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.

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.

Urticaria and the risk of cancer: a Danish population-based cohort study.

BACKGROUND: Urticaria has been tentatively linked to cancer, but epidemiological evidence supporting this link is sparse and conflicting. We therefore conducted a population-based cohort study using healthcare databases of the Danish population (January 1980-December 2022). We followed 87,507 people for a median of 10.1 years after first hospital contact for urticaria. OBJECTIVES: To examine associations of a hospital diagnosis of urticaria with incident cancer. METHODS: We computed absolute risk of cancer and standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) standardized to Danish national cancer rates. In a cross-sectional analysis, we examined whether the extent of cancer spread differed between people with vs. without a previous urticaria diagnosis. RESULTS: The overall SIR for all types of cancer was 1.09 (95% CI, 1.06-1.11) based on 7,788 observed vs. 7,161 expected cases. The risk for any cancer was 0.7% (95% CI, 0.6-0.7) for the first year of follow-up. Cancer was diagnosed in 588 people with urticaria during the first year of follow-up (SIR 1.49, 95% CI, 1.38-1.62) and in 7,200 people thereafter (SIR 1.06, 95% CI, 1.04-1.09). During the first year of follow-up, we found strong associations with hematological cancers (e.g., non-Hodgkin lymphoma SIR 2.91, 95% CI, 1.92-4.23). Cancer stage was similar in people with vs. without previous urticaria diagnosis. CONCLUSIONS: At the time of urticaria diagnosis, or in the first year afterwards, we found a large increase in the risk for cancer. In subsequent years, a persistent 6% increase in risk remained. Diagnostic efforts may partly explain the elevated short-term risk, but occult cancer may promote urticaria, or cancer and urticaria share common risk factors.

miRNA Signature in Early-stage Mycosis Fungoides.

Altered miRNA expressions are assigned pathogenic properties in several cancers including mycosis fungoides and could play a role in the early onset of the disease. The aim of this study was to examine disease-specific miRNA expression in early-stage mycosis fungoides patch and plaque lesions. A quantitative real-time PCR platform of 384 human miRNAs was used to study miRNA expression in 154 diagnostic mycosis fungoides biopsies. A total of 110 miRNAs were significantly differentially expressed (>2-fold, p < 0.05) between plaque lesions and healthy controls, and 90 miRNAs (>2-fold, p < 0.05) differed between patch lesions and healthy controls. Moreover, 13 miRNAs differed in expression between patch and plaque lesions. Early-stage mycosis fungoides exhibited miRNA features that overlapped with those of psoriasis. However, 39 miRNAs, including miR-142-3p, miR-150 and miR-146b, were specific to mycosis fungoides. In conclusion, early-stage mycosis fungoides expresses a distinct miRNA profile, indicating that miRNAs could play a role in the early development of mycosis fungoides.

Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Suppressed microRNA-195-5p expression in mycosis fungoides promotes tumor cell proliferation.

BACKGROUND: Several cancers, including mycosis fungoides (MF), have reported dysregulation of miR-195-5p. miR-195-5p plays a role in cell cycle regulation in several malignant diseases. OBJECTIVES: This study aimed to investigate: (a) the expression level of miR-195-5p in lesional MF skin biopsies and (b) the potential regulatory roles of miR-195-5p in MF. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine miR-195-5p expression in MF skin biopsies and cell lines. The effect of miR-195-5p and ADP-ribosylation factor-like protein 2 (ARL2) on cell cycle and apoptosis was measured by flow cytometry assays. Changes in ARL2 expression were determined by RT-qPCR and Western blotting (WB). RESULTS: We found lower expression levels of miR-195-5p in lesional skin from MF patients compared with non-lesional MF skin and skin from healthy volunteers. Additionally, miR-195-5p showed lower expression levels in the skin from patients with disease progression compared with patients with stable disease. In vitro studies showed that overexpression of miR-195-5p induced a cell cycle arrest in G0G1. Using microarray analysis, we identified several genes that were regulated after miR-195-5p overexpression. The most downregulated gene after miR-195-5p mimic transfection was ARL2. RT-qPCR and WB analyses confirmed downregulation of ARL2 following transfection with miR-195-5p mimic. Lastly, transfection with siRNA against ARL2 also induced a G0G1 arrest. CONCLUSION: Upregulation of miR-195-5p in MF inhibits cycle arrest by downregulation of ARL2. miR-195-5p may thus function as a tumor suppressor in MF and low miR-195-5p expression in lesional MF skin may promote disease progression.

MicroRNA-93 Targets p21 and Promotes Proliferation in Mycosis Fungoides T Cells.

