A metabolic function of FGFR3-TACC3 gene fusions in cancer

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Abstract

Chromosomal translocations that generate in-frame oncogenic gene fusions are notable examples of the success of targeted cancer therapies1,2,3. We have previously described gene fusions of FGFR3-TACC3 (F3–T3) in 3% of human glioblastoma cases4. Subsequent studies have reported similar frequencies of F3–T3 in many other cancers, indicating that F3–T3 is a commonly occuring fusion across all tumour types5,6. F3–T3 fusions are potent oncogenes that confer sensitivity to FGFR inhibitors, but the downstream oncogenic signalling pathways remain unknown2,4,5,6. Here we show that human tumours with F3–T3 fusions cluster within transcriptional subgroups that are characterized by the activation of mitochondrial functions. F3–T3 activates oxidative phosphorylation and mitochondrial biogenesis and induces sensitivity to inhibitors of oxidative metabolism. Phosphorylation of the phosphopeptide PIN4 is an intermediate step in the signalling pathway of the activation of mitochondrial metabolism. The F3–T3–PIN4 axis triggers the biogenesis of peroxisomes and the synthesis of new proteins. The anabolic response converges on the PGC1α coactivator through the production of intracellular reactive oxygen species, which enables mitochondrial respiration and tumour growth. These data illustrate the oncogenic circuit engaged by F3–T3 and show that F3–T3-positive tumours rely on mitochondrial respiration, highlighting this pathway as a therapeutic opportunity for the treatment of tumours with F3–T3 fusions. We also provide insights into the genetic alterations that initiate the chain of metabolic responses that drive mitochondrial metabolism in cancer.

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Figure 1: Activation of mitochondrial biogenesis and metabolism by F3–T3.
Figure 2: Phosphorylation of PIN4 at Y122 affects mitochondrial metabolism.
Figure 3: PGC1α and ERRγ are required for F3–T3-mediated mitochondrial metabolism and tumorigenesis.
Figure 4: Expression of F3–T3 fusion induces peroxisome biogenesis through phosphorylation of PIN4(Y122).

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Acknowledgements

We thank C. Scuoppo for donation of the pLCiG2 plasmid and support with the gRNA design, E. Chen for identification of PIN4 immunocomplexes and H. Li for high-content microscopy. This work was supported by NIH R01CA101644, U54CA193313 and R01CA131126 to A.L.; R01CA178546, U54CA193313, R01CA179044, R01CA190891, R01NS061776 and The Chemotherapy Foundation to A.I.; SickKids Garron Family Cancer Centre Pitblado Discovery and Ontario Institute for Cancer Research (OICR) Brain Translational Research Initiative to X.H.; American Brain Tumor Association (ABTA) and a Cancer Biology Taining Grant (T32CA009503) fellowship to V.Fra.; a NRF-2013R1A6A3A03063888 fellowship to S.B.L.; an Italian Association for Cancer Research (AIRC) fellowship to M.V.R.

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Authors

Contributions

A.I. and A.L. conceived and coordinated the studies and provided overall supervision. M.C. and S.M.P. developed and performed bioinformatics analyses and wrote the computational sections. V.Fra. performed cell, molecular biology and metabolic assays, with help of T., M.V.R., A.M.C. and S.B.L. J.J.F. and X.H. developed and analysed the Drosophila F3–T3 model. K.S.J.E.-J. and D.M.C.R. conducted the phosphoproteomics experiments. M.S. and K.M. provided GBM tissues and assisted with immunostaining. G.L., T., V.Fre. and H.S. performed immunostaining and protein analyses. T. and P.S. performed mouse experiments. L.G., J.Z., L.C. and R.M. conducted gene expression and bioinformatics analyses. A.I. and A.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Anna Lasorella or Antonio Iavarone.

