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Role of Dysregulated Cytokine Signaling and Bacterial Triggers in the Pathogenesis of Cutaneous T-Cell Lymphoma

  • Author Footnotes
    10 These authors contributed equally to this work.
    Melania H. Fanok
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA
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  • Author Footnotes
    10 These authors contributed equally to this work.
    Amy Sun
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA
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  • Laura K. Fogli
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA
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  • Vijay Narendran
    Affiliations
    Department of Medicine, Division of Hematology-Oncology, New York University School of Medicine, New York, New York, USA
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  • Miriam Eckstein
    Affiliations
    Department of Basic Science and Craniofacial Biology, NYU College of Dentistry, New York, New York, USA
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  • Kasthuri Kannan
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Office of Collaborative Science, New York University School of Medicine, New York, New York, USA
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  • Igor Dolgalev
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Office of Collaborative Science, New York University School of Medicine, New York, New York, USA
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  • Charalampos Lazaris
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Laura and Isaac Perlmutter Cancer Institute, New York University School of Medicine, New York, New York, USA
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  • Adriana Heguy
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Office of Collaborative Science, New York University School of Medicine, New York, New York, USA
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  • Mary E. Laird
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA
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  • Mark S. Sundrud
    Affiliations
    Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
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  • Cynthia Liu
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA
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  • Author Footnotes
    11 Current address: Biology and Translation Science, Infinity Pharmaceuticals, Incorporated, Cambridge, Massachusetts, USA
    Jeff Kutok
    Footnotes
    11 Current address: Biology and Translation Science, Infinity Pharmaceuticals, Incorporated, Cambridge, Massachusetts, USA
    Affiliations
    Department of Pathology, Brigham and Women’s Hospital; Boston, Massachusetts, USA
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  • Rodrigo S. Lacruz
    Affiliations
    Department of Basic Science and Craniofacial Biology, NYU College of Dentistry, New York, New York, USA
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  • Jo-Ann Latkowski
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA
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  • Iannis Aifantis
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Laura and Isaac Perlmutter Cancer Institute, New York University School of Medicine, New York, New York, USA
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  • Niels Ødum
    Affiliations
    Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
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  • Kenneth B. Hymes
    Affiliations
    Department of Medicine, Division of Hematology-Oncology, New York University School of Medicine, New York, New York, USA

    Department of Pathology, Brigham and Women’s Hospital; Boston, Massachusetts, USA
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  • Author Footnotes
    12 Current address: Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, Bronx, New York, USA
    Swati Goel
    Footnotes
    12 Current address: Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, Bronx, New York, USA
    Affiliations
    Department of Medicine, Division of Hematology-Oncology, New York University School of Medicine, New York, New York, USA
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  • Sergei B. Koralov
    Correspondence
    Correspondence: Sergei Koralov, 550 First Avenue, MSB 531, New York, New York, USA.
    Affiliations
    Department of Pathology, New York University School of Medicine, New York, New York, USA

    Laura and Isaac Perlmutter Cancer Institute, New York University School of Medicine, New York, New York, USA
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  • Author Footnotes
    10 These authors contributed equally to this work.
    11 Current address: Biology and Translation Science, Infinity Pharmaceuticals, Incorporated, Cambridge, Massachusetts, USA
    12 Current address: Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, Bronx, New York, USA
Open ArchivePublished:November 08, 2017DOI:https://doi.org/10.1016/j.jid.2017.10.028
      Cutaneous T-cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic, and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome showed a highly heterogeneous landscape of genetic perturbations, and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway previously implicated in cutaneous T-cell lymphoma pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of cutaneous T-cell lymphoma. Using this mouse model, we show that T-cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

      Abbreviations:

      CNV (copy number variation), CTCL (cutaneous T-cell lymphoma), MF (mycosis fungoides), SS (Sézary syndrome), TCR (T-cell receptor)

