TRAIL as the ATO-Target Gene Uniquely Activated In the Hyperdiploid Subtype of Myeloma with Prognostic Relevance, Resulting In Better Prognosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1904-1904
Author(s):  
Jumei Shi ◽  
Jun Hou ◽  
Yi Tao ◽  
Xiuqin Meng ◽  
Ying Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Wild Type ◽  
Apoptotic Pathway ◽  
Myeloma Cells ◽  
University Of Utah ◽  
Start Up ◽  
The University

Abstract Abstract 1904 Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying mechanism remains elusive. In this study, the molecular mechanisms of ATO-induced myeloma apoptosis were explored on four myeloma cell lines OPM2, U266, RPMI8226, and KMS28PE of wild type or mutant p53 status and six primary myeloma cells. Gene expression profiling (GEP) of CD138+ bone marrow plasma cells from 22 healthy individuals (NPC), 44 patients with monoclonal gammopathy of undetermined significance (MGUS), and 351 newly diagnosed MM patients were published previously (Zhan et al. Blood. 2006;108:2020-8. Shaughnessy et al. Blood. 2007;109:2276-84.); and GEP from 9 myeloma cell lines were used in this study from the unpublished data of the University of Utah. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. GEP was performed by using the Affymetrix U133Plus2.0 microarray. ATO induced potent inhibition of myeloma cell growth and myeloma cell apoptosis, compared with controls. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb while up-regulating p53, p21Cip1, and p27Kip1 proteins, resulting in G0/G1 or G2/M cell cycle arrest. ATO treatment increased mRNA levels of interferon regulatory factor-1 and TRAIL, as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway in the mutated p53 myeloma cells. ATO also activated caspases 3 and 9, indicating involvement of the intrinsic apoptotic pathway in the wild type p53 myeloma cells. The usage of ATO and TRAIL agonist together has a synergistic effect, indicated by a combination index of less than 1. More importantly, these molecular changes induced by ATO-treated myeloma cells are very similar to the baseline expression pattern of hyperdiploid myeloma, which has a relative good prognosis with high expression of TRAIL and interferon related genes. Together, our data suggest that ATO induces apoptosis in MM through either extrinsic or intrinsic signaling pathway depending on the p53 genetic background. These observations may be employed as prognostic tools and lead to novel therapies in primary myelomas. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (30973450 to JS), start-up funds from Shanghai Tenth People's Hospital (JS), institutional start-up funds from the University of Utah School of Medicine and the Huntsman Cancer Institute (FZ), the National Institutes of Health grant RO1 (CA115399 to GT, FZ) and Senior Award from the Multiple Myeloma Research Foundation (FZ). Disclosures: Zhan: University of Utah: Employment, patent Submission.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4478-4478 ◽  
Author(s):  
Noriyoshi Iriyama ◽  
Hirotsugu Hino ◽  
Shota Moriya ◽  
Masaki Hiramoto ◽  
Yoshihiro Hatta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Dose Dependent

Abstract Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival. Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor. Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib. Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Synergy between 17 AAG and Arsenic Trioxide in the Induction of Apoptosis in Myeloma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5055-5055
Author(s):  
Yair Gazitt ◽  
Cagla Akay ◽  
Fatih Kircelli
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Additive Effect ◽  
Mutant P53 ◽  
Apoptotic Pathway ◽  
Potent Inducer ◽  
Myeloma Cells ◽  
Induction Of Apoptosis ◽  
In Cells

