Activin a Inhibition Impaired Human AML Cell Engraftment and Extended Survival in Immunodeficient Mice

Introduction: Myelodysplastic neoplasms (MDS) and myelofibrosis (MF) are clonal hematological disorders that affect multi-lineage blood cell production and develop into AML in approximately 30% and 20% of such patients, respectively (Menssen et al., 2020; Mannelli, 2021). Activin A (ActA) is a ligand of the transforming growth factor-beta (TGF-β) superfamily that is involved in numerous biological processes, including inflammation, fibrosis, metabolism, proliferation, and differentiation and has been implicated in playing a key role in disease progression in inflammatory and autoimmune diseases, fibrotic diseases, metabolic disorders, and cancer. ActA has emerged as a critical regulator in the pathophysiology of leukemia and has been shown to play a multifaceted role in leukemia progression through its impact on hematopoiesis, leukemic cell motility and remodeling of the bone marrow microenvironment (Portale et al., 2019). Understanding the complex role of ActA in leukemia biology can inform the development of therapies aimed at disrupting ActA-mediated signaling to provide therapeutic benefit to patients with MDS and MF by potentially inhibiting transformation to AML.

Elritercept (KER-050) and its research form, RKER-050, are modified activin receptor type IIa (ActRIIa) ligand traps designed to inhibit select TGF-β superfamily ligands, including ActA, ActB, GDF8, and GDF11, to address ineffective hematopoiesis in MDS and MF. Elritercept has the potential to have disease-modifying effects by improving bone marrow health and function to promote normal hematopoiesis and potentially delaying transformation to AML.

Aim: The goal of this study was to examine the impact of RKER-050-mediated ActA inhibition on AML transformation using in vitro cell migration and in vivo engraftment as surrogate models.

Methods: In the transwell migration assays, C1498-GFP mouse AML cells were plated overnight in 1% serum-containing media with the vehicle, ActA (100ng/mL), RKER-050 (400ng/mL), or the combination of ActA and RKER-050. Overnight pre-treated C1498-GFP cells were added to the cell permeable membrane of the transwell plate and complete media containing the chemoattractant CXCL-12 was added to the plate bottom. After 4 hours, GFP+ cells that migrated through the transwell were counted via flow cytometry. In a human AML in vivo engraftment model, 10-12-week-old male immunodeficient NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac) mice were pre-treated twice weekly with RKER-050 for 2 weeks prior to human MOLM13-GFP+ leukemic cell transplant via tail vein injection and continued treatment twice weekly after transplantation. Seven days after the transplant, a cohort of mice (n=10 per group) was euthanized, and bone marrow was collected and assessed for GFP+ MOLM13 AML cell engraftment via flow cytometry. A second cohort of mice (n=10 per group) was assessed for survival.

Results: In transwell migration experiments, AML cells pre-treated with ActA migrated toward CXCL-12-containing media significantly more than vehicle pre-treated cells, whereas RKER-050 pre-treatment significantly reduced the migratory ability of the AML cells. AML cells pre-treated with the combination of ActA and RKER-050 significantly reduced migration compared to ActA-pre-treated cells, demonstrating that RKER-050 inhibited the ActA-induced migration of AML cells.

In the in vivo study, mice pre-treated with RKER-050 for 2 weeks prior to AML transplantation showed a significant reduction in AML cell engraftment 7 days after transplantation compared to vehicle pre-treated mice. Additionally, an improvement in survival was observed in mice pre-treated with RKER-050 compared to vehicle pre-treated mice. Taken together, RKER-050 reduced AML cell bone marrow engraftment and improved survival of AML cell-burdened mice.

Conclusions: Inhibition of ActA with RKER-050 reduced in vitro AML cell migration and decreased AML cell bone marrow engraftment, leading to extended survival in mice. The multifaceted role of ActA in disease and malignancies underscores its significance as a therapeutic target and the potential for ActA-modulating therapies to improve outcomes for patients with MDS or MF by reducing the risk of progression to AML.

Moses:Keros Therapeutics: Current Employment. Wheeler:Keros Therapeutics: Current Employment. Dills:Keros Therapeutics: Current Employment. Daman:Keros Therapeutics: Current Employment. Grenha:Keros Therapeutics: Current Employment. Lerner:Keros Therapeutics: Current Employment. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Lachey:Keros Therapeutics: Consultancy, Current equity holder in publicly-traded company. Cadena:Keros Therapeutics: Current Employment.