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.

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.

TGFb1 Antagonist Inhibits Fibrosis in a Murine Model of Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 605-605 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Pedro A. Martinez ◽  
Sharon M Cawley ◽  
Robert Li ◽  
Robert Scott Pearsall ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Murine Model ◽  
Jak2 V617f ◽  
Mutant Mice ◽  
Wild Type ◽  
Employment Equity ◽  
Bone Marrow Fibrosis ◽  
Equity Ownership ◽  
Marrow Fibrosis

Abstract Introduction: Myelofibrosis (MF) is a clonal stem cell disorder that originates from acquired mutations in the hematopoietic stem cells leading to abnormal kinase signaling, cell proliferation, cytokine expression, and splenomegaly and ultimately bone marrow (BM) fibrosis. Primary myelofibrosis (PMF), post-polycythemia vera (PV) MF and post-essential thrombocythemia MF are categorized under MF with overlapping disease phenotypes including progression to BM fibrosis. A genetic mutation in Janus kinase 2 (V617F) was identified as causative in ~95% PV, and ~50% of ET and PMF patients. Currently, treatment of MF patients with a JAK2 inhibitor offers symptomatic benefit, but does not alter the natural history of the disease or improve BM fibrosis. It is known that TGFβ1 is a critical regulator of fibrosis in many disease states. Elevated TGFβ1 levels were reported to be important for fibrosis in patients with MF. We hypothesize that inhibition of TGFβ1 signaling may prevent fibrosis and help reduce secondary morbidities associated with disease in MF patients. Therefore, we evaluated this hypothesis using a TGFβ1 antagonist in a murine model of MF. Methods: Transgenic JAK2 (V617F) mutant mice (MF model) and age-matched wild-type controls were used in the studies. Mice were dosed twice weekly with TGFβ1 antagonist (10 mg/kg). Complete blood counts (CBC), serum TGFβ1, bone metabolism and inflammatory cytokines levels were determined at different ages (2-12 months) during disease progression. Bone marrow and spleen cells were analyzed for different cell lineages by flow cytometry. Tissue sections were stained with H&E and reticulin to determine cellularity or degree of fibrosis respectively. Results: To understand the onset and progression of MF disease in JAK2 (V617F) mice, we initially analyzed the CBC and degree of fibrosis at various ages (2, 3, 4, 5, 8, 10 and 12 months) and compared the data with wild-type mice. These data were then correlated with the levels of TGFβ1 and other cytokines. As expected, red blood cells (RBC) and platelets were elevated in JAK2 mutant mice at all ages compared to wild-type mice, although a trend towards a progressive increase was observed between 2 to 5 months followed by a decrease from 8 to 14 months. Bone marrow fibrosis was detected starting at 5 months and worsened with age. JAK2 mutant mice displayed splenomegaly that increased as the disease progressed. Interestingly, serum levels of TGFβ1, TGFβ3 and bone metabolism cytokines (OPG, OPN, aFGF and Trance) displayed an increase at earlier ages (2-5 months) compared to the latter ages, a trend similar to RBC levels. These levels peaked during the initiation of fibrosis at 5 months. In contrast, inflammatory cytokines (such as IL6, IL-1β, and TNFα) were elevated at later ages consistent with disease progression. We initiated treatment with TGFβ1 antagonist in JAK2 (V617F) mice (N=8/treatment group) at 4 months of age, the age corresponding to elevated serum TGFβ1 levels and prior to the onset of fibrosis (at 5 months of age). Following 6 months of treatment, vehicle (VEH) treated JAK2 mutant mice displayed elevated RBC (+37.1%, P<0.001), platelets (+74.5%, P<0.001) and spleen weights (+9.5 fold, P<0.001) compared to wild-type mice. BM and spleen sections from VEH treated JAK2 mutant mice revealed severe fibrosis. TGFβ1 antagonist treatment of JAK2 mice displayed moderate effect on RBC (-8.4%, N.S) without any effect on platelet counts compared to VEH treatment. Flow-cytometry identified a reduced proportion of Ter119+ erythroid precursors in BM and spleen (-15%, P<0.05) and no change in CD41+ megakaryocytes. TGFβ1 antagonist treated mice displayed reduced spleen weights (-29%, P<0.01), and marked reduction in fibrosis in bone marrow (Figure) and spleen sections compared to VEH. Consistent with the reduction in fibrosis, TGFβ1 antagonist treated JAK2 mice displayed reduced IL-6 levels (-48.9%, P<0.05) compared to VEH treatment. Conclusion: Together, these data demonstrated that TGFβ1 levels were correlated with bone marrow fibrosis in a murine model of MF disease, and its inhibition using TGFβ antagonist reduces fibrosis, splenomegaly and inflammation in this murine model of myelofibrosis. Figure 1. Figure 1. Disclosures Suragani: Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Martinez:Acceleron Pharma: Employment. Cawley:Acceleron Pharma Inc: Employment. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

