Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas.

Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. By RNA-seq analysis, we identify a RET rearrangement in the tumour material of a patient who does not harbour any known RAS or BRAF mutations. This new gene fusion involves exons 1–4 from the 5′ end of the Trk fused Gene (TFG) fused to the 3′ end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase-dependent manner. TFG-RET oligomerises in a PB1 domain-dependent manner and oligomerisation of TFG-RET is required for oncogenic transformation. Quantitative proteomic analysis reveals the upregulation of E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in both tumor and metastatic lesions, which is further confirmed in additional patients. Expression of TFG-RET leads to the upregulation of HUWE1 and inhibition of HUWE1 significantly reduces RET-mediated oncogenesis.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The sample size is always given in the figure legends. We performed technical and biological replicates as indicated in the figure legends. No power calculations based on expected effect sizes were done.

not done
The experiments were performed multiple times and it is indicated in the legends now. When we present representative data from a single experiment, which was reproduced, it is indicated (for instance with Western blot experiments).
Samples/organisms/participants were not randomized. Established cell lines were used for in vitro experiments. In the animal experiments animals received either control or knock-out cells. Tumor bearing animals were not treated with any drug, so a randomization of the animals was not necessary. Patient material was obtained from the Biobank at the University Medicine Center. We received tissue from papillary thyroid carcinoma patients who did not harbor BRAF, KRAS, NRAS or HRAS mutations. BRAF as well as NRAS mutations are well known driver mutations and we wanted to identify novel mutations contributing to the tumorigenesis of PTCs. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Mycoplasma was tested routinely in the lab and the contaminant cell lines are either cured are culled from the analysis.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
NOD.CB17-Prkdcscid mice, female, age at the beginning of the experiment 8 to 10 weeks none none The animal experiments were granted by the Landesuntersuchungsbehörde Koblenz -the locally responsible authority for conducting animal studies.
We obtained material from the local biobank. We used patient material from duly consented PTC patients without known BRAF, KRAS, NRAS or HRAS mutations.
The tissues employed in this study were obtained from the biobank. as a donation with due consent following the ethical and legal guidelines of the institution.