Abstract Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32C and mutant p53 phenotype at 37C (Akay et al. Akay et al., AACR; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation, 2005). Employing the Affymetrix Microarray technology to compare global gene expression in myeloma cell lines we identified a number of new genes affected by ATO (Gazitt et al. ASH presentation, 2005). One of these genes was heat shock protein 90 (HSP90). We therefore hypothesized that treatment of myeloma cells with blockers of HSP90 such as geldanamycin or its newly discovered potent derivative; 17-allylamino-17-demethoxygeldanamycin (17-AAG) in combination with ATO will result with synergy in the induction of apoptosis in these cells. Indeed, treatment of IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) with 17 AAG (0 to 3uM) resulted with a time/dose induced apoptosis to a maximum of 25% apoptosis by annexin V. Treatment with ATO alone at 2.5 uM resulted with 22% apoptosis following 24 hours of treatment. However, ATO synergized with 17 AAD to induce 2–3 fold higher apoptosis compared to the sum of the individual drugs in each dose tested. In contrast, only additive effect was observed between 17 AAG and ATO in the induction of mitochondrial membrane (MM) depolarization as measured by TMRE fluorescence and in the depletion of glutathione measured by MCB fluorescence. Interestingly, 17 AAG did not have any effect on generation of reactive oxygen species as measured by DHR fluorescence. Finally, 17 AAG induced mild arrest of cells at G2/M with marked increase in G2/M arrest when combined with ATO, which by itself did not increase the percentage of cells at G2/M. Similar trend was observed in U266 cells, in which apoptosis, MM depolarization, depletion of glutathione and G2/M arrest were much higher with 17 AAG alone or with ATO alone, and hence only additive effect was observed between the 2 drugs, at the same dosing used for the IM9 cells. Western immunoblot analysis of the levels of HSP 90 -alpha (90kd) and beta (81kd) subunits revealed slight inhibition with 17 AAG alone and ATO alone with marked decreased in HSP90 inhibition observed in cells treated with both drugs. Furthermore, analysis of the proteins involved in the intrinsic and extrinsic apoptosic pathways revealed a shift from activation of the intrinsic to activation of the extrinsic apoptotic pathway in IM9 cells treated with the combination of 17 AAG and ATO, similar to the pattern observed in U266 treated with ATO alone. These results strongly suggest that 17 AAG could potentiate the effect of ATO, in myeloma patients treated with the combination of the 2 drugs.

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3417-3417
Author(s):  
Yutaka Okuno ◽  
Hiro Tatetsu ◽  
Shikiko Ueno ◽  
Hiroyuki Hata ◽  
Yasuhiro Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Promoter Region ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Upstream Region ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Expression Levels

Abstract It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

Microarray Analysis of PU.1-Induced Growth Arrest and Apoptosis of Myeloma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2499-2499
Author(s):  
Shikiko Ueno ◽  
Hiro Tatetsu ◽  
Naoko Harada ◽  
Hiroyuki Hata ◽  
Tadafumi Iino ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Down Regulation ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Enhancer Region

Abstract PU.1 is an Ets family transcription factor, which is important for differentiation of granulocytes, monocytes/macrophages, and B cells. In the Friend leukemia model, it is reported that the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest that leads to erythroleukemia. In conditional knockout mice of the 3.5 kb length of enhancer region located in14 kb 5′ of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells down to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like disease. In addition, a deletion of the 3.5 kb enhancer region, which also contains the suppressor region for PU.1 in T cells, results in ectopic expression of PU.1 in T cells, which leads to T cell lymphoma in those mice. Taken together, the failure of up-regulation or down-regulation of PU.1 in certain differentiation stages for each lineage appears to cause differentiation arrest and hematological malignancies. We recently reported that PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ of human PU.1 gene which is homologous to that in14 kb 5′ of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of those PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that down-regulation of PU.1 is necessary for myeloma cell growth. In addition, we reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of some myeloma patients. Myeloma patients with low-to-negative PU.1 expression (lower 25th percentile of PU.1 expression level distribution among 30 patients we examined) may have poor prognosis compared to those with high PU.1 expression, although more patient samples have to be examined to define the significance of the relationship of PU.1 expression levels and prognosis. To elucidate the mechanisms of PU.1 induced cell growth arrest and apoptosis of myeloma cells, we next performed DNA microarray analysis to compare gene expression levels before and after PU.1 induction. We utilized Illumina Sentrix® Human-6 Expression BeadChip. Of 47296 genes, 479 genes were up-regulated (>2fold) and 1697 genes down-regulated (<0.5 fold) either day 1 or 3 after PU.1 induction in U266 cells. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL partially inhibited apoptosis of U266 cells expressing PU.1, suggesting that TRAIL is related to PU.1 induced cell death of U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed with protein levels. We are now examining the roles of the observed up-regulated genes in both U266 and KMS12PE myeloma cell lines.

