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1
Hao, Mu, Yu Qin, Meirong Zang, et al. "Hypermethylation of TAp73 Suppresses ABL1-Involved DNA Damage Response in Multiple Myeloma." Blood 124, no. 21 (2014): 3374. http://dx.doi.org/10.1182/blood.v124.21.3374.3374.
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Abstract Background: More recently, multiple myeloma (MM) cells evade apoptosis despite pervasive DNA damage was demonstrated. However, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed remain to be fully elucidated. p53 deletion and mutations do not appear to be a pivotal event in the evolution from pre-malignancy toward malignancy in MM. The protooncogene ABL1 was an alternative pathway to p53 down stream of ATM/ATR, which is commonly translocated in Chronic Myelogenous Leukemia (CML). ABL1 forms a complex with the tumor suppressor gene TP73, which belongs to the p53 family. P73 is expressed as multiple isoforms due to the usage of two different promoters, P1 promoter of TAp73 inducing apoptosis and P2 promoter of Δ Np73 promoting survival. In this study, we explored the role of ABL1/p73 axis in MM cells evading ongoing DNA damage induced apoptosis. Materials and methods: Real-time-PCR was used to detect the ABL1 and miR-203a expression in MM primary samples and MM cell lines. Immunofluency staining was performed to detect the ɣ-H2A.X level in MM cells. Flow cytometry was performed to detect the apoptosis in MM cells. Bisulfite Pyrosequencing and Methylation Specific-PCR were used to detect the p73 promoter methylation. Results: Our results revealed that ABL1 level was up-regulated both in primary MM samples and MM cell lines (-1.25±0.28 vs. 0.06±0.24, p=0.02). MiR-203 which suppresses ABL1 expression was down-regulated (0.01±0.01 vs. 0.97±0.08, p=0.01). MM cell lines and primary cells showed high ɣ-H2A.X staining. Immunofluency staining showed that ABL1 relocalized in the nucleus of MM cells after treated with doxorubicin. The apoptosis of MM cells was significantly up-regulated (7.8±2.1)% vs. (25.4±4.5)%, p<0.05. Doxorubicin treatment combined with ABL1 inhibitor (STI571) suppressed the apoptosis significantly, (25.4±4.5)% vs. (12.2±3.4)%, p=0.03. Bisulfite Pyrosequencing and MS-PCR of 42 newly diagnosed MM patient sample revealed that the P1 promoter of p73 was hypermethylated compared with normal plasma cells (86% ±7% vs. 58%±4%, p=0.032). RT-qPCR and western blotting showed that Δ Np73 levels were significantly higher than TAp73 (148.6±19.3 vs. 6.8±2.4, p=0.021) in plasma cells of those patients and MM cell lines. Conclusion: Hypermethylation of p73 promoter suppresses TAp73 expression. The deregulated of ABL1-p73 pathway in MM cells resulted in marked reduction of apoptosis induced by ongoing DNA damage. Disclosures No relevant conflicts of interest to declare.
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Schultheis, B., A. Krämer, A. Willer, U. Hegenbart, H. Goldschmidt, and R. Hehlmann. "Analysis of p73 and p53 gene deletions in multiple myeloma." Leukemia 13, no. 12 (1999): 2099–103. http://dx.doi.org/10.1038/sj.leu.2401609.
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Lunghi, Paolo, Nicola Giuliani, Laura Mazzera, et al. "Targeting MEK/MAPK Signal Transduction Module Potentiates Arsenic Trioxide (ATO)-Induced Apoptosis in Multiple Myeloma Cells through Multiple Signaling Pathways." Blood 110, no. 11 (2007): 1517. http://dx.doi.org/10.1182/blood.v110.11.1517.1517.
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Abstract Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index &lt;1.0) induction of apoptosis in 7 human myeloma cell lines (HMCLs: XG1, XG6, OPM2, JJN3, RPMI, H929, Sultan) analyzed, irrespective of their p53 status. The combined treatment was also a highly potent inducer of apoptosis and mitochondrial damage in the majority of the primary multiple myeloma (MM) cell samples ex vivo analyzed at different disease stage (9 out of 12). Growth factors, IL-6 or insulin-like growth factor 1 (IGF-1), or a co-culture system with bone marrow stromal cells (BMSCs) failed to confer resistance to this combination regimen. The combination PD/ATO had a minimal effect on normal B cells in vitro. By investigating the molecular mechanisms involved in MM cells PD/ATO-induced apoptosis, we found that co-treatment with PD strikingly elevated the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of ATO-treated HMCLs that do not have a functional p53 pathway. In HMCLs carrying a functional p53 pathway, the treatment with PD greatly enhanced the ATO-induced p53 accumulation (two fold increase) and p73, a p53 paralogue, cooperated with p53 in the pro-apoptotic p53/p73 target genes up regulation, caspase-9, -3 activation and apoptosis induction; in these HMCLs the selective down-regulation of p53 or p73 demonstrated that both have a biological relevance in PD/ATO-induced caspase-3 activation, PARP fragmentation and apoptosis. In HMCLs carrying a functional p53 the extrinsic caspase-8 mediated pathway was partially activated by PD/ATO treatment. We also demonstrated that, in MM cells carrying or not a functional p53 pathway, the combined treatment PD/ATO increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated). The selective down-regulation of Bim significantly diminished caspase-8/-9/-3 cleavage/activation, PARP fragmentation and apoptosis of PD/ATO-treated MM cells, thereby indicating that Bim can play an important role not only in the intrinsic mitochondrial programmed cell death but also in the extrinsic caspase-8 mediated pathway. Accordingly, a physical interaction between Bim and DR4/DR5 TRAIL receptors in PD/ATO-treated MM cells carrying a non functional p53 was found by coimmunoprecipitation and Western blot studies. Our experiments have enlightened some relevant mechanisms that explain the apoptotic response of myeloma cells to ATO plus MEK inhibitor combination.
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Raab, Marc S., Klaus Podar, Jing Zhang, et al. "Targeting Proteinkinase C Alters ER-Stress and b-Catenin Signaling in Multiple Myeloma: Therapeutic Implications." Blood 110, no. 11 (2007): 258. http://dx.doi.org/10.1182/blood.v110.11.258.258.
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Abstract We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a &gt; 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.
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Lunghi, Paolo, Nicola Giuliani, Laura Mazzera, et al. "Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways." Blood 112, no. 6 (2008): 2450–62. http://dx.doi.org/10.1182/blood-2007-10-114348.
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Abstract We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)–induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.
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Shammas, Masood A., Paola Neri, Hemanta Koley, et al. "Specific killing of multiple myeloma cells by (-)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications." Blood 108, no. 8 (2006): 2804–10. http://dx.doi.org/10.1182/blood-2006-05-022814.
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AbstractEpigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-κB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.
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Shammas, Masood A., Ramesh B. Batchu, Hemanta Koley, et al. "A Green Tea Polyphenol, Epigallocatechin-3-Gallate, Induces Selective Apoptosis in Multiple Myeloma Cells: Mechanism of Action and Therapeutic Potential." Blood 106, no. 11 (2005): 1590. http://dx.doi.org/10.1182/blood.v106.11.1590.1590.
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Abstract Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, induces dose and time dependent cell death in both IL-6-dependent and independent multiple myeloma cell lines and primary patient cells, with minimal or no effect on the growth of normal cells. The cell death is apoptotic as determined by annexin V staining and is not inhibited by IL-6. Evaluation of molecular mechanism of action by gene expression profiling indicated that EGCG had a profound effect on transcription of major regulatory genes involved in distinct pathways controlling cell growth arrest and apoptosis: Exposure of myeloma cells to EGCG induced the expression of: 1) Fas ligand, Fas, and caspase 4, the initiators and mediators of death receptor dependent apoptosis; 2) death-associated protein kinase 2, a multifunctional pro-apoptotic protein kinase; 3) P53-like proteins, p73, p63; 4) CARD10 and CARD14, positive regulators of apoptosis and NF-kappaB activation; and 5) Cyclin-dependent kinase inhibitors, p16 and p18. In a subset of these selected genes, the expression data is also confirmed with western blot analyses. We have also demonstrated that the transcript and protein levels of a metastasis associated laminin receptor 1 are significantly elevated in myeloma cell lines and patient samples compared to normal cells. RNAi mediated inhibition of laminin receptor 1, abrogated EGCG-induced apoptosis in myeloma cells thus indicating that the profound anti-cancer effect of this compound is probably mediated through this receptor. The selective expression of this receptor explains the selective activity of EGCG in multiple myeloma cells without adversely affecting normal cells. Taken together these data confirm significant and selective anti-cancer activity of EGCG, a natural product, in MM and provides the basis for its clinical evaluation.
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Cottini, Francesca, Teru Hideshima, Martin Sattler, Federico Caligaris-Cappio, Kenneth C. Anderson, and Giovanni Tonon. "The Role of the ABL1/YAP1/P73 Axis in Prevention of DNA Damage-Mediated Apoptosis in Multiple Myeloma." Blood 120, no. 21 (2012): 725. http://dx.doi.org/10.1182/blood.v120.21.725.725.
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Abstract Abstract 725 Background: Genome integrity plays a crucial role in the development of normal plasma cells to eliminate aberrant ones. Multiple myeloma (MM) is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. MM cells show constitutive DNA Damage Response (DDR) and activate compensatory mechanisms to prevent DNA-damage mediated apoptosis. Here we define the molecular mechanisms of these protective effects. Methods: A panel of 15 MM cell lines was used. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and subjected to Ficoll-Paque density sedimentation to get mononuclear cells (MNCs). Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery system was used for expression and knock-down of YAP1 in KMS-18, KMS-20, MM.1S and UTMC-2 MM cell lines. The biologic impact of YAP1 phenotype was evaluated using cell growth, viability and apoptosis assays. Results: We confirmed that a wide range of MM patient cells and MM cell lines have markers of constitutive DDR, including phosphorylation of H2A.X, ATM, ATR, Chk2 and Chk1, assessed by western blot analysis and immunofluorescence. However, these MM cells do not show basal level of apoptosis. Specifically, cleaved forms of caspase 3 and PARP are lacking in non-treated cells, and the absence of co-expression of cleaved caspase 3 with phospho-H2A.X by immunofluorescence confirms that phospho-H2A.X positive cells are viable cells. Since DDR is present in both p53-wild-type (wt) and p53-mutated cell lines, we examined whether ABL1/YAP1/p73 axis represents an alternative and crucial pathway to avoid DNA-damage mediated apoptosis. Indeed, ABL1 is up-regulated and predominantly localizes in the nucleus, a potential apoptotic stimulus, in MM cells, as assessed by western blot and immunofluorescence. To define if ABL1 nuclear localization was triggered by DDR, we treated MM cells with a specific ATM inhibitor (Ku55933) and a DNA damaging agent, doxorubicin. Inhibition of DDR in both p53 wt cell lines (MM.1S and H929) and p53 mutated cell lines (UTMC-2, JJN-3 and KMS-20) causes ABL1 cytoplasmic retention, while doxorubicin increases ABL1 nuclear translocation. Co-treatment with doxorubicin and ABL1 inhibitor STI-571 rescues MM cells from doxorubicin-mediated cell death. In particular, apoptotic cells decrease from 47.2% to 21.5% in U266, from 55.3% to 12.4% in MM.1S, and from 57.9% to 19.1% in UTMC-2 cells in response to combination treatment. To delineate the molecular mechanisms whereby MM cells repress ABL1 pro-apoptotic function, we focused on YAP1, a downstream target of the Hippo pathway involved in ABL1 cascade. We explored a large dataset of aCGH data on MM patients and discovered that YAP1 genomic locus (chr. 11q22) is deleted, associated with BIRC2 and BIRC3 in 11% of patients. This genetic abnormality was also found in KMS-18 and KMS-20 cell lines. Although YAP1 expression was normal in peripheral blood MNCs (PBMCs), its expression was decreased in the majority of patient MM cells and MM cell lines regardless of the presence of focal deletion. Importantly, low expression of YAP1 is associated with poor prognosis of MM patients. To further delineate the biologic significance of YAP1 in MM cells, we re-expressed pLENTI4-YAP1-EGFP in MM cell lines with either YAP1 deletion (KMS-18, KMS-20) or YAP1 low expression (MM.1S). We also silenced YAP1 using two different specific shRNAs in UTMC-2 MM cell line. As expected, YAP1 re-expression reduces cellular growth and increases apoptosis in all cell lines tested (25.7%, 37.1% and 32.3% apoptotic KMS-20, KMS-18 and MM.1S cells, respectively), condition that was further enhanced by doxorubicin treatment. Previous studies have shown that P73 is expressed in MM even though at low levels and we here show that they inversely correlate with YAP1 protein expression. Importantly, YAP1 re-expression increases p73 stability and promotes transcription of p73-target genes including BAX, PUMA and p21. In contrast, UTMC-2-YAP1−/− cells show improved survival with lower levels of basal apoptosis and higher resistance to treatment with bortezomib or doxorubicin. Conclusion: YAP1 mediates a strong apoptotic signal for MM cells. Thus, activation and/or overexpression of YAP1 represent a novel therapeutic strategy to improve outcome of patients with MM. Disclosures: Anderson: Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder and Scientific Founder, Scientific Founder and Scientific Founder Other.
