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

Oncotarget. 2015 Oct 6;6(30):30178-93. doi: 10.18632/oncotarget.4903.

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.

Keywords: CDK4/6 inhibitor; Ewing sarcoma; cyclin D1; epigenetics; sarcoma/soft-tissue malignancies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Bone Neoplasms / drug therapy*
  • Bone Neoplasms / enzymology
  • Bone Neoplasms / genetics
  • Bone Neoplasms / pathology
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cyclin D1 / antagonists & inhibitors*
  • Cyclin D1 / genetics
  • Cyclin D1 / metabolism
  • Cyclin-Dependent Kinase 4 / antagonists & inhibitors*
  • Cyclin-Dependent Kinase 4 / genetics
  • Cyclin-Dependent Kinase 4 / metabolism
  • Cyclin-Dependent Kinase 6 / antagonists & inhibitors
  • Cyclin-Dependent Kinase 6 / metabolism
  • Drug Discovery / methods*
  • Female
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Neoplastic
  • High-Throughput Nucleotide Sequencing*
  • High-Throughput Screening Assays*
  • Humans
  • Mice, Inbred NOD
  • Mice, SCID
  • Molecular Targeted Therapy
  • Protein Kinase Inhibitors / pharmacology*
  • RNA Interference
  • RNA-Binding Protein EWS / genetics
  • RNA-Binding Protein EWS / metabolism
  • Sarcoma, Ewing / drug therapy*
  • Sarcoma, Ewing / enzymology
  • Sarcoma, Ewing / genetics
  • Sarcoma, Ewing / pathology
  • Signal Transduction / drug effects
  • Time Factors
  • Transfection
  • Tumor Burden / drug effects
  • Vascular Endothelial Growth Factor Receptor-1 / genetics
  • Vascular Endothelial Growth Factor Receptor-1 / metabolism
  • Xenograft Model Antitumor Assays

Substances

  • Antineoplastic Agents
  • CCND1 protein, human
  • Protein Kinase Inhibitors
  • RNA-Binding Protein EWS
  • Cyclin D1
  • FLT1 protein, human
  • Vascular Endothelial Growth Factor Receptor-1
  • CDK4 protein, human
  • CDK6 protein, human
  • Cyclin-Dependent Kinase 4
  • Cyclin-Dependent Kinase 6