Treatment Of Diamond-Blackfan Anemia Research Articles

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.

Ferritin Levels Do Not Reflect the Severity of Iron Overload in Diamond Blackfan Anemia

▪Introduction: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by hypoplastic anemia, congenital malformations and an increased risk to develop malignancies.Until now, treatment of DBA consists of red blood cell (RBC) transfusions, glucocorticoids (GC) and allogeneic hematopoietic stem cell transplantation in a selection of patients. Whereas RBC transfusions are the main cause of IO, elevated iron parameters have also been reported in non-transfusion-dependent DBA patients. Here we investigated the incidence and severity of IO in a well-described cohort of transfusion-dependent and non-transfusion-dependent DBA patients in order to gain more insight in the regulation of iron metabolism in DBA, and to provide clinical guiding to improve the diagnosis and management of IO in DBA.Methods: In this retrospective, observational study we have included twenty-nine pediatric and adult DBA patients for whom at least one serum ferritin level and/or MRI result was available. Ten patients (34%) were classified as transfusion-dependent (TD) (ten or more transfusions during the twelve months prior to evaluation). Non-transfusion-dependent (NTD) patients (66%) were treated with either GC, incidental transfusions or received no treatment. Transfusion burden (transfusion history) was assessed via medical records. Serum ferritin levels ≥250 ng/mL in males and ≥150 ng/mL in females were considered to be elevated. Results of MRI were expressed as liver iron content (LIC) and as cardiac T2* in milliseconds (ms). LIC ≥3 mg/g indicates significant hepatic IO, and LIC ≥7 mg/g is associated with clinical morbidity. Cardiac T2* ≤20 ms indicates significant cardiac IO.Results: In 15/29 (52%) MRI analysis of IO was performed. Hepatic IO (LIC >3 mg/g) was present in 9/29 (31%) of DBA patients, of which 8/9 (89%) had moderate to severe IO (LIC>7mg/g), despite the fact that all but one were treated with chelation therapy. Overall serum ferritin levels and LIC correlated significantly (r=0.7907, p<0.001), and all TD patients with LIC ≥7 mg/g had serum ferritin levels ³400 ng/mL, however, none of the patients had a serum ferritin >1000 ng/mL (Figure 1A). Interestingly, in the NTD group, hepatic IO was present in 2/7 patients (29%), who both only had mildly elevated serum ferritin levels (263 ng/mL and 277 ng/mL) and were not treated with iron chelation therapy. Based on total transfusion burden since birth, patients were classified in distinct groups: nine patients who received ³10 transfusions during life (9/10) were diagnosed with hepatic IO, whilst none of the patients who received <10 transfusions were diagnosed with hepatic IO. Both mean serum ferritin levels and mean LIC values were significantly higher in patients with ³10 transfusions compared to all with <10 transfusions (Figure 1B-C).Discussion: We demonstrate that IO is common in DBA yet can be easily overlooked in NTD patients that were treated with transfusions in the past. While serum ferritin levels significantly correlated with LIC values, this parameter cannot be used exclusively to screen for IO or titrate iron chelation therapy. We conclude that in clinical practice, biochemical parameters in combination with transfusion history justify a low-threshold to perform an MRI-based evaluation of IO, and to start adequate chelation therapy. [Display omitted] DisclosuresVan Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding. Wijk: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1αshort promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19- deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Metformin Upregulates Mir-26a to Improve Erythropoiesis in Preclinical Models of Diamond Blackfan Anemia through Suppression of Nlk Expression

