NVP-BEZ235, A Dual Inhibitor of Phosphoinositol-3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), Is a Potent Inhibitor of Lymphoma Cell Growth and Survival

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4965-4965 ◽  
Author(s):  
Daniela Buglio ◽  
Manuela Lemoine ◽  
Sattva S. Neelapu ◽  
Francisco Vega ◽  
Donald Berry ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Lymphoma Cell ◽  
Parp Cleavage ◽  
Lymphoma Cells ◽  
Dual Inhibitor ◽  
Growth And Survival ◽  
Stat Pathway ◽  
Cell Growth And Survival

Abstract Abstract 4965 The Phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway is frequently deregulated in Hodgkin (HL) and non-Hodgkin lymphoma (NHL), and has been linked with tumor cell growth and survival. Although several proteins/enzymes in this pathway can be targeted by a variety of small molecules in vitro and in vivo, it remains unclear which protein target is the ideal for clinical testing. Previous studies demonstrated that the clinical activity of mTOR inhibitors may be attenuated by a negative feedback loop that involves activation of AKT, suggesting that a dual inhibition of AKT and mTOR activation may produce a better therapeutic outcome. To test this hypothesis, we evaluated the in vitro activity of NVP-BEZ235, a dual inhibitor of PI3K and mTOR, in a panel of 13 HL and NHL cell lines. NVP-BEZ235 inhibited cell growth and induced apoptosis in lymphoma cell lines in a time and dose dependent manner. After 48 hours of incubation, the IC50 ranged between 50 and 100 nM, and it was equally effective in ABC and GCB-derived DLBCL cell lines. NVP-BEZ235 induced cell death was primarily due to induction of apoptosis, as evident by the annexin-V and PI dual staining method, and the induction of caspase 3 and PARP cleavage. NVP-BEZ235 effectively inhibited the activation of the PI3K pathway at several steps, including decreasing the phosphorylation level of p-Akt (Ser473), p-Akt (Thr308), p-mTOR, p-4-EBPI and pP70S6K. Because lymphoma cells frequently depend on multiple activated signaling pathways to promote their survival, including the JAK/STAT pathway, we investigated the potential synergy between PI3K and JAK/STAT pathway inhibitors. Lymphoma cells were variably sensitive to the JAK1/2 inhibitor INCB16562 in vitro. Submaximal concentrations of NVP-BEZ235 demonstrated a synergistic activity with INCB16562. Collectively, our data show that the PI3K/mTOR inhibitor NVP-BEZ235 is highly effective against a wide range of lymphoma cell lines, and warrants evaluating it alone and in combination with JAK/STAT inhibitors in phase I/II clinical trials in patients with relapsed lymphoma. Disclosures: No relevant conflicts of interest to declare.

Stromal Interaction of Lymphoma Cells Regulates Survival and Chemoresistance

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5195-5195
Author(s):  
Gyeongsin Park ◽  
Byunghoo Song ◽  
Yuji Lee ◽  
Ahwon Lee ◽  
Yang-Guk Chung ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Lymphoma Cell ◽  
Low Grade ◽  
High Grade ◽  
Lymphoma Cells ◽  
Growth And Survival ◽  
Stromal Microenvironment ◽  
Cell Growth And Survival

Abstract Abstract 5195 High-grade lymphomas are aggressive but largely curable, whereas low-grade lymphomas are indolent, but frequently recur to be incurable, paradoxically. Mesenchymal stromal cells (MSCs) have been known to participate for reconstituting microenvironment. Studies show that signalings between normal lymphoid cells and stromal cells were frequently altered in high grade lymphomas, but relatively conserved in low grade lymphomas. However, which cell and mechanism is responsible for lymphoma recurrence remains unclear. Here we hypothesized that the interaction with stroma may play a role for lymphoma cell growth and survival. For this, we investigated the effect of MSCs on lymphoma cell growth and chemo-resistance by using a coculture system with MSCs (derived from tonsil), as a stromal microenvironment for lymphoma cells. First, coculture of lymphoma cell line (Pfeiffer) and primary lymphoma cells with/without MSCs for 3 days showed that the MSC-cocultured cells grew more rapidly (1.4 times and 1.8 times for Pfeiffer and primary cells, respectively) than MSC-free lymphoma cells. To further investigate the underlying mechanism of promoting growth, lymphoma cells were cocultured with MSCs in the presence or absence of transwell filter. At day 4, the proliferation of lymphoma cells in the absence of transwell was 3.5 times higher than in the presence of transwell. Interestingly, the lymphoma cells cocultured with MSCs showed increased expression of CXCR4 compared with lymphoma cells cultured alone. When the cyto-protective effect of MSCs was examined by doxorubicin treatment (1ug/ml) in the presence or absence of MSCs, 91% of MSC-cocultured cells survived, whereas only 28% of stroma-free cultured cells survived. These results demonstrate that the direct contact interaction with stroma might be important for lymphoma cell growth and survival. In conclusion, our data suggest that the interaction with stromal microenvironment is an important factor for survival of lymphoma cells which cells might be responsible for chemoresistance, and raise the possibility that the stromal interaction could be a potential target for lymphoma treatment. Disclosures: No relevant conflicts of interest to declare. This research was supported by a grant (10172KFDA993) from Korea Food & Drug Administration in 2011. Disclosures: No relevant conflicts of interest to declare.

