RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in a Murine Model of Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3796-3796 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Dianne Sako ◽  
Asya Grinberg ◽  
R. Scott Pearsall ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Erythroid Differentiation ◽  
Severe Anemia ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Erythroid Hyperplasia ◽  
Erythroid Precursors ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation

Abstract Abstract 3796 Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients. Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts. We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control. Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

ACE-536 Improves Ineffective Erythropoiesis, Anemia and Co-Morbidities in β-Thalassemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 248-248 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Sharon Cawley ◽  
Stefano Rivella ◽  
R. Scott Pearsall ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Treatment Group ◽  
Research Funding ◽  
Erythroid Differentiation ◽  
Blue Staining ◽  
Wild Type ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation

Abstract Abstract 248 β-thalassemia, the most common congenital anemia, is caused by mutations in β-globin gene resulting in partial or complete absence of β-globin protein chains. In the absence of properly paired α- and β-globin chains, the α-globin protein accumulates causing proteotoxicity and apoptosis of erythroid cells. Hemolysis and ineffective erythropoiesis together cause severe anemia in thalassemia syndromes. Increased proliferation with arrest of terminal erythroid differentiation and accelerated apoptosis is the hallmark of ineffective erythropoiesis in β-thalassemia. In chronic patients, blood transfusions are required for survival, but result in severe iron overloading. Non-transfusion dependent thalassemia (NTDT) patients however, are also affected by ineffective erythropoiesis, anemia and iron overload. Recombinant EPO therapy is ineffective and rarely used for β-thalassemia patients, as it does not affect the later stages of erythroid differentiation. Therefore, a pharmacological approach is necessary that can increase hemoglobin levels, prevent splenomegaly, bone abnormalities and iron overloading in β-thalassemia patients. Several members of the TGFβ-superfamily are involved in erythropoiesis. ACE-536 is a modified activin type IIb (ActRIIb) receptor fusion protein that acts as a ligand trap. Unlike wild type ActRIIb, ACE-536 does not inhibit activin A induced signaling but inhibits signaling induced by other members of the TGF-β superfamily such as GDF11. While EPO increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. We hypothesized that ACE-536 treatment will promote terminal erythroid differentiation, as well as reduce anemia, ineffective erythropoiesis and associated co-morbidities in β-thalassemia. We investigated the efficacy of RAP-536 (murine ortholog of ACE-536) in a mouse model of β-thalassemia intermedia (Hbbth1/th1). β-thalassemic mice were severely anemic and had significantly decreased RBC (−31.6% p<0.001), hemoglobin (−35.0% p<0.001) and hematocrit (−34.8% p<0.001) compared to wild type littermates. β-thalassemic mice were treated subcutaneously twice a week with RAP-536 (1 mg/kg) or TBS vehicle (VEH) control for two months (N=7 per treatment group). Wild-type littermates were dosed with VEH or RAP-536 (1 mg/kg) and used as controls (N=13 per treatment group). Following two months of treatment, RAP-536 treated β-thalassemic mice had significantly increased RBC (+32.9%, p<0.01), hemoglobin (+17.4%, p<0.01) hematocrit (+11.0%, p<0.01) and displayed reduced reticulocytosis (−30.07%, p<0.05) compared to VEH treated β-thalassemic mice. Terminal erythroid differentiation analyses of bone marrow and spleen from β-thalassemic mice treated with RAP-536 revealed significant decreases in basophilic erythroblasts while increasing late stage orthochromatic erythroblasts. RAP-536 treated β-thalassemic mice had significantly decreased serum EPO levels (639.7±111 vs. 1769.7± 517 pg/mL, p<0.05), bone marrow erythroid precursors and spleen weights (418.3± 28 vs. 677.1± 65 mgs, p<0.01) compared to VEH treatment indicating decreased erythroid hyperplasia and extramedullary erythropoiesis. RAP-536 treatment also restored bone mineral density in β-thalassemic mice to levels observed in wild type mice. Furthermore, RAP-536 treatment resulted in decreased splenic, liver and kidney iron levels by Perl's Prussian blue staining indicating decreased iron overloading. Interestingly, serum bilirubin (0.41± 0.01 vs. 0.72± 0.09 mg/dL, p<0.05) and lactate dehydrogenase levels (334.6± 33 vs. 424.6± 76 IU/mL) were lower in β-thalassemic mice treated with RAP-536 compared to VEH treated mice demonstrating decreased hemolysis. Morphological assessment of blood smears also displayed decreased hemolysis, reduced α-globin inclusions and poikilocytosis compared to VEH treatment. RAP-536 treatment also extended RBC life span in β-thalassemic mice compared to VEH treated mice. In summary, these data demonstrate that RAP-536 attenuates ineffective erythropoiesis, ameliorates anemia and improved associated co-morbidities in a murine model of β-thalassemia. ACE-536 represents a novel potential therapy for patients with β-thalassemia and these preclinical data provide a rationale for clinical studies of ACE-536 in β-thalassemia patients. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Rap-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA-1 Function in Murine b-Thalassemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 751-751 ◽  
Author(s):  
Pedro A. Martinez ◽  
Rajasekhar NVS Suragani ◽  
Manoj Bhasin ◽  
Robert Li ◽  
Robert Scott Pearsall ◽  
...  
Keyword(s):  
Murine Model ◽  
Target Genes ◽  
Erythroid Differentiation ◽  
Heme Biosynthesis ◽  
Ineffective Erythropoiesis ◽  
Down Regulation ◽  
Employment Equity ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation ◽  
Tgfβ Superfamily

