Microrna Expression Profile Identifies Distinct Clinically Relevant Sub-Groups in Multiple Myeloma: Novel Prognostic Markers and Potential Targets for Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 96-96 ◽  
Author(s):  
Sophia Adamia ◽  
Herve AvetLoiseau ◽  
Samirkumar B Amin ◽  
Yu-Tzu Tai ◽  
Steven P. Treon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Differentiation ◽  
Clinical Outcome ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Plasma Cells ◽  
Normal Plasma ◽  
Group A ◽  
Group B

Abstract MicroRNA, an abundant class of small endogenous RNAs, regulate target genes through inducing translational inhibition and cleavage of targeted transcripts. To date, microRNAs have been implicated in normal biological processes, including development, cell differentiation, apoptosis and proliferation as well as in malignant transformation. However, their role in multiple myeloma (MM) remains unknown. Here we investigated role of microRNAs in myelomagenesis, and their influence on prognosis and clinical outcome. We evaluated profiles of 384 microRNAs in bone marrow derived CD138+ plasma cells (PC) from 79 uniformly treated MM patients, 11 MM cell lines and 9 healthy donors using qRT-PCR based microRNA array. The relative expression was calculated using comparative Ct method, and data was normalized using endogenous controls and analyzed using SDS, RQ manager, R and dChip softwares. MicroRNA expression profiles detected in MM patients were correlated with clinical outcome measures. We observed significant modulate expression of 61 microRNAs in myeloma cells compared to normal plasma cells. When more stringent criteria were used, we identified 24 differentially expressed microRNAs in patient samples. Further, unsupervised hierarchical clustering of filtered microRNAs, based on their DCt values, identified two major groups within the MM population (groups A and group B). Samples of Group A clusters with MM cell lines, indicating more proliferative nature of MM patient cells. Within B group, a second degree node group B2, clusters with normal plasma cells indicating more indolent course, while patients in an additional node B1 represented an assorted pattern. The unsupervised clustering of all MM samples showed consistent changes in miR-30b, -30c, -30d, -142-5p, -24, -191, -181d, -374, -146b, -140, -145, -125a, -151, -223, -155, let7b, indicative of a role of these microRNA in myelomagenesis; while supervised analysis of samples within groups A and B identified modulated expression of different sets of miRNAs. In group A miR-585 and let-7f were upregulated 8–12 fold, while miRs -125a, -126, -155, -223, -146a, -374 -19a, -20a, -26a, -30a -5p, -30b, and -30d were significantly downregulated; in group B, all differentially expressed microRNAs were downregulated (p<0.001) compared to normal plasma cells. These modulated miRNAs target critical signaling pathways including apoptosis, hematopoietic cell differentiation and proliferation, survival and angiogenesis by upregulating function of HOX9, c-myc, VCAM-1, Bcl-2, E2F1, SHP1, SHP2, VEGF, and DUSp6 molecules. We further analyzed the effect of microRNA on clinical outcome. We have observed significantly superior event free and overall survival of patients in group B2 compared to patients in group A (2 yr estimated EFS 79% versus 54% respectively; p=0.05; and 2 yr estimated OS 94% versus 70% respectively; p =0.017). Taken together this data identifies critical microRNAs as modulators of gene expression and signaling pathways and provides potential novel microRNA and gene targets in MM to both understand biological behavior and for therapeutic application.

Clinical and biological significance of microRNA profiling in patients with myeloma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8539-8539
Author(s):  
S. Adamia ◽  
H. Avet-Loiseau ◽  
S. B. Amin ◽  
P. Moreau ◽  
S. S. Minvielle ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Cell Lines ◽  
Plasma Cells ◽  
Biological Significance ◽  
Biological Behavior ◽  
Normal Plasma ◽  
Consistent Change ◽  
Group A ◽  
Group B