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Competing interests

A.I. and A.L. received research funds from AstraZeneca and Tahio Pharmaceutical Co., Ltd. M.S. is an investigator in two clinical trials using anti-FGFR therapies: AZD4547 (NCT02824133, funded by AstraZeneca) and TAS-120 (NCT02052778, funded by Tahio Pharmaceutical Co., Ltd). The remaining authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks R. Cagan, P. Mischel, M. Ochs and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Activation of mitosis and mitochondria by F3–T3.

a, Immunoblot analysis of FGFR3 and phospho-FGFR3 in F3–T3 human astrocytes treated with DMSO or PD173074, or human astrocytes expressing F3–T3(K508M) or vector. β-Actin is shown as a loading control. Experiment was repeated at least five times with similar results. b, Heat map of correlations among F3–T3 human astrocytes, F3–T3 human astrocytes treated with PD173074, and human astrocytes expressing vector or F3–T3(K508M). Top and right track colours represent sample type; left track colour scale represents correlation between each sample and the F3–T3 group. F3–T3 human astrocytes and F3–T3 human astrocytes treated with PD173074 (n = 5 biologically independent samples per group). Human astrocytes expressing vector or F3–T3(K508M) (n = 3 biologically independent samples per group). c, Enrichment map network of GO categories scoring as significant (Q < 10−6 in each comparison) from three independent GSEAs (F3–T3 human astrocytes versus F3–T3 human astrocytes treated with PD173074; F3–T3- versus F3–T3(K508M)-expressing human astrocytes; F3–T3- versus vector-expressing human astrocytes). Nodes represent GO terms and lines indicate their connectivity. Size of nodes is proportional to enrichment significance and thickness of lines indicates the fraction of genes shared between the groups. d, RT–qPCR of vector- or F3–T3-expressing human astrocytes treated with vehicle (DMSO) or PD173074 for 12 h. Data are fold change relative to vector (dotted line) of one representative experiment out of two independent experiments (data are mean ± s.d., n = 3 technical replicates). P values were calculated using a two-tailed t-test with unequal variance; *P < 0.05, **P < 0.01, ***P < 0.001. For a complete list of P values see Source Data. e, Left, analysis of mitochondrial mass by MitoTracker FACS analysis in human astrocytes expressing F3–T3, F3–T3(K508M) or vector. Right, quantification of mean fluorescence intensity (MFI). Data are mean ± s.d. of three (vector and F3–T3) and two (F3–T3(K508M)) independent experiments. *P < 0.05, **P < 0.01; two-tailed t-test with unequal variance. f, Immunoblot analysis of mitochondrial proteins in human astrocytes expressing F3–T3, F3–T3(K508M) and vector. Experiment was repeated independently three times with similar results. g, Representative micrographs of VDAC1 and NDUFS4 immunofluorescence (top, green) in F3–T3;shTrp53 and HRAS(12V);shTrp53 mGSCs. DAPI staining of nuclei is shown as an indication of cellular density (bottom, blue). Experiment was repeated independently twice with similar results. Molecular weights are indicated on all immunoblots. Source data

Extended Data Figure 2 F3–T3 induces sensitivity to inhibitors of mitochondrial metabolism.

