Table S1. Small molecule inhibitors. Listing of all 116 agents on the screening inhibitor panel with respective molecular targets and sources Table S2. Primers for QRT-PCR and plasmid construction. Table S3. IC50s of 116 small molecule inhibitors in EWS cells. Table S4. IC50s of small molecule inhibitors in other cancer types. Table S5. Patients clinical information and H-score numbers. Table S6. MALAT1 expression among different cancer types ( patient sample tissues from Baird Sarcoma database). Table S6. Overlaps of lists of genes significantly co-downregulated and co-upregulated by SYK inhibitor GS-9973 (SYK i) and SYK knockdown (shSYK) in TC71 cells. Table S7. MALAT1 expression among different cancer types (patient sample tissues from Baird Sarcoma database). Table S8. MALAT1 Expression among different cancer types ( cancer cell lines from CCLE database). Table S9. ChIP-seq data source information.
ARTICLE ABSTRACT
Purpose: Ewing sarcoma (EWS) is a devastating soft tissue sarcoma affecting predominantly young individuals. Tyrosine kinases (TK) and associated pathways are continuously activated in many malignancies, including EWS; these enzymes provide candidate therapeutic targets.Experimental Design: Two high-throughput screens (a siRNA library and a small-molecule inhibitor library) were performed in EWS cells to establish candidate targets. Spleen tyrosine kinase (SYK) phosphorylation was assessed in EWS patients and cell lines. SYK was inhibited by a variety of genetic and pharmacological approaches, and SYK-regulated pathways were investigated by cDNA microarrays. The transcriptional regulation of MALAT1 was examined by ChIP-qPCR, luciferase reporter, and qRT-PCR assays.Results: SYK was identified as a candidate actionable target through both high-throughput screens. SYK was highly phosphorylated in the majority of EWS cells, and SYK inhibition by a variety of genetic and pharmacologic approaches markedly inhibited EWS cells both in vitro and in vivo. Ectopic expression of SYK rescued the cytotoxicity triggered by SYK-depletion associated with the reactivation of both AKT and c-MYC. A long noncoding RNA, MALAT1, was identified to be dependent on SYK-mediated signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the promoter of MALAT1 and transcriptionally activated MALAT1, which further promoted the proliferation of EWS cells.Conclusions: This study identifies a novel signaling involving SYK/c-MYC/MALAT1 as a promising therapeutic target for the treatment of EWS. Clin Cancer Res; 23(15); 4376–87. ©2017 AACR.
