L428 Cells Research Articles

Combinatorial ixazomib and belinostat therapy induces NFE2L2-dependent apoptosis in Hodgkin and T-cell lymphoma.

Ixazomib activity and transcriptomic analyses previously established in T cell (TCL) and Hodgkin (HL) lymphoma models predicted synergistic activity for histone deacetylase (HDAC) inhibitory combination. In this present study, we determined the mechanistic basis for ixazomib combination with the HDAC inhibitor, belinostat, in HL and TCL cells lines (ixazomib-sensitive/resistant clones) and primary tumour cells. In ixazomib-treated TCL and HL cells, transient inhibition followed by full recovery of proteasomal activity observed was accompanied by induction of proteasomal gene expression with NFE2L2 (also termed NRF2) as a prominent upstream regulator. Downregulation of both NFE2L2 and proteasomal gene expression (validated by quantitative real time polymerase chain reaction) occurred with belinostat treatment in Jurkat and L428 cells. In addition, CRISPR/Cas9 mediated knockdown of NFE2L2 in Jurkat cells resulted in a significant decrease in cell viability with ixazomib compared with untreated control cells. Using transcriptomic and proteasomal activity evaluation of ixazomib, belinostat, or ixazomib+belinostat treated cells, we observed that NFE2L2, proteasome gene expression and functional recovery were abrogated by ixazomib+belinostat combination, resulting in synergistic drug activity in ixazomib-sensitive and -resistant cell lines and primary cells. Altogether, these results suggest that the synergistic activity of ixazomib+belinostat is mediated via inhibition NFE2L2-dependent proteasomal recovery and extended proteasomal inhibition culminating in increased cell death.

Proteomic analysis and verification of protein expression after upregulation of human CD99 in Hodgkin lymphoma cell line L428

目的探索稳定过表达CD99前后霍奇金淋巴瘤细胞株L428细胞中差异表达的蛋白，并分析验证差异蛋白的功能。方法采用荧光差异凝胶双向电泳和质谱分析技术检测L428细胞稳定过表达CD99前后蛋白表达的差异，采用GOfact开放软件对差异蛋白进行聚类分析，筛选在细胞转化过程中起重要作用的蛋白，并分析验证其功能。结果筛选鉴定得到38个差异蛋白，其中在稳定过表达CD99的霍奇金淋巴瘤细胞株L428（L428-CD99细胞）中与CD99表达呈正相关的蛋白21个，呈负相关的蛋白17个，所得的38个差异蛋白中有32个蛋白参与了细胞的生物过程，35个蛋白参与了细胞的组成和构建，28个参与了抗氧化、蛋白结合、催化活性、酶调节、信号转导、结构分子、翻译调节和离子转运等多个方面。结论筛选得到38个差异蛋白，其中关于细胞骨架、细胞分化、信号通路、调节基因表达等蛋白的改变与H/RS细胞和B淋巴瘤细胞转化相关。

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.

Nutritional status regulates algicidal activity of Aeromonas sp. L23 against cyanobacteria and green algae.

Algicidal bacteria have received broad acceptance as an ecofriendly tool for controlling harmful algal blooms. However, their practical application is still limited to the lab-scale tests due to the complex alga–bacterium interactions in different nutrient statuses. In this study, the Aeromonas sp. L23 that exhibit relatively wide-spectrum in algicidal activity was isolated from a eutrophic agricultural lake. The physiological response of cyanobacteria and green to the algicidal activity under varied nutritional status were studied in an alga-bacterial co-culture. The algicidal activities of L23 against Microcystis aeruginosa UTEX LB 2385, Microcystis aeruginosa NHSB, Anabaena variabilis AG10064, Scenedesmus quadricauda AG10003, and Chlorella vulgaris AG10034 were 88 ± 1.2%, 94 ± 2.6%, 93 ± 0.5%, 82 ± 1.1%, and 47 ± 0.9%, respectively. The L23 cells had low algicidal activity in cell pellet (3%–9%) compared with the cell-free supernatant (78%–93%), indicating that the activity is induced by extracellular substances. Adding glucose, NaNO3, NH4Cl, and KH2PO4 to the co-culture raised the algicidal activity of the L23 against green algae by 5%–50%. Conversely, a 10%–20% decrease in activity occurred against the target cyanobacteria except M. aeruginosa UTEX LB 2385. These results indicated that the interspecific algicidal activity changes according to the nutritional status, which means that the alga-bacterium interaction will be more complex in the field where the nutritional status changes from time to time.

