-deficient cells in comparison with control cells (mean intensity of fluorescence for RPL5-deficient cells: 2.63 ±0.34; RPS19-deficient cells: 3.29 ±0.41; and control cells: 1.02 ±0.22).Our results suggest that the defective ribosomal biogenesis is associated with the induction of inflammatory cytokine signature, oxidative stress, increased DNA damage and activation of DDR signaling. Further research is needed to elucidate the hierarchy of these processes.This study was supported by project GACR 15-13732S, AZV 16-32105A, IGA_LF_2017_015, and in part by LTAUSA17142 (BK). 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.

Erythropoiesis Failure in Diamond-Blackfan Anemia Starts at the Oligopotent Common Myeloid and Megakaryocyte-Erythroid Progenitor Stage

Diamond-Blackfan anemia (DBA) is a hereditary bone marrow failure disorder that is characterized by erythropoiesis failure and chronic anemia and is frequently associated with physical malformations. Considered a ribosomopathy, most patients harbor pathogenic mutations in one of at least 22 large or small ribosomal protein genes in an autosomal dominant manner, however rarer cases involving mutations in GATA1, TSR2 and EPO have also been described. A recent study of the architecture of the human hematopoietic hierarchy throughout development (Notta et al, Science 2016) showed that, after birth, unipotent progenitors can derive directly from multipotent progenitors without intermediate differentiation into oligopotent progenitors. Critically, this work identified novel progenitors for which the abundance, clonal capacity and progenitor function is currently unknown in many benign and malignant hematological disorders. Specifically, multipotent, common myeloid and megakaryocyte-erythroid progenitors (MPP, CMP and MEP) were found to be functionally heterogeneous and could be subdivided (F1, F2 and F3 subtypes) based on the combination of CD71 and BAH1 expression. We hypothesized that in-depth analysis of these novel and previously reported progenitors will better elucidate hematopoiesis in DBA, providing insights in to DBA pathogenesis and erythropoietic failure. Using flow cytometry, we quantified the frequencies of 11 hematopoietic stem and progenitor cell (HSPC) populations: HSC, MPP F1-F3, CMP F1-F3, MEP F1-F3 and granulocyte-monocyte progenitors (GMP), from the BM specimens of 8 DBA patients harboring ribosomal protein mutations and 6 healthy age-matched control donors. To characterize the function of HSPCs in DBA we then utilized a highly sensitive and quantitative single-cell in-vitro stromal feeder-based differentiation assay that supports the expansion and lineage commitment of single HSCPs towards mature cell types (myeloid, erythroid and megakaryocytes) with progeny immunophenotyping and quantification by flow cytometry. Our refined quantification of HSPCs by flow cytometry showed that the defect of erythropoiesis in DBA patients starts at the CMP/MEP compartment. Namely, CMP-F2 and CMP-F3 as well as MEP-F2 and MEP-F3 sub-populations that are predicted to form erythroid cells were significantly reduced in DBA patients compared to healthy aged-matched individuals (Figure 1A-B). In contrast, CMP/MEP-F1 sub-populations that are predicted to form only myeloid cells (BAH1-/CD71-) showed no significant difference compared to healthy controls. Importantly, the multipotent compartment (HSC and MPP) did not significantly differ from that of healthy controls. Functional analysis of F2/F3 CMP/MEP HSCP sub-populations in single-cell colony assays showed that they have a high propensity to produce erythroid progeny in healthy control samples (Figure 1C). In contrast, in DBA patients these HSCPs had limited erythroid forming potential, but maintain a high propensity to develop myeloid colonies (Figure 1D). To our knowledge this is the first study that the HSCP sub-populations F1/F2/F3 HSCP fractions have been analyzed in DBA. Collectively, these data indicate that the hematopoietic defect in DBA occurs in erythroid-producing CMP/MEP populations. Our data show that these sub-populations are both reduced and dysfunctional in DBA patients and we suggest these abnormalities lead to the pure red cell aplasia seen in these patients. Figure 1: DBA patients have reduced CMP and MEP sub-populations that behave dysfunctional in-vitro. A-B) DBA patients have reduced CMP-F2/3 (A) and MEP-F2/3 (B) sub-populations in their bone marrow. C) CMP F2-F3 and MEP F2-F3 have a high propensity to produce erythroid cell types in healthy controls. D) Single-cells isolated from the CMP-F2/3 and MEP-F2 population of DBA patients produced proportionally fewer erythroid and greater myeloid colonies in in-vitro colony assays (plot shows the mean respective proportions from all healthy controls and DBA patients tested, each N=5). Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

A Review of Diamond-Blackfan Anemia: Current Evidence on Involved Genes and Treatment Modalities.

