HUT-78 Cells Research Articles

5-Azacitidine induces demethylation of PTPL1 and inhibits growth in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) consists of various lymphoid malignancies with a diverse clinical pathology and biological characteristics. Methylation of cytosine residues by DNA methyltransferases at CpG dinucleotides in the promoter region of the genes is a major epigenetic modification in mammalian genomes that can have profound effects on gene expression. The PTPL1 methylation pattern was screened by methylation-specific polymerase chain reaction (MSP) in 7 lymphoma-derived cell lines and in 47 samples of diffuse large B cell lymphoma (DLBCL). The PTPL1 gene was hypermethylated in the CA46, Raji, Jurkat and DB cell lines; however, it was unmethylated in the Hut78, Maver and Z138 cell lines. The expression of PTPL1 mRNA was re-inducible by 5-azacytidine (5-Aza), an agent of DNA demethylation. The methylations were detected in 59.6% of DLBCL versus 6.3% in reactive lymph node proliferation. Therefore, the present data showed that PTPL1 was epigenetically regulated in NHL suggesting an involvement of the PTPL1 tumor-suppressor genes in NHL, and highlights 5-Aza as a potential therapeutic candidate for NHL.

Synthesis of structural analogues of hexadecylphosphocholine and their antineoplastic, antimicrobial and amoebicidal activity

Twelve derivatives of hexadecylphosphocholine (miltefosine) were synthesized to determine how the position and length of the alkyl chain within the molecule influence their biological activities. The prepared alkylphosphocholines have the same molecular formula as miltefosine. Activity of the compounds was studied against a spectrum of tumour cells, two species of protozoans, bacteria and yeast. Antitumour efficacy of some alkylphosphocholines measured up on MCF-7, A2780, HUT-78 and THP-1 cell lines was higher than that of miltefosine. The compounds showed antiprotozoal activity against Acanthamoeba lugdunensis and Acanthamoeba quina. Some of them also possess fungicidal activity against Candida albicans equal to miltefosine. No antibacterial activity was observed against Staphylococcus aureus and Escherichia coli. A difference in position of a long hydrocarbon chain within the structure with maximum efficacy was observed for antitumour, antiprotozoal and antifungal activity.

Effect of ursolic acid on proliferation of T lymphoma cell lines Hut-78 cells and its mechanism

目的探讨乌索酸对T细胞淋巴瘤细胞株Hut-78细胞体外增殖的影响及其作用机制。方法采用MTT法观察不同浓度（10、20、40、80 µmol/L）乌索酸作用不同时间(4、12、24、48、72 h)对Hut-78细胞增殖的抑制作用，并采用流式细胞术分析其早期凋亡的情况。采用免疫印迹法检测乌索酸作用后Hut-78细胞细胞核因子-κB（NF-κB）p65、p50、p52和p100亚基蛋白，以及caspase-8、3、9蛋白表达变化，采用逆转录PCR法检测血管内皮生长因子(VEGF)和环氧合酶-2(COX-2）mRNA表达水平。结果与不加药对照组比较，不同浓度的乌索酸作用组Hut-78细胞的增殖均受抑制（P值均<0.05）；Annexin Ⅴ+/PI−细胞比例均升高（P值均<0.05）；经10、20、40、80 µmol/L乌索酸处理Hut-78细胞72 h后，Hut-78细胞NF-κB p65、p50蛋白和VEGF、COX-2 mRNA表达水平均下降（P值均<0.05），caspase-8、3、9蛋白表达均增加（P值均<0.05）；NF-κB p100、p52蛋白表达差异均无统计学意义（P值均>0.05）。上述作用均呈剂量和（或）时间依赖性（P值均<0.05）。结论乌索酸抑制Hut-78细胞增殖，作用可能与促其凋亡有关，经过死亡受体和线粒体途径完成；影响NF-κB经典信号路径可能是其机制之一，VEGF和COX-2可能也参与其中。

Bortezomib and Methotrexate Interfere with the DNA Repair Signaling Transduction Pathways and Induce Apoptosis in Cutaneous T-Cell Lymphoma

