Fig 2: Assessment of the TBXT regulome by T-DARPin expression in UM-Chor1 cells.(A) UpSet plot of RNA-seq data showing the number and intersections of genes significantly deregulated by T-DARPin expression as a matrix of six sets (T-DARPins A2, B1, and D4; up- or down-regulated). Rows show the number of genes per set. Columns show the number of intersecting genes at the top and the intersecting sets as connected dots at the bottom. DEGs, as defined in the text, are indicated in teal. Significantly deregulated genes were identified using generalized linear models (GLMs) with a Wald test (n = 3, FDR < 1%). (B) GSEA of RNA-seq data with the MSigDB Hallmark gene collection. Dots represent enrichments that are not statistically significant based on the permutation test. NES, normalized enrichment score. (C) UpSet plot of DDA-MS data showing the number and intersections of proteins significantly deregulated by T-DARPin expression as a matrix of six sets (T-DARPins A2, B1, and D4; up- or down-regulated). Rows show the number of proteins per set. Columns show the number of intersecting proteins at the top and the intersecting sets as connected dots at the bottom. DEPs, as defined in the text, are indicated in orange and yellow. Significantly deregulated proteins were identified using empirical Bayes moderated t test (n = 3, FDR < 5%). (D) GSEA of DDA-MS data with the MSigDB Hallmark gene collection. Dots represent enrichments that are not statistically significant based on the permutation test. (E) Circos plot of DEPs categorized according to their target development levels (TDLs). The inner heatmap shows the log2(fold change) of proteins deregulated by T-DARPins A2, B1, and D4. The outer heatmap shows the log2(fold change) of the corresponding RNA-seq data if the respective DEP-encoding gene was also among the DEGs.

Fig 3: Chordoma cells depend on JAK-STAT signaling.(A) ISG expression in UM-Chor1 cells as determined by RNA-seq, DDA-MS, and DIA-MS under different treatments shown at the top. Open circles indicate nonsignificant expression [RNA-seq: GLM with Wald test (n = 3, FDR < 1%); DDA-/DIA-MS: empirical Bayes moderated t test (n = 3, FDR < 5%)]. (B) GSEA of 1107 tumor transcriptomes (1106 patients) using 50 Hallmark gene sets. Mean gene set enrichment scores of 23 chordoma samples (red) versus 1084 nonchordoma samples (blue). Gene sets ranked by mean enrichment score in chordoma. Error bars: 95% confidence intervals (t distribution). (C) Western blots of chordoma cell lines U-CH1, U-CH2, JHC7, and UM-Chor1; the osteosarcoma cell line U2OS; the B cell leukemia cell line MEC-1; and the T cell leukemia cell line Jurkat showing JAK/STAT pathway members, TBXT, EGFR, and phospho-EGFR (Y1068). (D) GR analysis after 7 days of treatment with pan-JAK (filgotinib), JAK2 (AZD1480, fedratinib, and pacritinib), STAT1 (fludarabine), and FLT3 (gilteritinib) inhibitors (10 μM to 1 nM). GR < 1 and > 0: proliferation inhibition; GR = 0: cytostasis; GR < 0: cytotoxicity. One of three representative experiments shown; data points represent technical triplicates. (E) Apoptosis measured by annexin V/7-AAD staining and flow cytometry after 4-day treatment with 3 μM of indicated drugs. Mean + SEM of two biological replicates. (F) Colony formation of U2OS and chordoma cell lines after 14-day treatment with 1 μM of indicated drugs. Representative images of three independent experiments (fig. S6D).

Fig 4: T-DARPins are specific for TBXT and bind TBXT variants.(A) IP of Flag-tagged DARPins from U-CH2 cells with an anti-Flag antibody 5 days after transduction, followed by Western blotting (right). The whole-cell lysate input is shown on the left. One representative image of three independent experiments. (B) Enrichment of proteins identified by AP-MS of anti-Flag IPs for DARPins A2, B1, D4, and E3_5 and of anti-TBXT antibody (ab) IPs 4 days after transduction of U-CH2 and UM-Chor1 cells. Sphere sizes represent empirical Bayes moderated t statistic (n = 3) as an indicator of statistical certainty, and bait types and cell lines are indicated by colors. (C) Simulation of molecular interactions between T-DARPin structures predicted with AlphaFold2 and the TBXT DBD (PDB: 7HI8). T-DARPin:TBXT DBD (7H18) complexes were overlaid with the TBXT DBD:DNA PDB structure 6F58. The DNA is displayed as blue and red helices. (D) Anti-Flag bead pulldowns of DARPin E3_5 and T-DARPin D4 cotransfected into HEK293T cells with EV or a vector expressing HA-tagged WT TBXT, TBXT-R16L, TBXT-G177D, TBXT-H171R, or TBXT lacking the DBD (ΔDBD).

Fig 5: IGFBP3 modifies part of the TBXT regulome.(A) IGFBP3 protein levels quantified by ELISA of UM-Chor1-Cas9 cells transduced with sgNTC or sgTBXT. Mean ± SEM of three biological replicates. Unpaired t test. ***P ≤ 0.001. (B) IGFBP3 protein levels in lysates and culture medium of UM-Chor1-Cas9 cells transduced with sgNTC or sgTBXT, with or without PNGase F treatment. (C) Western blot of UM-Chor1-Cas9 cells transduced with sgNTC, sgTBXT, or two sgRNAs targeting YAP1. (D) Viability of UM-Chor1 and U-CH2 spheroids expressing doxycycline-inducible IGFBP3 or EV, treated with doxycycline (0.5 μg/ml) versus untreated controls after 28 days. Mean ± SEM of two biological replicates (five to six spheroids averaged per condition). Representative spheroid images shown on the right. (E) Viability (luminescence units × 107) after 14 days of UM-Chor1-Cas9 spheroids double-transduced with sgNTC/sgNTC, sgTBXT/sgNTC, sgIGFBP3/sgNTC, or sgIGFBP3/sgTBXT. Mean ± SEM of three biological replicates (four to six spheroids averaged per condition). One-way ANOVA with Dunnett’s test for multiple comparisons; ***P ≤ 0.001. (F) Schematic of DIA-MS analysis in UM-Chor1-Cas9 cells 14 days posttransduction with sgNTC or sgIGFBP3, followed by T-DARPin D4 or E3_5, resulting in four conditions: ① control, ② TBXT inhibition, ③ IGFBP3 knockout, and ④ combined TBXT inhibition and IGFBP3 knockout. Created in BioRender. S.F. (2025) BioRender.com/r29p687. (G) MSigDB Hallmark GSEA comparing sgNTC (orange) versus sgIGFBP3 (purple) UM-Chor1-Cas9 proteomes under TBXT inhibition. The x axis shows NESs for gene sets significantly enriched in at least one condition (FDR < 5%, permutation test with Benjamini-Hochberg correction). NES values for both conditions are shown; dashed lines indicate FDR > 5%. (H) Expression changes of IGFBP3-dependent genes induced by T-DARPin D4 in sgNTC (black) or sgIGFBP3 (red and blue) UM-Chor1-Cas9 cells. Vertical lines indicate differences in log2(fold change). Red indicates reduced, blue increased expression upon IGFBP3 knockout (GLM with Wald test, n = 3, FDR < 1%). Interferon response genes are indicated and data points are circled. (I) Western blot of UM-Chor1-Cas9 cells with (sgIGFBP3) or without (sgNTC) IGFBP3 knockout and expressing DARPins E3_5 or D4.