Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3224-3224
Author(s):  
Claire Torre ◽  
Yanyan Gu ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Feedback Loop ◽  
Myeloma Cell ◽  
Pi3k Signaling ◽  
Mtor Signaling Pathway ◽  
The Impact

Abstract Introduction The PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination. Methods Human myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination. Result 10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade. Conclusion The combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib. Disclosures: Boise: Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

MLN4924, An Investigational NAE Inhibitor, Suppresses AKT and mTOR Signaling Pathway Through up Regulating REDD1 in Human Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1870-1870
Author(s):  
Yanyan Gu ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Mtor Pathway ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Myeloma Cells

Abstract Abstract 1870 Introduction: The development and survival of normal plasma cells as well as multiple myeloma cells depend on an elaborately regulated ubiquitin proteasome system (UPS). Proteasome inhibitors such as bortezomib have proved to be highly active in the treatment of multiple myeloma. MLN4924, a newly developed investigational NEDD8 activating enzyme (NAE) inhibitor, exhibits promising anti-tumor effect through both clinical and laboratory observation. We sought to evaluate the individual signaling effects of MLN4924 in multiple myeloma, with the intent of further understanding the mechanism of action and identifying potential combinations. Methods: Human myeloma cell lines (MM.1S, MM.1R and U266) were treated with increasing concentrations of MLN4924 for 24, 48 and 72 hrs respectively or concurrently with bortezomib. Cell viability (MTT), apoptosis, western blot, RT-qPCR and siRNA assays were used to identify the cellular and molecular sequelae of MLN4924 treatment. Results: Single agent studies demonstrate that MLN4924 induces cytotoxicity in all three MM cell lines. Cytotoxicity is associated with increased apoptosis and suppression of AKT and mTOR signaling pathway, as detected by FACS and western blot. MLN4924 suppresses protein turnover of Cullin-ring ligases substrates leading to stabilization of specific proteins, such as p27, CDT1, NRF2. We find that REDD1, a substrate of CUL4 A–DDB 1–ROC1–β-TRCP ubiquitin ligase and negative regulator of mTOR pathway, increases in as early as 2 hours when treated with MLN4924. Knock-down of REDD1 using siRNA alleviates MLN4924 induced AKT, mTOR signaling suppression as well as the growth inhibition, which suggests that MLN4924 inhibits AKT, mTOR signaling through upregulating REDD1 protein leading to cytotoxicity. Combining MLN4924 with bortezomib synergistically enhances the antitumor effect of MLN4924. Western blot confirms the development of significantly increased procaspase and PARP cleavage, as well as substantial down regulation of AKT and mTOR signaling. Neither IL-6 nor IGF-1 is able to abrogate these combination effects. When we silence REDD1 in the combination assay in MM.1R, we find less cell apoptosis and suppression of AKT, mTOR pathway, which further support that REDD1 is an important regulator for MLN4924 induced cytotoxicity in MMs. Conclusion: MLN4924 is a potent investigational antitumor compound suppressing mTOR signaling pathways in myeloma cell lines. Down-regulation of the mTOR signaling pathway is associated with increased expression of REDD1. Combination of MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MMs cell lines which can overcome the prosurvival effects of growth factor (IL-6 and IGF-1). These findings could positively impact clinical combination strategies. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Onyx: Consultancy; Merck: Consultancy.

Bortezomib Induces Activation of b-Catenin/TCF Signaling Pathway in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1851-1851
Author(s):  
Ya-Wei Qiang ◽  
Bo Hu ◽  
Yu Chen ◽  
Wei Qiang ◽  
Christoph Heuck ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Dose Dependent