Introduction:Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the commonest types and together comprise 54% of all CTCL. MF evolves from patches to infiltrated plaques and eventually tumors. SS is a lymphoma-leukemia syndrome characterized by erythroderma and the presence of a malignant T-cell clone in the peripheral blood and the skin. At present, no curative treatment for CTCL is available. Therefore current CTCL research efforts are focused on elucidating the molecular mechanisms of the disease’s pathogenesis and on identifying new pharmacological targets. Several drugs have shown potentially significant activity either alone or in combination with conventional agents. Their effectiveness and their mechanisms of action comprise a current research challenge for the improvement of CTCL therapy. The aim of this study was to investigate the possible alterations in the gene expression profile (focusing on DNA Damage Signaling and DNA Repair pathways) and cell death in CTCL cell lines after treatment with two chemotherapeutic agents, Bortezomib and Methotrexate.Methods:Three CTCL cell lines were used. MyLa, (MF), SeAx and Hut-78 (both SS). Cells were cultured in RPMI 1640 and were treated with either Bortezomib (10nmol/L) or Methotrexate (10μM) for 24h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. Gene expression profiling following PCR arrays analysis was performed after total RNA extraction and purification from untreated and drug-treated cells. All RNA samples’ amplification, labeling and hybridization to RT2 Profiler PCR Arrays (DNA Damage Signaling and DNA Repair PCR array) (QIAGEN) were performed according to the manufacturer’s instructions. All data were analyzed using the appropriate RT2 Profiler PCR Array data analysis tool.Results:Hut-78, Seax and Myla cells responded with statistically significant enhanced apoptosis when treated for 24h with bortezomib, compared to untreated cells, while Methotrexate led to a rather moderate increase of apoptosis in Hut-78 and Seax cells and did not affect the apoptosis of Myla cells. Microarrays analysis after bortezomib treatment revealed a great effect in the expression profile of genes involved in almost all DNA repair pathways tested, in all three cell lines, with Hut-78 being affected the most. Specifically, in all cell lines, there was a significant down-regulation of a large number of genes involved in the Double Strand Breaks DNA Repair mechanism, (i.e. BRCA1, BRCA2, RAD50, RAD51, RAD51C, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6) as well as of genes involved in the Mismatch Repair pathway (i.e. MLH1, MLH3, MSH2, MSH5, MSH6) and the Nucleotide Excision Repair mechanism (i.e. DDB2, LIG1 and RAD23A), compared to untreated cells. On the contrary, bortezomib had a small effect on Base Excision repair mechanism, mostly downregulating the expression of XRCC1 gene in Hut-78 and Myla cells. Methotrexate treatment also led to a significant down-regulation of genes involved in the DSB (RAD50, XRCC4, XRCC6), MMR (MSH4) and NER (CDK7, RAD23A) repair mechanisms in Hut-78 cells but had a rather much more moderate effect on the expression profile of Seax and Myla cells, where fewer genes were affected.Conclusions:Our data clearly demonstrate a differential effect of bortezomib and methotrexate in terms of apoptosis induction on CTCL cells with bortezomib inducing apoptosis of both MF and SS derived cell lines and methotrexate being rather inactive on SS derived cells. We showed that both drugs, but mostly bortezomib significantly down-regulate a large number of genes involved in the DSB, MMR and NER mechanisms, suggesting a possible mechanism, among probably others, for the enhanced sensitivity to apoptosis of SS and MF cell lines after treatment. Bortezomib’s significant effect could be easily understood, since it is a well known proteasome inhibitor and has been previously related to inhibition of NF-kB and accumulation of pro-apoptotic proteins, while it has also been reported that cancer cells are more sensitive to proteasome inhibition than normal cells. Although these results need to be further confirmed, they appear very encouraging for understanding the mechanisms of action of these drugs in CTCL with the view to ameliorate their use in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Romidepsin Synergizes with Lenalidomide in T Cell Lymphoma Cell Lines By Increasing Reactive Oxygen Species and Modulating PI3K/AKT and MAPK/ERK Signaling Pathways

Abstract Background and purpose.T-cell lymphoma (TCL) is an heterogeneous and aggressive group of lymphoproliferative disorders which commonly relapse with current chemotherapy regimens and then difficult to treat. Therefore, novel treatment approach are urgently needed. Romidepsin is a potent and specific inhibitor of class I and II HDAC inhibitor that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide (IMiD), is an immunomodulatory agent highly effective for the treatment of a wide range of hematological malignancies with a low toxicity profile. In this study, we sought to define the potential anticancer effect of romidepsin (Celgene) in combination with lenalidomide (Celgene) in TCL preclinical model. Methods . The TCL cell lines Hut-78 and Karpas-299 were treated with increasing concentrations of romidepsin (0,5-25 nM) and lenalidomide (1-100 µM) as single agents from 24-72h to identify the IC50 of each drug. The interaction between romidepsin (0,5nM, 1nM, 2,5nM) and lenalidomide (2µM, 4µM, 10µM) was evaluated using the Chou-Talalay method. The cell proliferation was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. The cell cytotoxicity was quantified by MTT. Clonogenic survival was studied with the methylcellulose clonogenic assay. Apoptosis, reactive oxygen species (ROS), cell cycle analysis and Bcl-2 protein expression were measured by flow cytometry, whereas caspase activation, AKT/PI3K and MAPK/ERK pathways were analyzed by Western blot. Results. Treatment with romidepsin alone resulted in time- and dose-dependent increase in cytotoxicity in Hut-78 and Karpas-299 cell lines with an IC50 at 24h of 5.87 nM and 6.36 nM for Hut-78 and Karpas-299, respectively. Lenalidomide alone did not inhibit cells viability up to72 h of treatment in the two TCL cell lines, as we previously described in other cell lines probably due to a more pleitropic (or cytostatic) effects. However, the combination of lenalidomide (10µM) with a low dose of romidepsin (2.5 nM) showed a strong synergistic interaction with combination index (CI) of 0.14 in Hut-78 cells and an additive effect in Karpas-299 cells, (CI of 1.08) after 24h. In Hut-78 cells sequential treatment with romidespin for 6h followed by washout and the next addition of lenalidomide for 24h enhanced the cytotoxic effect of romidepsin and confirmed its irreversible effect. Treatment with romidepsin and lenalidomide for 24 h reduced clonogenic survival to approximately 15 % in HUT-78 and to 28 % in Karpas-299. The combination did not trigger relevant decreased in the viability of normal peripheral blood mononuclear cells and suppressed cell proliferation of TCL cell lines when co-cultured with bone marrow mesenchymal stromal cells in a system that mimics the bone marrow microenvironment. Romidepsin and lenalidomide in combination induced apoptosis with little effect on cell cycle. Interestingly, the treatment with the caspase inhibitor z-VAD-fmk allowed detection of cell cycle arrest in G0/G1 phase and a decrease of S phase. The induction of apoptosis by drugs combination was associated with an increase in ROS production. The co-administration of the antioxidant N-acetyl cysteine (NAC) blocked the increased ROS generation and apoptosis observed in the drugs combination compared with control or single-agent treatments. Cell death was associated with the activation of caspases -3, -9 -8 and PARP and was mediated by Bim, Bax, Bad, Bcl-xL and Mcl-1 but did not modify Bcl-2 expression. The drugs combination induced up-regulation of cell cycle protein p21, p27 and a slight decrease of cyclin E and cyclin D. These events were associated with the dephosphorylation of PI3K/Akt and MAPK/ERK pathways. Finally, the induction of histone acetylation (H3) and acetylated alpha-tubulin was confirmed. Conclusion . The combination of romidepsin with lenalidomide triggered cell death through interacting mechanisms including ROS generation, caspase activation and down-regulation of PI3K/AKT and MAPK/ERK pathways. These results provided a rational basis for the clinical development of the combination of romidepsin and lenalidomide for the treatment of TCL Disclosures No relevant conflicts of interest to declare.

