Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome.

ABSTRACT: Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus-specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Malignant T cells induce skin barrier defects through cytokine-mediated JAK/STAT signaling in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA-mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

Concomitant Inhibition of FASN and SREBP Provides a Promising Therapy for CTCL.

Cutaneous T cell lymphoma (CTCL) is a group of non-Hodgkin's primary cutaneous T cell lymphomas, with Mycosis Fungoides and Sézary syndrome (SS) being the two most common subtypes. Fatty acid synthase (FASN) is a crucial enzyme that catalyses the biosynthesis of fatty acids, which has been reported to play an oncogenic role in various malignancies but not in CTCL so far. Herein, we show that FASN is highly expressed in CTCL cell lines and in peripheral blood mononuclear cells (PBMCs) from CTCL patients, while it is not in PBMCs from healthy individuals. The inhibition of FASN in CTCL cell lines impairs cell viability, survival, and proliferation, but, interestingly, it also increases FASN expression. However, inhibiting sterol regulatory element binding protein (SREBP), a transcription factor that promotes the expression of FASN, partially reversed the upregulation of FASN induced by FASN inhibitors. Thus, the combination of FASN and SREBP inhibitors enhanced the effects on both CTCL cell lines and PBMCs from SS patients, where a valid inhibition on cell proliferation could be verified. Importantly, compared to non-malignant cells, primary malignant cells are more sensitive to the inhibition of FASN and SREBP, making the combination of FASN and SREBP inhibitors a promising novel therapeutic strategy in CTCL.

Bacterial genotoxins induce T cell senescence.

Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells-the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.

Generation of a tissue-specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo.

Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

JAK3 Is Expressed in the Nucleus of Malignant T Cells in Cutaneous T Cell Lymphoma (CTCL).

Perturbation in JAK-STAT signaling has been reported in the pathogenesis of cutaneous T cell lymphoma (CTCL). JAK3 is predominantly associated with the intra-cytoplasmic part of IL-2Rγc located in the plasma membrane of hematopoietic cells. Here we demonstrate that JAK3 is also ectopically expressed in the nucleus of malignant T cells. We detected nuclear JAK3 in various CTCL cell lines and primary malignant T cells from patients with Sézary syndrome, a leukemic variant of CTCL. Nuclear localization of JAK3 was independent of its kinase activity whereas STAT3 had a modest effect on nuclear JAK3 expression. Moreover, JAK3 nuclear localization was only weakly affected by blockage of nuclear export. An inhibitor of the nuclear export protein CRM1, Leptomycin B, induced an increased expression of SOCS3 in the nucleus, but only a weak increase in nuclear JAK3. Importantly, immunoprecipitation experiments indicated that JAK3 interacts with the nuclear protein POLR2A, the catalytic subunit of RNA Polymerase II. Kinase assays showed tyrosine phosphorylation of recombinant human Histone H3 by JAK3 in vitro-an effect which was blocked by the JAK inhibitor (Tofacitinib citrate). In conclusion, we provide the first evidence of nuclear localization of JAK3 in malignant T cells. Our findings suggest that JAK3 may have a cytokine-receptor independent function in the nucleus of malignant T cells, and thus a novel non-canonical role in CTCL.

Depletion of keratin 8/18 modulates oncogenic potential by governing multiple signaling pathways.

Keratin 8/18, the predominant keratin pair of simple epithelia, is often aberrantly expressed in various squamous cell carcinomas (SCCs) including skin SCC. Its aberrant expression is correlated with increased invasiveness and poor prognosis of the same, although the underlying mechanism is still unclear. A previous report from our laboratory has shown K8-mediated regulation of α6ß4 integrin signaling and thereby tumorigenic potential of oral SCC-derived cells. Another study on transgenic mouse model has shown that during skin carcinogenesis, K8 favors conversion of papillomas toward malignancy. In order to understand the role of K8 and allied mechanism in skin SCC, K8 was stably knocked down in a skin epidermoid carcinoma-derived A431 cells. K8 downregulation significantly reduced the tumorigenic potential of these cells. In agreement with our phenotypic data, differential quantitative proteomics followed by IPA analysis showed altered expression of many proteins associated with biological functions including 'Cancer', 'Cellular movement', 'Cell death and survival', and 'Cellular morphology'. Some of these proteins were TMS1, MARCKSL1, RanBP1, 14-3-3γ, Rho-GDI2, etc. Furthermore, to our surprise, there was a significant reduction in K17 protein stability upon loss of K8, probably due to its caspase-mediated degradation. This was supported by altered TMS1-NF-κB signaling, leading to increased apoptotic sensitivity of A431 cells which in turn affected 'Cell death and survival'. Moreover, MARCKSL1-Paxillin1-Rac axis was found to be deregulated bestowing a possible mechanism behind altered 'Cellular movement' pathway. Altogether our study unravels a much broader regulatory role of K8, governing multiple signaling pathways and consequently regulating oncogenic potential of skin SCC-derived cells. DATABASE: Proteome Xchange Consortium via PRIDE database (dataset identifier PXD007206).

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation of Keratin-8 in skin squamous cell carcinoma derived cell line.

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine73 (head-domain) and Serine431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1T14,Y15 , EIF4EBP1T46,T50 , EIF4BS422 , AKT1S1T246,S247 , CTTN1T401,S405,Y421 , and CAP1S307/309 in K8-S73A/D mutant and CTTN1T401,S405,Y421 , BUB1BS1043 , and CARHSP1S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176 , ZYXS344 , and PNNS692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.