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Raab, Marc S., Iris Breitkreutz, Giovanni Tonon, et al. "Targeting PKC: A Novel Role for Beta-catenin in ER Stress and Apoptotic Signaling." Blood 112, no. 11 (2008): 2763. http://dx.doi.org/10.1182/blood.v112.11.2763.2763.
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Abstract Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising pre-clinical activity in a wide range of tumor cells. In this study, we further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of b-catenin in regulating growth and survival of tumor cells. Inhibition of PKC leads to rapid accumulation of b-catenin by preventing the phosphorylation required for its proteasomal degradation. Specifically, b-catenin was dephosphorylated at Ser33,37,41 and accumulated in a dose- and time-dependent manner in all cell lines tested (including primary MM cells and 10 MM cell lines, 3 colon cancer, HeLa, as well as HEK 293 cells). Microarray analysis and siRNA-mediated gene silencing in MM cells revealed that accumulated b-catenin activates early ER stress signaling via eIF2a, CHOP and p21, leading to immediate inhibition of proliferation. Conversely, knock-down of components of the ER stress response pathway by siRNA (i.e., CHOP) abrogated the inhibitory effect of enzastaurin on MM cell proliferation. Importantly, accumulated b-catenin also contributes to enzastaurin-induced cell death, since inhibition of b-catenin by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a b-catenin -dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; b-catenin induction by enzastaurin led to p73 (but not p53) activation, which was also abrogated by b-catenin -specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of b-catenin in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. In summary, our data reveal a novel role of b-catenin in ER stress-mediated growth inhibition and a new pro-apoptotic mechanism triggered by b-catenin upon inhibition of PKC isoforms, and further demonstrate that p73 represents a novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies including MM.
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Raab, Marc S., Iris Breitkreutz, Giovanni Tonon, et al. "Targeting PKC: a novel role for beta-catenin in ER stress and apoptotic signaling." Blood 113, no. 7 (2009): 1513–21. http://dx.doi.org/10.1182/blood-2008-05-157040.
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Abstract Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising preclinical activity in a wide range of tumor cells. We further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of β-catenin in regulating growth and survival of tumor cells. Specifically, inhibition of PKC leads to rapid accumulation of β-catenin by preventing the phosphorylation required for its proteasomal degradation. Microarray analysis and small-interfering RNA (siRNA)–mediated gene silencing in MM cells revealed that accumulated β-catenin activates early endoplasmic reticulum stress signaling via eIF2α, C/EBP-homologous protein (CHOP), and p21, leading to immediate growth inhibition. Furthermore, accumulated β-catenin contributes to enzastaurin-induced cell death. Sequential knockdown of β-catenin, c-Jun, and p73, as well as overexpression of β-catenin or p73 confirmed that accumulated β-catenin triggers c-Jun–dependent induction of p73, thereby conferring MM cell apoptosis. Our data reveal a novel role of β-catenin in endoplasmic reticulum (ER) stress-mediated growth inhibition and a new proapoptotic mechanism triggered by β-catenin on inhibition of PKC isoforms. Moreover, we identify p73 as a potential novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies, including MM.
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Faruq, Faruq, Davidson Zhao, Jian Wu, Jonahunnatha Nesson George William, Min Zhang, and Hong Chang. "Downregulation of MDM2 Leads to Anti-Proliferative Effects through Activation of p53-Associated Pathway Mediated By Both Dual Inhibitor MX69 and Mir-548c-3p in Multiple Myeloma." Blood 134, Supplement_1 (2019): 4419. http://dx.doi.org/10.1182/blood-2019-129430.
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Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. MM remains an incurable disease with a high rate of relapse and development of drug resistance. Mouse double minute 2 homolog (MDM2) has been characterized as an oncogene that is associated with cancer development and radio/chemotherapy resistance in cancer. However, the mechanism(s) underlying MDM2 overexpression and its association with drug resistance in MM have not been fully explored. In addition, the effect of the newly discovered dual inhibitor MX69, which targets MDM2 and XIAP, is unknown in MM. To assess the effects of MDM2 overexpression in MM patients, we performed GEO data (GSE6477, GSE31161) differential expression analysis on bone marrow samples from three patient groups- normal donors, newly diagnosed MM and relapsed MM. We found that relapsed MM patients expressed high MDM2 compared to newly diagnosed MM patients, and both groups expressed higher MDM2 compared to normal donors (p<0.001). Furthermore, immunoblotting and qRT-PCR showed upregulated MDM2 expression in drug resistant MM cell lines (MM1R, 8226R5) as compared to their parental drug sensitive cells (MM1S, 8226S). XIAP is an important inhibitor of apoptotic proteins caspases 3, 7 and 9. Elevated expression of XIAP is involved in chemotherapy resistance in cancer. To determine whether MDM2 and XIAP downregulation by dual inhibitor MX69 could induce apoptosis in MM cells, we treated two drug resistant MM cell lines with MX69. Cell viability assay showed that MX69 reduced viability of MM cells in a time- and dose-dependent manner. Since MDM2 is an antagonist of tumor suppressor p53, we measured the expression of p53 following MX69 treatment and found that MX69-mediated downregulation of MDM2 significantly enhanced p53, PUMA and p21 expression in MM cell lines harboring wild-type p53. In MM p53 null cells, we found that p53 family pro-apoptotic factors p63 and p73 were increased upon MX69 treatment. To investigate the downstream mechanism of MDM2 targeting in MM drug resistance, we knocked down MDM2 in MM cells via siRNA-mediated silencing. Cell viability assay indicated that siMDM2 reduced cell proliferation compared to negative control (P=0.009). Following MDM2 silencing, XIAP expression was decreased and p53 expression was increased in p53 positive MM cell lines, whereas p63, p73 and p21 were increased in both p53 positive and p53 null MM cells. Furthermore, combined treatment of MX69 with dexamethasone (Dex), doxorubicin (Dox) or lenalidomide (Len) displayed synergistic toxic effects (combination index ranging from 0.437 to 0.8) in drug resistant MM cell lines. MX69 alone or in combination with Dex, Dox and Len significantly increased apoptosis and decreased proliferation of MM cells. To explore whether miRNAs play a role in regulating MDM2 overexpression and MM drug resistance, several miRNA target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Four miRNAs (miR-890, miR-211-5p, miR-429 and miR-548c-3p) were common among GSE49261, GSE39571, GSE16558 and GSE17498 GEO datasets. These four miRNAs were downregulated in MM patients and negatively correlated with MDM2. Furthermore, analyzing prediction score revealed that miR-548c-3p had the highest binding score with MDM2 3'UTR. Moreover, MiR-548c-3p showed a significant downregulation in drug resistant cells in comparison with their parental sensitive cells (p<0.0001). To determine whether MDM2 expression is selectively regulated by miR-548c-3p, we transfected two MM drug resistant cells with miR-548c-3p mimics and found that MDM2 mRNA and protein levels were suppressed and the cell viability was significantly reduced compared to the scrambled controls. In conclusion, our results indicate that MDM2 overexpression is associated with disease progression and drug resistance in MM. Treatment of drug resistant MM cells with MDM2/XIAP dual inhibitor MX69 sensitizes the cells to anti-myeloma drugs and induces apoptosis. Our finding of the regulatory interaction between miR-548c-3p and MDM2 reveals key insights into the dysregulation of MDM2 in MM and provides a new therapeutic approach to target MDM2 by restoration of miR-548c-3p. Disclosures No relevant conflicts of interest to declare.
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Saha, Manujendra N., Hua Jiang, Yijun Yang, Donna Reece, and Hong Chang. "PRIMA-1Met/APR-246 Displays High Antitumor Activity in Multiple Myeloma By Induction of p73 and Noxa." Molecular Cancer Therapeutics 12, no. 11 (2013): 2331–41. http://dx.doi.org/10.1158/1535-7163.mct-12-1166.
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Abdi, Jahangir, Manujendra N. Saha, Mona Sobhani, Qian Shi, and Hong Chang. "Bone Marrow Stromal Cells Induce Bortezomib Resistance Partly through a Transcriptome Modulation in Multiple Myeloma Cells." Blood 124, no. 21 (2014): 3392. http://dx.doi.org/10.1182/blood.v124.21.3392.3392.
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Abstract INTRODUCTION It has been suggested that environment-mediated drug resistance (EMDR) might account for the intrinsic (de novo) resistance to therapy in some myeloma patients developing in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Understanding the mechanisms exploited by bone marroe stromal cells (BMSCs) to generate such resistance has long been sought. Furthermore, knowledge of the role that changes in expression of various genes could play is scanty. In this study we explored how BMSCs could induce resistance to bortezomib and whether it would change the expression of an array of genes in MM cells in the presence of bortezomib. METHODS Using patient-derived BMSCs and a normal immortalized human BMSC line (HS-5), we set up a model in which several human myeloma cell lines (HMCLs), RPMI-8226, U266, MM1.S, OPM2 and H929, were co-cultured with stromal cells in the presence or absence of bortezomib. Drug cytotoxicity in HS-5 context was performed using XTT assay. Transwell system, Paraformaldehyde (PFA) fixation of BMSCs and treatment of BMSCs with Brefeldin-A (BFA, an inhibitor of intracellular protein transport) were used as controls to delineate the direct or indirect roles of BMSCs. Percentage of apoptotic cells was determined with anti-CD138/APC, annexin-V/FITC and PI staining in FACS. For gene expression array, co-cultures were applied to magnetic cell separation (EasySep, Stem Cell Technologies), total RNA was isolated from MM cell pellets and synthesized cDNAs were applied to real time PCR gene expression arrays. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in co-cultures compared to MM cell monocultures in the presence of bortezomib using the 2-ΔΔCtalgorithm. RESULTS In the XTT cytotoxicity assay, all tested HMCLs displayed a higher level of viability in the presence of stroma than in stroma-free conditions (average bortezomib IC50s: stroma 22nM vs no stroma 8.53nM, p<0.02). Moreover, FACS analysis for all HMCLs showed that the percentage of bortezomib-induced apoptosis in stroma context was significantly lower than that in stroma-free conditions (p<0.05). Notably, in BFA and PFA treatment settings the level of bortezomib-induced apoptosis was lower (but not significant) than that in Transwell system suggesting that both soluble factors secreted by BMSCs and direct adhesion contribute to stroma-induced resistance to bortezomib. Gene expression analyses in real time PCR showed that the induced resistance was associated with downregulation of some genes involved in cell cycle and proliferation control (CDKN2A, CDC6, CDK2, EGR1, PTEN), downregulation of some genes involved in p53 signaling pathway (PUMA, P73, P53, MDM2, NOXA, BAX) and downregulation of some genes involved in apoptosis (CASP3, PARP). CONCLUSION Our findings suggest that BMSCs induce resistance against cytotoxic and apoptotic effects of bortezomib in MM cells. To this end, in our tested HMCLs, the gene expression patterns may imply involvement of multiple pathways in controlling stroma-induced resistance to bortezomib. Although gene expression results were verified real time PCR readouts, changes in genes may not always correlate with protein changes. Hence, our study has given us the rationale to proceed with more in depth exploration as to whether BMSCs may render MM cells resistant to bortezomib by negative modulation of p53 signaling pathway controlling apoptosis and cell cycle. Disclosures No relevant conflicts of interest to declare.
