Cutaneous T-cell Lymphoma Cells Research Articles

Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL) is a heterogeneous neoplasm and patients with relapsed/refractory disease exhibit resistance to standard cytotoxic therapies. We have previously demonstrated that the MUC1-C oncoprotein plays a critical role in regulating apoptosis and protection from oxidative stress in CTCL. Targeting of MUC1-C with a pharmacologic inhibitor, GO-203, was associated with late apoptosis and early necrosis and slowing of tumor growth in in vitro and in vivo models of CTCL, respectively. However, disease responses following MUC1-C inhibition were incomplete underscoring the need for combinatorial strategies that could exploit the vulnerability of CTCL cells to oxidative signals. Decitabine is a DNA methyltransferase inhibitor, previously shown to modulate oxidative stress in acute myeloid leukemia models. We hypothesized that treatment with decitabine and GO-203 would present a potent novel therapeutic combination for patients with CTCL.Method/Results: Our studies demonstrate that exposure of CTCL cells to decitabine in combination with GO-203, increased the generation of reactive oxygen species (ROS) and decreased levels of scavenger molecules, NADPH, glutathione (GSH), and TIGAR, a p53-inducible regulator of glycolysis and apoptosis, critical to intracellular redox homeostasis. This in turn led to marked increase in the ratio of stress activated proteins; phospho-p38 and phospho-JNK to Total-p38 and Total-JNK repectively. Further dual exposure to GO-203 and decitabine resulted in near abrogation of DNA methyl transferase 1 and 3b crucial for maintaining DNA methylation, demonstrating significant synergy of these agents for inducing hypomethylation. In concert with these findings, treatment with decitabine and GO-203 upregulated the ROS generating enzyme, NADPH oxidase 4 (Nox4) potentially due to their effect on epigenomic regulation of this protein. In concert with these findings, exposure to decitabine and GO-203 resulted in heightened apoptotic death of CTCL cell lines, primary patient derived samples and in a aggressive murine xenograft model.Conclusion: These findings indicate that decitabine augments MUC1-C inhibition induced redox imbalance and provides a novel combination of targeted and epigenetic agents for treatment of patients with CTCL. Accordingly an early phase multi-institution clinical trial to to investigate this combinatorial strategy based on epigenetic modulation of redox homeostasis in patients with relapsed/refractory T cell lymphomas is being planned. DisclosuresRosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding. Arnason:Gilead: Consultancy. Kufe:Genus Oncology LLC: Consultancy, Equity Ownership.

A Functional Characterization of BCL2-Family Members Identifies BH3 Mimetics As Potential Therapeutics in T-Cell Lymphomas