Integrin αvβ3 Transduces Survival and Angiogenic Signals to T Cell Lymphomas and Is a Therapeutic Target

Malignant T cell proliferation, survival and drug resistant are dependent on a combination of external stimuli delivered by the microenvironment. Previous studies have shown that the transmembrane receptor integrin αVβ3 plays a critical role mediating the interaction of T cell lymphoma (TCL) cells with external signals. Integrin αVβ3 ligands include extracellular matrix-associated signaling proteins and soluble factors such as thyroid hormones (TH). We have also shown that THs stimulate the proliferation of TCLs through complimentary intracellular pathways involving the αVβ3 integrin. We therefore hypothesized that targeting integrin αVβ3 could represent a novel strategy to treat TCL patients. To determine survival pathways induced by TH on αVβ3 integrins, we evaluated the transcriptional changes (RNA-seq) induced by physiological concentrations of cell impermeable T3/T4 coupled to agarose (TH-AG) vs. control in the TCL cell line CUTLL1. We identified 123 up- and 5 down-regulated transcripts (p< 0.01), belonging to “angiogenesis” (e.g. VEFGB), “lymphocyte proliferation/differentiation” and “DNA replication/transcription” (e.g. DBP, IL4, EDF1, DOK2) pathways. Target-gene promoter analysis suggested that TH acting on αVβ3 integrin activated NFkB (that was later confirmed by EMSA-like assays). We then focused on the angiogenesis pathway since (i) VEGF expression and angiogenesis correlate with survival and prognosis in TCL patients, and (ii) we found a positive correlation between integrin αVβ3 and VEGFA and VEGFB expression in 169 cases of TCLs. We analyzed T3/T4-αVβ3-dependent increase of VEGFB and VEGFA in an extended panel of cell lines (n=5) representing the spectrum of immature and peripheral TCL. Similarly to CUTLL1, treatment with TH-AG increased VEGFB and VEGFA mRNA levels in Jurkat (TCL/L), HuT-78 (CTCL), OCI-Ly12 (PTCL-NOS) and Karpas299 (ALCL-ALK+) cells. Increase in VEGF production was completely abrogated by knocking-down either αV or β3components with specific si-RNAs (vs. siRNA control) in CUTLL1, HuT-78 and OCI-Ly12 cells. Moreover, exposing HMEC1 endothelial cells to conditioned medium from CUTLL1 cells treated with TH-AG vs control increased they proliferation and migration (to 481 ± 118 cells from 206 ± 82 cells, respectively, p= 0.01). Importantly, this higher migration was completely abrogated when conditioned medium was obtained from CUTLL1 cells knocked-down for either αV or β3 and treated with TH-AG. To determine whether targeting αVβ3integrin could be of therapeutic benefit for TCL, we developed TCL xenografts in SCID mice using CUTLL1 cells transfected with si-control, si-αV and si-β3, and monitored tumor growth and angiogenesis. We found that CUTLL1 transfected with si-αV and si-β3 developed significant smaller tumors than si-control. Also, tumors from integrin knocked-down cells, showed decreased tumor vascularization (by CD31) and VEGF expression. To determine the translational impact of this strategy, we assessed the effect of cilengitide, a selective αVβ3 integrin inhibitor in phase 3 for glioma, in pre-clinical models of PTCL-NOS and ALCL-ALK+. For PTCL-NOS we xenografted OCI-Ly12 cells in NOD/SCID mice (n=12) and for ALCL-ALK+ we developed a patient-derived tumorgraft (PDT) in NSG mice (n=8). Cilentide treatment for 10 days (vs. vehicle), at human equivalent dose, induced tumor remission in both models (p<0.001, T-test), without toxicity to normal tissues. Similarly to si-αV and si-β3 treated mice, the anti-lymphoma effect of cilengitide correlated with lower levels of angiogenesis and NFkB activation. In sum, we elucidated the mechanism by which integrin αVβ3 activation increases TCL proliferation by activating pro-survival pathways in malignant T cells while promoting angiogenesis. We also showed that genetic and pharmacological targeting of integrin αVβ3 induces anti-lymphoma effect in TCLs, including an ALCL-ALK+ PDT model obtained from an ALK inhibitor refractory patient. DisclosuresNo relevant conflicts of interest to declare.

