Functional and Clinical Impact of Splicing Factor Dysregulation in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 726-726 ◽  
Author(s):  
Mariateresa Fulciniti ◽  
Mehmet Kemal Samur ◽  
Naim Ur Rashid ◽  
Rajya Lakshmi Bandi ◽  
Manoj Bhasin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Board Of Directors ◽  
Plasma Cells ◽  
Splicing Factor ◽  
Alternate Splicing ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Transcriptome modifiers such as alternative pre-mRNA splicing (AS), long non-coding RNA and microRNA (miRNA) need to be considered in order to provide a more accurate genomic framework for clinical correlation, as well as for high value therapeutic target discovery. Aberrant splicing of numerous genes has been reported in other malignancies, including a small number of genes reported in MM. We have evaluated AS in MM by analyzing clinically annotated high throughput RNA-seq data from 410 newly-diagnosed patients and 18 normal donor plasma cells. We observed a profound and significant AS in MM with over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. Importantly, unsupervised analysis identified clinically relevant MM subgroups with high and low splicing index respectively and showed significant impact of alternate splicing on overall clinical outcome. Based on these data, we next focused on understanding the molecular mechanisms driving aberrant alternate splicing in myeloma. Several studies provide evidence that an abnormally expressed splicing factor (SF) can have oncogenic properties by impacting alternative splicing of cancer-associated genes. We detected dysregulated expression of several SFs, including SF3B1, Fox2, SRSF1, NONO, in patients with MM compared to normal plasma cells with impact on outcome, highlighting for the first time the prognostic significance of splicing related factors in myeloma. We further observed that overexpression of some of these SFs increased cell proliferation, enhanced anchorage independent growth in semi-solid medium, and affected tumorigenic potential. We have further investigated role of Serine/Arginine Splicing Factor 1 (SRSF1) in MM by gain of- and loss of- function studies. Enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells. Conversely, transient or stable downregulation of SRSF1 with specific siRNA and shRNAs respectively, significantly inhibited MM cell proliferation and cell survival. We have also investigated a small molecular inhibitor of SRSF1 (TG003) and observed inhibition of MM cell growth and survival. The impact of this inhibitor on allelic isoforms of specific gene targets is undergoing. To dissect the mechanisms involved in the SRSF1-mediated MM growth induction, we used SRSF1 mutants lacking either of the two RNA-recognition motifs (ΔRRM1 or ΔRRM2 mutants) or the serine/argine-rich C-terminal domain (ΔRS mutant) involved in protein-protein interactions, subcellular localization, and recruitment of spliceosome components. We also used a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. We surprisingly found that only NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation. Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of E2F1, a transcription factor with significant impact on MM cell growth and survival. A significant reduction in SRSF1 at mRNA and protein levels was observed after E2F1 and/or E2F1 heterodimerization partner Dp1 gene silencing. Moreover, peptide-based strategy to abrogate interaction between Dp1-E2F1 led to decreased SRSF1 expression levels. These results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM. The study highlights the need to further understand the splicing pattern in myeloma and also supports the emerging concept that splicing programs, together with transcriptional programs participate in the altered cellular function during tumor initiation and progression. Disclosures Munshi: onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.

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.

Biology and Therapeutic Targeting of Sp1 Transactivation In Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 134-134
Author(s):  
Mariateresa Fulciniti ◽  
Samir B. Amin ◽  
Puru Nanjappa ◽  
Scott J Rodig ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Luciferase Reporter ◽  
Parp Cleavage ◽  
Specific Inhibition ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Synergistic Cytotoxicity

Abstract Abstract 134 Alteration in expression and function of transcription factors has been frequently associated with neoplastic transformation. We here provide both experimental and clinical evidence that Sp1, a transcription factor that controls number of cellular processes, plays an important regulatory role in MM cell growth and survival. Although Sp1 is ubiquitously expressed, its nuclear localization observed in MM is functionally both relevant and important. We have confirmed high Sp1 activity in MM cells both by demonstrating increased DNA binding as well as increased Sp1-responsive promoter activity measured by luciferase reporter assay. MM cell-BMSC interaction led to Sp1 activation which was completely abrogated by the ERK pathway inhibitor U0126 but not by the AKT inhibitor LY29004. Furthermore, using both SiRNA and ShRNA mediated Sp1 knock-down, we have confirmed the growth and survival effects of Sp1 on MM cell growth. Using gene expression profile of MM cells from 172 uniformly treated patients, we have further confirmed these in vitro results by observing that overexpression of Sp1-related genes, including HIF-1a, HDAC1 and MAPK genes, correlate with poor prognosis in MM. This clinical correlation further suggests the role of Sp1 in MM biology, providing the rationale to preclinically target Sp1 in MM. We have investigated TMP, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA) which disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity. We have previously confirmed specific inhibition of both Sp1 binding and transcriptional activity in MM cells by TMP, including in the context of MM-BMSC interaction. Along with inhibition of Sp1 activity, we observed both in vitro and in vivo in murine models of human myeloma, anti-myeloma effect of TMP. Importantly, there was no significant synergistic effect when MM cells transfected with Sp1 siRNA were treated with TMP, confirming specificity of TMP's mechanism of action. We have further observed activation of the mitochondrial apoptotic pathway by TMP via activation of caspase-3, -9 and -7 and PARP cleavage while caspase-8 was not activated suggesting possible synergism with activators of alternate apoptotic pathways. We have also observed that lenalidomide and dexamethasone upregulate Sp1 activity, suggesting that Sp1 may be a common resistance mechanism. We have confirmed that the increased Sp1 activity by lenalidomide or dexamethasone is abrogated by TMP and the combination provides synergistic cytotoxicity, in MM cell lines as well as primary MM cells. In conclusion, we report significant role of Sp1 in myeloma cell growth, survival and drug resistance with its influence on clinical outcome in MM. Our results suggest that specific inhibition of Sp1 activity may be an important therapeutic target in MM. Disclosures: Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

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.

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.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin

2004 ◽  
Vol 287 (3) ◽  
pp. G715-G725 ◽  
Author(s):  
Vinzenz Stepan ◽  
Saravanan Ramamoorthy ◽  
Nonthalee Pausawasdi ◽  
Craig D. Logsdon ◽  
Frederick K. Askari ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Binding Proteins ◽  
Dominant Negative ◽  
Growth And Survival ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Growth Promoting ◽  
Stimulation Of ◽  
Cell Growth And Survival

G17 has growth promoting and antiapoptotic effects on the AR4–2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4–2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4–2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4–2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4–2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4–2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.

Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 951-951 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Costas M Pitsillides ◽  
John T Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

scholarly journals IL-17A-Mediated Notch Signaling in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3434-3434
Author(s):  
Rao H. Prabhala ◽  
Teru Hideshima ◽  
Mariateresa Fulciniti ◽  
Sophia Adamia ◽  
Rajya Lakshmi Bandi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Growth ◽  
Protein Expression ◽  
Notch Signaling ◽  
Cellular Protein ◽  
Full Length ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM. Disclosures No relevant conflicts of interest to declare.

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