Victoria Sung

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas–Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin

Sotatercept (ACE‐011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor ‐β (TGF‐β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP‐011 (murine ortholog of ACE‐011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late‐stage erythroid precursors in the bone marrow (BM). RAP‐011 also induces a significant increase in erythroid burst‐forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co‐culture studies demonstrate that BM accessory cells are required for RAP‐011 effects. To better understand which TGF‐β family ligand(s) mediate RAP‐011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP‐011 may act to rescue growth differentiation factor 11/Activin A‐induced inhibition of late‐stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Activin receptor antagonists for cancer-related anemia and bone disease

Introduction: Antagonists of activin receptor signaling may be beneficial for cancer-related anemia and bone disease caused by malignancies such as multiple myeloma and solid tumors. Areas covered: We review evidence of dysregulated signaling by activin receptor pathways in anemia, myeloma-associated osteolysis, and metastatic bone disease, as well as potential involvement in carcinogenesis. We then review properties of activin receptor antagonists in clinical development. Expert opinion: Sotatercept is a novel receptor fusion protein that functions as a soluble trap to sequester ligands of activin receptor type IIA (ActRIIA). Preclinically, the murine version of sotatercept increased red blood cells (RBC) in a model of chemotherapy-induced anemia, inhibited tumor growth and metastasis, and exerted anabolic effects on bone in diverse models of multiple myeloma. Clinically, sotatercept increases RBC markedly in healthy volunteers and patients with multiple myeloma. With a rapid onset of action differing from erythropoietin, sotatercept is in clinical development as a potential first-in-class therapeutic for cancer-related anemia, including those characterized by ineffective erythropoiesis as in myelodysplastic syndromes. Anabolic bone activity in early clinical studies and potential antitumor effects make sotatercept a promising therapeutic candidate for multiple myeloma and malignant bone diseases. Antitumor activity has been observed preclinically with small-molecule inhibitors of transforming growth factor-β receptor type I (ALK5) that also antagonize the closely related activin receptors ALK4 and ALK7. LY-2157299, the first such inhibitor to enter clinical studies, has shown an acceptable safety profile so far in patients with advanced cancer. Together, these data identify activin receptor antagonists as attractive therapeutic candidates for multiple diseases.

Pharmacokinetic characterization of amrubicin cardiac safety in an ex vivo human myocardial strip model II. Amrubicin shows metabolic advantages over doxorubicin and epirubicin

Anthracycline-related cardiotoxicity correlates with cardiac anthracycline accumulation and bioactivation to secondary alcohol metabolites or reactive oxygen species (ROS), such as superoxide anion (O2̇̄) and hydrogen peroxide (H2O2). We reported that in an ex vivo human myocardial strip model, 3 or 10 μM amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-d-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] accumulated to a lower level compared with equimolar doxorubicin or epirubicin (J Pharmacol Exp Ther 341:464–473, 2012). We have characterized how amrubicin converted to ROS or secondary alcohol metabolite in comparison with doxorubicin (that formed both toxic species) or epirubicin (that lacked ROS formation and showed an impaired conversion to alcohol metabolite). Amrubicin and doxorubicin partitioned to mitochondria and caused similar elevations of H2O2, but the mechanisms of H2O2 formation were different. Amrubicin produced H2O2 by enzymatic reduction-oxidation of its quinone moiety, whereas doxorubicin acted by inducing mitochondrial uncoupling. Moreover, mitochondrial aconitase assays showed that 3 μM amrubicin caused an O2̇̄-dependent reversible inactivation, whereas doxorubicin always caused an irreversible inactivation. Low concentrations of amrubicin therefore proved similar to epirubicin in sparing mitochondrial aconitase from irreversible inactivation. The soluble fraction of human myocardial strips converted doxorubicin and epirubicin to secondary alcohol metabolites that irreversibly inactivated cytoplasmic aconitase; in contrast, strips exposed to amrubicin failed to generate its secondary alcohol metabolite, amrubicinol, and only occasionally exhibited an irreversible inactivation of cytoplasmic aconitase. This was caused by competing pathways that favored formation and complete or near-to-complete elimination of 9-deaminoamrubicinol. These results characterize amrubicin metabolic advantages over doxorubicin and epirubicin, which may correlate with amrubicin cardiac safety in preclinical or clinical settings.

RAP-011, an activin receptor ligand trap, increases hemoglobin concentration in hepcidin transgenic mice

Over expression of hepcidin antimicrobial peptide is a common feature of iron‐restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP‐011, a “murinized” ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β‐thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor‐β superfamily members. We found that erythropoietin and RAP‐011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP‐011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin‐treated mice exhibited iron‐restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP‐011‐treated mice did not exhibit the same degree of iron‐restricted erythropoiesis. In conclusion, we have demonstrated that RAP‐011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP‐011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP‐011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron‐restricted erythropoiesis. Am. J. Hematol. 90:8–14, 2015.