Activated Ral and Mutated RAS Are Independent Drivers of Multiple Myeloma Cell Survival.

IntroductionRAS-associated pathways are promising therapeutic targets in RAS-driven tumors because oncogenic RAS itself is not druggable. Alongside the pathways via PI3K/AKT and RAF/MAPK, the small GTPase RAL is considered to represent a third route for oncogenic RAS signaling, which mediates malignant transformation and tumor cell survival. We performed shRNA-mediated knockdown studies in multiple myeloma (MM) cells to investigate the functional role of RAL activity and to analyze the putative functional link between oncogenic RAS and RAL. Moreover, we used screening approaches to identify possible RAL effectors and interacting partners.MethodsFirst, we analyzed expression of the isoforms RALA and RALB in bone marrow biopsies (n=24) and in CD138+ selected primary MM cell samples (n=10) by immunohistochemistry and Western blotting, respectively. Next, we generated two isoform-specific shRNA expression vectors for each of the RAL isoforms to perform RNAi-mediated RAL knockdown and to analyze the functional consequences of RAL abrogation. Experiments were also carried out in combination with clinical anti-myeloma drugs. Furthermore, we treated MM cells with a recently developed pharmacological RAL inhibitor. MM cell survival and apoptotic cells were measured by flow cytometry with annexin V/propidium iodide staining. Changes in RAF/MAPK, PI3K/AKT or RAL pathway activation were detected by Western analysis. We then combined RAL pulldown and knockdown of mutated RAS to investigate the functional link between RAL activation and oncogenic RAS signaling. In addition, we compared RAS- and RAL-mediated gene expression profiles by RNA sequencing. Last, we used mass spectrometry to identify potential RAL effectors and interaction partners.ResultsRAL protein was detected in all MM cells tested, with RALA showing ubiquitously strong expression and RALB showing more heterogeneous stainings. In contrast, RALA and RALB expression was weak or absent in MGUS or normal plasma cells. MM cell survival was strongly impaired by shRNA-mediated RALA or RALB knockdown, with cell survival rates of 25% or 40 % in L-363 cells and 32% or 52% in MM.1S cells, respectively. No cross-activation between RAL and PI3K/AKT or RAF/MAPK pathways could be detected in MM cell lines. Pharmacological RAL inhibition was achieved in an MM subgroup at concentrations of 20 µM. Combining RAL knockdown and treatment with carfilzomib, pomalidomide, or ixazomib led to enhanced cell death induction. Of note, knockdown of oncogenic RAS also strongly reduced MM cell survival, but did not change constitutive RAL activation as detected by pulldown assay. Moreover, RNA sequencing after RAS or RAL knockdown produced differential gene expression profiles (1473 KRAS-regulated genes versus 771 RALA-regulated genes, with an overlap of 235 genes), again suggesting that both targets represent distinct signaling pathways. Using mass spectrometry, we identified novel RAL interaction partners which are currently being evaluated.ConclusionOur data indicate that the RAL signaling pathway constitutes a promising therapeutic target in MM and mediates MM cell survival and apoptosis independently of oncogenic RAS. Clinical translation of novel pharmacological RAL inhibitors may therefore be a future therapeutic strategy to tackle MM. DisclosuresNo relevant conflicts of interest to declare.

Combination of Momelotinib and Citarinostat Show Strong Synergistic Effect in Lymphoid Cell Lines Co-Targeting JAK2/STAT3 and HDAC6

