Fusion transcripts FYN-TRAF3IP2 and KHDRBS1-LCK hijack T cell receptor signaling in peripheral T-cell lymphoma, not otherwise specified

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.


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Raw sequence data from clinical specimens has been deposited in the European Genome-Phenome Archive (accession EGAS00001004646 for 3 PTCL-TFH cases, accession EGAS00001005015 for 15 PTCL-NOS cases). Raw sequence data from murine CD4+ T cells is available in the European Nucleotide Archive (accession PRJEB42764). Sequence data from anaplastic large cell lymphoma was obtained from the Sequence Read Archive (accession SRP044708) and sequence data from healthy lymph nodes was obtained from ArrayExpress (accession E-MTAB-2836). Expression data for immune cells in healthy individuals used in Supplementary figure 2b were obtained from DICE. The source data underlying Figures 2,3,4,5,6,7,8,9,10 and Supplementary Figures 1,2,3,4,5,6 are provided as a Source Data file. All the other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request.
-The number of patient samples was determined by the availability of samples.
-For studies with mice, we used at least 5 mice per group. This sample size is adequate disease alleles with high penetrance will cause disease in a majority of the animals. The sample size was chosen based on previous experience in our laboratory (e.g. PMID 29496663), sample size in literature in the field of mouse models of T cell lymphoma (e.g. PMID 21926465, PMID 29398449) In the KHDRBS1-LCK group, one mouse developed an ulcerated squamous cell carcinoma of the skin and was censored in the survival curve. No other data were excluded from the analyses.
All in vitro experiments used at least 3 biological independent replicates. In vitro experiments were reproduced at least twice with congruent results. We used different batches of virus for repetitions of experiments involving viral transduction. The number of biological replicates is included the figure legends. The number of repetitions for every experiment is included under the header Statistics and reproducibility in the Methods section of the main article.
For drug treatments, drugs were dispensed randomly with a D300e digital dispenser (Tecan) in 96-well plates. For all other in vitro experiments, wells with identical number of cells were randomly allocated to each group. For bone marrow transplant models, mice of the same age were randomly allocated to be injected with lineage negative cells transduced with different constructs. Mouse samples used for flow cytometry, immunohistochemistry and flow cytometry were chosen randomly.
Ba/F3: The identity of the cell line was confirmed by karyotyping.
Once per year we control the identity of Jurkat cells with STR profiling. 293T cells were not further authenticated.
Cell lines were tested negative for mycoplasma on a regular basis (MycoAlert Mycoplasma Detection Kit, Westburg). Primary cells were cultured in Primocin (Invivogen, cat. code ant-pm-2), which contains 3 compounds with activity against mycoplasma species.

None
We used 8 to 10 week old C57BL/6J mice to harvest T cells for in vitro experiments. For bone marrow transplant experiments, we harvested lineage negative cells from 6 to 8 week old male C57BL/6J donor mice and we used 6 tot 8 week old female C57BL/6J mice as recipients. Mice were housed in individually ventilated cages with a temperature between 18 and 23°C and humidity between 40 and 60% No more than 5 mice were housed in a single cage. The room had a programmed 12 hour light-12 hour dark cycle.
The study did not involve wild animals.
The study did not involve field-collected samples.

April 2020
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Recruitment
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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. Patient samples for the discovery cohort were collected retrospectively from the tumor banks of the University Hospitals Leuven and the CHU Mont-Godinne and prospectively in the University Hospitals Leuven. For prospectively obtained samples, we obtained informed consent from all patients. Patient samples for the validation cohort were obtained from the T-cell lymphoma biobank (TENOMIC) of the Lymphoma Study Association (LYSA). Inclusion was based on the availability of sufficient material from a surgical biopsy. Therefore, patients with poor performance status or in critical condition that did not undergo a surgical biopsy, may have been missed due to the design of the study.
The study was approved by the Ethics Committee UZ/KU Leuven (S62100).
Cultured cells were washed and resuspended in staining buffer (PBS with 2% FBS). Spleens and lymph nodes were smashed with the plunger of a 10 ml syringe on a 40 µm cell strainer. Pellets were incubated red blood cell lysis buffer (RCL, 150 mM NH4Cl, 0.1 mM EDTA, 10 mM KHCO3) and resuspended in staining buffer. Bones were flushed with medium, red blood cells lysed with RCL and pellets resuspended in staining buffer. Whole blood was lysed with RCL and pellets resuspended in staining buffer. Single cell suspensions were blocked with unconjugated CD16/32 antibody and stained with Fixable viability dye eFluor506 in staining buffer for 10 minutes protected from light at room temperature. Next cells were washed with stain buffer. For surface marker staining, cells were resuspended in staining buffer with the appropriate antibody dilutions for 30 minutes at 4°C protected from light. For the detection of cytosolic proteins, cells were fixed with room temperature IC fixation buffer for 15 minutes and permeabilized with eBioscience Permeabilization buffer (both from ThermoFisher Scientific). For the detection of phosphorylated cytosolic proteins, cells were fixed with room temperature IC fixation buffer for 15 minutes and permeabilized with ice-cold methanol for 20 minutes. For the detection of phosphorylated transcription factors, cells were prepared with the Transcription Factor Buffer set (BD Biosciences) according to the manufacturer's instructions. For the detection of BCL6 and TdT, cells were processed with the FoxP3 transcription factor staining buffer set (ThermoFisher Scientific) according to the manufacturer's protocol. Cells were washed thoroughly and resuspended in permeabilization buffer or staining buffer (only methanol-permeabilized cells) with the appropriate antibody dilutions for 60 minutes or over night at 4°C protected from light. In case of unconjugated primary antibodies, cells were stained with secondary antibodies for 60 minutes at 4°C protected from light. Data was analyzed with FlowJo v10.6 (Becton, Dickinson & Company Biosciences).
Post-sort purity was verified with a MACSQuant Vyb cytometer (Miltenyi) for cultured cells. The purity of cells sorted prior to BCL6 staining was done with the Fortessa X-20 (Becton, Dickinson & Company Biosciences). Purity of the sorted populations was > 95%.