Human T-cell Lymphoma Cell Line Research Articles

P1293: COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA.

Background: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In a Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in preclinical studies. Aims: Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death, such as necroptosis, by rational combination of our clinical compounds in preclinical models of TCL. Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key necroptosis biomarkers (RIPK3, MLKL) were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. Karpas-299 cells genetically-manipulated to express RIPK3 were used to demonstrate involvement of necroptosis in drug-induced cell death (Cytotox NIR) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key immunomodulatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma. Levels of methylation in RIPK3 promoter regions were measured by pyrosequencing after bisulfite conversion. Comparative changes in gene expression were measured by RT-qPCR. Results: TCL cell lines treated with tolinapant, decitabine or both led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating target engagement of both agents. The combination of tolinapant and decitabine synergistically reduced viability of some human T-cell lymphoma cell lines. Some cell lines, including Karpas-299, were resistant to tolinapant treatment and showed low expression of RIPK3, which was found to be due to promoter methylation. Increased expression of RIPK3 in Karpas-299 by genetic manipulation or by decitabine treatment resulted in enhanced lytic cell death upon tolinapant treatment. Decitabine and tolinapant treatments resulted in expression of cytokines, chemokines and DAMPs, suggesting potential for immune activation and the effects were enhanced when combined. Furthermore, normally silenced cancer/testis antigen expression was increased by decitabine, potentially increasing the immunogenicity of the cells. Evaluation of the combination of agents in mouse models suggested that increased necroptosis signal and immune-potentiating biomarker modulation can be achieved in vivo. Summary/Conclusion: These data demonstrate that hypomethylating agents enhance immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. In addition, modulation of cytokine response and cancer/testis antigen expression could enhance anti-tumour response. These findings provide a strong rationale to explore this combination clinically.

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). [Display omitted] DisclosuresSmyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

Synthesis and antitumor activity in vitro of novel berbamine derivatives

A series of new berbamine derivatives were synthesized, and their cytotoxic activity was evaluated against Human T-cell lymphoma cell line H9 and multiple myeloma cell line RPMI8226 in vitro. Compared with berbamine, the cytotoxicity of the modified derivatives was enhanced, especially simultaneously substituted at OH and 5-position. Compounds 2a and 4b exhibited high antitumor activity. The IC50 value of compound 2a was 0.30 μM for RPMI8226 cells, and the IC50 value of compound 4b was 0.36 μM for H9 cells, whereas berbamine IC50 values were 4.0 μM for H9 cells and 6.19 μM for RPMI8226 cells, respectively.

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

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

Glutathione-garlic sulfur conjugates: slow hydrogen sulfide releasing agents for therapeutic applications.

Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.

Plumbagin induces apoptosis in lymphoma cells via oxidative stress mediated glutathionylation and inhibition of mitogen-activated protein kinase phosphatases (MKP1/2)

Maintaining cellular redox homeostasis is imperative for the survival and normal functioning of cells. This study describes the role and regulation of MAPKinases in oxidative stress mediated apoptosis. Plumbagin, a vitamin K3 analog and a pro-oxidant, was employed and it induced apoptosis in both mouse and human T-cell lymphoma cell lines via increased oxidative stress, caspase activity and loss of mitochondrial membrane potential. The pro-oxidant and cytotoxic effects of plumbagin were sensitive to antioxidants indicating a decisive role of cellular redox balance. Plumbagin induced persistent activation of JNK and pharmacological inhibition as well as shRNA-mediated JNK knock-down rescued cells from plumbagin-induced apoptosis. Further, plumbagin induced cytochrome c release, FasL expression and Bax levels via activation of JNK pathway. Exposure of lymphoma cells to plumbagin led to inhibition of total and specific phosphatase activity, increased total protein S-glutathionylation and induced glutathionylation of dual specific phosphatase- 1 and 4 (MKP-1 and MKP-2). The in vivo anti-tumor efficacy of plumbagin was demonstrated using a mouse model. In conclusion, oxidative stress mediated tumor cytotoxicity operates through sustained JNK activation via a novel redox-mediated regulation of MKP-1 and MKP-2.

Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro

BackgroundExtracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure. MethodsHuman T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy. ResultsCell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A. Conclusion8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.