8539 MicroRNA, a small endogenous RNA regulating specific expressed gene function has been implicated in normal biological processes as well as in malignant transformation. Here we have investigated the role of microRNAs in multiple myeloma (MM) biology, and their influence on prognosis and clinical outcome. We evaluated profiles of 384 microRNAs in CD138+ MM cells from 79 patients with MM, 11 cell lines and 9 healthy donors using qRT-PCR based microRNA array. We detected significant modulation of expression of 61 microRNAs in myeloma cells compared to normal plasma cells. Unsupervised hierarchical clustering of filtered matured microRNAs, identified two major groups within the MM population (groups A and group B). Group A clustered with MM cell lines, indicating more aggressive course of disease. Within B group, second degree node, group B2, clustered with normal plasma cells indicating indolent course. The unsupervised clustering of all MM samples showed consistent change in miR-146b, -140, -145, -125a, -151, -223, -155, let-7f, indicative of a role of these microRNA in myelomagenesis; while supervised analysis of samples within groups A and B identified modulated expression of different sets of miRNAs. In group A miR585 and let-7f were upregulated 8–12 fold; in group B, all differentially expressed microRNAs were downregulated (p<0.001) compared to normal plasma cells. These modulated miRNAs target critical signaling molecules such as HOX9, c-myc, Bcl-2, SHP1 and SHP2. We further analyzed the effect of microRNA on clinical outcome. We have observed significantly superior event free and overall survival of patients in group B compared to patients ingroup A (2 yr estimated EFS 79% versus 54% respectively; p=0.05; and 2 yr estimated OS 94% versus 70% respectively; p =0.017). Functional analysis by modulating miRNAs 585, 155 and let-7f showed change in levels of predicted genes with consequent biological effect on growth and apoptosis in MM cell line. Taken together, this data identifies miRNAs as critical modulators of gene expression and signaling pathways and provides potential novel targets in MM to both understand biological behavior and for therapeutic application. No significant financial relationships to disclose.

CD69, a New Potential Clinical Marker in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2027-2027 ◽  
Author(s):  
Gabriele Buda ◽  
Giovanni Carulli ◽  
Enrico Orciuolo ◽  
Paola Sammuri ◽  
Daniele Campa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Cell Phenotype ◽  
Clinical Report ◽  
Dependent Manner ◽  
Cd69 Expression ◽  
Group A ◽  
Group B

Abstract CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. CD69 has not been yet studied in human multiple myeloma, though it has been shown that human chronic lymphocytic lymphoma cells, when induced toward a plasma cell phenotype with tetradecanoyl phorbol acetate (TPA) have increased CD69 expression. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. Therefore, we analyzed the CD69 expression on pathological PCs, from bone marrow samples of 43 patients, by flow cytometry with two aims: to evaluate the real expression of CD69 on pathological PCs and to determine the clinico-pathological significance of this molecule. Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%. 22 of 43 pts (see table I, group A) were MM resistant/refractory to at least two different chemotherapy regimens (including bortezomib in all patients). 21 patients (table I, group B) were smouldering multiple myeloma (SMM) or MM in at least very good partial response (VGPR) after first line treatment. CD69 was detected on bone marrow PCs in 19 of the 43 patients evaluated (44%). Of the 19 patients with CD69+ (see table II) only 6 (27%) were in the group of refractory/resistant MM, while the majority of these advanced patients, 16/22 (73%), had an absent expression of CD69. On the contrary in the group of SMM/VGPR/CR MM 13 patients (62%) were CD69+ (p=0.04, using a Chi squared test with Yates correction). At the best of our Knowledge this is the first clinical report that confirms CD69 expression on pathological PCs of MM patients. Our preliminary data also suggest an intriguing role of CD69, this molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table IPts CharacteristicsGroup AGroup B2221SexMale8(36%)11(52%)Female14(64%)10 (48%)Clinical statusSMMMM inVGPR/CR9 (43%)12 (57%)Relapsed/refractory22(100%)Number of Previous Therapy (range)3,5 (2-6)1 (0-1)Previous Bor regimenSMM0MM inVGPR/CR12(100%)Relapsed/refractory22(100%)Previous Lena regimenSMM0MM inVGPR/CR0Relapsed/refractory17(77%) Table II Pts Results Group A Group B 22 21 CD69+ 19/43 (44%) 6 (27%) 13 (62%) CD69-24/43 (56%) 16 (73%) 8 (38%) Disclosures No relevant conflicts of interest to declare.

