Abemaciclib Is Active in Preclinical Models of Ewing Sarcoma via Multipronged Regulation of Cell Cycle, DNA Methylation, and Interferon Pathway Signaling.

Abstract

Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The <i>EWS/ETS</i> fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the <i>in vitro</i> response of ES cell lines to abemaciclib. We then evaluated abemaciclib <i>in vivo</i> in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. Abemaciclib induced quiescence in ES cell lines via a G₁ cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.