Pevonedistat, a Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Induces Cell Cycle Deregulation, Anaphase Catastrophe, and Apoptosis in T-Cell Lymphoma Cells

Abstract Introduction: Despite the significant progress of targeted therapies in B-cell malignancies, T-cell lymphomas remain an area of unmet medical need. Most patients are diagnosed at an advanced stage and have limited treatment options. Moreover, most patients who relapse following initial chemotherapy ultimately succumb to disease. Recent successes of targeting the proteasome (i.e., bortezomib) and E3 ligases (i.e., lenalidomide) identify the ubiquitin-proteasome system (UPS) as a tractable target in lymphoma. Pevonedistat, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE), interferes with activation of NEDD8, a ubiquitin-like modifier. This interference ultimately leads to decreased activity of cullin-RING (E3) ligases and accumulation of their substrates, including inhibitor of NFκB (IκB), the replication licensing protein Cdt1, and p27. We previously demonstrated that targeting NAE affected primary neoplastic B cells via several mechanisms: disruption of NFκB activity as well as induction of Cdt1, DNA damage, and cell cycle arrest. Here, we demonstrate that targeting NAE in T-cell lymphoma cells mediates apoptosis via cell cycle deregulation, accompanied by induction of Cdt1 and p27, and induction of anaphase catastrophe. Methods: Experiments were performed in T-cell lymphoma cell lines (SR, HH, Jurkat, and SUP-T1) as well as circulating primary cells from patients with peripheral T-cell lymphoma and Sezary syndrome. Pevonedistat (TAK-924) was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). Apoptosis was assessed by Annexin V staining. Results: SR (PTCL) cells and primary T-cell lymphoma cells were the most sensitive to pevonedistat (IC50of ~250nM at 24 hours); Jurkat and SUP-T1 cells demonstrated low/intermediate sensitivity, whereas HH (CTCL) cells were resistant. Targeting NAE disrupted cullin neddylation in a dose-dependent manner across all tested cell lines and primary neoplastic T cells, followed by accumulation of phospho-IκBα. Upregulation of phospho-IκBα was notable within 2 hours of pevonedistat treatment across both sensitive and resistant cell lines and primary cells. Concomitantly, we observed induction of p27 and Cdt1. Upregulation of Cdt1 was attenuated in HH cells compared with SR, consistent with the low proliferation rate of the former. Treatment of SR cells with pevonedistat led to DNA damage as evidenced by γH2AX and G2/M arrest. Chromosomal instability is a prominent feature in cancer and poorly studied as a therapeutic target. We have previously shown that cancer cells undergo multipolar anaphase in response to inhibition of cyclin-dependent kinase-2 (CDK2), an interphase CDK, followed by apoptosis and termed this event anaphase catastrophe (Hu et al., 2015; Danilov et al., 2016). As we observed robust accumulation of the endogenous CDK inhibitor p27 in cells treated with pevonedistat, an event presumed to lead to attenuated CDK2 activity, we studied anaphase catastrophe in this setting. We visualized anaphase catastrophe by immunofluorescent staining for nuclear material (DAPI) and γ-tubulin, and scored it in 50 cells per condition. NAE inhibition with pevonedistat induced anaphase catastrophe in SR and Jurkat cells. Upon 24-hour exposure to 250 nM pevonedistat, 9.8±6.0% of SR and 18±4.4% of Jurkat cells demonstrated multipolar anaphases, compared with 1±0.8% and 3.0±2.6% with vehicle control, respectively. Conclusions: Inhibiting NAE with pevonedistat induces apoptosis of T-cell lymphoma cells. We propose deregulation of Cdt1 and p27, followed by anaphase catastrophe, as a key mechanistic event implicated in pevonedistat-induced apoptosis in neoplastic T cells. Our work provides rationale to further investigate neddylation as a therapeutic target in T-cell lymphoma. Disclosures Danilov: Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy; Genentech: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding.