      Introduction

      Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of non-Hodgkin’s lymphomas characterized by the accumulation of malignant T lymphocytes in the skin (
      • Willemze R.
      WHO-EORTC classification for cutaneous lymphomas.
      ). CTCL patients typically present with erythematous, scaling skin patches, and plaques that can progress to tumors and widespread erythroderma (
      • Hwang S.T.
      • Janik J.E.
      • Jaffe E.S.
      • Wilson W.H.
      Mycosis fungoides and Sézary syndrome.
      ,
      • Jawed S.I.
      • Myskowski P.L.
      • Horwitz S.
      • Moskowitz A.
      • Querfeld C.
      Primary cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome): part I. Diagnosis: clinical and histopathologic features and new molecular and biologic markers.
      ). The most common variant of CTCL, mycosis fungoides (MF), is often indolent in its early stages and can be managed by topical agents or phototherapy. However, advanced stages of MF and the leukemic variant of the disease, Sézary syndrome (SS), have a more aggressive clinical course, prove difficult to treat, are debilitating, and have no cure.
      The age-adjusted incidence of CTCL is less than 10 cases per million people in the United States (
      • Jawed S.I.
      • Myskowski P.L.
      • Horwitz S.
      • Moskowitz A.
      • Querfeld C.
      Primary cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome): part I. Diagnosis: clinical and histopathologic features and new molecular and biologic markers.
      ). The rarity and heterogeneity of CTCL has made it difficult to understand the pathogenesis of this malignancy. Although several studies have investigated the genetic changes in CTCL, there is little consensus as to the molecular drivers of this disease. Array-based comparative genome hybridization and sequencing studies have shown that CTCL is genetically unstable, with various mutations and gains and losses of large parts of chromosomes (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ,
      • van Doorn R.
      • van Kester M.S.
      • Dijkman R.
      • Vermeer M.H.
      Oncogenomic analysis of mycosis fungoides reveals major differences with Sézary syndrome.
      ,
      • Vermeer M.H.
      • van Doorn R.
      • Dijkman R.
      • Mao X.
      • Whittaker S.
      • van Voorst Vader P.C.
      • et al.
      Novel and highly recurrent chromosomal alterations in Sezary syndrome.
      ). These aberrations have resulted in changes in cytokine signaling pathways and in the Rb, p53, and PTEN pathways (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ,
      • da Silva Almeida A.C.
      • Abate F.
      • Khiabanian H.
      • Martinez-Escala E.
      • Guitart J.
      • Tensen C.P.
      • et al.
      The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome.
      ,
      • Kiel M.J.
      • Sahasrabuddhe A.A.
      • Rolland D.C.M.
      • Velusamy T.
      • Chung F.
      • Schaller M.
      • et al.
      Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.
      ,
      • Lamprecht B.
      • Kreher S.
      • Möbs M.
      • Sterry W.
      • Dörken B.
      • Janz M.
      • et al.
      The tumour suppressor p53 is frequently nonfunctional in Sézary syndrome.
      ,
      • McGirt L.Y.
      • Jia P.
      • Baerenwald D.A.
      • Duszynski R.J.
      • Dahlman K.B.
      • Zic J.A.
      • et al.
      Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides.
      ,
      • Vermeer M.H.
      • van Doorn R.
      • Dijkman R.
      • Mao X.
      • Whittaker S.
      • van Voorst Vader P.C.
      • et al.
      Novel and highly recurrent chromosomal alterations in Sezary syndrome.
      ,
      • Wang L.
      • Ni X.
      • Covington K.R.
      • Yang B.Y.
      • Shiu J.
      • Zhang X.
      • et al.
      Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes.
      ,
      • Wong H.K.
      • Mishra A.
      • Hake T.
      • Porcu P.
      Evolving insights in the pathogenesis and therapy of cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome).
      ). Changes in genes within cell survival pathways, the NF-κB pathway, and those involved with chromatin remodeling and DNA damage response have also been observed (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ,
      • Kiel M.J.
      • Sahasrabuddhe A.A.
      • Rolland D.C.M.
      • Velusamy T.
      • Chung F.
      • Schaller M.
      • et al.
      Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.
      ). However, the contribution of the individual genetic perturbations to disease pathogenesis remains unclear.
      The characteristic skin homing of malignant T cells in CTCL is notable, in part, because as a barrier surface, the epithelium is host to antigens that can trigger inflammatory responses contributing to malignant transformation. T cells in MF patients typically show characteristic signs of chronic antigenic stimulation (
      • Girardi M.
      • Heald P.W.
      • Wilson L.D.
      The pathogenesis of mycosis fungoides.
      ). Coupled with notable increases in antigen-presenting cells in patient skin (
      • Pigozzi B.
      • Bordignon M.
      • Belloni Fortina A.
      • Michelotto G.
      • Alaibac M.
      Expression of the CD1a molecule in B- and T-lymphoproliferative skin conditions.
      ), evidence indicated a role for T-cell receptor (TCR) signaling in the etiology of this disease. Several studies showing that the incidence of CTCL is higher among certain professions further highlight the fact that exposure to specific environmental antigens may be a contributing factor (
      • Morales-Suárez-Varela M.M.
      • Olsen J.
      • Johansen P.
      • Kaerlev L.
      • Guénel P.
      • Arveux P.
      • et al.
      Occupational exposures and mycosis fungoides. A European multicentre case–control study (Europe).
      ).
      Commensal microbiota represent a primary source of antigenic exposure on the skin, and a role for the skin-resident commensal Staphylococcus epidermis in tuning the inflammatory milieu within this tissue has been established (
      • Naik S.
      • Bouladoux N.
      • Wilhelm C.
      • Molloy M.J.
      • Salcedo R.
      • Kastenmuller W.
      • et al.
      Compartmentalized control of skin immunity by resident commensals.
      ). Although S. epidermis promotes protective immunity at the skin barrier surface via enhancing T helper (Th) 17 differentiation, microbial dysbiosis has been shown to play a role in the initiation and progression of cancer in other contexts (
      • Hu B.
      • Elinav E.
      • Huber S.
      Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer.
      ). In addition to commensal microbes, pathogenic infections have also been implicated in malignancy (
      • Belkaid Y.
      • Hand T.W.
      Role of the microbiota in immunity and inflammation.
      ,
      • Elinav E.
      • Nowarski R.
      • Thaiss C.A.
      • Hu B.
      • Jin C.
      • Flavell R.A.
      Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms.
      ,
      • Polk D.B.
      • Peek R.M.
      Helicobacter pylori: gastric cancer and beyond.
      ). In line with this, CTCL patients frequently present with bacterial infections, particularly with Staphylococcus aureus, and antibiotic treatment to eliminate S. aureus often produces notable clinical improvements (
      • Jackow C.M.
      • Cather J.C.
      • Hearne V.
      • Asano A.T.
      • Musser J.M.
      • Duvic M.
      Association of erythrodermic cutaneous T-cell lymphoma, superantigen-positive Staphylococcus aureus, and oligoclonal T-cell receptor V beta gene expansion.
      ,
      • Nguyen V.
      • Huggins R.H.
      • Lertsburapa T.
      • Bauer K.
      • Rademaker A.
      • Gerami P.
      • et al.
      Cutaneous T-cell lymphoma and Staphylococcus aureus colonization.
      ,
      • Tokura Y.
      • Yagi H.
      • Ohshima A.
      • Kurokawa S.
      • Wakita H.
      • Yokote R.
      • et al.
      Cutaneous colonization with staphylococci influences the disease activity of Sézary syndrome: a potential role for bacterial superantigens.
      ). Although a link between microorganisms and CTCL initiation and/or progression has been noted (
      • Axelrod P.I.
      • Lorber B.
      • Vonderheid E.C.
      Infections complicating mycosis fungoides and Sézary syndrome.
      ,
      • Jackow C.M.
      • Cather J.C.
      • Hearne V.
      • Asano A.T.
      • Musser J.M.
      • Duvic M.
      Association of erythrodermic cutaneous T-cell lymphoma, superantigen-positive Staphylococcus aureus, and oligoclonal T-cell receptor V beta gene expansion.
      ,
      • Willerslev-Olsen A.
      • Krejsgaard T.
      • Lindahl L.
      • Bonefeld C.
      • Wasik M.
      • Koralov S.
      • et al.
      Bacterial toxins fuel disease progression in cutaneous T-cell lymphoma.
      ), establishing a causative relationship between skin-resident and pathogenic bacteria and disease progression is nearly impossible in the absence of a reliable animal model of this malignancy.
      In this study, whole-exome sequencing of a cohort of SS patients showed a heterogeneous spread of genetic alterations that converged on several oncogenic pathways, including PI3K signaling and the STAT3 pathway. Critically, gene set enrichment analysis of high-throughput RNA sequencing data from malignant T cells showed a distinct CTCL transcriptional signature validated with previously published transcriptome data. Using conditional gene targeting to express a hyperactive allele of STAT3 selectively in T lymphocytes, we generated an animal model of CTCL that recapitulates many of the key features of human disease. Generation of this model shows the causative role of deregulated STAT3 signaling in CTCL pathogenesis. Furthermore, by using this model we also show that antigenic signaling and the presence of microbiota are necessary for CTCL progression, establishing a preclinical model for evaluation of therapeutic strategies for CTCL.

