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

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.

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

Blood ◽

10.1182/blood.v120.21.3796.3796 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3796-3796 ◽

Cited By ~ 1

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.

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BRD4 Proteolysis Targeting Chimera (PROTAC) Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Leukemias and Overcomes Stroma Mediated Resistance By Modulating Surface Expression of CXCR4

Blood ◽

10.1182/blood.v126.23.675.675 ◽

2015 ◽

Vol 126 (23) ◽

pp. 675-675

Author(s):

Sujan Piya ◽

Teresa McQueen ◽

Duncan Mak ◽

Marina Konopleva ◽

Patrick Zweidler-McKay ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Trypan Blue ◽

Research Funding ◽

Annexin V ◽

Blue Staining ◽

Employment Equity ◽

Acute Leukemias ◽

Lower Chamber ◽

Equity Ownership

Abstract Background: Hematological neoplasms are characterized by aberrant epigenetic events that modify the chromatin regulatory machinery to enhance oncogene expression (Zuber et al. Nature 2011). BRD4, a member of the bromodomain and extra terminal domain (BET) family, is critical in the assembly of a 'super enhancer complex' that drives expression of oncogenes MYC, CCND1, SOX2 and NF-kB and anti-apoptotic proteins e.g. Bcl-2 and BCL-XL (Klapproth et al. Br J Haematol 2010; Wang et al. Cancer Res 2014; Zou et al. Oncogene 2014). Small molecule BRD4 inhibitors lead to rapid accumulation of BRD4 that may partially account for their moderate suppression of MYC (Lu et al. Chem Biol 2015). ARV-825 is hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 and sustained down-regulation of MYC (Lu et al. Chem Biol 2015). Aim: We investigated the activity of ARV-825 against leukemia cell lines representing acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) including gamma secretase inhibitor (GSI) resistant T-ALL and against primary AML blasts and compared it to that of the BRD4 inhibitor, JQ1. As stroma renders leukemia resistant, we tested if and through what mechanism ARV-825 can overcome stroma mediated drug resistance. Results: The IC50s for all tested cell lines at 72 hours were in very low nanomolar range; AML-OCIAML3-8.3 nM, HL60-2.4nM; ALL-KOPT-K1/SUPTI-1.3nM (GSI resistant T-ALL), LOUCY-2.3nM (early T-cell phenotype ALL), MOLT4-3.12nM and HPB-ALL-4.6nM (T- ALL) while the same for JQ1 was 102.6 nM, 5 nM, 1.5nM/8.8 nM, 4.5nM, 9.4 and 8.4 nM respectively. Consistent with sustained degradation of BRD4, the apoptotic effect of ARV-825 is sustained longer than that of JQ1 after the removal of compounds following a 24-hr treatment at IC50 (Annexin V positive 70% vs. 4% at 48 hrs post-washout respectively) (Fig. 1). Importantly, IC50 values for primary AML samples for ARV-825 were approximately 6-100 times lower than those for JQ1 (Fig. 2). ARV-825 led to sustained decreases of BRD4 and MYC proteins with corresponding increase in cleaved PARP, cleaved Caspase-3 and γH2AX in MOLT4 and OCIAML3 cells. While Bcl-2 level remained suppressed with ARV-825, it recovered at 48 hrs of treatment with JQ1. We co-cultured OCI-AML3 cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow microenvironment. While MSC protected OCI-AML3 cells from cytarabine (25 % reduction in cell death), there was no stromal protection for ARV-825. To gain mechanistic insight, we used a Mass cytometry (CyTOF) based approach to analyze the changes in apoptotic, cell adhesion and signaling proteins (panel of 24 antibodies) in OCI-AML3 cells after 12 and 24-hour treatment with ARV-825. MYC and surface CXCR4 were the two most down-regulated proteins. The reduction of surface CXCR4 was confirmed by conventional flow cytometry (Fig. 3A) and the functional relevance was confirmed in migration assays against the CXCR4 ligand SDF-1 (29.2% Control vs. 9.03% ARV-825) (Fig. 3B). Conclusion: ARV-825, a BRD4 PROTAC, has substantial anti-leukemia activity across a range of acute leukemias and importantly can overcome stroma-mediated drug resistance. ARV-825 promises to be an exciting molecule in the quest towards efficient BRD4 inhibition. Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay). Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. / Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. / (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay). Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Lu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.751.751 ◽

2015 ◽

Vol 126 (23) ◽

pp. 751-751 ◽

Cited By ~ 5

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.

