Molecular Basis of Genomic Instability and Progression in Multiple Myeloma: Potential Role of Apurinic (Apyrimidinic) Endonuclease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1561-1561
Author(s):  
Masood A. Shammas ◽  
Hemanta Koley ◽  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Ramesh B. Batchu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Genomic Instability ◽  
Cell Lines ◽  
Molecular Basis ◽  
Endonuclease Activity ◽  
Ap Endonuclease ◽  
Myeloma Cells ◽  
Genome Wide ◽  
New Mutations

Abstract Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.

Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1418-1418
Author(s):  
Masood A. Shammas ◽  
Hemant Koley ◽  
Sima Shah ◽  
Ramesh B. Batchu ◽  
Pierfrancesco Tassone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Cell Lines ◽  
Plasma Cells ◽  
Dna Recombination ◽  
Endonuclease Activity ◽  
Single Nucleotide ◽  
Genetic Changes

Abstract Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 276-276
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Genotoxic Agents ◽  
Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5333-5333
Author(s):  
Hiroshi Ikeda ◽  
Tadao Ishida ◽  
Toshiaki Hayashi ◽  
Yuka Aoki ◽  
Yasuhisa Shinomura
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Cell Contact ◽  
Drug Induced ◽  
Myeloma Cells

Abstract The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma　(MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Epigenetic inactivation of the MIR34B/C in multiple myeloma

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. 5901-5904 ◽  
Author(s):  
Kwan Yeung Wong ◽  
Rita Lok Hay Yim ◽  
Chi Chiu So ◽  
Dong-Yan Jin ◽  
Raymond Liang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Promoter Hypermethylation ◽  
Myeloma Cells ◽  
Specific Pcr ◽  
Paired Samples ◽  
Normal Controls ◽  
Transcriptional Target

Abstract We postulated that MIR34B/C, a direct transcriptional target of TP53, might be inactivated by promoter hypermethylation in multiple myeloma (MM). MIR34B/C promoter methylation was studied in 8 normal marrow controls, 8 MM cell lines, 95 diagnostic, and 23 relapsed/progressed MM samples by methylation-specific PCR. MIR34B/C was methylated in 6 (75.0%) MM cell lines but not normal controls. 5-Aza-2′-deoxycytidine led to MIR34B/C promoter demethylation and MIR34B reexpression. Moreover, restoration of MIR34B led to reduced cellular proliferation and enhanced apoptosis of myeloma cells. In primary samples, methylation of MIR34B/C occurred in 5.3% at diagnosis and 52.2% at relapse/disease progression (P < .001). In 12 MM patients with paired samples at diagnosis and relapse/progression, MIR34B/C methylation was acquired in 6 at relapse/progression. In conclusion, MIR34B/C is a tumor suppressor in myeloma. Hypermethylation of MIR34B/C is tumor-specific. Frequent MIR34B/C hypermethylation during relapse/progression but not at diagnosis implicated a role of MIR34B/C hypermethylation in myeloma relapse/progression.

scholarly journals Nucleotide Excision Repair (NER) Is Frequently Impaired and Affects Outcome in Multiple Myeloma (MM)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2055-2055
Author(s):  
Raphael Szalat ◽  
Matija Dreze ◽  
Mehmet Kemal Samur ◽  
Anne S. Calkins ◽  
Giovanni Parmigiani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Dna Repair ◽  
Genomic Instability ◽  
Nucleotide Excision Repair ◽  
Cell Lines ◽  
Excision Repair ◽  
Sequencing Data ◽  
Nucleotide Excision

