CS1, a New Surface Target on Multiple Myeloma (MM) Cells, Protects Myeloma Cells from Apoptosis Via Regulation of ERK1/2, AKT and STAT3 Signaling Cascades.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 109-109 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Giovanni Tonon ◽  
Merav Leiba ◽  
Xian-Feng Li ◽  
Peter Burger ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Surface ◽  
Cyclin B1 ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Growth And Survival ◽  
Downstream Target ◽  
Moderate Growth ◽  
Cell Growth And Survival

Abstract Cell surface glycoprotein CS1 is universally expressed in the majority of MM patients. CS1 gene is localized on chromosome 1q which amplified in many MM patients and in MM cell lines (i.e., H929 and OPM2). We recently further detected CS1 protein in MM patient sera, but not in individuals with monoclonal gammopathy of undetermined significance (MGUS) or in healthy donors (Abstract # 950059). Since circulating CS1 levels correlate with active MM (Abstract # 950059), we postulated that CS1 may regulate MM cell growth and survival. We here defined a role of CS1 in MM cell growth and survival by generating CS1-null OPM2 MM cells using lentiviral CS1 short interfering RNA (CS1 siRNA). Four CS1-null OPM2 transfectants were derived using 4 CS1 siRNA targeting different regions in CS1 gene. Specific knockdown was confirmed by complete abrogation of CS1 mRNA and protein expression. In contrast, CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cnt-OPM2). Significantly, immunoblotting analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in all 4 CS1-null OPM2 cells, compared with OPM2 and cnt-OPM2 cells. We next examined growth and survival of these cells using MTT and Alamar Blue colorimetric/fluorescence assays. Serum deprivation markedly induced apoptosis at earlier time points in CS1-null OPM2 cells, but not in cnt-OPM2 cells. Earlier apoptosis in CS1-null OPM2 cells was mediated by earlier activation and cleavage of caspases, i.e. caspase 3 (> 7-fold) and caspase 8 (> 10-fold). Microarray gene expression profiling of cells under normal cultures showed altered cell cycle regulators (i.e., decreased CDK1, cyclin B1, cdc25b, cyclin D2, and increased CDK inhibitor p18), reduced survival/anti-apoptotic proteins (i.e., Mcl-1), and increased proapoptotic molecules (i.e., BINP3, BIM) in CS1-null OPM2 vs. cnt-OPM2 cells (>4-fold alteration, p<0.001). Additionally, p-21-activated kinase 1 (PAK1) that regulates cell motility, invasiveness, and survival in breast cancer cells, was completely blocked in CS1-null OPM2 transfectants. These transcriptional changes were further confirmed by immunoblotting using specific Abs. Moreover, CS1-null OPM2 transfectants had defective IL-6/IL-6R signaling, since IL-6-induced pERK1/2, pAKT, and pSTAT3 was completely blocked in CS1-null cells, but not in cnt-OPM2 cells. FGFR3 was overexpressed in cnt-OPM2 cells but was significantly downregulated in CS1-null OPM2 cells. MIP-1alpha (CCL3, an osteoclasts-activating factor), a downstream target of FGFR3 and ERK1/2 signaling, was concomitantly and specifically downregulated in CS1-null OPM2 transfectants. Ingenuity analysis further revealed altered transcriptional factors (i.e., XBP-1 and its downstream target genes, IRF4, RUNX2, SMAD1), immunoproteasome components (i.e., NMI, PSMB9, PSME2), and cell surface receptors (i.e., CCR1, Syndecan 1, IL-6R, gp130) in CS1-null vs cnt-OPM2 cells. Finally, MM1S cells transfected with a CS1-overexpressing plasmid had a moderate growth increase and increased pERK1/2, pAKT, and pSTAT3, compared with parental MM1S cells. Together, these results suggest a role of CS1 in MM cell survival by cross-talking major growth and survival pathways, strongly supporting novel therapies targeting CS1 function 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.

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.

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.

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.

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