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Iron is essential for proper red blood cell development. During erythroid maturation in the bone marrow iron dependency starts at the basophilic stage, and lasts until the reticulocyte stage. Thereby the majority of iron is delivered in a Tf-TfR dependent manner and is incorporated in hemoglobin of developing red cells. Just recently, it has been discovered that erythroblasts and mature red blood cells not only incorporate iron but also express the sole known iron exporter, ferroportin (Fpn), throughout their development, and thus can also effectively export iron that is not used for heme synthesis. Fpn surface expression is regulated via hepcidin, a hepatocyte-derived peptide, which binds directly to surface-Fpn, inducing its internalization and consequent degradation in lysosomes. Among patients suffering from anemia of chronic disease (ACD), hepcidin is constantly induced due to long-lasting inflammation, leading to impaired iron export capacity. While splenic tissue iron overload, which is associated with the restricted capacity of red pulp macrophages to export recycled iron form degraded erythrocytes is well established, the effect of high hepcidin levels in ACD and its consequences on developing erythroblasts in the bone marrow has not been investigated. First we induced chronic kidney disease (CKD) in C57BL/6 mice via an Adenine - diet. Alongside microcytic anemia, reduced Tf-saturation, increased hepcidin levels and splenic tissue iron overload, we found a ~38% increase in tissue iron content in bone marrows of CKD mice compared to control mice. In parallel, Western blot analysis revealed massively reduced Fpn protein levels in the bone marrow. Moreover, the individual iron-dependent erythroblast precursor populations (i.e. basophilic, polychromatic, orthochromatic erythroblasts and reticulocytes) showed higher levels of intracellular iron as measured by Calcein fluorescence via flow cytometry. We could further corroborate these results by Western blot analysis and flow cytometric work-up of reticulocytes and mature red blood cells in the blood stream, both revealing highly reduced Fpn protein levels on these cells in mice suffering from CKD. Based on these findings we performed additional experiments to investigate the detrimental effect of iron overload on erythroid development and to exclude the possibility of a direct inflammation-regulated process in the CKD model. Therefor we established an iron-overload mouse model via repeated parenteral iron dextran applications. Despite significantly increased Tf-saturation levels as well as hepatic hepcidin levels (>4-fold) and reduced bone marrow Fpn protein levels among iron-treated mice, iron overload led to higher stress levels among erythroid precursor populations. In detail, we could demonstrate via flow cytometry that higher intracellular iron pools (measured by Calcein), correlated with higher levels of mitochondrial stress and higher levels of lipid peroxidation (determined by mean fluorescence intensity of MitoSOX and Bodipy 581/59, respectively). These data indicate that iron is a critical regulator of stress during erythroid development and can be regulated via the hepcidin-Fpn axis. In conclusion, we clearly show that Fpn expression on erythroid precursor cells is inversely regulated to systemic hepcidin levels and affects the erythropoietic bone marrow iron content. Moreover our results reveal a novel model for ineffective erythroid output in patients suffering from ACD due to hepcidin-triggered Fpn internalistation. Based on our data, anti-hepcidin treatment strategies are promising to overcome restricted erythropoiesis, which will be evaluated in future experiments. Disclosures Theurl: Kymab Ltd: Consultancy, Equity Ownership.

Every second more than 2 million new erythrocytes are released from the bone marrow of human adults, highlighting the tremendous turn-over of these cells. In parallel to hematopoietic stem cell niches, the last stages of erythropoiesis take place in specialized bone marrow niches, termed 'erythroid niches'. Concretely, the erythroid niche is composed of a 'central macrophage' which is surrounded by erythroid progenitor cells. Regardless of steady-state or stress erythropoiesis, iron availability is, beside erythropoietin, a key factor determining erythroid output and red blood cell quality, as reflected by hemoglobin content of these cells. It is well established that systemic iron availability for erythropoiesis, in the form of iron saturated transferrin (Tf), is mainly maintained via a recycling process of senescent red blood cells, which takes place in macrophages of the reticuloendothelial system. Yet, it is still a matter of debate if also central macrophages are involved in iron supply for red blood cell development in a more direct way due to their close proximity to developing red blood cells. Using a myeloid-specific knockout mouse strain, lacking the solely known iron exporter ferroportin (Fpn; Fpnfl/flLysMCre+/+ mice) and specific reporter mice (ROSA26tdTomatofl/fl Cx3cr1CreERT2 mice), we examined the connection between iron metabolism, erythropoiesis and central macrophages. Analysis of Fpnfl/flLysMCre+/+ animals at steady state revealed microcytic anemia, higher tissue iron loading, reduced hepatic hepcidin expression and distorted erythroid precursor population distribution in the bone marrow with no significant chances in Tf saturation (Tf-Sat). The latter is giving a first hint, that local bone marrow Fpn expression on macrophages may be important for iron supply for erythropoiesis. Strikingly, further work up via flow cytometry demonstrated that disturbances seen in bone marrow erythropoiesis were accompanied by nearby loss of resident bone marrow macrophages (defined as CD11blo, F4/80pos, MerTKpos). In parallel, a CD11bhi, F4/80pos, MerTKpos population came into existence, suggesting that these cells may compensate for the loss of 'canonical' central macrophages. Attempting to explain these intriguing results, we sought to investigate differentiation pathways and turnover of bone marrow central macrophages. First, we used the ROSA26tdTomatofl/fl Cx3cr1CreERT2 monocyte-specific reporter mice and