In-Vivo Activation Of STAT3 In Angioimmunoblastic T Cell Lymphoma, PTCL Not Otherwise Specified, and ALK Negative Anaplastic Large Cell Lymphoma: Implications For Therapy

T cell lymphoma (TCL) is a heterogeneous group of malignant lymphomas and is subdivided in three major subgroups, (i) cutaneous (mycosis fungoides (MF), sezary syndrome (SS) and primary CTCL), (ii) nodal (peripheral T-cell lymphoma, PTCL NOS, anaplastic large cell lymphoma (ALCL), angioimmunoblastic (AITL)) (iii) and extranodal. Because of this broad morphological spectrum and immunophenotypic variation the pathogenesis of most of the TCL is poorly understood. Constitutive STAT3 signaling provides growth-promoting signals in many forms of malignancy; however in-vivo activation of phosphorylated STAT3 and its clinical correlation in subtypes of TCLs has not been extensively studied. In this study we examined constitutive STAT3 activation in various subtypes of TCL tumors and performed pre-clinical testing to determine whether STAT3 inhibitors will be effective.First, we assessed the STAT3 activation (pSTAT3Tyr705) by immunohistochemistry (IHC) on a tissue microarray (TMA) from 169 untreated TCL patient samples. Using a cutoff of ≥30% pSTAT3 nuclear staining, 38% (64/169) of TCL tumors were pSTAT3+. Interestingly, pSTAT3 was differentially expressed among the various TCL subgroups. 27% (16/59) PTCL NOS, 29% (11/38) AITL, 93% (14/15) ALK+ ALCL, 57% (13/28) ALK- ALCL, 50% (6/12) extranodal NK/T cell tumors and 17% (1/6) primary cutaneous showed pSTAT3 positivity. Using either a 30% or 80% cut-off, pSTAT3 positivity was associated with inferior event free survival for AITL, PTCL NOS and ALK neg ALCLs TCLs. Due to small sample size within each group statistical significance (p=0.03) was met only for ALK neg ALCL (80% cutoff). Further analysis in the human TCL cell lines and normal control T-cells also revealed differential expression of pSTATs. ALCL cell lines, Karpas 299 and SR786 were pSTAT3 positive but pSTAT5 negative. Both the CTCL (SS) cell lines HuT78 and SeAx were pSTAT3 negative, however MyLa a cell line of MF origin was strongly pSTAT3+ and pSTAT5+. The normal control CD4 T-cells was pSTAT3- and pSTAT5+. These data clearly suggest that pSTAT3 is differentially expressed among the TCL tumors and is a potential therapeutic target. Potential mechanisms for STAT3 activation could be aberrant secretion of cytokines/chemokines produced by the tumor itself or the tumor microenvironment. We compared the cytokine profile of the culture supernatant from pSTAT3 positive SUDHL1 and pSTAT3 negative HuT78 TCL cell lines using a 30-plex ELISA. The most significantly different cytokines among these 2 cell lines were interleukin -10 (IL-10) and soluble interleukin-2 receptor alpha (sIL-2Ra). The SUDHL1 was found to secrete a very high level (1700 pg/ml) of IL-10, in contrast to only 167 pg/mL in the HuT78 supernatant. Moreover, SUDHL1 secretes 3.5 times more sIL-2Ra (6600 pg/ml) than the pSTAT3 negative cell line HuT78 (1800 pg/ml). In-vitro exposure to IL-10 but not sIL-2Ra was able to further upregulate pSTAT3 in SUDHL1 and Karpas 299. Overall, these data suggest that autocrine secretion of IL-10 but not sIL-2Ra is important for STAT3 activation in ALCL TCLs. Next we compared the cytokine profile of 2 pSTAT3 positive cells from different origin; SUDHL1 (ALCL) and MyLa (MF). In contrast to SUDHL1 (which produce high level of IL-10), MyLa was producing very little IL-10 (18pg/ml). The other most significantly different cytokines between these cells was RANTES with MyLa producing a very high level (28897 pg/ml) and SUDHL1 a very low level (8pg/ml). We also noticed differential level of IL-13 between these cells; MyLa producing 113pg/ml while SUDHL1 only 13 pg/ml. SAR302 (Sanofi, Cambridge, MA), a selective JAK2 inhibitor currently in clinical trial for myeloproliferative neoplasms was able to inhibit autocrine secretion of IL-10 but not sIL-2Ra in a dose-dependent manner followed by STAT3 dephosphorylation and decreased lymphoma cell survival.In summary, our data provide first evidence of in-vivo STAT3 activation in AITL, PTCL NOS, ALK negative ALCL TCLs using TMA from patient samples. Comprehensive cytokine analysis determined IL-10 as an autocrine factor driving STAT3 activation in ALCLTCLs, while RANTES and IL-13 might be important drivers for STAT3 activation in MF TCLs. Finally our data provide a mechanistic basis of selecting and targeting TCL tumor cells with high IL-10 levels and/or constitutive STAT3 activity with potent and novel JAK2 inhibitors such as SAR302 or other STAT3 inhibitors. Disclosures:No relevant conflicts of interest to declare.