Nemo-like Kinase (NLK) is an atypical member of the Mitogen-activated protein (MAP) kinase family and shares a highly conserved kinase domain with other MAP kinases. For this reason it is challenging to design small molecules that specifically inhibit NLK. In hematopoiesis, NLK expression is suppressed in all lineages except erythroid progenitors. In non-erythroid progenitors, micro-ribonucleic acid (miR)-181 is upregulated and binds to the 3' untranslated region (UTR) of NLK transcripts, resulting in dramatic degradation of NLK transcripts. NLK is not activated or required for healthy erythropoiesis, but in Diamond Blackfan Anemia (DBA), NLK is chronically hyper-activated, which contributes to failed erythroid expansion and disease pathogenesis. Given the sensitivity of NLK transcripts to 3'UTR binding-mediated degradation, we hypothesized that targeting NLK degradation by upregulating miRNAs that bind the NLK 3'-UTR could provide an alternative approach to directly inhibiting the kinase to suppress aberrant NLK activity in bone marrow progenitors from DBA patients. One miRNA binding site that is present in the NLK-3'UTR is miR-26a. We determined that miR-26a is upregulated 2.4-fold (p=0.009) in CD235+ erythrocytes by the Type 2 diabetes drug metformin. Metformin suppressed NLK expression 62.4% (p=0.006) and improved erythroid expansion 4-fold (p=0.001) in human and 6-fold (p=0.042) in zebrafish models of DBA. This effect required an intact miR-26a binding site within the NLK 3'UTR. However, metformin failed to induce miR-26a in murine models of DBA, however the exogenous expression of miR-26a suppressed NLK 36.4% (p=0.028) and improved erythropoiesis 2-fold (p=0.017). In summary, targeted suppression of NLK through metformin-mediated upregulation of NLK transcript-binding miRNAs effectively improved red blood cell production from 5.6% up to 33.3% of controls in a preclinical model of DBA. These results suggest that metformin is a potential drug for treatment of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy. OffLabel Disclosure: metformin. This drug is given to differentiating HSPCs in vitro to examine effect on specific kinases.

Metformin-induced suppression of Nemo-like kinase improves erythropoiesis in preclinical models of Diamond–Blackfan anemia through induction of miR-26a

Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

A CLINICALLY APPLICABLE LENTIVIRAL VECTOR CORRECTS THE ANEMIA AND BONE MARROW FAILURE IN A MOUSE MODEL FOR DIAMOND-BLACKFAN ANEMIA

Diamond-Blackfan Anemia (DBA) is a congenital erythroid hypoplasia caused by dysfunction of genes coding for ribosomal proteins. The ribosomal protein S19 (RPS19) gene is the most frequently mutated. Our previous studies showed that gene therapy with autologous HSCs modified to express RPS19 represents a potential therapeutic option. Here, we assessed the efficacy of a clinically applicable single gene lentiviral vector with a good safety profile for treatment of RPS19-deficient DBA. We designed a lentiviral vector harbouring a codon-optimized human RPS19 cDNA driven by the shortened version of the human elongation factor 1α (EFS) promoter. We transduced c-Kit+ bone marrow cells isolated from our established doxycycline inducible RPS19-deficient DBA mouse model and injected cells into lethally irradiated wild-type mice (Vector). The recipients transplanted with mock transduced c-Kit+ bone marrow cells from DBA mice were regarded as Mock group. After transplantation, cells were engrafted for 2 months, followed by doxycycline induction to recipients to induce DBA phenotype. The age matched wild-type mice receiving no irradiation and transplantation but same doxycycline induction concentration were regarded as control group (WT). The short-term effect of vector was analysed at 2 weeks after doxycycline induction. In Vector group, all recipients showed normal blood cellularity (RBC:10.79±1.05 × 109/L, Hb:162.75±5.26g/L) comparing with WT group (RBC:8.55±1.03 × 109/L, Hb:162±2.45g/L), while in Mock group, half of mice died due to dramatic BM failure and rest of mice showed DBA phenotype (RBC:4.74±2.2 × 109/L, Hb:79.13±34.24g/L). Our results demonstrate the feasibility and preclinical efficacy for treatment of DBA by using lentiviral vector. The long-term effects (16 weeks) and analysis of vector integration sites are in progress.