a, Immunoblot analysis using the FGFR3 antibody in human astrocytes expressing vector, F3–T3 or F3–T3(K508M). α-Tubulin is shown as a loading control. Experiment was repeated five times with similar results. b, OCR of GSC1123 cells expressing F3–T3 in the presence or absence of AZD4547. Data are mean ± s.d. (n = 6 technical replicates) of one representative experiment out of two independent experiments. P < 0.001 for rate 1–4 and 9–12, two-tailed t-test with unequal variance. c, OCR of RPE cells expressing F3–T3, F3–T3(K508M) or the empty vector in the presence or absence of AZD4547. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate with similar results. P < 0.05 for rate 1–4; P < 0.001 for rate 9–12; two-tailed t-test with unequal variance. d, OCR of U251 cells expressing F3–T3, F3–T3(K508M) or the empty vector in the presence or absence of AZD4547. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P < 0.01 for rate 1–4; P < 0.001 for rate 9–12; two-tailed t-test with unequal variance. e, ECAR of human astrocytes expressing F3–T3, F3–T3(K508M) or the empty vector. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P < 0.01 for rate 9–12; two-tailed t-test with unequal variance. f, Ratio between OCR (rate 4) and ECAR (rate 8) in human astrocytes expressing F3–T3, F3–T3(K508M) or vector. Data are mean ± s.d. (n = 6 replicates) of two independent experiments each performed in triplicate. P < 0.01; two-tailed t-test with unequal variance. g, Quantification of ATP production in human astrocytes expressing F3–T3 or vector following treatment with the indicated concentrations of oligomycin for 72 h. Data are independent technical replicates (n = 4) and means (connecting lines) of one representative experiment out of two independent experiments performed with similar results. **P < 0.01; ***P < 0.001; two tailed t-test with unequal variance. h, Time-course analysis of cellular growth of human astrocytes expressing F3–T3 or vector cultured in the presence of glucose (25 mM) or galactose (25 mM) with or without oligomycin (100 nM). Data are independent technical replicates (n = 3) of one representative experiment out of two independent experiments performed with similar results. ***P < 0.001; two-tailed t-test with unequal variance. ik, Survival ratio of F3–T3;shTrp53 and HRAS(12V);shTrp53 mGSCs treated for 72 h with vehicle or metformin (i), rotenone (j) or menadione (k) at the indicated concentrations. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of two independent experiments performed with similar results. **P < 0.01; ***P < 0.001; two-tailed t-test with unequal variance. l, Western blot analysis of COX1 and COX2 proteins in F3–T3;shTrp53 and HRAS(12V);shTrp53 mGSCs treated with vehicle or tigecycline at a concentration of 8 μM for 72 h. α-Tubulin is shown as a loading control. Experiment was independently repeated twice with similar results. m, Quantification of cellular ATP in F3–T3;shTrp53 (left) and HRAS(12V);shTrp53 (right) mGSCs treated with vehicle or metformin (1 mM), tigecycline (8 μM) or menadione (5 μM) for 16 h. Data are mean ± s.d. of one experiment (n = 6 technical replicates). **P < 0.01; ***P < 0.001; two-tailed t-test with unequal variance. n, Quantification of tumour volume of F3–T3;shTrp53 mGSCs in control and tigecycline-treated mice. Data are tumour volumes (median with interquartile range) at day 6 of treatment, a time when all mice were still in the study; n = 8 for control (median = 1,427 mm3) and n = 10 for tigecycline-treated mice (median = 843.4 mm3). *P < 0.05; two-sided Mann–Whitney U-test. Molecular weights are indicated in immunoblots. Source data

Extended Data Figure 3 Phosphorylation of Y122 of PIN4 by F3–T3.

a, Amino acid sequence flanking Y122 of PIN4 (in red) is evolutionarily conserved. b, Immunoprecipitation–western blot analysis of human astrocytes expressing F3–T3 or F3–T3(K508M) with or without silencing of endogenous PIN4. β-Actin is shown as a loading control. WCL, whole-cell lysate. c, Immunoblot analysis of phosphotyrosine immunoprecipitates (left) or whole-cell lysates (right from U87 glioma cells expressing empty vector, FGFR3, F3–T3 or F3–T3(K508M) using the indicated antibodies. The asterisk indicates a non-specific band. d, Immunoblot analysis of phosphotyrosine immunoprecipitates (left) or whole-cell lysates (right) from human astrocytes expressing empty vector, F3–T3 or F3–T3(K508M) using the indicated antibodies. FAK is shown as a loading control. e, Immunoblot analysis of phosphotyrosine immunoprecipitates (left) or whole-cell lysates (right) from GSC1123 cells expressing endogenous F3–T3 shows decreased phosphorylation of F3–T3 substrates following treatment with AZD4547 for the indicated times. Paxillin is shown as a loading control. f, Immunoblot analysis of canonical FGFR signalling proteins in GSC1123 cells treated with AZD4547 for the indicated time. β-Actin is shown as a loading control. g, Immunoblot analysis of phosphotyrosine immunoprecipitates from human astrocytes expressing F3–T3 or vector transduced with wild-type or the unphosphorylable Y to A F3–T3 kinase substrate mutants. Paxillin is shown as a loading control. The asterisk indicates a non-specific band. h, Confocal images of immunofluorescence staining using the phospho-PIN4(Y122)-specific antibody (red) in human astrocytes transduced with vector or F3–T3 without or with silencing of endogenous PIN4. Nuclei were stained with DAPI (blue). i, Immunoblot analysis of phospho-PIN4(Y122), total PIN4 and FGFR3 in SF126 cells transduced with FGFR3, F3–T3, F3–T3(K508M) or the empty vector. β-Actin is shown as a loading control. Molecular weights are indicated in all panels. Experiments in bg, i were repeated independently three times with similar results. Experiment in h was repeated independently four times with similar results.