techniques of transient monocyte labelling in utero and in adult phlebotomized animals to determine the origin of central macrophages. We could show that those cells undergo constant replenishment by circulating monocytes. Notably, the rate of this process got markedly increased upon recovery from blood loss and concomitant expansion of the central macrophage population. Second, by administration of a CCR2/CCR5 inhibitor (cenicriviroc), diminishing monocyte egress from the bone marrow and tissue infiltration, we could demonstrate decreased reticulocyte count during stress erythropoiesis, thus strengthening the direct impact of macrophages to support effective erythroid output. Next, effects of stress-induced erythropoiesis were investigated in Fpnfl/flLysMCre+/+ compared to Fpnfl/flLysMCre-/- mice. Amelioration of anemia after phlebotomy was extended, microcytosis was more pronounced and reticulocyte egress was diminished but prolonged. Of interest, Fpnfl/flLysMCre+/+ mice on a diet containing an 8-times higher iron content during phlebotomy, thus transiently increasing Tf-Sat, recovered from anemia wildtype-like. These results indicate that stress erythropoiesis with a high iron demand depends, under normal iron availability, in part on central macrophages and their nursing function to overcome the increased demand of iron. Ongoing experiments aim to identify how recruited bone marrow macrophages, i.e. central macrophages, contribute to erythropoiesis during stress - if central macrophages directly supply developing erythroid cells with iron in a Tf-free fashion or, if they are suppliers of additional growth factors that work synergistically with the Tf-bound iron to drive hemoglobin production. In summary our data clearly show that macrophages need to be recruited to the bone marrow for effective erythroid output during stress erythropoiesis. Disclosures Weiss: Kymab Ltd.: Consultancy. Theurl:Kymab Ltd.: Consultancy, Research Funding.

Anemia of chronic disease (ACD) is the most common cause of anemia in hospitalized patients. The underlying pathophysiological mechanisms are manifold, including reduced Erythropoietin (EPO) availability and sensitivity, direct negative effects of inflammatory cytokines on erythropoiesis and functional iron deficiency due to iron restriction mainly in the reticuloendothelial system (RES). The latter can be ascribed to increased hepcidin levels, a small liver derived peptide that has been found to be the key iron regulator. Up to date, most patients suffering from ACD are treated with a combination of both ESA (Erythropoietin Stimulating Agent) and intravenous (i.v.) iron to maintain hemoglobin (Hb) levels. Despite this combination therapy a significant number of patients require increasing doses of i.v. iron and ESA during their medical history - often resulting in continuous, potential toxic iron overload and ESA doses that exceed the acceptable range. As hepcidin is central to the iron-restrictive phenotype in ACD, several therapeutic approaches of hepcidin modulation have been investigated to overcome iron overload and EPO resistance. Some of these therapies are currently investigated in early clinical trials. We here report the effects of a fully human anti-BMP6 antibody on anemia, iron metabolism, erythropoiesis and ESA dosing in two different, well established rodent models of ACD. As BMPs, mainly BMP2 and BMP6, have been reported to be involved in hepcidin control, with knock out mice showing very low hepcidin levels even in an inflammatory setting, a fully human anti-BMP6 antibody has been developed to suppress hepcidin levels. We tested the antibody treatment in two distinct ACD animal models: First, a rat arthritis model (PG-APS in Lewis rats) and second, a Chronic Kidney Disease (CKD) mouse anemia model (Adenine model in C57BL/6 mice). Both models present with long-lasting anemia as seen in humans suffering from ACD. Mice and rats were treated with different doses of the anti-BMP6 antibody with and without ESA. Whole blood count, serum iron parameters (including hepcidin), bone marrow erythropoiesis determined by FACS analysis, cytokine expression and iron metabolism gene expression in spleen, liver and kidney were analyzed. Anti-BMP6 as well as ESA monotherapy resulted in a net increase in Hb level but only anti-BMP6 treatment significantly increased MCV and MCH, which can be ascribed to effective iron mobilization. In contrast, ESA therapy raised Hb levels by increasing red blood cell numbers. Of note, mere i.v. iron supplementation (sodium ferric gluconate), even at high doses, was not able to restore Hb levels to the same extent as the anti-BMP6 monotherapy. Strikingly, combination of both, ESA and anti-BMP6 treatment, had a synergistic effect on Hb levels, especially in the rat PG-APS model. Combination therapy of low ESA doses that only had a modest effect as a monotherapy, led to a dramatic increase in Hb levels, even exceeding those seen in healthy rats. Based on these results, additional experiments were performed to investigate the potential of this combination treatment to reduce ESA doses. Indeed, when anti-BMP6 was combined with a significantly reduced total ESA dose Hb levels were corrected to normal values in disease animals. Anti-BMP6 treatment also led to a significant decrease of iron deposition in the spleen with no iron deposition in parenchymal organs, indicating that the freed-up iron was effectively used for erythropoiesis and not just distributed elsewhere. In summary, anti-BMP6 therapy worked synergistically with ESA treatment in two different models of ACD leading to significantly increased Hb levels, a reduced ESA need as well as reduced iron overload in the RES. Furthermore, these experiments clearly show that treatment of ACD, being a complex multifactorial disease, benefits from using a combination of diversified approaches to overcome anemia and significantly reduce the dose of each therapeutic. Disclosures Wake: Kymab Ltd.: Employment. Bayliss:Kymab Ltd.: Employment. Papworth:Kymab Ltd.: Employment. Carvalho:Kymab Ltd.: Employment. Deantonio:Kymab Ltd.: Employment. Weiss:Kymab Ltd.: Consultancy. Germaschewski:Kymab Ltd.: Employment. Theurl:Kymab Ltd.: Consultancy, Research Funding.

