Proliferation Of Primary Myeloma Cells Research Articles

CRISPR/Cas9 mediated knock-out of VPREB1 gene induces a cytotoxic effect in myeloma cells.

BackgroundMultiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies.ObjectivesWe aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1”VPREB1” gene on the malignant proliferation of primary cultured myeloma cells.MethodsBioinformatics’ analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay.ResultsThere was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation.ConclusionCRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Abstract 3830Poster Board III-766Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures:Ecsedy:Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Inhibition of HIF1A Signaling by a Novel Class of Sulfonanilides for Targeted Treatment of Multiple Myeloma.

Abstract 2856Poster Board II-832 BACKGROUND.Molecular profiling of multiple myeloma allows the identification of novel targets, including HIF1A, and evaluation of their expression within large cohorts of patients. We report here the expression of HIF1A in myeloma and for the first time the preclinical testing of 4 members of a novel class of sulfonanilide HIF1A signaling inhibitors. PATIENTS AND METHODS.Expression of HIF1A was assessed using Affymetrix DNA-microarrays in 329 samples of CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the novel HIF1A signaling inhibitors ELR510490, ELR510454, ELR510444 and ELR105813 on the proliferation of 12 human myeloma cell lines and the first three on the survival of 5 primary myeloma cell-samples cultured within their microenvironment was tested, and their ability to inhibit HIF1A signaling was examined using a cell-based reporter assay. Studies were also conducted to determine in vitro stability (in plasma and microsomes), as well as single-dose PK (SDPK) parameters and maximum tolerated dose (MTD) levels after dosing in mice. RESULTS.We found (i) HIF1A to be expressed by 95.4% of CD138-purified primary myeloma cell samples from previously untreated patients. (ii) HIF1A expression shows a weak but significant correlation (r=0.3, p<0.001) with a gene expression based proliferation index. (iii) Of the chromosomal aberrations tested, myeloma cells of patients with presence of a translocation t(4,14) show a significantly higher expression of HIF1A (p<0.001) vs. patients without. Myeloma cells of hyperdiploid patients show a significantly lower expression of HIF1A (p=0.02) vs. non hyperdiploid patients. (iii) HIF1A expression does not show a correlation with event-free or overall survival. (iv) The sulfonanilides ELR510490, ELR510444, ELR510454 and ELR105813 completely inhibit proliferation of all tested myeloma cell lines at nM concentrations. (v) The compounds tested, i.e. ELR510490, ELR510444, ELR510454, are active on all primary myeloma cell-samples tested. (vi) The compounds show a pronounced effect on the HIF1A signaling pathway at EC50s of 1-25nM. (vii) Pre-clinical pharmacology data for the compounds ELR510444 and ELR510490 in mice indicate favorable absorption, distribution, metabolism, and excretion (ADME) profiles as well as exposure levels upon dosing at well-tolerated levels that are significantly above the in vitro EC50 in all the cell lines tested. CONCLUSION.HIF1A is expressed in almost all primary myeloma cells. The novel HIF1A signaling inhibitors tested are very active on myeloma cell lines as well as primary myeloma cells and show favorable in vivo profiles with exposure levels in mice significantly higher than the concentrations required for the inhibition of cell proliferation or apoptosis induction in vitro. This class of compounds thus represents a promising weapon in the therapeutic arsenal against multiple myeloma. Disclosures:Rebacz:ELARA Pharmaceuticals: Employment. Lewis:ELARA Pharmaceuticals: Employment. Schultes:ELARA Pharmaceuticals: Employment.

Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma

AbstractGenetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.