MUC1 As a Potential Therapeutic Target in Cutaneous T-Cell Lymphoma

Introduction: MUC1 is an oncoprotein that is aberrantly expressed in many epithelial tumors and hematologic malignancies, conferring cell growth and survival. Expression and functional significance of MUC1 in cutaneous T-cell lymphoma (CTCL) has not been previously characterized. In the present study, we evaluated the potential role that MUC1 mediated signaling plays in the pathogenesis of Mycosis Fungoides (MF) and Sezary Syndrome cells (SS), the 2 common subtypes of CTCL.Methods and results: MUC1 was strongly expressed on the SS cell lines HuT-78 (60%), H9 (77%) and SeAx (59%) and the MF cell lines, Myla (94%) and HuT-102 (87%) when incubated with anti-MUC1 antibody and analyzed by flow cytometry (FCM) . In contrast, B-cell lymphoma (BCL) cell lines (Val, K422, Toledo, SU-DHL-2, OCI-Ly7, OCI-Ly8, SU-DHL-5, SU-DHL-8, OCI-Ly3, RCK8, Ramos, Daudi, JVM-2) expressed minimal levels of MUC1 (0-7%), as did circulating T-cell populations isolated from healthy controls (0-3%). The finding of high MUC1 expression on CTCL and minimal expression on BCL and normal circulating T cells was confirmed byimmunoblotting (IB) with another antibody directed against MUC1-C, the oncogenic subunit. The expression profile of MUC1-C was found to be similar at the mRNA level by RT-PCR analysis. We subsequently examined MUC1 as a therapeutic target in CTCL. We have developed an inhibitor of the MUC1-C receptor subunit, designated GO-203, which blocks MUC1-C dimerization necessary for nuclear translocation and downstream signaling. Growth inhibition was determined using CellTiter-Glo that measures ATP produced by live and proliferating cells and demonstrated that the concentration required to inhibit growth by 50% (IC50) following 72-hour exposure ranged from 3 uM to 3.5 uM in most cell lines (HuT-78, H9 and Myla). Culture of HuT-78, H9 and Myla cells in the presence of 5 uM GO-203 concentration for 72-hours resulted in 70, 80 and 90% cell death respectively as determined by Annexin V and propidium iodide staining by FCM. Cell proliferation, as assessed by incorporation of tritiated thymidine, was inhibited by 70% and 80% in 2 cell lines (HuT-78 and Myla respectively) when treated with GO-203 at concentration of 5 uM for 72-hours. To determine mechanism of action of GO-203 induced apoptosis in vitro, level of hydrogen peroxide (H2O2), a reactive oxygen species (ROS) was measured using FCM based conversion of carboxy-2′7′ dichlorodihydrofluorescein diacetate to 2′7′-dichlorodihydrofluorescein. Cell lysates were analyzed for expression of the protein TIGAR, a fructose-2-6-bisphosphatase p53-inducible regulator of glycolysis and apoptosis by IB. Exposure of Hut-78, Myla and H9 cells to GO-203 for 72-hours was associated with an increase in ROS and down regulation of TIGAR expression. To confirm ROS mediated apoptosis by GO-203, in vitro treatment with the antioxidant N-acetylcysteine (NAC) was performed and levels of H2O2 were measured including IB with anti-TIGAR antibody. Treatment with NAC blocked the decreases in TIGAR levels, supporting the hypothesis that this response was mediated by increases in ROS. The ability of GO-203 to eradicate disease was evaluated in vivo across a spectrum of tumor volumes in 2 xenograft mouse models. Five million HuT-78 and 2.5 million Myla cells were injected subcutaneously (SC) into NOD-SCID IL2Rgammanull (NSG) mice. Treatment with GO-203 was administered daily at the dose of 14mg/kg SC for 3 weeks. Tumor volumetric analysis by three-dimensional ultrasound (3D-US) in HuT-78 and Myla xenograft NSG mouse models demonstrated a statistically significant decrease in tumor volume in mice treated with GO-203 compared to control (In the HuT-78 xenograft, average tumor volume was 707 mm3 in the GO-203 treated arm versus 1171 mm3 in the control arm rendering a p-value of 0.0029; whereas in the Myla xenograft average tumor volume was 184 mm3 in the GO-203 treated arm versus 1080 mm3 in the control arm rendering a p-value of 0.0001).Conclusion: The data demonstratesthat MUC1-C is highly expressed in CTCL cell lines, and that blockade of the oncogenic MUC1-C subunit with the peptide inhibitor GO-203 induces apoptosis in vitro and in vivo. Targeting MUC1-C in in vitro is associated with down-regulation of TIGAR and increases in ROS, indicating that MUC1-C functions in maintaining redox balance. Our results highlight the potential of MUC1-C to be an exciting new therapeutic target in CTCL. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

Preclinical Screening of the HDAC6 Inhibitor Rocilinostat (ACY-1215) Combined with Bendamustine in Lymphoma Cell Lines

