Abstract Novel targeted drug combinations have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, where tumor genomes are typically simple with infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The CDK4/6 pathway, which has been extensively validated as a target in estrogen receptor-positive breast cancer, recently emerged as a dependency in Ewing sarcoma, an aggressive pediatric bone tumor. Given the heightened efficacy of CDK4/6 inhibitors (CDK4/6i) in combination with other targeted drugs in breast cancer, as well as the known propensity of resistance to emerge with single agent targeted cancer therapy, we aimed to identify CDK4/6i resistance mechanisms and biologically relevant drug combinations in Ewing. To identify candidate resistance mechanisms, we performed a genome-wide lentiviral open-reading frame (ORF) rescue screen in two Ewing cell lines sensitive to the CDK4/6 inhibitors palbociclib and ribociclib and looked for genes that confer resistance to these drugs. This revealed IGF1R as a gene whose overexpression promoted drug escape. In parallel to the ORF screen, we established resistance to CDK4/6i in Ewing sarcoma cell lines by chronic ribociclib exposure. We found elevated levels of phospho-IGF1R in resistant cells by immunoblotting, also supporting that this pathway's activation is relevant to acquired resistance. We performed CRISPR knockout of IGF1R in these drug resistant cells and demonstrated rescue of sensitivity to CDK4/6i. Furthermore, resistant lines maintained high sensitivity to an IGF1R inhibitor. Concurrently, in a small molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6i in Ewing cells. We thus tested ribociclib and the IGF1R tool compound AEW541 in a panel of six Ewing cell lines in vitro. We observed strong synergy utilizing the Chou-Talalay method. This finding was next validated in an in vivo cell line xenograft mouse model, where we demonstrated prolonged survival and decreased tumor volumes with combination drug treatment. In order to investigate mechanism of CDK4/6-IGF1R inhibitor synergy, we used proteomic analysis by reverse phase protein array (RPPA) in single and combined drug treated Ewing cells. RPPA revealed enhanced suppression of the PI3K/mTOR pathway in the combination, a central cancer cell signaling axis. Taken together, these results suggest that IGF1R activation is an escape mechanism to CDK4/6i in Ewing and that dual targeting of CDK4/6 and IGF1R provides a candidate synergistic drug combination for this disease. Citation Format: Lillian M. Guenther, Neekesh V. Dharia, Linda Ross, Amy S. Conway, Amanda L. Robichaud, Alanna J. Church, Rajarshi Guha, Mindy I. Davis, Gabriela Alexe, Jaume Mora, Federica Piccioni, Kimberly Stegmaier. Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1629.