Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma

// Alyssa L. Kennedy 1, 2, * , Mounica Vallurupalli 1, 3, * , Liying Chen 1, * , Brian Crompton 1 , Glenn Cowley 4 , Francisca Vazquez 4 , Barbara A. Weir 4 , Aviad Tsherniak 4 , Sudha Parasuraman 5 , Sunkyu Kim 5 , Gabriela Alexe 1, 4, 6 , Kimberly Stegmaier 1, 4 1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children’s Hospital, Boston, Massachusetts, USA 2 Boston Combined Residency Program in Pediatrics, Boston, Massachusetts, USA 3 Department of Internal Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA 4 Broad Institute, Cambridge, Massachusetts, USA 5 Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA 6 Bioinformatics Graduate Program, Boston University, Boston, Massachusetts, USA * These authors have contributed equally to this work Correspondence to: Kimberly Stegmaier, e-mail: kimberly_stegmaier@dfci.harvard.edu Keywords: CDK4/6 inhibitor, cyclin D1, epigenetics, Ewing sarcoma, sarcoma/soft-tissue malignancies Received: June 29, 2015      Accepted: August 07, 2015      Published: August 18, 2015 ABSTRACT Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1 . While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease.