diamond blackfan anemia
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2022 ◽  
Author(s):  
Jimmy Hom ◽  
Theodoros Karnavas ◽  
Emily Hartman ◽  
Julien Papoin ◽  
Yuefeng Tang ◽  
...  

Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.


2021 ◽  
Author(s):  
Simon Lebaron ◽  
Marie‐Françoise O’Donohue ◽  
Scott C. Smith ◽  
Kendra L. Engleman ◽  
Jane Juusola ◽  
...  

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 56
Author(s):  
Jeffrey M. Lipton ◽  
Christine L. S. Molmenti ◽  
Pooja Desai ◽  
Alexander Lipton ◽  
Steven R. Ellis ◽  
...  

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome, the founding member of a class of disorders known as ribosomopathies. Most cases result from loss of function mutations or deletions in 1 of 23 genes encoding either a small or large subunit-associated ribosomal protein (RP), resulting in RP haploinsufficiency. DBA is characterized by red cell hypoplasia or aplasia, poor linear growth and congenital anomalies. Small case series and case reports demonstrate DBA to be a cancer predisposition syndrome. Recent analyses from the Diamond Blackfan Anemia Registry of North America (DBAR) have quantified the cancer risk in DBA. These studies reveal the most prevalent solid tumor, presenting in young adults and in children and adolescents, to be colorectal cancer (CRC) and osteogenic sarcoma, respectively. Of concern is that these cancers are typically detected at an advanced stage in patients who, because of their constitutional bone marrow failure, may not tolerate full-dose chemotherapy. Thus, the inability to provide optimal therapy contributes to poor outcomes. CRC screening in individuals over the age of 50 years, and now 45 years, has led to early detection and significant improvements in outcomes for non-DBA patients with CRC. These screening and surveillance strategies have been adapted to detect familial early onset CRC. With the recognition of DBA as a moderately penetrant cancer risk syndrome a rational screening and surveillance strategy will be implemented. The downstream molecular events, resulting from RP haploinsufficiency and leading to cancer, are the subject of significant scientific inquiry.


Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 353-360
Author(s):  
Lydie M. Da Costa ◽  
Isabelle Marie ◽  
Thierry M. Leblanc

Abstract Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized as a rare congenital bone marrow erythroid hypoplasia (OMIM#105650). Erythroid defect in DBA results in erythroblastopenia in bone marrow as a consequence of maturation blockade between the burst forming unit–erythroid and colony forming unit–erythroid developmental stages, leading to moderate to severe usually macrocytic aregenerative (<20 × 109/L of reticulocytes) anemia. Congenital malformations localized mostly in the cephalic area and in the extremities (thumbs), as well as short stature and cardiac and urogenital tract abnormalities, are a feature of 50% of the DBA-affected patients. A significant increased risk for malignancy has been reported. DBA is due to a defect in the ribosomal RNA (rRNA) maturation as a consequence of a heterozygous mutation in 1 of the 20 ribosomal protein genes. Besides classical DBA, some DBA-like diseases have been identified. The relation between the defect in rRNA maturation and the erythroid defect in DBA has yet to be fully defined. However, recent studies have identified a role for GATA1 either due to a specific defect in its translation or due to its defective regulation by its chaperone HSP70. In addition, excess free heme-induced reactive oxygen species and apoptosis have been implicated in the DBA erythroid phenotype. Current treatment options are either regular transfusions with appropriate iron chelation or treatment with corticosteroids starting at 1 year of age. The only curative treatment for the anemia of DBA to date is bone marrow transplantation. Use of gene therapy as a therapeutic strategy is currently being explored.


2021 ◽  
Vol 23 ◽  
Author(s):  
Evida Mars-Holt ◽  
Alex Murdoch ◽  
Amanda Frugoli ◽  
Brian Utz ◽  
Lynn Kong

Cureus ◽  
2021 ◽  
Author(s):  
Raffaele A Ruggiero ◽  
Qasim Z Iqbal ◽  
Ali Akram ◽  
Jared Dendy ◽  
Julie Zaidan

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2189-2189
Author(s):  
Mark C Wilkes ◽  
Aya Shibuya ◽  
Vanessa M Scanlon ◽  
Hee-Don Chae ◽  
Anupama Narla ◽  
...  

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2179-2179
Author(s):  
Aya Shibuya ◽  
Mark C Wilkes ◽  
Manuel Serrano ◽  
Johan Flygare ◽  
Bert Glader ◽  
...  

Abstract Diamond Blackfan Anemia (DBA) is associated with a hypoproliferative anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life with the majority of patients carrying mutations in one of at least 17 ribosomal proteins, with RPS19 being the most common. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions, chronic effects from long term corticosteroid use, or complications from stem cell transplantation. The serine threonine kinase Nemo-like Kinase (NLK) is an atypical member of the MAP kinase family of enzymes and has been shown to be chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as in erythroid progenitors from DBA patients. In RPS19-insufficient human hematopoietic stem and progenitor cells, genetic silencing of NLK by shRNA increased erythroid expansion by 220.3% (SD = 6.6%), indicating that aberrant NLK activation may contribute to the pathogenesis of the disease and is a potential target for DBA therapy. A number of clinically approved or advanced compounds have been developed to inhibit MAP kinases with various degrees of cross reactivity among its family members. We therefore screened a number of compounds that inhibit NLK as an off-target and found that these NLK inhibitors improved erythroid expansion in DBA models. Of these inhibitors, OTS-167 performed optimally, improving erythropoiesis by 2-fold at 300nM, with an EC50 of 146nM. Previous studies of OTS-167 in xenograft models of neuroblastoma for one month did not result in neutropenia, suggesting very little to no toxicity to myeloid cells. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or treatment with steroids. Our results suggest that pharmacologic inhibition of NLK is a potential approach to treat patients with DBA. We are currently investigating other NLK inhibitors in preclinical models for future clinical application. Disclosures Glader: Agios: Consultancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 859-859
Author(s):  
Senthil Velan Bhoopalan ◽  
Jonathan Yen ◽  
Thiyagaraj Mayuranathan ◽  
Yu Yao ◽  
Kalin Mayberry ◽  
...  

Abstract Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy. Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F). We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H). In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. Figure 1 Figure 1. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1129-1129
Author(s):  
Mark C Wilkes ◽  
Aya Shibuya ◽  
Jacqueline D Mercado ◽  
Anupama Narla ◽  
Bert Glader ◽  
...  

Abstract Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease. We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA. Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated. Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors. Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31). Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. Disclosures Glader: Agios: Consultancy.


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