EXTH-123. Novel Dyrk1a inhibitors impair glioblastoma proliferation and enhance the effectiveness of other cell-cycle-directed therapies

Abstract INTRODUCTION Dysregulated cell proliferation is a hallmark of glioblastoma, and DYRK1A kinase, a key cell cycle regulator, may provide a novel therapeutic target. METHODS The effects of two DYRK1A inhibitors (Leucettinib-21 and #423) were evaluated against SB28 mouse glioblastoma cells in vitro and in vivo. RESULTS Dyrk1a inhibition dramatically impairs SB28 cell proliferation with IC50 values in the micromolar range based on CFSE dilution and MTS cell viability assays. Treatment at IC50 concentrations reduces cell migration in a scratch assay and increases nuclear size compared to vehicle-treated controls. Western blot analyses indicate that inhibitor-treated SB28 cells do not undergo apoptosis, suggesting Dyrk1a inhibition has a cytostatic effect. Cytokine array experiments on conditioned media indicate that inhibitor-treated SB28 cells alter their secretion of numerous proinflammatory cytokines such as CCL5, IL6, and CXCL10. GSEA of bulk RNA-sequencing data demonstrates an upregulation of senescence pathway genes in treated SB28 cells. We find that Dyrk1a inhibition arrests tumor cells in the G2 phase of the cell cycle, and β-galactosidase staining confirms that prolonged treatment induces cellular senescence. Mice bearing SB28 allografts treated with Leucettinib-21 (10 mg/kg, daily oral gavage) show decreased recruitment of myeloid cells to the tumor microenvironment and a significant, but transient, decrease in tumor growth. The changes in cell cycle progression after Leucettinib-21 treatment led us to hypothesize that combining Dyrk1a inhibition with cyclin-dependent kinase (CDK) blockade may augment the anti-proliferative effects. Treatment with the FDA-approved CDK4/6 inhibitor, abemaciclib, which has an IC50 value in the micromolar range when used as a single agent, and Dyrk1a inhibition results in strikingly elevated apoptosis and shifts the IC50 value of abemaciclib to the nanomolar range. CONCLUSION Dyrk1a plays an underappreciated role in glioblastoma cell cycle regulation, and novel DYRK1a inhibitors are an exciting translational opportunity to sensitize GBM cells to FDA-approved cancer therapeutics.