Arsenic Trioxide-Mediated Growth Inhibition of Myeloma Cells Is Associated with An Extrinsic Signaling Pathway Involved in Caspase 3 and Caspase 8.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4899-4899
Author(s):  
Jumei Shi ◽  
Yi Wu ◽  
Siqing Wang ◽  
Xiuqin Meng ◽  
Rong Wei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Arsenic Trioxide ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Myeloma Cells

Abstract Abstract 4899 Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying molecular mechanism remains elusive. This study was performed to evaluate the molecular mechanism underlying its anti-myeloma activities. Cells from OPM2, U266, RPMI8226 myeloma cell lines and patients diagnosed with myeloma (n=6) were cultured with various concentrations of ATO for 4 days. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. ATO induced potent inhibition of myeloma cell growth compared with untreated control cells. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb, while it up-regulated p53, p21Clip1, and p27Kip1 proteins, resulting in G2/M cell cycle arrest and cell growth inhibition. ATO treatment increased mRNA levels of interferon regulatory factor-1 (IRF-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway. No significant change was detected in the expression levels of Bax, Bcl-xL caspase 9 and Bcl-2, indicating that the intrinsic signaling pathway was not involved. A pan-caspase inhibitor abrogated ATO-induced apoptosis of myeloma cells. Our data suggest that ATO induces apoptosis in MM cells most likely through an extracellular signaling pathway. Disclosures No relevant conflicts of interest to declare.

Aptamer TY04 Inhibits Multiple Myeloma Cell Growth Via Cell Cycle Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5390-5390
Author(s):  
Jing Liu ◽  
Hong-Juan Dai ◽  
Bian-Ying Ma ◽  
Jian-Hui Song ◽  
Hui-yong Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
M Phase ◽  
Target Molecules

Abstract Multiple myeloma (MM), also known as plasma cell myeloma, is characterized by accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin (Ig) in the blood or urine. MM accounts for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease in the vast majority. With conventional chemotherapy, the 5-year median survival rate for MM patients is approximately 25%. Aptamers are single-stranded RNA or DNA sequences that bind to target molecules with high affinity and specificity. Compared with antibodies, aptamers have unique advantages including easy chemical synthesis and modification, low toxicity, lack of immunogenicity, and rapid tissue penetration, Based on these advantages, aptamers show great potential for therapeutic application. The aptamer TY04 is a single-stranded DNA (ssDNA) generated by a method named cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), We found TY04 strongly inhibited the growth of multiple myeloma cell lines including MM1.S, NCI-H929, KM3 and OPM2,The concentration of TY04 to inhibit 50% cell growth (IC50) on MM1.S was 3.89 μM. In contrast, TY04 had no effect on the growth of non-tumor cell lines — immortal B lymphoblastoid cell lines. Next, we used MM1.S cell line as the model to study the mechanism of TY04 anti- multiple myeloma. Flow cytometry analysis showed that TY04 with the sequence specifically bind to MM1.S cells when compared with unselected ssDNA library control. To investigate whether the target molecules of TY04 are membrane proteins on cell surface, MM1.S cells were treated with trypsin and proteinase k for 2 or 10 minutes before incubation with TY04. The result revealed that TY04 lost partly recognition ability on treated cells, indicating that the target molecules were most likely membrane proteins. Furthermore, we evaluated the cell cycle distribution of MM1.S after TY04 treatment. We found that TY04 significantly caused cell-cycle arrest in G2/M phase. The percentage of G2/M phase cells increased from 30.1±1.56 to 53.2±6.36. To identify the underlying molecular mechanism, G2/M-related proteins were assayed by flow cytometry. Following TY04 treatment, a concomitant inhibition of ERK1/2, cyclin B, CDK1 and γ-tubulin expression occurred. Meanwhile, human cell cycle PCR array was used to analyze the expression of 84 genes key to cell cycle regulation in TY04-treated MM1.S cells. Our results indicated that aptamer TY04 decreased the genes expression of CCNB1, CCNB2, BIRC5, BRCA1 and CCNH, which were involved in the progress of G2/M phase. All these results are significant in that they provide a framework for further exploring the use of TY04 as a novel anti-multiple myeloma agent. Disclosures: No relevant conflicts of interest to declare.