Schedule-dependent inhibition of T-cell lymphoma cells by cotreatment with the mTOR inhibitor everolimus and anticancer drugs

Everolimus (RAD001) is a novel mammalian target of rapamycin (mTOR) inhibitor, and anti-proliferative activity in various malignancies has been reported. This study evaluated the anti-tumor effects and schedule-dependent synergism of everolimus in combination with other chemotherapeutic agents in T-cell lymphoma cell lines. Human T-cell lymphoma cell lines Hut-78 and Jurkat were treated with increasing doses of everolimus, alone or in combination with doxorubicin, etoposide, vincristine, or bortezomib, using different dosing schedules. Anti-tumor effects were measured by assays for cell proliferation, apoptosis, and cell cycle distribution. Drug interactions were determined by median effect analysis. Exposure to everolimus alone induced G1 phase cell cycle arrest without significant apoptosis. With certain dosing schedules, everolimus showed synergism with doxorubicin, etoposide, and bortezomib, but antagonism with vincristine. Cytotoxic synergism was observed following cotreatment with doxorubicin and everolimus, bortezomib and everolimus, doxorubicin followed by everolimus, and bortezomib followed by everolimus. By contrast, cell exposure to everolimus followed by doxorubicin or followed by bortezomib resulted in antagonistic effects. Sequential exposure to doxorubicin or bortezomib followed by everolimus effectively prevented potential negative interactions, and resulted in drug synergism. Drug combination synergisms or antagonisms were associated with variable effects on the cell cycle distribution. Everolimus effectively inhibited the growth of T-cell lymphoma cells in vitro. Specific schedule-dependent combinations of everolimus with other anti-tumor agents which avoid potential drug antagonism and produce effective synergism may lead to clinically effective treatments for T-cell lymphoma.

CUTLL1, a novel human T-cell lymphoma cell line with t(7;9) rearrangement, aberrant NOTCH1 activation and high sensitivity to γ-secretase inhibitors

Activating mutations in NOTCH1 are present in over 50% of human T-cell lymphoblastic leukemia (T-ALL) samples and inhibition of NOTCH1 signaling with gamma-secretase inhibitors (GSI) has emerged as a potential therapeutic strategy for the treatment of this disease. Here, we report a new human T-cell lymphoma line CUTLL1, which expresses high levels of activated NOTCH1 and is extremely sensitive to gamma-secretase inhibitors treatment. CUTLL1 cells harbor a t(7;9)(q34;q34) translocation which induces the expression of a TCRB-NOTCH1 fusion transcript encoding a membrane-bound truncated form of the NOTCH1 receptor. GSI treatment of CUTLL1 cells blocked NOTCH1 processing and caused rapid clearance of activated intracellular NOTCH1. Loss of NOTCH1 activity induced a gene expression signature characterized by the downregulation of NOTCH1 target genes such as HES1 and NOTCH3. In contrast with most human T-ALL cell lines with activating mutations in NOTCH1, CUTLL1 cells showed a robust cellular phenotype upon GSI treatment characterized by G1 cell cycle arrest and increased apoptosis. These results show that the CUTLL1 cell line has a strong dependence on NOTCH1 signaling for proliferation and survival and supports that T-ALL patients whose tumors harbor t(7;9) should be included in clinical trials testing the therapeutic efficacy NOTCH1 inhibition with GSIs.