Extended Data Figure 4 Functional analysis of tyrosine phosphorylation of F3–T3 kinase substrates.

a, Western blot analysis of phosphotyrosine immunoprecipitation of F3–T3;shTrp53 and HRAS(12V);shTrp53 mGSCs using the PIN4 antibody. F3–T3 and HRAS(12V) expression are shown. α-Tubulin is shown as a loading control. b, Immunofluorescence images using the phospho-PIN4(Y122)-specific antibody (red, top) in tumours from F3–T3;shTrp53 and HRAS(12V)shTrp53 mGSCs. Nuclei were counterstained with DAPI (blue, bottom). Experiment was repeated independently twice with similar results. c, Left, representative images of phospho-PIN4(Y122) immunofluorescence in F3–T3-positive (top) and F3–T3-negative (bottom) GBM (green). Right, higher magnification images of phospho-PIN4(Y122)–DAPI co-staining depicting cytoplasmic localization of phospho-PIN4(Y122). Middle, DAPI staining of nuclei is shown as an indication of cellular density. d, Analysis of OCR in human astrocytes F3–T3 transduced with wild-type or the unphosphorylable Y to A mutant of GOLGIN84, C1orf50 and DLG3. Human astrocytes expressing the empty vector are included as a control. Data are mean ± s.d. (n = 5 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P < 0.001, rate 9–12 for vector versus each F3–T3 combination, two-tailed t-test with unequal variance. e, Analysis of OCR of human astrocytes expressing F3–T3 transduced with PKM2(WT), PKM2(Y105A) or the empty vector. Human astrocytes expressing the empty vector are included as control. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of three independent experiments; P < 0.001, rate 9–12 for vector versus each F3–T3 combination, two-tailed t-test with unequal variance. f, Immunoblot analysis of GOLGIN84, C1orf50 and DLG3 wild-type or Y to A mutants in human astrocytes experessing F3–T3 or vector. g, Immunoblot analysis of human astrocytes transduced with empty vector or F3–T3 expressing PKM2(WT) or PKM2(Y105A). h, Immunoblot analysis of human astrocytes transduced with F3–T3 or the empty vector for the expression of PIN4(WT) or PIN4(Y122F). i, Immunoblot analysis of PIN4 proteins in human astrocytes expressing F3–T3 following silencing of endogenous PIN4 and reconstitution with PIN4(WT), PIN4(Y122A) or PIN4(Y122F). In fi, β-actin is shown as a loading control. Molecular weights are indicated on all immunoblots. j, Quantification of ATP levels in human astrocytes treated as in i. Data are mean ± s.d. (n = 4 technical replicates) of one out of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test with unequal variance. Experiments in a, fi were repeated independently three times with similar results. Source data

Extended Data Figure 5 Transcriptomic analysis of F3–T3 fusion-positive and fusion-like GBM and validation of ee-MWW using different cancer-driving alterations.

a, Consensus clustering on the Euclidean distance matrix based on the top and bottom 50 genes having the highest and lowest probability to be upregulated, respectively, in the nine F3–T3 fusion-positive GBM. The consensus matrix is obtained from 10,000 random samplings using 70% of the 544 samples. The nine F3–T3-positive GBM (in red) fall in one cluster (cyan). b, MWW enrichment plot of the ‘hallmark oxidative phosphorylation’ GO category in F3–T3-positive GBM. NES and P-values are indicated. c, Enrichment map network of statistically significant GO categories (Q < 0.001, NES > 0.6, upper-tailed MWW-GST) in nine fusion-like GBM. Nodes represent GO terms and lines demonstrate their connectivity. Size of nodes is proportional to number of genes in the GO category and thickness of lines indicates the fraction of genes shared between the groups. d, Analysis of copy number amplification of the POLRMT gene comparing fusion-like GBM with all other GBM at different thresholds for amplification detection on log-R ratio from single-nucleotide polymorphism arrays. P value and log-odds at different thresholds are indicated (Fisher’s exact test). e, POLRMT gene expression in fusion-like GBM (n = 9) and the remaining samples (n = 535). Box plot spans the first to third quartiles and whiskers show the 1.5× interquartile range. P value, two-sided MWW test. f, Representative images of VDAC1 immunofluorescence in F3–T3-positive (green, left) and F3–T3-negative (right) GBM. DAPI staining of nuclei is shown as an indication of cellular density (blue, bottom). g, Hierarchical clustering of two GBM samples with a KRAS mutation (red) out of 544 samples. Heat map of the two KRAS mutant samples is enlarged to the left. h, Hierarchical clustering of five KRAS-mutated samples (red) in the invasive breast carcinoma (BRCA) cohort (n = 1,093). i, Hierarchical clustering of six EGFR–SEPT14-positive GBM samples (red) out of 544 samples. Data in gi, were obtained using the Euclidean distance and Ward linkage method and are based on the top and bottom 50 genes having the highest and lowest probability to be upregulated, respectively.

