Vitamin B5 and Succinyl-CoA Improve Ineffective Erythropoiesis in SF3B1 Mutated Myelodysplasia

Abstract Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity. Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients. In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients. In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients. Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

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Canine red blood cell transfusion practice

Journal of the American Animal Hospital Association ◽

10.5326/15473317-32-4-303 ◽

1996 ◽

Vol 32 (4) ◽

pp. 303-311 ◽

Cited By ~ 57

Author(s):

MB Callan ◽

DA Oakley ◽

FS Shofer ◽

U Giger

Keyword(s):

Adverse Events ◽

Blood Cell ◽

Red Blood Cell ◽

Blood Cells ◽

Transfusion Practice ◽

Red Blood Cell Transfusion ◽

Ineffective Erythropoiesis ◽

Packed Red Blood Cells ◽

The Mean ◽

Rbc Transfusion

Red blood cell (RBC) transfusions in 307 dogs were reviewed. A total of 658 units of RBCs, including 474 (72%) units of packed red blood cells (PRBCs) and 184 (28%) units of whole blood (WB), were administered. Reasons for transfusion included hemorrhage (n = 222), hemolysis (n = 43), and ineffective erythropoiesis (n = 42). The mean pretransfusion packed cell volume (PCV) of dogs with hemolysis (13%) was significantly lower (p less than 0.0001) than the mean pretransfusion PCVs of dogs with hemorrhage (21%) or ineffective erythropoiesis (18%). The mean total volume of PRBCs transfused was significantly greater (p less than 0.03) in dogs with hemolysis. Overall, 187 (61%) of 307 dogs were discharged from the hospital. Cause of anemia, pretransfusion PCV, and total volume of blood administered did not appear to influence survival. However, the mean adjusted posttransfusion PCV of dogs with hemorrhage was significantly higher (p less than 0.001) in dogs that survived. Possible adverse events were observed during or shortly after RBC transfusion in 10 (3.3%) dogs; all reactions were mild and self-limiting, and none were hemolytic.

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Safety and Tolerability of Donor Type Red Blood Cell Transfusion before Allogeneic Stem Cell Transplantation in Children with Major ABO Mismatch

Blood ◽

10.1182/blood.v128.22.3415.3415 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3415-3415

Author(s):

Andrea Jarisch ◽

Jan Soerensen ◽

Emilia Salzmann ◽

Eva Rettinger ◽

Andre Manfred Willasch ◽

...

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Red Blood Cell ◽

Research Funding ◽

Pure Red Cell Aplasia ◽

Red Blood Cell Transfusion ◽

Red Cell ◽

Donor Type ◽

Transfusion Reactions ◽

Rbc Transfusion

Abstract Background Major ABO mismatch in allogeneic bone marrow transplant (BMT) can cause clinical problems like severe transfusion reactions, acute haemolysis, and delay of red blood cell (RBC) engraftment or manifestation of pure red cell aplasia, related to circulating isoagglutinin titers. Even there is no increased incidence of graft failure or slower engraftment of neutrophils and platelets ABO mismatch leads to an increase of transplant related mortality. To reduce circulating isoagglutinin titers pre transplant plasma exchange during conditioning period can be performed. This procedure can be wearing for the patient and rebound effects are observed. RBC depletion can cause a loss of CD34+stem cells in bone marrow. A further strategy to reduce circulating isoagglutinin titers is in vivo immunoadsorption due to donor type RBC transfusion pre transplant. Aim of the study The Aim of the retrospective single center study was to investigate the safety and tolerability of donor-type red blood cell transfusion prior to allogeneic stem cell transplantation in children with major ABO mismatch. Methods From 2007-2015 37 children (median age 7 years, range 0.6-18.4) received an ABO mismatched RBC transfusion (2-3 ml KG BW) pre transplant under antihistaminic and steroid cover. Reaction to donor type RBC and graft transfusion, number of donor type RBC transfusions, haemolysis parameters, and trend of isoagglutinin titers, and engraftment data were observed. Wilcoxon signed-rank test was used to examine the difference between the paired values of titers (Conventional test tube, CTT; titration method using nonspecific anti-IgG antibody; Anti-IgG gel titer) before and after donor type RBC transfusion. Age and time until engraftment were reported using median and ranges. The quantitative values were reported using frequency and percentage. All statistical tests were two-sided with a significance level of 5%. Data analysis was performed using commercial software R.Statistical methods. Results Safety of mismatched RBC transfusions Compared with accidentally transfused RBC no severe complications are observed in our cohort. 34 (92%) children presented no reactions (Grade 0), 1 child had a minimal oxygen demand,1 child showed a mild hypertension (Grade 1) and 1 child a moderate hypertension, requiring treatment. Neither severe reactions nor anaphylactic shock were observed. Efficiency of mismatched RBC transfusion A significant reduction of isoagglutinin titers was defined as a final titer of 1:4 or at least a reduction of 3 titer steps was achieved. 23 of 37 (64%) required 1 donor type RBC transfusion to reduce significant the isoagglutinin titers, 8 patients (22%) needed a second and 5 patients (14%) a third transfusion. Figure 1 showed the significant reduction of isoagglutinin titers in the different patients groups after donor type RBC transfusion. The pairwise analysis of isoagglutinin titers indicates that the initial titer of patients required one donor type transfusion of RBC was significant lower than in the patient group required 2 or 3 transfusions. Engraftment data The median time to WBC and platelet engraftment was 21.5 (range 19-24) and 28 (range 26-60) days respectively. Haemolysis parameter post mismatched RBC transfusion Haemolysis parameter lactat dehydrogenase (LDH) and bilirubin was determined on a daily base. In all patients no significant increase of bilirubin levels was observed. Conclusion Donor type RBC transfusion is a safe, tolerable and effective procedure to reduce the isoagglutinin titers prior to allogeneic ABO mismatched bone marrow transplantation in children. In our cohort no severe transfusion reactions, acute haemolysis, delay of engraftment or manifestation of pure red cell aplasia was observed. Figure Figure. Disclosures Jarisch: Novartis: Consultancy. Bader:Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Medac: Research Funding; Riemser: Research Funding; Neovii: Research Funding.

