Fig 2: Neuroblastoma-intrinsic oncogenic pathway activation correlates with the non-T cell-inflamed tumor microenvironment. (A) Activation of MYCN signaling network. Arrows indicate direction (from the upstream regulator to the downstream target molecules). Pink color indicates the gene is upregulated in non-T cell-inflamed tumors relative to inflamed. (B) Immunofluorescent IHC straining against CD8+ T cells and Batf3+ DCs in MYCN-amplified and non-amplified tumors. Scale bars, 20 µm. (C) Target molecule expression heatmap of ASCL1, SOX11, and KMT2A as upstream regulators in non-T cell-inflamed versus T cell-inflamed tumors. (D) The percentage of tumors harboring activation in MYCN, ASCL1, SOX11, and KMT2A transcriptional programs (defined as, pathway activation score >0.517) in non-T cell-inflamed and T cell-inflamed groups. An aggregation of the four pathways (MYCN, ASCL1, SOX11, and KMT2A) is shown on top. (E) Correlation between the T cell-inflamed gene signature and activation of each pathway at a continuous scale. Tumor samples were sorted by T cell-inflamed gene expression (left to right: lower to higher).

Fig 3: IL-2 and IL-15 promote ALCL proliferation and activate STAT1, STAT5 and ERK1/2.a Indicated 7 ALCL cell lines were treated with rhIL-2 (100 ng/ml). Control cell lines were SUP-M2 (ALK+, IL-2R?-negative) and Jurkat (T-cell leukemia-derived, IL-2Ra- and IL-2Rß-negative). Resazurine assay was used to measure metabolic activity as a marker of cell proliferation. Data are means ± SD of biological triplicates given as a percentage of the untreated control. P values were determined by two-tailed unpaired Student’s t test; n.s. not significant. Structure of IL-2 bound to the trimeric IL-2R complex is shown (PDB ID: 2B5I). b In BATF3-KO cells, no IL-2-induced proliferation in Karpas-299 (ALK+) and Mac-1 (ALK-) could be seen compared to CRISPR control. Proliferation was determined by resazurine assay. Data are normalized to untreated control and shown as means ± SD of biological triplicates. P values were determined by two-tailed unpaired Student’s t test. c Immunoblot analysis of total STAT1, STAT5 and ERK1/2 proteins and their phosphorylated (pY, pT/Y) forms in Mac-2A (ALK-), TLBR-2 (BIA) and Jurkat control cells stimulated with rhIL-2 (50 or 100 ng/ml), Neo-2/15 (100 ng/ml) or PBS (-) as control. GAPDH serves as loading control. Blots shown are representative of two independent experiments with similar results in different cell lines. d Limiting dilution of Karpas-299 (ALK+), Mac-1 (ALK-) and Jurkat control cells with or without rhIL-2 (100 ng/ml) in a 96-well plate containing RPMI1640 and 10% FCS. At day 0, the indicated number of cells were seeded and incubated for 2 weeks. Representative example of biological triplicates is shown. e Inhibition of IL-2-induced proliferation by the IL-2Ra-blocking antibody Basiliximab (1 µg/ml). Proliferation was determined by resazurine assay. Data are normalized to control-treated cells and shown as means ± SD of biological triplicates. P values were determined by two-tailed unpaired Student’s t test. f Representative images of 9 patient samples measured of IL-15 IHC staining in fresh-frozen primary ALCL tissue. g Effect of IL-15 (50 ng/ml) on cell proliferation of indicated 4 ALCL and Jurkat cell lines. SU-DHL-1 (ALK+, IL-2R?-negative) and Jurkat (T-cell leukemia-derived, IL-15Ra- and IL-2Rß-negative) serve as control cell lines. Proliferation was measured by [3H]-thymidine incorporation. Values represent means ± SD of biological triplicates as percentage of untreated control. P values were determined by two-tailed unpaired Student’s t test; n.s. not significant. h Immunoblot analysis of STAT1, STAT5 and ERK1/2 proteins and their phosphorylated (pY, pT/Y) forms in FE-PD (ALK-) and Jurkat control cell lines stimulated with rhIL-15 (50 or 100 ng/ml), Neo-2/15 (100 ng/ml) or PBS (-) as control. GAPDH protein expression serves as control.

Fig 4: BATF3 regulates expression of IL2R subunits and CD30 in ALCL.aIL2RA, IL2RB and IL2RG mRNA expression in cell line panel consisting of 13 ALCL cell lines (6 ALCL, ALK+, 4 ALCL, ALK- and 3 BIA-ALCL), 4 T-cell leukemia-derived control cell lines (T) and PBMCs, or in b CRISPR/Cas9-mediated BATF3-KO and CRISPR control ALCL cells, using quantitative RT-PCR. Data are indicated as means ± SD of biological triplicates. P values were determined by two-tailed unpaired Student’s t test. c Cell surface expression of CD30 in BATF3-KO (red) or CRISPR control (green) ALCL cells, isotype control (gray) and unstained cells (black). d BATF3 ChIP of the CD30 regulatory region as compared to a control region (Ref_Ch4). Combined data of two biological replicates are shown as mean ± SEM (see Supplementary Table 8 for ChIP primer sequences). e ChIP-seq tracks at the CD30 locus for BATF3 and H3K27ac in ALCL cell lines with indicated SE regions. f Electrophoretic mobility shift assay (EMSA) of AP-1 TRE complexes on the IL2RA promoter or IL2RB enhancer region (see Supplementary Table 8 for probe sequences) in 10 ALCL cell lines and 4 T-cell leukemia-derived control cell lines. The positions of AP-1 complexes, nonspecific bands (n.s.) and free probes are indicated. Blots shown are representative of three independent experiments with similar results. g BATF3 ChIP on IL2RA or IL2RB regulatory regions as compared to a control region (Ref_Ch4). Combined data of two biological replicates are shown as mean ± SEM (see Supplementary Table 8 for ChIP primer sequences).