B168 APRIL Induces Proliferation of Primary Myeloma Cells Expressing Cyclin D2

B168 APRIL Induces Proliferation of Primary Myeloma Cells Expressing Cyclin D2

Inhibition of Aurora-Kinases for Tailored Risk-Adapted Treatment of Multiple Myeloma

BACKGROUND. At the time of diagnosis, myeloma cells are characterized by a low proliferation rate that increases in relapse. Presence of proliferation correlates with adverse prognosis. At the same time, myeloma cells harbor a high median number of chromosomal aberrations, often associated with genetic instability. Cellular proliferation and genetic instability in turn have been associated with Aurora-kinase expression in several cancer entities, including multiple myeloma.PATIENTS AND METHODS. Expression of Aurora-A, -B and -C was assessed using Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14) (q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the clinical Aurora-kinase inhibitor VX680 on proliferation of 20 human myeloma cell lines and survival of 5 primary myeloma cell-samples was tested.RESULTS. We found Aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient-cohorts, including 23% for Aurora A in our first cohort of patients treated with high dose therapy (see figure shown below). Aurora-C expression was found in testis-samples only. Myeloma cell-samples with detectable Aurora-A expression show a significantly higher proliferation rate, whereas the number of chromosomal aberrations (aneuploidy) is not higher compared to myeloma-cells with absent Aurora-A expression. The same holds true for subclonal aberrations (i.e. genetic instability). The Aurora-kinase inhibitor VX680 induces apoptosis in all myeloma cell lines and primary myeloma cell-samples tested. Presence of Aurora-A expression delineates significantly inferior event-free and overall survival in two independent cohorts of patients undergoing high-dose chemotherapy and autologous stem cell transplantation. This observation is independent of conventional prognostic factors, i.e. serum-ß2-microglobulin or ISS-stage.CONCLUSION. Aurora-kinase inhibitors (including VX680 tested here) are very active on myeloma cell lines as well as primary myeloma cells and represent a promising weapon in the therapeutic arsenal against multiple myeloma. Gene expression profiling allows the assessment of Aurora-kinase expression and thus in turn a tailoring of treatment to patients expressing Aurora-A associated with adverse prognosis. [Display omitted]

A Gene Expression Based Proliferation Index as Independent Prognostic Factor in Multiple Myeloma.

BACKGROUND. The proliferation-rate of primary myeloma cells is a strong adverse prognostic factor in various trials, but not routinely assessed, partially due to effort in obtaining it.AIM. As gene-expression profiling is increasingly considered a standard diagnostics in myeloma, we investigated the possibility to develop a prognostically relevant gene-expression based proliferation index (GPI).PATIENTS AND METHODS. Gene expression was determined by Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. The GPI was derived by selecting genes associated with proliferation (in terms of gene ontology) differentially expressed in proliferating malignant (human myeloma cell lines) and benign (plasmablastic) cells compared to non-proliferating, non-malignant cells (normal plasma cells and memory B-cells). The GPI comprises the sum of the expression values of 50 genes (ASPM, AURKA, AURKB, BIRC5, BRCA1, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDC25C, CDC6, CDCA8, CDKN3, CEP55, CHEK1, CKS1B, CKS2, DLG7, ESPL1, GINS1, GTSE1, KIAA1794, KIF11, KIF15, KIF20A, KIF2C, KNTC2, MAD2L1, MCM10, MCM6, MKI67, NCAPD3, NCAPG, NCAPG2, NEK2, NPM1, PAK3, PCNA, PGAM1, PLK4, PTTG1, RACGAP1, SMC2, SPAG5. STIL, TPX2, ZWINT). Proliferation of primary myeloma cells was assessed by propidium iodinestaining (n=67). Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3).RESULTS. In the two groups, 39 and 32 percent of primary myeloma cells show a GPI above the median plus three standard deviations of normal bone marrow plasma cells, respectively. The GPI is significantly higher in advanced- compared to early-stage myeloma (P=.001) and in patients harboring a gain of 1q21 (n=95, P<0.001). It correlates significantly with proliferation as determined by propidium iodine in primary myeloma cells (rs=.52, P<.001, n=67). The GPI as continuous variable is significantly predictive for event-free survival (EFS, n=120, P<.001, n=345, P<.001, respectively) and overall survival (OAS, n=345, P<.001) in patients treated with high-dose chemotherapy, independent of serum-β2-microglobulin (B2M) or ISS-stage. A GPI above the median (GPIhigh) delineated significantly inferior EFS (n=168, 41.6 vs. 26 months, P=.04, HR 1.57, CI [1.02,2.42]; n=345, 68.6 vs. 45.2 months, HR 1.55, CI [1.16,2.09], P=.003) and OAS (n=345, P<.001) in two independent cohorts of patients undergoing high-dose chemotherapy. By using B2M above 3.5 mg/l and GPI as staging variables, four groups with difference in median EFS (n=345, B2M <3.5mg/l, GPIhigh/low 76.1 months; B2M < 3.5mg/l, GPIhigh 62.4 months, B2M ≥3.5mg/l, GPIlow 41.8 months, B2M ≥3.5mg/l, GPI 36.1 months, P<.001) and OAS can be delineated.CONCLUSION. The GPIrepresents a validated tool for the assessment of proliferation in multiple myeloma patients,allows a risk stratification in terms of proliferation either alone or in combination with B2M or ISS, respectively, andhas the potential to be used within a risk adapted targeting of anti-proliferative treatment.