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The Novel Tumor Suppressor SAMHD1 Is Differentially Expressed and Partly Regulated By MYC in Peripheral T-Cell Lymphomas (PTCL)

Blood ◽

10.1182/blood-2018-99-119229 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4130-4130

Author(s):

Pedro Farrajota Neves Da Silva ◽

Nikolaos Tsesmetzis ◽

Ioanna Xagoraris ◽

Agata Magdalena Wasik ◽

Georgia Kokaraki ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Differentially Expressed ◽

Lymphoma Cells ◽

T Cell Lymphomas ◽

Samhd1 Expression

Abstract Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates required for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. More recently, SAMHD1 mutations have been reported in T-prolymphocytic leukemia (T-PLL). Based on these findings and the fact that SAMHD1 limits the dNTP pool in the cell, it may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, with cytarabine in acute myeloid leukemia being an example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of peripheral T-cell lymphomas (PTCL) are not yet known. Methods: The patient cohort included 64 PTCLs of various histologic types which were diagnosed and treated at Karolinska University Hospital (Sweden). A control group of 4 reactive lymph nodes and 2 reactive tonsils was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on a PTCL tissue microarray (TMA) with duplicate tumor cores from each case or full tissue sections using dual immunostaining (SAMHD1 / CD68) and a monoclonal antibody against SAMHD1 (Bethyl Laboratories, San Antonio, TX). At least 500 lymphoma cells were counted to calculate the percentage of SAMHD1-positive tumor cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. Two T-cell lymphomas cell lines (Mac1, Mac2A) were used as an in vitro system. As our preliminary findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, we hypothesized that MYC might regulate SAMHD1 expression. Therefore, the T-cell lymphoma cell lines were treated with the selective BET / MYC inhibitor JQ-1 or transiently transfected with a MYC-overexpressing plasmid or MYC gene-specific siRNA constructs, respectively. Western blot analysis was used to assess the protein levels. Results: SAMHD1 protein was expressed in reactive T-cells and histiocytes (CD68+) in all reactive lymphoid tissues (lymph nodes and tonsils) with strong staining intensity. SAMHD was differentially expressed among PTCL subtypes generally with weaker staining intensity as compared to normal T-cells and histiocytes, thus being positive in all (100%) angioimmunoblastic T-cell lymphomas (AILT), 67% PTCL-NOS, 45% ALK+ ALCL, 20% of ALK+ ALCL, and none (0%) of T-lymphoblastic lymphomas (p=0.0017, chi-square test). Among the SAMHD1- positive cases, the percentage of positive lymphoma cells ranged from 0 to 100% and its highest median was observed in AILT. SAMHD1 expression inversely correlated with CD30 expression (% CD30+ positive lymphoma cells) (p=0.0025, Mann-Whitney test). No significant associations between SAMHD1 levels and other clinicopathologic parameters or clinical outcome (EFS or OS) were found, however, the number of patients analyzed in each histologic subtype was limited. Inhibition of MYC activity by JQ-1 or MYC gene silencing with specific siRNA resulted in a substantial increase in the SAMHD1 protein level in T-cell lymphoma cell lines. Inversely, transient transfection of the cell lines with a MYC overexpressing plasmid resulted in decreased levels of SAMHD1. Taken together, the in vitro data suggest a possible MYC-associated regulation (repression) of SAMHD1 gene expression in T-cell lymphoma. Conclusions: SAMHD1 is shown for the first time to be differentially expressed among PTCL types and its regulation may involve MYC. Preliminary survival analysis shows no significant associations of SAMHD1 expression with EFS and OS in this cohort of PTCL, however, analysis of a larger PTCL study group is underway to draw definite conclusions. Disclosures Österborg: Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding.

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Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v104.11.4637.4637 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4637-4637

Author(s):

Gerald G. Wulf ◽

Anita Boehnke ◽

Bertram Glass ◽

Lorenz Truemper

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cytolytic Activity ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Human T Cell

Abstract Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

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The Brd-Inhibitor OTX015 Shows Pre-Clinical Activity in Anaplastic Large T-Cell Lymphoma (ALCL)

Blood ◽

10.1182/blood.v120.21.4872.4872 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4872-4872 ◽

Cited By ~ 3

Author(s):

Michela Boi ◽

Paola Bonetti ◽

Maurilio Ponzoni ◽

Maria Grazia Tibiletti ◽

Anastasios Stathis ◽

...