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of lymphoid malignancies with generally poor outcomes when treated with current regimens. The pro-survival BCL-2 family members BCL-2, BCL-xL, and MCL-1 contribute to tumor maintenance, progression, and chemoresistance across a range of cancers but their contributions in distinct subtypes of PTCL are poorly understood. Immunohistochemical analyses of PTCL specimens have revealed a distinct expression pattern of BCL-2 family members, most notably the high level expression of BCL-2 in up to 50% of certain PTCL entities (Rassidakis et al., J Pathol 2003). In fact, high BCL-2 expression has been associated with unfavorable prognosis (Ling et al., Biomed Environ Sci 2011).We amassed a collection of 21 T-cell lymphoma cell lines (representing Alk+ ALCL, Alk- ALCL, PTCL-NOS, cutaneous T-cell lymphoma (CTCL) and rare subtypes) and 7 patient-derived xenograft (PDX) models of T-cell lymphoma. The latter include models of Alk+ ALCL, Alk- ALCL, ATLL, NK-T cell lymphoma and AITL (available at http://www.PRoXe.org) (Townsend et al. Cancer Cell 2016). To assess the expression level and protein abundance of BCL2 family members, we performed RNA-Seq and immunoblotting. To functionally characterize dependence on BCL-2 family members, we utilized BH3 profiling, a technique that allows for assessment for how "primed" or close to the cell death threshold cells are by evaluating the degree of mitochondrial outer membrane permeabilization (MOMP), induced by a panel of BH3 domain peptides (Ryan and Letai, Cell Death and Differentiation 2013). Binding specificity of BH3 domain peptides allows for determination of which pro-survival Bcl-2 family members cells are dependent on for survival and thus makes it a powerful tool to predict response to BH3 mimetics. Finally, we assessed in vitro the cytotoxicity induced by the BH3 mimetics venetocxlax (ABT-199, a BCL-2 specific agent) and navitoclax (ABT-263, which targets both BCL-2 and BCL-xL) in PTCL cell lines.Gene expression and protein levels of the anti-apoptotic BCL-2 family members showed a distinct pattern in the cell lines that closely recapitulated immunohistochemical analysis of clinical samples (Rassidakis et al., J Pathol 2003). Specifically, both MCL-1 and BCL-xL were ubiquitously expressed, with higher levels of MCL-1 in ALCL cell lines and the PTCL-NOS cell line SMZ-1, while BCL-xL was highly expressed predominately in CTCL cell lines. While cell lines and PDX models from Alk+ ALCL and CTCL universally did not express BCL-2, approximately two-thirds of cell lines and PDX models representing Alk- ALCL, PTCL-NOS, AITL, NK/T-cell lymphoma, ATLL and rare subtypes of T-cell lymphomas did express BCL-2. Despite this expression, only 3 of 8 BCL2-expressing cell lines were sensitive to ABT-199 (IC50<1 µM), indicating that BCL2 expression is an inadequate biomarker for ABT-199 sensitivity. In contrast, BH3 profiling of these models identified either exclusive BCL-2 dependence, which correlated with sensitivity to ABT-199 in vitro, or exclusive MCL-1 dependence, which correlated with resistance to ABT-199. Alk+ and Alk- ALCL cell lines and PDX models were predominately MCL-1 dependent, but some also showed co-dependence on BCL-xL that correlated with sensitivity to ABT-263 in vitro. Among CTCL cell lines, we identified a dominant BCL-xL dependence that correlated with low nanomolar IC50 to ABT-263. In line with this, primary samples of CTCL (n=3) also showed BCL-xL dependence, offering a novel therapeutic strategy for this disease. Table 1 shows a representative illustration of these data in a selection of cell lines.In summary, we have defined distinct classes of BCL-2 family member dependence that are revealed by BH3 profiling and predict sensitivity or resistance to available clinical agents. [Display omitted] DisclosuresLetai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Weinstock:Novartis: Consultancy, Research Funding.

Effects of acitretin and interferon on the proliferative activity of and interleukin-15 expression in a human cutaneous T-cell lymphoma cell line Hut78

Objective To evaluate effects of acitretin and interferon-α (INF-α) alone or in combination on the proliferative activity of and interleukin-15 expression in human cutaneous T-cell lymphoma Hut78 cells. Methods Cultured Hut78 cells were divided into several groups, including blank control group, negative control group, dimethyl sulphoxide (DMSO) group and experimental groups. Cells in experimental groups were additionally classified into several subgroups to be treated with acitretin (0.1-10 μmol/L, acitretin groups) or INF-α (5 000-20 000 IU/ml, INF-α groups) alone, or the combination of 1.0 μmol/L acitretin and IFN-α at concentrations of 5 000-20 000 IU/ml (combination groups) , for 24, 48 and 72 hours. Subsequently, cell counting kit 8 (CCK8) assay was performed to assess the proliferative activity of Hut78 cells, and enzyme-linked immunosorbent assay (ELISA) to measure the expression of IL-15 in these cells. Results The proliferative activity of and IL-15 expression in Hut78 cells were both obviously suppressed in the acitretin groups and combination groups compared with the DMSO group, as well as in the INF-α groups compared with the negative control group, and the inhibitory effects gradually increased with the increase in acitretin or INF-α concentrations and treatment durations. As repeated measures analysis of variance revealed, there was a significant difference in both proliferation inhibition rates and IL-15 expression among different treatment durations and among different concentrations of acitretin or INF-α (all P < 0.05) , and there was an interaction effect between treatment durations and drug concentrations (all P < 0.05). A significant difference was observed in both proliferation inhibition rates and IL-15 expression at 24, 48 and 72 hours when the 1.0-μmol/L acitretin+ 10 000/20 000-IU/ml IFN-α group was compared with the 1.0-μmol/L acitretin group and 10 000/20 000 IU/ml IFN-α group (all P < 0.05). There was also a significant difference in IL-15 expression at 24, 48 and 72 hours between the 1.0-μmol/L acitretin+ 50 000-IU/ml IFN-α group and 5 000-IU/ml IFN-α group (all P < 0.05) . Conclusions Acitretin and IFN-α both can inhibit the proliferation of and IL-15 expression in Hut78 cells, the inhibitory effects are enhanced with the increase in drug concentrations and treatment durations, and the combination of acitretin and IFN-α appears to have stronger inhibitory effects than acitretin or IFN-α alone. Key words: Lymphoma, T-cell, cutaneous; Acitretin; Interferon-alpha; Interleukin-15; Cell proliferation; Cell line, tumor; Hut78 cells