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Nagoshi, Hisao, Tomohiko Taki, Kazuhiro Nishida, et al. "Identification of the Novel Chimeric Gene, PVT1-WWOX, in Multiple Myeloma with 8q24 Abnormality." Blood 120, no. 21 (2012): 1823. http://dx.doi.org/10.1182/blood.v120.21.1823.1823.
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Abstract Abstract 1823 Genetic abnormalities play a crucial role in the pathogenesis of various malignancies, including multiple myeloma (MM). Secondary cytogenetic abnormalities implicated in MM progression include 8q24 rearrangements, gain of the long arm of chromosome 1 (1q+), and loss of the short arm of chromosome 17 (17p-). The 8q24 rearrangements, including MYC and PVT1, have been identified by conventional cytogenetic analysis in 3.5–5.0% of MM patients and by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) in 9.5–20%. 8q24 rearrangements are frequently associated with advanced disease in MM patients and MM cell lines. Ig chromosomal translocations, such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% of MMs with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14 and 16q22, in which no candidate genes have been delineated so far, have also been identified as translocation partners. MYC has long been a possible candidate target gene for 8q24 rearrangements; however, many of the breakpoints within 8q24 have been assigned to various regions that encompassed more than 2 Mb centromeric or telomeric to MYC. We have previously found frequent PVT1 rearrangements in MM. PVT1 rearrangements were detected in 7 of 12 patients (58.3%) and in 5 of 8 cell lines (62.5%) with 8q24 abnormalities. A combination of SKY, FISH, and oligonucleotide array identified several partner loci of PVT1 rearrangements, such as 4p16, 4q13, 13q13, 14q32 and 16q23–24, and identified a chimeric gene, PVT1 - NBEA, resulting in high expression of abnormal NBEA in a cell line with t(8;13)(q24;q13), AMU-MM1, suggesting PVT1 rearrangements play significant roles in myelomagenesis. In this study, we analyzed RPMI8226 cell line in detail to identify other partner genes of PVT1 in these partner loci. SKY analysis revealed a complex karyotype including der(16)t(16;22)ins(16;8)(q23;q24) in this cell line. Oligonucleotide array analysis clearly demonstrated that the copy number change at 8q24 occurred within intron 1 of PVT1, and at 16q23, the copy number change occurred within intron 8 of WWOX, indicating that the translocation breakpoints of 8q24 and 16q23 were within intron 1 of PVT1 and intron 8 of WWOX, respectively. Based on these results, RT-PCR analysis was performed to detect chimeric products and direct sequencing of this product revealed the fusion of 5'-PVT1 exon 1 with WWOX exon 9-3'. The expression level of WWOX exon 9 was higher than WWOX exon 8–9 detected by semi-quantitative RT-PCR in RPMI8226, suggesting that high expression of WWOX derived from PVT1 - WWOX chimeric transcript, like PVT1 - NBEA. WWOX is generally considered to be a candidate tumor suppressor gene, and known to have a proapoptotic effect by participating in the tumor necrosis factor (TNF) apoptotic pathway and via direct physical interaction with p53 and its homolog p73. However, immunohistochemistry revealed that WWOX protein level were rather elevated in gastric and breast carcinoma. Therefore, WWOX seemed not to act as tumor suppressor gene simply. Although both NBEA and WWOX are located at common fragile site, usually contributing to gene inactivation, FRA13A and FRA16D respectively, these genes highly express via fusion to PVT1. These findings indicate that PVT1 rearrangements play significant roles in myelomagenesis via translocation and fusion to cancer-related genes. Disclosures: No relevant conflicts of interest to declare.
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Doudican, Nicole A., Shireen Vali, Shweta Kapoor, et al. "Predictive Simulation Based Design and Validation Of Repurposed Novel Therapeutics With Multi-Target Mechanisms For Multiple Myeloma." Blood 122, no. 21 (2013): 3859. http://dx.doi.org/10.1182/blood.v122.21.3859.3859.
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Abstract Introduction Development of resistance to single agent therapy is a significant clinical obstacle in the treatment of multiple myeloma (MM). Genetic mutations and the bone marrow micro-environment are major determinants of MM resistance mechanisms. Given the complexity of MM, the need for combinatorial therapeutic regimens targeting multiple biological mechanisms of action is pressing. Repurposing has the advantage of using drugs with known clinical history. Methodology We used a predictive simulation-based approach that models MM disease physiology in plasma cells by integrating and aggregating signaling and metabolic networks across all disease phenotypes. We tested the efficacy of over 50 repurposed molecularly targeted agents both individually and in combination across simulation avatars of the MM cell lines OPM2 and U266. OPM2 harbors mutations in KRAS, CDKN2A/2C, PTEN, RASSF1A and P53, whereas U266’s mutational components include BRAF, CDKN2A, P53, P73, RASSF1A and RB1. These cell lines were used as models because they possess mutations in genes classically known to be involved in myeloma. The predicted activity of novel combinations of existing drug agents was validated in vitro using standard molecular assays. MTT and flow cytometry were used to assess cellular proliferation. Western blotting was used to monitor the combinatorial effects on apoptotic and cellular signaling pathways. Synergy was analyzed using isobologram plots and the Bliss independence model. Results Through simulation modeling, we identified two novel therapeutic regimens for MM using repurposed drugs: (1) AT101 (Bcl2 antagonist) and tesaglitazar (PPAR α/γ agonist) and (2) Ursolic acid (UA, inhibitor of NFκβ) and SP600125 (pan-JNK inhibitor). Simulation predictions showed that combining the IC30 concentrations with respect to viability of AT101 and tesaglitazar reduced proliferation by 40% and viability by 50%. Similarly simulation predictions showed that the combination of the IC30 concentrations of UA and SP600125 reduced proliferation by 50% and viability by 40%. Corroborating our predictive simulation assays, 10 µM tesaglitazar and 2 µM AT101 caused minimal growth inhibition as single agents in OPM2 and U266 MM cell lines. Growth inhibition in these cell lines is synergistically enhanced when the drugs are used in combination, reducing cellular viability by 88% and 77% in OPM2 and U266 cells, respectively. Similarly, proliferation was reduced by 34% with 7.5 μM UA and 25% with 10 μM SP600125 in OPM2 cells. When used in combination, cellular proliferation was synergistically reduced by 64%. In addition, isobologram analysis predicted synergy of lowered doses of the drugs in combination. Both combinations synergistically inhibited proliferation and induced apoptosis as evidenced by an increase in the percentage sub-G1 phase cells and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Conclusions These results highlight and validate the use of our predictive simulation approach to design therapeutic regimens with novel biological mechanisms using drugs with known chemistries. This allows for design of personalized treatments for patients using their tumor genomic signature beyond the “one-gene, one-drug” paradigm. The reuse of existing drugs with clinical data facilitates a rapid translational path into clinic and avoids the uncertainties associated with new chemistry. The corroboration of these results with patient derived cell lines will be pursued and discussed. Disclosures: No relevant conflicts of interest to declare.
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Teoh, Phaik Ju, Chonglei Bi, Chirackal Sintosebastian, Liang Seah Tay, Rafael Fonseca, and Wee Joo Chng. "PRIMA-1 targets the vulnerability of multiple myeloma of deregulated protein homeostasis through the perturbation of ER stress via p73 demethylation." Oncotarget 7, no. 38 (2016): 61806–19. http://dx.doi.org/10.18632/oncotarget.11241.
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Robak, Pawel, Anna Linke, Barbara Cebula, Lorenzo M. Leoni, Tadeusz Robak, and Piotr Smolewski. "Cytotoxic Effect of R-Etodolac (SDX-101) in Combination with Purine Analogues or Monoclonal Antibodies on Ex-Vivo B-Cell Chronic Lymphocytic Leukemia Cells." Blood 106, no. 11 (2005): 2122. http://dx.doi.org/10.1182/blood.v106.11.2122.2122.
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Abstract Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil. Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX). Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic (<0.8) interactions. Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p<0.03, vs. single drugs alone). The SDX-101+2-CdA combination reduced viability by 27.8% (p<0.006 vs. single drugs), SDX-101+RTX reduced viability by 22.3% (p<0.01 vs. single drugs), and that of SDX-101+ALT combination reduced it by 18.7% (n.s.). The CIs for those combinations were 0.89, 1.17, 0.95, and 1.25, respectively. Apoptosis analysis by sub-G1 fraction and caspase-3 activity confirmed the viability results for each tested combination. Analysis of apoptosis-regulating proteins showed, that SDX-101 combined with RTX or 2-CdA distinctly up-regulated Bax (p<0.02, vs. single drugs), whereas other combinations did not significantly increase its expression. The expression of p73 protein showed 2–4 fold increase after treatment with SDX-101 alone, and all combinations further up-regulated p73, especially that with RTX (up to 6-fold increase in p73 expression). Interestingly, although SDX-101 (400 μM at 24 hr) increased protein expression of Mcl-1, SDX-101 combination with RTX, 2-CdA or FA caused a 2-3-fold decrease of its expression. Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.
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Liao, Chengcheng, Srikanth Talluri, Subodh Kumar, et al. "Base Excision Repair and Homologous Recombination Pathway Intermediates Drive Genomic Instability and Evolution in Myeloma." Blood 136, Supplement 1 (2020): 27–28. http://dx.doi.org/10.1182/blood-2020-141042.