Keyword(s):

Cell Cycle ◽

T Cell ◽

Cell Lines ◽

Proliferative Activity ◽

Research Funding ◽

Cell Lymphoma ◽

G1 Arrest ◽

Apparent Difference ◽

Mrna Levels ◽

Rt Pcr

Abstract Abstract 4872 Background: ALCL, is clinically/biologically heterogeneous disease, including ALK+ and ALK- systemic forms. Despite the progresses in understanding the molecular pathogenesis of ALCL, the therapy is still based on chemotherapy, thus the identification of new treatment modalities is needed. Bromodomain-containing proteins are components of transcription factors complexes and determinants of epigenetic memory. Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family, have recently shown antitumor activity in different hematological malignancies models. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a panel of ALCL cell lines. Material and Methods: Eight established human cell lines derived from ALK+ and ALK- anaplastic large cell lymphoma (ALCL) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72h exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed on using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: We assessed OTX-015 anti-proliferative activity in eight ALCL cell lines. The majority (5/8) of the cell lines were sensitive, with IC50 between 36 and 546 nM. There was no apparent difference between ALK+(6) and ALK- (2) cell lines. Cell cycle analyses revealed G1 arrest and a concomitant decrease of the S phase after 24h OTX015 exposure in 4/4 ALCL cell lines, without an increase in cell death, suggesting a cytostatic effect of OTX015. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in the most sensitive ALK+ALCL cell line. To understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after treatment. We observed that OTX015 suppressed the transcription of MYCgene and some of its downstream target genes (such as NCL and CAD) in 4/4 ALCL cell lines, with less efficacy in the most resistant one. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several ALCL cell lines. The down-regulation of MYC gene, followed by cell cycle G1 arrest and increase of cellular senescence, was observed after OTX015 treatment, appearing one of the possible mechanisms of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in ALCL and in other mature T-cell tumors. Disclosures: Bonetti: OncoEthix SA: Research Funding. Cvitkovic:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Inghirami:OncoEthix SA: Research Funding. Bertoni:OncoEthix SA: Research Funding.

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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

Blood ◽

10.1182/blood-2021-153451 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4356-4356

Author(s):

John S Manavalan ◽

Ipsita Pal ◽

Aidan Pursley ◽

George A. Ward ◽

Tomoko Smyth ◽

...

Keyword(s):

T Cell ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Tnf Alpha ◽

Sensitive Cell ◽

Advisory Committees

Abstract 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). Figure 1 Figure 1. Disclosures Smyth: 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.

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Circulating Lymphoma Cells in Mycosis Fungoides/Sezary Syndrome Show Heterogeneity of the Cell-of-Origin and Variable PD-1 Expression By Flow Cytometric Analysis

Blood ◽

10.1182/blood-2018-99-119799 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1624-1624

Author(s):

Mariko Yabe ◽

Natasha Lewis ◽

Qi Gao ◽

Allison Sigler ◽

Jeeyeon Baik ◽

...

Keyword(s):

T Cell ◽

Research Funding ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Cd4 T Cell ◽