Diamond-Blackfan anemia (DBA) is a congenital cause of bone marrow failure predominantly involving the erythroid cell line, with occasional impact on other cell lines. In the vast majority of cases, it is diagnosed by one year of age. We looked at the existing literature on the disease presentation along with established as well as upcoming treatment options. Numerous genes have been identified and extensively studied in the context of their part in the pathogenesis of DBA. Treatment revolves around the use of steroids and regular blood transfusions, with hematopoietic stem cell transplantation reserved for steroid-resistant cases. Newer modalities such as gene therapy, l-leucine, sotatercept, trifluoperazine, SMER28, and danazol are also concisely discussed. The purpose of this article is to review the previous literature on DBA and weigh the role of newer therapeutic agents.

The nuclear gene rpl18 regulates erythroid maturation via JAK2-STAT3 signaling in zebrafish model of Diamond\u2013Blackfan anemia

Diamond–Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome, characterized by red blood cell aplasia, developmental abnormalities, and enhanced risk of malignancy. However, the underlying pathogenesis of DBA is yet to be understood. Recently, mutations in the gene encoding ribosomal protein (RP) L18 were identified in DBA patients. RPL18 is a crucial component of the ribosomal large subunit but its role in hematopoiesis remains unknown. To genetically model the ribosomal defect identified in DBA, we generated a rpl18 mutant line in zebrafish, using CRISPR/Cas9 system. Molecular characterization of this mutant line demonstrated that Rpl18 deficiency mirrored the erythroid defects of DBA, namely a lack of mature red blood cells. Rpl18 deficiency caused an increase in p53 activation and JAK2-STAT3 activity. Furthermore, we found inhibitors of JAK2 or STAT3 phosphorylation could rescue anemia in rpl18 mutants. Our research provides a new in vivo model of Rpl18 deficiency and suggests involvement of signal pathway of JAK2-STAT3 in the DBA pathogenesis.