Extended Data Figure 6 Pan-glioma and multi-cancer analysis of F3–T3 fusion-positive samples.

a, b, Hierarchical (a) and consensus clustering (b) of 11 F3–T3-positive samples (red) out of 627 pan-glioma samples. The 11 F3–T3-positive samples (red) in b fall in one cluster (blue). c, Enrichment map network of statistically significant GO categories (Q < 0.001, NES > 0.6; upper-tailed MWW-GST) in the 11 F3–T3 fusion-positive pan-glioma samples. Nodes represent GO terms and lines demonstrate their connectivity. Size of nodes is proportional to number of genes in the GO category and thickness of lines indicates the fraction of genes shared between the groups. d, MWW enrichment plot of the ‘hallmark oxidative phosphorylation’ GO category in F3–T3-positive samples in the pan-glioma cohort. e, Hierarchical clustering of four F3–T3-positive (red) samples out of 86 lung squamous cell carcinoma (LUSC) samples. f, Enrichment map network of statistically significant GO categories (Q < 0.001, NES > 0.6; upper-tailed MWW-GST) in four F3–T3-positive LUSC Nodes represent GO terms and lines demonstrate their connectivity. Size of nodes is proportional to number of genes in the GO category and thickness of lines indicates the fraction of genes shared between the groups. g, Hierarchical clustering of two F3–T3-positive, human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSC) samples (in red) out of 36 samples. h, Enrichment map network of statistically significant GO categories (Q < 0.001, NES > 0.6; upper-tailed MWW-GST) in two F3–T3-positive HNSC samples. Nodes represent GO terms and lines demonstrate their connectivity. Size of nodes is proportional to number of genes in the GO category and thickness of lines indicates the fraction of genes shared between the groups. i, Hierarchical clustering of two F3–T3-positive samples (red) out of 184 oesophageal carcinoma (ESCA) samples. Heat maps of the two F3–T3-positive samples are enlarged to the left. j, Hierarchical clustering of four F3–T3-positive samples (red) out of 305 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) samples. k, Hierarchical clustering of five F3–T3-positive samples (red) out of 408 urothelial bladder carcinoma (BLCA) samples. l, TDA network of pan-glioma samples (n = 627) reconstructed using variance normalized Euclidean distance and locally linear embedding as filter function. The nodes containing F3–T3-positive samples are highlighted in red. m, Correlation between the expression of F3–T3 (log2 of total fragment, x axis) and NES (y axis) of three top ranking mitochondrial functional categories in a multi-cancer cohort including F3–T3-positive samples (n = 19) from eight tumour types (r and P values are indicated, upper-tailed Spearman’s rank correlation test). n, Analysis of the activity of master regulators in the pan-glioma cohort (n = 627 glioma). Grey curves represent the activity of each master regulator with tumour samples ranked according to master regulator activity. Red and blue lines indicate individual F3-T3-positive GBM samples displaying high and low master regulator activity, respectively. P values, two-sided MWW test, for differential activity (left) and mean of the activity (right) of the master regulator in F3–T3-positive versus F3–T3-negative samples are indicated. o, Gene expression analysis of PPARGC1A and ESRRG genes in F3–T3-positive and F3–T3-negative GBM; n = 9 F3–T3-positive tumours; n = 525 F3–T3-negative tumours. Box plot spans the first to third quartiles and whiskers show the 1.5× interquartile range. P value, two-sided MWW test. Data in a, e, g, ik, were obtained using the Euclidean distance and Ward linkage method and are based on the top and bottom 50 genes having the highest and lowest probability to be upregulated, respectively.