Diagnosis and treatment of Diamond-Blackfan anemia

Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome, characterized by simplered hematopoietic failure, usually macrocytic anemia with an absence or less than 0.05 of erythroid precursors (erythroblastopenia) in an otherwise normal bone marrow.Patient management is therefore centered on accurate diagnosis, appropriate use of transfusions and iron chelation, corticosteroids, Cyclosporine A and hematopoietic stem cell transplantation. Key words: Diamond-Blackfan anemia; Diagnosis; Treatment

Investigation on long-term complications of glucocorticoid in treatment of Diamond-Blackfan anemia

Objective To collect and summarize long-term complications of glucocorticoid (GC) in treatment of Diamond-Blackfan anemia (DBA). Methods A total of 17 DBA patients, treated with GC more than 1 year from December 2009 to November 2012 in Being Children's Hospital, Capital Medical University, were retrospectively investigated.The data of general information, drug treatment, treatment response, height and body mass in different the-rapy periods, and therapy related adverse reaction were collected.The data entry and the statistical analysis were performed using SPSS 16.0 software. Results Seventeen cases which fulfilled the research criteria were enrolled.The 58.8 percent of cases (10/17) began GC therapy from the age younger than 6 months.The 76.4 percent of the cases(14/17) started prednisone therapy with the dosage≥2 mg/(kg·d), and the median time of maintenance therapy with this dosage was 2 months (1-5 months). The median time of prednisone dosage greater than 0.5 mg/(kg·d) was 6 months (3-48 months). Patients were divided into 2 groups at the beginning of therapy according to whether their age was younger or older than 6 months.The median height of younger age group was -1.0 SD (-3.5-1.0 SD) of corresponded age- sex- standard height at the beginning of prednisone therapy, and was dropped to -3.5 SD(-3.5--2.0 SD)after half year treatment.For older age group, it was 0.0 SD(-1.5-2.0 SD)and -0.5 SD (-1.5-0.5 SD) respectively.During the therapy, there were 1 fracture, 2 measles pneumonia, 3 pneumonia, 3 hi-rsuitisms, 5 thrushes, and 12 central obesity cases. Conclusions GC related adverse reaction might appear when app-lying prednisone for the treatment of DBA in long term.It was suggested that GC therapy should start after 6 months old if possible, and the duration of 0.5 mg/(kg·d) GC treatment should be reduced as short as possible. Key words: Diamond-Blackfan anemia; Glucocorticoid; Complication; Growth status

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

Progress towards Mechanism-Based Treatment for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplastic anemia, characterized by macrocytic anemia, reticulocytopenia, and severely reduced numbers of erythroid precursors in the bone marrow. For more than fifty years, glucocorticoids have remained the main option for pharmacological treatment of DBA. While continuous glucocorticoid administration increases hemoglobin levels in a majority of DBA patients, it also causes severe side effects. There is therefore a great need for more specific and effective treatments to boost or replace the use of glucocorticoids. Over the years, many alternative therapies have been tried out, but most of them have shown to be ineffective. Here we review previous and current attempts to develop such alternative therapies for DBA. We further discuss how emerging knowledge regarding the pathological mechanism in DBA and the therapeutic mechanism of glucocorticoids treatment may reveal novel drug targets for DBA treatment.

Treatment of Zebrafish Models of Ribosomopathies (Diamond Blackfan Anemia (DBA) and 5q- Syndrome) with L-Leucine Results In An Improvement of Anemia and Developmental Defects: Evidence for a Common Pathway?