Cell Of Origin ◽

Lymphoma Cells ◽

T Cell Subset ◽

Subset Analysis ◽

Flow Cytometric

Abstract Introduction: Mycosis fungoides (MF) is an epidermotropic primary cutaneous T-cell lymphoma. Its leukemic variant is recognized as Sezary syndrome (SS), although a study suggests that MF and SS may be distinct entities arising from different T-cell subsets; effector memory T-cells and central memory T-cells, respectively (Campbell JJ et al. Blood. 2010;116(5):767-771). Recent studies show bright PD-1 (T follicular helper (TFH)-like), and CD25/FOXP3 (Treg-like) expression in a subset of MF/SS cases by immunohistochemistry, and these findings raise the possibility that the cell-of-origin of MF/SS may be heterogeneous (Cetinozman F et al. Arch Dermatol. 2012;148(12):1379-1385, Prince HM et al. J Am Acad Dermatol. 2012;67(5):867-875, Satou A et al. Histopathology. 2016;68(7):1099-1108). In order to address this question comprehensively, we evaluated the expression levels of PD-1 on lymphoma cells of MF/SS patients by flow cytometry in peripheral blood (PB), and the results were compared to other T-cell lymphomas including angioimmunoblastic T-cell lymphoma (AITL). We also performed extensive flow cytometric immunophenotyping to algorithmically assess cell-of-origin of MF/SS with a subset of patients (Maecker HT et al. Nat Rev Immunol. 2012;12(3):191-200). Methods: Patients who were diagnosed with T-cell lymphoma at Memorial Sloan Kettering Cancer Center between August 2015 and August 2018, and have circulating lymphoma cells in PB were selected for this study. Diagnosis of MF/SS was confirmed by skin biopsy along with the clinical presentation. PD-1 expression levels on lymphoma cells in PB were evaluated by 10-color flow cytometry including anti-CD279 (PD-1) antibody. Immunophenotyping to assess cell-of-origin of lymphoma cells (T-cell subset analysis) was performed with 3 tube/10-color flow cytometry, using the following antibodies: CD3, CD4, CD8, CD25, CD27, CD45RA, CD45RO, CD127, CD279, HLA-DR, CCR4, CCR6, CCR7, and CXCR3. Results: Our study group is composed of 82 patients, including 34 MF/SS, 22 AITL, 2 anaplastic large cell lymphoma, ALK-negative (ALCL, ALK-), 8 adult T-cell leukemia/ lymphoma (ATLL), 5 peripheral T-cell lymphoma, NOS (PTCL-NOS), 5 T-cell large granular lymphocytic leukemia (T-LGL), and 6 T-cell prolymphocytic leukemia (T-PLL). The expression levels of PD-1 of lymphoma cells of the patients with MF/SS were widely variable (mean fluorescence intensity (MFI); Mean 949.0; range 101.0 - 3188.6) (Fig 1). 32.4% (11/34) of MF/SS cases showed high PD-1 expression equivalent to that of AITL cases. T-cell subset analysis to assess cell-of-origin was performed in 14 patients, including 4 patients with MF and 10 patients with SS (Table 1). While routine flow cytometric analysis showed similar immunophenotype other than the variation of PD-1, by flow cytometric T-cell subset analysis, we identified 3 patients with lymphoma cells showing T follicular helper (TFH) immunophenotype with CCR4+/CXCR3-/CCR6- and bright CD279 expression (case 2, 6, 8) (Table 2). Lymphoma cells of 8 patients showed effector memory CD4+ T-cell immunophenotype defined as CCR7-/CD45RA- (case 1, 3, 4, 5, 9, 10, 12, 13). Among these 8 patients, 2 patients had a subset of lymphoma cells showing central memory CD4+ T-cell immunophenotype defined as CCR7+/CD45RA- (case 5 and 12), and 2 patients had a subset with effector CD4+ cell immunophenotype defined as CCR7-/CD45RA+ (case 9, 13). 2 patients (case 7, 14) showed central memory CD4+ T-cell immunophenotype only, and 1 patient showed effector CD4+ T-cell immunophenotype only (case 11) (Table 2, Fig 2, 3). No cases showed Treg immunophenotype. The small sample size limited any subset analysis but we did not see obvious association between cell-of-origin of lymphoma cells and clinical features including prognosis, nodal presentation, number of circulating tumor cells, and histological findings in this study with limited number of cases. Conclusions: Lymphoma cells in MF/SS show marked heterogeneity of expression of PD-1 including clear subset arising from TFH. This suggests MF/SS may represent multiple biological entities. Further studies will be necessary to investigate the clinical significance of cell-of-origin of MF/SS. Disclosures Yabe: Y-mAbs Therapeutics: Consultancy. Moskowitz:ADC Therapeutics: Research Funding; Incyte: Research Funding; Bristol Myers-Squibb: Consultancy, Research Funding; Takeda: Honoraria; Merck: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. Horwitz:Infinity/Verastem: Consultancy, Research Funding; Trillium: Consultancy; Innate Pharma: Consultancy; Portola: Consultancy; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Aileron Therapeutics: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Mundipharma: Consultancy; Millennium/Takeda: Consultancy, Research Funding; Forty Seven: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum: Research Funding; Corvus: Consultancy.

