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posted on 2023-03-31, 20:27 authored by Haibo Sun, De-Chen Lin, Qi Cao, Brendan Pang, David D. Gae, Victor Kwan Min Lee, Huey Jin Lim, Ngan Doan, Jonathan W. Said, Sigal Gery, Marilynn Chow, Anand Mayakonda, Charles Forscher, Jeffrey W. Tyner, H. Phillip Koeffler Fig. S1. GSEA analysis revealed significant enrichment of gene associated with genomic unstable EWS phenotype (A), cell cycle (B) and NF-κB (C) in SYK knockdown cells, and gene associated with metastasis (D) in cells treated with the SYK inhibitor. Fig. S2. Representative IHC photos of p-SYK expression in TC71 control and shSYK cells. No Ab, no antibody control; NC, scramble shRNA control. Fig. S3. TC71 cells were treated with 4 μmol/L GS-9973 or PRT062607 for 48 h, and cell cycle distribution was analyzed by PI staining. Fig. S4. TC71 and SKES1 cells were infected with control (NC) or sh-MYC lentivirus for 72h, and c-MYC protein level was evaluated by immunoblotting assay. Fig. S5.Total nuclear RNA was immunoprecipitated by the antibody-coated beads (either nonspecific IgG or anti-EZH2 antibody), reverse-transcribed and the cDNA was further amplified by qRT-PCR. Bar graphs displayed the relative MALAT1 levels (Y-axis) in different groups. Protein lysates (input, IgG and anti-EZH2 antibodies) were immunoblotted and probed with anti- EZH2 antibody (lower panel). Data represent mean {plus minus} SD, n=3.
Funding
National Center for Advancing Translational Sciences
NUS
History
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.