      Results

      High-throughput sequencing of malignant cells highlights the genetic heterogeneity of SS

      To gain insight into the molecular etiology of CTCL, we isolated T lymphocytes from the blood of SS patients. Transformed T lymphocytes were isolated at high purity based on the characteristic expression of CD7 and CD26 (see Supplementary Figure S1 online). Despite a large number of mutations found in malignant T cells, few genes were mutated in more than one patient, and no genes were mutated in three or more patients, emphasizing the molecular heterogeneity of this disease (Figure 1a). In contrast, copy number variation (CNV) analysis of exomes showed notable recurrent losses and gains of whole sections of chromosomes (Figure 1b). Patients with a larger burden of circulating tumor cells tended to have more gross CNVs, indicating an escalation in chromosomal instability with disease progression. The most common chromosomal changes include the loss of 17p, gain of 17q, loss of parts of chromosome 10, and gain of 8q, with most of these changes previously observed (
      • Cristofoletti C.
      • Picchio M.C.
      • Lazzeri C.
      • Tocco V.
      • Pagani E.
      • Bresin A.
      • et al.
      Comprehensive analysis of PTEN status in Sezary syndrome.
      ,
      • Fischer T.C.
      • Gellrich S.
      • Muche J.M.
      • Sherev T.
      • Audring H.
      • Neitzel H.
      • et al.
      Genomic aberrations and survival in cutaneous T cell lymphomas.
      ,
      • Kiel M.J.
      • Sahasrabuddhe A.A.
      • Rolland D.C.M.
      • Velusamy T.
      • Chung F.
      • Schaller M.
      • et al.
      Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.
      ,
      • Lamprecht B.
      • Kreher S.
      • Möbs M.
      • Sterry W.
      • Dörken B.
      • Janz M.
      • et al.
      The tumour suppressor p53 is frequently nonfunctional in Sézary syndrome.
      ,
      • Vermeer M.H.
      • van Doorn R.
      • Dijkman R.
      • Mao X.
      • Whittaker S.
      • van Voorst Vader P.C.
      • et al.
      Novel and highly recurrent chromosomal alterations in Sezary syndrome.
      ). Notable genes lost in 17p and chromosome 10 include the tumor suppressors TP53 and PTEN, respectively, and gene copy numbers gained include STAT3 and STAT5 in 17q and MYC in 8q, all well established as associated with lymphomagenesis (Figure 1b). Copy number amplifications of STAT3 and MYC and loss of PTEN and TP53 were verified using digital droplet PCR (see Supplementary Figure S2 online). The observed diverse genetic aberrations converged on common cancer-associated signaling pathways in SS, with many CNVs associated with the ERK/MAPK, NF-κB, PI3K-AKT, TP53, and STAT3 signaling pathways (see Supplementary Figure S3 online).
      Figure 1
      Figure 1Genetic landscape of CTCL. (a) Circos plot of SNVs identified by WES. Mutations were identified by comparing the tumor cells with the patient’s B cells. Individual chromosomes are marked on the outer ring. Concentric circles represent patient genomes. Black pips indicate deleterious SNVs filtered for coverage by more than 14 reads and predicted deleteriousness score of greater than 0.5 by PolyPhen-2 (
      • Adzhubei I.A.
      • Schmidt S.
      • Peshkin L.
      • Ramensky V.E.
      • Gerasimova A.
      • Bork P.
      • et al.
      A method and server for predicting damaging missense mutations.
      ). Eight patient biospecimens are ordered from outside-in by decreasing tumor burden. (b) Copy number gains (red) and losses (blue) detected in SS genomes. Green indicates normal copy number. Each row represents analysis from an individual patient, with samples ordered by decreasing tumor burden. (c) PCA plot (top) and silhouette analysis (bottom) of RNA-sequencing data from seven SS samples and T naïve and T memory cells from healthy individuals. (d) Heat map displaying differential gene expression of malignant T cells (tumor) to memory T cells (memCD4). Genes displayed have Q-value ≤ 0.05. (e) GSEA performed on published transcriptome of sorted T cells from SS patients and healthy individuals using our CTCL gene signature. CTCL, cutaneous T-cell lymphoma; GSEA, gene set enrichment analysis; FWER, family-wise error rate; PCA, principal component analysis; SNV, single nucleotide variation; SS, Sézary syndrome; Tmem, memory T cell; WES, whole-exome sequencing.

      Transcriptome analysis shows a distinct CTCL gene expression signature

      We next examined the gene expression profile of sorted T cells from patient tumor samples compared with naïve and memory T cells from healthy individuals. Principal component analysis showed that although the memory and naïve T-cell populations from healthy individuals clustered with their respective cell types, tumor samples were widely spread across the principal component analysis plot, highlighting the molecular heterogeneity of this disease (Figure 1c). Silhouette analysis performed to measure the degree of similarity between clusters underscored the heterogeneity of the tumor cluster (Figure 1c). Both the MF and SS forms of CTCL are thought to arise from malignant transformation of memory T lymphocytes, and indeed this is consistent with our principal component analysis, because the malignant cells are closer to memory T cells along PC1 than they are to naïve T lymphocytes. Analysis of differential gene expression between the transformed T cells and memory T cells yielded a gene expression signature, which we trimmed down to 124 genes up-regulated in SS cells, based on Q-value (≤0.05) and fold difference in expression (>4) (Figure 1d, and see Supplementary Table S1 online). To validate the robustness of this signature, we performed gene set enrichment analysis (
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • et al.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      ) on a previously published transcriptome of SS cells from 32 patients and sorted T lymphocytes from healthy individuals (
      • Wang L.
      • Ni X.
      • Covington K.R.
      • Yang B.Y.
      • Shiu J.
      • Zhang X.
      • et al.
      Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes.
      ). Our CTCL signature was dramatically enriched in the malignant T cells from the published dataset, despite the differences in cell isolation, library preparation, and sequencing methodology (Figure 1e, and see Supplementary Figure S4 online), highlighting the potential of using this gene expression signature for diagnosis.

      STAT3 inhibition results in decreased cell proliferation and survival in CTCL cell lines