BACKGROUND: Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL), is a lymphoproliferative disorder characterized by proliferation of malignant T cells in a chronic inflammatory environment in the skin. The nature of MF is still not fully understood, but aberrant microRNA (miR) expression and function seem to play an important role in the pathogenesis and disease progression and have been proposed as a putative disease marker. Recent studies have reported aberrant expression of miR-93 in situin MF lesions and linked dysregulated miR-93 expression to advanced stages of MF. However, the pathophysiological role of miR-93 in MF is unknown. OBJECTIVE: Here, we provide the first evidence that miR-93 targets the cell cycle regulator cyclin-dependent kinase inhibitor p21 and promotes growth of malignant T cells in MF. METHODS/RESULTS: Thus, inhibition of miR-93 in MF patient-derived malignant T-cell lines increases expression of p21 and inhibition of malignant proliferation. Notably, treatment with the histone deacetylase inhibitor Vorinostat (SAHA) reduces miR-93 expression and enhances p21 expression in the malignant T cells. Importantly, transfection with an miR-93 mimic partly blocks SAHA-induced p21 expression. CONCLUSIONS: we provide evidence that enhanced expression of the putative oncogenic miR, miR-93, represses the cell cycle inhibitor p21 and promotes proliferation of malignant T cells. Moreover, we demonstrate that SAHA triggers p21 expression - at least partly - through an inhibition of miR-93.

The Thioredoxin-Interacting Protein TXNIP Is a Putative Tumour Suppressor in Cutaneous T-Cell Lymphoma.

BACKGROUND: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients. OBJECTIVES: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL). METHODS: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 - an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry. RESULTS: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells. CONCLUSIONS: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL.

Role of B-cells in Mycosis Fungoides.

Mycosis fungoides is the most common type of cutaneous T-cell lymphoma. The inflammatory micro-environment in mycosis fungoides is complex. There is accumulating evidence that the neoplastic T-cells take control of the microenvironment and thereby promote their own expansion by suppressing cellular immunity. B-cells have proved to be upregulated in large-cell transformed mycosis fungoides, and could potentially play a role in disease progression. To investigate the presence of B-cells in mycosis fungoides compared with controls, this study analysed 85 formalin-fixed and paraffin-embedded mycosis fungoides biopsies. MS4A1 gene expression was significantly upregulated in mycosis fungoides compared with controls (p < 0.0001) and further upregulated in disease progression, (p = 0.001). Digital quantification of PAX5+/CD20+ cells confirmed the increased presence of B-cells in mycosis fungoides compared with controls. No co-labelling of CD3/CD20 was observed in the neoplastic T-cells. This study found a significantly increased presence of B-cells in the tumour-associated microenvironment in mycosis fungoides. These findings could potentially lead to new treatment strategies for mycosis fungoides.

Prognostic miRNA classifier in early-stage mycosis fungoides: development and validation in a Danish nationwide study.

Mycosis fungoides (MF) is the most frequent form of cutaneous T-cell lymphoma. The disease often takes an indolent course, but in approximately one-third of the patients, the disease progresses to an aggressive malignancy with a poor prognosis. At the time of diagnosis, it is impossible to predict which patients develop severe disease and are in need of aggressive treatment. Accordingly, we investigated the prognostic potential of microRNAs (miRNAs) at the time of diagnosis in MF. Using a quantitative reverse transcription polymerase chain reaction platform, we analyzed miRNA expression in diagnostic skin biopsies from 154 Danish patients with early-stage MF. The patients were subdivided into a discovery cohort (n = 82) and an independent validation cohort (n = 72). The miRNA classifier was built using a LASSO (least absolute shrinkage and selection operator) Cox regression to predict progression-free survival (PFS). We developed a 3-miRNA classifier, based on miR-106b-5p, miR-148a-3p, and miR-338-3p, which successfully separated patients into high-risk and low-risk groups of disease progression. PFS was significantly different between these groups in both the discovery cohort and the validation cohort. The classifier was stronger than existing clinical prognostic factors and remained a strong independent prognostic tool after stratification and adjustment for these factors. Importantly, patients in the high-risk group had a significantly reduced overall survival. The 3-miRNA classifier is an effective tool to predict disease progression of early-stage MF at the time of diagnosis. The classifier adds significant prognostic value to existing clinical prognostic factors and may facilitate more individualized treatment of these patients.

MicroRNA-106b Regulates Expression of the Tumour Suppressors p21 and TXNIP and Promotes Tumour Cell Proliferation in Mycosis Fungoides.

A prognostic 3-miRNA classifier for early-stage mycosis fungoides has been developed recently, with miR-106b providing the strongest prognostic power. The aim of this study was to investigate the molecular function of miR-106b in mycosis fungoides disease progression. The cellular localization of miR-106b in mycosis fungoides skin biopsies was determined by in situ hybridization. The regulatory role of miR-106b was assessed by transient miR-106b inhibitor/mimic transfection of 2 mycosis fungoides derived cell lines, followed by quantitative real-time PCR (RT-qPCR), western blotting and a proliferation assay. MiR-106b was found to be expressed by dermal T-lymphocytes in mycosis fungoides skin lesions, and miR-106b expression increased with advancing mycosis fungoides stage. Transfection of miR-106b in 2 mycosis fungoides derived cell lines showed that miR-106b represses the tumour suppressors cyclin-dependent kinase inhibitor 1 (p21) and thioredoxin-interacting protein (TXNIP) and promotes mycosis fungoides tumour cell proliferation. In conclusion, these results substantiate that miR-106b has both a functional and prognostic role in progression of mycosis fungoides.