AbstractAbstract 195A growing number of disorders associated with impaired ribosome biogenesis and function have been recently recognized and termed ribosomopathies. The first of these to be identified was Diamond Blackfan Anemia (DBA), a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound macrocytic anemia. DBA is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stress erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and are at an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. A second ribosomopathy, 5q- syndrome is a subtype of myelodysplasia, with an erythroid phenotype strikingly similar to DBA. The erythroid phenotype of 5q- syndrome has been recently demonstrated to be attributable to acquired haploinsufficiency of the ribosomal protein RPS14 gene associated with clonal loss of the long arm of chromosome 5. In both DBA and 5q- syndrome, it is unclear why defects in ribosomal proteins, which would be expected to have widespread consequences, have such a specific effect on erythroid maturation. It has been demonstrated that the efficiency of mRNA translation is significantly depressed in cells derived from DBA patients, consistent with a pathogenic ribosomal defect. L-leucine is an essential branched chain amino acid that is known to modulate protein synthesis by enhancing translation. L-leucine has been used to treat DBA patients, and has been shown to lead to improvement in hemoglobin levels and to transfusion independence in some patients. Based on this observation we hypothesized that a common pathway associated with defective mRNA translation underlies both DBA and 5q- MDS, and that activation of translation by L-Leucine would alleviate the anemia associated with both ribosomopathies. In order to address this hypothesis, we have modeled DBA and 5q- MDS in zebrafish embryos using an antisense morpholino appraoach and have demonstrated that similar to the human diseases, Rps19 and Rps14 deficiency lead to defective erythropoiesis and to developmental abnormalities (specific to DBA). Treatment of Rps19 and Rps14 deficient embryos with L-Leucine resulted in partial reversal of both the anemia and the developmental defects. L-Leucine is thought to regulate protein synthesis by acting as a nutrient signal involving the mTOR (mammalian target of rapamycin) signaling pathway. It has been suggested that L-Leucine enhances translation by activating translation initiation factors and by specific up-regulation of the ribosomal protein S6 kinase. Activation of the mTOR pathway is currently being analyzed in the L-Leucine treated Rps19 and Rps14 deficient zebrafish embryos. Additionally, our observations are also being validated in in vitro human models for DBA and 5q-MDS using human hematopoietic progenitor CD34+ cells and shRNAs specific for Rps19 and Rps14 genes followed by L-Leucine treatment. Our studies should provide evidence for activation of pathways involved in mRNA translation and validate the use of the orally administered amino acid L-Leucine as a therapeutic agent in the treatment of DBA as well as 5q- syndrome without the side-effects associated with current therapies for both diseases. Disclosures:No relevant conflicts of interest to declare.

Stress Erythropoiesis: HIF-1 Alpha Synergizes with Glucocorticoids to Induce BFU-E Progenitor Self-Renewal