Abstract We have previously reported that Smad2/3 signaling (of the TGFβ superfamily) is elevated in myelodysplastic syndromes (MDS) and β-thalassemia, diseases that are characterized by ineffective erythropoiesis (Suragani et al. 2014). Smad2/3 pathway inhibition using RAP-536 (murine version of ACE-536/luspatercept), a modified activin receptor type IIB ligand trap, decreased ineffective erythropoiesis (IE) and alleviated disease pathology in a murine model of β-thalassemia. In this study, we investigated the a) potential role of different Smad2/3 ligands that bind to luspatercept in the regulation of erythropoiesis and b) molecular mechanism of RAP-536 therapy in the murine model of β-thalassemia. Wild-type (WT) mice were treated with neutralizing antibodies against activin B, GDF8 or GDF8/11 (10mg/kg, s.c, twice weekly for 2- weeks, N=5/group) either as a single agent or in combination, and compared with RAP-536 (10 mg/kg, s.c) treatment. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours following administration the splenic basophilic erythroblasts (CD71+ Ter119+ FSChigh) were sorted by flow cytometry and RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis. a) Surface plasmon analysis revealed that ACE-536 binds Smad2/3 signaling ligands GDF11 and GDF8 with high affinity and activin B with lower affinity. There was minimal binding detected with Activin A, TGFβ1 or TGFβ3 ligands. Wt mice treated with RAP-536 increased RBC (+19%, P<0.001) and Hgb (+15.2%, P<0.001) compared to VEH treated mice. Treatment with anti-GDF8 or anti-activin B antibodies marginally affected RBC parameters (~2-4%, N.S) where as anti-GDF8/11 treatment alone increased RBC (+6.1%, P<0.05) and Hgb (6.9%, P<0.05) compared to VEH treatment. A combination treatment of anti-GDF8/11 and activin B antibodies synergistically increased RBC (10.7%, P<0.001) and Hgb (11%, P<0.001) compared to VEH treated mice. These data suggests that in addition to GDF11 and activin B, other TGFβ superfamily ligands are possibly involved in the stimulation of erythropoiesis by luspatercept. b) Transcriptome analysis of β-thalassemic erythroblasts revealed a total of 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples compared to VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on data from RAP-536 and VEH treated samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1 (erythroid differentiation), NFE2 and heat shock factor (involved in globin expression and protein quality-control). Previously, multiple studies established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. The individual gene symbols based comparative analysis revealed up-regulation of 53/478 GATA-1 activators and down regulation of 9/342 GATA-1 repressors. The GATA-1 target genes that were up regulated by RAP-536 treatment are involved in heme biosynthesis (such as Ppox, Fech, Alas2 and Abcb10) and erythroid differentiation (such as Klf1, Nfe2, Gypa, Bcl2l, Bnip3l, Bach1, and Ank1). Further GSEA of GATA-1 activator and repressor signatures against RAP-536 treatment data revealed a significant up-regulation of 158/328 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis, and cell cycle regulation whereas there was no statistically significant down regulation of GATA-1 repressed genes. Consistent with this data, our preliminary results in differentiating mouse erythroleukemic (MEL) cells showed increased Smad2/3 phosphorylation that is correlated with reduced GATA-1 protein levels suggesting that pSmad2/3 may negatively regulate terminal erythroid differentiation by decreasing GATA-1 availability. These data provide a potential mechanistic role for luspatercept treatment in β-thalassemia, by transcriptionally upregulating genes that promote erythroid differentiation and processing of unpaired α-globins. By inhibiting SMAD2/3 signaling, luspatercept relieves the block of terminal erythroid maturation and decreases ineffective erythropoiesis in diseases such as β-thalassemia and MDS. Disclosures Martinez: Acceleron Pharma: Employment. Suragani:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

