Diamond-Blackfan Anemia Patients Research Articles

Treatment of refractory/relapsed Diamond-Blackfan anaemia with eltrombopag.

Diamond-Blackfan anaemia (DBA) is a rare, inherited bone marrow failure syndrome with a ribosomal defect causing slowed globin chain production with normal haem synthesis, causing an overabundance of reactive iron/haem and erythroid-specific cellular toxicity. Eltrombopag, a non-peptide thrombopoietin receptor agonist, is a potent intracellular iron chelator and induced a robust durable response in an RPS19-mutated DBA patient on another trial. We hypothesized eltrombopag would improve RBC production in DBA patients. We conducted a single-centre, single-arm pilot study (NCT04269889) assessing safety and erythroid response of 6 months of daily, fixed-dose eltrombopag for DBA patients. Fifteen transfusion-dependent (every 3-5 weeks) patients (median age 18 [range 2-56]) were treated. One responder had sustained haemoglobin improvement and >50% reduction in RBC transfusion frequency. Of note, 7/15 (41%) patients required dose reductions or sustained discontinuation of eltrombopag due to asymptomatic thrombocytosis. Despite the low response rate, eltrombopag has now improved erythropoiesis in several patients with DBA with a favourable safety profile. Dosing restrictions due to thrombocytosis may cause insufficient iron chelation to decrease haem production and improve anaemia in most patients. Future work will focus on erythropoiesis dynamics in patients and use of haem synthesis inhibitors without an impact on other haematopoietic lineages.

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

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

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

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

Preclinical Studies of the GlyT1 Inhibitor Bitopertin in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) results from the haploinsufficiency of 1 of 20 different ribosomal proteins. While DBA patients have hematopoietic stem/progenitor cell defects which broadly impact hematopoiesis, the dominant phenotype is ineffective erythropoiesis and severe anemia. In ~60% of patients, the anemia responds to corticosteroid therapy, leaving an unmet need for safe and effective treatment. In DBA, the synthesis of heme (a chemical chelate) proceeds at a near normal rate while the ribosomal haploinsufficiency leads to slowed translation and delayed globin protein synthesis, resulting in a heme-globin imbalance. DBA patient colony forming unit-erythroid (CFU-E)/proerythroblasts and murine Rpl11 haploinsufficient CFU-E/proerythroblasts show increased heme- and reactive oxygen species (ROS)-mediated cell damage, apoptosis, and ferroptosis. Single cell RNAseq analyses also suggest that excessive heme is a major factor in DBA erythroid marrow failure. When patient marrow cells are cultured in the presence of succinylacetone to reduce heme synthesis or hemopexin to facilitate heme export, erythroid output significantly increases. Succinylacetone competitively inhibits the 2 nd step in heme synthesis, while hemopexin enhances heme export 100-fold and scavenges the extracellular heme. Neither would be a pragmatic and safe therapy for DBA patients. In the first step of heme biosynthesis, glycine and succinyl-CoA combine to form aminolevulinic acid. Erythroid precursors are unable to synthesize enough glycine to support their robust heme synthesis and must import glycine via glycine transporter 1 (GlyT1) (PMID 28495915). Bitopertin is an investigational oral small-molecule inhibitor of GlyT1. In Phase 3 clinical studies of schizophrenia, it was found to be ineffective but exhibited a favorable safety profile (PMID 27816567). To determine whether bitopertin could rebalance heme and globin synthesis, reduce ineffective erythropoiesis, and improve red cell production in DBA, we cultured marrow cells from patients with DBA without and with 10 nM bitopertin (n=3). Following 7 days of culture, bitopertin increased the mean number of cells (21%) and the number of CD71+ erythroid precursors (31%) without impacting erythroid maturation. We next made human cord blood CD34+ deficient for RPS19 (44±2% reduction) with RPS19 shRNA transduction (or control luciferase shRNA) to model RPS19 haploinsufficient DBA. Treatment with bitopertin improved erythroid expansion of RPS19-deficient CD34+ cells 60% (P<0.01) but not control CD34+ cells. Additionally, to assess erythroid differentiation in vitro in the context of an erythroblastic island (EBI) niche, we cultured the CD34+ cell-derived erythroblasts with cord blood-derived macrophages. Erythroid expansion is 7.9±1.3-fold higher (P<0.02) in EBI cultures compared to cultures without macrophages. Treatment of the EBI cultures with bitopertin increased the expansion of RPS19-deficient CD34+ cell-derived erythroblasts 3.9±0.3-fold (P<0.002) but did not increase the expansion of control CD34+ cell-derived erythroblasts. As a final study, Rpl11 haploinsufficient mice were treated with bitopertin (10 to 150 ppm in chow, 2-30 mg/kg/day) for 8 weeks. Bitopertin treatment resulted in a dose-dependent improvement in anemia. After treatment with an optimal dose of bitopertin (100 ppm in chow, 20 mg/kg/day), there was an 8.8% increase in the hemoglobin values, and there was significant improvement in red cell numbers (7.5±1.1 M/µL to 8.9±1.3 M/µL, p=0.005), hematocrit (41.6±4.9% to 45.5±4.2%, p<0.05), and mean corpuscular volume (55.7±3.5 fL to 51.8±4.2 fL, p=0.01). Additionally, bitopertin treatment diminished the developmental block at the CFU-E/proerythroblast stage and reduced erythroblast ROS. The improved in vitro erythroid differentiation of DBA patient marrow and RPS19-deficient CD34+ cells and the improved anemia of RPL11 haploinsufficient mice suggest that bitopertin might mitigate anemia in DBA patients. These encouraging preclinical data supported the initiation of a Phase 1/2 clinical trial (NCT05828108: Young et al, abstract this meeting).