Abstract Introduction Multiple Myeloma (MM) is a heterogeneous disease characterized by genomic instability and eventual poor outcome. Aberrations in DNA repair-related pathways have been considered to explain the instability. Nucleotide excision repair (NER) is an important pathway involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins. Little is known about the relationship between NER in MM biology and patient outcomes. Here we assess the role of NER in MM. Methods We evaluated NER efficiency in a panel of MM cell lines (n=18), with a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) complex (DDB2 proteo-probe, Dreze et al. 2014). NER proficiency was correlated with cytogenetic characteristics, p53 status, sequencing data, gene expression profile, and with melphalan (MLP) sensitivity evaluated by CellTiterGlo (CTG). We then evaluated NER efficiency in patient samples and interrogated the role of NER in MM patients by correlating expression of NER genes with survival (OS) in a cohort of 170 patients (IFM 2005-01) homogeneously treated with alkylating agents. Results NER, measured as the amount of (6-4) photoproducts remaining 2 hours after UV irradiation, showed variability between MM cell lines. Out of 18 cell lines, 7 exhibited various levels of NER deficiencies, defined as less than 90% repair at 2 hours (4 cell lines 90-70% and 3 cell lines <60%). The other 11 cell lines presented more than 90% of repair. P53 loss of function did not associate with NER deficiency. Notably, all t(4;14) cell lines tested (n=5) showed a NER repair rate > 90%. NER deficient cell lines (NER <90%) were sensitive to melphalan. However all melphalan sensitive cells did not exhibit NER deficiency, This suggests that other DNA repair pathways are involved in the repair of melphalan-induced lesions. Furthermore, we performed the assay in patient samples showing variable levels of NER, which may reflect different disease status and prognosis. Whole genome sequencing data from 6 NER deficient cell lines revealed missense mutations in critical NER genes in 2 of these cell lines. MM1S and MM1R cells showed mutations in the Xeroderma Pigmentosum Complementation Group A (XPA) gene (mutation D70H), and MM1R was also mutated in the Cockayne syndrome, ERCC6 gene (mutation L682I). Gene expression profile comparison in 12 of these showed a positive correlation between expression of NER genes and NER efficiency. We next studied expression of 20 NER genes in 170 patients treated with high dose melphalan (IFM 2005-01). The analysis revealed a significant negative correlation between 5 overexpressed NER genes (ERCC3, ERCC4, ERCC6, MMS19 and NTHL1) and overall survival (OS). Conclusion NER efficiency is heterogeneous in MM, in part due to acquired mutations. Impairment of NER is associated with outcome as well as may contribute to genomic instability. Ability to proficiently measure NER in patient samples provides us an opportunity to now evaluate NER as a prognostic marker in myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4335-4335
Author(s):  
Kim De Veirman ◽  
Siyang Yan ◽  
Ken Maes ◽  
Nathan De Beule ◽  
Sylvia Faict ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Overall Survival ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Single Agent ◽  
Myeloid Cells ◽  
Myeloma Cell ◽  
Myeloma Cells

Introduction The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment. Material & Methods To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP+cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP+DiD+('dormant', non-proliferating) and GFP+DiD-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&D). Results Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p<0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p<0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (>10%), U266 (>20%) and LP-1 (>10%) cells compared to single agent therapy (n=6) (p<0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p<0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p>0.01). Conclusion We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients. Disclosures No relevant conflicts of interest to declare.

N-Cadherin Expressing MM Cells Form Calcium-Dependent, Adherent Junctions That Likely Contribute to Focal Lesions and Nodular Growth of Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 979-979
Author(s):  
Ya-Wei Qiang ◽  
Bo Hu ◽  
Yu Chen ◽  
Erming Tian ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bone Destruction ◽  
Dependent Manner ◽  
Focal Lesions ◽  
Myeloma Cells ◽  
Calcium Dependent ◽  
Nodular Growth ◽  
E Cadherin