Modified Activin Receptor Type IIb-Fc Fusion Protein (RAP-536) Decreases Anemia In a Murine Model Of Myelodysplastic Syndrome and Improves Overall Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 749-749
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Sharon Cawley ◽  
R. Scott Pearsall ◽  
Ravi Kumar
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Fusion Protein ◽  
Murine Model ◽  
Wild Type ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Fc Fusion Protein ◽  
Erythroid Precursors ◽  
Equity Ownership

Abstract Myelodysplastic syndromes (MDS) are caused due to abnormal proliferation and differentiation of pluripotent hematopoietic stem cells leading to peripheral cytopenias including anemia and an increased risk of progression to acute myelogenous leukemia (AML). The mainstay of anemia treatments for majority of non-del (5q) patients are frequent transfusions but often leads to iron overloading and enhanced progression to AML, causing a negative impact on overall survival (OS). Therefore, alternative therapies that promote effective erythropoiesis, decrease anemia, and improve OS, are needed. Members of the TGF-β superfamily are known regulators of erythropoiesis. ACE-536 is a modified soluble activin receptor type IIB-Fc fusion protein that acts as a ligand trap for certain TGF-β family ligands and prevents Smad 2/3 signaling. ACE-536 has shown a robust increase in RBCs in mice, rats and monkeys. In normal mice, ACE-536 promotes maturation but not proliferation of late stage erythroid precursors. Additionally, we have shown that RAP-536 (murine ortholog of ACE-536) corrected anemia in the NUP98-HOXD13 (NHD13) murine model of MDS. In this study, we evaluated the progression of MDS disease and OS of NHD13 mice administered RAP-536. NHD13 mice begin to develop anemia, neutropenia and lymphopenia at four months of age. NHD13 mice die by 14 months due to severe pancytopenia or progression to AML. In this study, 4-month old NHD13 mice (N=12-16/group) were dosed with RAP-536 (10 mg/kg) or vehicle (VEH) twice per week for 5 or 10 months. Age matched wild type mice were used as controls. At each time point, blood samples were collected for CBCs. Bone marrow and splenic hematopoietic precursors of various cell lineages were immuno-stained and analyzed by flow cytometry (FCM). Spleen sections, blood and bone marrow smears were also analyzed for histopathological changes. After 5 months of treatment, VEH treated NHD13 mice had decreased RBC (-19.6%, P<0.001), WBC (-30.8%, P<0.001), lymphocytes (-63.2%, P<0.001) and increased platelet counts (+89.2%, P<0.05) compared to wild type mice. Treatment with RAP-536 increased RBC (+7.2%, P<0.05) and reduced platelet counts compared to VEH control. No significant changes in other blood lineages were observed following RAP-536 treatment, demonstrating that RAP-536 is selective of the erythroid lineage. After 10 months of treatment, VEH treated NHD13 mice had severely decreased RBC (-32.9%) and hemoglobin (-21.8%) compared to wild-type mice. RAP-536 treatment increased RBC (+21.4%) and hemoglobin (+16.6%) compared to VEH treatment. FCM evaluation of erythroid precursors from bone marrow of NHD13 mice demonstrated increased immature CD71+Ter119+ erythroblasts (from 13.1% to 18.3%), and decreased mature CD71-Ter119+ erythroblasts (from 13.2% to 3.8%) compared to wild-type mice. Treatment with RAP-536 increased mature erythroblasts (from 3.8% to 9.6%) consistent with improved RBC parameters, indicating the stimulation of erythroid differentiation. Additionally, bone marrow from NHD13 mice had significantly elevated Gr1+ & CD11b+ (from 33.6% to 62.6%) and CD4+ & CD8+(from 19.1% to 32.3%) precursors, while peripheral blood displayed a concomitant decreases in granulocytes (-22.5%), WBC (-37.5%) and lymphocytes (-45%) compared to wild type mice, demonstrating ineffective hematopoiesis. Treatment with RAP-536 displayed a non-statistical decrease in these precursors in bone marrow and a similar increase in peripheral blood compared to VEH control. No changes in platelets were observed after ten months of treatment. These data suggests that the effect of RAP-536 on other hematopoietic lineages is likely secondary to its effect on erythropoiesis. Importantly, histopathological findings revealed no indication of increased leukemic progression in RAP-536 treated NHD13 mice compared to VEH treated mice. Furthermore, RAP-536 treated NHD13 mice demonstrated a trend for increased median survival compared to VEH treated mice, from 238 days to 277 days (P=0.08). Together, these data demonstrate that RAP-536 corrects anemia associated with ineffective erythropoiesis in NHD13 mouse model of MDS. RAP-536 does not enhance progression to AML, and may increase overall survival of NHD13 mice. ACE-536 is currently being evaluated for the treatment of anemia in patients with MDS and β-thalassemia, conditions characterized by ineffective erythropoiesis. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