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Multiple myeloma (MM) cells demonstrate significant genomic instability with acquisition of new genomic events over time. In an effort to decipher the pathways utilized by MM cells to genomically evolve and to acquire proliferative advantage as well as develop drug resistance, we have investigated role of various pathway intermediates. Based on our prior results showing elevated homologous recombination (HR) contributing to genomic instability and development of drug resistance, we have further studied RAD51, the key HR gene. Also based on the dysfunctional base excision repair (BER) that can increase DNA damage and dysregulate genome stability, we have investigated apurinic/apyrimidinic endonuclease (APEX)1/2 in MM. We first observed that both APEX1 and APEX2 interact with multiple HR genes in MM. APEX1 interacts with P73, a known transcriptional regulator of RAD51 at RAD51 promoter, and contributes to its transcriptional upregulation and increase in HR activity. To further extend these observations, we conducted sequential chromatin immunoprecipitation (ChIP-reChIP) assays, using MM.1S cells. For the first ChIP, anti-APEX1 and for the reChIP anti-p73 antibody was used. DNA recovered was used for real-time Q-PCR assays, using promoter-specific primers. Colocalization of APEX1 and p73 was thus confirmed on the promoters of three HR related genes (RAD51, BRCA1 and BLM), whereas interaction was not observed for CDK2 promoter. These data suggested that APEX1 may be involved in the overall regulation of HR machinery. To investigate the role of APEX1 in genomewide transcriptional regulation in the context of HR, we cultured MM (H929 and MM.1S) cells in the presence or absence of camptothecin (CPT; a topoisomerase I inhibitor that induces DSBs which are mainly repaired by HR), and conducted ChIP assays using anti-APEX1 antibody followed by DNA sequencing. We have confirmed the interaction of APEX1 with RAD51 promoter and the impact of CPT, using Q-PCR and observe that the occupancy of RAD51 promoter by APEX1 increases following treatment with CPT. The sequencing data to be presented will identify genomic regions occupied by APEX1 under spontaneous as well as modulated HR conditions. We have further confirmed interrelationship between these two key pathway intermediates in MM. We observe that overexpression of APEX1 increases abasic sites in MM as well as another cancer (esophageal adenocarcinoma) cells, while RAD51-knockdown in these cells prevents the increase in abasic sites by APEX1. Similarly, APEX1-overexpression increased HR activity which was prevented by RAD51 inhibition, indicating that APEX-induced RAD51 contributes to dysregulation of both the HR and base excision repair.We also demonstrated thatAPEX-overexpression in normal hematopoietic cells increased HR and genomic instability (as assessed by micronuclei), which was reversed by RAD51 inhibitor. The treatment of control plasmid transfected CD138+ normal plasma cells with RAD51 inhibitor reduced micronuclei by 53.5%. Importantly, APEX1-overexpression in these cells increased micronuclei by 34.7%, whereas treatment of these cells with RAD51 inhibitor completely reversed this increase. Similarly, the treatment of bone marrow/stromal (HS5) cells with RAD51 inhibitor reduced micronuclei in APEX1-overexpressing cells by 55%. This unique functional interaction between APEX nuclease and RAD51 also provides rationale for combining APEX inhibitors with chemotherapeutic agents. Consistently, we observe thatAPEX inhibition synergistically increases the efficacy of chemotherapeutic agents in MM cells. We also investigated the impact of simultaneous suppression of APEX1 and RAD51. Cancer cells (FLO-1) were transduced with control shRNAs or those targeting APEX1, RAD51 or both, and following puromycin selection evaluated for HR and evidence of apoptosis. Combined RAD51/APEX1 knockdown led to a significantly increased apoptosis (P=0.0003), relative to RAD51 or APEX1 inhibition alone. In summary, we report an interesting interaction/crosstalk between 2 major DNA repair pathway intermediates, APEX1 and RAD51 recombinase, driving genomic evolution in MM. These results provide insight into processes driving genomic progression and suggest possible avenues to control evolution of the disease and its clinical consequences in MM. Disclosures Munshi: Janssen: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.
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Lunghi, Paolo, Laura Mazzera, Attilio Corradi, et al. "Arsenic Trioxide (ATO) Interacts Synergistically with MEK Inhibitors to Induce Apoptosis in STI571-Resistant Bcr-Abl Mutants." Blood 114, no. 22 (2009): 2177. http://dx.doi.org/10.1182/blood.v114.22.2177.2177.
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Abstract Abstract 2177 Poster Board II-154 Resistance toward Imatinib and other Bcr-Abl tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. Previous preclinical studies reported that the MEK inhibitors PD184352 or PD0325901 (Pfizer), strikingly enhances ATO-mediated apoptosis in Acute Myelogenous Leukemia and in Multiple myeloma. The aim of this study was to investigate whether the combined treatment with PD184352 (PD) and ATO has cytotoxic effects on murine Ba/F3 cells expressing wild-type (wt) or various imatinib-resistant mutant forms of Bcr-Abl, including T315I. We first analyzed the pharmacologic interactions between PD and ATO using a fixed-ratio experimental design in Bcr-Abl Ba/F3p210wt, Ba/F3p210T315I and Ba/F3p210Y253F cell lines and found that the combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in all cell lines tested (Chou-Talalay method): the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in PD plus ATO treated cells were 0.72± 0.19, 0.61± 0.04 and 0.69± 0.09 in BCR-ABL Ba/F3p210wt, Ba/F3p210T315I, and Ba/F3p210Y253F respectively. Synergistic interaction between ATO and PD0325901, a derivative of PD184352, was demonstrated in all tested cell lines. In order to investigate the molecular effectors involved in PD/ATO-induced apoptosis we first evaluated its effects on Bcr-Abl protein expression and CrkL phosphorylation, a well-known downstream target of Bcr-Abl. Immunoblotting analyses demonstrated that treatment for 24 to 48 hours of Ba/F3p210wt and Ba/F3p210T315I cells with PD (1μM) or ATO (1μM) alone or in combination had no effects on levels of total Bcr-Abl or phospho—CrkL thereby indicating that the combination PD/ATO does not act via Bcr-Abl oncogenic signaling. Therefore, we studied whether p53 and the p53-related gene p73 are molecular targets of the combined treatment in Ba/F3p210wt and Ba/F3p210T315I cell lines. We found that monotreatment with neither PD nor ATO 1 μM (or their combination) was able to induce p53 accumulation, whereas the combination PD/ATO promoted the accumulation of the proapoptotic and antiproliferative TA-p73α protein and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔN-p73α in both cell lines. Consistent with these results, we found that PD greatly enhanced the ATO-induced Puma expression, mitochondrial depolarization, caspase-3 activation, and apoptosis in Ba/F3p210wt and Ba/F3p210T315I cells and functional knock-out of p73 gene expression by small interfering (si)RNAs significantly reduced (P< .05 Dunnett test) the PD/ATO induced mitochondrial depolarization. To determine whether the PD plus ATO efficacy observed in vitro for BaF3 cells expressing mutant forms of Bcr-Abl was recapitulated in vivo, we studied PD/ATO combination in a mouse model of Imatinib-resistant, Bcr-Abl—dependent disease. Severe combined immunodeficient mice were injected intravenously with Ba/F3 cells expressing Bcr-Abl-T315I isoform. Mice with Bcr-Abl-T315I—induced leukemia were treated with the MEK inhibitor PD0325901 (10 mg/kg; orally) plus ATO (3.75 mg/kg; intraperitoneally) or Imatinib (50 mg/kg, twice daily; intraperitoneally) or vehicle for three weeks. Untreated or Imatinib-treated mice harboring the T315I isoform developed aggressive disease, with massive liver and splenic infiltration, typically resulting in death in 32 days. However, mice harboring the T315I isoform showed significantly prolonged survival when treated with PD/ATO (43 days, P=.001, Kaplan-Meier method and compared using the log-rank test). Moreover, histopathological analysis of 20 days Imatinib-treated mice revealed infiltration of the liver and spleen. In contrast, histopathological analysis of organs from PD/ATO-treated mice demonstrated normal tissue architecture. Consistent with these results immumoblottig analysis of the lysates from livers and spleens revealed a marked expression of Bcr-Abl protein in mice treated with Imatinib or vehicle. Our preclinical in vitro and in vivo studies suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl-positive leukemias, including those harboring the T315I mutation. Disclosures: No relevant conflicts of interest to declare.
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Terragna, Carolina, Marina Martello, Sandra Durante, et al. "High Number of Copy Number Alterations and Over-Expression of Genes Involved in the Response Mechanisms to Genotoxic Stress Both Characterize Newly Diagnosed Multiple Myeloma (MM) Patients Carrying Amplified MDM4 and/or Deleted p53,." Blood 118, no. 21 (2011): 3935. http://dx.doi.org/10.1182/blood.v118.21.3935.3935.
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Abstract Abstract 3935 Background The p53 tumor suppressor pathway is tightly kept in check, or completely silenced in cancer cells. A potent inhibitor of p53 is represented by MDM4, which is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. TP53 mutations are rare in newly diagnosed MM, while occur more frequently as late event in the course of the disease and are related to survival. Recently, the adverse prognostic impact of chr. 1q amplification, described in almost 40% of newly diagnosed MM pts, has been reported. The minimal amplified region on chr. 1q harbors MDM4. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) incorporated into autologous stem cell transplantation, in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. Methods Thirty eight pts treated with VTD incorporated into autologous stem cell transplantation were analysed by means of gene expression profile (Affymetrix U133 Plus2.0 array) and unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array). Both GEP and SNP arrays experiments were performed on highly purified CD138+ bone marrow plasma cells obtained at diagnosis from each pts. The presence of CNAs in chr.1 and 17 was evaluated to identify pts carrying amp(1q) and del(17p). Results Eighteen out of 38 pts (42%) carried a minimal amplification region of 1,1 Mb on chr.1q, which harbors MDM4. Five out of 38 pts (13%) carried a minimal deletion region of 482 Kb on chr.17, which harbors TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 18 pts) or the absence of both these abnormalities (group B, 20 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≥ 3 bone lesions in pts carrying amplified MDM4 and/or deleted p53. The rate of best complete or near complete response was 89% in group A and 75% in group B. With a median follow-up of 36 months, the risk of relapse or progression was 50% for pts in group A and 25% for those in group B. The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03); indeed, the presence of amplified MDM4 and/or deleted p53 was significantly associated with a list of 95 CNAs (clustered on chr. 1, 2, 6, 8, 11, 13, 16 and 18), which included del16q (with a minimal area of deletion including WWOX), observed in 39% vs. 5% cases from group A and B, respectively (p<0.05) and chr.8 aberrations (with amplifications and or deletions, both including TRPS1) observed in 61% vs. 15% cases from group A and B, respectively (p<0.05). A comparison of expression profiles of patients carrying or not amplified MDM4 and/or deleted p53 confirmed the over-expression of MDM4 in the former group of pts and highlighted in these pts an overall activation of genes involved in the response mechanisms to genotoxic stress: indeed a significant over-expression of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) was observed in pts with amplified MDM4 and/or deleted p53 (one-way ANOVA plus multiple-test correction with FDR <0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. Conclusions Pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The amplification of MDM4 locus and the over-expression of YY1 might contribute to maintain p53 in an OFF state by an indirect mechanism. Additional data on the role of both direct and indirect control of p53 pathway on VTD-treated MM pts prognosis, extended to an higher number of pts, will be presented during the meeting. Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Disclosures: Cavo: Genzyme: Honoraria.
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Gaken, Joop, Louisa Pericleous, Farzin Farzaneh, Ghulam J. Mufti, and Mahvash Tavassoli. "TAT-Apoptin Mediated Induction of Apoptosis in Leukaemic Cells." Blood 108, no. 11 (2006): 1900. http://dx.doi.org/10.1182/blood.v108.11.1900.1900.
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Abstract We have studied the specific targeting of leukaemic cells using the Chicken Anaemia Virus (CAV)-derived protein VP3 (Apoptin) linked to the protein transduction domain (PTD) from HIV TAT with the aim of using this strategy for in vitro purging. Apoptin is a 13.6 kDa protein which induces apoptosis specifically in cancer cells whilst leaving normal cells unaffected. Expression of Apoptin in normal cells results in its cytoplasmic localisation. In tumour cells Apoptin resides initially in the cytoplasm and subsequently translocates to the nucleus and induces apoptosis. Apoptin is phosphorylated both in vitro and in vivo in tumour cells but negligibly in normal cells at threonine 108. A gain-of-function point mutation (T108E) results in accumulation of Apoptin in the nucleus and the killing of normal cells, implying that phosphorylation is a key factor of the tumour-specific properties of Apoptin. We have demonstrated that Apoptin induces apoptosis in a variety of human solid tumour cell lines, but not in normal fibroblast and epithelial cells. Apoptin induced apoptosis in HSC3 head and neck cancer cells acts through the mitochondrial pathway and was blocked (>75%) by shRNA against PUMA, a BH3 Only protein which induces Bax and BAK resulting in loss of mitochondrial membrane potential and release of cytochrome C. Furthermore, activation of the p53 family member, p73, substantially increased (5–10 fold for p73 β and γ) sensitivity of Saos2 tumour cells to Apoptin-induced killing. For efficient protein delivery, Apoptin was fused to a TAT PTD and addition of this protein to normal and tumour cells resulted in the selective killing of tumour cells. To increase the stability and solubility of TAT-Apoptin we have fused it to the maltose binding protein (MBP), this modification significantly increases both yield and the solubility of Apoptin while retaining its biological function. Apoptin tumour specific toxicity was assessed in a range of leukaemic and solid tumour cell lines. Addition of MBP-TAT-Apoptin protein to HL60, K562 and Jurkat cells resulted in 50%, 55% and 75% cell death by apoptosis as judged by PARP cleavage, respectively, at day 4 as compared to MBP-TAT control whilst normal B cells, fibroblasts and epithelial cells are unaffected. Fluorescent microscopy demonstrated that MBP-TAT-Apoptin was rapidly internalised in almost 100% of cells within 24hrs in all cell types tested. Direct injection of Apoptin expressing Ad vectors also showed clear regression of established tumours in mice. The cancer specific toxicity of Apoptin has potential value for a range of therapeutic applications such as purging of autologous bone marrow as used for the treatment of multiple myeloma and possibly direct treatment of leukaemias either alone or linked to antibodies for targeting of specific types of leukaemias.