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 &gt; 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.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

LDH serum levels as a predictive factor for global outcome in pretreated colorectal cancer patients receiving regorafenib: Implications for clinical management.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 497-497 ◽  
Author(s):  
Michela Del Prete ◽  
Mario Scartozzi ◽  
Tiziana Prochilo ◽  
Luca Faloppi ◽  
Riccardo Giampieri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Outcome ◽  
Cancer Patients ◽  
Progression Free Survival ◽  
Serum Levels ◽  
Roc Curve Analysis ◽  
Group A ◽  
Group B ◽  
Colorectal Cancer Patients

497 Background: Although a demonstrated clinical efficacy, a non negligible proportion of colorectal cancer patients does not seem to benefit from regorafenib and are consequently exposed to unnecessary toxicity. LDH serum levels represent an indirect marker of tumour hypoxia, neo-angiogenesis and worse prognosis in many tumour types. In colorectal cancer LDH showed a correlation with treatment outcome for patients receiving antiangiogenetic treatment, thus suggesting a possible interaction with the activity profile of these drugs. We analyzed the role of LDH serum levels in predicting clinical outcome for pre-treated metastatic colorectal cancer patients receiving regorafenib. The final aim was to individuate a potentially reliable and easy to use marker for patients stratification. Methods: 118 colorectal cancer patients treated with regorafenib were available for our analysis. For all patients, LDH values were collected within one month before the procedure and after treatment end. LDH cutoff value was determined by ROC curve analysis, patients were then divided into two groups (A and B, below and above cut-off level respectively). Patients were also classified according to the variation in LDH serum levels pre- and post-treatment (increased patients vs. decreased patients). Results: Patients in group A and B proved homogeneous for all clinical characteristics analyzed. In group A patients median progression free survival (PFS) was 3.18 months, whereas it was 1.87 months in group B patients (p = 0.0018). Median overall survival (OS) was 6.23 months and 3.28 months in group A and B respectively (p = 0.048). Significant differences were not noted among the 2 groups for response rate. All the other clinical variables analyzed failed to show any correlation with patients outcome. Conclusions: Our observations seem to suggest a role of LDH as a marker of clinical outcome in colorectal cancer patients receiving regorafenib. We can then speculate that high LDH patients may not be optimal candidates for regorafenib. After further confirmation in larger trial, these findings may be relevant for a better patients stratification and selection.

Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2926-2926
Author(s):  
Antonella Caivano ◽  
Francesco La Rocca ◽  
Alessandra Favole ◽  
Sonia Carturan ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Plasma Cells ◽  
Molecular Target ◽  
Over Expression ◽  
In Vitro Angiogenesis ◽  
Novel Therapeutic

Abstract Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Compromised Nuclear Sirtuins Activity Sensitizes BRCA-Proficient multiple Myeloma Cells to DNA Damage Agents

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 723-723
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Aditya Munshi ◽  
Yu-Tzu Tai ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Lines ◽  
Plasma Cells ◽  
Dsb Repair ◽  
Dna Repair Mechanisms ◽  
Dna Damaging Agents ◽  
Normal Plasma ◽  
Repair Mechanisms

