P1410: RKER-050, A NOVEL INHIBITOR OF TGF-Β SUPERFAMILY SIGNALING, INDUCED PLATELET PRODUCTION IN HEALTHY MOUSE MEGAKARYOCYTES

Abstract

Background: Transforming growth factor-beta (TGF-β) superfamily signaling is vital in regulating hematopoiesis, including normal platelet (PLT) production. Megakaryocytes (MKs) are platelet producing cells that are controlled, in part, by this signaling pathway. Dysfunctional signaling can lead to abnormal PLT production causing thrombocytopenia as well as myeloproliferative diseases. KER-050 is an investigational modified ActRIIA ligand trap designed to inhibit a subset of TGF-β superfamily ligands, including activin A (ActA), activin B, GDF8, and GDF11. In a clinical study in healthy volunteers, KER-050 increased red blood cell and PLT levels. Understanding the mechanism of action of KER-050 in the context of PLT production is important to ascertain its potential clinical benefits. Aims: To investigate the effects of KER-050 on PLT biology in mice. Methods: All studies used RKER-050, a research form of KER-050. Murine studies were carried out in 8-14-week-old C57BL/6 mice. For PLT number, % CD41+ (MK marker) composition and ploidy assessment, a single intraperitoneal dose of RKER-050 (10 mg/kg) was given with multiple timepoints post-dose assessed. Ploidy levels of the CD41+ population were analyzed using a DNA stain. For in vitro ploidy assays, bone marrow (BM) from untreated mice was isolated and cultured with vehicle, ActA, RKER-050, or RKER-050+ActA for 6 days and analyzed as described above. For proplatelet formation assays, lin- (lineage negative) cells were isolated from BM of untreated mice and cultured with thrombopoietin and hirudin for 3 days. MKs were enriched using a BSA gradient and then cultured with vehicle, RKER-050, ActA, or RKER-050+ActA for 16-18 hrs and the percentage of MKs producing proplatelets assessed. For gene expression analysis, qPCR was performed on RNA from BM-derived CD41+ cells isolated from untreated mice. Results: Circulating PLT counts and percent CD41+ BM cells were elevated >2-fold relative to vehicle 12 hrs post-RKER-050 treatment. The rapidity of these changes suggests that RKER-050 may affect terminal stages of platelet production, while the increase in CD41+ cells suggests that RKER-050 may also expand MKs in BM. At 24 hr post-dose, CD41+ cell ploidy was increased, suggesting RKER-050 may promote MK maturation. To more directly assess changes elicited by RKER-050 on the terminal stages of platelet formation, proplatelet formation was assessed. RKER-050 increased the percentage of MKs producing proplatelets, indicative of RKER-050 enhancing these terminal stages. Treatment with ActA reduced proplatelet formation. In contrast, co-treatment of ActA with RKER-050 partially rescued the inhibitory effects of ActA on proplatelet formation, resulting in more mature, proplatelet-forming MKs. Additionally, a qPCR panel was conducted on CD41+ cells isolated from untreated mouse BM to assess the TGF-β superfamily ligands and receptors that may be involved in regulating thrombopoiesis. Summary/Conclusion: In these preclinical studies, we determined that RKER-050 acts on multiple stages of thrombopoiesis resulting in increased PLTs in mice. Concordant with the increase in PLT output, the percentage and ploidy of PLT progenitor CD41+ cells were also increased. In addition, we demonstrated that the effect of RKER-050 could be, at least in part, through its ActA inhibition. These data support that RKER-050 may be promoting MK maturation and the terminal stages of thrombopoiesis. Overall, these data support that KER-050 has the potential to treat diseases of thrombocytopenia.