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.

Landmark Analyses of DNMT3A Mutations in Hematological Malignancies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 407-407
Author(s):  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Melanie Wild ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mutation Rate ◽  
Mutation Frequency ◽  
Cpg Methylation ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Equity Ownership

Abstract Abstract 407 CpG methylation is an epigenetic modification that is important for cellular development. The DNMT3A gene, located on chromosome 2p23.3, encodes for a DNA methyltransferase and plays a central role in de novo CpG methylation. Recently, DNMT3A has been reported to be mutated in 22% of AML and 8% of MDS (Ley et al., N Engl J Med, 2010; Walter et al., Leukemia, 2011). Further, DNMT3A mutations were observed to be associated with a short overall survival in both diseases, respectively. In order to determine the role of DNMT3A mutations in leukemia we investigated two different entities by next-generation sequencing: 145 AML patients and 83 cases harboring a T-cell acute lymphoblastic leukemia (T-ALL). We applied an amplicon based deep-sequencing assay (454 Life Sciences, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA). The peripheral blood or bone marrow samples were obtained from untreated patients. The AML cohort was restricted to cases with normal karyotype (CN-AML). 87/145 (60%) cases were specifically selected to be wild-type for NPM1, FLT3-ITD, CEBPA, and MLL-PTD, whereas 58/145 (40%) samples were mutated in NPM1 (n=33) or double-mutated in NPM1 and FLT3-ITD (n=25). In our cohort of AML cases without mutations in NPM1, FLT3-ITD, CEBPA, and MLL-PTD, we observed a DNMT3A mutation frequency of 17.2% (15/87 cases). The DNMT3A mutation rate in the NPM1 mutated/FLT3 wild-type cases (16/33, 48.5%, P=0.001) and NPM1/FLT3-ITD mutated cases (19/25, 76%, P<0.001) was significantly higher, confirming the association of DNMT3A mutations with NPM1 and FLT3-ITD mutations that had been reported previously (Ley et al.). Interestingly, also in the cohort of T-ALL we detected patients that carried a DNMT3A mutation (16/83, 19.3%), which is very similar to the mutation frequency in AML, and has not been described yet. To further address the biology of DNMT3A mutations in acute leukemias we combined the AML and T-ALL cohorts and identified in total 31 distinct missense mutations in 65 patients (49 AML, 16 T-ALL). Most frequently, amino acid R882 located in exon 23 was mutated (n=29 cases). In addition, we identified 7 frame-shift alterations, 5 nonsense and 2 splice-site mutations. Moreover, 9 of the 65 mutated cases had two independent mutations. Focusing on AML, only three (6.1%) of the 49 DNMT3A-mutated cases were observed to harbor two different mutations concomitantly. In contrast, in the cohort of T-ALL we detected two different mutations in 6/16 (37.5%, P=0.003) cases. Further, in the cohort of AML, no homozygous mutation was detected, however, in the T-ALL group, two cases harbored a homozygous mutation. Therefore, only 3/49 AML (6.1%) cases, but 8/16 T-ALL (50%) cases showed biallelic mutation status (P<0.001). With respect to overall survival, no association was seen in the complete cohort of CN-AML cases (n=145). After limiting this cohort to the cases without mutations in NPM1, FLT3-ITD, CEBPA and MLL-PTD (n=87), an inferior survival was observed for DNMT3A-mutated patients as compared to DNMT3A wild-type patients (n=15 vs. n=72; alive at 2 years: 27.9% vs. 56.6%; P=0.048). Remarkably, also in the cohort of T-ALL a worse survival for patients with DNMT3A mutations was seen which has not been reported thus far (n=13 vs. n=64; alive at 1 years: 28.6% vs. 80.9%; P=0.001). Subsequently, we were interested whether gain-of-function mutations of the DNMT3A gene were associated with trisomy 2 and acquired uniparental disomy (aUPDs) of the short arm of chromosome 2 where DNMT3A is located. As such, we investigated 9 cases harboring a trisomy 2 (AML n=4, MDS n=4, and CMML n=1) and one MDS patient harboring an aUPD 2p, as confirmed by SNP microarray analyses (SNP Array 6.0, Affymetrix, Santa Clara, CA). Not all, but 3/9 cases with trisomy 2 harbored a DNMT3A mutation (one AML, MDS, and CMML case each), suggesting that duplication of DNMT3A mutations can enhance the effect of the mutation. Moreover, the single case with aUPD 2p also showed a mutation, further suggesting that LOH leading to loss of the wild-type DNMT3A may be another mechanism of disease leading to progression of leukemia. In conclusion, we here report on a high mutation rate of DNMT3A in both AML and T-ALL and independently confirmed an inferior overall survival in these two entities, respectively. This indicates a significant role of DNMT3A alterations in myeloid as well as in lymphoid neoplasms. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Wild:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Erythroid Factor Erythroferrone and Its Role In Iron Homeostasis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4-4 ◽  
Author(s):  
Léon Kautz ◽  
Grace Jung ◽  
Elizabeta Nemeth ◽  
Tomas Ganz
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Iron Availability ◽  
Erythropoietic Activity ◽  
Wild Type ◽  
Advisory Committees ◽  
Hepcidin Expression ◽  
Erythroid Precursors ◽  
Equity Ownership ◽  
Hepcidin Mrna

