Simon Fredholm

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 β 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.

Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.

B-lymphoid tyrosine kinase (Blk) is an oncogene and a potential target for therapy with dasatinib in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphoma (CTCL) is the most frequent primary lymphoma of the skin. Patients diagnosed in early stages often experience an indolent disease course and have a favorable prognosis. Yet, the disease follows an aggressive course in a substantial fraction (15–20%) of patients and despite recent progress in novel therapies, advanced disease remains a major challenge as relapses are common and cure is rare.1 Recently, it was discovered,2 and independently confirmed in a meta-analysis study,3 that malignant T cells in the majority of patients display ectopic expression of the B-lymphoid tyrosine kinase (Blk), a member of the Src kinase family. Importantly, gene knockdown experiments showed that Blk promoted the proliferation of malignant T cells from CTCL patients,2 suggesting that Blk—in analogy with other Src family members—may function as an oncogene. In support, Montero-Ruiz et al.4 provided evidence that Blk is implicated in childhood acute lymphoblastic leukemia. However, studies in mice suggested that murine Blk also has tumor-suppressive functions depending on the specific cellular context.5 To study the oncogenic potential of human Blk, we therefore transfected a cytokine (IL-3)-dependent lymphoid cell line (Ba/F3) with plasmids expressing either wild-type (wt) Blk or a constitutively active form of Blk lacking the kinase-inhibitory site due to a tyrosine-to-phenylalanine substitution at amino-acid position 501 (Y501F). Stable transfectants were established by selecting for the plasmid-encoded blasticidin resistance gene, and before experimentation, transformed cells were maintained in blasticidin- and IL-3-supplemented growth media. As shown in Figure 1a, the constitutively active form of Blk (Y501F) was fully able to transform growth factor (IL-3)-dependent Ba/F3 cells into IL-3-independent cells, whereas non-transfected and Blk-wt-transfected Ba/F3 cells remained dependent on exogenous IL-3 to survive and proliferate. In accordance, IL-3 deprivation induced massive apoptosis in non-transfected and Blk-wt-transfected Ba/F3 cells, whereas no increase in apoptosis was observed in Blk(Y501F)-transfected Ba/F3 cells following IL-3 withdrawal (Figure 1b). As expected, Blk(Y501F) was phosphorylated on the activating tyrosine (Y388) and not on the inhibitory tyrosine phosphorylation site (Y501), whereas Blk-wt was heavily phosphorylated on the kinase-inhibitory site (Y501) (Figures 1c and d). The well-characterized Src family kinase inhibitor, Lck inhibitor (LckI, Calbiochem, San Diego, CA, USA), selectively inhibited the proliferation of Blk(Y501F)-transfected Ba/F3 cells, whereas an inhibitor of mitogen-activated protein kinase p38 (SB203580, Selleck Chemicals, Houston, TX, USA) did not (Figure 1e). Likewise, the dual-specificity inhibitor of Bcr-Abl and the Src family kinases, dasatinib (Sprycel, Selleck Chemicals), inhibited Y388 phosphorylation and proliferation of the Blk(Y501F)-transfected Ba/F3 cells (Figures 1f and g). Taken together, these results indicate that the active form of human Blk is able and sufficient to transform cytokine-dependent lymphoid cells into cytokine-independent cells. These findings support the previous observation made by others6 that murine Blk(Y495F) is lymphomagenic in mice. In keeping, enzymatic inhibition by LckI and other Src family kinase inhibitors, as well as siRNA-mediated knockdown of Blk, inhibits proliferation of Blk-positive malignant T cells including MyLa2059 and MyLa2000 (ref. 2 and data not shown). As dasatinib profoundly inhibited Blk(Y501F)-transformed Ba/F3 cells and tyrosine phosphorylation of Blk in the MyLa2059 cells (Figure 2a), we hypothesized that dasatinib—which is used for treatment of chronic myelogenous leukemia and other malignancies7—has a potential for treatment of CTCL. To address this hypothesis, we initially studied the effect of dasatinib on malignant proliferation in vitro. As shown in Figure 2b, dasatinib inhibited the spontaneous proliferation of the malignant CTCL T-cell line MyLa2059 in a concentration-dependent manner. Likewise, the Blk-positive CTCL cell lines MyLa2000 and PB2B2 were also inhibited by dasatinib, whereas the Blk-negative Sezary Syndrome cell line (SeAx) was resistant (Supplementary Figure S1). As malignant T cells, including some cell lines obtained from CTCL2 and peripheral T-cell lymphoma patients,8 often express Fyn (another Src kinase), we cannot exclude the possibility that the effect of dasatinib was partially mediated through an inhibition of both Blk and Fyn. However, Fyn is not tyrosine phosphorylated in the malignant MyLa cells suggesting that Fyn may not be functionally active in these cells (data not shown). The observation that dasatinib inhibited Blk-positive tumor cells prompted us to examine the effect of dasatinib on tumor growth in a xenograft transplantation model of CTCL.9, 10 In a preliminary experiment, mice were inoculated subcutaneously (s.c.) with MyLa2059 cells and treated orally with different concentrations of dasatinib (or vehicle as a control) to evaluate the effect on tumor formation in vivo. The average time of tumor onset was significantly (P<0.05) delayed from day 13 in the control group (N=5) to day 20 in animals (N=6) treated with dasatinib (data not shown). Next, we addressed whether dasatinib inhibited growth of already established tumors. Accordingly, eight mice were inoculated s.c. with MyLa2059 cells and following detection of palpable tumors (day 1) the mice were treated with either dasatinib (Sprycel) (40 mg/kg) or vehicle as control. Tumor dimensions were measured in each group on days 3, 5, 8 and 10. As shown in Figure 2c, dasatinib significantly inhibited tumor growth. Likewise, volume of the resected tumors harvested on day 10 was significantly lower in the dasatinib-treated mice when compared with the control mice, confirming that dasatinib does inhibit CTCL tumor growth in vivo (Figure 2d). As the malignant T cells do not express the Bcr-Abl oncogene (data not shown), the present finding suggests that Blk functions as an oncogene in the CTCL cells. As NF-κB is active in CTCL2 and supports growth of dasatinib/imatinib-resistant cells,11, 12 we tested dasatinib in combination with NF-κB inhibitors. Interestingly, dasatinib and NF-κB inhibitors had an additive effect on malignant proliferation in vitro (data not shown), which might explain why Blk(Y501F)-transformed Ba/F3 cells were more sensitive to dasatinib than the malignant MyLa cells. In summary, (i) Blk is enzymatically active in malignant CTCL cells and expressed in situ,2 (ii) its constitutively active form confers cytokine independence (Figure 1) and (iii) it promotes tumor growth in vivo as indicated by the effect of dasatinib on tumor growth in mice (Figure 2). Taken together, these findings strongly suggest that Blk is a potential therapeutic target in CTCL for dasatinib and other clinical-grade dual-specificity Bcr-Abl and Src family kinase inhibitors. As dasatinib and other dual-specific inhibitors are already used in treatment of other hematological malignancies with a high efficacy, tolerability and compliance,7 these drugs are attractive novel candidates for the treatment of CTCL expressing Blk.