Abstract Abstract 979 E-cadherin-mediated adhesion regulates homeostasis in tissues of epithelial origin and homotypic N-cadherin interactions are central to the interaction of hematopoietic stem cells and the endosteal niche. Loss of E-cadherin in carcinomas is characteristic of the epithelial-to-mesenchymal transition and metastasis. The loss of E-cadherin, causes the release of b-catenin from the adherent complex, increased nuclear translocation, and increased transcriptional activity of b-catenin/TCF. We and others have demonstrated that alterations in the Wnt/b-catenin pathway exists in multiple myeloma, a malignancy of terminally differentiated antibody secreting plasma cells. The first evidence of this deregulation came from studies showing that MM cells secrete the potent Wnt/b-catenin signaling inhibitor DKK1. While it is now clear that DKK1 mediated suppression of Wnt/b-catenin in the bone marrow contributes to the decoupling of bone formation, the role of Wnt/b-catenin in normal plasma cell development and myelomagenesis is less clear and often controversial. Myeloma cells grow exclusively in the bone marrow. This growth is characterized as being interstitial or nodular, with most disease exhibiting a mixed pattern. Nodular growth, recognized as focal lesions (FL) on MRI, characterizes the conversion of MGUS to symptomatic MM. Consistently, DKK1 levels are highest in CD138 cells isolated from FL where nodular tumor growth and bone destruction may be linked by Wnt/b-catenin suppression in MM cells and local microenvironment. Given the central role of classical cadherins in promoting cell-cell adhesion and regulating b-catenin, we hypothesized that abnormal expression of cadherins might play a direct role in myelomagenesis. Cell lysates were prepared from 24 MM cell lines and from CD138+ cells from the BM of eight patients with MM. Immunoblotting was performed with antibodies specific to human N-cadherin protein. N-cadherin protein was observed in more than 82% of MM cell lines, with high protein levels in 55% of the cell lines. Similar levels of N-cadherin protein were seen in primary myeloma cells from the eight MM patients: two patients showed the highest levels of N-cadherin protein, two showed intermediate levels, and three weak levels; N-cadherin protein was absent in one patient. Similar results were obtained with three color-flow cytometry analysis of primary MM BM. To determine whether N-cadherin mediated MM cell interactions, a cell aggregation assay using GFP-expressing, N-cadherin+ MM cells observed under fluorescence microscopy was employed. When N-cadherin+ JJN3 cells were cultured in normal growth medium, they aggregated to form clusters. Similar results were observed in other MM cell lines, including OPM-2 and KMS-28-BM. Addition of a neutralizing N-cadherin antibody to these cultures significantly attenuated aggregation of JJN3 cells compared to control cells in normal medium or cells treated with control IgG. Homotypic N-cadherin interaction forms adherent junctions in a calcium dependent manner. To see if aggregation of MM cells results in the formation of adherent junctions, immunochemical staining was preformed to visualize the N-cadherin protein in OPM-2 cells. N-cadherin protein was clearly observed between myeloma cells in aggregation clusters that were significantly diminished in calcium-free medium. Taken together, these data suggest that N-cadherin induces homotypic adhesion of myeloma cells in a calcium-dependent manner and suggests that calcium release during bone resorption may enhance adherent junctions in MM that may in turn enhance plasma membrane localization of b-catenin. Studies are currently underway to determine whether DKK1 and N-cadherin adherent junctions cooperate to suppress b-catenin nuclear activity in MM cells and all converge to induce the nodular growth pattern and bone destruction often seen in N-cadherin-positive MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Smenamide A Analogues. Synthesis and Biological Activity on Multiple Myeloma Cells

2018 ◽  
Author(s):  
Alessia Caso ◽  
Ilaria Laurenzana ◽  
Daniela Lamorte ◽  
Stefania Trino ◽  
Germana Esposito ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiple Myeloma ◽  
Biological Activity ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Structural Features ◽  
Synthetic Route ◽  
Myeloma Cells ◽  
Apoptotic Mechanism

Smenamides are an intriguing class of peptide/polyketide molecules of marine origin showing antiproliferative activity against lung cancer Calu-1 cells at nanomolar concentrations through a clear pro-apoptotic mechanism. To probe the role of the activity-determining structural features, the 16-epi-analogue of smenamide A and eight simplified analogues in the 16-epi series were prepared using a flexible synthetic route. The synthetic analogues were tested on multiple myeloma (MM) cell lines showing that the configuration at C-16 slightly affects the activity, since the 16-epi-derivative is still active at nanomolar concentrations. Interestingly, it was found that the truncated compound 8, mainly composed of the pyrrolinone terminus, was not active while compound 17, essentially lacking the pyrrolinone moiety, was 1000-fold less active than the intact substance and was the most active among all the synthesized compounds.

scholarly journals Interleukin-6 Adversely Impacts Genomic Stability Via Targeting Multiple Pathways in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4467-4467
Author(s):  
Chaitanya Yenumula ◽  
Srikanth Talluri ◽  
Tommaso Perini ◽  
Subodh Kumar ◽  
Jialan Shi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Genomic Instability ◽  
Cell Lines ◽  
Genome Stability ◽  
Fold Increase ◽  
Dna Breaks ◽  
Soluble Factors ◽  
Abasic Sites ◽  
The Impact

Abstract In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6. Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM. Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells. Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression. Disclosures Munshi: OncoPep: Other: Board of director.

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