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.

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.

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.

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 .

Hematopoietic cell transplantation and gene therapy for Diamond-Blackfan anemia: state of the art and science.

Diamond-Blackfan anemia (DBA) is one of the most common inherited causes of bone marrow failure in children. DBA typically presents with isolated erythroid hypoplasia and anemia in infants. Congenital anomalies are seen in 50% of the patients. Over time, many patients experience panhematopoietic defects resulting in immunodeficiency and multilineage hematopoietic cytopenias. Additionally, DBA is associated with increased risk of myelodysplastic syndrome, acute myeloid leukemia and solid organ cancers. As a prototypical ribosomopathy, DBA is caused by heterozygous loss-of-function mutations or deletions in over 20 ribosomal protein genes, with RPS19 being involved in 25% of patients. Corticosteroids are the only effective initial pharmacotherapy offered to transfusion-dependent patients aged 1 year or older. However, despite good initial response, only ~20-30% remain steroid-responsive while the majority of the remaining patients will require life-long red blood cell transfusions. Despite continuous chelation, iron overload and related toxicities pose a significant morbidity problem. Allogeneic hematopoietic cell transplantation (HCT) performed to completely replace the dysfunctional hematopoietic stem and progenitor cells is a curative option associated with potentially uncontrollable risks. Advances in HLA-typing, conditioning regimens, infection management, and graft-versus-host-disease prophylaxis have led to improved transplant outcomes in DBA patients, though survival is suboptimal for adolescents and adults with long transfusion-history and patients lacking well-matched donors. Additionally, many patients lack a suitable donor. To address this gap and to mitigate the risk of graft-versus-host disease, several groups are working towards developing autologous genetic therapies to provide another curative option for DBA patients across the whole age spectrum. In this review, we summarize the results of HCT studies and review advances and potential future directions in hematopoietic stem cell-based therapies for DBA.

Establishment and characterization of CSCRi006-A: an induced pluripotent stem cell line generated from a patient with Diamond-Blackfan Anemia (DBA) carrying ribosomal protein S19 (RPS19) mutation.

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes (Gadhiya and Wills in Diamond-Blackfan Anemia, https://www.statpearls.com/ ; 2023). A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Toward this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA-iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments.

Identification of novel mutations in patients with Diamond-Blackfan anemia and literature review of RPS10 and RPS26 mutations.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia, physical malformation, and cancer predisposition. Twenty ribosomal protein genes and three non-ribosomal protein genes have been identified associated with DBA. To investigate the presence of novel mutations and gain a deeper understanding of the molecular mechanisms of disease, targeted next-generation sequencing was performed in 12 patients with clinically suspected DBA. Literatures were retrieved with complete clinical information published in English by November 2022. The clinical features, treatment, and RPS10/RPS26 mutations were analyzed. Among the 12 patients, 11 mutations were identified and 5 of them were novel (RPS19, p.W52S; RPS10, p.P106Qfs*11; RPS26, p.R28*; RPL5, p.R35*; RPL11, p.T44Lfs*40). Including 2 patients in this study, 13 patients with RPS10 mutations and 38 patients with RPS26 mutations were reported from 4 and 6 countries, respectively. The incidences of physical malformation in patients with RPS10 and RPS26 mutations (22% and 36%, respectively) were lower than the overall incidence in DBA patients (~50%). Patients with RPS26 mutations had a worse response rate of steroid therapy than RPS10 (47% vs. 87.5%), but preferred RBC transfusions (67% vs. 44%, p = 0.0253). Our findings add to the DBA pathogenic variant database and demonstrate the clinical presentations of the DBA patients with RPS10/RPS26 mutations. It shows that next-generation sequencing is a powerful tool for the diagnosis of genetic diseases such as DBA.