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

Blood ◽

10.1182/blood.v122.21.749.749 ◽

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.

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EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1528-1528

Author(s):

Sebastian Stasik ◽

Jan Moritz Middeke ◽

Michael Kramer ◽

Christoph Rollig ◽

Alwin Krämer ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Mutational Status ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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Randomized Phase II Study Evaluating the Efficacy and Safety of Romiplostim Treatment of Patients with Low or Intermediate Risk Myelodysplastic Syndrome (MDS) Receiving Lenalidomide.

Blood ◽

10.1182/blood.v114.22.1770.1770 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1770-1770 ◽

Cited By ~ 6

Author(s):

Roger M Lyons ◽

Richard A. Larson ◽

Michael A. Kosmo ◽

Sunil Gandhi ◽

Delong Liu ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Intermediate Risk ◽

Employment Equity ◽

Treatment Groups ◽

Platelet Counts ◽

Equity Ownership ◽

Clinically Significant ◽

Dose Reductions ◽

Platelet Transfusions

Abstract Abstract 1770 Poster Board I-796 Introduction Romiplostim is a peptibody protein designed to increase platelet production by binding to and activating the thrombopoietin receptor. Low platelet counts in patients with myelodysplastic syndromes (MDS) may be due to the underlying disease or to treatment with disease-modifying agents, and platelet transfusions are often the only treatment for clinically significant thrombocytopenia (CST) or bleeding. This was a phase 2 multi-center, randomized, double-blind, placebo-controlled, dose-finding study that evaluated the effect of romiplostim on the incidence of clinically significant thrombocytopenic events (grade 3 or 4 thrombocytopenia and/or receipt of platelet transfusions) and the safety of romiplostim in patients with low or intermediate risk MDS receiving lenalidomide. Patients and Methods Patients who were ≥18 years old, had MDS by bone marrow exam and WHO criteria, had low or Intermediate-1 risk category MDS using the IPSS, and were planning to receive lenalidomide were eligible. Patients were randomized 1:1:1 into treatment groups receiving placebo, 500 μg romiplostim, or 750 μg romiplostim by weekly subcutaneous injections in combination with lenalidomide (one 10 mg capsule by mouth daily for each 28-day cycle). Treatments continued for a total of four cycles. Results The median age of the 39 randomized patients was 74 years (range, 39 to 90); 15 (39%) had platelet counts <50 × 109/L, and 7 (18%) had del(5q). We report trends due to baseline imbalances between treatment groups, likely due to the limited sample size. The overall incidence rates of CST appeared to be greater in the placebo group than either romiplostim group (Table). In contrast to the placebo patients, median platelet counts remained above 50 × 109/L in both the 500 μg and 750 μg romiplostim groups for the treatment period. The incidence of platelet transfusions appeared to be lower in the 500 μg romiplostim group, and the incidence of adverse events was comparable between all of the groups. No deaths were reported during the treatment period. Twelve patients (31%) discontinued the study. Disease progression to AML was reported in 1 patient in the romiplostim 500 μg group. The patient withdrew consent and discontinued the study. No bone marrow was available to confirm AML by protocol-defined criteria. Fewer lenalidomide dose reductions and delays due to thrombocytopenia were seen in both of the romiplostim treated groups. The proportion of patients who achieved an MDS treatment response was 8%, 36% and 15% for the placebo, 500 μg romiplostim, and 750 μg romiplostim groups, respectively. MDS response rates appeared higher in the romiplostim group, regardless of baseline del(5q) status. Baseline imbalance between groups due to the small sample size limited our interpretation of the data. Conclusions Romiplostim appeared to be well-tolerated in low and intermediate risk MDS patients receiving lenalidomide. This preliminary information suggests that romiplostim may reduce the rate of clinically significant thrombocytopenic events in these patients while increasing platelet counts and decreasing the incidence of lenalidomide dose reductions and delays due to thrombocytopenia Disclosures Lyons: GlaxoSmithKline: Consultancy, Speakers Bureau; Johnson&Johnson: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy; Amgen Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau. Off Label Use: Use of romiplostim to treat Thrombocytopenia in MDS. Larson:Amgen Inc.: Equity Ownership, Research Funding. Liu:Amgen Inc.: Honoraria, Research Funding. Hu:Amgen Inc.: Employment, Equity Ownership. Franklin:Amgen Inc.: Employment, Equity Ownership. Berger:Amgen Inc.: Employment, Equity Ownership.