Dexmethasone Down-Regulates Stromal Cell-Derived Factor-1 Production in Bone Marrow Stromal Cells: Implications in Myeloma Cell Biology.

Stromal cell-derived factor-1 (SDF-1) plays a role in the homing of myeloma cells to bone marrow. In addition, SDF-1 modestly enhances the proliferation of myeloma cells and inhibits Dexmethasone (Dex)-induced apoptosis of the cells. Dex is currently used to treat multiple myeloma, based on its apoptic effects. In this study, we investigated the regulatory effects of Dex on SDF-1 production in bone marrow stromal cells (BMSCs) and on CXCR4 expression in myeloma cells. As previously reported, it was evident that primary myeloma cells (CD138+ cells obtained from patients with multiple myeloma) and Dex-resistant myeloma cell line RPMI8226 expressed CXCR4 and responded to SDF-1, resulting in chemotaxis. SDF-1 modestly stimulated the proliferation of primary myeloma cells and RPMI8226 cells and protected the cells from Dex-induced apoptosis. Human umbilical vein endothelial cells transduced with the SDF-1 gene using adenoviral vectors better supported the formation of cobblestone areas of primary myeloma cells and RPMI8226 cells in co-culture, similar to hematopoietic progenitor cells; this was blocked by pretreating the myeloma cells with pertussis toxin, indicating that SDF-1 plays a critical role not only in migration of the cells underneath the SDF-1-producing stromal cells but also in proliferation of the cells in contact. Dex up-regulated CXCR4 expression in RPMI8226 cells; however, its regulatory effects on CXCR4 in primary myeloma cells differed among patients. RT-PCR and Northern blot analyses revealed that Dex down-regulated SDF-1 mRNA expression in both primary BMSCs and murine stromal MS-5 cells in a dose-dependent manner. Western blot analysis and ELISA assay confirmed that Dex inhibited SDF-1 production in BMSCs. Furthermore, Dex inhibited cobblestone area formation of RPMI8226 cells in co-culture with MS-5. Interestingly, Dex up-regulated CXCR4 mRNA expression and cytoplasmic CXCR4 in BMSCs. These results indicate that Dexamethasone induces the down-regulation of SDF-1 production in BMSCs, which might mediate, at least in part, its anti-myeloma effects in vivo.

Agonist anti-gp130 transducer monoclonal antibodies are human myeloma cell survival and growth factors.

We have previously reported obtaining two monoclonal antibodies (mAb) against the human gp130 interleukin-6 (IL-6) transducer which made possible the dimerization of gp130 and the activation of several IL-6-driven functions when used together. We report here that these mAb induce gp130-mediated signaling in human myeloma cells and support the survival and the long-term growth of five IL-6-dependent human myeloma cell lines. Their agonist activity is not affected by neutralizing antibodies to IL-6 or IL-6R. These mAb induce a transient proliferation of primary myeloma cells from most patients with multiple myeloma. Again, IL-6 inhibitors do not affect this agonist activity. By using highly purified primary myeloma cells, we found that these anti-gp130 mAb supported the long-term survival of primary myeloma cells from five patients with primary plasma cell leukemia but failed to induce their long-term growth. For patients with fulminant disease and secondary extramedullary proliferation, the antibodies supported a long-term survival and growth, and anti-gp130 mAb-dependent cell lines were obtained. For patients with medullary involvement only, a co-stimulatory signal is necessary, together with gp130 activation, to trigger cell survival and cycling. Leukemia (2000) 14, 188-197.