Identification of FAM46C As a Multiple Myeloma Repressor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 836-836 ◽  
Author(s):  
Yuan Xiao Zhu ◽  
Chang-Xin Shi ◽  
Patrick Jedlowski ◽  
Klaus Martin Kortum ◽  
Laura Ann Bruins ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Cell Viability ◽  
Cell Survival ◽  
Pathway Analysis ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Wild Type ◽  
Myeloma Cells

Abstract Introduction: Partial loss of chromosome arm 1p frequently occurs in multiple myeloma (MM), and is associated with a poor prognosis. Several minimally altered regions on 1p have been identified, including 1p32.3, 1p31.3, 1p22.1-1p21.3, and 1p12. Cytoband 1p12 was deleted in 19% of cases, and this deletion was associated with shorter overall survival (OS) in univariate analysis. The target of homozygous deletion 1p12 was FAM46C. In addition, mutations of FAM46C were identified in 3.4% to 13% of primary MM tumors and 25% of 16 human myeloma cell lines (HMCLs), implying its potential pathogenic relevance. In other work we have suggested that FAM46C mutation is a progression event and have shown that it is rarely seen in newly diagnosed del17 patients, inferring some overlap in function. However, there is no published functional annotation of FAM46C and its role in MM remains unknown. In the present study, we aimed to identify the biological role of FAM46C in myeloma cells. Materials/Method: The expression of FAM46C in HMCLs was analyzed by western blot. Lentiviral constructs expressing wild type and mutated FAM46C were generated and transduced into HMCLs, followed by cell viability assay and cell cycle analysis. Cells were harvested and processed to measure gene expression and cell signaling changes after introduction of FAM46C by mRNAseq, pathway analysis and immunoblotting assay. Results: The expression of FAM46C protein is generally low in most HMCLs. Introduction of wild type FAM46C in HMCLs induced a substantial cell growth inhibition and apoptosis, especially in two HMCLs including MM1.S and KMS11. Cell viability of KMS11 and MM1.S was reduced by 50% to 80% at day 6 after introduction of FAM46C, compared to 0-30% growth retardation detected in HMCLs and non-myeloma cell lines that do not carry FAM46C deletion. We identified 88 genes whose mRNA expression was significantly altered after enforced expression of FAM46C in MM1.S cells. Pathway analysis revealed that FAM46C-regulated genes are enriched in the canonical pathways associated with unfolded protein response, cell cycle control and DNA damage repair. Critical MM genes that are downregulated by FAM46C expression include IRF4 and MYC, which are also downstream targets of immunomodulatory drugs (IMiDs). Consistently, some HMCLs such as KMS11 and OPM2 show an enhanced sensitivity to lenalidomide after introduction of FAM46C. Next, lentiviral constructs expressing various FAM46C mutants were generated in order to understand the consequence of FAM46C mutation. The mutant constructs mimic mutations identified in MM patients or HMCLs. Those mutants and wild type FAM46C were transduced and tested together in MM1.S cells. We found that three published misssense mutations, one frame-shift mutation and deletion of the sequence between aa172 and aa186 of FAM46C (which has been found in previous studies as a hot spot of mutation) all abolished FAM46C-mediated anti-myeloma activity, thus would be expected to confer a MM cell survival advantage. Conclusion: Our data demonstrated that enforced FAM46C expression in myeloma cells induced myeloma growth inhibition and apoptosis. Mutations in FAM46C and TP53 in newly diagnosed patients seem mutually exclusive but not in relapsed patients from our patients sequencing studies, suggesting it may associate with disease progression. Together, these studies suggest that FAM46C may function as a tumor suppressor in myeloma. We also found that published mutations of FAM46C confer a survival advantage to MM cells, and that FAM46C overexpression downregulates IRF4 and MYC and is thus associated with loss of myeloma cell survival. Disclosures Stewart: Novartis: Consultancy; Oncospire Inc.: Equity Ownership; Celgene: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

Arsenic trioxide–induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4078-4087 ◽  
Author(s):  
Qun Liu ◽  
Susan Hilsenbeck ◽  
Yair Gazitt
Keyword(s):  
Cell Cycle ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Myeloma Cells ◽  
Induced Apoptosis ◽  
In Cells