T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance

AbstractDepsipeptide (FK228) is a novel histone deacetylase inhibitor currently in clinical trials and the first to demonstrate clinical activity in patients. Responses have been observed in patients with T-cell lymphomas, despite prior treatment with multiple chemotherapeutic agents. To better understand the effects of histone deacetylase inhibitors on T-cell lymphoma, the human T-cell lymphoma cell line HUT78 was tested for sensitivity and molecular response to depsipeptide. Treatment with depsipeptide, as well as other histone deacetylase inhibitors, caused induction of histone acetylation, induction of p21 expression, and substantial apoptosis without significant cell cycle arrest. Treatment with the caspase inhibitor z-VAD-fmk significantly inhibited depsipeptide-induced apoptosis, enabling detection of cell cycle arrest. Treatment with depsipeptide increased expression of the interleukin-2 (IL-2) receptor, and combination with the IL-2 toxin conjugate denileukin diftitox resulted in more than additive toxicity. Cells selected for resistance to depsipeptide overexpressed the multidrug resistance pump, P-glycoprotein (Pgp). However, cells selected for resistance to depsipeptide in the presence of a Pgp inhibitor had a Pgp-independent mechanism of resistance. These studies confirm the activity of depsipeptide in a T-cell lymphoma model and suggest a general sensitivity of T-cell lymphoma to histone deacetylase inhibitors, an emerging new class of anticancer agents. (Blood. 2004;103:4636-4643)

Morphological changes in the nucleus and actin cytoskeleton in the process of Fas-induced apoptosis in Jurkat T cells.

To investigate the early event of apoptosis, we monitored the morphological changes in the early stage of Fas-induced apoptosis in the human T-cell lymphoma cell line Jurkat, using confocal microscopy. Morphological changes in the nuclei were observed from 30 min after stimulation, and preceded the changes in the cytoskeleton. This kind of change was enhanced in the presence of EGTA but decreased in the presence of dihydrocytochalasin B, without any changes in caspase-3 activation. During the changes in shape of the cells, the actin cytoskeleton collapsed and shrank in the center. Even though nuclei also changed their shapes in apoptotic cells, they were partially TUNEL-negative, suggesting that they were not yet damaged at the DNA level. Our results suggest that, in the process of apoptosis in Jurkat cells, cell nuclei and cytoskeleton are changed first, then membrane blebbing and caspase-3 activation occur, and fragmentation of chromosomal DNA is last.

Neuronal (type I) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type II) nitric oxide synthase expression.

Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor kappaB (NF kappaB) as NOS inhibitors enhance basal NF kappaB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NF kappaB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-alpha (TNF-alpha) induces NF kappaB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-alpha-induced NF kappaB activation in astrocytes; instead PDTC itself induces NF kappaB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NF kappaB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NF kappaB. NF kappaB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NF kappaB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NF kappaB-regulated genes.

An exon 5-deleted mrna encodes a functional interleukin 2 receptor alpha-subunit

Two polypeptides are involved in interleukin 2 binding: a low-affinity receptor of 55 kD (IL2-R alpha) and an intermediate affinity component of 75 kD (IL2-R beta). We describe the cloning by the Polymerase Chain Reaction of the coding region of IL2-R alpha from a human T-cell lymphoma cell line. One clone presented a 72-bp deletion that precisely corresponds to exon 5. The deleted form and the normal IL2-R alpha cDNA were expressed CHO cells. Stable transfected cellular clones were compared for their immunoreactivity to monoclonal antibodies directed against IL2-R alpha and for their ability to bind radiolabeled IL2. The presence or absence of the protein region encoded by exon 5 did not modify the IL2-binding capacity of the receptor.

Characterization of a T-cell determinant defined by a monoclonal antibody (TH5.2) which is involved in the interleukin-2-producing and proliferative capabilities of T cells