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Genetic Loss of Tmprss6 Increases Effective Erythropoiesis in a Mouse Model of β-Thalassemia

Blood ◽

10.1182/blood.v120.21.482.482 ◽

2012 ◽

Vol 120 (21) ◽

pp. 482-482

Author(s):

David B. Stagg ◽

Sara Gardenghi ◽

Stefano Rivella ◽

Nancy C. Andrews ◽

Karin E. Finberg

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Red Blood Cell ◽

Research Funding ◽

Erythroid Cells ◽

Wild Type ◽

Ineffective Erythropoiesis ◽

Mature Erythrocyte ◽

Globin Chains ◽

Erythroid Precursors

Abstract Abstract 482 β-thalassemia is a disorder of ineffective erythropoiesis in which oxidative damage caused by unpaired α-globin chains leads to erythroid apoptosis, increased proliferation of erythroid precursors, and impaired erythroid differentiation. Patients develop systemic iron overload that is caused by red blood cell transfusions and by insufficient inhibition of gastrointestinal iron absorption by the iron regulatory hormone hepcidin. Previously we reported that homozygous genetic loss of Tmprss6, a hepatic transmembrane serine protease that inhibits hepcidin expression by the liver, led to hepcidin elevation and systemic iron deficiency in Hbbth3/+ mice, a model of β-thalassemia intermedia. Interestingly, we also found that while maintaining similar hemoglobin levels, Hbbth3/+mice with homozygous loss of Tmprss6 showed a significant reduction in splenomegaly and marked improvement in peripheral red blood cell (RBC) morphology. Here, we investigated the effects of genetic loss of Tmprss6 on erythropoiesis in Hbbth3/+ mice. In mice of different Tmprss6-Hbb genotypes, we used flow cytometry to quantify the proportion of total bone marrow cells of the erythroid lineage by measuring expression of TER119, an antigen expressed from the pro-erythroblast through the mature erythrocyte stage. Additionally, within the TER119+ population, we quantified the different erythroblast subpopulations by analyzing the intensity of forward scatter and CD44 expression. Compared to wild type (Tmprss6+/+Hbb+/+) controls, Hbbth3/+ mice with 2 wild-type Tmprss6 alleles (Tmprss6+/+Hbbth3/+) showed a significant increase in the proportion of total erythroid cells in the bone marrow, significant increases in the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population, and a significant decrease in the proportion of mature RBCs, resulting in a marrow profile consistent with ineffective erythropoiesis. In Hbbth3/+ mice with homozygous Tmprss6 disruption (Tmprss6−/−Hbbth3/+), the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population remained significantly elevated; however, the proportion of total erythroid cells in the bone marrow was no longer increased. Compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a significant increase in the proportion of mature RBCs; this was accompanied by a reduction in reactive oxygen species (ROS) production (as assessed by the indicator CM-H2DCFDA) and apoptotic cells (as assessed by annexin V binding) within both the orthochromatic erythroblast/reticulocyte and mature red cell subpopulations. Additionally, compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a marked reduction in α-globin precipitates in membrane fractions prepared from peripheral RBCs. Interestingly, when normalized to α-globin mRNA expression, bone marrow mRNA encoding α-hemoglobin stabilizing protein (AHSP), an α-globin chaperone, was significantly higher in Tmprss6−/−Hbbth3/+ mice compared to Tmprss6+/+Hbbth3/+ mice, compatible with the known stabilization of AHSP mRNA under low iron conditions. Together, these findings suggest a model in which genetic loss of Tmprss6 in Hbbth3/+ mice leads to a systemically iron-deficient state in which reduced iron availability to erythroid precursors leads to stabilization of free α-globin chains, a reduction in both ROS formation and erythroid apoptosis, and ultimately more effective erythropoiesis. In the context of previous findings, these results indicate that hepcidin-elevating strategies based on pharmacological inhibition of Tmprss6 might alter the clinical phenotype of β-thalassemia not only by reducing systemic iron loading but also by altering erythroid maturation. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