Abstract Abstract 723 Background: Multiple myeloma (MM) is a clonal malignancy of plasma cells with hallmark genetic instability resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. Inhibition of DNA repair mechanisms leads to significant reduction in acquisition of new genetic changes and associated progression of MM. Mammalian sirtuins are class III NAD+-dependent deacetylases emerging as innovative proteins involved in multiple pathways, including genome maintenance. Methods: A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The antitumor effect of a pan-sirtuins inhibitor, Nicotinamide (Nam), alone and combined with DNA-damaging agents, was investigated by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with thymidine incorporation, Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results: We analyzed an Affymetrix GeneChip (GSE6477) array of patient MM cells (n=162) compared with normal plasma cells, and found that transcript levels of two nuclear sirtuins (SIRT6 and SIRT7) were significantly higher in monoclonal gammopathy of undetermined significance (MGUS), smoldering MM, active MM, and relapsed MM compared with normal plasma cells. Importantly, protein analysis confirmed increased nuclear levels of these deacetylases in MM cell lines, including those resistant to DNA-damaging agents (MM.1R, LR-5, Dox40), as well as in patient CD138+ MM cells compared to PBMCs from healthy donors. Next we evaluated the functional role of these Sirtuins in MM cells by using loss of function approaches with RNAi. SIRT6 and SIRT7 silencing by knockdown reduced MM cell proliferation compared with control scrambled cells, with only a modest induction of cytotoxicity. We also examined the effects of Nam on DNA-damage response signaling triggered by conventional anti-MM agents melphalan and doxorubicin. Nam treatment did not appreciably affect MM cell viability; however, pretreatment with Nam impaired DNA double-strand breaks (DSBs) repair as well as DNA repair mechanisms triggered by conventional DNA damaging agents, evidenced by γH2AX and RPA phosphorylation, respectively. Consistent with these findings, Nam-pretreated MM cells formed fewer RAD51 foci in response to Doxorubicin and Melphalan, thereby conferring sensitivity to these agents. Importantly, this sensitizing effect was also observed in MM cells resistant to doxorubicin (RPMI-Doxo40) or melphalan (LR5), indicating that Nam increases chemosensitivity in both drug-sensitive and –resistant MM cells. Similarly, lentivirus-mediated shRNA knockdown of SIRT-6 and −7 sensitized MM cells to melphalan and doxorubicin. Finally, both chemical and genetic approaches improved the efficiency of DNA DSB repair mechanisms (Homologous and non-Homologous end-joining Recombination) in MM cell lines containing chromosomally integrated green fluorescent protein-based reporter constructs. Ongoing in vivo experiments are assessing how the chemical susceptibility of SIRT6 and/or 7-deficient cells can be exploited therapeutically. Conclusion: Our study demonstrates a link between nuclear sirtuins and DNA instability in MM cells, providing the basis for incorporation of inhibitors of these SIRTs into innovative anti-MM therapeutic approaches. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Patterns of Microrna in Plasma Cells: From Normal Differentiation to Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2069-2069
Author(s):  
Alboukadel Kassambara ◽  
Angelique Bruyer ◽  
Michel Jourdan ◽  
Nicolas Robert ◽  
Véronique Pantesco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Differentiation ◽  
Plasma Cell ◽  
Mirna Target ◽  
Plasma Cells ◽  
Protein Translation ◽  
Fine Tuning ◽  
Published Data ◽  
Target Mrnas