Histone deacetylase inhibitor MGCD0103 synergizes with gemcitabine in human pancreatic cells

Histone deacetylase inhibitors are a group of recently developed compounds that modulate cell growth and survival. We evaluated the effects of the histone deacetylase inhibitor MGCD0103 on growth of pancreatic carcinoma models following single agent treatment and in combination with gemcitabine. MGCD0103 inhibited tumor cell growth and acted synergistically with gemcitabine to enhance its cytotoxic effects. Gene expression analysis identified the cell cycle pathway as one of the most highly modulated gene groups. Our data suggest that MGCD0103 + gemcitabine might be an effective treatment for gemcitabine‐refractory pancreatic cancer. (Cancer Sci 2011; 102: 1201–1207)

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure.

5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

Pharmacokinetic characterization of amrubicin cardiac safety in an ex vivo human myocardial strip model I. Amrubicin accumulates to a lower level than doxorubicin or epirubicin

Antitumor anthracyclines such as doxorubicin and epirubicin are known to cause cardiotoxicity that correlates with anthracycline accumulation in the heart. The anthracycline amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-d-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] has not shown cardiotoxicity in laboratory animals or patients in approved or investigational settings; therefore, we conducted preclinical work to characterize whether amrubicin attained lower levels than doxorubicin or epirubicin in the heart. Anthracyclines were evaluated in ex vivo human myocardial strips incubated in plasma to which anthracycline concentrations of 3 or 10 μM were added. Four-hour incubations were performed to characterize myocardial anthracycline accumulation derived from anthracycline uptake in equilibrium with anthracycline clearance. Short-term incubations followed by multiple washouts were performed to obtain independent measurements of anthracycline uptake or clearance. In comparison with doxorubicin or epirubicin, amrubicin attained very low levels in the soluble and membrane fractions of human myocardial strips. This occurred at both 3 and 10 μM anthracycline concentrations and was caused primarily by a highly favorable clearance of amrubicin. Amrubicin clearance was facilitated by formation and elimination of sizeable levels of 9-deaminoamrubicin and 9-deaminoamrubicinol. Amrubicin clearance was not mediated by P glycoprotein or other drug efflux pumps, as judged from the lack of effect of verapamil on the partitioning of amrubicin and its deaminated metabolites across myocardial strips and plasma. Limited accumulation of amrubicin in an ex vivo human myocardial strip model may therefore correlate with the improved cardiac tolerability observed with the use of amrubicin in preclinical or clinical settings.

Increased cellular accumulation and distribution of amrubicin contribute to its activity in anthracycline-resistant cancer cells.

PurposeMulti-drug resistance and cumulative cardiotoxicity are major limitations for the clinical use of anthracyclines. Here, we evaluated and compared the cross-resistance of amrubicin, a third-generation synthetic anthracycline and potent topoisomerase (topo)-II inhibitor with little or no observed cardiotoxicity to other anthracyclines and the topo-II inhibitor etoposide in drug-resistant tumor models in order to elucidate its potential mechanisms of action.MethodsAmrubicin activity was assessed in multi-drug-resistant cell lines and human tumor explants using cytotoxicity assays, confocal microscopy, fluorescence time-lapse imaging, flow cytometry, immunoblotting, and gene expression profiling techniques.ResultsWe demonstrate that both doxorubicin-resistant tumor cell lines and several drug-resistant human ovarian and breast tumor explants retain sensitivity to amrubicin. In addition, we observed similar levels of amrubicin uptake and accumulation in doxorubicin-sensitive versus doxorubicin-resistant cell lines. Although amrubicin is a weak P-glycoprotein substrate, transport and retention of amrubicin were not solely modulated by P-glycoprotein in the resistant cell lines overexpressing drug efflux pumps. The cellular retention of amrubicin is likely to be a result of rapid influx due to its high intrinsic permeability and lipophilic properties, and this may explain why amrubicin overcomes pleiotropic drug resistance. Consistent with drug accumulation studies, amrubicin induced DNA damage, G2–M cell cycle arrest, and apoptosis in both doxorubicin-sensitive and doxorubicin-resistant lines. Using gene expression profiling studies, several classes of genes were significantly and uniquely regulated following amrubicin, but not doxorubicin or etoposide, treatment.ConclusionsAmrubicin appears to have a distinct mode of action that overcomes typical anthracycline resistance mechanisms. Therefore, amrubicin may be useful in the treatment of anthracycline-refractory or anthracycline-resistant tumors.