scholarly journals Novel Therapeutic Rationale for Targeting HDAC1 and PIM2 in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3111-3111 ◽  
Author(s):  
Takeshi Harada ◽  
Asuka Oda ◽  
Hiroto Ohguchi ◽  
Yohann Grondin ◽  
Hirofumi Tenshin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Survival ◽  
Cell Lines ◽  
Histone Deacetylases ◽  
Class I ◽  
Combination Strategy ◽  
Growth And Survival ◽  
Cell Growth And Survival

Multiple myeloma (MM) cells preferentially grow and expand in the bone marrow (BM) to elicit the alteration of gene expression thereby acquiring drug resistance. The serine/threonine kinase PIM2 is constitutively overexpressed which is further up-regulated as a critical anti-apoptotic mediator in MM cells by interacting with BM stromal cells (BMSCs) and/or osteoclasts (Leukemia 2011, 2015). Histone deacetylases (HDACs) generally repress gene expression through deacetylation of lysine residues in histone tails. Therefore, HDAC inhibitors are able to restore the expression of tumor suppressor genes, and utilized as anti-cancer agents for various types of malignancies, including MM. Importantly, class-I and a class-IIb (HDAC6) HDACs have been shown as important therapeutic targets in MM (Nat Chem Biol 2010). Among class-I HDAC isoforms, HDAC1 and HDAC3 are highly expressed in MM cells (GSE5900 and GSE2113) and we have already reported that the HDAC3-DNMT1 axis is a critical therapeutic target (Leukemia 2017). However, the significance of HDAC1 expression in MM cell growth and survival is still largely unknown. In the present study, we aimed to clarify the epigenetic regulation of PIM2 and the therapeutic implication of HDAC1 in MM cells. We observed that HDAC1- and HDAC3-selective inhibitor MS-275 (Entinostat) inhibited MM cell growth in a dose-dependent fashion. HDAC1 knockdown using a lentiviral shRNA system induced apoptosis in MM cell lines, indicating a crucial role of HDAC1 in MM cell growth and survival. To identify downstream targets of HDAC1 mediating MM cell survival, we next carried out RNA-Seq using RPMI 8226 cells after HDAC1 knockdown. Expression of a number of genes were altered (adjusted P values < 0.05, log fold change > 0.5). Among these genes, we found that PIM2 and IRF4 were significantly downregulated in HDAC1 knocked down cells. The downregulation of IRF4 and PIM2 was further confirmed at mRNA and protein levels in additional MM cell lines. It has been shown that MS-275 impaired the viability of primary MM cells associated with downregulation of IRF4 and PIM2 expression. However, importantly, HDAC1 knocked down-induced growth inhibition was not observed in RPMI8226 cells with IRF4 overexpression, indicating that IRF4 is a key MM cell survival mediator targeted by HDAC1 inhibition. Previous study shows that HDAC1 is abundantly enriched around at H3K27 acetylation or RNA Pol II- binding sites compared to HDAC2 or HDAC3 (GSE86450), However, our data assessed by ChIP-Seq indicated that HDAC1-occupied genes were not completely upregulated but rather downregulated in HDAC1-knockdown cells. Indeed, MS-275 and a histone acetyltransferase inhibitor C646 downregulated IRF4 and PIM2 expression in MM cells despite upregulation and downregulation of histone H3 acetylation, respectively. The ChIP-Seq data showed HDAC1 binding is enriched around the promotor regions of IRF4 and PIM2 in MM cells; however, MS-275 significantly reduced the HDAC1 enrichment as determined in ChIP-Q-PCR assays, suggesting that IRF4 and PIM2 expression is regulated by the balance between acetylation and deacetylation status of histones in MM cells. In addition, we found that IRF4 binds to the promoter of PIM2 and IRF4 knockdown reduced PIM2 expression, suggesting that IRF4 transcriptionally regulates PIM2. Although PIM2 expression is robustly upregulated in MM cells in an ambient microenvironment with BMSCs and/or osteoclasts, MS-275 and the PIM inhibitor SMI-16a cooperatively induce MM cell death. In conclusion, our data provides a basis of rationale combination strategy targeting of class-I HDAC and PIM2 to improve MM patient outcome. Disclosures Anderson: Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board.

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.