Background and Purpose. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new therapeutic option for lymphoid malignancies. Rocilinostat (ACY-1215) is a novel selective histone deacetylase 6 (HDAC6) inhibitor. HDAC6 is a class IIB histone deacetylase that plays an important role in cellular response to environmental stress. The purpose of the present study was to evaluate the preclinical activity of HDAC6 inhibitor rocilinostat (Acetylon) alone and the potential of combining of rocilinostat with bendamustine (alkylating agent) in lymphoma cell lines.Methods. Anti-tumor activity of rocilinostat was investigated using a panel of six lymphoma cell lines: two follicular lymphoma (FL) (WSU-NHL, RL), two mantle cell lymphoma (MCL) (Granta-519, Jeko-1) and two T-cell lymphoma (TCL) (HUT-78 - cutaneous T cell lymphoma and Karpas-299 - anaplastic lymphoma cells). IC50 values of each drug were calculated from curves based on rocilinostat concentrations (0,01 - 100 µM), and bendamustine (25 - 300 µM) after 24, 48 and 72 h. The cell proliferation was determined by using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit and cell cytotoxicity with MTT-assay. The interaction between drugs was evaluated by isobologram analysis based upon the Chou-Talalay method to determine if the combination were additive or synergistic. Apoptosis and cell cycle analysis were measured by flow cytometry.Results. Exposure of lymphoma cell lines for 24 - 72 h resulted in time- and dose-dependent inhibition of cell growth with IC50 values ranging from 0.17 to 8.65 μmol/L. Significant cytotoxic effect was evident after 48 hours of rocilinostat incubation by MTT assays with the most sensitive cell lines being WSU-NHL and Hut-78 (IC50: 1.97 – 1.5 μmol/L) and the least sensitive being Granta-519 (IC50: 20 μmol/L). Rocilinostat alone induced time- and dose-dependent increases in apoptosis. After 48 h of treatment with doses ranging from 1 to 20 μmol/L, the percentage of apoptotic cells in early and late apoptosis increased from 11% to 56% and induced an increase in the percentage of cells in the G0/G1 phase of the cell cycle compared with untreated controls.Synergy analyses were done using WSU-NHL, Hut-78 and Granta-519 cells treated with different concentrations of rocilinostat (0, 2, 4 and 8 μmol/L) in combination with bendamustine (0, 10, 20, 40, 50 and 100 μmol/L) and lymphoma cells were assayed by MTT at 24 and 48 h. A clear synergistic interaction, confirmed by the Chou-Talalay method, was observed after 24 h using low concentrations of two drugs which are lower than their IC50 values. Acy-1215 (4 and 8 μmol/L) and bendamustine (20 and 40 μmol/L) showed a synergistic interaction with CI (combination index) values ranging between 0.13 and 0,34 in Hut-78 and WSU-NHL cells. The treatment of Granta-519 cells with rocilinostat (20 and 40 μmol/L) with bendamustine (50 and 100 μmol/L) showed CI values of 0,26 and 0,21 respectively. The drug combinations enhanced apoptosis as assessed by AnnexinV/PI staining. The percentage of apoptosis after 24 h ranged from 55 to 80 %. Furthermore, rocilinostat with bendamustine reduced the proportion of cells in the G0/G1 and S phases and caused an increase of “sub-G0/G1” peak. Finally, the combination of rocilinostat with bendamustine did not trigger relevant decreases in the viability of normal peripheral blood mononuclear cells (PBMNCs).Conclusion. These preclinical results indicate that rocilinostat can have marked activity in lymphoma cell lines in combination with bendamustine. Further investigation is required to continue to study the activity of rocilinostat in lymphoma both as a single agent and in combinations. DisclosuresNo relevant conflicts of interest to declare.

Evidence of an Oncogenic Role for Aberrant Tox Activation in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) consists of two main subtypes: mycosis fungoides (MF), which primarily affects the skin; and Sézary syndrome (SS), which is characterized by the presence of circulating malignant Sézary cells. Although defective apoptosis and cell cycle progression have been implicated in CTCL disease development, the precise molecular mechanism underlying these abnormalities is only partially understood. Recently our group showed that TOX, a transcription factor transiently expressed during thymocyte development, was ectopically expressed in the malignant CD4+ T cells in MF skin lesions. Normally in mature CD4+ T cells, TOX is expressed at very low levels. Not only is TOX highly expressed in the infiltrating CD4+ T cells in MF skin lesions, but its expression levels also inversely correlate with the clinical outcome of CTCL patients. However, its involvement in other types of CTCL remains unknown and its biological effects on CTCL pathogenesis have not been explored. In this study, we sought to examine if aberrant TOX expression occurs in SS (a leukemic variant of CTCL), and whether it contributes to CTCL pathogenesis. We found that TOX expression is also enhanced significantly in primary CD4+CD7- cells from SS patients (n=12), and its enhanced transcript levels correlate with higher disease-specific mortality (P=0.039). To investigate the role of TOX in CTCL, we corrected the ectopic TOX expression by lentiviral-shRNA-mediated TOX gene knock-down in CTCL cell lines (Hut78, HH, and SZ4). In all three cell lines, this resulted in markedly increased apoptosis (measured using Annexin V apoptosis assay, 2-3 fold, P<0.05) and reduced cellular proliferation (assessed by BrdU incorporation assay and colony formation in 3D cultures, 1.5-12 fold, P<0.05). In line with these phenotypes, TOX knock-down led to an increase in cleaved caspases (3, 9) and enhanced expression of multiple cell cycle inhibitors (CDKN1B, CDKN1C). Moreover, to test the effect of TOX suppression on the tumor-inducing ability of CTCL cells in vivo, we injected TOX-suppressed cells or control CTCL cells (1x106 per injection site) subcutaneously into NOD-SCID IL2rγ null (NSG) mice and monitored local tumor formation. Control CTCL cells (Hut78, HH) induced local tumor growth in six out of six mice within 2 weeks of injection, with enlargement progressively to 1.5cm in diameter within 4 weeks of injection. In contrast, TOX silencing effectively reduced the tumor volume induced by HH cells (n=6) and abolished the tumor-forming ability of Hut78 cells (n=6). Since TOX is a DNA-binding nuclear protein, we next sought to explore the potential downstream molecular partners of TOX by performing comparative transcriptome analysis between TOX-suppressed cells and control CTCL cells (Hut78, HH). The expression of several tumor suppressor genes, including SMAD3 and FOXO3, was increased upon TOX knock-down in both Hut78 and HH cell lines. Pathway analyses of the differentially-expressed genes uncovered activation of apoptotic pathways and cell cycle arrest processes upon TOX suppression. These findings were validated using quantitative RT-PCR. In conclusion, our results indicate that ectopic expression of TOX in CTCL cells causes the proliferation/apoptosis balance to shift toward proliferation through suppression of the transcription of multiple tumor suppressors. Therefore abolishing TOX activity may be a promising treatment strategy for CTCL. DisclosuresNo relevant conflicts of interest to declare.