Thymic Stromal Chemokine TSLP Acts through Th2 Cytokine Production to Induce Cutaneous T-cell Lymphoma.

Thymic stromal lymphopoietin (TSLP) activates dendritic cells to induce Th2-mediated inflammation. Periostin, an extracellular matrix protein produced by fibroblasts, induces chronic inflammation by stimulating TSLP production. Recently, a reinforcing cycle linking Th2-type immune responses with periostin-induced keratinocyte activation has been proposed in atopic dermatitis pathogenesis. In this study, we investigated the role of TSLP and periostin in the development of cutaneous T-cell lymphoma (CTCL), where Th2 cytokines and chemokines are also dominant. TSLP and periostin mRNA expression levels were elevated in CTCL lesional skin, both of which correlated with IL4 expression levels. In vitro and ex vivo, IL4 or IL13 stimulated periostin expression by dermal fibroblasts, and fibroblasts from CTCL lesional skin expressed higher levels of periostin than those from control skin. Serum periostin levels of CTCL patients were also significantly higher than those of healthy individuals. Hut78 and MJ, CTCL cell lines, and peripheral blood mononuclear cells from leukemic CTCL patients expressed the TSLP receptor. TSLP induced production of IL4 and IL13 by Hut78 and MJ cells through the activation of STAT5. Moreover, TSLP induced proliferation of CTCL cells both in vitro and in vivo These data suggest that periostin-mediated TSLP production by keratinocytes directly stimulates CTCL tumor cell growth in addition to inducing a Th2-dominant tumor environment in CTCL. Cancer Res; 76(21); 6241-52. ©2016 AACR.

Increased serum soluble DNAM-1 levels and decreased membrane DNAM-1 expression on NK cells and CD8+ cells in cutaneous T-cell lymphoma

Increased serum soluble DNAM-1 levels and decreased membrane DNAM-1 expression on NK cells and CD8+ cells in cutaneous T-cell lymphoma

Modeling, Synthesis, and Biological Evaluation of Potential Retinoid X Receptor (RXR)-Selective Agonists: Analogues of 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and 6-(Ethyl(5,5,8,8-tetrahydronaphthalen-2-yl)amino)nicotinic Acid (NEt-TMN).

Sulfonic acid analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene, 1) as well as seven novel and two reported analogues of 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN) were synthesized and assessed for selective retinoid X receptor (RXR) agonism. Compound 1 is FDA-approved for treatment of cutaneous T-cell lymphoma (CTCL); however, 1 can provoke side effects by impacting RXR-dependent receptor pathways. All of the analogues in this study were evaluated for their potential to bind RXR through modeling and then assayed in an RXR-RXR mammalian-2-hybrid (M2H) system and in RXR-responsive element (RXRE)-mediated transcriptional experiments. The EC50 profiles for these unique analogues and their analogous effectiveness to inhibit proliferation in CTCL cells relative to 1 suggest that these compounds possess similar or even enhanced therapeutic potential. Several compounds also displayed more selective RXR activation with minimal cross-signaling of the retinoic acid receptor. These results suggest that modifications of potent RXR agonists such as NEt-TMN can lead to improved biological selectivity and potency compared with the known therapeutic.