Alternate Splicing Factor Fox2 Affects Growth and Survival of Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1810-1810
Author(s):  
Weihua Song ◽  
Yiguo Hu ◽  
Guang Yang ◽  
Parantu K Shah ◽  
Weisong Shan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Rna Binding ◽  
Exon Array ◽  
Newly Diagnosed ◽  
Alternate Splicing ◽  
Inhibition Of Proliferation ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 1810 Alternative splicing of pre-mRNA plays a critical role in number of cellular processes including cell growth and survival. We have previously demonstrated significant frequency of alternate splicing in multiple myeloma (MM) that points to the need for evaluation of not only the expression level of genes but also post translational modifications. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. We have now further studied the molecular mechanisms that may be involved in differential splicing in myeloma. We have evaluated the expression data from 170 uniformly treated newly diagnosed patients with MM and identified Fox-2, an RNA binding protein that is thought to be a key regulator of alternative exon splicing as one of the most important gene predicting clinical outcome in MM. We have now investigated the molecular effects of Fox2 and its role on myeloma cell growth and survival. Using Fox-2-directed shRNA lentivirus, we knocked-out (KO) the expression of Fox2 in MM1S and RPMI8226 cell lines. Real-time PCR and western blot analysis confirmed KO of over 90% of Fox2 expression in both cell lines. We observed significant inhibition of proliferation of both MM1S and RPMI8226 cell lines in Fox-2- KO compared to control cell lines. Using annexin V and PI staining and flow cytometric analysis we also observed induction of apoptosis in Fox-2-KO-MM1S and RPMI8226 cells. The apoptosis was Caspase 3 and caspase 8-mediated in the Fox-2-KO. A similar effect of Fox-2-KO was observed on cell survival and proliferation in Hela and 293 cells. Fox-2 also plays a crucial role in the maintenance of cytoskeleton of MM cells. We observed that actin was significantly downregulated in both MM1S and RPMI8226 knockdown cell lines compare to controls, whereas GAPDH expressions are equal in all conditions. It's been recognized that actin derangement is associated with cell apoptosis. The RNA binding specificity of Fox-2 in MM cells by CLIP-seq and changes in alternate splicing in Fox-2-KO by exon array is underway to understand the molecular events mediated by this transcriptome modifier affecting MM pathobiology. These results identify Fox-2 as a biologically important intermediate with essential function in myeloma with potential biological and clinical implications. Disclosures: No relevant conflicts of interest to declare.

scholarly journals KDM6B modulates MAPK pathway mediating multiple myeloma cell growth and survival

Leukemia ◽  
2017 ◽  
Vol 31 (12) ◽  
pp. 2661-2669 ◽  
Author(s):  
H Ohguchi ◽  
T Harada ◽  
M Sagawa ◽  
S Kikuchi ◽  
Y-T Tai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Mapk Pathway ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Multiple Myeloma Cell ◽  
Cell Growth And Survival

CD27-Mediated Apoptosis Is Dependent on Siva-Induced Caspase Activation in Human Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3398-3398 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Rory Coffey ◽  
Iris Breitkreutz ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Plasma Cells ◽  
Death Domain ◽  
Caspase Activation ◽  
Growth And Survival ◽  
Induced Apoptosis ◽  
Cell Growth And Survival

Abstract CD27, a member of tumor necrosis factor receptor superfamily that lacks a death domain in its cytoplasmic region, and its interaction with its ligand, CD70, is crucial for differentiation into plasma cells. In malignant B cells, aberrant expression and reverse signaling of CD70 might contribute to disease progression. Recent studies showed that CD27 is heterogeneously expressed on multiple myeloma (MM) plasma cells and the expression is reduced with the progression of MM. However, a possible role for the loss of CD27-CD70 interaction in myelomagenesis was never defined. In this study, we identify functional significance of CD27-CD70 interaction in 4 CD27-expressing MM lines and define mechanisms regulating CD27-mediated MM cell death. Using RT-PCR and flow cytometric analysis, we first found that all of MM lines highly express CD70 (n=10) and 4 MM lines 12BM, 12PE, 28BM, 28PE express CD27 on the cell surface. We next evaluated the effect of CD27 ligation, by CD70-transfected NIH3T3 cells (CD70 transfectant), on [3H] thymidine incorporation by CD27-expressing MM lines. CD27 ligation by CD70 transfectants inhibited DNA synthesis in these 4 CD27-expressing MM lines, but not the control transfectants. Conversely, a blocking anti-CD70 mAb blocked CD27-mediated growth inhibition in a dose-dependent manner, indicating induced growth inhibition specific triggered by CD27-CD70 interaction. Using MTT assay, CD27 ligation by CD70 transfectant also inhibited MM cell survival. IL-6 (20 ng/ml) could overcome the inhibitory effect triggered by CD27 ligation on MM cell growth and survival. In addition, CD27 ligation further enhanced Dex-induced MM cell death. Importantly, CD27-mediated MM cell death was also observed in 2 CD27-expressing patient MM cells. Since Siva is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27, we investigated its role in CD27-mediated apoptosis in MM cells. Overexpression of Siva by transducing adenovirus-expressing Siva (Ad-Siva-GFP) in 12BM MM line is sufficient to induce cell death whereas control adenovirus (Ad-GFP) transduction did not alter 12BM cell growth and survival. CD27 ligation by CD70 transfectants on Siva-overexpressing 12BM cells further enhanced Siva-induced apoptosis, as evidenced by increased subG0 fraction in cell cycle analysis. Thus, the apoptosis triggered by Siva overexpression was related to the CD27-mediated apoptotic pathway. We further determined caspase involvement in the Siva-induced apoptosis in the absence and presence of CD70 transfectants. Caspase 8 and caspase 9 activities were detected 24h following Ad-Siva-GFP transduction in 12BM cells, whereas caspas-3 activity was detected 48h after transduction. Coculture of Ad-Siva-GFP-transduced 12BM cells with CD70 transfectant further enhanced caspase activities. Therefore, overexpression of Siva is sufficient to induce apoptosis and CD27-mediated apoptosis is mediated by Siva-dependent caspase activation in MM. Furthermore, these results suggest that lack of CD27 may lead to evasion of apoptosis in human MM.

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.

HIF-1 Supports the Survival of Multiple Myeloma Cells through the Induction of Survivin Gene.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 110-110 ◽  
Author(s):  
Keita Kirito ◽  
Hu Yongzhen ◽  
Kozue Yoshida ◽  
Toru Mitsumori ◽  
Kei Nakajima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cancer Cells ◽  
Cell Lines ◽  
Dependent Manner ◽  
Survivin Mrna ◽  
Myeloma Cells ◽  
Survivin Gene ◽  
Growth And Survival ◽  
Induced Apoptosis

Abstract In spite of the recent development of therapeutic strategies, multiple myeloma (MM) still remains incurable. Several cytokines and chemokines contribute to progression of the disease and acquisition of resistance to chemotherapy. These humoral factors support the growth and survival of myeloma cells through the regulation of transcription factors including NF-κB, Stat3 and FOXO3a. Hypoxia inducible factor-1 (HIF-1) is an important transcription factor that is activated under low oxygen tension and controls dozens of genes involved in angiogenesis, energy production and resistance to apoptosis. Interestingly, HIF-1 is frequently activated in cancer cells even under normoxic condition and it is well established that HIF-1 expression and activation correlates with tumor progression and resistance to cancer treatments. In this study, we investigated whether HIF-1 is involved in the biology of multiple myeloma. To this end, we used three MM cell lines U266, RPMI8226 and KMM-1. After informed consent, we also prepared primary MM cells from bone marrow samples of patients (n=5) using anti-CD138 magnetic beads. Initially, we treated MM cells with insulin-like growth factor-1 (IGF-1) and IL-6, both of which are major growth and survival factors for myeloma cells. Treatment with IGF-1 and, to be a lesser degree, IL-6 clearly enhanced expression of HIF-1α, a subunit of HIF-1, in all three cell lines. Similar results were obtained from isolated primary MM cells. Based on several lines of evidence that survivin, a member of inhibitor of apoptosis (IAP) family protein, is transcriptionally regulated by HIF-1 in breast cancer cells, and that this anti-apoptotic factor is important for growth of MM cells, we examined whether HIF-1 supports the survival of MM cells through the induction of survivin. Quantitative RT-PCR assay revealed that IGF-1 increased survivin mRNA both in MM cell lines and in primary MM cells. In addition, IGF-1 activated survivin gene promoter containing a HIF-1-binding site. To confirm that IGF-1-induced activation of survivin gene is mediated by HIF-1, we treated MM cell lines with echinomycin, an inhibitor of DNA-binding activity of HIF-1. As expected, echinomycin inhibited IGF-1-induced survivin gene expression in a dose-dependent manner. The inhibitor also induced apoptosis of MM cells, and IGF-1 could not rescue the MM cells from echinomycin-induced apoptosis. Furthermore, echinomycin enhanced melphalan-induced apoptosis of MM cells. To further examine the involvement of HIF-1 in IGF-1-induced survivin gene expression, we generated three independent HIF-1α knockdown KMM-1 clones using siRNA system. Survivin mRNA was not detected in the HIF-1α knockdown cells, and these clones easily underwent apoptosis even in the presence of IGF-1, compared to the parental cells. Taken together, HIF-1 plays a pivotal role in survival of MM cells through the induction of survivin gene. In conclusion, HIF-1 might be an attractive therapeutic target for MM.