A Role of JAK/STAT Pathway in Cutaneous T-Cell Lymphomas: Exploring Its Effects for Targeted Therapy

INTRODUCTIONDevelopment of targeted therapy in Cutaneous T-Cell Lymphoma (CTCL) patients still requires actionable mutated genes and deregulated pathways to be identified. We have recently published the mutational status of a number of human CTCL lesions, and found JAK/STAT signaling pathway to frequently harbor somatic mutations (Vaqué et al 2014). In this line of evidence, activating mutations in JAK kinases have been reported in human hematological malignancies (Kameda T1 2010) and may serve as indicators for targeted therapy. Therefore, we decided to analyze the mutational status of JAK/STAT pathway in a greater cohort of human CTCL patient samples and cell lines, by using massive parallel sequencing techniques, to shed light on its possible role in the pathogenesis and to uncover its potential as a new therapeutic target for this disease. To this end, we have studied the biological and molecular effects that targeted inhibition of the JAK/STAT signaling pathway, exerts in human CTCL cells, and explore its potential use as a targeted therapy.MATERIAL AND METHODSNGS: We searched for mutations in the catalytic domain of JAK and STAT genes in 39 CTCL patients using targeted NGS techniques: HaloPlex (Agilent) and sequencing in IonTorrent (LifeTech) and Illumina sequencers. Proliferation, apoptosis, cell cycle and DNA synthesis assays: CTCL cell lines were: My-La (MF), HH (MF) and HUT-78 (SS) (ECACC,Salisbury, UK). Effects in proliferation was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA). To analyze the effects on cell survival, we evaluated early and delayed cell death provoked by FACS using FlowCellect Annexin Red Kit (EMD Millipore Corporation, USA). The distribution of cells among different phases of the cell cycle was evaluated by FACS using propidium iodide (PI, Sigma-Aldrich, USA) and Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Technologies-Thermo Fisher Scientific, USA) according to manufacturer’s instruction. Gene expression (GEP) studies: mRNA was extracted with Trizol and mRNA Array-based expression analysis was perfomed using a Whole Human Genome Agilent 4 × 44K v1 Oligonucleotide Microarray.RESULTS:Using our approach we found JAK/STAT pathway mutated in up to 24% of the CTCL samples. 3 mutations were found in JAK1, 5 in JAK3 and 1 in STAT5A genes. Interestingly, most of these mutations affected the tyrosine kinase domain of JAK kinases, a hotspot for activating mutations described in multiple types of human cancer.Thereafter, we decided to explore the biological effects of targeted JAK/STAT inhibition, using specific JAK inhibitors (JAKi) in preclinical CTCL models. To this end, CTCL cell lines were incubated at different time points with increasing concentrations of JAKi. These treatments inhibited JAK/STAT signaling in CTCL cells, as assessed by western-blot using P-STAT-1,-3 and -5 antibodies. Biologically, JAKi provoked a marked inhibition of cell proliferation by a mechanism impinging the control of DNA replication. This was accompanied with a modest increase in cell death. To better understand the molecular mechanisms controlled by aberrant JAK/STAT signaling, we also performed an array-based mRNA expression analysis (GEP) in HUT78 cell line (JAK1Y654F) treated at different time points (0, 0.5 and 3h) with JAKi (at a concentration of IC50). Our results showed a number of genes regulated by JAK/STAT signaling which activity has been described as T-cell activation, differentiation and proliferation (i.e. PAG1,TNF or EGR1).These genes may serve as potential indicators for therapy using JAK/STAT inhibitors or as surrogate markers for drug response.CONCLUSIONSOur results show frequent mutations affecting JAK/STAT downstream signaling in samples from patients with CTCL. Targeted inhibition of this pathway reveals an aberrant CTCL growing controlled by this signaling. Thus, these results provide evidence for the use of JAK/STAT inhibitors on the basis of a targeted massive sequencing analysis in specific cases of CTCL. DisclosuresNo relevant conflicts of interest to declare.

Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo

[Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab.[Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014).[Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. DisclosuresOkamoto:CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

Depletion of M2-Like Tumor-Associated Macrophages Delays Cutaneous T-Cell Lymphoma Development In Vivo

Macrophages have key roles in tumor development and invasion in several human cancers, but little is known about their pathogenic role in cutaneous T-cell lymphoma (CTCL). Herein, we used PCR arrays to profile the expression of inflammatory cytokines in 12 patients with mycosis fungoides (MF), the most common variant of CTCL. Compared with normal controls, MF skin displayed increased mRNA levels of macrophage-related cytokines. Moreover, we detected CD163, a reliable marker of tumor-associated macrophages, in the tumor microenvironment of MF biopsies. To demonstrate that macrophages had a role in CTCL tumorigenesis, we xenografted human CTCL tumor cells in immunocompromised mice and compared tumor development using clodronate-containing liposomes to deplete macrophages in mice. Mice treated with clodronate-containing liposomes show markedly less tumor growth compared with mice treated with phosphate-buffered saline-containing liposomes (P<0.001). We also noted a strong correlation between macrophage depletion and decreased expression of vascular marker, CD31, and lymphatic marker, podoplanin, suggesting a role for macrophages in angiogenesis. In vitro, clodronate-containing liposomes killed activated murine M2 macrophages, but not Hut78 cells, demonstrating selective ability to induce apoptosis in macrophages. Our data indicate that macrophages have a critical role in the progression of Hut78 cell tumor formation in skin, thus providing a new therapeutic strategy for CTCL.