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Use of a New Rotary Powered Device for Bone Marrow Aspiration and Biopsy Yields Excellent Specimens Quickly and Efficiently.

Blood ◽

10.1182/blood.v114.22.4544.4544 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4544-4544

Author(s):

Ronan T. Swords ◽

Kevin R. Kelly ◽

Devalingam Mahalingam ◽

Stephen C. Cohen ◽

Larry J. Miller ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsy ◽

Clinical Evaluation ◽

Research Funding ◽

Bone Marrow Aspiration ◽

Swine Model ◽

Employment Equity ◽

Equity Ownership ◽

Specimen Quality ◽

The Mean

Abstract Abstract 4544 Background The importance of bone marrow aspiration and biopsy in the evaluation of hematopoietic and non-hematopoietic disorders is well established. Recently, a new FDA-cleared battery powered bone marrow biopsy system was developed to allow operators access to the bone marrow space quickly and efficiently. Aims The first aim of this study was to evaluate the quality of core specimens using the new powered device compared to specimens obtained using the traditional manual technique in a swine model. The second aim was to evaluate the safety and efficacy of the device in patients presenting for outpatient hematology clinic visits. Materials and Methods For the pre-clinical evaluation of the device, three anesthetized pigs were used for the study. The powered device (OnControl, Vidacare Corporation, San Antonio, TX, USA) was comprised of a battery powered driver and needle set. The manual device used was a T-Handle Jamshidi bone marrow biopsy needle (Cardinal Health, Dublin, OH, USA). Core biopsy samples obtained were assessed for length and sample quality and then submitted for analysis to a pathologist blinded to the device used. The clinical evaluation of the device was conducted in accordance with practice guidelines and directions for use. Data collection included insertion success, time from insertion to removal, specimen quality, operator satisfaction with control/function of the device and overall operator satisfaction based on a scoring system (0-5; 0=totally unacceptable, 5=outstanding). Results Twenty six samples were collected from the swine model (19 samples using the powered device and 9 using the manual technique). No cellular artifact or thermal damage was reported in any of the samples obtained. The mean lengths for samples obtained using the powered and manual techniques were respectively 19.4mm±1.6mm and 18.6mm±5.3mm. For the clinical evaluation of the device, 16 patients were recruited from 2 centers. Mean insertion time was 11.25±3.39 seconds and mean time from needle contact with skin to needle removal was 38.5±13.94 seconds. No complications were reported. Five operators rated the overall use of the device as outstanding in 75% of cases. Conclusions In this study, the manual and powered samples were equivalent in specimen quality. The powered device however, captured longer biopsies when compared to the manual technique. In the patients evaluated, the device was easy to use as well as being safe and effective. The mean procedural time was significantly faster than previously reported with a manual technique. A randomized study of the powered device compared to the manual technique is underway. Disclosures: Swords: Vidacare Corporation: Research Funding. Kelly:Vidacare Corporation: Research Funding. Mahalingam:Vidacare Corporation: Research Funding. Cohen:Vidacare Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Miller:Vidacare Corporation: Employment, Equity Ownership. Philbeck:Vidacare Corporation: Employment, Equity Ownership. Brenner:Vidacare Corporation: Consultancy, Research Funding. Giles:Vidacare Corporation: Research Funding.

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Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽

10.1182/blood.v118.21.3279.3279 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3279-3279 ◽

Cited By ~ 6

Author(s):

Ann Janssens ◽

Michael D. Tarantino ◽

Robert Bird ◽

Maria Gabriella Mazzucconi ◽

Ralph Vincent V. Boccia ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Platelet Response ◽

Employment Equity ◽

Advisory Committees ◽

Platelet Production ◽

Platelet Counts ◽

Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

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Results of the OPAL Study: A Phase II Study to Evaluate the Efficacy, Safety and Tolerability of Tosedostat (CHR-2797) in Elderly Subjects with Treatment Refractory or Relapsed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.767.767 ◽

2011 ◽

Vol 118 (21) ◽

pp. 767-767

Author(s):

Jorge E. Cortes ◽

Eric J Feldman ◽

Karen Yee ◽

David A. Rizzieri ◽

Anjali S. Advani ◽

...

