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FYN–TRAF3IP2 induces NF-κB signaling-driven peripheral T-cell lymphoma

Nature Cancer volume 2, pages 98–113 (2021)Cite this article

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Abstract

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma not otherwise specified (PTCL, NOS) have poor prognosis and, in most cases, lack driver actionable targets for directed therapies. Here we identify FYN–TRAF3IP2 as a recurrent oncogenic gene fusion in AITL and PTCL, NOS tumors. Mechanistically, we show that FYN–TRAF3IP2 leads to aberrant NF-κB signaling downstream of T-cell antigen receptor activation. Consistent with a driver oncogenic role, FYN–TRAF3IP2 expression in hematopoietic progenitors induces NF-κB-driven T-cell transformation in mice and cooperates with loss of the Tet2 tumor suppressor in PTCL development. Moreover, abrogation of NF-κB signaling in FYN–TRAF3IP2-induced tumors with IκB kinase inhibitors delivers strong anti-lymphoma effects in vitro and in vivo. These results demonstrate an oncogenic and pharmacologically targetable role for FYN–TRAF3IP2 in PTCLs and call for the clinical testing of anti-NF-κB targeted therapies in these diseases.

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Fig. 1: Identification of the FYN–TRAF3IP2 gene fusion in PTCL.
Fig. 2: FYN–TRAF3IP2 drives NF-κB activation in response to TCR-induced PKC signaling.
Fig. 3: FYN–TRAF3IP2 signaling requires membrane localization.
Fig. 4: FYN–TRAF3IP2-induced NF-κB activation is mediated by TRAF6.
Fig. 5: Expression of FYN–TRAF3IP2 in mouse hematopoietic progenitors induces PTCL, NOS.
Fig. 6: Histopathologic and clonality features of FYN–TRAF3IP2-induced PTCL, NOS tumors.
Fig. 7: NF-κB activation in FYN–TRAF3IP2-induced mouse PTCL, NOS.
Fig. 8: Anti-lymphoma effects of NF-κB inhibition in FYN–TRAF3IP2-induced PTCLs.

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Data availability

DNA and RNA sequencing data that support the findings of this study have been deposited in the database of Genotypes and Phenotypes (dbGaP), (phs001962.v1.p1), and Gene Expression Omnibus (GEO), GSE138416. Previously published datasets that were reanalyzed during this study include RNA-seq data from individuals with AITL and PTCL, NOS in dbGaP (phs000689.v1.p1) and in the Sequence Read Archive (SRP029591), as well as RNA-seq data from mouse AITL with RhoA G17V in the GEO (GSE83918). We performed GSEA with gene sets available in the Molecular Signatures Database (MSigDB), https://www.gsea-msigdb.org/gsea/msigdb, and T. Gilmore’s NF-κB target database (Boston University; http://www.bu.edu/nf-kb/gene-resources/target-genes/). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank R. Levine (Memorial Sloan Kettering Cancer Center) for kindly providing the Tet2fl/fl mouse line and the Digital Computational Pathology Laboratory in the Department of Pathology and Cell Biology at Columbia University Irving Medical Center. This work was supported by St. Baldrick’s Foundation (A.A.F.); NIH grants P30 CA013696 (Confocal and Specialized Microscopy Shared Resource, Molecular Pathology Shared Resource, Oncology Precision Therapeutics and Imaging Core (OPTIC), Flow Cytometry Shared Resource, Genomics Shared Resource, Herbert Irving Comprehensive Cancer Center), R01 CA197945 (T.P.), R35 CA210065 (A.A.F.), R01 CA185486 (R.R.), R01 CA179044 (R.R.), U54 CA121852 (R.R.) and F30 CA225052 (C.S.M.); the Stewart Foundation (R.R.); Leukemia & Lymphoma Society grants TRP-6507-17 (T.P.), TRP-6163-12 (A.A.F.) and Special Fellow Award 3395-20 (S.A.); and Spanish Ministerio de Ciencia, Innovación y Universidades grant RTI2018-094274-B-I00 (E.C.). E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats (ICREA) of the Generalitat de Catalunya. J.R.C. is supported by a Lady Tata Memorial Trust fellowship.