SiRNA therapy in cutaneous T-cell lymphoma cells using polymeric carriers

Cutaneous T-cell lymphomas (CTCLs) arise from specific molecular aberrations that lead to uncontrolled cell proliferation. RNA interference (RNAi) with short interfering RNAs (siRNAs) is a feasible approach to interrupt aberrant signal processing in CTCL cells, but functional biomaterial carriers are needed to effectively deliver siRNAs intracellularly. Towards this goal, we explored the utility of lipid-substituted polyethylenimines (PEI) carriers in a cell model of CTCL. Using caprylic and linoleic acid substituted 2 kDa PEI (PEI-CA and PEI-LA, respectively), we showed effective delivery of siRNA to T-lymphocyte Hut78 and Jurkat cells, but silencing of a model protein (Green Fluorescent Protein, GFP) was possible only in the Hut78 cells. To enhance siRNA delivery to Hut78 cells, a high siRNA: carrier ratio used to assemble the complexes and centrifugation of cells in the presence of complexes were found effective. The toxicities of PEI-CA and PEI-LA were significantly lower than other commercial carriers, 25 kDa PEI and Lipofectamine® RNAiMAX. This might have contributed to reduced siRNA delivery efficiency of the latter carriers. Screening several endogenous targets led us to identify phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and cyclin-dependent kinase 18 (CDK18) as viable targets to induce siRNA-mediated cell growth inhibition. The results of this study identified promising polymeric carriers and molecular targets that could control proliferation of CTCL cells based on RNAi therapy.

Interaction between tumor-associated macrophages (TAMs) and HUT-78 lymphoma cells

This study was aimed to explore the effects of lymphoma cells on the differentiation of monocytes from peripheral blood to tumor-associated macrophages (TAM) and the effect of TAM on proliferation of lymphoma cells in vitro, and investigate the difference between newly diagnosed lymphoma patients and healthy volunteers. Blood samples were obtained from 15 newly diagnosed lymphoma patients and 8 healthy volunteers. Monocytes from peripheral blood were isolated by Ficoll- Hypaque density gradient centrifugation and CD14 immuno-magnetic beads. Then monocytes were directly co-cultured with HUT-78 lymphoma cells by using Transwell apparatus in vitro. Expression of the markers of TAM (CD68 and CD163) were detected by flow cytometry to analyse the proportion of differentiated TAM. Growth curve of HUT-78 cells was made by direct cell count. The IL-10 and VEGF levels in the co-culture system were detected by ELISA. The detection results of newly diagnosed lymphoma patients were compared with that of healthy controls. The results showed that the proportion of CD68(+), CD163(+) and CD68+CD163 (+) cells were significantly up-regulated after co-cultured with HUT-78 lymphoma cells in both patients and healthy controls (P < 0.05). There was no statistical significance in the increasing degree between patients and healthy controls. TAM differentiated from peripheral blood monocytes showed no significant promotion or inhibition on the growth of co-cultured lymphoma cells. For patients, the IL-10 and VEGF levels in the co-culture group were significantly lower than those in two single culture groups (P < 0.05) . For healthy controls, there was no significant difference between these two. It is concluded that lymphoma cells can promote the differentiation of monocytes to macrophages with M2-like phenotype. There is no difference in the promoting degree between patients and healthy controls. TAM differentiated from patients' monocytes significantly down-regulate levels of IL-10 and VEGF in the co-culture system, exhibited functions more like M1 macrophages. In contrast, TAM differentiated from monocytes of healthy controls show no such effects on the co-culture system.

Bcl3 regulates pro-survival and pro-inflammatory gene expression in cutaneous T-cell lymphoma

The advanced stages of cutaneous T cell lymphoma (CTCL) are characterized not only by decreased levels of pro-inflammatory cytokines, resulting in high susceptibility to infections, but also by high constitutive activity of NFκB, which promotes cell survival and resistance to apoptosis. The increased expression of the proto-oncogene Bcl3 belonging to IκB family is associated with the pathogenesis of the different types of human cancer, yet, the function and regulation of Bcl3 in CTCL have not been studied. Here, we show that Bcl3 is highly expressed in CTCL Hut-78 and HH cells. The suppression of Bcl3 levels decreases the expression of the pro-survival genes cIAP1 and cIAP2, reduces cell viability, and increases CTCL apoptosis. Interestingly, Bcl3 suppression concomitantly increases expression and the release of the pro-inflammatory cytokines IL-8 and IL-17 in CTCL cells. Chromatin immunoprecipitation studies show that Bcl3 regulates cIAP1, cIAP2, IL-8 and IL-17 gene expression through direct binding to their promoters. Bcl3 expression is regulated by bortezomib (BZ)-mediated proteasome inhibition, and BZ inhibits Bcl3 recruitment to its target promoters, resulting in decreased expression of cIAP1 and cIAP2, but increased expression of IL-8 and IL-17. The Bcl3 expression is regulated through NFκB subunit exchange on Bcl3 promoter. In untreated cells, the Bcl3 promoter is occupied predominantly by p65/p50 heterodimers, inducing Bcl3 expression; however, in BZ-treated cells, the p65/50 heterodimers are replaced by p52 subunits, resulting in Bcl3 transcriptional repression. These data provide the first insights into the function and regulation of Bcl3 in CTCL, and indicate that Bcl3 has an important pro-survival and immunosuppressive role in these cells.

