Fig 2: EZH2 suppresses ERVs, LINE-1, and an interferon response downstream of NELL2 signaling.(A) Extracellular NELL2 signals to maintain histone H3K27me3 modification in ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. Histone H3K27me3 modification in ERVs and LINE-1 was analyzed by chromatin immunoprecipitation. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (B) EZH2 expression is regulated by NELL2 signaling in Ewing sarcoma. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of EZH2 was analyzed by qRT-PCR. NELL2 silencing resulted in reduced EZH2 expression, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (C) The CD133low population displays low EZH2 expression, which was rescued by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of EZH2 by qRT-PCR. *p < 0.05 compared with the CD133high population and with the CD133low population expressing exogenous CD133. (D) NELL2 signaling maintains EZH2 binding to ERVs and LINE-1. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. The binding of EZH2 to ERVs and LINE-1 was assessed by chromatin immunoprecipitation. NELL2 silencing reduced EZH2 binding to ERVs and LINE-1, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (E) EZH2 suppresses an interferon response induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The expression of the indicated genes was analyzed by qRT-PCR. *p < 0.05 compared with cells transfected with control siRNA and empty vector and with cells transfected with EZH2. (F) EZH2 suppresses growth arrest induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. Cell proliferation was assessed using the IncuCyte live-cell imaging system. (G) EZH2 expression does not affect siRNA-mediated NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The protein levels of NELL2, IFNB1, Mx1, OAS1, and p21 were assessed by immunoblotting. Tubulin serves as a loading control.

Fig 3: Suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons in Ewing sarcoma.(A) Extracellular NELL2 signals to suppress ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of the indicated genes was analyzed by qRT-PCR. NELL2 silencing induced the expression of ERVs and LINE-1, which was suppressed by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. **p < 0.05 compared with cells transfected with NELL2 siRNA and left untreated or treated with recombinant NELL2. (B) The CD133low population expresses high levels of ERVs and LINE-1, which can be suppressed by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of the indicated genes by qRT-PCR. *p < 0.05 compared with the CD133high population and with the CD133low population expressing exogenous CD133.

Fig 4: Suppression of NELL2 signaling induces an interferon response in Ewing sarcoma.(A–E) siRNA-mediated silencing of NELL2 induces an interferon response in Ewing sarcoma cells. Ewing sarcoma cells were transfected with NELL2 siRNA pool or control siRNA pool and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by quantitative RT-PCR (left) and immunoblotting (right). *p < 0.05 compared with control siRNA transfected cells. (A) A673 cells, (B) EW8 cells, (C) TC32 cells, (D) TC71 cells, and (E) SK-N-MC cells. (F) shRNA-mediated silencing of NELL2 induces an interferon response in A673 cells. NELL2 was silenced by lentiviral expression of shRNA in A673 cells and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. *p < 0.05 compared with control shRNA expressing cells. (G) Extracellular NELL2 signals to suppress an interferon response in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. **p < 0.05 compared with cells transfected with NELL2 siRNA and left untreated or treated with recombinant NELL2.

Fig 5: Ewing sarcoma is dependent on NELL2, a target of EWS-FLI1(A) Outline of secretome proteomic analysis. Note that EWS-FLI1 (FLI1 C terminus) shRNA could also silence untranslocated FLI1, which is expressed at low levels.(B) shRNA-mediated silencing of EWS-FLI1 in A-673 cells.(C) EWS-FLI1 silencing reduces NELL2 protein levels in A-673 secretome. The quantification based on spectral counting by mass spectrometry (left); the immunoblotting data (right).(D) EWS-FLI1 silencing reduces NELL2 RNA levels in A-673 cells. *p < 0.05 (n = 3).(E) EWS-FLI1 induces NELL2 RNA expression in human mesenchymal stem cells (hMSCs), the putative cells of origin of Ewing sarcoma. The quantitative real-time RT-PCR data (left). *p < 0.05 (n = 3); the immunoblotting data (right).(F) EWS-FLI1 binds to the NELL2 gene promoter. Chromatin immunoprecipitation analysis for EWS-FLI1 binding to the promoter of NELL2 and known EWS-FLI1 target genes (NR0B1, GLI-1, and FOXO1), as well as control (GAPDH), with and without EWS-FLI1 silencing (n = 3).(G) NELL2 is highly expressed in Ewing sarcoma tumors and cell lines. NELL2 RNA expression was analyzed by qRT-PCR and was normalized to the levels in hMSCs (n = 3).(H) NELL2 silencing inhibits Ewing sarcoma proliferation. NELL2 was silenced by siRNAs and cell proliferation was assessed by the IncuCyte live-cell imaging system. NELL2 silencing was verified by immunoblotting (bottom).(I) Recombinant NELL2 rescues proliferation arrest induced by NELL2 silencing. A-673 cells were transfected with NELL2 or control siRNAs and were treated with or without recombinant NELL2 (250 ng/ml).(J) NELL2 siRNAs have little effect on the proliferation of 293 and HeLa cells.(K) NELL2 RNA expression in A-673, 293, and HeLa cells, which was normalized to the levels in hMSCs (n = 3).(L) siRNA-mediated silencing of NELL2 RNA in 293 and HeLa cells (n = 3).(M) NELL2 silencing inhibits anchorage-independent growth of A-673 cells. shRNA-mediated silencing of NELL2 was verified by immunoblotting (right). *p < 0.05 (6 independent experiments).(N) NELL2 silencing inhibits xenograft tumorigenicity of A-673 cells (n = 5, p < 0.05).(O) Ewing sarcoma depends on autocrine signaling by NELL2, an EWS-FLI1 target.