AbstractAbstract 814The goal of this study was to find small molecules that stimulate erythropoiesis at an earlier stage than erythropoietin (Epo). We therefore studied the mechanisms by which glucocorticoids (GCs) promote erythroblast formation during stress erythropoiesis (SE). Since the target cell of GCs in SE was not known we first established a FACS-based method to purify burst-forming unit erythroid (BFU-E) and colony-forming unit of erythroid lineage (CFU-E) cells from mouse fetal liver. BFU-E and CFU-E cells were resolved by differences in CD71 or CD24a expression (BFU-E = CD71/CD24a10% low; CFU-E = CD71/CD24a20% high) from a population of cells positive for Kit, and negative for the cell surface markers Ter119, B220, Mac-1, CD3, Gr-1, Sca-1, FcGR, CD41 and CD34. Purified “BFU-E” cells formed 94% BFU-E colonies and “CFU-E” cells formed 95% CFU-E colonies at cloning efficiencies of 55–70%. Using pure CFU-E and BFU-E progenitors we demonstrated that while 100nM Dexamethasone (Dex) does not increase proliferation or self-renewal of CFU-E cells, Dex stimulates the earlier BFU-E progenitors to undergo limited self-renewal, which increases formation of CFU-E cells and erythroblasts >20-fold. After identifying BFU-E progenitors as the target cells of GCs during SE, we next determined how DEX changes miRNA and mRNA expression in BFU-Es by next generation sequencing. No miRNAs changed the level of expression more than 50%. Of the 10,000 most expressed mRNAs 83 were up-regulated and 112 mRNAs down-regulated more than 2-fold in response to Dex. Since previous studies show that dimerization (trans activation) of the GC receptor is necessary for GCs to induce erythroblast production, the most important GC target genes are likely up- rather than down-regulated. We hypothesized that other molecules that are able to enhance the expression of the 83 up-regulated genes could enhance or replace the stimulatory effect of Dex. We therefore computationally analyzed the promoter regions of the up-regulated genes using motif enrichment analysis. We found that Dex induces expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1 alpha (HIF1a) binding sites (p-value = 1e–27). Analysis of six published gene expression datasets, where the effect of GCs on gene expression had been determined in cell types other than BFU-E cells, showed very low or no enrichment of HIF1a targets among genes up-regulated by Dex. We confirmed the suggested overlapping effects of HIF1a and GCs on gene expression in BFU-E cells by comparing the effect of Dex with that of pharmacological HIF1a activation by the prolyl hydroxylase inhibitor (PHI) Dimethyloxalylglycine (DMOG). As determined by next generation mRNA sequencing, DMOG increased expression of 98 genes more than 50%, of which 28 were among 109 genes up-regulated >50% by Dex (p-value for overlap = 1e–26). Importantly the overlap in effects on gene expression also resulted in overlapping biological effects. Similar to Dex, DMOG had little effect on CFU-E cells (Figure A). In contrast DMOG synergized with Dexamethasone to promote BFU-E self-renewal and prevent BFU-E cell exhaustion, enhancing the production of CFU-E cells 170-fold and maximum erythroblast production 300-fold (Figure B). DMOG thus enhanced the stimulatory effect of 100nM Dex on erythroblast production 7-fold. In similar experiments where BFU-E cells were cultured in GCs levels corresponding to physiological cortisol levels (1 nM Dex), DMOG enhanced erythroblast production 10-fold. We further showed that DMOG and Dex also synergistically enhance proliferation of adult mouse and human erythroid progenitors. Our results support a physiological model of SE where increased systemic levels of free cortisol, in combination with local anoxia, induce BFU-E self-renewal and increase erythroblast production. This suggests that the therapeutic potential for PHIs in treatment for anemia goes beyond replacing recombinant Epo. Here we show that in addition to enhancing Epo production from the kidneys, PHIs have an intrinsic stimulatory effect on BFU-E progenitors, leading to increased production of Epo-responsive CFU-E cells. Thus PHIs provide a new window for treating Epo-resistant anemia. The synergistic effect of GCs and PHIs further suggests that PHIs may enhance or replace the effect of Prednisone in the treatment of Diamond-Blackfan Anemia and other bone marrow failure syndromes. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Imbalance of p53 Family Members as a New Target of Therapeutics for Treatment of Diamond Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is characterized by defective erythropoiesis, congenital anomalies and risk of malignancies. Ribosomal protein (RP) S19 is mutated in about 25% of patients and RPS24 in 2%. RPS19 deficiency impairs ribosomal biogenesis. It still remains unknown how impaired ribosome biogenesis and function lead to DBA. We found that RPS19 deficiency in zebrafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the RPS19-deficient phenotype is mediated by upregulation of the p53 family genes. DeltaNp63 is BMP4 target and during gastrulation, required for specification of non-neural ectoderm. Its upregulation suppresses the development of neural structures and is likely to contribute to brain and craniofacial defects observed in RPS19 deficient zebrafish and DBA. In response to ribosomal stress from RPS19 deficiency, deltaNp63 expression is induced in erythroid progenitors suggesting a role for deltaNp63 in hematopoiesis. Similar to other vertebrates, zebrafish experience two waves of blood formation, primitive, producing mostly erythrocytes and macrophages and definitive producing all types of blood cells. Erythroid progenitors proliferate and differentiate into mature erythrocytes in the Intermediate Cell Mass (ICM), a region between the dorsal aorta and axial vein. We found that number of blood cells in the ICM was reduced in RPS19-deficient embryos and their differentiation was delayed. Morphologically, erythrocytes from morphants differed from wild-type cells. They varied in size and some cells retained a blast-like phenotype. The hemoglobin level was also decreased in morphants. The immature phenotype of RPS19-deficient erythroid cells and their inability to differentiate into mature erythrocytes may be explained by DNp63 induction. Importantly, downregulation of expression of both p53 and DNp63 can rescue the RPS19 deficient phenotype in zebrafish. Our findings suggest that suppression of p53 pathway might be beneficial for DBA patients with RPS19 mutations. Several drugs were reported to be effective in the treatment of DBA although the mechanism of their action is unknown. They include such dissimilar compounds as corticosteroids, cyclosporine A, and valproic acid. Metoxyclopramide was reported as effective by some authors but not others. We treated RPS19-deficient embryos with these drugs and found that dexamethasone, cyclosporine A, and valproic acid improve RPS19-deficient phenotype. We further found that treatment of embryos with dexamethasone and cyclosporine A influences p53 level. Metoxyclopramide was not effective in rescuing RPS19-deficient embryos and had no effect on p53 level. We next questioned if known compounds inhibiting p53 pathway can improve the phenotype and survival of RPS19-deficient zebrafish. Pifithrin m inhibits p53 binding to mitochondria by reducing its affinity to antiapoptotic proteins Bcl-xL and Bcl-2. It specifically suppresses the mitochondria branch of the p53 pathway but does not affect other p53 functions. Pifithrin has no effect of the p53 level in zebrafish embryos but was efficient in improving RPS19-deficient phenotype. These data suggest that suppression of p53 activity might be a mechanism of action of some drugs currently used for DBA treatment and open new directions for development of novel drugs.