▪Introduction. Janus kinases (JAKs) are well described signaling kinases comprising four family members JAK1, JAK2, JAK3 and TYK2 that are essential in hematological malignancy, as JAK mutations have been shown to contribute to the pathogenesis of myeloproliferative disorders. Momelotinib is a potent inhibitor of JAK1/JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Citarinostat, a second generation HDAC6 selective inhibitor, is a well-tolerated compound compared with nonselective HDAC inhibitors, with reduced potency against Class I HDACs while retaining anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available.Methods. Momelotinib and citarinostat alone and in combination were tested in 12 lymphoid cell lines: WSU-NHL, RL, Karpas422 (follicular lymphoma), Granta519, Jeko1 (mantle cell lymphoma), Hut78 (cutaneous T cell lymphoma), Karpas299 (anaplastic large cell lymphoma), L540, L1236 (Hodgkin's lymphoma), U266, RPMI8266 (multiple myeloma) and MEC1 (chronic lymphocytic leukemia).Synergistic interaction was evaluated using the Chou-Talalay method. Annexin V staining, ROS generation, cell cycle and migration assay were determined by flow cytometry. ATP levels and Mitochondrial Membrane Potential (ΔΨm) were evaluated by fluorometric assay. Lactate levels and Cyt-C were evaluated by colorimetric assay. Activity of caspases-8,-9 and 3 was measured using colorimetric assay. ER stress and apoptosis-related proteins and JAK2/STAT3 were detected by western blotting. Clonogenic survival was studied with the methylcellulose clonogenic assay. Co-cultures with bone marrow stromal cells were also performed.Results. The combination of momelotinib (1 μM) and citarinostat (4 μM) at 24 h showed a synergistic effect in WSU-NHL, RL, Karpas422, Jeko1, Hut78, Karpas299, L540, RPMI8226 and U266 cells with CI values < 1 and antagonist effect in L1236, Granta519 and Mec1 cells with CI > 1. We studied seven lymphoid cell lines (WSU-NHL, RL, Karpas422, Jeko1, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. Drug combination exhibited a strong cytotoxicity, evidenced by reduction of mitochondrial depolarization, depletion of ATP and lactate levels and Cyt-C release from the mitochondria but also by increase in cellular apoptotic index and reactive oxygen species levels, leading to arrest in the sub-G0/G1 phase of the cell cycle. Apoptosis induced by the drug combination was exerted via the mitochondrial apoptotic pathway as demonstrated by upregulation of caspase-9 that was especially evident in WSU-NHL and Karpas422 with a fold increase of 3.2 and 3.8. The extrinsic apoptotic pathway was active in Karpas422 and Jeko1 cells as evaluated by upregulation of caspase-8 but not in WSU-NHL, RL and L540. The apoptosis was associated with activation of caspase-3, PARP and with increased hallmarks of ER stress and was mediated by the increased expression of the pro-apoptotic proteins Bad, Bax and Bim and downregulation of Bcl2, Bcl-xL and Mcl-1. Drug combination inhibited the migration induced by CXCL12 (chemoattractant known as stromal-cell derived factor-1, SDF-1α), reduced clonogenic survival and suppressed cell viability of lymphoid cells when co-cultured with bone marrow mesenchymal stromal cells targeting JAK2/STAT3 pathway and confirming acetylation of acetyl-α tubulin.Conclusion. Due to the good toxicity profile and the oral administration, combined therapy with momelotinib and citarinostat may represent a promising novel therapeutic modality for hematological malignancies. The study is ongoing and further investigation is required. DisclosuresNo relevant conflicts of interest to declare.

Myeloma-Specific Oncolytic Adenovirus Induces Significant Tumour Oncolysis In Vitro and In Vivo and Prevents Cell Line Regrowth