Extended Data Figure 7 PGC1α and ERRγ are required for mitochondrial metabolism and tumorigenesis of cells transformed by F3–T3.

a, Immunoblot of endogenous PGC1α in human astrocytes expressing F3–T3 following silencing of PIN4 and reconstitution with wild-type or PIN4(Y122F). Exogenous expression of PGC1α in human astrocytes is included as positive control. Experiment was independently repeated three times with similar results. b, RT–qPCR of PPARGC1A in human astrocytes expressing F3–T3 or vector. Data are mean ± s.d. (n = 6 replicates) from two independent experiments each performed in triplicate. c, RT–qPCR of PPARGC1A in human astrocytes expressing F3–T3 treated as in a. Data are mean ± s.d. (n = 4 biological replicates) from four independent experiments. d, GSEA shows upregulation of ROS detoxification genes in human astrocytes expressing F3–T3 (n = 5 biological replicates) compared with vector (n = 3 biological replicates). Nominal P value is indicated. e, Immunoblot of Flag-PIN4 (wild-type and Y122F) and PGC1α (wild-type and L2L3A) in human astrocytes expressing F3–T3 after silencing of PIN4. Experiment was repeated twice independently with similar results. f, Soft agar colony-forming assay of human astrocytesF3–T3 following silencing of PIN4 and reconstitution with wild-type or Y122F Flag-PIN4 in the presence or the absence of PGC1α. Data are mean ± s.d. (n = 3 technical replicates) from one representative experiment out of two independent experiments. g, RT–qPCR of PPARGC1A in human astrocytes expressing vector or F3–T3 transduced with PPARGC1A shRNA1 or PPARGC1A shRNA2 lentivirus. Data are mean ± s.d. (n = 3 technical replicates) from one representative experiment. h, Immunoblot analysis of PGC1α in HA-F3–T3 treated as in g. Experiment was repeated two times independently with similar results. Exogenous expression of PGC1α is included as positive control. Experiment was repeated twice independently with similar results. i, RT–qPCR of PPARGC1A in F3–T3 human astrocytes expressing two independent gRNAs against PPARGC1A (PPARGC1A gRNA1, two clones; PPARGC1A gRNA2, 1 clone) or the empty vector. Data are mean ± s.d. (n = 3 technical replicates) from one representative experiment. j, Western blot of cells treated as in (i) using the indicated antibodies. Experiment was repeated twice independently with similar results. k, OCR of human astrocytes expressing vector or F3–T3 transduced with PPARGC1A gRNA1 or gRNA2. Data are mean ± s.d. (n = 5 technical replicates) from one representative experiment out of two independent experiments. P < 0.001 for rate 1–4 and 9–12; two-tailed t-test with unequal variance. l, RT–qPCR of ESRRG in human astrocytes expressing vector or F3–T3 infected with ESRRG shRNA1 or ESRRG shRNA2 lentiviruses. Data are mean ± s.d. (n = 3 technical replicates) from one representative experiment. m, Immunoblot analysis of ERRγ in human astrocytes expressing F3–T3 treated as in l. Experiment was repeated twice independently with similar results. n, Soft agar colony-forming assay of human astrocytes treated as in Fig. 3g. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of two independent experiments performed in triplicate. o, GSC1123 cells were transduced with PPARGC1A shRNA lentiviruses or the empty vector. Cells were analysed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in 96-well cultures from three independent infections. p, Bar graph shows the frequency of gliomaspheres from three independent infections analysed by LDA as shown in o. Data are mean ± s.d. (n = 3 biological replicates). q, The photograph shows tumours generated by human astrocytes F3–T3 transduced with PPARGC1A shRNA1, ESRRG shRNA1 or vector lentivirus in Fig. 3i at the time of mouse euthanasia. sh-P, PPARGC1A shRNA1; sh-E, ESRRG shRNA1. r, RT–qPCR of Ppargc1a in F3–T3-shTrp53 and HRAS(12V)shTrp53 mGSCs transduced with Ppargc1a shRNA1 or Ppargc1a shRNA2 lentivirus. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment. s, Tumour volume of F3–T3-shTrp53 mGSCs expressing a pLKO-vector (n = 5), Ppargc1a shRNA1 (n = 5) or Ppargc1a shRNA2 (n = 5). Data are the tumour growth curve of individual mice. t, Tumour volume of mice injected subcutaneously with HRAS(12V);shTrp53 mGSCs expressing pLKO-vector (n = 5) or Ppargc1a shRNA1 (n = 5) or Ppargc1a shRNA2 (n = 5). Data are tumour growth curve of individual mice; NS, not significant, two-tailed t-test with unequal variance (time points 1–7). u, Photograph shows tumours generated from F3–T3;shTrp53 mGSCs transduced with Ppargc1a shRNA1 or Ppargc1a shRNA2 or vector lentivirus in s at the time of mouse euthanasia. v, Photograph shows tumours generated by HRAS(12V)shTrp53 mGSCs transduced with Ppargc1a shRNA1 or Ppargc1a shRNA2 or vector lentivirus in t at the time of mouse euthanasia. Molecular weights are indicated and β-actin or α-tubulin is shown as a loading control in all immunoblots. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed t-test with unequal variance. Source data