Successful Treatment of Diamond Blackfan Anemia With Metoclopramide

Successful Treatment of Diamond Blackfan Anemia With Metoclopramide

Bone Marrow Transplantation for Diamond-Blackfan Anemia

Patients with Diamond-Blackfan anemia (DBA) who are unresponsive to or intolerant of corticosteroids, experience treatment failure with other treatments, develop additional cytopenias or clonal disease, or opt for curative therapy are often treated with allogeneic bone marrow transplantation. We studied the transplantation outcomes of 61 DBA patients whose data were reported to the International Bone Marrow Transplant Registry between 1984 and 2000. The median age was 7 years (range, 1-32 years). Among 55 patients with available transfusion information, 35 (64%) had received ≥20 units of blood before transplantation. Most patients (67%) received their bone marrow grafts from an HLA-matched related donor. The median time to neutrophil recovery was 17 days (range, 10-119 days) and to platelet recovery was 23 days (range, 9-119 days). Five patients did not achieve neutrophil engraftment. The 100-day mortality was 18% (95% confidence interval, 10%-29%). Grade II to IV acute graft-versus-host disease occurred in 28% (range, 17%-39%) and chronic graft-versus-host disease in 26% (range, 15%-39%). The 3-year probability of overall survival was 64% (range, 50%-74%). In univariate analysis, a Karnofsky score ≥90 and transplantation from an HLA-identical sibling donor were associated with better survival. These data suggest that allogeneic bone marrow transplantation is effective for the treatment of DBA. Transplantation before deterioration of the performance status and from an HLA-identical sibling donor may improve survival.

Treatment of Diamond-Blackfan anemia with recombinant human interleukin- 3 [see comments

This report describes the response of eighteen Diamond-Blackfan anemia (DBA) patients to recombinant human interleukin-3 (rhIL-3). rhIL-3 was administered subcutaneously once daily on an escalating dose schedule (0.5 to 10 micrograms/kg/d). The rhIL-3 dose was escalated every 21 days until erythroid response was attained, grade III or IV nonhematologic toxicity was observed, or the maximum rhIL-3 dose was reached. Four patients experienced clinically significant erythroid responses. Two of the responders were steroid-dependent and transfusion-independent, while two were steroid-independent and transfusion-dependent. Baseline clinical or laboratory parameters, in particular in vitro bone marrow erythroid progenitor assays, were not useful in predicting rhIL-3 response. rhIL-3 administered at 5 to 10 micrograms/kg/d was associated with an increase in total white blood cell count, secondary to increases in neutrophils, eosinophils, and lymphocytes. Patients experienced a dose-dependent elevation in absolute eosinophils across the entire dose range. Two of the responding patients remain on maintenance rhIL-3, without diminution of effect at 244 and 370 + days. rhIL-3 was discontinued in the other two responders, because of the development of deep venous thrombi.