Abstract Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression either via the degradation of target mRNAs or the inhibition of protein translation. miRNAs have been involved in fine-tuning critical cellular processes and play critical roles in cell differentiation and tumor development. Furthermore, there is an increasing recognition of the role of miRNAs in multiple myeloma, a plasma cell (PC) malignancy characterized by an accumulation of malignant PCs within the bone marrow. However, little is known about miRNA expression during human PCD as well as about the full extent to which individual miRNAs regulate fundamental processes during PCD. A complete delineation of miRNA and their target expression during normal PCD is essential to understand the role of miRNAs in plasma cell malignancies. Methods: We analyzed the expression profile of miRNAs and mRNAs during human plasma cell differentiation (PCD) to infer miRNA-target relationships, as well as in multiple myeloma tumor plasma cells. We developed a method and an R package, that uses miRNA and mRNA expression profiles across PCD cell subpopulations to infer candidate miRNA-target interactions that could be active and functional in PCD. We inferred miRNA-target relationships from sequence-based prediction, experimentally validated target interactions curated from the literature, published data from miRNA perturbation experiments and inverse expression relationships between miRNAs and their target mRNAs. Results: Our results reveal 63 miRNAs with significant temporal changes in their expression during normal PCD. We derived a high-confidence network of 295 target relationships comprising 47 miRNAs and 147 targets. These relationships include new examples of miRNAs that are likely to coordinately regulate multiple members of critical pathways associated with PCD. Notably, we identify new miRNAs that coordinately regulate important pathways in PCD, including members of miR-30 and miR-29 families, miR-106b and miR-16, which regulates IRF4/PRDM1 axis, active DNA methylation pathway, TGF-b signaling pathway, autophagy, ZBTB4/EZH2 axis and cell cycle progression. Furthermore, our work demonstrates that 28 PCD stage-specific miRNAs are aberrantly overexpressed in multiple myeloma cells (MMCs) compared to their normal counterpart and/or are associated with high risk myeloma, suggesting that MMCs frequently acquired expression changes in miRNAs already undergoing dynamic expression modulation during normal PCD. Finally, expression of some targets of these PCD - MM miRNAs is correlated with clinical outcome of uniformly treated MM patients. Conclusions: Altogether, our results demonstrate that miRNAs may be important in controlling PCD and malignant plasma cell biology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of N6-Methyladenosine (m6A) RNA Modification in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5601-5601
Author(s):  
Christian Bach ◽  
Magdalena Leffler ◽  
Cindy Flamann ◽  
Jan Kronke ◽  
Dimitrios Mougiakakos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Messenger Rna ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Mrna Levels ◽  
Molecular Abnormalities ◽  
Gene Expression Control

Abstract Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities. In recent years, integrating proteasome inhibitors and immunomodulatory drugs into MM frontline therapy has significantly improved treatment efficacy with a median overall survival (OS) being prolonged from 3-4 to 7 years. Despite this progress, patients refractory to the aforementioned agent classes display a median OS of only 9 months. Thus, the clinical necessity for developing novel therapeutic alternative approaches is self-evident. Methylation of N6-adenosine (m6A) is known to be important for diverse biological processes including gene expression control, translation of protein, and messenger RNA (mRNA) splicing. m6A regulatory enzymes consist of "writers" METTL3 and METTL14, "readers" YTHDF1 and YTHDF2, and "erasers" FTO and ALKBH5. However, the functions of m6A mRNA modification and the specific role of these enzymes in MM remain unknown. Here we report that METTL3, a key component of the m6A methyltransferase complex, is highly expressed in MM cell lines and in isolated patient's MM cells. In contrast, we found no significant differences in the expression of the m6A demethylases FTO and ALKBH5. Accordingly, compared to plasma cells from healthy donors, global PolyA+ RNA showed a significant increase in m6A content in patient's MM plasma cells. In MM cell lines, global m6A profiling by methylated RNA-immunoprecipitation sequencing revealed m6A peaks near the stop codon in mRNAs of multiple oncogenes including MAF and CCND1. Cross-linking immunoprecipitation showed that METTL3 bound to the m6A peak within MAF and CCND1 mRNA. Depletion of METTL3 by shRNA had little effect on global mRNA levels, but specifically reduced protein levels of c-Maf and Cyclin D1. Moreover, downregulation of METTL3 in several MM cell lines results in cell cycle arrest and apoptosis. Together, these results describe a role for METTL3 in promoting translation of a subset of oncogenes in MM and identify this enzyme as a potential therapeutic target for multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phenotypic and functional analysis of B cell lines from patients with multiple myeloma

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 444-446
Author(s):  
M Goldstein ◽  
J Hoxie ◽  
D Zembryki ◽  
D Matthews ◽  
AI Levinson
Keyword(s):  
Multiple Myeloma ◽  
Functional Analysis ◽  
Cell Differentiation ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Plaque Assay ◽  
Functional Markers ◽  
B Cell Differentiation

We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.

Sign in / Sign up

Export Citation Format

Share Document