STAT3/5-Dependent IL9 Overexpression Contributes to Neoplastic Cell Survival in Mycosis Fungoides.

Purpose: Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9–producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown. Experimental Design: We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice. Results: Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro. IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes. Conclusions: Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328–39. ©2016 AACR.

IL-15 and IL-17F are differentially regulated and expressed in mycosis fungoides (MF)

Skin lesions from mycosis fungoides (MF) patients display an increased expression of interleukin-15 (IL-15), IL-17F, and other cytokines implicated in inflammation and malignant cell proliferation in cutaneous T-cell lymphoma (CTCL). In the leukemic variant of CTCL, Sézary syndrome (SS), IL-2 and IL-15 trigger activation of the Jak-3/STAT3 pathway and transcription of IL17A gene, whereas it is unknown what causes IL-15 expression, Jak3/STAT3 activation, and production of IL-17F in MF. Here, we studied the expression and regulation of IL-15 and its relation to IL-17F in MF cell lines and skin lesions from 60 MF patients. We show that: (1) the spontaneous IL-15 mRNA expression is resistant to Jak3 and STAT3 inhibitors at concentrations that profoundly inhibit STAT3 activation and IL-17F mRNA expression; (2) anti-IL-15 antibody blocks STAT3 activation induced by exogenous IL-15 in non-malignant MF T cells, whereas the spontaneous STAT3 activation and IL-17F expression in malignant T cells is not inhibited; (3) patients display heterogeneous IL-15/IL-17F mRNA expression patterns in skin lesions; and (4) IL-15 expression (in contrast to IL-17F) is not associated with progressive disease. Taken together, these findings indicate that IL-15 and IL-17F are differentially regulated and expressed in MF. We propose that IL-15 and IL-17F are markers for different inflammatory environments and play distinct roles in the development and progression of MF.