scholarly journals Decreased PGC1β expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taha Sen ◽  
Jun Chen ◽  
Sofie Singbrant
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mitochondrial Function ◽  
Iron Homeostasis ◽  
Erythroid Differentiation ◽  
Refractory Anemia ◽  
Cell Cycle Exit ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Terminal Erythroid Differentiation

AbstractProduction of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.

Pharmacological Inhibition Of Histone Deacetylase (HDAC) 1, 2 Or 3 Have Distinct Effects On Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 564-564
Author(s):  
Jeffrey R Shearstone ◽  
John H van Duzer ◽  
Simon S Jones ◽  
Matthew Jarpe
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Employment Equity ◽  
Human Cd34 ◽  
Ic50 Values ◽  
Equity Ownership ◽  
Marrow Cells

Abstract Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells. Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2. To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010). Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

Evaluation Of The Effects Of Long-Term Treatment With Eltrombopag On Bone Marrow In Patients With Chronic Immune Thrombocytopenia (ITP) — Data From The EXTEND Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 326-326 ◽  
Author(s):  
Russell K. Brynes ◽  
Attilio Orazi ◽  
Kalpana Bakshi ◽  
Christine K Bailey ◽  
Geoffrey W. Chan
Keyword(s):  
Bone Marrow ◽  
Trabecular Bone ◽  
East Asian ◽  
Myeloproliferative Neoplasm ◽  
Clinical Signs ◽  
Employment Equity ◽  
European Consensus ◽  
Erythroid Hyperplasia ◽  
Long Term Treatment ◽  
Equity Ownership