The HCK/BTK inhibitor KIN-8194 is active in MYD88 driven lymphomas and overcomes mutated BTKCys481 ibrutinib resistance.

Blood ◽  
2021 ◽  
Author(s):  
Guang Yang ◽  
Jinhua Wang ◽  
Li Tan ◽  
Manit Munshi ◽  
Xia Liu ◽  
...  
Keyword(s):  
B Cell ◽  
Malignant Cell ◽  
Pharmacokinetic Parameters ◽  
B Cell Malignancies ◽  
Lymphoma Cells ◽  
Dual Inhibitor ◽  
Growth And Survival ◽  
Activating Mutations ◽  
Ibrutinib Resistance

Activating mutations in MYD88 promote malignant cell growth and survival through HCK mediated BTK activation. Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481 particularly BTKCys481Ser are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88 mutated lymphoma cells, including ibrutinib resistant BTKCys481Ser expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained HCK and BTK inhibition for 24 hours following single oral administration of KIN-8194 in MYD88 mutated TMD-8 ABC DLBCL xenografted mice with either wild-type BTK (BTKWT) or BTKCys481Ser expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT and BTKCys481Ser expressing TMD-8 DLBCL xenografted mice, including sustained complete responses >12 weeks off treatment in mice with BTKWT expressing TMD-8 tumors. The Bcl-2 inhibitor venetoclax enhanced the anti-tumor activity of KIN-8194 in BTKWT and BTKCys481Ser expressing MYD88 mutated lymphoma cells, and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Maria Cosenza ◽  
Stefano Sacchi ◽  
Samantha Pozzi
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Biological Effects ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Induction Of Apoptosis ◽  
Preclinical Efficacy

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI < 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI > 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3526-3526
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Sophia Adamia ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Genomic Alteration ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

Recruitment of the SWI/SNF Chromatin Remodeling Complex by NFATc1 in the Transcriptional Regulation of the C-MYC Oncogene in Aggressive B-Cell Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3808-3808 ◽  
Author(s):  
Lan V. Pham ◽  
Archito Tamayo ◽  
Hai-Jun Zhou ◽  
Yen-Chiu Lin-Lee ◽  
Lingchen Fu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Chromatin Remodeling ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Growth And Survival ◽  
Myc Oncogene ◽  
Cell Growth And Survival

Abstract The NFAT (nuclear factor of activated T-cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway for involvement in the pathogenesis of various malignancies, including large B-cell lymphoma (LBCL), a non-Hodgkin’s lymphoma subgroup that is generally responsive to conventional cancer therapies (R-CHOP), but relapse is common that subsequently leads to therapeutic resistance. Although we have shown previously that NFAT family member NFATc1 is constitutively activated and has the ability to maintain cell growth and survival in LBCL cell lines and primary cells, the molecular mechanism(s) underlying how NFATc1 regulates cell growth and survival in LBCL is still unclear. In this study, we demonstrate that the well-known oncogene c-myc is transcriptionally regulated by the transcription factor NFATc1 in LBCL, through a chromatin remodeling mechanism that involves the recruitment of the SWI/SNF chromatin-remodeling complex. In aggressive B-cell lymphoma cell lines, c-myc oncogene protein expression was shown to correlate with NFATc1 protein expression. We further showed that NFATc1 binds to a specific DNA binding element within the proximal c-myc promoter and up-regulates c-myc transcription. The SWI/SNF proteins Brg-1 and Brm, chromatin-remodeling proteins that utilize ATP hydrolysis for energy to modify chromatin structure in order to regulate gene expression, also were shown to bind to the NFAT binding site on the c-myc promoter. Confocal microscopic analysis showed that NFATc1 colocalizes with Brg-1, and co-immunoprecipitation assays showed that Brg-1 interacts with NFATc1. Both proteins interact with the c-myc promoter within the NFAT binding site, as demonstrated by chromatin-immunoprecipitation (ChIP) analysis. Induction of a constitutively active mutant of NFATc1 (caNFATc1) in an NFATc1 negative lymphoma cell line induces c-myc protein expression. Constitutively active NFATc1 also enhances Brg-1 binding to the c-myc promoter when analyzed by ChIP-qPCR assays, suggesting that NFATc1 recruits Brg-1 to the NFAT binding site in the c-myc promoter. Down-regulation of NFATc1 by chemical inhibitors (FK-506) or by validated shRNA of NFATc1, inhibited c-myc protein expression and in-vitro lymphoma cell growth. Our data indicates a novel control mechanism for the transcriptional regulation of c-myc in the pathophysiology of aggressive lymphoma B cells and suggests that targeting NFATc1 could have therapeutic value.