A specific DNA methylation profile correlates with a high risk of disease progression in stage I classical (Alibert-Bazin type) mycosis fungoides

Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma; in its classical presentation it evolves slowly, but it can have an aggressive course in a subset of patients. To investigate the impact of epigenetic mechanisms on the progression of early stage MF. We analysed DNA methylation at 12 different loci and long interspersed nucleotide elements-1 (LINE-1), as a surrogate marker of global methylation, on tissue samples from 41 patients with stage I MF followed up for at least 12years or until disease progression. The methylation profiles were also analysed in two T-cell lymphoma cell lines and correlated with gene expression. The selected loci were methylated in a tumour-specific manner; concomitant hypermethylation of at least four loci was more frequent in cases progressing within 1-3 and 3-6years than in late-progressive or non-progressive cases. LINE-1 methylation was significantly lower in rapidly progressive MF at 3years (61%, P<0·001) than in those at 12years (67%). PPARG, SOCS1 and NEUROG1 methylation showed remarkable differences among the prognostic groups, but only PPARG was a significant predictor of disease progression within 6years, after adjustment for patients' age or gender. Strikingly, a methylation profile similar to progressive cases was found in highly proliferative Sézary-derived HUT78 cells but not in MF-derived HUT102 cells. Exposure to a DNA demethylating agent restored sensitivity to apoptosis and cell cycle arrest. Epigenetic silencing of specific biomarkers can predict the risk of disease progression in early-stage MF, providing insights into its pathogenesis, prognosis and therapy.

NFκB‐dependent Bcl3 regulates survival in cutaneous T‐cell lymphoma (762.1)

Cutaneous T cell lymphoma (CTCL) is characterized by high constitutive activity of NFκB, which promotes cell survival and resistance to apoptosis. Increased expression of the proto‐oncogene Bcl3 belonging to IκB family is associated with the pathogenesis of different types of human cancer, yet, the regulation and function of Bcl3 in CTCL have not been previously studied. Here, we show that human CTCL Hut‐78 and HH cells express high protein levels of Bcl3, which is localized both in the cytoplasm and in the nucleus. NFκB subunits p65 and p50 are highly recruited to the Bcl3 promoter, indicating that Bcl3 expression is regulated by NFκB p65/p50 heterodimers in CTCL cells. Proteasome inhibition by bortezomib (BZ), which induces apoptosis in CTCL cells, inhibits Bcl3 expression. The BZ‐decreased Bcl3 expression is associated with decreased recruitment of NFκB p65 and p50 subunits to the endogenous Bcl3 promoter, but with increased recruitment of NFκB p52. Importantly, suppression of Bcl3 levels by siRNA induces apoptosis and down‐regulates expression of the anti‐apoptotic genes cIAP1 and cIAP2 in CTCL cells. Bcl3 is specifically recruited to the endogenous cIAP1 and cIAP2 promoters, and BZ inhibits this recruitment. These data provide first insights into the regulation and function of Bcl3 in CTCL, and indicate that Bcl3 has an important pro‐survival role in these cells.Grant Funding Source: Support by NIH grants AI085497 and CA173452 to I.V.

Epigallocatechin-3-gallate Inhibits Tax-dependent Activation of Nuclear Factor Kappa B and of Matrix Metalloproteinase 9 in Human T-cell Lymphotropic Virus-1 Positive Leukemia Cells

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol molecule from green tea and is known to exhibit antioxidative as well as tumor suppressing activity. In order to examine EGCG tumor invasion and suppressing activity against adult T-cell leukemia (ATL), two HTLV-1 positive leukemia cells (HuT-102 and C91- PL) were treated with non-cytotoxic concentrations of EGCG for 2 and 4 days. Proliferation was significantly inhibited by 100 μM at 4 days, with low cell lysis or cytotoxicity. HTLV-1 oncoprotein (Tax) expression in HuT- 102 and C91-PL cells was inhibited by 25 μM and 125 μM respectively. The same concentrations of EGCG inhibited NF-kB nuclearization and stimulation of matrix metalloproteinase-9 (MMP-9) expression in both cell lines. These results indicate that EGCG can inhibit proliferation and reduce the invasive potential of HTLV-1- positive leukemia cells. It apparently exerted its effects by suppressing Tax expression, manifested by inhibiting the activation of NF-kB pathway and induction of MMP-9 transcription in HTLV-1 positive cells.

Cap-Modified Hydroxamate Analogues as Histone Deacetylases Inhibitors and Antitumor Agents

Two series of SAHA-liked hydroxamate analogues were designed, synthesized and evaluated for their biological activities against nuclear HDACs. Compounds of Series I were found to be very effective inhibitors of cancer cell growth in the PC-3, Hut78, K562 and Jurkat E6-1 cancer cell lines with mean IC50 values from 0.54 M (Ic, Jurkat E6-1) to 7.73 M (Ib, K562), indicating that they are cell permeable and the benzimidazolyl-based ligands are flexible enough to occupy the binding site of HDAC.

Depsipeptide and roxithromycin induce apoptosis of lymphoma cells by blocking extracellular signal‐regulated kinase activation

Depsipeptide (FK228), a histone deacetylase inhibitor, was recently approved for use in cutaneous T-cell lymphoma. Roxithromycin (RXM) is a macrolide antibiotic that can induce apoptosis of some T-cell lines. In this study, we investigated whether combination of FK228 and RXM had a synergistic inhibitory effect on cell survival of various lymphoma cells and which signaling pathway was affected by the drugs in the presence or absence of chemokines, which were reported to inhibit apoptosis of some tumor cells. FK228 and RXM additively decreased the number of HUT-78, Ki-JK and EL-4 lymphoma cells at doses over 50nmol/L and 50μmol/L, respectively. These drugs inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK) of EL-4 cells in a dose-dependent manner. Significant association between ERK phosphorylation and cell number or annexin V(+) cells suggested that the ERK pathway may be critical for survival of EL-4 cells. Combination of 10 or 50nmol/L of FK228 and 10μmol/L of RXM decreased cell number of HUT78 and EL-4 compared to a single use of each drug. Our in vitro study suggested that combination of FK228 and RXM may be helpful for enhancing tumor killing effects. Although further study is necessary, this combination may be applicable to patients with cutaneous T-cell lymphoma in the future.