      Although our analysis of the genetic landscape, along with other recently published whole-exome sequencing studies (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ,
      • da Silva Almeida A.C.
      • Abate F.
      • Khiabanian H.
      • Martinez-Escala E.
      • Guitart J.
      • Tensen C.P.
      • et al.
      The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome.
      ,
      • Kiel M.J.
      • Sahasrabuddhe A.A.
      • Rolland D.C.M.
      • Velusamy T.
      • Chung F.
      • Schaller M.
      • et al.
      Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.
      ,
      • McGirt L.Y.
      • Jia P.
      • Baerenwald D.A.
      • Duszynski R.J.
      • Dahlman K.B.
      • Zic J.A.
      • et al.
      Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides.
      ,
      • Wang L.
      • Ni X.
      • Covington K.R.
      • Yang B.Y.
      • Shiu J.
      • Zhang X.
      • et al.
      Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes.
      ), highlighted the molecular heterogeneity of CTCL, one pathway that is consistently up-regulated in this disease is the STAT3 cytokine signaling pathway. Constitutive activation of STAT3 is an omnipresent feature of cell lines established from CTCL patients (
      • Krejsgaard T.O.R.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3-Stat3 signaling pathway.
      ,
      • Netchiporouk E.
      • Litvinov I.V.
      • Moreau L.
      • Gilbert M.
      • Sasseville D.
      • Duvic M.
      Deregulation in STAT signaling is important for cutaneous T-cell lymphoma (CTCL) pathogenesis and cancer progression.
      ,
      • Sommer V.H.
      • Clemmensen O.J.
      • Nielsen O.
      • Wasik M.
      • Lovato P.
      • Brender C.
      • et al.
      In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.
      ). Many cytokines can trigger STAT3 phosphorylation, and the subsequent activation of this signaling pathway contributes to the regulation of genes important in survival and proliferation. Our analysis of CNVs showed that STAT3 duplications are observed in nearly half of the patients (Figure 1b, and see Supplementary Figure S2), with CNVs and single nucleotide variations also observed in phosphatases and kinases known to regulate STAT3 activity (see Supplementary Figure S3). These results are consistent with reports of cytogenetic amplifications of STAT3 from other genome-wide studies of CTCL (
      • da Silva Almeida A.C.
      • Abate F.
      • Khiabanian H.
      • Martinez-Escala E.
      • Guitart J.
      • Tensen C.P.
      • et al.
      The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome.
      ,
      • Woollard W.J.
      • Pullabhatla V.
      • Lorenc A.
      • Patel V.M.
      • Butler R.M.
      • Bayega A.
      • et al.
      Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome.
      ). Two recent studies further emphasize the potential role of STAT3 in CTCL pathogenesis by identifying rare somatic mutations in the STAT3 SH2 domain, responsible for mediating dimerization, in malignant T lymphocytes from CTCL patients (
      • da Silva Almeida A.C.
      • Abate F.
      • Khiabanian H.
      • Martinez-Escala E.
      • Guitart J.
      • Tensen C.P.
      • et al.
      The mutational landscape of cutaneous T cell lymphoma and Sézary syndrome.
      ,
      • Kiel M.J.
      • Sahasrabuddhe A.A.
      • Rolland D.C.M.
      • Velusamy T.
      • Chung F.
      • Schaller M.
      • et al.
      Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.
      ). To test the reliance of CTCL cells on STAT3 activity, we treated CTCL cell lines with STA-21, a selective STAT3 inhibitor (
      • Song H.
      • Wang R.
      • Wang S.
      • Lin J.
      A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells.
      ). The Sez4 cell line is derived from the blood of an SS patient (
      • Abrams J.T.
      • Lessin S.
      • Ghosh S.K.
      • Ju W.
      A clonal CD4-positive T-cell line established from the blood of a patient with Sézary syndrome.
      ), and the MyLa 2059 line is derived from a plaque biopsy sample of an MF patient (
      • Kaltoft K.
      • Bisballe S.
      • Dyrberg T.
      • Boel E.
      • Rasmussen P.B.
      • Thestrup-Pedersen K.
      Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.
      ). The two patient-derived cell lines that we tested showed decreased cell number and increased cell death after STAT3 inhibition (Figure 2). These results are consistent with previous studies that have shown apoptosis of CTCL cell lines after inhibition of STAT3 activity by small interfering RNA or the dominant negative form of STAT3 and together highlight the importance of this signaling pathway for survival of the malignant cells (
      • Krejsgaard T.O.R.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3-Stat3 signaling pathway.
      ,
      • Sommer V.H.
      • Clemmensen O.J.
      • Nielsen O.
      • Wasik M.
      • Lovato P.
      • Brender C.
      • et al.
      In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.
      ,
      • Verma N.K.
      • Davies A.M.
      • Long A.
      • Kelleher D.
      • Volkov Y.
      STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via downregulation of Bcl-xL.
      ).
      Figure 2
      Figure 2STAT3 regulates cell survival in CTCL. Cultured Sez4 or MyLa cells were treated with 80 μmol/L STA-21 or DMSO. Total cell number and percentage of dead cells using trypan blue staining were determined at set time points. n = 3 for all conditions. Three independent experiments with multiple technical duplicates. Two-way analysis of variance with Sidak multiple comparison posttest. ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001. Values are shown as mean ± standard error of the mean. CTCL, cutaneous T-cell lymphoma.

      An autochthonous mouse model of CTCL pathogenesis shows augmented Th17 differentiation and T-cell transcriptional signature mirroring human CTCL