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Laane, Edward, Purushothama Nanjappa, Subodh Kumar, et al. "XRCC5 Plays an Important Role in Homologous Recombination, Genome Stability and Survival of Myeloma Cells." Blood 126, no. 23 (2015): 1218. http://dx.doi.org/10.1182/blood.v126.23.1218.1218.
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Abstract Understanding mechanisms underlying genomic instability is critical in delineating pathogenesis and development of new treatments for prevention and treatment of cancer. We have previously shown that dysregulated homologous recombination (HR) significantly contributes to genomic instability and progression in multiple myeloma (MM). To identify the regulators of HR and genome stability in MM, we conducted a functional shRNA screen and identified XRCC5 (Ku80) as a novel regulator of HR in MM cells. XRCC5 has been known to work as part of DNA ligase IV-XRCC4 complex in the repair of DNA breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events. Evaluation by Western blotting showed that all myeloma cell lines tested (RPMI, MM1S, OPM2, MM1R, U266, ARP, H929) had elevated expression of XRCC5, ranging from 3- to 10-fold elevation relative to average expression in two normal PBMC samples. Expression profiling showed a wide range of XRCC5 expression in myeloma patients, with a subset of patients with very high expression. To investigate the role of XRCC5 in ongoing acquisition of genomic changes, we investigated the association of XRCC5 with genomic instability using two different patient datasets (gse26863, n=246 and IFM 170 pt dataset) in which both the gene expression and genomic copy number information for each patient was available. Copy events were defined as changes observed in ≥ 3 and/or 5 consecutive SNPs. Higher XRCC5 expression significantly correlated with increase in the number of copy number change events in both the 170 dataset (p ≤ 0.005 for amplifications and p = 0.0001 for deletions) as well as in gse26863 dataset (p ≤ 0.004 for amplifications and p ≤ 0.00003 for deletions). To understand mechanisms by which XRCC5 regulates HR in myeloma cells, we investigatedprotein-protein interactions using a custom protein array coated with antibodies against major DNA repair and cell cycle proteins. Array was sequentially incubated with MM cell lysate and HRP-conjugated anti-XRCC5 antibody, and interacting partners were then identified by their address on the array. Investigation in two different cell lines (RPMI and U266) showed that XRCC5 in myeloma interacts with XRCC4 (an NHEJ protein), a panel of major HR regulators (RAD51, RAD52, BRCA2, BRCA1, BARD1, P73, P53, C-ABL) and with components of cell cycle including CDC42, CDK1 (which controls entry from G2 to mitosis), CDK4, CDK6, CHK, CDC36, CDC34, and cyclins E and H. Consistent with these data, knockdown (KD) of XRCC5 was associated with reduced HR as well as reduced proliferation rate followed by a complete cell death over a period of two to three weeks in different experiments, in all 3 myeloma cell lines tested. Moreover, the investigation in U266 cells showed that XRCC5-KD is associated with 3-fold increase in the fraction of cells in G2 phase of cell cycle. Importantly, the elevated expression of XRCC5 was associated with shorter event free (p < 0.013) as well as poor overall survival (p < 0.008) in 170 patient dataset. We evaluted the expression and clinical correlation of XRCC5 in RNA-seq data from 311 newly-diagnosed MM patients and observed that the elevated expression of XRCC5 also correlated with event free survival (p = 0.03). In summary, we report that XRCC5, besides its known role in NHEJ, has important roles in HR, cell cycle and may be involved in the crosstalk among these DNA repair pathways. Elevated XRCC5 expression is associated with dysregulation of HR with consequent impact on survival of myeloma patients. Elevated XRCC5 is, therefore, a promising new target to inhibit/reduce genomic evolution as well as MM cell growth. Disclosures Avet-Loiseau: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.
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Mello, Mariana Rezende Bandeira, Dulcinéia Martins Albuquerque, Krizzia Borges Albanez, et al. "Chromatin Texture and Molecular Features Are Independent Prognostic Factors In AML." Blood 116, no. 21 (2010): 4850. http://dx.doi.org/10.1182/blood.v116.21.4850.4850.
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Abstract Abstract 4850 Introduction: The WHO 2008 classification of acute myeloid leukemia (AML) is based on morphology, cytogenetics and molecular features. Among them, mutations and internal tandem duplications of FLT3 in AML with a normal karyotype have been associated to a poor prognosis. Mutated NPM1 in the absence of FLT3-ITD is associated to a favorable. On the other hand, variables of nuclear chromatin texture have been described as independent risk factors in several malignancies (ALL, melanoma and multiple myeloma). Aim: To compare the influence on overall survival of the chromatin fractal dimension and molecular features in adult patients with AML. Patients and Methods: We analyzed 106 consecutive cases diagnosed at our Institution between 2007 and 2009. Diagnosis was made by bone marrow (BM) cytology and karyotype, and cases were classified by WHO criteria. Genomic DNA was extracted by phenol-chloroform. Genotyping was made with the MegaBACE 1000 equipment and analyzed in the Fragment Profiler v1.2. For detection of the FLT3-TKD mutation, genomic DNA was amplified by PCR followed by restriction analysis. Blasts from the diagnostic BM cytology were digitalized, segmented and nuclear morphometric variables were examined. Their influence on overall survival was analyzed in the Cox model. Results: Median age: 52 years; peripheral blood (PB) leukocytes: 24.0×109/l (0.7-281.3). In the univariate analysis were significant: PB leukocyte count (p=0.005), low-risk karyotype (p=0.002), FLT3 ITD+ (p=0.002), FLT3+NPM1- (p=0.029) and “goodness of fit” (R245) of the chromatin fractal dimension (Minkowski) (p=0.03). Age, fractal dimension and methylation status of p15, p16, p57, p73, ER and MDR1 were not significant. In the multivariate analyses including age, PB leukocytes, R245 and mutations, high leukocyte counts (p=0.03) and low R245 (p=0.01) were independent unfavorable and FLT3-NPM1- (p=0.04) and FLT3-NPM1+ (p=0.02) were favorable prognostic variables. Conclusions: The blast chromatin texture measured by R245 was an independent prognostic factor together with known risk variables in AML. Supported by: FAPESP and CNPq Disclosures: No relevant conflicts of interest to declare.
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Martínez-Baños, Déborah, Beatríz Sánchez-Hernández, Guadalupe Jiménez, Georgina Barrera-Lumbreras, and Olga Barrales-Benítez. "Global methylation and promoter-specific methylation of the P16, SOCS-1, E-cadherin, P73 and SHP-1 genes and their expression in patients with multiple myeloma during active disease and remission." Experimental and Therapeutic Medicine 13, no. 5 (2017): 2442–50. http://dx.doi.org/10.3892/etm.2017.4274.
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Levy, Daphna, Amos M. Cohen, and Jeremy Don. "Dual Role of Pim-2 Depends on the Associated Proteome." Blood 108, no. 11 (2006): 4334. http://dx.doi.org/10.1182/blood.v108.11.4334.4334.
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Abstract Pim-2, a Ser/Thr kinase, is a proto-oncogene originally identified as common proviral insertion sites in T and B cell lymphomas in mice. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, multiple myeloma and prostate cancer. In human non-Hodgkin’s lymphomas and in chronic lymphocytic leukemia Pim-2 is up-regulated and its expression correlates with disease activity. Pim-2 promotes cell survival in response to a wide variety of proliferative signals. Pim-2 promotes cell survival by phosphorylation of Bad and Cot. The goal of this study was to clarify the significance of our new unanticipated aspect of Pim-2′s function, namely, that over-expression of Pim-2 in HeLa cells led to cell cycle arrest at G1 and to increased apoptosis. We found that the G1 arrest was associated with increased (T14/Y15) phosphorylation of CDK2, increased proteosomic degradation of CDC25A, and increased levels of the CDK inhibitor p57. In addition, we found increased E2F-1 levels, which suggested the usage of the E2F-1 dependent apoptotic pathway. Using dominant negative forms of either E2F-1 or p73, which were co-expressed with Pim-2 in HeLa cells, revealed significant rescue of the G1 arrest and apoptotic phenomena. Silencing of Pim-2 in these cells, via siRNA, reversed the G1 arrest and pro-apoptotic effects, and verified the Pim-2 dependent specificity. We conclude that Pim-2 might play a dual role. Our data suggest that under certain environmental circumstances and in various cell types, Pim-2 appears to increase cell survival by abrogating some pro-apoptotic substrates, but under different proteomic associations Pim-2 might favor G1 arrest and apoptosis.
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Paydas, S., B. Sahin, E. Seyrek, and S. Zorludemir. "p53 mutations in multiple myeloma." Molecular Pathology 50, no. 6 (1997): 329. http://dx.doi.org/10.1136/mp.50.6.329-a.
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Teoh, P. J., and W. J. Chng. "p53 Abnormalities and Potential Therapeutic Targeting in Multiple Myeloma." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/717919.
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p53 abnormalities are regarded as an independent prognostic marker in multiple myeloma. Patients harbouring this genetic anomaly are commonly resistant to standard therapy. Thus, various p53 reactivating agents have been developed in order to restore its tumour suppressive abilities. Small molecular compounds, especially, have gained popularity in its efficacy against myeloma cells. For instance, promising preclinical results have steered both nutlin-3 and PRIMA-1 into phase I/II clinical trials. This review summarizes different modes of p53 inactivation in myeloma and highlights the current p53-based therapies that are being utilized in the clinic. Finally, we discuss the potential and promise that the novel small molecules possess for clinical application in improving the treatment outcome of myeloma.
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Sundan, Anders, Torstein B. Ro, Janne Bonhorst, Anders Waage, and Magne Borset. "Bmps(Bone Morphogenetic Proteins) Inhibit Growth in Multiple Myeloma Cells by p53 Activation." Blood 104, no. 11 (2004): 3354. http://dx.doi.org/10.1182/blood.v104.11.3354.3354.
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Abstract Only a few naturally occurring cytokines are able to inhibit myeloma cell growth, and among them BMP-4 is a potent inhibitor of growth as well as an inductor of apoptosis in myeloma cells. To study the mechanism behind BMP-4-induced growth inhibition, we performed gene expression profiling by microarrays in two human myeloma cell lines after BMP-4 stimulation for 4 hours. We found that BMP-4 upregulated several known p53 target genes like p21/Cip1, Bax, cyclin G, Gadd45 and dual specificity phosphatases, suggesting a role for p53 in BMP-mediated growth inhibition of myeloma cells. p53 is activated by several post-translational modifications, including phosphorylation at several serine/threonine residues. We found that BMP-4 treatment lead to rapid phosphorylation of p53 at Serine 15. Furthermore, we were able to co-precipitate phosphorylated Smad-1/5/8 and p53 from nuclear extracts of cells treated with BMP by applying a p53 binding element from the Gadd45 promoter. Because some myeloma cell lines are resistant for BMP-4-induced growth inhibition, we examined whether this BMP-4 resistance was associated with p53 mutations in the BMP-4-resistant cell lines. We sequenced the p53 cDNA from eight myeloma cell lines, and resistant cells all had mutated or deleted p53. Introduction of normal p53 by adenoviral constructs in the p53 mutated cell lines restored BMP-4 responsiveness to some degree. Furthermore, in a myeloma cell line with a temperature-sensitive p53 mutation, the cells were sensitive towards BMP-induced growth inhibition at the permissive temperature but resistant when p53 was inactive. In conclusion, we show that normal p53 activity is involved in BMP-4-induced growth inhibition of multiple myeloma cells.