Utilizing an OKT-4-positive human T-cell lymphoma cell line as immunogen, we have produced a monoclonal antibody (MAb), designated TH5.2, which is capable of augmenting the interleukin-2 (IL-2) production and proliferation of antigen-activated T cells. TH5.2 MAb by itself is not mitogenic for T cells; the enhanced IL-2 production and proliferation requires costimulation with either antigen or mitogen. TH.2 MAb recognizes all peripheral blood T cells at varying intensities, but does not react with monocytes. Using dual-color fluorescence analysis, it was determined that TH5.2 MAb reacts at a higher intensity on Leu-3a-positive T cells compared to Leu-2a-positive cells. Sorting T cells on the basis of fluorescent intensity with TH5.2 MAb demonstrated that the T cells reacting at high TH5.2 intensity were able to respond to mitogen at a higher rate (5–10×), as well as produce higher concentrations of IL-2 in response to mitogen as compared to the low IL-2 intensity cells. Cytotoxically treating peripheral blood mononuclear cells (PBMC) with TH5.2 MAb plus complement, which resulted in an approximate 8% decrease in the total population, significantly reduced the ability of the cells to proliferate to both mitogen and antigen. Addition of recombinant IL-2 to the cultures was able to restore the proliferative capability of the cells. In further analyzing the ability of TH5.2 MAb to augment the proliferative capability of PBMC to antigen and mitogen stimulation in vitro, it was determined that TH5.2 MAb was capable of acting synergistically with antigen or mitogen in increasing the Il-2-producing capability of T cells. Taken together the data suggest that the TH5.2 determinant is involved in the proliferative capability of T cells and is functioning at the level of IL-2 synthesis and/or secretion.

Quantification of soluble E-receptor in the serum of patients with various diseases and its accompanying in vitro immunosuppression in neoplasia.

Evidence in the literature indicates that soluble E-receptor in the serum may modulate T-dependent immune response. We have developed a solid-phase radioimmunoassay to measure the soluble form of E-receptor from various sources. The assay detects solubilized antigens derived from E-receptor-bearing T lymphocytes and not with non-E-receptor-bearing B lymphocytes. The sensitivity limit of the assay is 0.1 ng/ml of purified E-receptor antigen. Using this assay, one can show that the T-cell mitogen phytohaemagglutinin stimulated both lymphocytes and cells of the resting human T-cell lymphoma cell line Jurkat to shed or secrete E-receptor into the culture medium. Results of the radioimmunoassay performed on human sera indicated that some patients with Hodgkin's disease, melanoma, sarcoma, or acute or chronic lymphocytic leukaemia had elevated levels of this antigen in their serum, whereas normal human sera registered lower levels. Elevated levels of the soluble form of E-receptor in the serum were suggestive of an in vitro assessment of their immunosuppressive activity. These results indicate that activation of T-cell immunity in vivo may result in humoral immunosuppression.

Purification and chemical characterization of the receptor for interleukin 2 from activated human T lymphocytes and from a human T-cell lymphoma cell line.

The cell surface receptor for interleukin 2 plays a central role in the biology of this T-cell growth factor. A combination of affinity chromatography, high-performance liquid chromatography, and NH2-terminal protein sequencing was used to purify and chemically characterize the interleukin 2 receptor both from phytohemagglutinin-activated T cells and from the human T-cell lymphoma cell line HuT-102. The receptor isolated from HuT-102 cells was purified 16,000-fold to homogeneity as evidenced by (i) a final specific activity close to the theoretical specific activity of 18,182 fmol of receptor per microgram of protein, (ii) a single band on polyacrylamide gel electrophoresis with an Mr of 55,000, and (iii) a unique, unambiguous NH2-terminal protein sequence. The receptor purified from phytohemagglutinin-activated T lymphocytes had an Mr of 60,000 but it had the same NH2-terminal protein sequence.

Abundant transcription of a cellular gene in T cells infected with human T-cell leukemia-lymphoma virus.

Human T-cell leukemia-lymphoma virus (HTLV) is a type C retrovirus associated with a subtype of mature T-cell malignancy in humans. HTLV also infects normal human cord blood mature T lymphocytes in vitro and induces a number of phenotypic changes in these cells, including their continuous growth and partial or complete independence of T-cell growth factor (TCGF). As part of our initial study designed to analyze gene(s) specifically activated by HTLV infection, we have isolated a recombinant DNA clone by differential screening of a cDNA library made from mRNA of a human T-cell lymphoma cell line producing HTLV. This cDNA identifies a single-copy gene in all human DNAs and a single mRNA species of 2.3 kilobases expressed at several hundred copies per cell in five HTLV-positive neoplastic T-cell lines. In addition, cord blood T lymphocytes infected with HTLV, but not the uninfected counterparts, express high levels of mRNA from this gene. A survey of different human hematopoietic cell types showed that this gene is expressed at low or undetectable levels (less than 10 copies) in human T, B, myeloid, or erythroid cell lines; in moderate amounts in lymphoid precursor (immature) cell lines; and in high amounts in lectin-activated mature T-cells, comparable to those of HTLV-infected T-cell lines. The precise function of this gene has not yet been determined.