Abstract Introduction In humans and other mammals, erythroid precursors in the bone marrow are the main consumers of iron. The availability of iron for erythropoiesis is controlled by hepcidin-induced endocytosis and degradation of ferroportin, the iron exporter which delivers iron to plasma from absorptive enterocytes and erythrocyte-recycling macrophages. In humans, within less than a day after hemorrhage or the administration of erythropoietin, duodenal iron absorption is increased by a mechanism which presumably evolved to provide for the iron requirement of increased erythropoiesis. Increased iron availability appears to be mediated by the suppression of the hormone hepcidin, thereby increasing ferroportin and delivering more iron to plasma. Increased erythropoietic activity is known to suppress hepcidin, but the molecular mechanism is not understood despite extensive investigation. We report that bleeding or administration of erythropoietin leads to the release of an erythroid factor made by erythroblasts which acts on hepatocytes to suppress hepcidin. Results We examined the mouse hepcidin mRNA response to hemorrhage in wild-type mice or mice lacking hemojuvelin or TfR2, two of the critical mediators of hepcidin synthesis. Wild-type, hemojuvelin and iron-depleted TfR2 mutant mice all responded to hemorrhage by similar suppression of hepcidin mRNA within 9-15h, indicating that hemojuvelin and TfR2 are not essential for this response. We therefore initiated an unbiased search for potential suppressors of hepcidin by examining the time course of bone marrow response to hemorrhage (500 μl) using gene chip-based expression profiling. We identified less than a dozen erythroid-specific transcripts that change prior to the suppression of hepcidin mRNA. Searching for secreted proteins, we focused on a previously unidentified transcript that is highly induced prior to hepcidin suppression, and provisionally named it “erythroferrone” (Erfe). Erfe mRNA expression was greatly increased in the bone marrow and the spleen 4h after phlebotomy or EPO stimulation, preceding hepcidin suppression. Erfe-deficient mice did not suppress hepcidin mRNA after phlebotomy (Figure) or EPO injection and recovered more slowly from phlebotomy-induced anemia than their wild-type counterparts. We did not observe any significant defects in their baseline erythropoiesis or the composition or maturation of erythroid precursors suggesting that Erfe exerts its effect specifically on hepcidin for the regulation of iron availability. Hepcidin expression was reduced by injection of recombinant Erfe (2 μg/g) in wild-type mice. Moreover, treatment of mouse primary hepatocytes with supernatants of HEK293T cells overexpressing Erfe led to a significant decrease in hepcidin expression suggesting that Erfe can act directly on the liver to suppress hepcidin. Importantly, we also found that Erfe mRNA is greatly increased in the marrow and spleen of the mouse model of β-thalassemia Hbbth3/+ compared to wild-type controls. Conclusion Erythroferrone may be the long-sought erythroid factor repressing hepcidin during increased erythropoietic activity, and may contribute to the pathogenesis of iron-loading anemias including β-thalassemia. Disclosures: Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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.

Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4288-4288 ◽  
Author(s):  
Claudia Haferlach ◽  
Anna Stengel ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Overall Survival ◽  
Relative Risk ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Complex Karyotype ◽  
Wild Type ◽  
Employment Equity ◽  
Total Cohort ◽  
Equity Ownership

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p<0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p<0.001), SRSF2 (27% vs 12%, p<0.001), and ZRSR2 (15% vs 3%, p<0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p<0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p<0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was < 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p<0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽  
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.

scholarly journals Higher Percentages of Ring Sideroblasts and SF3B1 Mutations in Patients with AML Correlate with Mutations in TP53 and Adverse Cytogenetics, but Have No Independent Impact on Outcome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3847-3847
Author(s):  
Pedro Martin-Cabrera ◽  
Sabine Jeromin ◽  
Tamara Alpermann ◽  
Karolína Perglerová ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
De Novo ◽  
Wild Type ◽  
Employment Equity ◽  
Tp53 Mutations ◽  
Ring Sideroblasts ◽  
Impact On Survival ◽  
Equity Ownership ◽  
Kras Codon ◽  
Codon 61

Abstract Introduction Ring sideroblasts (RS) are common findings in myeloid malignancies as MDS or MDS/MPN, especially in RARS-T, and are associated with SF3B1 mutations. However, the incidence of both RS and SF3B1 mutations have not yet been assessed properly in acute myeloid leukemia (AML). Aim Determine the frequency of RS and SF3B1 mutations in 1857 patients with de novo and therapy-related AML (t-AML). Define their impact on survival and association with other frequently mutated genes, as well as with cytogenetic abnormalities. Patients and Methods From a total of 1857 AML patients (excluding those with cytogenetically-defined entities according to WHO), bone marrow assessment revealed 473 (25%) with RS ≥ 1% and thereof 183 (10% of all) with ≥15 RS. Sequencing or melting curve analysis were performed in a subcohort of 340/473 patients for the detection of mutations in: SF3B1, ASXL1, DNMT3A, FLT3- TKD, IDH1 R132, IDH2 R140, IDH2 R172, KRAS, NPM1, NRAS, RUNX1, TET2, FLT3 -ITD and MLL -PTD. These 340 cases were subject to the study. Out of these 340, 141 cases (42%) had RS ≥ 15% and the remaining 199/340 (59%) had RS ≥1 to <15%. The cohort consisted of 303 (89%) de novo AML (FAB: M0 n=18, M1 n=67, M2 n=165, M4 n=30, M5 n=3, M6 n=20) and 37 (11%) t-AML. 148 were female and 192 were male, with median age 74 years, range: 20-93 years. Chromosome banding analysis (assisted by FISH if needed) was performed in all 340 cases. Results The percentage of bone marrow blasts correlated inversely with the percentage of RS present (r 0.213, p<0.001). 