PF345 THE INVOLVEMENT OF OXIDATIVE STRESS, INFLAMMATION, AND DNA DAMAGE RESPONSE IN PATHOPHYSIOLOGY OF DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia mostly caused by different pathogenic variants in ribosomal protein (RP) genes. The ribosomal stress and subsequent activation of the p53 tumor suppressor pathway result in increased apoptosis of erythroid precursors and/or induction of cell cycle arrest.Aims:We studied the contribution of p53 pathway, oxidative stress, and inflammation to the DBA pathophysiology.Methods:We analyzed the bone marrow and peripheral blood samples from DBA patients listed in the Czech DBA Registry. We also prepared the RPL5‐ and RPS19‐deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. Activation of p53 pathway, oxidative DNA damage, and pro‐inflammatory cytokines were assessed in undifferentiated and differentiated RPL5‐ and RPS19‐deficient cells; the differentiation was induced by dimethyl sulfoxide (DMSO) for at least 48 hours.Results:Created RP‐deficient MEL cells presented the main phenotypic features of DBA patients’ erythroid cells: decreased levels of RPL5 or RPS19 proteins, decreased proliferation capacity, increased apoptosis, and abnormal GATA1 expression (2‐times lower than in control cells) upon differentiation. The increased p53 activation was also confirmed in RPL5‐ and RPS19‐deficient cells consistent with increased p53 expression in the bone marrow of DBA patients (p53+ cells: 8.4% in RPL5‐ and 7.3% in RPS19‐mutant patient vs. 0.4% in normal controls). One of the known inducer of p53 is oxidative stress causing oxidative DNA damage. Indeed, elevated positivity for 8‐oxoguanine (8‐oxoG), a marker of oxidative DNA damage, was detected in differentiated RP‐deficient MEL clones and in DBA patients’ samples in contrast to control samples. It is known, that reactive oxygen species (ROS) are produced under inflammation resulting in ROS‐mediated DNA damage response (DDR). The upregulation of pro‐inflammatory cytokines TNF‐α, IL6, and IL1b was found in RPL5‐ and RPS19‐deficient cells and also in the serum from DBA patients along with the upregulation of other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF‐γ, and GM‐CSF). Moreover, elevated pro‐inflammatory cytokines play a role in cellular senescence which was confirmed in RP‐deficient MEL clones by significantly elevated β‐galactosidase activity. In addition, increased ROS levels were detected in all differentiated RPL5‐ and RPS19‐deficient cells and in the serum of all DBA patients with elevated inflammatory cytokines. Finally, the DNA double‐strand breaks can occur as an outcome of ROS production responding by phosphorylation of Ser‐139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RP‐deficient MEL clones in comparison with control cells (mean intensity of fluorescence for RPL5‐deficient cells: 2.63 ± 0.34; RPS19‐deficient cells: 3.29 ± 0.41; control cells: 1.02 ± 0.22).Summary/Conclusion:The defective ribosomal biogenesis is associated with abnormal GATA1 expression and augmented apoptosis of erythroid cells. Our results show that the upregulation of inflammatory cytokines, senescence, oxidative stress, increased DNA damage, and activation of DDR signaling are also playing an important role in DBA pathogenesis. Our results also suggest the possibility of using the anti‐oxidative and anti‐inflammation therapy in DBA patients as the additive treatment for moderating DBA phenotype.Grants support: AZV 16–32105A, IGA_LF_2019_006, and by LTAUSA17142 (BK,LL).

Fetal Erythropoiesis Is Defective in Rpl11 Heterozygous Mice and Increases in Severity in Young Animals

Diamond Blackfan Anemia (DBA) is a congenital red blood cell aplasia usually caused by haploinsufficiency of selected ribosomal protein genes. Approximately 10% of DBA patients are anemic at birth, and 80% of DBA patients present by 6 months. These and other data suggest that the DBA erythroid defect is exacerbated by the switch from fetal to adult hematopoiesis. We hypothesize that unique molecular or cellular features initiate DBA pathogenesis during fetal hematopoiesis that manifests after the switch to adult erythropoiesis as severe anemia.To test this hypothesis, we developed a mouse model with constitutive Rpl11 haploinsufficiency in fetal and adult hematopoietic tissues driven by VavCre. Rpl11 is a commonly mutated ribosomal gene in DBA. In adult mice, reduced Rpl11 expression results in severe anemia by 6 weeks, with markedly reduced hemoglobin, increased red cell volume and elevated erythroid adenosine deaminase. Flow cytometric analysis of the bone marrow (BM) revealed no change in the frequency of megakaryocyte-erythroid progenitors (MEP) but a significant increase in both BFU-E and CFU-E, with a corresponding decrease in maturing Ter119+ cells. Functional progenitor assays showed reduced and delayed proliferation with an impaired ability to produce viable CD71+ Ter119+ cells. Treatment of purified Rpl11+/- BFU-E with dexamethasone rescued this proliferative defect. Thus, our data indicate that a differentiation block and functional impairment at the BFU-E stage of adult erythropoiesis contribute to Rpl11 haploinsufficient anemia.At the molecular level, Rpl11 haploinsufficiency in BM resulted in the accumulation of 32S and 12S pre-rRNA. Polysome profiling of total BM suggested reduced polysome assembly and impaired translation. Transcriptional profiling of normal and Rpl11+/- MEP, BFU-E, CFU-E and Ter119+ cells identified 13 differentially expressed genes across DBA erythropoiesis including known and novel targets such as CDKN1A, TNFSF4 and TSPAN14 . Enrichment of the p53 pathway was unique to BFU-E and CFU-E progenitors. Interestingly, TGFB1 and EGF pathways were also selectively affected within Rpl11+/- BFU-E and CFU-E progenitors, suggesting p53-independent self-renewal and proliferative defects in these populations.In contrast to adult hematopoiesis, fetal hematopoiesis defects are less severe. For example, fetal BFU-E are not significantly increased within E14 fetal livers and functionally generate a similar number of maturing erythroblasts in vitro . Lodish and colleagues have established that fetal liver BFU-E comprise at least two distinct subpopulations, termed “early” and “late”. Early BFU-E have high self-renewing capacity whereas late BFU-E have low self-renewing capacity. To determine whether the balance between early and late BFU-E is affected by Rpl11 haploinsufficiency, and to identify gene expression changes in early and late fetal Rpl11+/- BFU-E, we performed single cell RNA-sequencing.Normal fetal livers contained similar proportions of early and late BFU-E, whereas normal adult bone marrows only contained late BFU-E. Surprisingly, Rpl11+/- fetal livers primarily contained late BFU-E. One explanation for this observation is that early BFU-E are depleted from Rpl11+/- fetal livers as this population compensates for reduced function of non-renewing late BFU-E. Interestingly, late BFU-E from Rpl11+/- BM have a stronger “late” signature than their normal counterparts suggesting a reduced self-renewing capacity of BM BFU-E in DBA. We are currently testing this hypothesis by overexpressing early genes associated with BFU-E self-renewal in Rpl11+/- BFU-E and reassessing erythroid phenotypes.In conclusion, our studies show that erythropoiesis is defective in Rpl11+/- fetal liver at the cellular and molecular level, although functional impairment is not as marked as in adult mice. Moreover, single cell RNA-sequencing of BM and fetal BFU-E in our Rpl11+/- mouse model has uncovered a novel hypothesis for DBA pathogenesis that the loss of early, self-renewing BFU-E in fetal liver is a harbinger of adult erythroid failure. DisclosuresGlader:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Neuberg:Synta Pharmaceuticals: Other: Stock shares.

Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements.

We describe a novel approach to separate two ribosome populations from the same cells and use this method, and RNA-seq, to identify the mRNAs bound to S. cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond Blackfan Anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs, and that Rps26-deficient ribosomes preferentially translate mRNA from select stress response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm, the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed forward loop in gene-expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.

Whole exome sequencing in the differential diagnosis of Diamond-Blackfan anemia: Clinical and molecular study of three patients with novel RPL5 and mosaic RPS19 mutations

Diamond-Blackfan anemia (DBA) is a rare congenital disorder presenting remarkable phenotypic overlap with other inherited bone marrow failure syndromes, making differential diagnosis challenging and its confirmation often reached with great delay.By whole exome sequencing, we unraveled the presence of pathogenic variants affecting genes already known to be involved in DBA pathogenesis (RPL5 and RPS19) in three patients with otherwise uncertain clinical diagnosis, and provided new insights on DBA genotype-phenotype correlations. Remarkably, the RPL5 c.482del frameshift mutation has never been reported before, whereas the RPS19 c.3G>T missense mutation, although previously described in a 2-month-old DBA patient without malformations and refractory to steroid therapy, was detected here in the mosaic state in different bodily tissues for the first time in DBA patients.

A new in-frame deletion in ribosomal protein S19 in a Chinese infant with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia that usually presents as macrocytic anemia during infancy. Ribosomal protein S19 (RPS19) is identified as the first gene associated with DBA. RPS19 is mutated in 25% of DBA patients, but its role in DBA pathogenesis remains to be elucidated. We have identified a novel heterozygous frameshift mutation in RPS19 gene in a DBA child presenting with profound anemia after birth. A single nucleotide heterozygous deletion (C.251delG) results in frameshift in RPS19 gene in exon 4 at codon 84 with possible premature stop codon (p.Arg84LysfsX21). The mutant allele was not detected in her parents, indicating de novo mutation. Both alleles were expressed at the same level. Using an immunofluorescence technique, the mutated-type RPS19 expressions were mostly localized to entire nuclei with little staining for nucleoli and its intracellular localization significantly differed from the wild-type RPS19, which was localized to both nuclei and nucleoli. This type of a mutation could be very helpful in further understanding the role of the RPS19 protein in DBA pathogenesis.