Multiple myeloma is a largely incurable disease and despite current therapies achieving good initial responses, patients frequently relapse. Therefore, new approaches are required that not only reduce the tumour load, but also prevent the growth of residual disease. One such approach is the use of oncolytic viruses. We developed an oncolytic adenovirus that utilizes transcriptional control of E1A under the myeloma-specific promoter CS1 (ADCE1A). We hypothesised that ADCE1A would be myeloma-specific, inducing tumour oncolysis and preventing tumour regrowth.A panel of myeloma cell lines (JJN3, L-363, OPM-2, U-266, RMPI-8226, NCI-H929, KMS-11, KMS-12-BM) were assessed for expression of CS1 by flow cytometry (FC). The same panel were treated with ADCE1A and cell death was monitored after 72 hours using FC and propidium iodide staining. Apoptosis was assessed following ADCE1A infection using annexin V staining and analysed by FC at 6 and 24 hours post treatment. CD138+ plasma cells from bone marrow aspirates were obtained from myeloma and plasma cell leukaemia patients and from the peripheral blood from healthy donors. The CD138+ and CD138- populations from these samples were treated with ADCE1A and cell death was monitored after 4 days using FC and propidium iodide staining. Myeloma cell regrowth was assessed after bortezomib (0.56-2.81nM) or bortezomib in combination with ADCE1A treatment using cell counts. Viral efficacy was tested in a xenograft model of myeloma, where 5 weeks after tumour cell injection (106 U266 cells intravenously), mice were treated with ADCE1A (1x107 pfu, 2x/wk) or control (PBS) for 3 weeks. Tumour burden was measured ex vivo in bone marrow flushes of the long bones by FC.CS1 was expressed in all myeloma cell lines, except KMS-12-BM. ADCE1A infected, replicated and caused oncolysis in JJN-3, L-363, OPM-2, U-266, RPMI-8226, NCI-H929, and KMS-11 myeloma cell lines. However, KMS-12-BM had the lowest sensitivity to ADCE1A. This correlated with CS1 expression, as CS1 was not expressed at the protein level in this cell line. Apoptosis, as detected by annexin V staining, was found to be a cell death mechanism involved in ADCE1A oncolysis. Importantly, ADCE1A induced oncolysis in primary patient malignant CD138+ plasma cells, but not in the non-malignant CD138- bone marrow mononuclear population from these patients. Additionally, ADCE1A had no effect on cell death in non-malignant CD138+ plasma cells and non-malignant CD138- peripheral blood mononuclear population from healthy donors. ADCE1A prevented regrowth of myeloma cell lines following treatment with bortezomib in vitro. In the U266 xenograft model, tumour load was significantly reduced (p<0.05) compared to control treated mice.In summary, ADCE1A has potential clinical efficacy as shown by preclinical models and patient tumour samples. Additionally, ADCE1A was able to stop tumour cell regrowth after chemotherapy in vitro, therefore, the use of oncolytic adenoviruses to target minimal residual disease may be a novel yet promising approach for the treatment of myeloma. DisclosuresSnowden:Jannssen/J&J: Other: Speaker fees; Jazz & Sanofi: Other: Speaker fees at ASH.

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30−/CD15− cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (−/−)(NSG) mice. Using cell sorting, we demonstrate that CD30−/CD15− subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30−/CD15− cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 103 cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30−/CD15− cells exhibiting high telomerase activity and telomere dysfunction.

MGCD0103 induces apoptosis and simultaneously increases the expression of NF-κB and PD-L1 in classical Hodgkin's lymphoma.

At present there is no consensus on the treatment of classical Hodgkin's lymphoma (CHL) following relapse. The aim of the present study was to access the class I-selective histone deacetylase (HDAC) inhibitor (HDACI) MGCD0103 on the expression levels of Bcl-2, nuclear factor (NF)-κB and programmed death-ligand 1 (PD-L1) in CHL, to explore the possible therapeutic value of MGCD0103 in combined relative target drugs for patients with CHL. In L1236 and L428 cell lines, apoptosis and cell cycle stage were identified using flow cytometry, and the effects of HDACI on CHL were assessed in terms of Bcl-2, NF-κB and PD-L1 expression levels, which were detected by western blotting and co-focusing experiments. The results demonstrated that MGCD0103 could induce cell apoptosis and cell cycle arrest, down-regulate Bcl-2 and increase NF-κB and PD-L1 expression levels in L1236 and L428 cell lines. MGCD0103 decreases Bcl-2 levels and upregulates PD-L1, which indicates that the combined use of HDACIs and a PD-L1 inhibitor in theory may improve treatment outcomes in patients with CHL. MGCD0103 may also up-regulate NF-κB, which seems to induce resistance towards anti-apoptotic drugs. Clinical trials combining HDACIs with NF-κB and/or PD-L1 inhibitors should be designed to further improve treatment outcomes for patients with CHL.

Abstract 2921: Co-inhibition of BET and proteasome enhances ER stress and Bim-dependent apoptosis with augmented cancer therapeutic efficacy