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Yang, Yonghui, Joseph Gera, and Alan Lichtenstein. "Targeting Deptor in Multiple Myeloma." Blood 120, no. 21 (2012): 575. http://dx.doi.org/10.1182/blood.v120.21.575.575.
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Abstract Abstract 575 DEPTOR is an mTOR binding protein specifically over-expressed in some genetic subtypes of multiple myeloma (MM). DEPTOR binds mTOR in TORC1 and TORC2 complexes and inhibits their kinase activity. As a result, p70 and 4E-BP1 phosphorylation, substrates of TORC1, are decreased. However, TORC1/p70 down-regulation results in marked feedback activation of the PI3K/AKT pathway and AKT becomes activated even though DEPTOR restrains TORC2. Most importantly, DEPTOR knockdown is deleterious to over-expressing MM cell lines, resulting in apoptosis. We hypothesized that compounds preventing DEPTOR-mTOR association would have cytotoxic effects in these cell lines, comparable to DEPTOR knockdown. We, thus, developed a yeast two-hybrid assay where survival of yeast in histidine-depleted media depended upon an association between DEPTOR and mTOR in genetically engineered yeast. After demonstrating a robust yeast two-hybrid protein interaction, yeast were screened against the NCI small inhibitor library (>150,000 compounds) and six compounds were identified that inhibited the interaction. These compounds rapidly increased p70 phosphorylation in MM cell lines in a concentration-dependent fashion, consistent with an inhibition of DEPTOR's inhibitory effect on mTORC1. Co-immunoprecipitation confirmed the compounds inhibited binding of DEPTOR to mTOR without effects on an mTOR-RICTOR interaction. The compounds also significantly inhibited cell survival of cell lines and they were at least 10 × fold more effective against DEPTOR over-expressing MM lines (8226, OPM-2, MM1.S) versus non-expressing lines (U266, FR4, delta 47). These results support a drug development strategy for myeloma that focuses on preventing DEPTOR binding to mTOR within TORC complexes for subsequent anti-myeloma cytotoxic effects. Disclosures: No relevant conflicts of interest to declare.
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Owen, R. G., S. A. Davis, J. Randerson, et al. "p53 gene mutations in multiple myeloma." Molecular Pathology 50, no. 1 (1997): 18–20. http://dx.doi.org/10.1136/mp.50.1.18.
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Markovic, Olivera, Dragomir Marisavljevic, Vesna Cemerikic, Maja Perunicic, and Milica Colovic. "The expression of p53 protein in patients with multiple myeloma." Srpski arhiv za celokupno lekarstvo 135, no. 1-2 (2007): 43–47. http://dx.doi.org/10.2298/sarh0702043m.
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Introduction: Although mutations of p53 are one of the most often acquired genetic changes in malignant tumors, these mutations are rare events in patients with newly diagnosed multiple myeloma (MM). Moreover, there are a few literature data about clinical significance of p53 overexpression in multiple myeloma. Objective The aim of our study was to evaluate the clinical significance of p53 immunoexpression in multiple myeloma. Method A total of 58 patients with newly diagnosed MM (26 females and 32 males, mean age 62 years) were enrolled in the study. The diagnosis of MM was made according to criteria of Chronic Leukemia-Myeloma Task Force. Clinical staging was done according to Durie and Salmon classification (4 patients had disease stage I, 15 patients stage II and 39 patients stage III). The histological grade and histological stage were determined according to predominant plasma cell morphology and volume of myeloma infiltration, respectively. Standard immunohistochemical analysis with p53 antibody in B5-fixed and paraffin- embedded bone marrow specimens was used to evaluate the expression of p53 in myeloma cells. The specimens were considered positive when ?5% of plasma cells exhibited clear nuclear positivity. Results Out of 58 patients, p53 expression was detected in 9 (15.52%). No significant correlation was found between p53 expression and clinical stage (I+II vs. III), ?2-microglobulin level (?6 mg/L vs. >6mg/L), histological grade (I vs. II+III), histological stage (<20% vs. 21-50% vs. >50%) and the extent of osteolytic lesions (?3 vs. >3 lesions). Median survival of patients with p53 immunoreactivity in =>5% of plasma cells was 10 months, whilst median survival of patients with p53 immunoreactivity in <5% of plasma cells was 36 months. However, such difference was not significant (p=0.2). Conclusion The frequency of p53 immunoexpression in our group of newly diagnosed MM was relatively low. Although p53 immunoexpression was not associated with clinical and histological features of more aggressive disease, or with shorter survival, further investigations of larger group of patients will lead to final conclusions.
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Stühmer, Thorsten, Manik Chatterjee, Martin Hildebrandt, et al. "Nongenotoxic activation of the p53 pathway as a therapeutic strategy for multiple myeloma." Blood 106, no. 10 (2005): 3609–17. http://dx.doi.org/10.1182/blood-2005-04-1489.
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AbstractMutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53–murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.
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Stühmer, Thorsten, Angela Zöllinger, Manik Chatterjee, Kurt Bommert, and Ralf C. Bargou. "Selective Induction of the p53-Pathway as Treatment for Multiple Myeloma." Blood 106, no. 11 (2005): 3382. http://dx.doi.org/10.1182/blood.v106.11.3382.3382.
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Abstract The p53 pathway is central to cellular defences against neoplastic transformation, and mutations that impair p53 function are prominent oncogenic events. However, certain malignancies, such as multiple myeloma (MM), rarely present with p53 defects except in late stages. MM might therefore be vulnerable to p53 induction therapy, but it is unknown if the p53 pathway remains actually functional in this disease, and if it can be sufficiently well triggered to induce tumor cell death. We have used nutlin-3, a newly developed small-molecule antagonist against the p53 suppressor murine double minute 2 (MDM2), to analyze the effect on myeloma cell lines and a large panel of patient tumor samples. Nutlin-3 specifically and exclusively induced p53 downstream targets in myeloma cells with wild-type p53, and a large majority of primary medullary myeloma samples was susceptible to nutlin-3-mediated apoptosis. Comparison with the clinically relevant genotoxic drugs melphalan and etoposide showed that nutlin-3 was as effective or better at inducing apoptosis of primary tumor cells, and that combinations of nutlin-3 with genotoxic cytostatics at low doses produced better than additive anti-tumor effects. Importantly, broad anti-tumor activity of nutlin-3 persisted even when primary tumor cells were kept in coculture with bone marrow stromal cells (BMSCs). However, primary tumor isolates often contain a fraction of cells apparently unaffected by drug treatment, and some primary samples were conspicuously less sensitive to nutlin exposure when the cells were kept in coculture with BMSCs, as compared to culture in medium supplemented with interleukin-6. We have begun to analyze to what extent heterogeneity for p53 induction within the tumor cell population may underlie these differences, and if the capacity to activate intracellular growth and survival pathways has an influence on the result of p53 pathway activation. Nutlin-3, as a non-genotoxic agent to specifically induce or strengthen p53-mediated apoptotic responses may be a promising agent to complement standard genotoxic regimens for the treatment of MM.
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Turner, Joel G., Jana L. Dawson, Steven Grant, et al. "Melphalan and XPO1 Inhibitor Combination Therapy for the Treatment of Multiple Myeloma." Blood 124, no. 21 (2014): 2084. http://dx.doi.org/10.1182/blood.v124.21.2084.2084.
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Abstract Introduction High-dose melphalan chemotherapy with autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma. However, patients eventually develop drug resistance and die from progressive disease despite the introduction of therapies using proteosome inhibitors (PIs) and immunomodulatory drugs (IMIDs). The incurable nature of multiple myeloma clearly demonstrates the need for novel agents and treatments. Here, our aim was to investigate whether the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) could sensitize de novo and acquired drug-resistant multiple myeloma cells both in vitro and ex vivo to the alkylating agent melphalan. Materials and Methods Human multiple myeloma cell lines NCI-H929, RPMI-8226, U266 and PBMC controls were treated in vitro with the XPO1i KOS-2464 and the orally available Selective Inhibitor of Nuclear Export (SINE) selinexor (KPT-330) or) +/- melphalan. Multiple myeloma cells were grown at high-density conditions (>3-5x106 cells/mL). High-density multiple myeloma cells have been shown to possess de novo drug resistance. Sensitivity of the XPO1i/melphalan-treated NCI-H929 cells was measured by cell viability assay (CellTiter-Blue). Apoptosis in XPO1i/melphalan-treated NCI-H929, RPMI-8226, and U266 cells was assayed using flow cytometry (activated caspase 3). Proximity ligation assays were performed to assess XPO1-p53 binding in the presence of an XPO1i. Western blots of XPO1i-treated myeloma cells were performed for nuclear and total p53. Drug-resistant U266 (PSR) and 8226 (8226/B25) myeloma cell lines were developed by incremental exposure to bortezomib. PSR cells are able to grow in 15 nM bortezomib and the 8226/B25 in 25 nM. These resistant myeloma cells were treated in vitro with XPO1i +/- melphalan. Sensitivity to therapy was measured by apoptosis and cell viability assay. Multiple myeloma cells isolated from patients with newly diagnosed, relapsed, or refractory disease were treated with XPO1i +/- melphalan and CD138+/light chain+ myeloma cells and assayed for apoptosis. Results Multiple myeloma cell (NCI-H929) viability was decreased synergistically by XPO1i when used in combination with melphalan, as shown by the calculated combinatorial index (CI) values. We examined sequencing of the drugs and found that concurrent treatment with melphalan (10 µM) and selinexor (300 nM) for 48 hours produced the best results (CI value 0.370, n=6). Sequential treatment (selinexor for 24 hours followed by melphalan for an additional 24 hours) or the reverse sequence had slightly less synergy, with CI values of 0.491 (n=9) and 0.565 (n=3), respectively. Normal PBMC control cells were unaffected by XPO1i/melphalan treatment as shown by viability and apoptotic assays. Proximity ligation assay demonstrated that XPO1i blocks XPO1/p53 binding. Western blot showed that the XPO1i treatment of myeloma cells increased nuclear and total p53. Drug-resistant 8226/B25 myeloma cells but not PSR cells were found to be resistant to melphalan when compared to parental cell lines. Both resistant myeloma cell lines were sensitized by XPO1i to melphalan as shown by apoptosis assay (3- to 10-fold). CD138+/light chain+ myeloma cells derived from newly diagnosed, relapsed, and refractory myeloma patients were also sensitized by XPO1 inhibitors to melphalan as demonstrated by apoptotic assays (e.g. activated caspase 3). Conclusions XPO1i synergistically improved the response of de novo and acquired drug-resistant myeloma cells to melphalan in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by XPO1i and the reported activation of p53 by melphalan. Future studies include in vitro experiments using drug-resistant human U266 myeloma cells in NOD-SCID-gamma mice and clinical trials using melphalan in combination with the SINE selinexor. Combination therapies using selinexor and melphalan may significantly improve the treatment of myeloma. Disclosures Kauffman: Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment.
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Ikeda, H., T. Hideshima, G. Perrone, et al. "Effect of the specific P53 stabilizer CBS9106 on multiple myeloma (MM)." Journal of Clinical Oncology 27, no. 15_suppl (2009): 8601. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.8601.