      To further investigate the role of STAT3 in malignant transformation of T lymphocytes in which a hyperactive version of STAT3, STAT3C (
      • Bromberg J.F.
      • Wrzeszczynska M.H.
      • Devgan G.
      • Zhao Y.
      • Pestell R.G.
      • Albanese C.
      • et al.
      Stat3 as an oncogene.
      ) was knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3Cstopfl). Excision of the stop cassette mediated by Cre recombinase leads to expression of a flag-tagged STAT3C and concomitant expression of EGFP from the IRES-GFP cassette (
      • Casola S.
      • Cattoretti G.
      • Uyttersprot N.
      • Koralov S.B.
      • Seagal J.
      • Segal J.
      • et al.
      Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting.
      ). Analysis of thymocytes from young R26STAT3Cstopfl/+CD4Cre mice and littermate controls showed no notable differences in thymic T-cell development (see Supplementary Figure S5 online). However, with age, R26STAT3Cstopfl/+CD4Cre mice developed a lymphoproliferative-like disease with characteristic hair loss and scaly skin plaques (Figure 3a). The skin phenotype of the mice progressed with age and was scored based on severity of disease. The mice on the lower end of the scale displayed dry skin and a range of hair loss, and on the more severe end of the scale animals developed obvious sores and lesions (see Methods section for disease scale). By 8 months, most mice displayed a visible skin phenotype (Figure 3b) with rare atypical lymphocytes with irregular, cerebriform nuclear appearance reminiscent of SS cells present in some blood smears from older mice (see Supplementary Figure S6 online).
      Figure 3
      Figure 3R26STAT3Cstopfl/+CD4Cre mice develop a skin phenotype highly reminiscent of CTCL. (a) Representative image of 6-month-old R26STAT3Cstopfl/+CD4Cre and littermate control mice. (b) Phenotype progression in R26STAT3Cstopfl/+ CD4Cre mice. Darker shading indicates more severe phenotype. Scale ranges from 0 (no phenotype) to 5 (moribund condition). See for a full description. n = 132 mice. (c) Skin sections from approximately 10-month-old control and R26STAT3Cstopfl/ CD4Cre mice, with a cluster of T cells, reminiscent of Pautrier microabscess in the knock-in animal. Scale bar = 50 μm. (d) Fold difference of CD3+CD4+ T cells isolated from skin of R26STAT3Cstopfl/+CD4Cre and age-matched control mice. Mean ± standard error of the mean from 30 independent experiments, n ≥ 32 for each genotype. P-value was determined using a Wilcoxon signed rank test. (e) Number of CD3+CD4+ T cells from peripheral lymph nodes of control and R26STAT3Cstopfl/+CD4Cre mice. Mean ± SEM from 24 independent experiments, n ≥ 29 for each genotype. Significance assessed using the nonparametric two-tailed Mann-Whitney U test. (f, left) Representative Ki67 staining of CD3+CD4+ T cells from peripheral lymph nodes of control and R26STAT3Cstopfl/+CD4Cre mice. (f, right) Quantification of Ki67+CD3+CD4+ cells. Data are from 14 independent experiments. n ≥ 16 for each genotype. For d–f, significance values are as follows: ns, P > 0.05; P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001. CTCL, cutaneous T-cell lymphoma.
      Immunofluorescent staining for CD3 in skin sections from older mice highlighted clustering of T cells reminiscent of Pautrier microabscesses at affected sites (Figure 3c, and see Supplementary Figure S7a and b online), a pathognomonic clinical feature of CTCL (
      • Jawed S.I.
      • Myskowski P.L.
      • Horwitz S.
      • Moskowitz A.
      • Querfeld C.
      Primary cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome): part I. Diagnosis: clinical and histopathologic features and new molecular and biologic markers.
      ). Additionally, acanthosis, hyperkeratosis, and parakeratosis reminiscent of CTCL (
      • Shapiro P.E.
      • Pinto F.J.
      The histologic spectrum of mycosis fungoides/Sézary syndrome (cutaneous T-cell lymphoma). A review of 222 biopsies, including newly described patterns and the earliest pathologic changes.
      ) accompanied T-cell accumulation and proliferation in the skin (see Supplementary Figure S7). Supporting these observations, flow cytometry of the skin showed a nearly 10-fold increase in CD4+ T-cell number in R26STAT3Cstopfl/+CD4Cre mice compared with control animals (Figure 3d). Deep sequencing of the T-cell repertoire suggested that the disease remained polyclonal (see Supplementary Figure S8 online), consistent with early MF (
      • Whittaker S.J.
      • Smith N.P.
      • Jones R.R.
      • Luzzatto L.
      Analysis of beta, gamma, and delta T-cell receptor genes in mycosis fungoides and Sézary syndrome.
      ), since advanced CTCL often shows expansion of a clonal subpopulation (
      • Kirsch I.R.
      • Watanabe R.
      • O'Malley J.T.
      • Williamson D.W.
      • Scott L.-L.
      • Elco C.P.
      • et al.
      TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
      ).
      Along with the augmented number of T cells in the skin, enlarged lymph nodes of R26STAT3Cstopfl/+CD4Cre mice had dramatically increased numbers of CD4+ T cells (Figure 3e). T cells isolated from secondary lymphoid organs of R26STAT3Cstopfl/+CD4Cre mice exhibited an increase in activated/memory CD4+ T cells (CD44hiCD62Llo) (see Supplementary Figure S9a and b online) and had a greater percentage of proliferating CD4+ T cells as indicated by positive staining for the Ki67 antigen (Figure 3f). Flow cytometry analysis of cytokine expression in CD4+ T cells from the skin of R26STAT3Cstopfl/+ CD4Cre mice showed a dramatic increase in IL-17– and IL-22–producing T cells compared with control animals (Figure 4a and b ). This is consistent with observations of increased IL-17A production from skin biopsy samples of MF patients (
      • Cirée A.
      • Michel L.
      • Camilleri-Bröet S.
      • Jean Louis F.
      • Oster M.
      • Flageul B.
      • et al.
      Expression and activity of IL-17 in cutaneous T-cell lymphomas (mycosis fungoides and Sézary syndrome).
      ). This trend was also observed in T cells isolated from peripheral lymph nodes (Figure 4a and b). The frequency of IFN-γ–producing cells in the skin was not significantly different; however, we observed a lower frequency of IL-4–producing cells in the skin of mutant mice (see Supplementary Figure S9c and d). Consistent with the previously reported finding that STAT3 directly promoted RORγt transcription (
      • Yang X.O.
      • Pappu B.P.
      • Nurieva R.
      • Akimzhanov A.
      • Kang H.S.
      • Chung Y.
      • et al.
      T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ.
      ), we observed a greater number of RORγt+IL-17 and RORγt+IL-17+ cells in the skin and lymph nodes of R26STAT3Cstopfl/+CD4Cre compared with littermate controls (see Supplementary Figure S9e and f). Indeed, up-regulation of STAT3 in malignant T cells of CTCL patients may explain the high frequency of Th17 cells often observed in this malignancy (
      • Krejsgaard T.O.R.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3-Stat3 signaling pathway.
      ,
      • Krejsgaard T.
      • Litvinov I.V.
      • Wang Y.
      • Xia L.
      • Willerslev-Olsen A.
      • Koralov S.B.
      • et al.
      Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma.
      ) and may contribute to the inflammatory skin microenvironment in this disease.
      Figure 4
      Figure 4Expanded population of Th17 cells in R26STAT3Cstopfl/+CD4Cre mice have a distinct CTCL transcriptional signature. (a) Representative intracellular flow cytometry analysis of CD3+CD4+ T cells from the skin and peripheral lymph nodes of approximately 10-month-old R26STAT3Cstopfl/+CD4Cre and control mice (b, top) Quantification of cytokine production from CD3+CD4+ T cells isolated from skin of R26STAT3Cstopfl/+CD4Cre and control animals for 23 independent experiments. n ≥ 25 for each genotype. (b, bottom) Th17 cytokine production in CD4+ T cells from peripheral lymph nodes for 25 or more independent experiments. n ≥ 28 for each genotype. Statistical significance was assessed using the nonparametric two-tailed Mann-Whitney U test. Significance values are as follows: ns, P > 0.05; P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001. Values are shown as mean ± standard error of the mean. (c) GSEA comparing the transcriptional profile of T cells from the skin of R26STAT3Cstopfl/+CD4Cre mice versus controls using the established human CTCL gene signature. CTCL, cutaneous T-cell lymphoma; FWER, family-wise error rate; GSEA, gene set enrichment analysis; Th, T helper.
      To further assess the relevancy of our mouse model to human disease, we examined the transcriptional profile of CD4+ T cells isolated from the skin of R26STAT3Cstopfl/+CD4Cre mice. As shown in Figure 4c, gene set enrichment analysis reflected that malignant T cells from the skin of R26STAT3Cstopfl/+ CD4Cre mice had a distinct, altered transcriptional pattern compared with CD4+ T cells sorted from the skin of control animals and that our previously characterized (see Supplementary Table S1) human CTCL gene expression signature was readily identifiable in the lymphocytes from mutant mice.

      Disease progression in a CTCL mouse model is dependent on TCR signaling and the presence of microbiota