Abstract Background: Agents that inhibit bromodomain and extra-terminal domain (BET) protein, a promising cancer therapeutic target which imposes epigenetic modulation of gene expression, have been actively tested in the clinic as potential anticancer drugs. Some proteasome inhibitors such as carfilzomib (CFZ) are FDA-approved drugs for the treatment of patients with advanced multiple myeloma. The current study focuses on studying the combination of a BET inhibitor (e.g., JQ1) and a proteasome inhibitor (e.g., CFZ) as a novel cancer therapeutic strategy. Methods: Diverse solid and hematologic cancer cell lines were used to test the effects of the combinations on cell survival and apoptosis via SRB assay, colony formation assay, flow cytometric analysis, and Western blotting. Bim knockout cell lines were generated using a CRISPR-based method for mechanistic study. Transcriptional regulation of target genes was examined by quantitative RT-PCR. The anti-tumor effects were also examined in a xenograft tumor model. Results: The tested combinations (e.g., JQ1 with CFZ) synergistically decreased cancer cell survival and enhanced apoptosis in vitro and inhibited tumor growth in vivo. The combinations dramatically induced apoptosis accompanying with enhanced elevation of Bim and several ER stress marker proteins. Bim knockout significantly blocked apoptosis induced by the combinations in both HCT116 and L363 cells. Transcriptional upregulation of Bim by the combinations was observed. Conclusions: The enhanced anti-tumor effects of dual inhibition of BET and proteasome is likely due to augmented induction of ER stress and Bim-dependent apoptosis. Our findings warrant further investigation of this combinatorial strategy as an effective regimen against cancer in the clinic. Citation Format: Guoqing Qian, Weilong Yao, Williams D. Hall, Mala Shanmugam, Sagar Lonial, Shi-Yong Sun. Co-inhibition of BET and proteasome enhances ER stress and Bim-dependent apoptosis with augmented cancer therapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2921.

Phosphatase of regenerating liver-3 (PRL-3) is overexpressed in classical Hodgkin lymphoma and promotes survival and migration

BackgroundPhosphatase of regenerating liver-3 (PRL-3) is implicated in oncogenesis of hematological and solid cancers. PRL-3 expression increases metastatic potential, invasiveness and is associated with poor prognosis. With this study, we aimed to show a possible oncogenic role of PRL-3 in classical Hodgkin lymphoma (cHL).MethodsPRL-3 expression was measured in 25 cHL patients by immunohistochemistry and gene expression was analyzed from microdissected malignant cells. We knocked down PRL-3 in the cHL cell lines L1236 and HDLM2 and used small molecular inhibitors against PRL-3 to investigate proliferation, migration and cytokine production.ResultsPRL-3 protein was expressed in 16% of patient samples. In three different gene expression datasets, PRL-3 was significantly overexpressed compared to normal controls. PRL-3 knockdown reduced proliferation, viability and Mcl-1 expression in L1236, but not in HDLM2 cells. Thienopyridone, a small molecule inhibitor of PRL-3, reduced proliferation of both L1236 and HDLM2. PRL-3 affected IL-13 secretion and enhanced STAT6 signaling. IL-13 stimulation partially rescued proliferation in L1236 cells after knockdown of PRL-3. PRL-3 knockdown reduced migration in both L1236 and HDLM2 cells.ConclusionPRL-3 was overexpressed in a subset of cHL patients. Inhibition of PRL-3 increased IL-13 cytokine production and reduced migration, proliferation and viability. The effects could be mediated through regulation of the anti-apoptotic molecule Mcl-1 and a feedback loop of IL-13 mediated activation of STAT6. This point to a role for PRL-3 in the pathogenesis of Hodgkin lymphoma, and PRL-3 could be a possible new drug target.

Tumor necrosis factor receptor 2 may promote the proliferation and drug resistance of Kapras299 and L428 lymphoma cells via the AKT and WNT/β-catenin signaling pathways.