Extended Data Figure 8 Drosophila PGC1α-homologue spargel (srl) mediates F3–T3-induced tumour growth.

a, Optical projections of whole brain–ventral nerve cord complexes from Drosophila larvae. Expression of the F3–T3 fusion oncogene using the repo-Gal4 (repo-Gal4>F3–T3) pan-glial driver induced pathological changes in brain and ventral nerve cord with ectopic tissue protrusions (yellow arrows) due to excessive glial cell proliferation and accumulation. b, Survival of larvae bearing F3–T3-driven glial tumours. Larvae bearing F3–T3-driven glial tumours die before developing into adulthood (biologically independent samples: n = 87, Repo-Gal4 > mRFP; n = 77, Repo-Gal4 > mRFP-F3-T3). Data are shown as mean ± s.e.m. *P < 0.05; two-tailed t-test with unequal variance. Individual dots represent the fraction of surviving animals. c, Glial expression of F3–T3 resulted in increased total glial cell number (Repo+mRFP+ cells) compared to controls. Note the excessive accumulation of glial cells in the brain lobe (white arrows) and ventral nerve cord (yellow arrows). d, Glial expression of F3–T3 increases glial cell proliferation (mRFP+ phosphorylated histone H3+ (phospho-HH3+) cells) compared to control. Note the excessive accumulation of glial cells in the brain lobe (white arrows) and ventral nerve cord (yellow arrows). e, Glia-specific srl knockdown in F3–T3-induced glial tumours resulted in decreased total glial number (Repo+eGFP+ cells) compared to controls. f, Quantification of glia number in control and srl-deficient tumours. n = 15 for repo-Gal4>F3–T3; n = 15 for repo-Gal4>F3–T3 RNAi-KK100201; n = 16 for repo-Gal4>F3–T3;RNAi-GL01019; n = 11, for repo-Gal4>F3–T3;RNAi-HMS00857; n = 6 for repo-Gal4>F3–T3;RNAi-HMS00858. Data are shown as mean ± s.e.m. ***P < 0.001; two-tailed t-test with unequal variance. g, Western blot analysis of the F3–T3 protein in repo-Gal4>F3–T3 and repo-Gal4>F3–T3;RNAi-srl Drosophila brains. The expression of F3–T3 in human GSC1123 cells is shown as a positive control for F3–T3 and α-tubulin is shown as a loading control. Experiments in ce, g were performed twice. Source data

Extended Data Figure 9 Glia-specific knockdown of srl has little to no effect on EGFR–PI3K-induced tumour growth but glia-specific knockdown of ERR inhibits F3–T3-induced tumour growth.