STAT5 induces miR-21 expression in cutaneous T cell lymphoma

In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.

The Expression of IL-21 Is Promoted by MEKK4 in Malignant T Cells and Associated with Increased Progression Risk in Cutaneous T-Cell Lymphoma

Please cite this article as: Fredholm S, Livinov I, Mongan NP, Schiele S, Willerslev-Olsen A, Petersen DL, Krejsgaard T, Sibbesen N, Nastasi C, Bonefeld CM, Persson JL, Thor Straten P, Andersen MH, Koralov SB, Wasik M, Geisler C, Sasseville D, Woetmann A, Ødum N, The expression of IL-21 is promoted by MEKK4 in malignant T cells and associated with increased progression risk in cutaneous Tcell lymphoma, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2015.12.033.

SATB1 in Malignant T Cells

Deficient expression of SATB1 hampers thymocyte development and results in inept T-cell lineages. Recent data implicate dysregulated SATB1 expression in the pathogenesis of mycosis fungoides, the most frequent variant of cutaneous T-cell lymphoma. Here, we report on a disease stage-associated decrease of SATB1 expression and an inverse expression of STAT5 and SATB1 in situ. STAT5 inhibited SATB1 expression through induction of microRNA-155. Decreased SATB1 expression triggered enhanced expression of IL-5 and IL-9 (but not IL-6 and IL-32), whereas increased SATB1 expression had the opposite effect, indicating that the microRNA-155 target SATB1 is a repressor of IL-5 and IL-9 in malignant T cells. In accordance, inhibition of STAT5 and its upstream activator JAK3 triggered increased SATB1 expression and a concomitant suppression of IL-5 and IL-9 expression in malignant T cells. In conclusion, we provide a mechanistic link between the proto-oncogenic JAK3/STAT5/microRNA-155 pathway, SATB1, and cytokines linked to CTCL severity and progression, indicating that SATB1 dysregulation is involved in cutaneous T-cell lymphoma pathogenesis.

A novel BLK-induced tumor model:

B-lymphoid tyrosine kinase (BLK) is a non-receptor tyrosine kinase belonging to the SRC family kinases. BLK is known to be functionally involved in B-cell receptor signaling and B-cell development. New evidence suggests that B-lymphoid tyrosine kinase is ectopically expressed and is a putative oncogene in cutaneous T-cell lymphoma and other T-cell malignancies. However, little is known about the role of BLK in lymphomagenesis, and the oncogenic function seems to depend on the cellular context. Importantly, BLK is also ectopically expressed in other hematological and multiple non-hematological malignancies including breast, kidney, and lung cancers, suggesting that BLK could be a new potential target for therapy. Here, we studied the oncogenic potential of human BLK. We found that engrafted Ba/F3 cells stably expressing constitutive active human BLK formed tumors in mice, whereas neither Ba/F3 cells expressing wild type BLK nor non-transfected Ba/F3 cells did. Inhibition of BLK with the clinical grade and broadly reacting SRC family kinase inhibitor dasatinib inhibited growth of BLK–induced tumors. In conclusion, our study provides evidence that human BLK is a true proto-oncogene capable of inducing tumors, and we demonstrate a novel BLK activity–dependent tumor model suitable for studies of BLK–driven lymphomagenesis and screening of novel BLK inhibitors in vivo.

Butyrate and propionate inhibit antigen-specific CD8+ T cell activation by suppressing IL-12 production by antigen-presenting cells

Short chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are products of microbial macronutrients fermentation that distribute systemically and are believed to modulate host immune responses. Recent data have indicated that certain SCFAs, such as butyrate and propionate, directly modulate human dendritic cell (DC) function. Given the role of DCs in initiating and shaping the adaptive immune response, we now explore how SCFAs affect the activation of antigen-specific CD8+ T cells stimulated with autologous, MART1 peptide-pulsed DC. We show that butyrate reduces the frequency of peptide-specific CD8+ T cells and, together with propionate, inhibit the activity of those cells. On the contrary, acetate does not affect them. Importantly, butyrate and propionate inhibit the production of IL-12 and IL-23 in the DCs and exogenous IL-12 fully restores the activation of the MART-1-specific CD8+ T cells, whereas IL-23 has no effect. In conclusion, these results point to a pivotal role of butyrate and propionate in modulating CD8+ T cell activation via the inhibition of IL-12 secretion from DCs. These findings reveal a novel mechanism whereby bacterial fermentation products may modulate CD8+ T cell function with possible implications in anti-cancer immunotherapy.