Circulating soluble tumor necrosis factor receptor 2 (sTNFR2) has been associated with a relatively poor prognosis in various types of lymphoma. However, the specific role of TNFR2 expression in lymphoma cells remains uncharacterized. In the present study, TNFR2 expression was quantified in the Hodgkin lymphoma cell line, L428, and the anaplastic large-cell lymphoma cell line, Karpas299, using RT-PCR and western blot analyses. Karpas299 cells exhibited higher TNFR2 expression than L428 cells. Proliferation and drug resistance experiments demonstrated that Karpas299 cells also possessed a greater proliferative ability and resistance to adriamycin (ADM) than L428 cells, with 50% inhibitory concentrations (IC50) of 1.423±0.24 µmol/l for Karpas299 cells, compared with 0.728±0.15 µmol/l for L428 cells (P<0.05). The knockdown of TNFR2 in Karpas299 cells significantly reduced their proliferative ability; when treated with ADM, the cell inhibition rate increased from 49.34±5.42% to 74.13±6.81% (P<0.05). The upregulation of TNFR2 in L428 cells significantly increased their proliferative ability; when treated with ADM, the cell inhibition rate decreased from 47.03±5.25% to 28.71±4.90% (P<0.05). Investigation of the underlying molecular mechanism indicated that the upregulation of TNFR2 expression in L428 cells increased the expression of β-catenin and the phosphorylation of AKT. In L428 cells overexpressing TNFR2, the β-catenin blocker, DKK1, or the AKT inhibitor, LY294002, abrogated the increase in proliferation induced by TNFR2 and increased cell inhibition rate upon treatment with ADM. In summary, the present study demonstrated that TNFR2 promoted the proliferative and drug resistance abilities of lymphoma cells via the AKT and WNT/β-catenin signaling pathways. This may provide the experimental basis for the further study of TNFR2 activity in lymphoma cells and warrant its investigation as a therapeutic target for lymphoma.

Targeting Hodgkin and Reed-Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance.

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed–Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol’s toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.

Aberrant expression of NKL homeobox gene HLX in Hodgkin lymphoma.

NKL homeobox genes are basic regulators of cell and tissue differentiation, many acting as oncogenes in T-cell leukemia. Recently, we described an hematopoietic NKL-code comprising six particular NKL homeobox genes expressed in hematopoietic stem cells and lymphoid progenitors, unmasking their physiological roles in the development of these cell types. Hodgkin lymphoma (HL) is a B-cell malignancy showing aberrant activity of several developmental genes resulting in disturbed B-cell differentiation. To examine potential concordances in abnormal lymphoid differentiation of T- and B-cell malignancies we analyzed the expression of the hematopoietic NKL-code associated genes in HL, comprising HHEX, HLX, MSX1, NKX2-3, NKX3-1 and NKX6-3. Our approach revealed aberrant HLX activity in 8 % of classical HL patients and additionally in HL cell line L-540. Accordingly, to identify upstream regulators and downstream target genes of HLX we used L-540 cells as a model and performed chromosome and genome analyses, comparative expression profiling and functional assays via knockdown and overexpression experiments therein. These investigations excluded chromosomal rearrangements of the HLX locus at 1q41 and demonstrated that STAT3 operated directly as transcriptional activator of the HLX gene. Moreover, subcellular analyses showed highly enriched STAT3 protein in the nucleus of L-540 cells which underwent cytoplasmic translocation by repressing deacetylation. Finally, HLX inhibited transcription of B-cell differentiation factors MSX1, BCL11A and SPIB and of pro-apoptotic factor BCL2L11/BIM, thereby suppressing Etoposide-induced cell death. Collectively, we propose that aberrantly expressed NKL homeobox gene HLX is part of a pathological gene network in HL, driving deregulated B-cell differentiation and survival.

Monoamine oxidase A is highly expressed in classical Hodgkin lymphoma.

Monoamine oxidase A (MAOA) is a mitochondrial enzyme that catalyzes oxidative deamination of neurotransmitters and dietary amines and produces H2 O2 . It facilitates the progression of gliomas and prostate cancer, but its expression and functional relevance have not been studied in lymphoma. Here, we evaluated MAOA in 427 cases of Hodgkin and non-Hodgkin lymphoma and in a spectrum of reactive lymphoid tissues by immunohistochemistry on formalin-fixed, paraffin-embedded specimens. MAOA was expressed by Hodgkin Reed-Sternberg (HRS) cells in the majority of classical Hodgkin lymphomas (cHLs) (181/241; 75%), with 34.8% showing strong expression. Weak MAOA was also noted in a minority of primary mediastinal large B-cell lymphomas (8/47; 17%) and in a mediastinal gray-zone lymphoma. In contrast, no MAOA was found in non-neoplastic lymphoid tissues, nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL; 0/8) or any other non-Hodgkin lymphomas studied (0/123). MAOA was more common in Epstein-Barr virus (EBV)-negative compared to EBV-positive cHL (p < 0.0001) and was especially prevalent in the EBV-negative nodular sclerosing subtype. Similar to primary human lymphoma specimens, most cHL-derived cell lines displayed MAOA activity, whereas non-Hodgkin-lymphoma-derived cell lines did not. The MAOA inhibitor clorgyline reduced the growth of L1236 cells and U-HO1 cells, and shRNA knockdown of MAOA reduced the growth of L1236 cells. Conversely, ectopic overexpression of MAOA increased the growth of MAOA-negative HDLM2 cells. Combined treatment with clorgyline and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) was more effective in reducing cell growth than either regimen alone. In summary, MAOA is highly expressed in cHL and may reflect the distinct biology of this lymphoma. Further studies on the potential utility of MAOA as a diagnostic marker and therapeutic target are warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 2514: Tetra-O-methyl nordihydroguaiaretic acid (M4N) broadly suppresses cancer metabolism and synergistically induces strong anticancer activity in combination with etoposide, rapamycin and UCN-01