a, Optical projections of whole brain–ventral nerve cord complexes from larvae with control and srl-deficient glia. b, Glia-specific srl knockdown in larval brains did not significantly affect the overall glial population (Repo+ cells) nor the mitotic index of glial cells (Repo+ phospho-HH3+ cells, yellow arrows). c, Quantification of glia volume in larval brains with control and srl-deficient glia; n = 13 for repo-Gal4>eGFP; n = 14 for repo-Gal4>eGFP;RNAi-KK100201; and n = 16, for repo-Gal4>eGFP;RNAi-HMS00857. Data are mean ± s.e.m. NS, not significant; two-tailed t-test with unequal variance. d, Quantification of proliferating glia number (Repo+; phospho-HH3+ cells) in larval brains with control and srl-deficient glia; n = 13 for repo-Gal4>eGFP; n = 14 for repo-Gal4> eGFP;RNAi-KK100201; and n = 16 for repo-Gal4> eGFP;RNAi-HMS00857. Data are mean ± s.e.m. NS, not significant; two-tailed t-test with unequal variance. e, Adult lethality in repo-Gal4>F3–T3 and repo-Gal4>F3–T3;RNAi-srl larvae (n > 100). f, Optical projections of control and srl-deficient brain tumours from repo-Gal4>Dp110CAAX;dEGFRλ;mRFP larvae. g, Quantification of tumour volume in control and srl-deficient tumours; n = 15 for repo-Gal4>Dp110CAAX;dEGFRλ;mRFP; n = 16 for repo-Gal4>Dp110CAAX;dEGFRλ;mRFP;RNAi-KK100201; n = 19 for repo-Gal4>-Dp110CAAX;dEGFRλ;mRFP;RNAi-HMS00857. Data are mean ± s.e.m. NS, not significant; two-tailed t-test with unequal variance. h, Optical projections of brain tumours from Drosophila larvae repo-Gal4>F3–T3 and repo-Gal4>F3–T3;RNAi-ERR. RNAi-mediated knockdown of ERR reduces the volume of F3–T3-induced glial tumours. i, Quantification of tumour volume in the control and ERR-deficient tumours; n = 20 for repo-Gal4>F3–T3; n = 16 for repo-Gal4>F3–T3;RNAi-JF02431; n = 19, for repo-Gal4>F3–T3;RNAi-HMC03087; n = 19, for repo-Gal4>F3–T3;RNAi-KK10839). ***P < 0.001; two-tailed t-test with unequal variance. In all experiments n are biologically independent animals. Experiments in a, b, f, h, were repeated twice with similar results. Source data

Extended Data Figure 10 Acute expression of F3–T3 fusion induces peroxisome biogenesis through phosphorylation of PIN4(Y122).

a, Representative confocal images (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left) and phospho-PIN4(Y122) (p-PIN4, red, middle panel) in human astrocytes expressing the empty vector and F3–T3. Right, higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently twice with similar results. b, Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle) and phospho-PIN4(Y122) (red, right) in human astrocytes expressing F3–T3. Arrows indicate protein co-localization. Experiment was repeated independently twice with similar results. c, Co-immunoprecipitation from H1299 cells using the PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analysed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d, Western blot analysis of co-immunoprecipitation of exogenous Flag-PEX1 in human astrocytes expressing F3–T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e, RT–qPCR of PEX1 in human astrocytes expressing F3–T3 or vector. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f, Western blot analysis of PEX1 expression in human astrocytes transduced with F3–T3, F3–T3(K508M) or the empty vector. β-Actin is shown as a loading control. Experiment was repeated independently three times with similar results. g, Time-course analysis of F3–T3 expression in human astrocytes by western blot. α-Tubulin is shown as a loading control. Experiment was repeated independently twice with similar results. h, Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in human astrocytes reconstituted with PIN4(WT) or PIN4(Y122F) after silencing of the endogenous PIN4 and acutely transduced with F3–T3 or vector. Representative bar plots (n = 4 technical replicates) from one out of three independent experiments. *P < 0.05, ***P < 0.001; two-tailed t-test with unequal variance. CHX-treated cultures were used as negative controls. i, Time-course expression analysis by RT–qPCR of the indicated mitochondrial genes in human astrocytes expressing F3–T3 or empty vector. Data are mean ± s.d. (n = 3 technical replicates) of one representative experiment out of two independent experiments performed in triplicate. Values were normalized to vector (dotted line). *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed t-test with unequal variance. j, Quantification of cellular ROS (measured by high-content microscopy) in human astrocytes reconstituted with PIN4(WT) or PIN4(Y122F) after silencing of the endogenous PIN4 and acutely transduced with F3–T3 or vector. Representative bar plots from one out of three independent experiments. Data are mean ± s.d. (n = 3 technical replicates). *P < 0.05; two-tailed t-test with unequal variance. N-acetyl-l-cysteine-treated cultures were used as negative controls. Molecular weights are indicated in all immunoblots. Source data

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Frattini, V., Pagnotta, S., Tala et al. A metabolic function of FGFR3-TACC3 gene fusions in cancer. Nature 553, 222–227 (2018). https://doi.org/10.1038/nature25171

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