Fig 2: Filgotinib or MRX-2843 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo.a–f Representative heatmaps for cell viability using MHH-ES-1 (a, b, c, e) or TC-71 cells (d, f) treated with indicated doses of compounds for 2 days. g Tumor volume measurements at indicated days post-injection of MHH-ES-1 cells subcutaneously with indicated compounds (irinotecan (10 mg/kg) and filgotinib (10 mg/kg)) injected by IP. Error bars were calculated as mean ± SD, n = 5 tumors. p values are calculated by two-way ANOVA followed by the Tukey multiple comparison test. h Isolated tumors from (g) and weighed in (i). Error bars were calculated as mean ± SD, n = 5 tumors. p values are calculated by a two-tailed student’s t-test. j, k Representative heatmaps for cell viability in MHH-ES-1 cells treated with indicated doses of compounds for 2 days. l Tumor volume measurements at indicated days post-injection of MHH-ES-1 cells with indicated compounds IP injected or by oral gavage at the indicated days. Error bars were calculated as mean ± SD, n = 5 tumors. p values are calculated as indicated (two-way ANOVA followed by Tukey multiple comparison test). m Isolated tumors from (l) and weighed in (n). Error bars were calculated as mean ± SD, n = 5 tumors. p values are calculated as indicated (two-tailed student’s t- test). o Animal body weight measurements at indicated days. Error bars were calculated as mean ± SD, n = 5 animals in each group. p A cartoon illustration for a proposed model. Chemotherapy triggers DNA damage and activates DNA-damaging kinases to phosphorylate the JAK1-S571Q motif leading to JAK1 activation and subsequent STAT6-Y641 phosphorylation. This causes STAT6 nuclear translocation to promote GAS6 transcription and secretion that binds and activates TAM kinases via either autocrine or paracrine manners, which further activates Akt and ERK signaling contributing to chemoresistance. Blocking either JAK1 by filgotinib or TAM kinases by clinical inhibitor MRX-2843 sensitizes Ewing sarcoma to conventional chemotherapy.

Fig 3: STAT6 governs chemotherapy-induced GAS6 transcription in Ewing sarcoma.a A cartoon illustration to reveal the involvement of transcription factors in mediating chemotherapy-induced GAS6 transcription in Ewing sarcoma. b, c IB analysis of WCL from control or STAT6-depleted MHH-ES-1 cells treated with or without indicated doses of SN-38 (b) or etoposide (c) for 24 h. d RT-PCR analyses of mRNA expression changes of GAS6 in control or STAT6-depleted MHH-ES-1 cells treated with SN-38 (1 μM) for 24 h. p values are labeled and represent differences between experimental groups compared to the control group (one-way ANOVA test). e IB analysis of both WCL and BCA-precipitated culture media from control or STAT6-depleted MHH-ES-1 cells treated with SN-38 (1 μM) for 24 h. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). f upper: an illustration of predicted STAT6 binding sites on human GAS6 promoter. Lower: representative PCR analysis for STAT6 cut&run from MHH-ES-1 cells treated with control or 1 μM SN-38 for 24 h. g Cell viability assays using control or STAT6-depleted MHH-ES-1 cells treated with the indicated dose of SN-38 (1 μM) for 48 h. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). *p value of each point of g: sgSTAT6/sgctrl: 0.0003&0.0008 (30 μM), 0.0001&0.0007 (100 μM), 0.0001&0.0015 (300 μM). h Cell viability assays using control or STAT6-depleted MHH-ES-1 cells treated with the indicated dose of SN-38 (1 μM) for 48 h. Where indicated, STAT6-depleted MHH-ES-1 cells were pre-treated with GAS6-conditioned media for 24 h prior to SN-38 treatment. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). *p value of each point of h: sgSTAT6-GAS6-conditioned media/sgctrl-normal media: <0.0001 (300 μM), sgSTAT6-GAS6-conditioned media/ sgSTAT6-normal media: 0.0118 (300 μM). WB data presented in this figure are representative data from biological duplicates.

Fig 4: IL13Rα2-derived peptide inhibits IL13Rα1 and IL13Rα2-mediated signalling. a Cell lines were lysed and analysed by western blot to detect IL13Rα1 expression. RhoGDI was used as loading control. b IL13Rα1-positive cells were subjected to flow cytometry to detect IL13Rα1 expression on the cell surface. c HT-29 colon cancer and glioblastoma U251 cells were transfected with IL13Rα1 and IL13Rα2 siRNAs or control. At 48 h, transfected cells were lysed and subjected to western blot to confirm the silencing of expression. RhoGDI was used as loading control. d The same transfectants were used for IL-13 triggered invasion in the presence of Rα1 or D1 peptides. IL-13-mediated cell invasion (**p < 0.01; ***p < 0.001) was inhibited after IL13Rα2 silencing (§§p < 0.01; §§§p < 0.001) or by the addition of D1 peptide (◊◊p < 0.01; ◊◊◊p < 0.001). e Cells treated as in (d) were analysed by western blot to detect Src, phospho-Src, STAT6 and phospho-STAT6. Total Src and STAT6 were used as loading controls

Fig 5: Chemotherapy induces JAK1-dependent STAT6-Y641 phosphorylation and nuclear translocation.a, b IB analysis of cytoplasmic and nuclear fractions from MHH-ES-1 cells treated with SN-38 (a) or etoposide (b) for 24 h. Cyto cytoplasm, Nuc nucleus. For quantification, STAT6 localization in cytoplasm or nuclei is determined by relative fluorescent intensity and at least 100 cells were used in each quantification. c Representative IF images from biological triplicates using MHH-ES-1 cells stably expressing EV or HA-STAT6 treated with or without SN-38 (1 μM) for 24 h. The scale bar represents 20 µm. The scale bar represents 20 µm. d, e IB analysis of WCL from MHH-ES-1 cells treated with indicated doses of SN-38 (d) or 1 μM SN-38 for indicated time periods (e). f–h IB analysis of WCL from MHH-ES-1 cells treated with indicated compounds for 24 h. IL-4, 1 ng/mL; AS1517499, 5 μM; SN-38, 1 μM; etoposide, 10 μM. i–k IB analysis of WCL from MHH-ES-1 cells treated with indicated compounds for 24 h. IL-4, 1 ng/mL; filgotinib, 5 μM; SN-38, 1 μM; etoposide, 10 μM. WB data presented in this figure are representative data from experimental duplicates.