Abstract The ability of Tetra-O-methyl nordihydroguaiaretic acid (M4N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M4N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M4N treatment. For instance, among the amino acids, the content of aspartate, a key metabolite in electron transport system in the mitochondria, was specifically suppressed by M4N. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M4N combination for cancer treatments. Citation Format: Kotohiko Kimura, Ru Chih C. Huang. Tetra-O-methyl nordihydroguaiaretic acid (M4N) broadly suppresses cancer metabolism and synergistically induces strong anticancer activity in combination with etoposide, rapamycin and UCN-01 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2017-2514

Abstract 2124: Differentiation of myeloid and multiple myeloma cell lines by inecalcitol involves a marked decrease in CD44 and CD49d cell surface labeling

Abstract Inecalcitol is a vitamin D receptor (VDR) agonist characterized by a high antiproliferative activity on various cancer cell lines (Okamoto et al., 2012; Ma et al., 2013) and a low calcemic potential as demonstrated by the high maximal tolerated oral daily dose of 4 mg in human patients (Medioni et al., 2014). On myeloid cells, inhibition of proliferation by vitamin D derivatives is paralleled by induction of differentiation into a monocyte/macrophage phenotype, mostly described as gain of functions (increase in lysozyme activity, phagocytosis and cell motility; Abe et al., 1981) and of the cell surface CD11b and CD14 biomarkers. The stimulation of the secretion of cathelicidin, a natural human antimicrobial peptide, is another specific activity of vitamin D and its derivatives (Wang et al., 2004). During a screening of 48 CD biomarkers on 4 human myeloid cell lines (HL-60, U-937, MOLM-13 and THP1) and 4 human multiple myeloma cell lines (MM.1R, MM.1S, L-363 and RPMI-8226) treated for 72 hours at 10 nM, we recently discovered that inecalcitol reproducibly induces CD38 expression (Benjamin et al., 2016), another gain of function. We now report the decrease in CD44 and CD49d cell surface labeling in all 8 cell lines (except CD44 in L-363 cells). This loss of functions reached -60% to -70% in MOLM-13 and HL-60 cells. We have then determined the EC50 (mean ± SEM; n = 3 or 4) of inecalcitol for several differentiation parameters on HL-60 cells. The most sensitive gains or loss of functions were the secretion of cathelicidin (0.2 ± 0.1 nM), the induction of CD38 (0.3 ± 0.1 nM) and the decrease in CD44 (0.8 ± 0.4 nM). Inhibition of proliferation (1.9 ± 0.8 nM) was exactly parallel to induction of CD11b (1.8 ± 0.8 nM). Decrease in CD49d (6.6 ± 1.1 nM) and increase in CD14 (8.0 ± 0.5 nM) occurred as final markers of differentiation by inecalcitol. CD44 is the receptor for hyaluronic acid; it is found on the surface of acute myeloid leukemia stem cells and suspected to favor the spread and relapse of the disease. CD49d is the integrin α4-subunit involved in the interactions of tumor cells with their microenvironment resulting in their “hiding” and survival. Therefore, decreasing both CD44 and CD49d represents newly identified potential beneficial pro-differentiation effects of inecalcitol. Citation Format: Susan Benjamin, Cecile Planquette, Remi Delansorne. Differentiation of myeloid and multiple myeloma cell lines by inecalcitol involves a marked decrease in CD44 and CD49d cell surface labeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2124. doi:10.1158/1538-7445.AM2017-2124

Quorum sensing modulators exhibit cytotoxicity in Hodgkin’s lymphoma cells and interfere with NF-κB signaling