Dimethyl fumarate restores apoptosis sensitivity and inhibits tumor growth and metastasis in CTCL by targeting NF-κB

AbstractDespite intensive efforts in recent years, a curative therapy for cutaneous T-cell lymphoma (CTCL) has not yet been developed. Therefore, the establishment of new therapeutic approaches with higher efficacy rates and milder side effects is strongly desired. A characteristic feature of the malignant T-cell population in CTCL is resistance toward cell death resulting from constitutive NF-κB activation. Therefore, NF-κB–dependent cell death resistance represents an interesting therapeutic target in CTCL because an NF-κB–directed therapy would leave bystander T cells widely unaffected. We investigated the effects of dimethyl fumarate (DMF) on CTCL cells in vitro and in vivo. DMF induced cell death in primary patient-derived CD4+ cells and CTCL cell lines, but hardly in T cells from healthy donors. DMF-induced cell death was linked specifically to NF-κB inhibition. To study the impact of DMF in vivo, we developed 2 CTCL xenograft mouse models with different cutaneous localizations of the T-cell infiltrate. DMF treatment delayed the growth of CTCL tumors and prevented formation of distant metastases. In addition, DMF induced increased cell death in primary CTCL tumors and in liver metastases. In summary, DMF treatment represents a remarkable therapeutic option in CTCL because it restores CTCL apoptosis in vitro and in preclinical models in vivo and prevents spreading of the disease to distant sites. DMF treatment is of particular promise in CTCL because DMF is already in successful clinical use in the treatment of psoriasis and multiple sclerosis allowing fast translation into clinical studies in CTCL.

Abstract 124: Molecular pathogenesis of an advanced cutaneous T-cell lymphoma

Abstract Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma (CTCL). MF is primarily a benign disease often confined to skin. MF, in about 10% of cases, can progress to SS or transform to large-cell histology. SS, the leukemic variant of CTCL, is defined as erythroderma on ≥ 80% of the skin plus ≥1000/μL circulating malignant T-cells. SS is very aggressive with a median overall survival of only 2-5 years. So far, effective treatment options for CTCLs are still very limited and the underlying genetic basis remains incompletely characterized. Here, we performed integrated genomic analyses of 105 patients with de novo or transformed SS, using whole-exome, targeted exon, transcriptome sequencing and SNP array. Frequent somatic alterations were identified in TP53, CARD11, CCR4, PLCG1, TNFRSF1B, CDKN2A, ARID1A, RPS6KA1 and ZEB1. Activating CCR4 and CARD11 mutations were detected in nearly one third of patients. CCR4 is important for T-cell migration into the skin. All CCR4 mutations were either nonsense or frameshift and located at the C-terminus. CARD11 is required for T cell receptor (TCR)-mediated activation of NF-κB signaling. About half of the CARD11 mutations were located in the domain previously reported in diffuse large B cell lymphoma (DLBCL). The remaining mutations were located at a highly conserved new hotspot. As was previously suggested in DLBCL, therapies targeting CARD11 signaling could benefit SS patients. PLCG1 is a key modulator of TCR signaling. In addition to the recently reported S345F hotspot mutation, we identified a new hotspot located in the C-terminal of the protein. The mutation frequency of the new hotspot was significantly higher in patients with prior history of MF, suggesting the possibility that PLCG1 may play a role in transformation of MF to SS. Focal deletion of ZEB1 was observed in over half of the patients. ZEB1 is suggested to be critical for early T-cell development. ZEB1 downregulation contributes to resistance to TGF-β1-mediated growth suppression in adult T-cell leukemia/lymphoma derived cell lines. Loss of ZEB1 may play a similar role in SS pathogenesis. IL32 and IL2RG were strongly upregulated in nearly all patients. IL32 protein expression is correlated with the mRNA expression and the density of Sézary cells in patients’ blood. IL32 accelerates the proliferation of CTCL cell lines through mitogen-activated protein kinase and NF-κB-mediated signaling. IL32 could become a target that is specific to SS patients. Surprisingly, the UVB mutation signature was detected in most of the cases, indicating that the malignant clonal population arose from a cell that spent significant time in the superficial layers of the skin and challenging the view that SS originated from the circulating memory T-cell rather than those resident in the skin. Our results showed profound disruption of key T-cell signaling in SS patients and suggested potential targets for novel therapies. Citation Format: Linghua Wang, Xiao Ni, Kyle R. Covington, Liu Xi, David A. Wheeler, Madeleine Duvic. Molecular pathogenesis of an advanced cutaneous T-cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 124.