The MicroRNA Expression Profile Differs Between Erythrodermic Mycosis Fungoides and Sézary Syndrome.

It is difficult to distinguish erythrodermic mycosis fungoides from Sézary syndrome due to their similar clinical and histological features. The main purpose of this study was to investigate whether microRNA expression profiles in lesional skin could discriminate patients with erythrodermic mycosis fungoides from those with Sézary syndrome. A further aim was to assess whether the microRNA expression profiles in erythrodermic mycosis fungoides skin was more comparable to microRNA expression profiles of Sézary syndrome or early-stage mycosis fungoides. RNA was extracted from diagnostic skin biopsies, followed by quantitative reverse transcription polymerase chain reaction analysis of 383 microRNAs. Twenty-seven microRNAs were significantly differentially expressed between erythro-dermic mycosis fungoides and Sézary syndrome. More-over, erythrodermic mycosis fungoides showed microRNA features overlapping with Sézary syndrome and early-stage mycosis fungoides, although hierarchical cluster analysis co-clustered erythrodermic mycosis fungoides with early-stage mycosis fungoides rather than with Sézary syndrome. These findings underscore that erythrodermic mycosis fungoides and Sézary syndrome are different diseases.

Clinical and Histological Characteristics of Mycosis Fungoides and Sézary Syndrome: A Retrospective, Single-centre Study of 43 Patients from Eastern Denmark.

Diagnosis of mycosis fungoides and Sézary syndrome can be very challenging. Clinical and histopathological data for patients with mycosis fungoides and Sézary syndrome in Denmark are limited. A retrospective study was performed in Region Zealand, Denmark from 1990 to 2016. A total of 43 patients with mycosis fungoides or Sézary syndrome were identified during the period. At the time of diagnosis the patients' mean age was 64.3 years and 74.5% had early-stage (≤IIA) disease. The mean time from onset of skin disease to diagnosis was 4.4 years. Surprisingly, 43% progressed to a higher disease stage, and risk of disease progression was higher for stage IB than IA (p = 0.01). All cases displayed some degree of epidermotropism and the infiltrates consisted of pleomorphic lymphocytes with a T-helper (CD4+/CD8-) phenotype. This study describes, for the first time, all aspects of clinical and histopathological findings in patients with mycosis fungoides and Sézary syndrome in a well-characterized Danish cohort.

Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region ß chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

Risk of venous thromboembolism in patients with mycosis fungoides and parapsoriasis: A Danish nationwide population-based cohort study.

BACKGROUND: Mycosis fungoides (MF) and parapsoriasis are characterized by malignant proliferation and chronic inflammation, which may affect the risk for venous thromboembolism (VTE). OBJECTIVES: To examine the risk for VTE in patients with MF and parapsoriasis. METHODS: We conducted a nationwide population-based cohort study in Denmark to examine the relative risk (RR) of VTE in 525 patients with MF and 634 patients with parapsoriasis compared with that in sex- and age-matched controls from the general population. RESULTS: In patients with MF, the 10-year absolute risk for VTE was 3.4% (95% confidence interval [CI], 2.0-5.4). The adjusted RRs were 2.41 (95% CI, 1.49-3.90) for VTE and 4.01 (95% CI, 2.16-7.46) for pulmonary embolism. Notably, within the first 5 years after diagnosis with MF, the RR of pulmonary embolism was increased 6.7-fold (to 6.71 [95% CI, 2.86-15.72]). Patients with parapsoriasis had a 2.7-fold increased RR of VTE (to 2.67 [95% CI, 1.32-5.40]) in the absence of other established VTE risk factors. LIMITATIONS: We had no information regarding disease stage of MF and prescribed drugs. CONCLUSION: Patients with MF and parapsoriasis had an increased RR of VTE, although the absolute risk remained low. These findings should increase awareness of comorbidities in patients with MF and parapsoriasis.

Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation.

Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus (SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.

Risk of Acute Myocardial Infarction or Stroke in Patients with Mycosis Fungoides and Parapsoriasis.

Mycosis fungoides (MF) and parapsoriasis display increased inflammation, which may be associated with increased risk of arterial cardiovascular events. The aim of this Danish nationwide population-based cohort study was to assess the relative risk (RR) of acute myocardial infarction (AMI) or stroke in patients with MF and parapsoriasis. In patients with MF, the RR of AMI or stroke was 1.0 (95% confidence interval (95% CI) 0.7-1.3). In the second half of the study period, the RR was 1.8 (95% CI 1.1-2.9) during the first 5 years of follow-up. In men with parapsoriasis, the RR of AMI or stroke was 1.7 (95% CI 1.1-2.7) within the first 5 years of follow-up, whereas the RR of AMI during the first 5 years of follow-up was 2.0 (95% CI 1.2-3.4). In conclusion, patients with MF and parapsoriasis have an increased RR of AMI or stroke within the first 5 years of follow-up.