In recent years it has become evident that bacteria can modulate signaling pathways in host cells through the secretion of small signaling molecules. We have evaluated the cytotoxic effects and NF-κB inhibitory activities of a panel of quorum sensing molecules and their reactive analogs on Hodgkin's lymphoma cells (L428). We found that several molecules inhibited NF-κB signaling in a dose dependent manner. Three inhibitors (ITC-12, ITC-Cl and Br-Furanone) showed 50% NF-κB inhibition at concentrations less than 10µM (4.1µM, 12.8µM and 9.9µM, respectively). Furthermore, all three molecules displayed cytotoxic effects against L428 cells with IC50 values of 12.4µM, 18.3µM and 3.1µM respectively after 48h incubation. They also showed inhibition of A549 adenocarcinoma cell migration at low concentrations 5.6µM, 2.6µM and 7.9µM respectively. Further analysis showed that these molecules significantly decrease the degree of expression of proteins of NF-κB subunits p50, p65 and RelB both in cytosolic and nuclear fractions. This confirms that these compounds have the potential to modulate the NF-κB pathway by suppressing their subunits and thus exhibit cytotoxicity and inactivation of NF-κB signaling in Hodgkin's lymphoma cells.

A novel PTPN1 splice variant upregulates JAK/STAT activity in classical Hodgkin lymphoma cells

AbstractChronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways is a hallmark of a variety of B-cell lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive JAK/STAT signaling is crucial for survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of cHL. Although the molecular basis of this constitutive JAK/STAT signaling in cHL has not been completely understood, accumulating reports highlight the role of an inactivation or reduced expression of negative JAK/STAT regulators such as silencer of cell signaling 1 (SOCS1) or protein-tyrosine phosphatase 1B (PTP1B) in this process. Here, we report the expression of truncated PTP1B mRNA variants identified in cHL cell lines and primary cHL tumor samples lacking either 1 or several exon sequences. One of these novel PTP1B variants, a splice variant lacking exon 6 (PTP1BΔ6), was found expressed at low levels in cHL cell lines. However, serum stimulation of cHL augmented the expression of PTP1BΔ6 significantly. Functional characterization of PTP1BΔ6 revealed a positive effect on interferon-γ- and interleukin-4-induced JAK/STAT activity in HEK293 or HEK293-STAT6 cells, and on the basal STAT activity in stably transfected L-428 and U-HO1 cHL cell lines. Furthermore, PTP1BΔ6 expression increased the proliferation of L-428 and U-HO1 cells and reduced cytotoxic effects of the chemotherapeutical agents gemcitabine and etoposide distinctively. Collectively, these data indicate that PTP1BΔ6 is a positive regulator of JAK/STAT signaling in cHL.

BCL-2 inhibitor combined with low dose cytarabine in the treatment of relapsed acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation: a case report and literature review

目的探索GTP结合蛋白氯苄乙胺2（Septin2）在霍奇金淋巴瘤细胞株L428细胞中的表达及其在H/RS（Hodgkin/Reed-Sternberg）细胞向B细胞再分化中的作用。方法采用实时荧光定量PCR（RT-qPCR）、Western blot、激光共聚焦显微镜、免疫细胞化学法检测霍奇金淋巴瘤细胞株L428细胞过表达CD99前后Septin2在mRNA和蛋白水平的表达；采用RT-qPCR和Western blot检测L428细胞转染Septin2干扰片段（Septin2-siRNA）后Septin2的表达；采用激光共聚焦显微镜、CCK8法和流式细胞术检测干扰Septin2基因对L428细胞骨架蛋白、细胞增殖活性和免疫表型的影响。结果与L428细胞空白对照组比较，L428细胞Septin2 mRNA表达水平在CD99过表达后（0.329±0.019对1.000，P=0.001）和转染Septin2-siRNA后（0.276±0.025对1.000，P=0.000）均下降，蛋白表达水平也下降。与转染空载体组比较，转染Septin2-siRNA后的L428细胞较前者胞体缩小，细胞增殖活性下降（F=204.927，P<0.001)，细胞中的微丝骨架发生了重建，CD30和CD15表达下降，CD19、CD10、CD38表达增高。结论Septin2在L428细胞中高表达，干扰Septin2可促进H/RS细胞向B细胞方向再分化。