      Cutaneous lymphomas are unique in that the malignant cells localize to surfaces, where environmental exposure in the form of pathogens or irritants may contribute to CTCL pathogenesis. Previous epidemiological studies have noted geographical clustering of this malignancy (
      • Ghazawi F.M.
      • Netchiporouk E.
      • Rahme E.
      • Tsang M.
      • Moreau L.
      • Glassman S.
      • et al.
      Comprehensive analysis of cutaneous T-cell lymphoma (CTCL) incidence and mortality in Canada reveals changing trends and geographic clustering for this malignancy.
      ,
      • Litvinov I.V.
      • Tetzlaff M.T.
      • Rahme E.
      • Habel Y.
      • Risser D.R.
      • Gangar P.
      • et al.
      Identification of geographic clustering and regions spared by cutaneous T-cell lymphoma in Texas using 2 distinct cancer registries.
      ,
      • Moreau J.F.
      • Buchanich J.M.
      • Geskin J.Z.
      • Akilov O.E.
      • Geskin L.J.
      Non-random geographic distribution of patients with cutaneous T-cell lymphoma in the greater Pittsburgh area.
      ), suggesting an environmental trigger in disease initiation, and a number of studies have implicated microbial contribution to CTCL initiation and progression (
      • Axelrod P.I.
      • Lorber B.
      • Vonderheid E.C.
      Infections complicating mycosis fungoides and Sézary syndrome.
      ,
      • Jackow C.M.
      • Cather J.C.
      • Hearne V.
      • Asano A.T.
      • Musser J.M.
      • Duvic M.
      Association of erythrodermic cutaneous T-cell lymphoma, superantigen-positive Staphylococcus aureus, and oligoclonal T-cell receptor V beta gene expansion.
      ,
      • Tokura Y.
      • Yagi H.
      • Ohshima A.
      • Kurokawa S.
      • Wakita H.
      • Yokote R.
      • et al.
      Cutaneous colonization with staphylococci influences the disease activity of Sézary syndrome: a potential role for bacterial superantigens.
      ). In particular, S. aureus is reported to be found on affected skin of 44–63% of CTCL patients across two prior studies (
      • Nguyen V.
      • Huggins R.H.
      • Lertsburapa T.
      • Bauer K.
      • Rademaker A.
      • Gerami P.
      • et al.
      Cutaneous T-cell lymphoma and Staphylococcus aureus colonization.
      ,
      • Talpur R.
      • Bassett R.
      • Duvic M.
      Prevalence and treatment of Staphylococcus aureus colonization in patients with mycosis fungoides and Sézary syndrome.
      ). Persistent activation of T cells via the antigen receptor by bacterial antigens and/or superantigens may contribute to CTCL pathogenesis. To examine if TCR engagement is critical for CTCL pathogenesis, we crossed the R26STAT3Cstopfl/+CD4Cre mice onto an OTII Rag2 knockout background to restrict the TCR repertoire. The OTII transgene encodes the TCRα and TCRβ chains of the T-cell antigen receptor specific for the chicken ovalbumin peptide, and the absence of the Rag2 enzyme ensures that no other TCR specificities are present in these animals. Analysis of R26STAT3Cstopfl/+ CD4Cre OTII Rag2KO mice showed no expansion of T cells in the skin and, in contrast to older R26STAT3Cstopfl/+CD4Cre littermates, these animals failed to develop any signs of clinical disease (Figure 5a).
      Figure 5
      Figure 5Disease symptoms of R26STAT3Cstopfl/+CD4Cre mice are ameliorated in TCR-limited and germ-free settings. (a) Average phenotype score of R26STAT3Cstopfl/+CD4Cre (red line), R26STAT3Cstopfl/+CD4Cre Rag2KO OTII (blue line), and control mice (black line). Scale ranges from 0 (no phenotype) to 5 (moribund condition). See for a full description. Results are mean ± standard error of the mean. (b) Average phenotype score of R26STAT3Cstopfl/+CD4Cre and control animals housed under specific pathogen-free (SPF) or germ-free (GF) conditions. Results are mean ± standard error of the mean. (c) Evaluation of pruritus over time normalized to average of control mice at each time point. n = 3 per genotype aged less than 5 months; n = 5 mice for each genotype above 5 months. See for details of video monitoring protocol. Two-way analysis of variance with Bonferroni posttest. P ≤ 0.05, ∗∗∗∗P ≤ 0.0001. KO, knockout; TCR, T-cell receptor.
      Given the often-noted bias in TCR Vβ repertoire in patient biospecimens (
      • Linnemann T.
      • Gellrich S.
      • Lukowsky A.
      • Mielke A.
      • Audring H.
      • Sterry W.
      • et al.
      Polyclonal expansion of T cells with the TCR Vbeta type of the tumour cell in lesions of cutaneous T-cell lymphoma: evidence for possible superantigen involvement.
      ) and the vast clinical experience with CTCL that suggests that worsening of symptoms is often associated with bacterial sepsis, with improvement of disease parameters after antibiotic therapy (
      • Talpur R.
      • Bassett R.
      • Duvic M.
      Prevalence and treatment of Staphylococcus aureus colonization in patients with mycosis fungoides and Sézary syndrome.
      ,
      • Tokura Y.
      • Yagi H.
      • Ohshima A.
      • Kurokawa S.
      • Wakita H.
      • Yokote R.
      • et al.
      Cutaneous colonization with staphylococci influences the disease activity of Sézary syndrome: a potential role for bacterial superantigens.
      ), we sought to examine the contribution of microbiota to disease initiation and progression in the R26STAT3Cstopfl/+CD4Cre model. We rederived our CTCL mouse model into germ-free isolators via hysterectomy and cross-fostering to generate mice that we confirmed to be free of bacteria, viruses, and fungi via culture, quantitative PCR, and sequencing-based approaches. Remarkably, we observed that although the clinical signs of disease started at the same age in germ-free and in conventionally housed animals (under specific pathogen-free conditions), the disease in germ-free animals never progressed to fulminant malignant disease (Figure 5b) observed in the specific pathogen-free animals. The course of disease was readily restored once the R26STAT3Cstopfl/+CD4Cre animals were cohoused with specific pathogen-free animals (Figure 5b).
      The incendiary role of skin-resident bacteria and the antigens they produce in CTCL pathogenesis is supported by the fact that S. aureus colonized the skin of CTCL patients at a higher rate than the general population (
      • Talpur R.
      • Bassett R.
      • Duvic M.
      Prevalence and treatment of Staphylococcus aureus colonization in patients with mycosis fungoides and Sézary syndrome.
      ) and by the association between S. aureus sepsis or colonization and CTCL progression (
      • Krejsgaard T.
      • Willerslev-Olsen A.
      • Lindahl L.M.
      • Bonefeld C.M.
      • Koralov S.B.
      • Geisler C.
      • et al.
      Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation.
      ). S. aureus and other skin-associated opportunistic infections may contribute to disease progression through stimulation of T cells or via induction of cytokine production in bystander cells, thereby contributing to the tumor microenvironment. We observed that much like MF patients, R26STAT3Cstopfl/+CD4Cre animals present with notable pruritus (
      • Ahern K.
      • Gilmore E.S.
      • Poligone B.
      Pruritus in cutaneous T-cell lymphoma: a review.
      ) (Figure 5c), and the persistent scratching is likely to result in introduction of opportunistic infections. Given these observations, it is worth considering whether aggressive treatment of pruritus and prevention of S. aureus colonization in patients with early stage MF would reduce the chance of progression to more advanced CTCL disease.