Abstract Abstract 1851 Background: The proteasome inhibitor Bortezomib (Bz) shows significant activity in Multiple Myeloma (MM) by acting on MM cell directly as well as by augmenting bone formation in vitro and in vivo. Its effect on the bone could be traced to promoting differentiation of mesenchymal stem cells into osteoblast cells by regulating BMP2 and canonical Wnt signaling. However, the molecular mechanism mediating the direct anti-MM activity of Bz remains to be fully understood. Initially the rationale for the use of Bz in MM was inhibition of NF-kB signaling, yet subsequent studies showed that Bz actually induces activation of this pathway. In this study, we examined whether Bz regulates the activity of canonical Wnt signaling pathway in MM and whether the growth-inhibition effect of Bz was associated with activation of this pathway by using multiple MM cell lines including EJM, H929, INA6, KMS28BM, JJN3, L363, OPM1, OPM2, RPMI8226, UTMC, XG2 and XG6 as well as primary plasma cells (PC) from six patients with newly diagnosed MM. Methods/Results: Immunoblotting demonstrated that Bz induces stabilization of b-catenin protein in three MM cell lines (H929, OPM2 and UTMC) in a time- and dose-dependent manner. These changes were not seen when the same cell lysate were immunoblotted for other catenin family members, a-catenin and g-catenin. Increased levels of b-catenin protein response to Bz treatment were observed in other 9 MM cell lines (EJM, INA6, KMS28BM, JJN3, L363, OPM1, RPMI8226, XG2 and XG6) and in the 6 CD138+ sorted bone marrow PC from patients with MM. To determine if Bz regulation of b-catenin level is a specific effect of the inhibition of 26S proteasome subunit we treated the same MM cell lines with another proteasome inhibitor, MG132. Similar results were observed in response to MG132 for all four MM cell lines, suggesting the effect of Bz on b-catenin protein is 26S proteosome inhibitor specific. Increases in b-catenin protein levels in MM cells were not due to increased Ctnnb1/CTNNB (b-catenin) gene transcription as b-catenin mRNA did not change in these cells treated with Bz. These results indicate that proteasome inhibition increases b-catenin is independent of transcriptional upregulation. To determine whether Bz induces the nuclear localization and transcriptional activity of b-catenin, cells were incubated with Bz for 6 hours and then fractionated to separate the nuclear and cytoplasmic fractions. Treatment with Bz resulted an increase in nuclear b-catenin as well as b-catenin in cytoplasm in four cell lines including H929, INA6, OPM1 and MM144. Increase in cytoplasmic and nuclear b-catenin was further confirmed by immunofluorescence with antibodies specific for active form of b-catenin. To determine whether Bz affects b-catenin-mediated transcriptional activity, we used a TCF/LEF luciferase reporter construct cloned in lentiviral vector. OPM2 cells were infected with lentiviral particle containing the TCF reporter or containing empty vector and were then treated with serial concentrations of Bz. The luciferase activity exhibited a dose-dependent response to Bz analogous to the stabilization of b-catenin. Similar results were observed in 7 out of 8 MM cell lines compared with untreated control. Stimulation of TCF transcriptional activity by Bz was independent of modifiers of extracellular Wnt ligands, such as Frizzled receptors, LRP5/6 co-receptors and sFRPs or the activation of intracellular GSK3b. Conclusion: These results indicate that Bz augments activation of canonical Wnt signaling by preventing b-catenin protein from proteosome-mediated degradation in MM cells. Concentrations of Bz for stimulating TCF transcriptional activity are comparable to those being used to induce inhibition of MM proliferation. Experiments modulating cytoplasmic as well as the nuclear players and interactions of the Wnt-pathway are ongoing to determine if Bz mediated activation of b-catenin signaling is responsible for its direct anti-MM effect. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

scholarly journals Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

PLoS ONE ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. e27707 ◽  
Author(s):  
Fabricio de Carvalho ◽  
Erico T. Costa ◽  
Anamaria A. Camargo ◽  
Juliana C. Gregorio ◽  
Cibele Masotti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Myeloma Cell ◽  
Cell Sensitivity ◽  
Multiple Myeloma Cell

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 Elevation of miR-302b prevents multiple myeloma cell growth and bone destruction by blocking DKK1 secretion

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zheyu Wu ◽  
Yufeng Zhang ◽  
Zhiqiang Yang ◽  
Yufan Zhu ◽  
Yuanlong Xie ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Bone Destruction ◽  
Bioinformatic Analysis ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Rpmi 8226