Utility of Whole Exome Sequencing in the Early Diagnosis of Atypical Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome, with a hallmark of erythroblastopenia and congenital anomalies. DBA demonstrates genetic heterogeneity and variable phenotypic expression. We present two cases of atypical DBA harboring de novo mutations in the RPS-19 gene with c.49 G>C and c.357-1G>T allelic variants. The two cases presented confounding critical illness demonstrated by multiorgan failure, aplastic crisis, with case 2 meeting the criteria for hemophagocytic lymphohistiocytosis. We highlight the utility of genetic testing in the early diagnosis of DBA and the associated complexities and burden of disease in caring for DBA patients.

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.

The Molecular Pathophysiology of Ineffective Erythropoiesis at Single Cell Resolution

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic, always reflect ineffective erythropoiesis, and yet result from diverse genetic mutations. To delineate the shared mechanisms that lead to the death of maturing erythroid cells, we studied single marrow cells from MDS-5q patients, DBA patients, and normal individuals with CITE-seq (antibody barcoding of cell surface proteins) and RNA sequencing. After testing several approaches, we opted to align transcriptomes with SCVelo pseudotime analysis, which includes incompletely processed mRNAs, since these are highly prevalent and regulated during erythropoiesis. We identified 38,296 erythroid lineage cells then plotted CITE-seq protein quantitation data versus the velocity pseudotime to identify BFU-E through orthochromatic erythroblasts based upon the sequential CD117, CD36, CD71, and GlyA expression levels, using well-established criteria. The analysis identified two distinct transcriptional trajectories. These diverge during the CFU-E stage, following upregulation of the transferrin receptor, CD71, and initiation of iron import, and right after upregulation of ALAS2, the first and rate-limiting step of heme synthesis. The normal (healthy) trajectory (trajectory A) contains cells at all stages, including the late stages of erythroid maturation, and there is high expression of mitochondrial oxidative metabolism and erythroid differentiation pathway genes. In contrast, the second trajectory (trajectory B) ends abruptly near the start of terminal erythroid differentiation as defined by cell surface proteins, and highly expresses gene pathways associated with excess heme and with apoptosis. There is loss of mitochondrial gene expression, premature loss of nuclear gene expression (increased % mitochondrial genes), and elevated levels of BAX and other apoptosis markers, implying that this is a death pathway. The significant increase in expression of genes in the heme-responsive dataset defined by Liao et al (Cell Rep. 2020. 31:107832) in trajectory B cells along with the pathway analysis, suggests that DBA and MDS-5q erythroid cells died from heme toxicity, a finding supportive of our earlier work (Yang et al. Sci Transl Med 2016. 8:338ra67). Cells along each trajectory were present in all samples but at different frequencies: 57±4% of MDS-5q, 73±2% of DBA, and 24±12% of normal cells followed trajectory B. That 24±12% of normal cells followed trajectory B was an unexpected finding, and indicated that heme might regulate the ineffective vs. effective differentiation of normal, as well as DBA and MDS-5q cells. As a corollary, drugs or interventions decreasing heme synthesis might modulate trajectory choice and be therapeutically beneficial. Also notably, fate decisions occur well before cell death, allowing time for therapeutic intervention. In addition, since we can independently track the fate and transcriptomes of cells with an intact chromosome 5q vs. cells with chromosome 5q deletion, these experiments provide insight into why anemia occurs early in MDS-5q (i.e., at presentation) often only 50-75% of the marrow is replaced by neoplastic cells. The analysis revealed both 5q- and 5q+ cells show early upregulation of HIF1A targets, ROS response, cell cycle regulation and spliceosome pathways and down regulation of erythroid differentiation and mTOR signaling pathways, indicating both 5q- and 5q+ cells share common pathologies. Additionally, the 5q- cells upregulate p53 pathways. The fact that the 5q+ cells are not normal implies that the presence of neoplastic 5q- cells influence the erythroid differentiation of the 5q+ cells. That they too have transcriptomic findings attributable to excessive heme may suggest a cell-extrinsic role for heme toxicity, which has led us to study heme processing by the central macrophage of erythroblastic islands (EBIs). Initial studies suggest EBI macrophage dysfunction contributes to the poor differentiation of 5q+ cells. This abstract includes data available on the medRxiv preprint server. https://doi.org/10.1101/2022.08.01.22278133