Abstract 936: Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome

Abstract Introduction Sézary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients. Methods Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement. Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines. Results Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P = 0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc = 4.83), mTOR (Fc = 4.61) mP70S6K (Fc = 4.64) BAD (Fc = 7.09) and 4EBP1 (Fc = 5.40)in skin-SS cells respect to circulating ones. We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients.. Conclusion: Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome. Citation Format: Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 936.

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

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. 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. 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. 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.

TOX expression and role in CTCL.

Cutaneous T-cell lymphomas (CTCL) are skin malignancies including mycosis fungoides (MF) and CD30(+) lymphoproliferative disorders (LPD). In early disease, CTCL can be difficult to diagnose, especially in MF for which there is no reliable diagnostic marker. MF/CTCL have increased expression of thymocyte selection-associated HMG box protein (TOX). Although TOX has been proposed to be a diagnostic marker for MF, further validation studies are needed. Moreover, it is unclear what drives TOX expression or its role in MF/CTCL. We hypothesize evaluation of TOX levels across a spectrum of CTCL, including MF precursor (large plaque parapsoriasis, LPP), will help elucidate the implications of altered TOX expression. TOX staining was performed in MF, CD30(+) LPD, LPP as well as benign inflammatory dermatoses (BID) and normal skin (NS). CTCL cell lines were utilized to evaluate the regulation of TOX. Positive TOX expression was identified in 73.6% of MF cases and in 31.6% of BID/NS. TOX had a positive predictive value (PPV) for MF of 86.7% and a negative predictive value (NPV) of 48.1%. TOX expression in MF was detected more commonly in Black patients (P = 0.015) and less commonly in transformed MF (P = 0.045). LPP had positive TOX staining in 70.0%. In CTCL cells, GATA3 knockdown decreased TOX mRNA and protein expression. TOX expression also decreased in the presence of CTCL therapeutics. Our data indicate that TOX is useful as a diagnostic marker in MF. Moreover, TOX expression was evident in LPP, indicating it may have a previously unappreciated role in the development of MF. Finally, our data suggest that GATA3 regulates TOX, revealing insight into TOX regulation.

Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma.

Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53−/−). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.

Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines: A Useful Model for Rapid Biological and Therapeutic Evaluation

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for invivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for invivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and invivo drug testing.

Th17 cytokine differentiation and loss of plasticity after SOCS1 inactivation in a cutaneous T-cell lymphoma.

We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 – STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.

086 Combination of retinoid and histone deacetylase inhibitor produced an anti-tumor effect in cutaneous T-cell lymphoma by restoring tumor suppressor gene, retinoic acid receptorβ2, via histone acetylation

