Fig 2: JAK2-V617F correlated with increased IL-1 expression in MPN patients.a Upper panel: Serum IL-1ß (pg/ml) in normal controls (NC; n = 20) and MPN patients (n = 120); ET (n = 42), PV (n = 44), PMF (n = 34). Correlation (r) between % JAK2-V617F in granulocytes and log transformed serum IL-1ß in MPN patients. Limit of detection is shown by dashed green line at y = 0.01 pg/ml. Lower panel: IL-1ß mRNA expression relative to ß-actin in granulocytes of NC (n = 12) and MPN patients (n = 46); ET (n = 11), PV (n = 18), PMF (n = 17). Correlation between log transformed IL1B mRNA expression and % JAK2-V617F. b Upper panel: Serum IL-1RA (pg/ml) in NC (n = 20) and MPN patients (n = 120); ET (n = 42), PV (n = 44), PMF (n = 34). Correlation (r) between % JAK2-V617F and log transformed serum IL-1RA. Lower panel: IL1RN (IL-1RA) mRNA expression relative to ß-actin in NC and MPN patients. Correlation between log transformed IL1RN mRNA expression and % JAK2-V617F. Two-tailed unpaired non-parametric Mann–Whitney t-test was performed in a and b. c Representative histogram showing the expression of interleukin 1 receptor type 1 (IL-1R1) in peripheral blood hematopoietic stem cells (HSCs) from isotype control, NC (n = 5), ET (n = 6), PV (n = 5), and PMF (n = 7). Bar graph showing the percentages of IL-1R1+ HSC), common myeloid progenitors (CMP), granulocyte macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP) and megakaryocyte progenitor (MkP). Graph showing correlation (r) between % JAK2-V617F and percentages of IL-1R1 + HSCs. d Representative histogram showing the expression of interleukin 1 receptor accessory protein (IL-1RAcP) in peripheral blood HSC from NC and MPN patients. Bar graph showing the percentages of IL1RAcP+ HSC, CMP, GMP, MEP, and MkP in NC (n = 5), ET (n = 6), PV (n = 5), and PMF (n = 7). Correlation (r) between % JAK2-V617F and percentages of IL-1RAcP+ HSPCs. Two-tailed unpaired t-test was performed for statistical comparisons in c and d. Spearman correlation (r) and two-tailed t-test was performed for correlation analysis in a–d. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also Supplementary Figs. 1–3. Source data and exact p values are provided as a Source Data file.

Fig 3: IL1RAP is a potential cell-surface therapeutic target in Ewing sarcoma (EwS). A, IHC analysis of IL1RAP in pediatric normal organ TMAs. Scale bars, 50 µm in high-power images and 200 µm in low-power images. B, Median asinh (signal/150) differences in IL1RAP intensity of the indicated stained cells from their corresponding FMO (fluorescence minus one) control. C and D, Binding of the IL1RAP antibody VH–Fc X1 to a panel of human cancer cell lines was determined by flow cytometry (C) and IF analysis (D). Scale bar, 10 µm. E, The ADCC reporter assay was performed using the Jurkat T-NFAT-Luc-CD16A effector cells encoding the luciferase reporter gene driven by the nuclear factor of activated T cells response element (NFAT-RE). A673 Ewing sarcoma cells were used as target cells. Target:effector cell ratio, 1:6. An isotype control or no antibody was added in the control groups. Baseline reporter activity was determined by omitting the Ewing sarcoma target cells in the assay. F, ADCC assays using PBMCs as effector cells and a panel of Ewing sarcoma cells as target cells (n = 3–4). Target:effector cell ratio, 1:15. All data presented are means ± SD. In B, one-sample t test was performed, and two-tailed unpaired Student t test was used in other panels. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: IL1RAP forms a complex with CD98 and xCT of the system Xc- cystine transporter and promotes cystine uptake. A, Volcano plots of proteins identified in the IL1RAP interactome in A673. B, Representative immunoblots of the indicated co-IP samples in A673 cells. The experiments were repeated twice with similar results. C, Representative IF staining of IL1RAP and CD98 in A673 cells. D, PLA of IL1RAP and CD98 in A673 cells (n = 5; >100 cells were analyzed for each condition). E, PLA of IL1RAP–CD98 or IL1RAP–xCT in TC32-derived Ewing sarcoma (EwS) xenografts in the murine renal subcapsular implantation model. Scale bar, 30 µm; red arrows indicate positive PLA signals in Ewing sarcoma tumor cells. F, Representative immunoblots of the indicated co-IP samples in A673 cells expressing wild-type (WT) or mutant CD98 (C109S or C330S). Rabbit (Rb) IgG and goat (Gt) IgG were used as isotype controls for the co-IP experiments. The experiments were repeated twice with similar results. G, Representative immunoblots of the indicated co-IP samples in HEK293 cells transfected with WT or mutant IL1RAP (C362S) and WT HA-tagged CD98. H and I, Representative images showing the IF staining of IL1RAP and xCT (H) and PLA assay of IL1RAP and xCT (I) in A673 cells. Scale bars, 10 µm. For I, n = 5–7; =50 cells were analyzed for each condition. J, The impact of IL1RAP knockdown (by sh#39 or #40) on cystine uptake was measured by cystine consumption assay in media containing regular (200 µmol/L) or low (10 µmol/L) levels of cystine. K and L, Intracellular cysteine and GSH pools were measured in the indicated cell lines (K) or Ewing sarcoma xenografts derived from TC32 (L) with or without IL1RAP knockdown (by sh#39 or #40). M, Rescue of WT or mutant IL1RAP (C362S) in Ewing sarcoma cells with IL1RAP depletion was determined by immunoblot analysis. N, Cystine uptake and glutamate secretion were determined in the indicated Ewing sarcoma cells cultured in media containing 10 µmol/L cystine. Data presented are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001;P values were determined by two-tailed unpaired Student t test.

Fig 5: The IL1RAP–CTH axis promotes the TSS pathway. A, Schematic showing the pathway that mediates de novo cysteine synthesis and GSH production via TSS. B, Schematic showing the global proteome analysis in A673 Ewing sarcoma cells with control knockdown (shCtrl) or IL1RAP knockdown (sh#39 and sh#40) grown in 3-D cultures. C, Global proteome changes induced by IL1RAP depletion with two independent shRNAs (n = 3 biologically independent samples). D, Immunoblot analysis of the indicated proteins. Densitometry analysis of CTH normalized to the vinculin loading control is shown. The experiment was repeated twice with similar results. E, Cell death and cell growth were measured by Incucyte in the indicated cells cultured in media containing different concentrations of cystine supplemented with/without 0.4 mmol/L homocysteine (Hcy). F, Intracellular levels of cystathionine and GSH were determined in cells cultured in media ± 200 µmol/L cystine or 0.4 mmol/L Hcy for 12 hours. G,De novo cysteine synthesis and GSH production in the indicated cells were determined in cystine-depleted cultures by flux analysis of 13C-serine, a key substrate for the TSS pathway as shown in A. 12C-labeled (M + 0) and 13C-labeled (M+1) metabolites were measured after 24 hours. H and I, The effects of CTH reexpression on GSH pools (12 hours; H), lipid ROS accumulation (22 hours; I, left), and cell death (48 hours; I, right) in IL1RAP-depleted cells with the indicated treatments. 2 and 5 µmol/L erastin was used in H and I, respectively; and 2 µmol/L Fer-1 was used in I. P values were determined by two-tailed unpaired Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.