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8601 Background: The mutations of P53 tumor suppressor protein are associated with progressive in Multiple Myeloma (MM), conversely, stabilization of P53 leads to cell cycle arrest and apoptosis. In this study, we examined p53 protein expression and demonstrated the effect of P53 stabilization using a novel specific P53 stabilizer CBS9106 in MM. Method: We examined P53 protein expression using Immunoblot analysis, as well as the growth inhibitory effect of CBS9106 in MM cell lines and primary tumor cells from MM patients. We also defined whether CBS9106 can overcome the growth promoting effect of exogenous cytokines and bone marrow stroma cells (BMSCs) using [3H]-thymidine uptake assay. Results: Expression of P53 protein was observed in 3/3 primary tumor cells from MM patients and 6/6 MM cell lines. CBS9106 at low nM levels triggered cytotoxicity against p53 wild type MM cell lines and primary tumor cells from MM patients, associated with phosphorylation of P53 (serine15 and 20). In contrast, CBS9106 did not affect the survival of normal peripheral blood mononuclear cells from healthy volunteers at concentrations as high as 10 μM. This agent also induced G1 cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, -8, -9 and PARP. Neither growth stimulating cytokines (IL-6 and IGF-1) nor BMSCs protected against apoptotic effect of CBS9106. Moreover, we demonstrate that combination of CBS9106 with MDM2 inhibitor Nutrin3 or proteasome inhibitor bortezomib induces synergistic anti-MM activity in both P53 wild type MM cell lines and primary tumor cells from MM patients. Conclusions: Stabilizing P53 by CBS9106 represents a novel promising p53-based therapy in MM. These results provide the preclinical framework supporting evaluation of CBS9106 in clinical trials to improve patient outcome in MM. No significant financial relationships to disclose.
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Kumar, Shaji, Michael Timm, Michael P. Kline, et al. "Mi-63, a Small Molecule Inhibitor of MDM2-p53 Interaction, Has Significant In Vitro Activity in Multiple Myeloma." Blood 108, no. 11 (2006): 3464. http://dx.doi.org/10.1182/blood.v108.11.3464.3464.
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Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.
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Sagawa, Morihiko, Hiroto Ohguchi, Takeshi Harada, et al. "Targeting Ribonucleotide Reductase M1 and M2 in Multiple Myeloma." Blood 128, no. 22 (2016): 360. http://dx.doi.org/10.1182/blood.v128.22.360.360.
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Abstract Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of ribonucleotide diphosphate to deoxyribonucleotide diphosphate and is essential for de novo DNA synthesis, DNA repair, and cell growth. RR primarily exists as a heterodimer tetramer composed of regulatory subunit RRM1, and catalytic subunit RRM2. Overexpression or polymorphisms of RR are described non-small cell lung, pancreas, breast, and ovarian cancers. Moreover, RRM1 and RRM2 expression is highly correlated with patient survival in non-small cell lung and pancreas cancers. To date, the biologic significance in multiple myeloma (MM) has not yet been elucidated. In this study, we characterized the role of RRM1 and RRM2 in MM pathogenesis. By examining 3 independent expression data from GEO database, we found that RR (especially RRM1) expression is higher in MM plasma cells than normal plasma cells. We examined RRM1 and RRM2 expression in 6 MM cell lines and 3 MM patients' tumor cells by quantitative real-time PCR and immunoblotting and confirmed that both RRM1 and RRM2 are highly expressed in these cells. We next knocked down both RRM1 and RRM2 in 4 MM cell lines (NCI-H929, MM.1S, RPMI8226, and KMS-11) using siRNA. Knockdown of RRM1 and RRM2 triggered significant growth inhibition, associated with apoptotic cells death evidenced by AnnexinV/PI staining, in NCI-H929 and MM.1S, but not RPMI8226 or KMS-11 cells. We next examined molecular mechanisms whereby RRM1 downregulation triggers MM cell death. Gene expression profiling showed that p53 regulated genes were overexpressed after RRM1 knockdown. We therefore further examined DNA damage response (phosphorylated-ATM, -ATR, -Chk1, and -Chk2, and gamma-Histone H2A.X) and p53 (phosphorylated-p53, p21, Noxa, Puma, Bax) signaling pathways and found that these pathways were activated in NCI-H929 and MM.1S (both p53 wild-type), but not in RPMI8226 (p53 mutant) or KMS-11 (p53 null) cells after RRM1 knockdown. To validate the role of RRM1 of in vivo, we subcutaneously injected MM.1S cells transduced with shRNA against RRM1 or shLuc into our mouse xenograft model and observed that tumor growth was significantly reduced in shRRM1-MM.1S cells versus shLuc-MM.1S cells. Clofarabine (CLO), a purine nucleoside analog, which allosterically inhibits both DNA polymerases and RRM1 is used to treat acute leukemia and chronic lymphocytic leukemia and has been studied preclinically in MM (Valdez et. al. Experimental Hematology 2013). We therefore next examined CLO as a potential therapeutic agent in MM. Consistent with RRM1 knockdown, CLO induced growth arrest in p53 wild-type cell lines (NCI-H929, MM.1S, and MOLP-8), but not in p53 mutant (RPMI8226, OPM2, U266) or null (KMS-11) cells. Moreover, CLO treatment combined with DNA damaging agents (Melphalan, Doxorubicin) triggered synergetic cell death in p53 wild-type MM cells. Our results therefore demonstrate that RR, especially RRM1, is a novel therapeutic target in patients with wild-type p53 MM, and provide the basis for clinical evaluation of CLO, alone or in combination with DNA damaging agents, to improve patient outcome. Disclosures Hideshima: Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.
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Drach, Johannes, Jutta Ackerman, Hannes Kaufmann, Robert Königsberg, and Heinz Huber. "Deletions Of The p53 Gene In Multiple Myeloma." British Journal of Haematology 108, no. 4 (2000): 886. http://dx.doi.org/10.1046/j.1365-2141.2000.01908.x.
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Vadikoliou, C., C. Lalayianni, A. Papalexadri, et al. "P073 Treatment-related AML/MDS in multiple myeloma." Leukemia Research 33 (May 2009): S101. http://dx.doi.org/10.1016/s0145-2126(09)70153-5.
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Teoh, P. J., T. H. Chung, S. Sebastian, et al. "p53 haploinsufficiency and functional abnormalities in multiple myeloma." Leukemia 28, no. 10 (2014): 2066–74. http://dx.doi.org/10.1038/leu.2014.102.
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Nardiello, Tricia, Achim A. Jungbluth, Anna Mei, et al. "MAGE-A Inhibits Apoptosis In Proliferating Multiple Myeloma Cells." Blood 116, no. 21 (2010): 785. http://dx.doi.org/10.1182/blood.v116.21.785.785.
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Abstract Abstract 785 The type I Melanoma Antigen GEne (MAGE) MAGE-A3 is commonly present in primary multiple myeloma cells and its expression is correlated with advanced disease and proliferation. MAGE-A3 belongs to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins, which are present in many cancers, but their normal expression is limited to developing germ cells and placental trophoblast. This unique expression pattern fuels speculation on a role for CTAg in oncogenesis; however, very little is known about their function. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome, suggesting they contribute to the pathogenesis or progression of this disease through effects on survival and/or proliferation of myeloma cells. To investigate this, we examined the impact of MAGE-A on disease progression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). MAGE-A3 protein expression was examined by immunohistochemistry in a new, independent set of myeloma bone marrow specimens from two critical clinical milestones, newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003). The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating a correlation between MAGE-A3, progression of disease and proliferation. The mechanisms for MAGE-A3 activity were investigated by silencing this gene in primary myeloma cells and HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA and protein in MM.1r (p53+/+) and ARP-1 (p53−/−) HMCL and in primary myeloma cells by 48 hours, and this effect was maintained up to 96 hours. Silencing of MAGE-A did not affect cell cycling, as this intervention did not affect the phosphorylation of the Retinoblastoma gene product (Rb) that is required for progression through the G1 cell cycle checkpoints and entry into S phase. In contrast, MAGE-A was required for survival of proliferating myeloma cells. Silencing of MAGE-A led to a precipitous loss of viable cells within 48–72 hrs compared to controls. This was due to activation of intrinsic apoptosis, as demonstrated by increased annexin V staining, loss of mitochondrial membrane polarization, and cleavage/activation of caspase-9. These effects of MAGE-A knock-down were completely reversed by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh. Apoptosis after MAGE-A silencing appeared to be mediated by at least two distinct mechanisms; p53-dependent activation of pro-apoptotic Bax and Bak expression and reduced expression of the Inhibitor of Apoptosis Protein survivin through both p53-dependent and independent mechanisms. These results demonstrate that MAGE-A plays a role in the survival of proliferating multiple myeloma cells through the regulation of two critical apoptotic mechanisms. Disclosures: No relevant conflicts of interest to declare.
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Chng, W. J., T. Price-Troska, S. Van Wier, et al. "Clinical and biological implication of defective p53 pathway in multiple myeloma (MM)." Journal of Clinical Oncology 24, no. 18_suppl (2006): 17516. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.17516.
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17516 Background: The p53 tumor suppressor is commonly inactivated by mutations. Even in tumors without mutations, there are defects in the response to p53 activation. In MM, the prognostic significance of p53 mutation is unknown, while there has been no systematic study of p53 function. We seek to address these issues in this study. Methods: p53 mutation was studied by conformation sensitive gel electrophoresis with primers encompassing exons 1 to 10 followed by sequencing of DNA fragments with altered electrophoretic pattern in newly diagnosed MM patients entered into ECOG E9486 trial where patients were randomized to receive variations of melphalan-based conventional chemotherapy (VBMCP). Fisher’s exact tests were used to compare variables between patients. Kaplan-Meier survival curves were compared using the log-rank test. To investigate p53 function, we analyzed the expression of p53, and 3 of its transcriptional targets, APAF1, p21 and MDM2, in a separate cohort of 15 normal plasma cells (PC), 14 MGUS, 13 smoldering myeloma (SMM) and 101 MM (73 new and 28 relapsed) from the Mayo Clinic who had gene expression profiling performed on the Affymetrix U133A chip (Santa Clara, Ca). Results: Two hundred and sixty-eight patients had enough materials for study and were included in the analysis. The prevalence of p53 mutation was 3% (n = 9). Five of the 9 patients (56%, p = 0.001) with mutations also had p53 deletion (studied by fluorescent in-situ hybridization) resulting in bi-allelic loss of p53. Soft tissue plasmacytomas (37% v 7%, p = 0.018), and among the common translocations, t(4;14) and t(14;16) (50% v 18%) were more common in patients with p53 mutations. Despite similar response to treatment, those with p53 mutation had very short OS (16.7 v 41.4 months, p < 0.001). There was induction of p53 expression in MGUS and SMM with concurrent induction of APAF1, p21 and MDM2 whereas loss of this pattern was frequent in MM (45% in new MM and 60% in relapse MM compared to 15% in MGUS/SMM (p = 0.03)). Conclusions: p53 mutations are relatively rare in newly diagnosed MM patients but portend a short survival. However, functional abnormalities of p53 are prevalent and may be important in progression from MGUS/SMM to MM. [Table: see text]
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Coyne, Mark R. E., Seán Naughton, Patrick J. Hayden, Alessia Montagnoli, Michael E. O'Dwyer, and Corrado Santocanale. "Targeting Cdc7 Kinase in Multiple Myeloma." Blood 114, no. 22 (2009): 3847. http://dx.doi.org/10.1182/blood.v114.22.3847.3847.