Abstract Introduction Eltrombopag, a thrombopoietin receptor agonist (TPO-RA), increases platelet counts in patients with cITP. TPO-RAs have been associated with varying degrees of bone marrow (BM) reticulin increases. Patients in the EXTEND study, an ongoing, open-label extension study of eltrombopag in patients with heavily pretreated cITP who received eltrombopag or placebo in a previous eltrombopag study, underwent annual BM biopsies while on study. Methods Central morphology review was done by 2 hematopathologists blinded to clinical information who assessed specimens for cellularity, megakaryocyte, erythroid, and myeloid quantity and appearance; trabecular bone changes; and reticulin and collagen deposition (European Consensus scale). Results In EXTEND, 302 patients with cITP were treated with eltrombopag for ≤6.5 years as of February 2013. Of these, 232 BM biopsy specimens collected from 117 patients from pretreatment and up to 5.5 y on treatment were available at the time of this analysis, and were centrally reviewed (Table). Of these, 5 patients provided a biopsy sample within <10 mo on treatment, 48 patients at 1 y, 68 at 2 y, 51 at 3 y, 30 at 4 y, and 11 at 5 y on treatment. Median age (range) of the 117 patients was 50 (20-78) y; 69 (59%) patients were female; 85% were Caucasian/European, 9% were East Asian, 2% were South East Asian, and 4% were Arabic/North African. Median time since ITP diagnosis was 58.6 (8.5-421) mo. Two hundred and nine on-treatment biopsies, collected from 115 patients, were graded for reticulin using the European Consensus scale. Most (63-80%) of the on-treatment biopsies, collected between 1 y and 5 y, were MF-0 or MF-1 (17-33%) (Figure). Reticulin fibers were increased (MF ≥2) in 5 specimens from 2/115 (1.7%) patients. All 5 specimens were positive for collagen, and 3 of the 5 specimens in the 2 patients were judged as hypercellular. Of the 2 patients, 1 patient (East Asian) had reticulin documented after 2 y (970 days) of treatment, and was preemptively withdrawn from the study. Per protocol, a post-treatment biopsy was performed; a BM specimen taken 7 mo following withdrawal was graded as MF-0 for reticulin and negative for collagen. The second patient (Caucasian) had MF-2 at 1 y, which increased to MF-3 at 2 y and contained megakaryocytes with myeloproliferative neoplasm-like features, and then returned to MF-2 at the 3 y and 4 y assessments; however, these biopsies at 3 y and 4 y showed no signs of myeloproliferative neoplasm-like features and had reverted to normal. The patient remains on treatment at the time of this analysis. Neither patient experienced any adverse event or abnormality in hematologic parameters potentially related to impaired BM function. Cellularity was normal in the majority of patients at each assessment timepoint. Most (82-99%) patients demonstrated megakaryocytic hyperplasia. Megakaryocyte morphology was normal in all patients in all years except for 1 patient in year 1. Seven patients (6%) had megakaryocytes reminiscent of those seen with polycythemia vera or essential thrombocythemia. Erythroid hyperplasia was observed in 13-27% and myeloid hyperplasia in 6-13% of patients. Trabecular bone thinning was found in 39-70% of patients over the 5-year observation period. No pathologic pattern of changes was identified among the on-treatment specimens. Discussion Reticulin fibrosis was considered a potential risk for cITP patients receiving TPO-RAs. In the ongoing EXTEND study, at the time of this analysis, reticulin was either absent or mildly increased in 113/115 (98%) patients treated with eltrombopag up to 5.5 years at the time of their biopsy. No patient with reticulin or collagen increases showed clinical signs or symptoms indicative of BM dysfunction, and the findings were reversible upon discontinuation of treatment. No pattern of increasing reticulin deposition with longer treatment periods was observed based on BM biopsy data available to date. These data suggest that for most cITP patients, treatment with eltrombopag is not associated with the development of BM reticulin or collagen fibrosis; however, the potential association of TPO-RAs and increased BM fibrosis in cITP still needs to be better understood. Disclosures: Brynes: GlaxoSmithKline: Research Funding. Bakshi:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Chan:GlaxoSmithKline: Employment.

Genetic Investigation of the Role of GDF11 in the Treatment of β-Thalassemia and MDS

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2439-2439 ◽  
Author(s):  
Paraskevi Rea Oikonomidou ◽  
Ping La ◽  
Ritama Gupta ◽  
Vania Lo Presti ◽  
Carla Casu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Erythroid Cells ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Equity Ownership ◽  
Murine Erythroleukemia

