A novel combination of CDK4/6 and PI3K inhibitors exhibits highly synergistic activity and translational potential in Ewing sarcoma.

The results of the screen revealed that a PI3K inhibitor, copanlisib, combined with a CDK4/6 inhibitor, ribociclib, exhibited strong synergistic anti-Ewing sarcoma activity. Using proteomic methods such as a reverse-phase protein array and western immunoblotting, we demonstrated that this combination induced a downregulation of the PI3K/AKT pathway as well as proteins involved in cell cycle regulation. We further confirmed these in vitro data using bulk RNA-sequencing.

To evaluate the phenotypic effect of the PI3K/CDK4/6 inhibition in Ewing sarcoma lines, we performed apoptosis and cell cycle analyses using flow cytometry and demonstrated that ribociclib primarily induced a G0/G1 arrest with minimal effect on Ewing cell viability but significantly enhanced the apoptotic effect of copanlisib treatment. In two xenograft models of Ewing sarcoma, we demonstrated that the combination significantly prolonged survival compared to treatment with either vehicle or single-agent therapy alone.

Our findings identify a new candidate therapy combination for Ewing sarcoma and provide a resource of additional potential synergistic combinations for future validation.