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Author notes

  1. These authors contributed equally: Christine S. Moon, Clara Reglero, Jose R. Cortes.

Authors and Affiliations

  1. Institute for Cancer Genetics, Columbia University, New York, NY, USA

    Christine S. Moon, Clara Reglero, Jose R. Cortes, S. Aidan Quinn, Silvia Alvarez, Anisha J. Cooke, Claudia Fiñana, Teresa Palomero & Adolfo A. Ferrando

  2. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA

    S. Aidan Quinn, Wen-Hsuan W. Lin, Craig R. Soderquist, Govind Bhagat, Teresa Palomero & Adolfo A. Ferrando

  3. Department of Systems Biology, Columbia University, New York, NY, USA

    Junfei Zhao, Francesco Abate, Raul Rabadan & Adolfo A. Ferrando

  4. Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA

    Giorgio Inghirami

  5. Department of Pathology, Hospital Clinic of Barcelona, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain

    Elias Campo

  6. Department of Biomedical Informatics, Columbia University, New York, NY, USA

    Raul Rabadan

  7. Department of Pediatrics, Columbia University Medical Center, New York, NY, USA

    Adolfo A. Ferrando

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Contributions

C.S.M. performed molecular biology, cellular and animal experiments and wrote the manuscript. C.R. performed molecular biology and cellular experiments and wrote the manuscript. J.R.C. designed and contributed to mouse in vivo pharmacological experiments and wrote the manuscript. C.S.M., S.A.Q., J.Z. and F.A. analyzed RNA-seq and whole-genome sequencing data. A.J.C. contributed to mouse in vivo pharmacological experiments. C.F. and S.A. performed molecular biology and cellular experiments. W.-H.W.L., C.R.S. and G.B. provided histopathological analysis of mouse tumors and analyzed data. G.B, C.R.S, E.C. and G.I. contributed clinical samples. R.R. supervised clinical sample RNA-seq and whole-genome sequencing analyses. T.P. and A.A.F. designed the study, supervised the research and wrote the manuscript with C.S.M.

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Correspondence to Teresa Palomero or Adolfo A. Ferrando.

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Peer review information Nature Cancer thanks Kojo Elenitoba-Johnson, Daniel Krappmann and Tak W. Mak for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 FYN-TRAF3IP2 detection by RT-PCR and dideoxynucleotide sequencing.

a, Reverse-transcription PCR (RT-PCR) amplification results of an independent panel of 31 PTCL RNA samples spanning the region of FYN-TRAF3IP2 fusion breakpoint. b, DNA sequencing chromatograms of the RT-PCR amplicons generated in a.

Source data

Extended Data Fig. 2 RT-PCR analysis of FYN-TRAF3IP2 in B cell malignancies.

a, Reverse-transcription PCR (RT-PCR) amplification results of a panel of 92 B cell malignancies RNA samples spanning the region of FYN-TRAF3IP2 fusion breakpoint. + indicates a PTCL positive control sample, - indicates a PTCL negative control sample. No PCR products corresponding to the size of the specific FYN-TRAF3IP2 amplicon were detected. b, DNA sequencing chromatogram of the RT-PCR amplicons generated in a for the positive control PTCL sample and in sample 66 which showed a weak band slightly smaller than the specific FYN-TRAF3IP2 product. Lack of priming in this sample ruled out the presence of a specific FYN-TRAF3IP2 RT-PCR product in sample 66.

