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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4872-4872 ◽  
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.

The Brd-Inhibitor OTX015 Is Active in Pre-Clinical Models of Mature B-Cell Lymphoid Tumors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1657-1657 ◽  
Author(s):  
Paola Bonetti ◽  
Michela Boi ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stahis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Rt Pcr ◽  
Down Regulation ◽  
Myc Gene ◽  
Dose Dependent

Abstract Abstract 1657 Background: Bromodomain-containing proteins play an important role in gene expression regulation, via chromatin structure remodelling. Antitumor activity has been reported in acute and chronic hematological malignancies using inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a large panel of cell lines derived from mature B-cell lymphoid tumors. Material and Methods: Established human cell lines derived from 13 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphoma (MCL), three splenic marginal zone lymphoma (SMZL) and from three multiple myeloma (MM) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72 hours 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 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 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: OTX015 demonstrated anti-proliferative activity in DLBCL cell lines (median IC50 0.192μM; range 0.069–12.68μM). Similar results were obtained on SMZL (median IC50 0.165μM, range 0.105–0.24μM), and on MM cell lines (median IC50 0.449μM; range 0.06–0.7μM). Conversely, MCL cell lines appeared less sensitive to OTX015 (median IC50 2.01μM; range 1.22- >15μM). Among DLBCL cell lines, there was no significant difference based upon the cell of origin of the cell lines. OTX105 caused a cell cycle arrest in G1 in a dose-dependent manner in 5/5 DLBCL and 3/3 MM cell lines, without an increase in cell death. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in 1/1 sensitive DLBCL cell line. In order to understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after 24h treatment with increasing doses. We observed a dose-dependent suppression of MYC mRNA by OTX015 in 4/5 DLBCL and in 2/2 MM cell lines. In DLBCL, down-regulation of MYC mRNA was observed within 1h after treatment with OTX015, suggesting a direct effect of the compound on the MYC gene. To determine whether the suppression of MYC gene by OTX015 was reversible, DLBCL cell lines were treated for 2h with OTX015 and then the inhibitor was removed from the media. MYC mRNA suppression appeared reversible, as shown in DLBCL cell lines, which, after 2h exposure to OTX015, showed a time-dependent restoration of MYC mRNA expression to untreated levels after 2–3h. In one of the most sensitive DLBCL cell lines no MYC mRNA down-regulation was observed after treatment, suggesting that alternative pathways can be affected by BRD-inhibition. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several cell lines representative of mature B-cell tumors. An apparently reversible down-regulation of MYC mRNA was commonly observed, appearing as a possible mechanism of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in mature B-cell origin malignancies. A phase I trial is scheduled to start in 2012. Disclosures: Bonetti: OncoEthix SA: Research Funding. Inghirami:OncoEthix SA: Research Funding. Noel:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Bertoni:OncoEthix SA: Research Funding.

scholarly journals Identification of the Epigenetic Reader BRD4 As a Novel Therapeutic Target in JAK2 V617F+ MPN Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2829-2829
Author(s):  
Alexandra Keller ◽  
Barbara Peter ◽  
Johannes Zuber ◽  
Philipp Bernhard Staber ◽  
Peter Bettelheim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Jak2 V617f ◽  
G1 Arrest ◽  
Mrna Levels ◽  
Neoplastic Cells ◽  
Hel Cells ◽  
Ic50 Values ◽  
Brd4 Inhibition