Keyword(s):

Bone Marrow ◽

Phase Ii ◽

Research Funding ◽

Phase Iii ◽

Elderly Subjects ◽

Primary Therapy ◽

Primary Analysis ◽

Employment Equity ◽

Once Daily ◽

Equity Ownership

Abstract Abstract 767 Background: Tosedostat is a novel oral inhibitor of the M1/17 family of aminopeptidases which induces an amino acid deprivation response that is selectively toxic for myeloid blasts (Leuk Res. 2011: 5:677-81) and has shown promising activity in elderly relapsed/refractory AML patients (J Clin Oncol 2010:28:4333-8). Aims: The OPAL study was undertaken to compare the activity of tosedostat at a once-daily dose of 120 mg for 24 weeks compared to 240 mg once daily for 8 weeks followed by 120 mg once daily for a further 16 weeks., as measured by bone marrow and hematology responses at 24 weeks. Methods: This was a phase II randomized (1:1) multi-center study. Patients were eligible if aged 60 years or older with previous CR lasting <12 months, or no CR after primary therapy, had a peripheral blast count <30,000/μl, PS<2 and adequate renal, hepatic and cardiac function. The primary analysis was performed at 24 weeks using IWG 2003 criteria. Results: Seventy-three patients were randomized and received tosedostat, 38 at 120 mg and 35 at 240 mg. Median age was 72 (range, 64 to 86), and 59% were male. Twenty-six patients (36%) had secondary or therapy-related AML, of which 19 (26%) had prior MDS. Median time since AML diagnosis was 211 days and 38% had received primary therapy with cytarabine/anthracyclines; 36% with a hypomethylating agent (HMA) and 23% with other cytarabine regimes. Fifty-two percent had been refractory to primary therapy, 19% had previously had a remission of up to 6 months and 29% a 6–12 month remission (mean 97 days including refractory). Twenty-three patients (32%) had no post-treatment bone marrow sample taken, predominantly due to early progression: 34% completed 12 weeks on study and 14% completed 24 weeks and were eligible to enter an extension study which is ongoing. The overall response rate was 22%; (CR/CRp/MLFS 12%; PR 10%) and an additional 29% had a best response of stable disease. The most common adverse events which occurred (total; grade 3 or worse) were diarrhea (58%; 4.1%), peripheral edema (55%; 0%), fatigue (49%; 21%), dyspnea (41%; 16%), nausea (38%; 0%), decreased appetite (37%; 3%), febrile neutropenia (36%; 29%) and hypotension (36%, 10%). Median overall survival (OS) (at 15 July 2011) was 126 days. Median OS in patients with CR/CRp/MLFS, PR and SD were 280, 195 and 162 days respectively, and 261.5 days for patients with a response of PR or better. Median OS for patients with progression of disease or who were unevaluable was 61 days. Similar responses were seen in the two dose groups. Additional non protocol-specified analyses showed that the following types of patient appeared to respond well: AML NOC vs other AML types 16% vs 29% response, median OS 75 vs 168 days; patients with poor risk cytogenetics compared to intermediate/better, median OS 159 vs 107 days; those who received prior HMA compared to others, 38% vs 13% response, median OS 171 vs 104 days; and absence of prior CR 29% vs 14% response and median OS 169 vs 103 days. Conclusions: These results provide further encouraging evidence of efficacy and a favorable toxicity profile in a difficult to treat patient population. A phase III program of pivotal studies with tosedostat in AML and MDS will start in the near future. Disclosures: Cortes: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Feldman:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Yee:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Rizzieri:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Advani:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Charman:Chroma Therapeutics Ltd.: Employment, Equity Ownership. Toal:Chroma Therapeutics Ltd.: Employment, Equity Ownership. Kantarjian:Chroma Therapeutics Ltd.: Consultancy, Research Funding.

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

Blood ◽

10.1182/blood.v122.21.564.564 ◽

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.

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