Source data

Extended Data Fig. 3 Expression and functional analysis of IL17 receptor and CARD 11 dependent signaling in T cells.

a, RNA expression level of IL17RC, essential for a functional IL17 receptor68, in T cells and monocytes represented from DICE (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics). b, NF-κB-GFP reporter activity in transduced Jurkat cells after stimulation with 200 ng/mL IL17A. Results are reported as mean of technical replicate values (bar) from 1 independent experiment with individual values (white circles). c, Immunoblot analysis of JPM50.6 CARD11 knockout and JPM50.6 cells reconstituted by CARD11-HA expression. Expression levels were verified for each independent experiment. d, NF-κB-GFP reporter activity after stimulation with 25 nM of PMA or 20 ng/mL TNFα as in c. e, Western blot analysis of FYN-TRAF3IP2-V5 and BCL10 expression in Jurkat cells and Jurkat BCL10 knockout cells infected with empty vector or FYN-TRAF3IP2-V5 expressing lentiviruses; and RT-PCR analysis of FYN-TRAF3IP2-V5 mRNA in the same Jurkat BCL10 knockout cells infected with empty vector or FYN-TRAF3IP2-V5 expressing lentiviruses. * and ** indicate non-specific bands detected by the V5 antibody, while the arrowhead indicates the location of the specific FYN-TRAF3IP2-V5 band detected with the same antibody. Expression levels were verified for each independent experiment.

Source data

Extended Data Fig. 4 Generation and characterization of FYN-TRAF3IP2-induced mouse PTCLs.

a. We transduced hematopoietic progenitors from CD4 Cre-ERT2 Tet2fl/fl mice with bicistronic retroviruses driving the expression of GFP, FYN-TRAF3IP2-V5 and GFP or wild type TRAF3IP2-V5 and GFP. We intravenously transplanted the infected cells into lethally irradiated C57BL/6 recipient mice and treated them 8 weeks post-transplant with vehicle only or with tamoxifen to preserve or delete Tet2 in CD4+ T cells, respectively. These cohorts of mice were then immunized with sheep red blood cells every 4-5 weeks to induce peripheral T cell activation. b, Representative histological micrographs of hematoxylin-eosin stained liver, lung, and kidney of Tet2fl/fl or Tet2-/- FYN-TRAF3IP2-induced lymphoma-bearing animals and the GFP only control. Tissues from 3 mice per group showed similar results. Scale bar = 200 μm. c, Representative FACS plots of mononuclear cells collected from liver, lung, and kidney of FYN-TRAF3IP2-induced lymphoma-bearing animals, showing GFP+ CD4+ cell infiltration. d, Quantitative analysis of lymphoma liver infiltration in FYN-TRAF3IP2 Tet2fl/fl and FYN-TRAF3IP2 Tet2-/- tumors. Results are reported as mean of values (bar) ± standard deviation (error bar) with individual values (white circles), n = 5 tumors from 5 different mice per condition. The P value was calculated using two-tailed Student’s t-test. e, Representative flow cytometry analyses of PD1, ICOS, BCL6 and CXCR5 Tfh cell marker expression in CD4+ GFP+ spleen tumor cells compared to CD4+ GFP− non-tumor cells from the same spleen. f, Flow cytometry analysis of PD1 and CXCR5 Tfh cell marker expression in CD4+ GFP− non-tumor cells and CD4+ GFP+ tumor cells from a representative FYN-TRAF3IP2-induced lymphoma-bearing spleen. g, Heatmap representation of Tfh-associated marker expression in CD4+ naïve wild type T cells, FYN-TRAF3IP2-induced CD4+ GFP+ lymphoma cells and RHOA G17V Tet2-/- AITL-like mouse tumor cells53. h, GSEA enrichment plots of differentially expressed genes associated with FYN-TRAF3IP2-induced mouse lymphoma cells compared to wild type naïve CD4+ T cells. AITL geneset: top differentially upregulated genes in AITL compared with PTCL, NOS (fold change 1.5, p < 0.002)38. Tfh geneset: top 100 genes associated with Tfh cells37.

Source data

Extended Data Fig. 5 Analysis of the Rhoa G17V mutation in FYN-TRAIF3I2-induced mouse PTCL samples.