Many patients with myelofibrosis (MF) develop anemia and are, or become, dependent on frequent red blood cell transfusions [1]. Results from the phase 2 studies for the treatment of myelofibrosis (MF) with the Jak1/2 inhibitor momelotinib (MMB) demonstrated that MMB treatment provided an anemia benefit, in addition to providing a durable spleen response and improvement in constitutional symptoms in MF patients [2]. MMB's anemia benefit was an unexpected outcome as erythropoietin (EPO)-mediated JAK2 signaling is essential for erythropoiesis [3, 4]. Systemic iron homeostasis is controlled by the peptide hormone hepcidin produced by the liver. Hepcidin reduces iron export from duodenal enterocytes and splenic and hepatic macrophages by binding to and down-regulating the iron exporter ferroportin [5-7]. In chronic disease, there is a significant increase in hepcidin levels which can result in severe anemia; this pathologic condition is termed anemia of chronic disease (ACD). Recently, MF patients have been shown to have elevated serum hepcidin levels and this increase is associated with inferior overall survival in these patients [8]. This study aimed to determine whether MMB's clinical anemia benefit is driven by direct activity of MMB on the hepcidin pathway. We assayed MMB inhibitory activity on the BMP-receptor kinase pathway (the central driver of hepcidin transcription in hepatocytes) and assessed the activity of MMB in a rodent model of anemia of chronic disease (ACD). We demonstrate that MMB inhibits BMP6-induced in vitro production of hepcidin in cultured hepatocytes (HepG2 cells) with an EC50 = 651 +/- 203 nM (n=3). This inhibitory activity is mediated by direct suppresion of the BMP-receptor kinase Alk2 as MMB inhibits Alk2 enzymatic activity with an IC50 = 8.4 +/- 1.5 nM (n=3). Ruxolitinib has no activity on either Alk2 or the BMP-receptor kinase pathway. To understand whether MMB could modulate hepcidin levels in vivo and ameliorate anemia in vivo we assessed the effect of MMB in a peptidoglycan-polysaccharide fragment (PG-APS)-induced rat ACD model. Treatment with clinically relevant exposure levels of MMB for 3 days resulted in a dose dependent reduction in both liver RNA and serum protein hepcidin levels and caused an increase in serum iron. Furthermore, long-term treatment with MMB for 21 days increased the numbers and percent of reticulocytes and mature red blood cells in the bone marrow and increased Hgb and hematocrit to normal levels in the blood. Our data suggest that MMB's clinical anemia benefit results from inhibition of ALK2-mediated expression of hepcidin in the liver, which results in increased release of iron from sequestered cellular stores and enhanced erythropoiesis. A phase 2 translational study in anemic subjects with MPNs is scheduled to confirm this mechanism. The Alk2-mediated activity on iron metabolism through hepcidin could prove beneficial in a number of additional indications, and facilitate the combination of MMB with myelosuppressive agents. 1. Tefferi, A., et al., One thousand patients with primary myelofibrosis: the mayo clinic experience. Mayo Clin Proc, 2012. 87 (1): p. 25-33. 2. Pardanani, A., et al., Update On The Long-Term Efficacy and Safety Of Momelotinib, a JAK1 and JAK2 Inhibitor, For The Treatment Of Myelofibrosis. Vol. 122. 2013. 108-108. 3. Verstovsek, S., et al., Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 2010. 363 (12): p. 1117-27. 4. Verstovsek, S., et al., A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012. 366 (9): p. 799-807. 5. Nemeth, E., et al., Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004. 306 (5704): p. 2090-3. 6. Theurl, I., et al., Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease. Blood, 2006. 107 (10): p. 4142-8. 7. Theurl, I., et al., Autocrine formation of hepcidin induces iron retention in human monocytes. Blood, 2008. 111 (4): p. 2392-9. 8. Pardanani, A., et al., Associations and prognostic interactions between circulating levels of hepcidin, ferritin and inflammatory cytokines in primary myelofibrosis. Am J Hematol, 2013. 88 (4): p. 312-6. Disclosures Warr: Gilead Science: Employment. Maciejewski:Gilead Science: Employment. Fowles:Gilead Science: Employment. Whitney:Gilead Sciences: Employment. Theurl:Gilead Science: Research Funding.