      Discussion

      Results of our genome- and transcriptome-wide analyses of sorted T cells from SS patients underscore the genetic heterogeneity of this malignancy. In agreement with a recent study that also highlighted a predominance of CNVs compared with single nucleotide variations in CTCL (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ), our whole-exome sequencing results suggest that CTCL may be a disease driven by CNVs instead of transformative point mutations. A few of the cytogenetic changes recurred in our cohort, including loss of 10q and 17p and gain of 8q and 17q. Despite the diversity of individual mutations and cytogenetic alterations that the landscape analysis of CTCL showed, the genetic perturbations consistently converged on the JAK/STAT pathway, along with the p53, MAPK, NF-κB, and PI3K pathways, in the malignant T cells. Changes to various genes within these pathways have previously been observed by whole-exome sequencing and comparative genome hybridization studies; nonetheless, there is a lack of consensus regarding the specific molecular drivers of CTCL (
      • Choi J.
      • Goh G.
      • Walradt T.
      • Hong B.S.
      • Bunick C.G.
      • Chen K.
      • et al.
      Genomic landscape of cutaneous T cell lymphoma.
      ,
      • de Leval L.
      • Bisig B.
      • Thielen C.
      • Boniver J.
      • Gaulard P.
      Molecular classification of T-cell lymphomas.
      ,
      • Vermeer M.H.
      • van Doorn R.
      • Dijkman R.
      • Mao X.
      • Whittaker S.
      • van Voorst Vader P.C.
      • et al.
      Novel and highly recurrent chromosomal alterations in Sezary syndrome.
      ). We posit that rather than a single genetic driver of malignant transformation, the collective single nucleotide variations and CNVs alter several pathways relevant to lymphomagenesis. Because most of these mutational changes were copy number aberrations, the cumulative chromosomal instability may be a key mechanism of transformation in this disease.
      Although constitutive phosphorylation of STAT3 and the key role of STAT3 in the survival of CTCL cell lines has previously been documented (
      • Krejsgaard T.O.R.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3-Stat3 signaling pathway.
      ,
      • McKenzie R.C.T.
      • Jones C.L.
      • Tosi I.
      • Caesar J.A.
      • Whittaker S.J.
      • Mitchell T.J.
      Constitutive activation of STAT3 in Sézary syndrome is independent of SHP-1.
      ,
      • Sommer V.H.
      • Clemmensen O.J.
      • Nielsen O.
      • Wasik M.
      • Lovato P.
      • Brender C.
      • et al.
      In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.
      ), our results help elucidate the molecular basis for the persistent STAT3 activation observed in CTCL and establish that this pathway is a driver of this malignancy. We found that STAT3 was duplicated in three out of eight patients, but we also observed amplification of several kinases known to phosphorylate STAT3, such as JAK3 and SRC family kinases (
      • Bromberg J.
      • Darnell J.E.
      The role of STATs in transcriptional control and their impact on cellular function.
      ), as well as loss of some of the negative regulators of STAT3. Our analysis of the heterogeneous genetic landscape and transcriptome of this enigmatic malignant disease yielded a defining transcriptional signature of CTCL. This presents a compelling potential diagnostic tool that warrants further investigation.
      Using conditional gene targeting, we showed the critical role of cytokine signaling, namely STAT3, in promoting CTCL pathogenesis and established a tractable and spontaneous model of CTCL. Previously published mouse models of CTCL relied on xenograft transplantation of cell lines established from human CTCL patients or on injection of virally transduced mouse cells into immunocompromised mice (
      • Adachi T.
      • Kobayashi T.
      • Sugihara E.
      • Yamada T.
      • Ikuta K.
      • Pittaluga S.
      • et al.
      Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma.
      ,
      • Charley M.R.
      • Tharp M.
      • Locker J.
      • Deng J.S.
      • Goslen J.B.
      • Mauro T.
      • et al.
      Establishment of a human cutaneous T-cell lymphoma in C.B-17 SCID mice.
      ,
      • Han T.
      • Abdel-Motal U.M.
      • Chang D.-K.
      • Sui J.
      • Muvaffak A.
      • Campbell J.
      • et al.
      Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma.
      ,
      • Ito M.
      • Teshima K.
      • Ikeda S.
      • Kitadate A.
      • Watanabe A.
      • Nara M.
      • et al.
      MicroRNA-150 inhibits tumor invasion and metastasis by targeting the chemokine receptor CCR6, in advanced cutaneous T-cell lymphoma.
      ,
      • Kittipongdaja W.
      • Wu X.
      • Garner J.
      • Liu X.
      • Komas S.M.
      • Hwang S.T.
      • et al.
      Rapamycin suppresses tumor growth and alters the metabolic phenotype in T-cell lymphoma.
      ,
      • Krejsgaard T.
      • Kopp K.
      • Ralfkiaer E.
      • Willumsgaard A.E.
      • Eriksen K.W.
      • Labuda T.
      • et al.
      A novel xenograft model of cutaneous T-cell lymphoma.
      ,
      • Thaler S.
      • Burger A.M.
      • Schulz T.
      • Brill B.
      • Bittner A.
      • Oberholzer P.A.
      • et al.
      Establishment of a mouse xenograft model for mycosis fungoides.
      ,
      • Wu X.
      • Wang T.W.
      • Lessmann G.M.
      • Saleh J.
      • Liu X.
      • Chitambar C.R.
      • et al.
      Gallium maltolate inhibits human cutaneous T-cell lymphoma tumor development in mice.
      ), while another model implanted a murine T-cell lymphoma line into a syngeneic host to generate malignant T-cell disease after DNFB stimulation (
      • Wu X.
      • Sells R.E.
      • Hwang S.T.
      Upregulation of inflammatory cytokines and oncogenic signal pathways preceding tumor formation in a murine model of T-cell lymphoma in skin.
      ). Although our genomic landscape analysis that highlighted the important role of STAT3 in human malignancy focused on genomic material from SS patients, our small animal model more closely mimicked the MF form of CTCL. Furthermore, our genetic approach of expressing the hyperactive mutant of STAT3 in all T lymphocytes precludes us from making any inference regarding the precise cell of origin for this malignant disease—a topic that is hotly debated in the field (
      • Campbell J.J.
      • Clark R.A.
      • Watanabe R.
      • Kupper T.S.
      Sézary syndrome and mycosis fungoides arise from distinct T-cell subsets: a biologic rationale for their distinct clinical behaviors.
      ,
      • Krejsgaard T.
      • Lindahl L.M.
      • Mongan N.P.
      • Wasik M.A.
      • Litvinov I.V.
      • Iversen L.
      • et al.
      Malignant inflammation in cutaneous T-cell lymphoma—a hostile takeover.
      ). Nonetheless, the relevant genetic trigger, the characteristic histopathological presentation of the disease, and the presence of a transcriptional signature of human malignancy in T cells from the mutant animals all distinguish the autochthonous mouse model generated here. Furthermore, because this model does not rely on injection or grafting of already malignantly transformed tissue, the development and progression of CTCL, along with genetic and pharmacological interventions, can be assessed.
      Pathogenesis in this model is linked to the expression of proinflammatory Th17 cytokines. This is intriguing in light of the normally characterized clinical progression of CTCL. Skin lesions from patients in early stages of disease show enhanced expression of Th1 cytokines, such as IFN-γ, thought to be linked to an antitumor immune response (
      • Bagot M.
      • Echchakir H.
      • Mami-Chouaib F.
      • Delfau-Larue M.H.
      • Charue D.
      • Bernheim A.
      • et al.
      Isolation of tumor-specific cytotoxic CD4+ and CD4+CD8dim+ T-cell clones infiltrating a cutaneous T-cell lymphoma.
      ,
      • Echchakir H.
      • Bagot M.
      • Dorothée G.
      • Martinvalet D.
      • Le Gouvello S.
      • Boumsell L.
      • et al.
      Cutaneous T cell lymphoma reactive CD4+ cytotoxic T lymphocyte clones display a Th1 cytokine profile and use a fas-independent pathway for specific tumor cell lysis.
      ,
      • Kim E.J.
      • Hess S.
      • Richardson S.K.
      • Newton S.
      Immunopathogenesis and therapy of cutaneous T cell lymphoma.
      ). As the disease progresses, the tumor microenvironment takes on a markedly different profile, characterized by an increase in Th2 cytokines, such as IL-4, IL-5, and IL-13, accompanied by a reduction in antitumor Th1 cells (
      • Krejsgaard T.
      • Lindahl L.M.
      • Mongan N.P.
      • Wasik M.A.
      • Litvinov I.V.
      • Iversen L.
      • et al.
      Malignant inflammation in cutaneous T-cell lymphoma—a hostile takeover.
      ). However, several studies find enhanced expression of the cytokines IL-17A and IL-17F in lesional skin driven by STAT3 hyperactivity (
      • Cirée A.
      • Michel L.
      • Camilleri-Bröet S.
      • Jean Louis F.
      • Oster M.
      • Flageul B.
      • et al.
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      ,
      • Krejsgaard T.O.R.
      • Ralfkiaer U.
      • Clasen-Linde E.
      • Eriksen K.W.
      • Kopp K.L.
      • Bonefeld C.M.
      • et al.
      Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3-Stat3 signaling pathway.
      ,
      • Krejsgaard T.
      • Litvinov I.V.
      • Wang Y.
      • Xia L.
      • Willerslev-Olsen A.
      • Koralov S.B.
      • et al.
      Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma.
      ). Enhanced expression of IL-17 is associated with an aggressive disease course in the subset of patients where it is seen (
      • Krejsgaard T.
      • Litvinov I.V.
      • Wang Y.
      • Xia L.
      • Willerslev-Olsen A.
      • Koralov S.B.
      • et al.
      Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma.
      ,
      • Krejsgaard T.
      • Lindahl L.M.
      • Mongan N.P.
      • Wasik M.A.
      • Litvinov I.V.
      • Iversen L.
      • et al.
      Malignant inflammation in cutaneous T-cell lymphoma—a hostile takeover.
      ). Our observation that mice expressing the hyperactive STAT3 allele display a Th17-biased inflammatory environment is consistent with the critical role that STAT3 plays in Th17 differentiation, and thus the model may represent the more aggressive form of the disease.
      Additionally, we took advantage of the CTCL animal model to show the central role of TCR signaling in the development of CTCL. This will facilitate further explorations of the role of T-cell interaction with antigen-presenting cells in the development of the disease. Furthermore, we confirm the necessity for microbial triggers in CTCL disease progression, thus validating the many epidemiological and clinical observations that have previously linked CTCL pathogenesis and bacterial infections. Use of this model can facilitate exploration of interventions that modify the composition of the skin microbiome as potential therapies. Given the molecular heterogeneity of this malignancy, we believe that targeting of the tumor microenvironment has to be considered along with inhibition of specific signaling networks found to be aberrantly expressed in the individual tumors. These interventions would have to truly reflect personalized medicine, with skin microbiome analysis complementing tumor RNA and DNA sequencing.

