Generation of a Predictive Score for Ixazomib Response in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5695-5695
Author(s):  
Amit Kumar Mitra ◽  
Sanjoy Dey ◽  
Andrew Hangsleben ◽  
Michael Steinbach ◽  
Vipin Kumar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
The United States ◽  
Myeloma Cell ◽  
Chemotherapeutic Agents ◽  
Predictive Score ◽  
Molecular Characteristics

Abstract Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States accounting for 1% of all cancers and 10% of all hematologic malignancies. Despite recent improvements in treatment strategies including the emergence of proteasome inhibitors (PIs) as effective chemotherapeutic agents, MM still remains difficult to cure with median survival rate of around 7 years, primarily due to wide inter-individual variation in response to treatment. We believe such heterogeneity in response to PIs is governed by the underlying molecular characteristics of the tumor including alterations in gene expression profile (GEP). In the current study, we used a panel of Human Myeloma Cell Lines (HMCLs) representing the gamut of biological and genetic heterogeneity in MM to evaluate the gene expression signatures associated with response to the second-generation PI Ixazomib and produced a predictive score (PI score) for Ix response. HMCLs (n=45) were treated with increasing concentrations of Ixazomib used as single agent and half maximal inhibitory concentration (IC50) values were determined using cell viability equation. Gene expression profiling data was obtained as publicly available data from the Keats lab website at TGen (http://www.keatslab.org/myeloma-cell-lines). Genes with high expression value and high standard deviation beyond the median values were pre-filtered and log expression values were normalized by subtracting mean expression of individual genes across all the samples and the housekeeping genes (GAPDH). Subsequently, analysis of correlation between Ix IC50 data and GEP data and the False Discovery Rate (FDR) based on 1000 random permutations were performed to identify true patterns of genes that are highly predictive of Ix response and to look for the top genes that could discriminate between the top sensitive and top resistant cell lines. Gene clusters were identified that correlated with response and will be presented. Our results will demonstrate in vitro modeling of response using GEP approaches that may provide predictive scoring algorithms of a defined set of genes that will be useful in clinical evaluation of drug choice in treating individual patients. Disclosures No relevant conflicts of interest to declare.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 832-832 ◽  
Author(s):  
Michael A Chapman ◽  
Jean-Philippe Brunet ◽  
Jonathan J Keats ◽  
Angela Baker ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Histone Modification ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Specific Expression ◽  
Aberrant Expression ◽  
High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

A Selective PIM Kinase Inhibitor Is Highly Active In Multiple Myeloma: Mechanism of Action and Signal Transduction Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4084-4084 ◽  
Author(s):  
Veerendra Munugalavadla ◽  
Leanne Berry ◽  
Yung-Hsiang Chen ◽  
Gauri Deshmukh ◽  
Jake Drummond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Myeloma Cell ◽  
Negative Regulator ◽  
Combination Drug ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3962-3962
Author(s):  
Xing-Ding Zhang ◽  
Robert Z. Orlowski ◽  
Lin Yang
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Hela Cells ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Cancer Center ◽  
In Vivo Models ◽  
Junction Protein ◽  
Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

Celastrol Overcomes Chemoresistance in Vitro and Potentiates the Effect of Bortezomib Against Human Multiple Myeloma in Nude Mice Mode

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5017-5017
Author(s):  
Radhamani Kannaiyan ◽  
Manu Kanjoormana Aryan ◽  
Muthu K Shanmugam ◽  
Feng Li ◽  
Gautam Sethi
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Nude Mice ◽  
Tumor Volume ◽  
Corn Oil ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Antitumor Effects ◽  
Group I

Abstract Abstract 5017 Introduction: Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of B cell in the bone marrow with low proliferative index. Despite the advent of novel therapeutics in addition to conventional chemotherapeutics, MM remains incurable because of the development of chemoresistance. Persistent activation of NF-κB/STAT3 signaling pathways and deregulation of apoptosis is considered to play an important role in the development of chemoresistance. The use of anticancer drugs derived from natural sources may be able to overcome resistance without some of the debilitating side effects of conventional chemotherapy. Celastrol is one such compound that has gained substantial attention recently for its anti-inflammatory and anticancer activities and is derived from the Chinese medicinal plant ‘Tripterygium wilfordii. We have demonstrated that celastrol overcomes the chemoresistance and induce apoptosis in MM cells by inhibiting NF-κB and STAT 3 pathways cell lines sensitive and resistant to various chemotherapeutic agents and Bortezomib. Our experimental findings have indicated that celastrol in combination with bortezomib/thalidomide can inhibit proliferation, induce apoptosis and overcome chemoresistance in MM cells in synergistic manner. We also observed that celastrol inhibited the activation of NF-κB and STAT3 and downregulated the expression of various genes involved in MM proliferation, survival and angiogenesis. Materials and Methods: Male athymic balb/c nude mice were implanted with 2×106 cells with either Human MM U266 cell lines subcutaneously. When tumors have reached more than 0. 3 cm in diameter, the mice were randomized into four groups. Group I (control) received corn oil 100 ul i. p. for five days a week, group II received 0. 25 mg/kg celastrol in 100ul corn oil for five days a week, group III received 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly and group IV received 0. 25mg/kg celastrol in 100 ul corn oil i. p. 5 days a week and 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly for 3 consecutive weeks. The tumor volume and body weight of the mice were monitored twice a week for the duration of the experiment. On completion of the treatment period, mice were euthanized by i. p. phentobarbital (40 mg/kg b. w) followed by cervical dislocation and then tumors were dissected and diameters measured. The tumor volume was calculated using the formula [L × W2]/2, where W and L are the width (short diameter) and the length (long diameter) of the tumor and the tumors were subjected to histological examination. Results: In the MM xenograft mice model, we observed that celastrol potentiated the antitumor effects of bortezomib and this correlated with significant suppression of NF-κB, STAT3, COX-2 and VEGF which was demonstrated by IHC. Overall, our data indicates that celastrol could be a potential therapeutic agent for the treatment of MM, especially in combination with the novel anti-myeloma agents. Disclosures: No relevant conflicts of interest to declare.

Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 939-939
Author(s):  
Esther Yoo ◽  
Alex Vasuthasawat ◽  
Danh Tran ◽  
Alan Lichtenstein ◽  
Sherie Morrison
Keyword(s):  
Multiple Myeloma ◽  
Fusion Protein ◽  
Cell Lines ◽  
Dna Content ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Inhibition Of Proliferation ◽  
Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2047-2047
Author(s):  
Nao Nishimura ◽  
Shinya Endo ◽  
Niina Ueno ◽  
Shikiko Ueno ◽  
Hiromichi Yuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Suppressor Activity ◽  
Enhancer Region ◽  
Tumor Suppressor Activity

Abstract PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4337-4337
Author(s):  
Chang-Xin Shi ◽  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Cecilia Bonolo De Campos ◽  
William Stewart ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Resistance Function ◽  
Proteasome Subunits ◽  
Underlying Mechanisms

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

scholarly journals De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4818-4818 ◽  
Author(s):  
Pavla Latalova ◽  
Jiri Minarik ◽  
Katerina Smesny Trtkova
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Dna Methyltransferases ◽  
Rt Pcr ◽  
Cpg Dinucleotides

Abstract Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer. Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used. Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation. Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a. With support of the grant NT14393. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Oncolytic Reovirus Immune Priming Strategy Dramatically Enhances the Efficacy of Anti-PD-L1 Antibody Therapy Against Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3960-3960 ◽  
Author(s):  
Kevin R. Kelly ◽  
Claudia M. Espitia ◽  
Weiguo Zhao ◽  
Valeria Visconte ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Study ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Single Agent ◽  
Bone Destruction ◽  
The United States ◽  
Significant Activity ◽  
Immune Priming

Abstract Multiple myeloma (MM) is the second most common adult hematologic malignancy in the United States and is characterized by a unique form of progressive bone destruction. Despite the development of new treatments such as proteasome inhibitors and immunomodulatory agents, patients with high-risk disease have a median survival of only 2-3 years, highlighting the demand for more effective therapeutic strategies. Oncolytic viral formulations represent a promising new class of anticancer agents that may have important applications in precision medicine. Recent studies have demonstrated that reoviruses specifically replicate only in cancer cells and this led to the development of a reovirus-based oncolytic viral therapy called Reolysin. We have demonstrated that reoviruses selectively replicate in MM cells and Reolysin possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274(programmed death 1 ligand, PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. Adaptive resistance mediated by inhibitory ligands such as PD-L1 has emerged as an important mechanism of malignant cell survival and has led to the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. These agents have exhibited dramatic efficacy in certain forms of cancer including melanoma and lung cancer. Analysis of specimens from patients treated on clinical trials with these agents indicates that high basal expression of PD-L1 on tumor cells may be necessary to elicit significant clinical benefit. Notably, most MM cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefiting from immune checkpoint therapy. However, novel immune priming strategies that stimulate transient upregulation of PD-L1 on malignant cells could potentially render agents that target the PD-L1/PD-1 axis significantly more effective for a broader range of malignancies including MM. We hypothesized that Reolysin could be used as a precision immune priming agent to potentiate the anti-MM efficacy of PD-L1 targeted therapy by rendering MM cells vulnerable to PD-L1 inhibition through the transient upregulation of target expression. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 108 TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P<0.01). Analysis of bone marrow specimens from mice in all experimental groups showed that Reolysin stimulated elevated PD-L1 levels in vivo in a manner that was directly linked to the enhanced efficacy of the combination. Our findings demonstrate that Reolysin has dramatic PD-L1-related immune priming effects in clinically relevant models of MM and support its use as a precision agent to sensitize MM cells to immune checkpoint therapy. Based on these promising data, we are currently planning a clinical study of Reolysin in combination with bortezomib and a PD-1 inhibitor in patients with relapsed/refractory MM. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment.

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