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Increased IL-6 production induces, via STAT3 phosphorylation, hepatic transcription of the gene encoding the iron-regulatory hormone, hepcidin, leading to development of anemia of chronic disease (ACD). Inhibition of bone morphogenetic protein (BMP) signaling prevents the induction of hepcidin gene expression by IL-6 and ameliorates ACD. Using mice with hepatocyte-specific deficiency of Alk2 or Alk3, we sought to identify the BMP type I receptor that participates in IL-6–mediated induction of hepcidin gene expression. Mice were injected with adenovirus specifying IL-6 (Ad.IL-6) or control adenovirus. Seventy-two hours later, serum iron concentrations and hepatic levels of STAT3 phosphorylation and hepcidin messenger RNA were measured. Additional mice were injected with recombinant murine IL-6 (mIL-6) or vehicle, and hepatic hepcidin gene expression was measured 4 hours later. Deficiency of Alk2 or Alk3 did not alter the ability of Ad.IL-6 injection to induce hepatic STAT3 phosphorylation. Ad.IL-6 increased hepatic hepcidin messenger RNA levels and decreased serum iron concentrations in Alk2- but not Alk3-deficient mice. Similarly, administration of mIL-6 induced hepatic hepcidin gene expression in Alk2- but not Alk3-deficient mice. These results demonstrate that the ability of IL-6 to induce hepatic hepcidin gene expression and reduce serum iron concentrations is dependent on the BMP type I receptor Alk3.

Increased IL-6 production induces, via STAT3 phosphorylation, hepatic transcription of the gene encoding the iron-regulatory hormone, hepcidin, leading to development of anemia of chronic disease (ACD). Inhibition of bone morphogenetic protein (BMP) signaling prevents the induction of hepcidin gene expression by IL-6 and ameliorates ACD. Using mice with hepatocyte-specific deficiency of Alk2 or Alk3, we sought to identify the BMP type I receptor that participates in IL-6–mediated induction of hepcidin gene expression. Mice were injected with adenovirus specifying IL-6 (Ad.IL-6) or control adenovirus. Seventy-two hours later, serum iron concentrations and hepatic levels of STAT3 phosphorylation and hepcidin messenger RNA were measured. Additional mice were injected with recombinant murine IL-6 (mIL-6) or vehicle, and hepatic hepcidin gene expression was measured 4 hours later. Deficiency of Alk2 or Alk3 did not alter the ability of Ad.IL-6 injection to induce hepatic STAT3 phosphorylation. Ad.IL-6 increased hepatic hepcidin messenger RNA levels and decreased serum iron concentrations in Alk2- but not Alk3-deficient mice. Similarly, administration of mIL-6 induced hepatic hepcidin gene expression in Alk2- but not Alk3-deficient mice. These results demonstrate that the ability of IL-6 to induce hepatic hepcidin gene expression and reduce serum iron concentrations is dependent on the BMP type I receptor Alk3.

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin–dependent pathway. Here, we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE+ mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE+ mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin–dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin–dependent pathway. Here, we show that AE also induces expression of the Cox-2gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE+mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE+mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin–dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6–induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6–induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETOfusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1and TLE4were the only shRNAs capable of rescuing AML1-ETOexpressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1or TLE4levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1or TLE4caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETOto cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression.The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques.Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice.Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD.Peterson: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.

Abstract 686

Anemia of chronic disease (ACD) describes anemia associated with diverse chronic inflammatory, infectious, or neoplastic processes. These conditions are frequently associated with increased circulating levels of inflammatory cytokines such as interleukin 6 (IL-6). IL-6 regulates expression of the hormone hepcidin, which inhibits the release of iron from hepatocytes, macrophages, and enterocytes into the circulation. In addition to IL-6, hepcidin gene expression is known to be transcriptionally regulated by bone morphogenetic protein (BMP) signaling.We hypothesized that BMP signaling is required for the induction of hepcidin gene expression by IL-6 and plays a critical role in the pathogenesis of ACD.We used a turpentine-dependent model of ACD in mice. Mice were challenged with weekly subcutaneous injections of turpentine, which induces anemia in an IL-6 dependent manner. This model was studied to determine hepcidin gene expression and rescue ACD using BMP inhibition. Moreover, we examined hepcidin gene expression in zebrafish injected with Pseudomonas aeruginosa, and in transgenic zebrafish overexpressing human IL-6. The regulation of hepcidin gene expression was also studied in the human hepatocarcinoma cell line (HepG2).Injections of mice with IL-6 (0.8 μg/g ip) increased hepatic hepcidin mRNA levels expression at 24 hours and decreased serum iron concentrations. Both effects were prevented by a small molecule BMP type I receptor kinase inhibitor, LDN-193189, or protein BMP antagonists. Weekly turpentine injections induced microcytic anemia after 3 weeks with a decrease in hemoglobin levels from 12.8±0.3 to 9.7±1.7 g/dL (*p<0.01). Concurrent treatment with LDN-193189 prevented turpentine-induced anemia and microcytosis (*p<0.01 for both). In mice challenged with turpentine for 6 weeks, treatment with LDN-193189, beginning after anemia was established at week 3, led to an increase in hemoglobin levels at week 6 (10.9±0.1 vs 9.5±0.2 g/dL, LDN193189 vs vehicle, respectively; *p<0.05).In zebrafish, microinjection with Pseudomonas aeruginosa or overexpression of human IL-6 induced hepatic hepcidin expression, an effect which was blocked by LDN-193189.Incubation of HepG2 cells with IL-6 (100 ng/ml) increased hepcidin mRNA levels 2 to 5 fold. Pretreatment with LDN-193189, or recombinant protein BMP antagonists such as noggin, abrogated the induction of hepcidin expression by IL-6. Incubation of HepG2 cells with BMP6 (2.5 to 10 ng/ml) modestly increased hepcidin mRNA levels. However, the combination of IL-6 and BMP6 synergistically increased hepcidin gene expression (*p<0.05).BMP signaling appears to play a critical role in the pathogenesis of anemia in a mouse ACD model. Our findings support the concept that BMP signaling is required for the induction of hepcidin gene expression by IL-6 in vitro and in vivo. Moreover, manipulation of BMP signaling represents a potentially novel therapeutic approach to the treatment of anemia associated with inflammation.Steinbicker: Deutsche Forschungsgemeinschaft DFG: Research Funding. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Peterson:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Bloch:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Yu:Harvard Stem Cell Institute Seed Grant and the Howard Hughes Medical Institute Early Career Physician-Scientist Award: Honoraria, Research Funding; NHLBI 5K08HL079943: Research Funding.

Abstract 2043