      Materials and Methods

      For detailed Materials and Methods, please see the Supplementary Materials online.

      Clinical samples

      Eight patients with SS and four healthy volunteers were identified at New York University Langone Medical Center and were included in this study in accordance with protocols approved by the New York University School of Medicine Institutional Review Board and Bellevue Facility Research Review Committee (ClinicalTrials.gov identification: NCT01663571). CTCL patients were diagnosed according to the WHO classification criteria. Patients with history of other hematopoietic malignancies were not included in this study. After written informed consent was obtained, peripheral blood samples were harvested.

      Quantification of disease progression in mouse model

      Monthly phenotype scoring was performed in a blinded fashion. Skin phenotype was assessed, and mice were assigned a score of 0–5 as follows: score of 0 = wild type appearance; 1 = hair loss around eyes; 2 = dry skin, obvious scratching, thinning of hair on neck; 3 = extensive hair loss on face or back of neck; 4 = large bald or scaly patches of skin; 5 = large scabs, sores, or open lesions. Mice with a score of 5 were killed, and the score of 5 was carried throughout the remainder of the analysis.

      Conflict of Interest

      The authors state no conflict of interest. JK is currently employed at Infinity Pharmaceuticals. His contribution to this work was before his employment there, while he was faculty at Brigham and Women’s Hospital.

      Acknowledgments

      We thank Markus Schober and Cindy Loomis for technical advice and for thoughtful discussions on skin biology. We thank Klaus Rajewsky for his thoughtful insight. We also thank the following New York University School of Medicine Core Facilities for expert assistance: Histopathology, Immunohistochemistry, Flow Cytometry, Biorepository, and the Genome Technology Center. We thank Harini Babu from HistoWiz, Inc., for her CD4 immunohistochemistry work.
      Work in SBK’s laboratory was supported by the National Institutes of Health (NIH) (R01HL125816), the NYUCI Pilot Grant, the Feinberg Lymphoma Research Grant, and grants from the Spatz Charitable Foundation, the Cutaneous Lymphoma Foundation, the Concern Foundation, and Hirschl/Weill-Caulier Trust. Additionally, MHF was supported by NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases award number F31AR070094. AS was supported by the William Randolph Hearst Foundation. AS and MHF were supported by NIH training grants T32-GM007308, T32-CA009161, and T32-AI 100853-3. LKF was supported by NIH F31 CA171596-02. The New York University Experimental Pathology Immunohistochemistry Core Laboratory is supported in part by the Laura and Isaac Perlmutter Cancer Center support grant, NIH/National Cancer Institute P30CA016087 and NIH S10 grants, and NIH/Office of Research Infrastructure Programs S10OD01058 and S10OD018338. NØ was supported by the Novo Nordic Foundation Tandem program and the Danish Cancer Society Knaek Cancer Program. ME and RSL were supported by R01 National Institute of Dental and Craniofacial Research (DE025639)

      Author Contributions

      MHF, AS, LKF, and SBK were directly involved in the design and execution of the experiments and writing of the manuscript. MHF performed all mouse experiments. AS, VN, SG, MEL, JL, and KBH were responsible for patient recruitment and biospecimen collection and processing. KK, ID, CL, and AH were responsible for sequencing and bioinformatics, together with VN, AS, and SBK. ME and RSL performed immunofluorescence studies. MSS, JK, CL, NØ, and KBH contributed to the interpretation of the results and direction of the project.

      Supplementary Material

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      Linked Article

      • A Microbiota-Dependent, STAT3-Driven Mouse Model of Cutaneous T-Cell Lymphoma
        Journal of Investigative DermatologyVol. 138Issue 5
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          In recent years, much has been learned about the molecular genetics of cutaneous T-cell lymphomas. Fanok et al. (2018) translate knowledge from systematic genomic and transcriptomic analyses to develop a mouse model that tests the hypothesis that activated STAT3 in CD4+ T cells may be a driver of cutaneous T-cell lymphomas. The transgenic mouse that they developed exhibits clinical features of mycosis fungoides, as well as Sezary syndrome, two well-known entities in the cutaneous T-cell lymphoma spectrum.
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