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Relationship Between Chelation and Clinical Outcomes in 600 Lower-Risk MDS Patients: Registry Analysis At 36 Months

Blood ◽

10.1182/blood.v120.21.3800.3800 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3800-3800

Author(s):

Roger M. Lyons ◽

Billie J. Marek ◽

Carole Paley ◽

Jason Esposito ◽

Lawrence Garbo ◽

...

Keyword(s):

Blood Cell ◽

Clinical Outcomes ◽

Red Blood Cell ◽

Lower Risk ◽

Research Funding ◽

Causes Of Death ◽

Disease Status ◽

Red Blood Cell Transfusion ◽

Cardiac Events ◽

Risk Status

Abstract Abstract 3800 Introduction: Treatment of anemia in pts with myelodysplastic syndromes (MDS) may require packed red blood cell transfusion. Transfusion dependence in MDS is associated with poorer clinical outcomes and reduced overall survival (OS). This US registry prospectively collected data on clinical outcomes in chelated and non-chelated, transfused, lower-risk MDS pts. OS, leukemic transformation, and clinical events are reported for non-chelated and chelated pts at 36 mos on study. Methods: This 5-year, non-interventional registry enrolled 600 pts from 107 US centers. Pts were ≥18 years old with lower-risk MDS (WHO, FAB, and/or IPSS) and transfusional iron overload (serum ferritin ≥1000 μg/L and/or ≥20 packed red blood cell units and/or ≥6 units every 12 weeks). The chelated group included all pts who had ever used iron chelation; sub-analysis was performed on pts with ≥6 mos chelation. Assessments were every 6 mos for 5 years or until death and included demographics, survival, disease status, comorbidities, causes of death, and MDS therapy. Results: Baseline demographics and IPSS risk status were similar between groups, although transfusion burden trended higher in chelated pts (Table 1). As of April 30, 2012, 169 pts continued on registry, and 431 discontinued (345 died, 57.5%; 61 lost to follow-up, 10.2%; and 25 other, 4.2%). In all, 264 (44%) pts received chelation therapy; 200 had ≥6 mos chelation. OS and time to acute myeloid leukemia (AML) transformation were significantly longer, and percentage of deaths was significantly lower in chelated ≥6 mos vs. non-chelated pts (P<0.0001, P=0.011 [median not reached in either group], P=0.0002, respectively; Table 2). AML transformations were also lower in chelated ≥6 mos pts (not significant [NS]). Cardiac (non-chelated, 51.5%; ≥6 mos chelation, 30.5%) and vascular disorders (non-chelated, 59.2%; ≥6 mos chelation, 45.5%) were more prevalent in non-chelated pts at baseline; this trend continued on study: cardiac (non-chelated, 49.7%; ≥6 mos chelation, 42.5%); vascular (non-chelated, 55.7%; ≥6 mos chelation, 48.5%; NS, all comparisons). Most frequent causes of death were MDS/AML, cardiac events, and infection. The percentage of pts who had ever received MDS therapy was lower among non-chelated pts (non-chelated, 88.4%; ≥6 mos chelation, 93.5%; NS). Conclusions: At 36 mos, chelated pts had significantly longer OS and time to AML transformation, as well as significantly fewer deaths. Trends toward fewer AML transformations and cardiac disorders were observed in chelated pts. Baseline characteristics and IPSS risk status were similar between groups, with the exception of more prevalent cardiac and vascular comorbidities in non-chelated pts. Additional assessments over the 5-year duration of this registry will provide further information on the association between chelation and clinical outcomes. Disclosures: Lyons: Novartis: Research Funding; Amgen: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Telik: Research Funding. Paley:Novartis: Employment. Esposito:Novartis: Employment. Garcia-Manero:Novartis: Research Funding.

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