Abstract Background Myeloma bone disease (MBD) is a severe complication of multiple myeloma (MM) mainly due to an imbalance between enhanced osteoclast activity and reduced osteoblast function. Previous studies have demonstrated that miRNAs play a vital role in the osteogenic differentiation of mesenchymal stromal cells (MSCs) in MM. However, the value of miR‑302b in MBD remains to be further elucidated. The aim of this study is to explore the role of miR‑302b in the regulation of MBD osteogenic differentiation and evaluate the potential of a new therapeutic strategy for the clinical treatment of MBD. Method Our previous research demonstrated that MiR-302b belongs to the miR-302 cluster and is able to inhibit tumor growth and osteolysis in an orthotopic osteosarcoma xenograft tumor mouse model. In this study, we first transfected miR-302b mimics, miR-302b inhibitor, and miR-302b NC into MM1.S and RPMI8226 MM cells to detect the correlation between miR-302b expression in the pathological specimens and the clinicopathological features by qPCR, the target correlation between miR-302b and DKK1 by immunohistochemistry, qPCR and Western blot, and the correlation between miR-302b and the Wnt/β-catenin signaling pathway by Western blot. The effect of miR-302b on osteoblastogenesis was also studied in a subperiosteal tumorigenesis model of NOD/SCID nude mice. Results We found that increased miR-302b suppressed cell proliferation and induced cell apoptosis in RPMI 8226 and MM1.S cells. TargetScan online bioinformatic analysis predicted that miR-302b is able to bind to 3′UTR of DKK1 mRNA. Target binding of miR-302b to DKK1 was demonstrated by dual-luciferase reporter assay, qPCR, Western blot and immunohistochemistry, indicating that miR-302b is able to degrade DKK1 in RPMI 8226 and MM1.S cells. The model of co-culturing MM cells with preosteoblast MC3T3-E1 cells showed that miR-302b inhibits MM-induced suppression of osteoblast differentiation. Western blotting showed that miR-302b promotes the Wnt/β-catenin signaling pathway in MM cells. Micro-CT and immunohistochemistry results showed that miR-302b suppresses myeloma bone destruction in vivo. Conclusion miR-302b is able to target DKK1 and promote the Wnt/β-catenin signaling pathway in MM.

scholarly journals Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wenfeng He ◽  
Yonghui Fu ◽  
Yongliang Zheng ◽  
Xiaoping Wang ◽  
Bin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Side Population ◽  
Mtor Signaling Pathway ◽  
Cycle Arrest

Abstract Background Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. Methods SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. Results Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. Conclusion We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.

scholarly journals Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4344-4344
Author(s):  
Seiichi Okabe ◽  
Yuko Tanaka ◽  
Mitsuru Moriyama ◽  
Akihiko Gotoh
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
New Agents ◽  
Myeloma Cells ◽  
Hypoxic Conditions ◽  
Inhibitor Treatment

Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis. Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition. Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells. Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

scholarly journals C-Myc-Driven and PRMT5-Dependent Regulation of Multiple Myeloma Cell Proliferation through TNRC6B Gene

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5520-5520
Author(s):  
Peipei Xu ◽  
Guo Dan ◽  
Bing Chen ◽  
Quan Zhao ◽  
Rong-Fu Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Suppressor Gene ◽  
Rna Seq ◽  
Multiple Myeloma Cell ◽  
Arginine Residues

Objective: The protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetrical bimethylation of arginine residues, which plays an important regulatory role in the life process. Transcription factor c-Myc is widely proved involved in the alteration and development of tumors, while the regulatory mechanism of c-Myc-mediated gene expression has not been fully understood. In this article we try to investigate the functional role of PRMT5 and c-Myc in regulating multiple myeloma cell proliferation. Methods: The protein and mRNA expression levels of PRMT5 in myeloma cells and normal cells were detected by Western Blot and qPCR. Lentivirally transduced shRNAs targeting PRMT5 (sh-PRMT5) was constructed using lentivirus mediated RNAi technology, and was packaged to infect MM cells to select positive colonies. The effect of PRMT5 on the proliferation of MM cells was detected by cck-8 assay. Annexin V/7-AAD double staining flow cytometry was used to detect cell survival and apoptosis rate. The expression of apoptotic suppressor gene c-Myc in sh-PRMT5 cell lines was detected by Western Blot and qPCR. Finally, double-knockdown of PRMT5 and c-myc was performed and gene expression differentiation were identified by RNA sequencing (RNA-seq). Results: PRMT5 expression in MM cell lines (RPMI8226 and U266) was relatively high compared with peripheral blood mononuclear cells (PBMCs), which was correlated with progression-free survival (PFS) and overall survival (OS) in MM patients. After sh-PRMT5 infection to MM cell lines, puromycin selection was performed and efficient gene knockdown was evaluated by Western Blot and qPCR. After targeted silencing of PRMT5 expression, the proliferation of MM cells was decreased and apoptosis was significantly increased (P < 0.05). C-Myc protein and mRNA levels were significantly downregulated after PRMT5 gene silencing. The results showed that PRMT5 promoted the proliferation of MM cells and inhibited the apoptosis of MM cells by regulating the expression of apoptotic suppressor gene c-Myc. Subsequently, RNA-seq confirmed SMCHD1 as the common candidate target gene of c-myc and PRMT5. Therefore, we speculated that PRMT5 and c-Myc may jointly regulate TNRC6B gene to promote the proliferation of MM cells. Conclusion: Altogether, our study not only proposed the mechanism PRMT5 and c-myc in regulating multiple myeloma related gene expression, but also provided a new strategy and theoretical basis for clinical treatment of multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2922-2922
Author(s):  
Liang Nong ◽  
Linda Mathews ◽  
Mark B Meads ◽  
William Dalton ◽  
Kenneth H. Shain
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Dna Repair ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Myeloma Cell ◽  
Sequential Treatment ◽  
Parp Inhibition ◽  
Repair Pathway ◽  
Myeloma Cells