Regulated Expression of GATA1 As a Gene Therapy Cure for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure disorder characterized by impaired red blood cell production leading to severe anemia. The only treatment options are regular transfusions, corticosteroids, or bone marrow transplant, and each has variable effectiveness and considerable morbidity. More effective, safer, and permanent cures for DBA are lacking, but few drugs are in active development. DBA is genetically heterogenous, characterized by loss-of-function mutations in one copy of at least 26 different ribosomal protein (RP) genes that alters the mRNA translation of a select group of genes, most notably the hematopoietic master regulator GATA1. Overexpression of GATA1 in hematopoietic stem and progenitor cells (HSPCs) from patients with DBA rescues the erythroid differentiation defects in vitro, but unregulated GATA1 expression in hematopoietic stem cells (HSCs) promotes premature erythroid differentiation at the expense of long-term (LT-) HSC maintenance. Gene therapy is an attractive strategy to achieve a cure for DBA, but a traditional gene therapy approach that overexpresses a functional copy of a mutated gene is not the ideal option, as it would require the development and validation of dozens of gene therapy vectors, each with a copy of one of the mutated DBA genes. Here, we report a unified gene therapy strategy, using developmentally-regulated and highly restricted expression of GATA1 that would be curative in most, if not all, patients with DBA, regardless of the underlying disease-causing mutation. To be a cure for DBA, a GATA1 gene therapy vector must incorporate into the genome of undifferentiated LT-HSCs but only drive robust GATA1 expression in early erythroid progenitors after lineage commitment. To design a vector with lineage-restricted expression, we identified accessible chromatin regions upstream of GATA1 that only gain accessibility in differentiating erythroid cells, but not in upstream HSPCs. Using these selectively accessible regions concatenated together, we constructed a human GATA1 enhancer (hG1E) element and used it to drive GATA1 and/or GFP expression from a lentiviral backbone. To ensure erythroid-restricted expression of the hG1E-GATA1-GFP vector, we infected CD34+ HSCs from healthy human donors and cultured them in conditions that either allowed HSC maintenance or promoted erythrocyte differentiation. We observed very low GFP expression in bulk HSCs and essentially no expression in the subpopulation of immunophenotypic LT-HSCs. However, more than 80% of committed erythroid cells demonstrated high levels of GFP expression, confirming the fidelity of lineage-restricted expression from the hG1E-GATA1 vector. To examine whether lineage-restricted GATA1 expression confers a functional correction of the erythroid maturation block in DBA, we used CRISPR editing to recapitulate RPS19 haploinsufficiency in human HSPCs. In RPS19 edited samples that were infected with a control GFP virus, we observed a progressive depletion over time of RPS19 edits as the cells underwent erythroid differentiation, consistent with the notion that RPS19 editing leads to impaired erythroid differentiation and are selected against. In contrast, RPS19-edited and hG1E-GATA1 treated samples had a more than 10-fold higher preservation of RPS19 edits in the bulk population and in erythroid colonies derived from single progenitor clones, indicating that regulated GATA1 expression could overcome RPS19 haploinsufficiency. To validate the results from the CRISPR model of DBA, we sought to correct the erythroid maturation block in primary HSCs from a DBA patient with a pathogenic mutation in RPL5. There was skewed differentiation of GFP-infected RPL5+/- HSCs away from the erythroid lineage and toward myeloid and megakaryocyte lineages, but regulated GATA1 expression from the hG1E-GATA1 vector reversed that skewing. There was a 10-fold increase in the ratio of erythroid to non-erythroid cells, and a significant expansion of erythroid cells in the hG1E-GATA1 treated patient cells. Preclinical assessment in additional patient samples both in vitro and in vivo is ongoing. Collectively, our preclinical data demonstrate that lineage-restricted expression of GATA1 is sufficient to rescue the impaired erythroid differentiation in DBA regardless of the underlying genetic lesion. These results will set the stage for the first in-human gene therapy trial for DBA.

Transfusion burden in early childhood plays an important role in iron overload in Diamond-Blackfan anaemia.

In Diamond-Blackfan anaemia (DBA), iron overload (IO) is common in transfusion-dependent patients, yet has also been reported in non-transfusion-dependent patients. We explored the incidence of IO in transfusion-dependent and non-transfusion-dependent DBA patients. We observed hepatic IO in 65% of patients analysed with MRI, including three patients that were only treated with transfusions in the past. Whereas overall ferritin levels and liver iron content correlated, ferritin levels did not reflect total body iron adequately. Our data suggest that transfusion burden in the past plays an important role in IO in DBA, and should be taken into account during follow up.

Diamond–Blackfan anaemia with iron overload: A serious issue

Transfusion-dependent Diamond-Blackfan anaemia (DBA) patients rapidly develop iron overload and frequently experience cardiac complications. The report by Lecornec and colleagues offers useful details on indications and the management of deferiprone, a highly efficient chelator in removing excess cardiac iron but associated with a high risk of agranulocytosis in DBA patients. Commentary on: Lecornec et al. Agranulocytosis in patients with Diamond-Blackfan anaemia (DBA) treated with deferiprone for post-transfusion iron overload: A retrospective study of the French DBA cohort. British Journal of Haematology 2022;199:285-288.

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.

Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell–extrinsic contributions to erythropoiesis

AbstractWe follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.