136 (40%) patients had a normal karyotype. Intermediate cytogenetics according to MRC criteria were found in 193 (57%), and adverse in 147 (43%). Patients with RS ≥15% more frequently had adverse cytogenetics in comparison to those with RS between 1-14% (54% vs 36%, p=0.001). The frequencies of gene mutations were as follows: TP53 103/331 (31%), RUNX1 84/315 (26%), DNMT3A 86/337 (25%), TET2 68/330 (20%), ASXL1 58/334 (17%), IDH2 R140 53/338 (15%), NPM1 43/340 (12%), SF3B1 34/334 (10%), FLT3 -ITD 33/340 (10%), NRAS 29/340 (8%), IDH1 R132 21/339 (6%), MLL -PTD 22/337 (6%), FLT3 -TKD 18/333 (5%), KRAS codon 12 13/299 (4%), IDH2 R172 13/338 (3%) and KRAS codon 61 3/299 (1%). Moreover, in total 30 variants in 28 patients were identified in DNMT3A, RUNX1, TET2 and TP53, which according to current knowledge cannot be assigned to mutations or SNPs yet. Patients with ≥15% more frequently had TP53 mutations (mut) (44% vs 22%, p<0.001) and less frequently IDH2 R140 mut (11% vs 19%, p=0.094) and MLL -PTD (2% vs 6%, p=0.006). Accordingly, patients with TP53 mutations had higher percentages of RS as compared to those without (28% vs 16%, p<0.001) and patients with IDH2 R140 mut and MLL -PTD, respectively, had lower percentages of RS as compared to those without (15% vs 21%, p=0.043 and 11% vs 21%, p=0.025, respectively). Furthermore, patients with mutations in the following genes had fewer RS than patients with the respective wild-types: ASXL1 (15% vs 21%, p=0.040), FLT3 -ITD (14% vs 21%, p= 0.049), IDH2 R140 (15% vs 21%, p=0.043), MLL -PTD (11% vs 21%, p=0.025), NPM1 (13% vs 21%, p=0.018) and KRAS codon 61 (3% vs 20%, p<0.001). Conversely, patients with mutated SF3B1 had more RS than patients with wild type (27% vs 19%, p=0.054). However, the number of RS did not translate into an increase in the mutational burden of SF3B1. Given the limited degree of overlap between mutations of the four most frequently mutated genes, we hierarchically separated the patients into 5 groups: TP53 mut, ASXL1 mut, NPM1 mut, SF3B1 mut and patients without any of these mutations. Interestingly, the percentage of RS was very similar in the two groups, TP53 mut and SF3B1 mut, and significantly higher as compared to all other groups (TP53 mut/SF3B 1mut: 28% vs ASXL1 mut/NPM1 mut/others 14%, p<0.001). The number of RS did not have an impact on the overall survival (OS) and event free survival (EFS) of patients. Conclusion 1. Ring sideroblasts ≥ 15% are present in 10% of de novo and t-AML. 2. The blast count correlates inversely with the number of RS. 3. Patients with ≥ 15% RS more frequently carry TP53 mutations and adverse cytogenetics. 4. Although patients with ≥ 15% RS have worse molecular (TP53 mut) and cytogenetic features, there is no statistically significant impact on survival when compared to patients with <15% RS. 5. Patients with mutated ASXL1, FLT3 -ITD, IDH2 R140, MLL -PTD and NPM1 have less RS than wild type patients while those with TP53 or SF3B1 mutations have higher RS. Figure 1. Figure 1. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Administration of the Dual E-Selectin/CXCR4 Antagonist, GMI-1359, Results in a Unique Profile of Tumor Mobilization from the Bone Marrow and Facilitation of Chemotherapy in a Murine Model of FLT3 ITD AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2826-2826 ◽  
Author(s):  
William E. Fogler ◽  
Henry Flanner ◽  
Curt Wolfgang ◽  
Jeffrey Allen Smith ◽  
Helen M. Thackray ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Murine Model ◽  
Standard Chemotherapy ◽  
Cxcr4 Antagonist ◽  
Employment Equity ◽  
Tumor Bearing ◽  
Equity Ownership ◽  
Cell Mobilization