Abstract Abstract 2922 The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A High-Throughput Drug Screen Reveals a Novel Compound Class That Significantly Depletes IRF4 Expression in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5545-5545 ◽  
Author(s):  
Elizabeth D. Lightbody ◽  
Mairead Reidy ◽  
Michael P. Agius ◽  
Salma El-Behaedi ◽  
Romanos Sklavenitis-Pistofidis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Western Blot ◽  
Cell Lines ◽  
High Throughput ◽  
Standard Of Care ◽  
Drug Screen ◽  
Dependent Manner ◽  
The Impact

Introduction Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal expansion of malignant plasma cells (PCs) within the bone marrow. MM is genetically heterogeneous with aberrations including hyperdiploidy and chromosomal translocations commonly involving the immunoglobulin heavy chain (IgH) region. Many transcription factors can revoke their normal processes and act as oncogenes when they are brought under the control of IgH regulatory regions by a chromosomal translocation. Interferon Regulatory Factor 4 (IRF4) is a transcription factor which controls plasma cell differentiation and possesses many regulatory roles including interferon response, immune cell response, cell proliferation, apoptosis, and metabolism. IRF4 has proven to be a genetic vulnerability in MM as silencing studies in a large panel of MM cell lines with various genetic etiologies have demonstrated IRF4 expression is essential for MM cell survival. Standard of care treatments that indirectly suppress IRF4 including Proteasome inhibitors and Cereblon modulators have provided the greatest clinical outcomes for patients. However, like many other transcription factors, IRF4 has been notoriously difficult to target due to the protein's lack of amenable binding pockets favored for small molecule inhibitor development. Thus, identifying novel mechanisms and compounds to target IRF4 (directly or indirectly) can provide significant clinical impacts for MM patients. Methods To discover compounds capable of depleting IRF4 levels, we performed a high-throughput drug screen utilizing the Selleckchem Drug Repurposing Library on a widely accepted IRF4-dependent cell line. This library consists of over 2,000 diverse compounds that have well validated mechanisms of actions and have additionally passed clinical phase 1 safety trials for accelerated translational use. MM.1S cells were treated for 48 hours in duplicate (n = 2) with 10 μM compound. Following treatment, the cells were fixed, permeabilized, and stained for viability and IRF4 levels. IRF4 expression and viability was acquired by using flow cytometry, with high dose lenalidomide and shRNA for IRF4 as positive controls. Compounds that reduced IRF4 levels and cell viability across both experimental runs were ranked and selected with a cutoff of 40% as promising candidate compounds for further validation. Results Our drug screen results revealed 20 compounds (undisclosed) which met our cutoff of a decrease of IRF4 levels by 40% or greater. Ten hits were selected as having greater or equal to IRF4 depleting properties of lenalidomide and moved forward to be validated by western blot. Six drugs were shown to deplete IRF4 by western blot in MM.1S and KMS-18 cells at 10 μM doses. Interestingly, 4 out of the 10 hits all belong to same compound class that selectively bind to the same target receptor (undisclosed). Additional experiments confirmed these class of compounds deplete IRF4 levels in a dose dependent manner (EC50 = 1 μM). A time course revealed that IRF4 levels decrease shortly after the binding of these drugs to their widely reported target receptor, suggesting this is a selective drug/target receptor-mediated mechanism directly altering levels of IRF4. In vitro studies demonstrated the ability to both halt cell growth and decrease the viability of a panel of 8 MM cell lines, with IC50's ranging from 1.6 - 8.5 μM. Synergy studies with Lenolidomide and Bortezomib are underway to determine any synergistic combinations with standard of care therapies. In vivo studies and RNA-sequencing are also currently underway to determine the impact of these compounds on MM tumor growth and overall survival, as well as better define the mechanism of action driving this novel class of IRF4 targeting compounds. Conclusions Despite knowledge that IRF4 is a biologically potent target in MM there have been no extensive studies highlighting drugs capable of targeting this transcription factor and its oncogenic signaling network. This screen has revealed novel compounds, some of which are clinically used, that are capable of depleting a highly dependent gene in MM. Notably, these compounds are able to deplete IRF4 in a novel mechanism which is capable of affecting survival of MM cell lines that represent the heterogeneity of myeloma, and thus holds potential for significant clinical impact. Disclosures Ghobrial: Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; BMS: Consultancy.

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