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Curcumin Reverses Resistance to Histone Deacetylase Inhibitors In Cutaneous T-Cell Lymphoma Cells: a Potential Role for NF-κB Signaling

Blood ◽

10.1182/blood.v116.21.3973.3973 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3973-3973

Author(s):

Chunlei Zhang ◽

Xiang Zhang ◽

Madeleine Duvic

Keyword(s):

T Cell ◽

Clin Oncol ◽

Histone Deacetylase ◽

Cell Lines ◽

Research Funding ◽

Histone Deacetylase Inhibitors ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Cell Cycle Distribution ◽

Cutaneous T Cell Lymphoma

Abstract Abstract 3973 Histone deacetylase inhibitors (HDACi), including vorinostat (SAHA), depsipeptide (FK228), panobinostat (LBH589), belinostat (PXD101), and entinostat (SNDX275), show in-vitro and clinical activity against cutaneous T-cell lymphoma (CTCL) cell lines and patients' skin lesions [Zhang & Duvic, Expert Rev Dermatol 5: 393–401, 2010]. Vorinostat and depsipeptide were recently approved [Duvic et al, Blood 109: 31-9, 2007; Olsen et al, J Clin Oncol 25: 3109-15, 2007; Piekarz et al, J Clin Oncol 27: 5410-7, 2009], at response rates of 29% and 42%, respectively, but development of resistance remains an important clinical problem. Because we have shown that curcumin, the active ingredient of turmeric, exhibits anti-cancer activity through selective induction of tumor T-cell apoptosis and inhibition of NF-κB signaling in CTCL [Zhang et al, J Invest Dermatol 130: 2110-9, 2010], we now investigated whether curcumin combined with HDACi has synergistic anti-tumor effects in CTCL. HDACi-resistant MJ, HDACi-sensitive HH and HDACi cross-resistant HH/VOR CTCL cells were treated with HDACi (panobinostat, vorinostat, or enlinostat) plus or minus curcumin for up to 48 hrs. Cell viability was examined by the MTS assay and apoptosis by FACS analysis of annexin V/PI binding populations and/or cell cycle distribution. The NF-κB signaling pathway was analyzed by electrophoretic mobility gel shift assay and Western blotting. In MJ and HH cell lines, 5 nM panobinostat induced 1.4% and 11.4% apoptosis and 10 μM curcumin induced 24.5% and 29% apoptosis compared to vehicle controls. Panobinostat combined with curcumin induced 46.9% and 83.4% apoptosis in MJ and HH cell lines, respectively. Of interest, the HDACi cross-resistant HH/VOR CTCL cells were sensitive to curcumin alone and curcumin further enhanced panobinostat-induced apoptosis by 30% in the HH/VOR CTCL cells. Moreover, panobinostat combined with curcumin synergistically suppressed the DNA binding of NF-κB and decreased protein expression of the NF-κB activator RANK and NF-κBp65. Synergism was associated with down-regulation of NF-κB-regulated anti-apoptotic proteins (bcl-2, bcl-xL, and survivin), anti-proliferative proteins (c-myc and cyclooxygenase-2), and pro-invasive protein matrix metalloproteinase-9. Similar synergism was also seen when vorinostat or entinostat was combined with curcumin. These results suggest that HDACi could be combined with curcumin to enhance apoptosis of malignant T-cells through inhibition of NF-κB signaling in CTCL. Curcumin alone and in combination with HDACi may be an attractive strategy for the treatment of HDACi-refractory CTCL patients. Disclosures: Zhang: Novartis: Research Funding. Duvic:Novartis: Research Funding.

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Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines,

Blood ◽

10.1182/blood.v118.21.3734.3734 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3734-3734

Author(s):

Cory Mavis ◽

Sarah Frys ◽

Juan Gu ◽

John Gibbs ◽

Myron S. Czuczman ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

In Vitro Exposure ◽

Anti Tumor Activity

Abstract Abstract 3734 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials. Disclosures: Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.

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Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Blood ◽

10.1182/blood.v130.suppl_1.720.720 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 720-720

Author(s):

Akihiro Kitadate ◽

Sho Ikeda ◽

Fumito Abe ◽

Naoto Takahashi ◽

Norio Shimizu ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Cell Lymphoma ◽

Selective Inhibitor ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Class I ◽

T Cell Lymphomas

Abstract Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice. Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6). Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat. Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. Disclosures Kitadate: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa: TaNeDS (Daiichi Sankyo): Research Funding.

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Romidepsin and Lenalidomide Show a Synergistic Effect In T–Cell Lymphoma Cell Lines

Blood ◽

10.1182/blood.v122.21.5148.5148 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5148-5148 ◽

Cited By ~ 2

Author(s):

Maria Cosenza ◽

Monica Civallero ◽

Stefania Fiorcari ◽

Samantha Pozzi ◽

Luigi Marcheselli ◽

...