DNA chromatograms corresponding to the sequence of Rhoa exon 3 in DNA from tumor CD4+ T cells sorted from five independent FYN-TRAIF3I2-induced lymphomas. Highlighted area indicates the sequence corresponding to Rhoa codon 17 which is wild type (G17) in every case. No mutations were detected in these analyses.

Extended Data Fig. 6 Characterization of FYN-TRAF3IP2-induced mouse PTCLs: transplantability and transcriptional profiling.

a, Kaplan-Meier survival curve of n = 9 mice transplanted with cell suspension containing FYN-TRAF3IP2 GFP+ lymphoma infiltrate. b, Spleens of secondary recipients transplanted with FYN-TRAF3IP2-induced lymphoma infiltrate. c, Representative histological micrographs of H&E stained spleen, liver, and lung of lymphoma transplanted mice at the endpoint showing lymphoma infiltration. Tissues from 3 mice showed similar results. Scale bar = 400 μm. d, Representative FACS plot showing GFP+ CD4+ lymphoma infiltrates in spleens from diseased secondary recipients. e, Representative flow cytometry analyses of PD1, ICOS and BCL6 Tfh cell marker expression in CD4+ GFP+ spleen tumor cells compared to CD4+ GFP− non-tumor cells from the same spleen. f, Tcrb gene clonal analysis of three secondary Tet2-/- FYN-TRAF3IP2 GFP+ lymphomas illustrated by side-by-side representation of Tcrb sequence reads from primary and secondary Tet2-/- FYN-TRAF3IP2 GFP+ lymphomas, indicating the retention and expansion of specific lymphoma clone with unique Tcrb rearrangements after transplantation. g, RNAseq data of six independent FYN-TRAF3IP2-induced mouse CD4+ GFP+ lymphomas and wild type isogenic mouse naïve CD4+ T cells represented as heat map of the unselected top 100 differentially expressed genes between FYN-TRAF3IP2-induced tumors and wild type CD4+ naïve T cells (mouse genes without known human orthologues excluded; scale bar shows color-coded differential expression, with red indicating higher levels of expression and blue indicating lower levels of expression). h, Volcano plot of all differentially expressed genes as in g (P value <0.001 and |log2(fold change)|> 1 deemed significant). Blue, significantly downregulated genes. Red, significantly upregulated genes. The number of significantly downregulated or upregulated genes is indicated. i, Principal component analysis plot of RNAseq data as in g. j-k, GSEA analyses of differentially expressed genes associated with FYN-TRAF3IP2-induced mouse lymphomas based on the RNAseq data and the Hallmark signatures69 from MSigDB. The top 6 upregulated signatures j and top 6 downregulated signatures k are represented as bar graphs of normalized enrichment scores and P values.

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Extended Data Fig. 7 IKK inhibitor treatment in normal T-cells.

a, Representative flow cytometry plot and quantification of the analysis of apoptosis in normal CD4+ T cells and CD4 FYN-TRAF3IP2 lymphoma cells treated with vehicle only (DMSO) or the IKK16 inhibitor. b, Representative flow cytometry plot and quantification of analysis of apoptosis in normal CD4+ T cells and CD4 FYN-TRAF3IP2 lymphoma cells treated with vehicle only (DMSO) or the BMS345541 IKK inhibitor. c, Representative flow cytometry plot and quantification of normal lymphocytes in spleen and lymph nodes of mice treated with vehicle only or with the BMS-345541 IKK inhibitor in vivo. Results are reported as mean of replicate values from n = 3 mice (bar) ± standard deviation (error bar) with individual values (black circles). P values were calculated using two-tailed Student’s t-test.

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Moon, C.S., Reglero, C., Cortes, J.R. et al. FYN–TRAF3IP2 induces NF-κB signaling-driven peripheral T-cell lymphoma. Nat Cancer 2, 98–113 (2021). https://doi.org/10.1038/s43018-020-00161-w

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