Abstract Arsenic trioxide (ATO) has been shown to induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells concomitant with down-regulation of the PML-RARα fusion protein, a product of the t(15:17) translocation characteristic of APL leukemic cells. However, ATO is also a potent inducer of apoptosis in a number of other cancer cells lacking the t(15:17) translocation. The exact mechanism of ATO-induced apoptosis in these cells is not yet clear. We tested the effect of ATO on 7 myeloma cell lines with varying p53 status and report that in cells with mutated p53, ATO induced rapid and extensive (more than 90%) apoptosis in a time- and dose-dependent manner concomitant with arrest of cells in G2/M phase of the cell cycle. Myeloma cells with wild-type (wt) p53 were relatively resistant to ATO with maximal apoptosis of about 40% concomitant with partial arrest of cells in G1 and up-regulation of p21. The use of caspase blocking peptides, fluorescence-tagged caspase-specific substrate peptides, and Western immunoblotting confirmed the involvement of primarily caspase-8 and -3 in ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in cells expressing wt p53. We also observed up-regulation by ATO of R1 and R2 APO2/TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) receptors. Most important, however, we observed a synergy between ATO and APO2/TRAIL in the induction of apoptosis in the partially resistant myeloma cell lines and in myeloma cells freshly isolated from myeloma patients. Our results justify the use of the combination of these 2 drugs in clinical setting in myeloma patients.

Enhanced Levels of Apoptosis by Combination of Farnesyl Transferase Inhibition (Tipifarnib) and Proteasome Inhibition (Bortezomib) in Myeloma Cell Lines and Primary Myeloma Cells and Its Mechanistic Effects on Akt and Caspase Pathways.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1573-1573 ◽  
Author(s):  
Jonathan L. Kaufman ◽  
Ebenezer David2 ◽  
Claire Torre3 ◽  
Rajni Sinha4 ◽  
Sagar Lonial5
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Annexin V ◽  
Myeloma Cell ◽  
Caspase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells ◽  
Farnesyl Transferase ◽  
Apoptotic Protein

Abstract Introduction: Despite significant advances in the treatment of myeloma, patients invariably become resistant to therapy. Therefore, novel treatment strategies are needed to overcome resistance. Overexpression of the anti-apoptotic protein Akt has been associated with resistance to bortezomib induced apoptosis. We have previously shown that treatment with farnesyl transferase inhibitors (FTI) is associated with synergisitic myeloma cell apoptosis when combined with bortezomib. In this study we explored the mechanism of action of the combination of bortezomib with tipifarnib, a FTI with previously demonstrated clinical activity in patients with hematologic malignancies. Our hypothesis is that the combination of bortezomib and tipifarnib will result in synergistic cell death by overcoming the anti-apoptotic effects of Akt. Methods: MM.1S, MM.1R, RPMI8226 and U266 cell lines were used in addition to fresh unmanipulated human myeloma cells from patients with relapsed MM. Cell proliferation was measured using the MTT assay. Cell death was measured by flow cytometric analysis of Annexin V and propidium iodide staining in the presence or absence of both agents and the broad spectrum caspase inhibitor Z-VAD-FMK (ZVAD). Caspase activity was assessed by Western blot ananlysis of cleaved caspases. Transient transfection of cell lines of using activated Akt, wild type Akt and BCL2 was also performed. Results: Dose escalation in vitro demonstrated that 8nM was a subtherapeutic dose of bortezomib, and 20nm bortezomib was an effective dose as a single agent. Doses of tipifarnib alone up to 5μM had modest effects on MM cell death. When 8nM or 20nM of bortezomib are combined with tipifarnib at doses of 5μM, cell death increases significantly in MM cell lines. Combination resulted in increased caspase 3, 8, and 9 activities in MM cell lines. Inhibition of caspase activities were confirmed with the broad spectrum caspase inhibitor ZVAD. Individual caspase inhibitor studies after 18 hours of combination treatment suggested that the inhibition of apoptosis is mainly mediated through caspase 8 and caspase 6 as measured by Annexin-V staining in MM.1S cells. Additionally, similar studies with the pan-caspase inhibitor ZVAD also suggested that there are caspase independent pathways resulting in inducing apoptosis of MM.1S cells. Combination therapy significantly reduces phos-Akt as early as 24hrs in MM cells, although, complete inhibition of phos-473-Akt varies between cell lines. Overexpression of activated Akt or wild type Akt and the anti-apoptotic protein Bcl2 in MM.1S did not abrogate the effect of combination on apoptosis. Primary human MM cells also demonstrated synergistic cell death when exposed to the combination at clinically achievable levels. Conclusion: The combination of tipifarnib with bortezomib is associated with greater cell death than either agent alone in both myeloma cell lines and patient myeloma cells. Therefore, we propose that the use of combined tipifarnib and bortezomib represents a novel and potentially active approach to MM therapy. The synergistic mechanism involved in the combination warrants further investigation.

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