Keyword(s):

Cell Cycle ◽

T Cell ◽

Synergistic Effect ◽

Western Blot ◽

Cyclin D1 ◽

Cell Lines ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Histone Proteins ◽

Alpha Tubulin

Abstract Background and purpose Successful treatment of T cell lymphoma (TCL) is still problematic and identification of new pharmacological targets in this malignancy is urgently needed. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new therapeutic option for lymphoid malignancies. These drugs increase the acetylation status and modulate the activity of a wide range of non-histone proteins, and effects on both histone and non-histone proteins may contribute to their anti-cancer activity. Romidepsin (depsipeptide) is a potent and specific inhibitor of class 1 HDACs that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide belongs to the immunomodulatory agents (IMID®) and it is has been demonstrated to be very active for the treatment of several types of hematological neoplasia. Purpose of the present study was to determine whether lenalidomide potentiates romidepsin activity in TCL cell lines and if so, by which mechanisms. Methods TCL cell lines (Hut-78: cutaneous TCL cells and Karpas-299 anaplastic lymphoma cells) were treated with increasing concentrations of either romidepsin (0.5 - 25 nM) or lenalidomide (1 - 100 µM), and IC50 at 24-48 and 72 hours was calculated. The interaction between romidepsin (0.5, 1, 2.5 nM) and lenalidomide (2, 4, 10 µM) was evaluated using the Chou-Talalay method to determine if the combination had additive or synergistic effects. The cell cytotoxicity was assessed by MTT assay and apoptosis was measured with annexin-V/propidium iodide (PI) by flow cytometry. Caspase activation was confirmed by Western blot analysis. The effect of the combination on AKT/PI3K and MAPK/ERK signaling pathways and cyclin D1 expression was evaluated by Western blot. Results Treatment with romidepsin alone resulted in time- and dose-dependent cytotoxicity in both cell lines. The IC50 of romidepsin at 24-hour was 5.87 nM and 6.36 nM in Hut-78 and Karpas-299 respectively. Lenalidomide alone did not induce a cytotoxic effect, and we were unable to reach IC50 even after 72 h of treatment. However, after 24 hours, the combination of romidepsin (2.5 nM) and lenalidomide (10µM) (ratio 1:4) showed a strong synergistic interaction with a CI (combination index) of 0.14 in Hut-78 cells, and an additive effect with a CI of 1.08 in Karpas-299 cells. In HUT-78 cells, the combination of romidepsin and lenalidomide enhanced apoptosis compared with each drug alone by the activation of caspases-3, -9 and -8. No change in the expression of Bcl-2 was observed with either treatment alone, or in combination. These events were associated with dephosphorylation of PI3K/Akt and MAPK/ERK pathways, decreased expression of cyclin D1 and Bcl-xL, and an accumulation of acetylated alpha-tubulin. The combination had relatively modest effect on cell cycle parameters. The analysis of the cell cycle showed an increase percentage of cells in sub G0/G1 and G2/M phase, and decrease of S phase. Conclusion These preliminary results indicate that the combination of romidepsin with lenalidomide has a synergistic effect in TCL cell lines and induces apoptosis through signaling events involving pro-survival pathways PI3K/AKT and MAPK/ERK, down-regulation of cyclin D1 and accumulation of acetylated alpha-tubulin. Data look promising and further investigations are required to better define the molecular mechanisms of cell death induced by the combination of romidepsin and lenalidomide in T cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Synergistic Anti-Tumor Efficacy of BET Inhibitors JQ1 and Otx-015 in Combination with Dasatinib in Preclinical Models of T-Cell Lymphomas

Blood ◽

10.1182/blood.v128.22.3967.3967 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3967-3967 ◽

Cited By ~ 1

Author(s):

