Hepcidin is an endocrine regulator of iron metabolism that, when elevated, can decrease levels of iron available for erythropoiesis and, as a result, decrease red blood cell production. Signaling though activin-like kinase-2 (ALK2), a TGFβ type 1 receptor, has been implicated in regulation of hepcidin-mediated iron regulation and mobilization; however, to date, ALK2's specific degree of involvement has not been convincingly elucidated, mainly due to the redundant effects of the type 1 receptors ALK3 and ALK5. Activation of type 1 receptors including ALK2, ALK3, and ALK5 via ligand BMPs and co-receptor hemojuvelin (m-HJV), results in downstream SMAD phosphorylation, increased hepcidin, and decreased serum iron while suppression of the receptor signaling would have the opposite effects. In order to assess the specific effect of ALK2 inhibition on hepcidin and iron mobilization, we utilized multiple modalities of inhibition and tested inhibitors in both naive and diseased animals. KTI-2338, a small molecule ALK2 kinase inhibitor, has been characterized to inhibit ALK2 signaling potently and selectively in in-vitro assays. To further assess the specific contribution of ALK2 to hepcidin expression and iron mobilization, we evaluated the effect of the novel neutralizing antibody KTI-A2.0MAb. This fully human antibody is targeted against the extracellular domain of ALK2 with no affinity for the other type 1 receptors and provides a unique tool for understanding ALK2 involvement in this system.

In wild-type animals, targeting ALK2 signaling with either a small molecule or biologic therapeutic leads to decreased serum hepcidin and increased serum iron. To assess the efficacy of ALK2 inhibition in a disease state, we utilized an siRNA-based model of Iron Refractory Iron Deficiency Anemia (IRIDA). In IRIDA, patients exhibit a loss of functional TMPRSS6, a gene that encodes the transmembrane type II serine protease Matriptase-2 (MT-2). MT-2 suppresses hepcidin secretion by cleaving m-HJV, interrupting ALK2 signaling and downstream SMAD activation. Failure to cleave m-HJV allows continued activation of BMPRs, increased hepcidin, and decreased serum iron. Phenocopying what is observed in IRIDA patients, intravenous dosing of TMPRSS6 targeted siRNA results in suppressed TMPRSS6 expression and functional MT-2, increases in serum hepcidin, and decreases in serum iron. Therapeutic dosing of either a small molecule or biologic ALK2 inhibitor in the siRNA based IRIDA model resulted in rescue of hemoglobin, hematocrit, serum hepcidin, and serum iron in the disease state. Following treatment, hemoglobin, hematocrit, and serum iron were increased and serum hepcidin was decreased in treated groups compared to control cohorts receiving vehicle.

Herein, we have evaluated multiple modalities of ALK2 inhibition in both healthy and disease states. We have characterized that inhibition of ALK2 signaling via either modality in both naïve and anemic mice contributes to a decrease in serum hepcidin and increase in serum iron levels. Though the use of a selective ALK2 targeted biologic does not completely preclude involvement of other BMP receptors such as ALK3, these data support our assertion that ALK2 signaling is an integral part of hepcidin-mediated iron mobilization, and illustrate the potential therapeutic benefit of ALK2 inhibition (with a small molecule inhibitor or a neutralizing monoclonal antibody) in anemia of high hepcidin including IRIDA and anemia of inflammation.

Disclosures

Backus:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Medeiros:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Author notes

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Asterisk with author names denotes non-ASH members.