Targeting Sp1 Transactivation In Waldenstrom's Macroglobulinemia: a Novel Therapeutic Option

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 120-120
Author(s):  
Mariateresa Fulciniti ◽  
Samir B. Amin ◽  
Varuna Mohan ◽  
Guang Yang ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Board Of Directors ◽  
Research Funding ◽  
Luciferase Reporter ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Reversing Metabolic and Epigenetic Cellular Alterations to Overcome Chemo-Resistance in Aggressive B Cell Lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1305-1305
Author(s):  
Kejie Zhang ◽  
Lan V Pham ◽  
Archito T. Tamayo ◽  
John Lee ◽  
Jerry Bryant ◽  
...  
Keyword(s):  
Cell Growth ◽  
Glucose Metabolism ◽  
Tumor Cell ◽  
Cell Lines ◽  
Histone Methylation ◽  
Ros Production ◽  
Tumor Cell Growth ◽  
Growth And Survival ◽  
Methylation Inhibitor ◽  
Cell Growth And Survival

Abstract Abstract 1305 Cancer cells exist in a stressed environment, mainly due to lack of nutrients and oxygen, particularly during chemotherapy, and rely on metabolic homeostatic regulatory mechanisms for protection against these lethal challenges. Increasing glucose metabolism and continuous reactive oxygen species (ROS) production is one strategy of metabolic adaptation utilized by tumor cells to relieve this stress. Thioredoxin interacting protein (TXNIP) is a negative regulator for both redox thioredoxin (ROS production) and cellular glucose uptake, not well understood but found to be repressed in various cancers, including diffuse large B-cell lymphomas (DLBCL), the most common form of non-Hodgkin lymphoma that continues increasing in incidence and remains incurable in many cases, primarily due to development of chemo-resistance. The molecular mechanisms by which TXNIP expression is down-regulated during cancer progression and chemo-resistance development have not been completely elucidated. Since key gene silencing events have now been identified in the pathogenesis of DLBCL, recent therapeutic interest has focused on dysregulated histone modifications as potentially important therapeutic targets, for developing strategies that can reactivate silenced tumor suppressor genes. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), is a highly conserved histone methyltransferase that targets lysine-27 of histone H3 (H3K27). Studies in human tumors show that EZH2 is frequently over-expressed in a wide variety of tumors, including lymphomas. More importantly, recent studies using whole-genome sequencing in primary DLBCL, identified frequent mutations in the EZH2 gene that leads to critical gene silencing in DLBCL pathophysiology. Our study showed that EZH2 is either over-expressed or mutated in representative DLBCL cell lines and primary DLBCL cells, and that down-regulation of EZH2 with siRNA leads to the reactivation of TXNIP, with subsequent inhibition of tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL. We also found that histone deacetylation (HDAC) is also involved in EZH2-mediated silencing of TXNIP in DLBCL. Pharmacologic agents aimed at reactivating TXNIP genes include histone methylation inhibitor 3-Deazaneplanocin A (DZNep) that targets EZH2, as well as HDAC inhibitor Vorinostat. DZNep is currently the only histone methylation inhibitor that is commercially available. Our data indicated that DZNep is highly effective in inhibiting cell growth in various DLBCL cell lines, particularly in chemo-resistant DLBCL cell lines. Vorinostat, on the other hand, has been a good drug and is currently in clinical trial for relapsed DLBCL and has been FDA approved for treating cutaneous T-cell lymphoma patients. Our data showed synergistic activity of DZNep and Vorinostat in reactivating TXNIP gene expression and inhibiting DLBCL cell growth and survival. We also discovered that EZH2 controls constitutive NF-κB activity through both, the canonical and alternative NF-κB pathways in DLBCL. This function of EZH2 is independent of its histone methyltransferase activity. These findings reveal that EZH2 and NF-κB, the two oncogenic factors display functional crosstalk in DLBCL cells. Our findings have indicated that deregulated EZH2 leads to constitutive NF-kB activation and to epigenetic silencing of TXNIP, resulting in uncontrolled tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL, and that targeting this pathway represents a novel, rational, and effective therapeutic approach to selectively reverse chemoresistance in DLBCL patients, particularly relapsed/refractory patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1311-1318 ◽  
Author(s):  
RJ Ford ◽  
A Goodacre ◽  
I Ramirez ◽  
SR Mehta ◽  
F Cabanillas
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoma Cells ◽  
B Cell Growth Factor ◽  
Hodgkin's Lymphomas ◽  
Dose Dependent Increase ◽  
Dose Dependent

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose- dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.

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