Abstract The current treatment of β-thalassemia only partially mitigates the phenotype of the disease, making the need for novel therapeutic agents imperative. The investigational drug Luspatercept (ACE-536) is a ligand trap that contains the modified extracellular domain of activin receptor IIB (ACVR2B) and induces red blood cell production in an erythropoietin independent fashion. ACE-536 binds with high affinity to members of the transforming growth factor (TGF) β superfamily and therefore alters activin/GDF signaling through the intracellular SMAD complex. In search of the specific ligands, recent studies in a mouse model of β thalassemia intermedia identified growth differentiation factor 11 (GDF11) as a possible target of the drug. It has been proposed that GDF11 is overexpressed in thalassemic erythroblasts and inhibits terminal erythroid maturation via SMAD complex phosphorylation. A negative role of GDF11 in erythropoiesis has been postulated also in myelodysplastic syndrome (MDS). We recapitulate, by a genetic approach, the phenotype of thalassemic and MDS mice treated with RAP-536, the murine counterpart of ACE-536. We generated and analyzed animals with GDF11 deletion in erythroid cells (Hbbth3/+ Gdf11fl/flEpoR-Cre and NUP98-HOXD13 Gdf11fl/flEpoR-Cre) and in all hematopoietic tissues (Hbbth3/+Gdf11fl/flVav-Cre and NUP98-HOXD13 Gdf11fl/flVav-Cre). We did not detect any changes in red blood cell number, reticulocyte count, hemoglobin or hematocrit levels compared to thalassemic or MDS mice in absence of the floxed gene. Focusing on thalassemic mice, administration of RAP-536 significantly improved the anemia and other hematopoietic parameters in the peripheral blood, decreased spleen size and ameliorated ineffective erythropoiesis as indicated by an increased ratio of mature to immature splenic erythroblasts analyzed by flow cytometry. Similar endpoints were seen comparing floxed and non-floxed animals treated with RAP-536. Therefore, the lack of GDF11 in erythroid or bone marrow derived cells did not prevent a response to the drug. To assess the effect of a pancellular absence of GDF11, we are currently in process of generating a model of β-thalassemia with total Gdf11 deletion. To investigate the possible effects of RAP-536, we treated erythroid cells derived from normal or thalassemic patients with the drug. Erythroid cell viability, number, differentiation and cell cycle remained unvaried. Of note, we did not detect significant expression of GDF11/Gdf11 in human and mouse erythroid cells. To investigate the effect of an exogenous source of GDF11 production on erythroid cells, we treated murine erythroleukemia (MEL) cells with recombinant GDF11. Upon treatment we observed phosphorylation of the SMAD2/3 complex by western blot. This effect was hindered by co-treatment of GDF11 with RAP-536. We further assessed the effects of RAP-536 on the mouse erythroid transcriptome, using RNA seq analysis in splenic erythroid populations. After administration of a single dose of RAP-536, thalassemic mice were euthanized. We used flow cytometry to identify possible alterations on differentiating erythroid populations in the spleen. Notably, between 60 and 72h we observed reduced numbers of basophilic and increased numbers of polychromatophilic erythroblasts. Analysis at 60h revealed that signal transducer and activator of transcription 5a (Stat5a), cyclin-dependent kinase 6 (Cdk6) and other cell cycle-related and metabolic genes were increased in the basophilic erythroid progenitors treated with RAP-536. This effect suggests that RAP-536 promotes proliferation and/or differentiation of erythroblasts. Thus, our genetic analyses suggest that lack of GDF11 may be required but not sufficient to improve erythropoiesis. Furthermore, erythroid cells do not produce but can respond to exogenous GDF11, likely synthesized by non-erythroid cells and under conditions of ineffective erythropoiesis. Even though we detected in vitro effects, these may not mimic physiological effects, as experimental conditions may not correlate with GDF11 concentrations in vivo. As additional ligands have been proposed (such as GDF8 and Activin B), our future studies will focus on the potential role of these molecules. Altogether, these results reveal a potential alternative target of action for ACE-536 and may lead to the discovery of new therapeutic molecules. Disclosures Suragani: Acceleron Pharma: Employment, Equity Ownership. Kumar:Acceleron Pharma: Employment, Equity Ownership.

Inhibition of HDACs and Aminopeptidases Is Highly Synergistic in Myeloma Cells Resulting in Cell Death Via the Upregulation of BIRC3, a Key Mediator of NF-KappaB Signalling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 607-607 ◽  
Author(s):  
Emma M Smith ◽  
Brian A Walker ◽  
Emma L Davenport ◽  
Lauren I Aronson ◽  
David Krige ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Viability ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Equity Ownership ◽  
Nf Kappab ◽  
High Degree