Retinoids exert anti-proliferative, differentiative, and apoptosis-inducing effects through their receptors. Retinoic acid receptor (RAR) β2 behaves as a tumor suppressor gene, and its expression is suppressible by DNA methylation in many malignancies. We aimed to determine whether combining a retinoid, Am 80, with a histone deacetylase inhibitor, MS-275, could suppress tumor growth in a RARβ2-negative human cutaneous T cell lymphoma (CTCL) cell lines and freshly isolated primary CTCL cells, and to elucidate the epigenetic mechanism behind the phenomena. SeAx cells were implanted subcutaneously in NOD-SCID mice which were randomly divided into four groups and treated with either Am80, MS-275 by oral gavage (five days/week), or a combination of the two agents. Cell proliferation assay, methylation-specific PCR, flow cytometric analysis of cell cycle and apoptosis and chromatin immunoprecipitation assay were employed.Quantitative PCR analysis revealed that RARβ2 gene expression was restored only by this combination rather than by either of the agents singly. Restored retinoid sensitivity was observed in combining retinoid with a histone deacetylase inhibitor significantly inhibited cell growth in vitro, suppressed subcutaneously transplanted tumor growth, and prolonged survival of tumor-bearing mice in vivo by more strongly inducing apoptosis and p21 expression in CTCL cells than either agent alone. In the combination treatment, the histone H4 acetylation level at lysine 12 and 16 in the promoter region increased after restoration of RARβ2 expression although the DNA methylation of RARβ2 remained unchanged. This is the first report of histone acetylation as the primary event in the restoration of RARβ2. Inducible RARβ2 expression may serve as a reliable predictor for tumor response in patients undergoing ‘epigenetic & differentiation’ therapy.

Romidepsin and Azacitidine Synergize in their Epigenetic Modulatory Effects to Induce Apoptosis in CTCL.

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of malignancies that despite available therapies commonly relapse. The emergence of combination epigenetic therapies in other hematologic malignancies have made investigation of such combinations in CTCL a priority. Here, we explore the synergistic antiproliferative effects of romidepsin, an HDAC inhibitor, and azacitidine, a demethylating agent, combination in CTCL. The growth inhibition under combination treatment and single agent was explored by the MTT cell viability assay and the Annexin V/propidium iodide (PI) apoptosis assay in different CTCL cell lines and tumor cells derived from Sézary syndrome patients. Quantitative analysis of a dose-effect relationship of romidepsin and azacitidine was done by the CompuSyn software. Investigation of mechanism of action was performed by flow cytometry, immunoblotting, qRT-PCR arrays, and chromatin immunoprecipitation. Global CpG methylation sequencing was utilized to study genome methylation alteration under the treatment modalities. The combination of romidepsin and azacitidine exerts synergistic antiproliferative effects and induction of apoptosis involving activation of the caspase cascade in CTCL cell lines and tumor cells derived from Sézary syndrome patients. We identified genes that were selectively induced by the combination treatment, such as the tumor suppressor geneRhoBthat is linked to enhanced histone acetylation at its promoter region in parallel with pronounced expression of p21. Global CpG methylation sequencing in a CTCL cell line and tumor cells demonstrated a subset of genes with a unique change in methylation profile in the combination treatment. The synergistic antiproliferative effects of romidepsin and azacitidine combination treatment justify further exploration in clinical trials for advanced CTCL.

The Effects of Arsenic Trioxide in Combination with Retinoic Acids on Cutaneous T-Cell Lymphoma Cell Lines

Cutaneous T-cell lymphomas (CTCL) are characterized by an infiltration of the skin with malignant T cells. Curative treatments for aggressive entities such as Sézary syndrome have not been identified yet. Arsenic trioxide (AsO₃) is used for the treatment of acute promyelocytic leukemia in combination with retinoids. As the latter are established treatment options in CTCL, we sought to evaluate the efficacy of AsO₃ for mono- and combination therapy in vitro. Analyses for apoptosis, cell cycle inhibition, cytotoxicity and cell viability were made after incubation of CTCL cells with AsO₃ alone or in combination with the retinoids all-trans-retinoic acid or bexarotene. While AsO₃ induced apoptosis, retinoids did not at the time point of analysis. However, retinoids strongly reduced cell viability. Due to the efficient apoptosis induction, AsO₃ might be a potentially suitable agent for CTCL treatment, although this effect was not increased by retinoids.