Abstract FLT3 mutated AML confers poor prognosis despite treatment with standard chemotherapy, attributable to bone marrow (BM) induced tumor cell resistance in the microenvironment via molecules such as CXCR4 and E-selectin. We recently reported that simultaneous targeting of CXCR4 and E-selectin with GMI-1359, a novel small molecule dual antagonist, mobilized leukemic cells into the circulation; further, combination with AraC and DNR resulted in prolonged survival in a FLT3-ITD AML murine xenograft model. Here we report a novel profile of leukemic cell mobilization induced by GMI-1359, its impact on survival when combined with chemotherapy, and toxicity profile supporting entry to the clinic. A comparison of tumor mobilization by GMI-1359 to E-selectin or CXCR4 antagonists was determined in NCR mice with established BM infiltrative MV4-11 FLT3 ITD human AML tumor (modified with mCherry and luciferase). Mice (n=18/group) were injected with GMI-1359 (40 mg/kg), a potent E-selectin antagonist (40 mg/kg), or plerixafor (5 mg/kg) and blood samples were assessed by flow cytometry at multiple times for human AML cells and murine WBC. Appearance of human AML cells in peripheral blood increased ~16-fold compared to untreated mice 8 h post treatment with GMI-1359, and remained elevated for the duration of the study (72 h). Tumor cell mobilization following treatment with the E-selectin antagonist was gradual, reaching a 9.7-fold increase 48 h post injection and trending down at 72 h. Administration of plerixafor induced a rapid 11-fold mobilization of tumor cells by 1 h returning to baseline at 24 h. Murine WBC count did not change in any group at the time points assessed. These data establish that the profile of GMI-1359 induced tumor cell mobilization is distinct from both plerixafor (rapid mobilization of short duration) and an E-selectin antagonist (gradual mobilization of long duration). As E-selectin but not CXCR4 has been reported to be critical for tumor cell entry into the BM, the sustained presence of tumor cells in circulation after GMI-1359 treatment could be the result of blocking not only CXCR4 but also E-selectin, thereby inhibiting their re-entry into the BM. The therapeutic impact of tumor mobilization by GMI-1359 was evaluated for therapeutic consequence in combination with AraC and DNR in MV4.11 tumor bearing mice. Mice (n=10/group) were treated with saline; GMI-1359 (40 mg/kg) alone; chemotherapy alone; or GMI-1359 and AraC/DNR. GMI-1359 was initiated at onset of chemotherapy and given for 1, 3 or 14 days. Treatment of mice with GMI-1359 alone or together with AraC/DNR was well-tolerated. Median survival times (MST) of mice treated with saline, GMI-1359 or AraC/DNR was 47, 50 and 50 days, respectively; all mice succumbed to progressive disease by study conclusion (Day 71). In contrast, the MST of tumor bearing mice treated with GMI-1359 qdx3 or qdx14 and AraC/DNR was 63.5 days (p<0.001 vs. AraC/DNR) with a 40% survival rate. The MST of mice given a single dose of GMI-1359 at the start of AraC/DNR was statistically indistinguishable from chemotherapy alone (49.5 and 50 days, respectively) even though a 20% survival advantage was noted for the GMI-1359 combination group. GMI-1359 was tested for off-target interactions against 87 potential targets, with no significant activity observed. Toxicology studies showed a safety profile consistent with that of plerixafor. The pharmacokinetic (PK) profile was characterized in mice, rats, dogs, and monkeys, and was linear with t1/2 <2h in monkey and no accumulation in plasma. GMI-1359 is not mutagenic or clastogenic. Based on microsomal and liver S9 stability testing, GMI-1359 is not expected to be highly metabolized in any species tested. GMI-1359 did not inhibit P-gp, is moderately bound to human plasma proteins, and is stable in human plasma at 37C. In summary, GMI-1359, an innovative dual E-selectin and CXCR4 antagonist, more rapidly mobilized tumor cells and maintained them in circulation for a longer time when compared to either an antagonist of E-selectin or CXCR4 alone in a murine model of FLT3 ITD AML. This novel kinetic signature of mobilization by dual inhibition of two important adhesion molecules and accompanying improvement in survival over standard chemotherapy support the potential for combination with current treatment to improve outcomes for patients with FLT3-mutated AML. First-in-human trials are being initiated. Disclosures Fogler: GlycoMimetics, Inc.: Employment. Flanner:GlycoMimetics, Inc.: Employment. Wolfgang:GlycoMimetics, Inc.: Employment. Smith:GlycoMimetics, Inc.: Employment. Thackray:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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