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 The Critical Role of HDAC1-IRF4-Pim-2 Axis in Myeloma Cell Growth and Survival: Therapeutic Impacts of Targeting the HDAC1-IRF4-Pim-2 Axis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1939-1939 ◽  
Author(s):  
Takeshi Harada ◽  
Asuka Oda ◽  
Yohann Grondin ◽  
Jumpei Teramachi ◽  
Ariunzaya Bat-Erdene ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Cell Growth ◽  
Critical Role ◽  
Class I ◽  
Growth And Survival ◽  
Class I Hdacs ◽  
Rpmi 8226 ◽  
Cell Growth And Survival

Abstract Multiple myeloma (MM) is a heterogeneous clonal plasma cell proliferative disorder with CRAB features. Although survival of MM patients has been greatly prolonged by recent implementation of various combinatory treatments with novel anti-MM agents, MM still remains incurable. MM cells preferentially grow and expand in the bone marrow to elicit the alteration of gene expression and thereby drug resistance. To improve the therapeutic efficacy, we urgently need to develop novel treatment strategies targeting the BM microenvironment-mediated drug resistance. The serine/threonine kinase Pim-2 is constitutively over-expressed and acts as a pro-survival mediator in MM cells. We have reported that cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) further up-regulate Pim-2 expression in MM cells to confer drug resistance (Leukemia 2011, 2015). Therefore, Pim-2 appears to be an important therapeutic target to impair the BM microenvironment-mediated drug resistance in MM. Histone deacetylases (HDACs) are generally accepted to be therapeutic targets for MM treatment. However, clinical application of currently available pan-HDAC inhibitors is limited with their adverse effects induced by a non-selective HDAC inhibition. To develop safe and effective HDAC inhibitor-based treatment, the therapeutic roles of HDAC isoform-specific inhibition should be elucidated. In this regard, we have recently reported therapeutic impacts on MM cells of inhibition of class-I HDACs, especially HDAC1 and HDAC3. HDAC3-selective inhibitor BG45 induces anti-MM activity in combination with DNA methyltransferase (DNMT) inhibitor azacytidine (Leukemia 2017). In the present study, we aimed to clarify the underlying mechanisms for impairment of MM cell growth and survival by HDAC1 inhibition. We first referenced the expression of class-I HDACs using a publicly available GSE6691 data set. Among class-I HDACs, HDAC1 and HDAC3 were highly expressed in MM cells. We then knockdowned HDAC1 gene using lentiviral shRNA system in MM cell lines. The HDAC1 gene silencing induced MM cell death with caspase-3 activation, indicating the critical role of HDAC1 in MM cell growth and survival. To determine target molecules of HDAC1, we carried out RNA-sequencing with and without the HDAC1 gene silencing in RPMI 8226 cells. Among genes whose expression significantly changed by the HDAC1 knockdown (adjusted P values < 0.05, log fold change > 0.5), we focused on IRF4 together with PIM2, because MM cell has been demonstrated to addict to aberrant IRF4-c-Myc regulatory network (Nature 2008). Downregulation of IRF4 and Pim-2 by the HDAC1 knockdown was further confirmed by quantitative PCR (Q-PCR) and immunoblotting in RPMI 8226 and MM.1S cells. Treatment with the class I HDAC-selective inhibitor MS-275 (entinostat) also induced MM cell death along with reduction of IRF4 and Pim-2 expression. Since previous study has shown that IRF4 binds to PIM2 promoter in MM cells (Nature 2008), we examined whether IRF4 regulates PIM2 expression. We found that IRF4 binds to the PIM2 promoter region by analyzing ChIP-Seq data in KMS-12 cells (GSE22901). We further confirmed the binding of IRF4 on PIM2 promoter by ChIP-Q-PCR. Indeed, the IRF4 knockdown downregulated Pim-2 expression in RPMI 8226 cells. These results suggest that HDAC1 inhibition downregulates IRF4 expression, thereby transcriptionally reducing PIM2 expression in MM cells. Pim-2 expression can also be augmented by multiple signaling pathways, including HIF-1a, JAK-STAT and NF-kB-mediated ones in MM cells through the interaction with BM microenvironment. Interestingly, the Pim inhibitor SMI-16a and MS-275 cooperatively induced apoptotic cell death in MM cell lines and CD138-positive primary MM cells even in the presence of BMSCs. Taken together, our results demonstrate the critical role of the HDAC1-IRF4-Pim-2 axis in MM cell growth and survival, and provoke the novel treatment strategy targeting the HDAC1-IRF4-Pim-2 axis in MM cells. Disclosures Anderson: Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

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.

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