Effects of chidamide combined with curcumin on human cutaneous T-cell lymphoma cell line Hut78 and their molecular mechanisms

Objective To evaluate the inhibitory effect of chidamide combined with curcumin on the proliferation of cutaneous T-cell lymphoma (CTCL) cell line Hut78, as well as their promotive effect on its apoptosis, and to explore their therapeutic mechanisms in CTCL. Methods Some Hut78 cells were treated with different concentrations of chidamide (0.3, 0.6, 1.2, 2.4 μmol/L) and 10 μmol/L curcumin alone or the combination of 1.2 μmol/L chidamide and 10 μmol/L curcumin for 24, 48 and 72 hours separately. MTS assay was conducted to estimate cell viability at each time point. After selection of chidamide concentrations, some Hut78 cells were treated with chidamide (0.6 and 1.2 μmol/L) and curcumin (10 μmol/L) alone or in combination (1.2 μmol/L chidamide and 10 μmol/L curcumin) for 24 hours, then, flow cytometry was performed to detect cell apoptosis and analyze cell cycle, real-time (RT) -PCR and Western-blot analysis were conducted to quantify the mRNA and protein expressions of apoptosis-associated genes Fas, caspase 8, nuclear factor (NF) -κB p65 as well as cell cycle-associated genes P21, CDK2 and cyclin E respectively. Statistical analysis was carried out by repeated-measures analysis of variance, one-way analysis of variance and the least significant difference (LSD) -t test. Results Chidamide could significantly inhibit the proliferation of Hut78 cells in a dose-dependent and time-dependent manner (F = 266.558, 564.966, respectively, both P 0.05) . Western-blot analysis showed that protein expressions of Fas, caspase 8 and P21 significantly increased, but those of NF-κB p65, CDK2 and cyclin E significantly decreased in the combined treatment group compared with the 0.6-, 1.2-μmol/L chidamide groups and blank control group receiving no treatment, which were in accordance with the above changes in mRNA expressions of these genes. Conclusion Chidamide can inhibit the growth of the CTCL cell line Hut78 by directly decelerating cell proliferation and inducing cell apoptosis, and the combibation with curcumin can markedly enhance the inhibitory effect of chidamide on the growth of Hut78 cells. Key words: Lymphoma, T-cell, cutaneous; Curcumin; Cell proliferation; Apoptosis; Chidamide; Hut-78 cell

Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma

Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells fromSS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAPexpression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment.

Disruption of CCL20-CCR6 interaction inhibits metastasis of advanced cutaneous T-cell lymphoma.

We recently demonstrated that upregulation of a chemokine receptor CCR6 and its ligand CCL20 led to metastasis of advanced cutaneous T-cell lymphoma (CTCL) cells, suggesting the involvement of CCL20-CCR6 interaction in initiating CTCL cell metastasis. In this study, we determined whether this interaction is functional in metastatic CTCL cells. We first demonstrated increased STAT3 expression during the progression of primary CTCL. STAT3 was spontaneously activated and mediated the transcription of CCL20 in CTCL cell lines. Next, to determine whether the transient knockdown of STAT3, CCL20, or CCR6 or treatment with neutralizing antibody against CCL20 (neutralizing CCL20 antibody) could reduce the migration ability of CTCL cells, we conducted an in vitro migration assay. All treatments reduced the nutrition-dependent migration activity of CTCL cells. Notably, treatment with neutralizing CCL20 antibody reduced the migration ability of the cells without decreasing the expression of CCL20 and CCR6. This demonstrated that the CCL20-CCR6 interaction is actually functional in metastatic CTCL cells. Finally, to examine the in vivo effect of neutralizing CCL20 antibody, we used NOD/Shi-scid IL-2γnul mice inoculated with CTCL cells. These mice were expected to die due to metastasis of CTCL cells into multiple organs. However, administration of neutralizing CCL20 antibody significantly prolonged the survival of the xenografted mice. These findings suggested that automatic activation of the STAT3/CCL20/CCR6 cascade was involved in CTCL lymphomagenesis and that disruption of CCL20-CCR6 interaction could be a key therapeutic strategy against advanced CTCL.