Abstract Abstract 607 Myeloma cells rely on a number of mechanisms to maintain their survival, including the silencing of genes that would normally check uncontrolled proliferation and lead to apoptosis. Epigenetic alterations such as histone deacetylation contribute significantly to the pathogenesis of both solid and haematological malignancies and are associated with the silencing of tumour suppressor genes. Pan-histone deacetylase inhibitors (HDACs), such as SAHA, panobinostat, depsipeptide, and numerous others have been published to have anti-myeloma activity. Here we report the effects of a novel pan HDAC inhibitor, CHR-3996, on multiple myeloma cells. Results: CHR-3996 was potently cytotoxic against a panel of myeloma cell lines (H929, KMS11, LP-1, MM1s, RPMI-8226, and U266) with a GI50 ranging from 9 to 65 nM and was equally as potent in primary patient myeloma cells. The loss of cell viability was associated with an increase in apoptotic cells; EndonucleaseG and Noxa were both up-regulated and caspase 9 was cleaved. Furthermore a decrease in apoptosis was demonstrated in the presence of a pan-caspase inhibitor indicating cell death is largely dependent on caspase-mediated apoptosis. There was no effect on bone marrow stromal (BMS) cell viability but there was an observed decrease in the IL-6 and VEGF the BMS cells secrete, both of which promote the growth and survival of myeloma cells in the bone marrow microenvironment. CHR-3996 caused an increase in acetylated H3K9 but there was minimal change in the levels of ubiquitinated proteins in the cell or acetylated alpha-tubulin, indicating low activity against HDAC6 or the proteasome (also demonstrated by an assay to specifically measure proteasome function). In addition to induction of apoptosis, cell cycle analysis showed an increased proportion of cells in G0/G1 indicating cell cycle arrest. In-keeping with data from treating myeloma cells with SAHA and panobinostat, an increase in cell cycle inhibitor p21 was observed. Gene expression profiling (GEP) identified changes in cell cycle regulators, indications of increased cell stress (elevated CHOP, ATF3, and TAO kinase 3), repression of Wnt (up-regulation of NLK, GSK3beta) and mTOR (decreased 4E-BP1) signalling, and changes in key pro- and anti-apoptotic proteins (for example SMAD3, BCL2, BIM, BID, and BIRC5). When used in combination studies CHR-3996 was highly synergistic in vitro with tosedostat, an aminopeptidase inhibitor, which we have previously shown to have anti-myeloma activity via the induction of the amino acid deprivation response and autophagy. One of the largest changes the GEP analysis identified was BIRC3 (CIAP2), an inhibitor of NF-kappaB signalling, which increased 23.5 fold with CHR-3996 as a single agent and over 100-fold when added to H929 cells in combination with tosedostat. Additionally both CHR-3996 and todestat independently up-regulated expression of members of the IkappaB family. Altered expression and nuclear localisation of canonical and non-canonical NF-kappaB family members were observed by immuno-fluorescence and immunoblotting, suggesting targeting of NF-kappaB signalling as a reason for the high degree of synergy between these two compounds. Early data suggest oral CHR-3996 is effective in a subcutaneous in vivo myeloma model and, reflecting the in vitro data, there is a degree of synergy when administered with tosedostat. Conclusions: The novel compound CHR-3996 is a potent HDAC inhibitor that leads to increased H3K9 acetylation but has no detectable activity against HDAC6 or proteasome activity in myeloma. It induces apoptosis of myeloma cells without affecting BMS cell viability. CHR-3996 shows a very high degree of synergy with an aminopeptidase inhibitor, tosedostat, potentially through targeting the NF-kappaB pathway. It has exciting therapeutic potential either as a mono-therapy or in combination with other agents. Disclosures: Krige: Chroma Therapeutics Ltd: Employment, Equity Ownership. Hooftman:Chroma Therapeutic Ltd: Employment, Equity Ownership. Drummond:Chroma therapuetics: Employment, Equity Ownership.

scholarly journals Ineffective Erythropoiesis inβ-Thalassemia

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Jean-Antoine Ribeil ◽  
Jean-Benoit Arlet ◽  
Michael Dussiot ◽  
Ivan Cruz Moura ◽  
Geneviève Courtois ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Premature Death ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Molecular Defects ◽  
Erythroid Precursors ◽  
Underlying Mechanisms

In humans,β-thalassemia dyserythropoiesis is characterized by expansion of early erythroid precursors and erythroid progenitors and then ineffective erythropoiesis. This ineffective erythropoiesis is defined as a suboptimal production of mature erythrocytes originating from a proliferating pool of immature erythroblasts. It is characterized by (1) accelerated erythroid differentiation, (2) maturation blockade at the polychromatophilic stage, and (3) death of erythroid precursors. Despite extensive knowledge of molecular defects causingβ-thalassemia, less is known about the mechanisms responsible for ineffective erythropoiesis. In this paper, we will focus on the underlying mechanisms leading to premature death of thalassemic erythroid precursors in the bone marrow.

scholarly journals RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 920-920
Author(s):  
Elena C. Brindley ◽  
Emily Hartman ◽  
Julien Papoin ◽  
Brian Dulmovits ◽  
Steven L. Ciciotte ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Ras Signaling ◽  
Cycle Progression ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation

Abstract RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage. The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p&lt;0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway. Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis. Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

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