Abstract Myeloproliferative neoplasms (MPN) are characterized by clonal expansion and accumulation of erythrocytes, platelets, and myeloid cells in the bone marrow (BM) and other organs. Classical MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation is frequently detected in neoplastic cells in patients with MPN. Although MPN are chronic and indolent diseases in most patients, fatal progression may occur. So far, the only curative approach for these patients is hematopoietic stem cell transplantation. Therefore, current research is evaluating new therapeutic targets and the effects of various targeted drugs. The epigenetic reader bromodomain-containing protein 4 (BRD4) has recently been identified as a promising target in acute myeloid leukemia. In the present study, we investigated the potential value of BRD4 as a molecular target in MPN. We employed two JAK2 V617F+ cell lines, SET-2 and HEL, as well as BM samples obtained from 18 MPN patients (ET: n=7; PV: n=7; PMF: n=4). Three BRD4 inhibitors were applied: JQ1, BI2536, and BI6727. As assessed by qPCR, primary MPN cells as well as SET-2 cells and HEL cells were found to express BRD4 mRNA. In 3 H-thymidine uptake experiments, all three BRD4 blockers were found to suppress the proliferation of the two MPN cell lines and of primary MPN cells in 8/8 patients tested. The effects of these drugs were dose-dependent, with the following IC50 values obtained in SET-2 cells: JQ1, 50-100 nM; BI2536, 20-40 nM; BI6727, 50-75 nM; and in HEL cells: JQ1, 100-500 nM; BI2536, 20-40 nM; BI6727, 30-50 nM. In primary MPN cells, all three agents tested produced IC50 values between 500 and 1000 nM. In normal BM cells, JQ1 did not produce a reasonable IC50 value (>5000 nM). In one patient sample (PMF), we analyzed the effect of JQ1 on the percentage of putative (neoplastic) stem cells (CD34+/CD38-). In this experiment, exposure to JQ1 was followed by a decrease in the percentage of CD34+/CD38- cells compared to control medium (control: 0.16% vs JQ1: 0.045%). To confirm the role of BRD4 as a potential target in MPN cells, we performed target-knockdown experiments in SET-2 cells and HEL cells using two different BRD4 shRNAs (#602 and #1817) and a random shRNA as control. In these experiments, the shRNA-induced knockdown of BRD4 was found to block proliferation in SET2 cells and HEL cells when compared to untransfected cells or random shRNA-transduced cells. In a next step, we examined the mechanism of drug-induced growth inhibition. In cell cycle experiments, BI2536 and BI6727 were found to induce a G2/M phase arrest in both cell lines. By contrast, JQ1 induced a G1 arrest in HEL cells, but did not show a significant effect on cell cycle progression in SET-2 cells. We also asked whether BRD4 inhibition is associated with induction of apoptosis in MPN cells. All three BRD4 blockers induced apoptosis in SET-2 cells and HEL cells at relatively high concentrations after 48 hours, with ED50 values of >5 µM for JQ1 and 0.5-5.0 µM for BI2536 and BI6727. Finally, we asked whether exposure to BRD4 inhibitors is associated with modulation of BRD4 mRNA or MYC mRNA expression. As assessed by qPCR, JQ1, BI2536, and BI6727 were found to downregulate BRD4 mRNA levels as well as MYC mRNA levels in SET-2 cells and HEL cells. In conclusion, our data show that BRD4 is expressed in JAK2 V617F+ MPN cells and that BRD4 inhibition is associated with decreased proliferation and survival of neoplastic cells. The clinical value of BRD4 as a novel target in MPN cells remains to be determined. Disclosures Zuber: Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Staber:Genactis: Research Funding; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1667-1667
Author(s):  
Adam Kittai ◽  
Scott R Best ◽  
Taylor Rowland ◽  
Nur Bruss ◽  
Craig Okada ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Jurkat Cells ◽  
Lymphoma Cells ◽  
E3 Ligases ◽  
Cell Cycle Deregulation

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.

Alternation of Cellular Morphology and Proliferation Induced by microRNA in Human Leukemia Cell Line, UT-7/EPO.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4203-4203
Author(s):  
Nobuyoshi Kosaka ◽  
Yusuke Yamamoto ◽  
Nami Nogawa ◽  
Keiichi Sugiura ◽  
Hiroshi Miyazaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Macrocytic Anemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Morphological Studies

Abstract Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals 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 ◽  
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.

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

scholarly journals CD55 and CD59 Can Limit the Anti-Tumor Efficacy of Daratumumab in Natural Killer/T-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1663-1663 ◽  
Author(s):  
Nurulhuda Mustafa ◽  
Adina Huey Fang Nee ◽  
Jing Yuan Chooi ◽  
Sabrina Hui Min Toh ◽  
Yan Ting Hee ◽  
...  
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
Correlation Coefficient ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Natural Killer T Cell ◽  
Cd38 Expression ◽  
Killer T Cell

Abstract Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38. In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and 'Sensitive' tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL. FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either. This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC. To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment. Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. Disclosures Zhou: Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

scholarly journals PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1509-1509
Author(s):  
Katsuyoshi Takata ◽  
Lauren C. Chong ◽  
Avinash Thakur ◽  
Tomohiro Aoki ◽  
Anja Mottok ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Sequencing Data ◽  
And Function ◽  
Inhibitor Treatment

Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

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

Blood ◽  
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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