Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment.

Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

Genetic screens reveal CCDC115 as a modulator of erythroid iron and heme trafficking.

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Anemia of Inflammation: A Review.

Impaired iron homeostasis and the suppressive effects of proinflammatory cytokines on erythropoiesis, together with alterations of the erythrocyte membrane that impair its survival, cause anemia of inflammation. Recent epidemiologic studies have connected inflammatory anemia with critical illness, obesity, aging, kidney failure, cancer, chronic infection, and autoimmune disease. The proinflammatory cytokine, interleukin-6, the iron regulatory hormone, hepcidin, and the iron exporter, ferroportin, interact to cause iron sequestration in the setting of inflammation. Although severe anemia is associated with adverse outcomes in critical illness, experimental models suggest that iron sequestration is part of a natural defense against pathogens.

Nicotinamide N-methyltransferase expression decreases in iron overload, exacerbating toxicity in mouse hepatocytes.

Iron overload causes the generation of reactive oxygen species that can lead to lasting damage to the liver and other organs. The goal of this study was to identify genes that modify the toxicity of iron overload. We studied the effect of iron overload on the hepatic transcriptional and metabolomic profile in mouse models using a dietary model of iron overload and a genetic model, the hemojuvelin knockout mouse. We then evaluated the correlation of nicotinamide N-methyltransferase (NNMT) expression with body iron stores in human patients and the effect of NNMT knockdown on gene expression and viability in primary mouse hepatocytes. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism and that NNMT, an enzyme that methylates nicotinamide and regulates hepatic glucose and cholesterol metabolism, is one of the most strongly down-regulated genes in the liver in both genetic and dietary iron overload. We found that hepatic NNMT expression is inversely correlated with serum ferritin levels and serum transferrin saturation in patients who are obese, suggesting that body iron stores regulate human liver NNMT expression. Furthermore, we demonstrated that adenoviral knockdown of NNMT in primary mouse hepatocytes exacerbates iron-induced hepatocyte toxicity and increases expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while overexpression of NNMT partially reversed these effects. Conclusion: Iron overload alters glucose and nicotinamide transcriptional and metabolic pathways in mouse hepatocytes and decreases NNMT expression, while NNMT deficiency worsens the toxic effect of iron overload. For these reasons, NNMT may be a drug target for the prevention of iron-induced hepatotoxicity. (Hepatology Communications 2017;1:803-815).

Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia.

Understanding anemia of chronic disease.

The anemia of chronic disease is an old disease concept, but contemporary research in the role of proinflammatory cytokines and iron biology has shed new light on the pathophysiology of the condition. Recent epidemiologic studies have connected the anemia of chronic disease with critical illness, obesity, aging, and kidney failure, as well as with the well-established associations of cancer, chronic infection, and autoimmune disease. Functional iron deficiency, mediated principally by the interaction of interleukin-6, the iron regulatory hormone hepcidin, and the iron exporter ferroportin, is a major contributor to the anemia of chronic disease. Although anemia is associated with adverse outcomes, experimental models suggest that iron sequestration is desirable in the setting of severe infection. Experimental therapeutic approaches targeting interleukin-6 or the ferroportin-hepcidin axis have shown efficacy in reversing anemia in either animal models or human patients, although these agents have not yet been approved for the treatment of the anemia of chronic disease.

The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro.

Previous studies have shown that administration of ferristatin II to rats is associated with decreased serum iron, reduced transferrin saturation, and increased hepatic hepcidin expression. BMP and IL-6 signaling act via Smad and Stat3 transcription factors, respectively, to increase expression of hepcidin, the master regulator of iron metabolism. In this study, we aimed to explore the underlying mechanism of ferristatin II action on hepcidin production. We found that ferristatin II greatly increased hepcidin expression both in vivo and in vitro. In the rat liver, ferristatin II treatment decreased expression of Smad downstream targets Smad7 and Id1 and increased expression of Stat3 downstream targets α-2-macroglobulin, α-1-acid glycoprotein, and C-reactive peptide. Ferristatin II also increased Stat3 phosphorylation in the rat liver without affecting serum or hepatic IL-6 levels. It is unclear whether the Stat3 activation observed in vivo is a cause or a consequence to hepcidin induction. Reporter gene expression studies demonstrated that ferristatin II synergized with BMP6 and IL-6 to enhance hepcidin expression in vitro. However, this synergy was not due to activation of either Smad or Stat3 signaling, raising the possibility that ferristatin II may activate a novel pathway for hepcidin regulation.

A chemical screen identifies small molecules that regulate hepcidin expression.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.

The small molecule, genistein, increases hepcidin expression in human hepatocytes.

UNLABELLED: Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Transferrin-a modulates hepcidin expression in zebrafish embryos.

The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo.

montalcino, A zebrafish model for variegate porphyria.

OBJECTIVE: Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. MATERIALS AND METHODS: Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. RESULTS: Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. CONCLUSION: In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.

Functional conservation of erythropoietin signaling in zebrafish.

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.

Ferroportin1 is required for normal iron cycling in zebrafish.

Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (weh(Tp85c-/-)) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, weh(Tp85c-/-) zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only weh(Tp85c-/-) zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, weh(Tp85c-/-) zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic weh(Tp85c-/-) adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1's function as an iron exporter.

Induction of myasthenia gravis, myositis, and insulin-dependent diabetes mellitus by high-dose interleukin-2 in a patient with renal cell cancer.

Interleukin-2 is an effective agent against renal cell carcinoma and melanoma, but it has been associated with autoimmune sequelae such as hypothyroidism and vitiligo. A 64-year-old man with non-insulin-dependent diabetes and metastatic renal cell carcinoma developed insulin-dependent diabetes after his first cycle of therapy with high-dose (HD) interleukin-2. After additional therapy with interleukin-2, the patient developed generalized myasthenia gravis (MG) and polymyositis, both of which responded to treatment with corticosteroids and plasmapheresis. To investigate the role of IL-2 in the development of these autoimmune complications, autoantibody titers were assayed from serum obtained before and after IL-2 treatment and after treatment with corticosteroids plus plasmapheresis. Before IL-2 treatment, the patient had antibodies directed against insulin, islet cell antigens, and striated muscle. Acetylcholine receptor antibody levels were normal before starting IL-2. After treatment with IL-2, the patient developed acetylcholine receptor binding antibodies and exhibited an increase in the striated muscle antibody titer from 1:40 to 1:160. Recovery from the MG and polymyositis was associated with substantial decreases in the acetylcholine receptor and striated muscle antibody titers. These findings suggest that HD IL-2 accelerated the progression of latent autoimmune diabetes and myositis in this patient whose tolerance to islet cell antigens and striated muscle had already been broken and precipitated a break in tolerance to the acetylcholine receptor resulting in the development of MG. This case demonstrates the importance of prompt recognition of IL-2-induced MG and shows how this complication can be successfully managed with aggressive therapy.