Sara Rizzitano ◽

Alessandra Cavanè ◽

Marco Piazzoni ◽

Antonio Vendramin ◽

Silvia Gimondi ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

T Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Leukemia Cell ◽

T Cell Lymphoma ◽

Dependent Manner ◽

Mitochondrial Depolarization ◽

Bet Inhibitors

Abstract Background: Approximately 50% of patients with peripheral T-cell lymphoma (PTCL) enter long-term remission after standard chemotherapy and stem cell transplantation. Patients who do not respond to chemotherapy have few treatment options highlighting the critical need for new effective and targeted therapeutics. Aberrant T cell receptor (TCR) and tyrosine kinase (TK) signaling have been described in PTCL (Agostinelli 2014;Netchiporouka 2014). Single-agent TK inhibitors (TKIs) have significantly improved patient outcomes across multiple tumor subtypes. However, TKI therapy is rarely curative. The recent discovery of a subgroup of PTCL characterized by high levels of GATA3 and c-Myc expression and poor prognosis (Iqbal 2014; Manso 2016), establishes the rationale of targeting c-Myc in PTCLs. Based on the demonstration that pharmacologic inhibition of c-Myc is achievable through targeting bromodomain and extra terminal (BET) family of chromatin adapters, the therapeutic potential of BET inhibition was assessed in a panel of T cell lymphoma and leukemia cell lines. Since expression of c-Myc is regulated by the TCR, we also hypothesized that simultaneous targeting of c-Myc and TCR would significantly enhance the antiproliferative effects of BET inhibitors (BETis) and TKI alone in preclinical models of PTCL. Methods: Five T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR-2, Karpas 299, Sup-T1, HH) were incubated with escalating doses of JQ1 (a small-molecule BETi with the highest affinity for BRD4) and OTX-015 (a BETi with a broader affinity for BRD2, BRD3, BRD4) and the tyrosine-kinase-inhibitor Dasatinib. Analysis of cell viability, cell cycle distribution, apoptosis and mitochondrial depolarization was performed using flow cytometry. Effects of treatments were assessed using gene expression profiling (GEP) and western blotting (WB). Combinations were evaluated using the Chou-Talalay Combination Index (CI), calculated with CompuSyn software (CompuSyn Inc, Paramus, NJ). Results: JQ1 and OTX-015 show antiproliferative activity with IC50 at nanomolar concentrations in all cell lines. As assessed determining viable cells by PI exclusion and flow cytometry, JQ1 and OTX-015 are similarly active in a dose-dependent manner in all cell lines. To understand the activity of JQ1 and OTX-015, we analyzed cell-cycle distribution using flow cytometry. JQ1 and OTX-015 induce a cell cycle arrest with G1-phase accumulation and decrease S-phase with the exception of SUPT1 cells that are characterized by a cell cycle arrest in G2-phase. Minimal increase in the sub-G1 population is observed in all cell lines, suggesting that JQ1 and OTX-015 mainly exert a cytostatic effect. We then examined GATA3 and c-Myc protein levels in all cell lines: varying amounts of GATA3 and c-Myc proteins were observed but a strong correlation between GATA3 and c-Myc expression was detected. After JQ1 and OTX-015 exposure, c-Myc protein level decrease in all cell lines apart from SUP-T1 cell line. Here c-Myc level do not change significantly upon BETis exposure, suggesting that BETis target other pathways relevant for SUP-T1 survival. Dasatinib efficiently inhibits the proliferation in all cell lines at micromolar concentrations in a dose-dependent manner. Dasatinib induces G0/G1-phase arrest and an increase in sub-G1 population indicating a modest induction of apoptosis confirmed by caspase-9 activation and mitochondrial depolarization. Compared to all single agents, combined treatments with sub-optimal concentrations of Dasatinib and JQ1 or OTX-015 exert synergistic lethal activity against all tested cell lines (C.I.<1). To uncover the main biological processes behind the synergistic interactions of BETis and Dasatinib, cell cycle analysis was assessed indicating that both combinations induce a significant increase of sub-G1 population associated with massive mitochondrial depolarization and cleavage of Caspase-9 and PARP. Conclusions: The experiments presented here support the combination of BET inhibitors with the TK inhibitor Dasatinib for PTCLs. Our data suggest a synergistic interaction for the combination of both BETis and Dasatinib in vitro. Mechanistically, combined treatments exert synergistic anti-tumor effects in all cell lines through growth inhibitory effects, direct induction of cell death by promotion of caspase-dependent apoptosis and mitochondrial depolarization. Disclosures No relevant conflicts of interest to declare.

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