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Abstract Abstract 3847 Poster Board III-783 Introduction Myeloma remains an incurable cancer and the development of new therapeutic approaches is of fundamental importance. A key feature of progressive myeloma is deregulation of the mechanisms that control progression through the cell cycle, in particular, the increase in the activity of S-phase promoting factors and the loss of cell cycle brakes such as the tumour suppressor protein p53. Moreover, high expression of Mcl-1, a potent anti-apoptotic Bcl2 family member, and defects in the p53 pathway are now known to be instrumental in inhibiting responsiveness to current and emerging myeloma therapeutics. The Cell division cyle 7 (Cdc7) kinase is an essential protein for the initiation of DNA replication and for cell cycle progression. Previous studies have indicated that Cdc7 inhibition can cause p53 independent apoptosis in several cancer models both in vitro and in vivo and currently Cdc7 inhibitors are entering clinical trials as experimental drugs. In this work we study the effects of the prototype Cdc7 protein kinase inhibitor, PHA-767491, in myeloma cells. In addition to targeting Cdc7, PHA-767491 also targets cyclin dependent kinase 9 (CDK9) and can downregulate Mcl-1 in a variety of cancer cell lines. Hypothesis: Given the molecular characteristics of progressive myeloma and the spectrum of activity of PHA-767491, we hypothesized that Cdc7 inhibition could be an effective treatment strategy in myeloma. Methods The Multiple Myeloma Research Consortium Validation-Core cell lines were treated with increasing doses of PHA-767491 as a single agent and in combination with drugs that currently constitute standards of care in myeloma treatment. These lines included lines resistant to conventional anti-myeloma agents (e.g. melphalan, dexamethasone, and doxorubicin) as well as novel anti-MM agents (e.g. immunomodulatory thalidomide derivatives). A number of lines were TP53 mutants. Similarly, CD138+ purified primary cells with clincal resistance to lenalidomide and bortezomib were treated with increasing doses of PHA-767491. Cell viability was measured by Cell TitreGlo 72 hours post treatment allowing IC50 calcuations. Combination indices were calculated as described by Chou et Talalay. Apoptosis was assessed by PI and Annexin V staining using flow cytometry and immunoblotting with relevant antibodies was performed for cell cycle and apoptotic proteins of interest. Results We found that PHA-767491 blocks proliferation and induces apoptosis with an IC50 in the low micromolar range in six out of seven MMRC cell lines and in primary cells from patient (s) with clinical resistance to bortezomib and lenalidomide. The averaqe and median IC50 value of MMRC cell lines was 2.25μM and 2μM respectively. TP53 mutation did not alter sensitivity to PHA-767491. Following treatment of myeloma cells with PHA-767491 there was potent inhibition of phosphorylation of the replicative helicase Mcm2 at serine 40/41 (a specific biomarker of Cdc7 activity) and complete downregulation of Mcl-1 by 6 hours, with concomitant caspase 3 cleavage and apoptosis as assessed by accumulation of sub-G1 cells. Annexin V/PI staining confirmed these cells to be apoptotic. When used in combination, PHA-767491 showed synergistic activity with melphalan and doxorubicin (Combination Index IC50 – 0.3, 0.7 respectively), even in the highly doxorubicin-resistant(p170/MDR1 mediated) line RPMI-8226-Dox-40 (Combination IndexIC50 – 0.3). Conclusion We suggest that Cdc7 inhibition with PHA-767491 or with small molecules that have similar properties could be an effective strategy in myeloma treatment and merits further evaluation. Disclosures: Montagnoli: Nerviano Medical Sciences Oncology: Employment.
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Zhuang, Junling, Fazal Shirazi, Ram Kumar Singh, et al. "Ubiquitin-activating enzyme inhibition induces an unfolded protein response and overcomes drug resistance in myeloma." Blood 133, no. 14 (2019): 1572–84. http://dx.doi.org/10.1182/blood-2018-06-859686.
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Abstract Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.
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John, Lukas, Maria Theresa Krauth, Klaus Podar, and Marc-Steffen Raab. "Pathway-Directed Therapy in Multiple Myeloma." Cancers 13, no. 7 (2021): 1668. http://dx.doi.org/10.3390/cancers13071668.
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Multiple Myeloma (MM) is a malignant plasma cell disorder with an unmet medical need, in particular for relapsed and refractory patients. Molecules within deregulated signaling pathways, including the RAS/RAF/MEK/ERK, but also the PI3K/AKT-pathway belong to the most promising evolving therapeutic targets. Rationally derived compounds hold great therapeutic promise to target tumor-specific abnormalities rather than general MM-associated vulnerabilities. This paradigm is probably best depicted by targeting mutated BRAF: while well-tolerated, remarkable responses have been achieved in selected patients by inhibition of BRAFV600E alone or in combination with MEK. Targeting of AKT has also shown promising results in a subset of patients as monotherapy or to resensitize MM-cells to conventional treatment. Approaches to target transcription factors, convergence points of signaling cascades such as p53 or c-MYC, are emerging as yet another exciting strategy for pathway-directed therapy. Informed by our increasing knowledge on the impact of signaling pathways in MM pathophysiology, rationally derived Precision-Medicine trials are ongoing. Their results are likely to once more fundamentally change treatment strategies in MM.
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Stanganelli, Carmen, Jorge Arbelbide, Juliana Zimerman, Dorotea Beatriz Fantl, Claudia Corrado, and Irma Slavutsky. "Aberrant Gene Promoter Methylation of Tumor Suppressor Genes in Plasma Cell Disorders." Blood 110, no. 11 (2007): 2487. http://dx.doi.org/10.1182/blood.v110.11.2487.2487.
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Abstract There is increasing evidence that, in addition to genetic aberrations, epigenetic processes play a major role in carcinogenesis. Particularly, hypermethylation of CpG islands of the promoter regions of tumor suppressor genes (TSG) is now considered as an important epigenetic mechanism for gene inactivation. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. During this process, multiple genetic alterations are sequentially acquired and aberrant promoter hypermethylation might be one of the steps involved in this progression. In this study, we have evaluated methylation status of the following TSG: p15INK4b, p16INK4a, p14ARF, SOCS-1, p27KIP1, RASSF1A and p73 genes, in order to determine if they were involved in the evolution of MGUS to MM. Forty four MM (21 males; mean age 67.5 years; Durie-Salmon clinical stages: I: 20%, II:14% and III: 66%) and 21 MGUS patients (6 males; mean age 68 years) were study. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Peripheral blood samples from 10 normal individuals and CpGenome Universal Methylated DNA (Chemicon International) were used as negative and positive controls, respectively. DNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls using phenol/chloroform method. Methylation status was performed using Methylation Specific PCR (MSP) technique. For statistical analysis, Student t and Fisher exact tests were used. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was also calculated. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS patients (14%) (p=0,006). Frequencies of methylation of p14ARF, p15INK4b, p16INK4a and RASSF1A were comparable in both entities: 29%, 32%, 7% and 2%, respectively, for MM; and 29%, 29%, 5% and 0%, respectively, for MGUS. TP73 gene showed a tendency of higher methylation in MM (45%) than in MGUS (33%). All patients lacked methylation at p27KIP1 gene. Whereas the percentage of MM with at least one gene methylated (84%) did not showed differences to that of MGUS (66%), the mean MI of MGUS was lower (0.16; range 0.14-0.43) than that of MM (0.24; range 0.14-0.71) (p&lt;0.05). None of the target genes were methylated in normal samples. No statistical significant correlation with clinical characteristics: gender, age, isotype, level of M-component, type of light chain, stage of the disease, haemoglobin, serum albumin level, calcium, β2 microglobulin and LDH, were observed. To our knowledge, this is the first report of methylation in MM and MGUS from Argentina. The similar frequency of p14ARF, p15INK4b, p16INK4a and RASSF1A gene methylation observed in MM and MGUS would suggest that they are probably not involved in the progression of MGUS. However, SOCS1 gene methylation was significantly more frequent in MM than in MGUS suggesting that methylation of this gene might be involved in clonal evolution of MGUS to MM. SOCS1 is a negative regulator of cytokine signaling, being important in normal lymphocyte development and differentiation. Silencing of SOCS1 may result in greater responsiveness to cytokines, which may favour the neoplastic development.
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Munawar, Umair, Santiago Barrio, Markus Roth, et al. "Implications of TP53 alterations for Therapy Response in Multiple Myeloma." Blood 132, Supplement 1 (2018): 3189. http://dx.doi.org/10.1182/blood-2018-99-118658.
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Abstract Deletion of the tumor suppressor gene TP53is significantly associated with an unfavorable clinical course of Multiple Myeloma (MM). In addition, point mutations that abrogate p53 function similarly shorten survival. Most recently 'double hit', bi-allelic TP53inactivated MM was identified as an ultimate high risk feature of MM, affecting 3.7% of newly diagnosed MM patients (NDMM, Walker et al., Leukemia 2018). For TP53genetic analysis we combined data from two targeted sequencing M3P cohorts with targeted sequencing and FISH data, one of NDMM (n=142, Kortüm et al. Blood Cancer J. 2016), and one of multirefractory patients (rMM, n=40; Kortüm et al. Blood 2016). We also included two independent cohorts with paired NDMM and rMM, one with mutational data (n=43; Corre J et al 2017) and one with paired FISH analysis (Merz M et al. Haematologica 2017). We confirmed an increase of mutations in TP53 (8% NDMM / 16.9% rrMM), as well as del17p (12.6% / 22%). Similarly, mono- (12.7% / 22.5%) and bi-allelic events (5.6% / 17.5%) both demonstrated a significant increase over time (Figure, top). Importantly we observed deletion after mutation and vice versa in our cohorts. Next we established an AMO-1 MM cell line model (TP53+/+) mimicking mono and bi-allelic TP53-inativation. After CRISPR/Cas9-mediated TP53destruction, we introduced a modified Sleeping Beauty (SB) vector with two separate expression cassettes (p53 wt / wt and mutant p53 (R282W or R175H). Functionality of the p53 system was confirmed using nutlin-3, as inhibitor of MDM2-p53 interaction, as described elsewhere. Results: Doxorubicin and Melphalan are commonly used compounds in the treatment of MM. The IC50 of Melphalan in AMO1 naïve cells was 5µM and 5.5 µM after the reintroduction of 2 copies of wt TP53 in the KO model, with cell viability at 10µM of 30% and 31% respectively, measured by AlamarBlue Assay. Strikingly, we observed significant resistance induction in our hemizygous systems (del/wt p53; cell viability at 10µM 60% and mut/wt (62%). This furthermore increased within our homozygous models (TP53 del/del (85%); TP53 mut/del 80%) (Figure, bottom). Similar results were observed under doxorubicin treatment. Remarkably, this effect was absent against proteasome inhibition. Conclusions Here we present first evidence of TP53 inactivation impacting drug response to Melphalan and Doxorubicin, which might lead to the clonal selection of MM subclones harboring increased risk. The fact, that response to proteasome inhibition was not affected in our model might, at least in part, might explain their ability to confine high risk in MM. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Liu, Qun, and Yair Gazitt. "Potentiation of dexamethasone-, paclitaxel-, and Ad-p53–induced apoptosis by Bcl-2 antisense oligodeoxynucleotides in drug-resistant multiple myeloma cells." Blood 101, no. 10 (2003): 4105–14. http://dx.doi.org/10.1182/blood-2002-10-3067.
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Abstract Overexpression of Bcl-2 in myeloma cells results in resistance to drugs such as dexamethasone (DEX), adenovirus-mediated delivery of p53 (Ad-p53), and paclitaxel (TAX), which work through the intrinsic apoptotic pathway. Bcl-2 antisense oligodeoxynucleotides (Bcl-2-ASO) have been shown to induce apoptosis in cancer cells, as a single agent or, better, in combination with chemotherapy. We hypothesized that down-regulation of Bcl-2 by Bcl-2-ASO will sensitize drug-resistant myeloma cells to undergo apoptosis. In this paper we report a detailed time/dose study of the effect of Bcl-2-ASO on myeloma cells with varying levels of Bcl-2. Treatment of myeloma cells expressing relatively low levels of Bcl-2 with Bcl-2-ASO resulted in a substantial apoptosis concomitant with a substantial depletion of Bcl-2 protein. Maximal apoptosis was observed at 5 to 10 μg/mL Bcl-2-ASO, following 4 days of treatment. Down-regulation of Bcl-2 and apoptosis were time and dose dependent and were sequence specific. In these cell lines, apoptosis was accompanied by activation of caspase-9 and caspase-3 and by release of cytochrome c to the cytosol. In contrast, high Bcl-2–expressing myeloma cells were practically resistant to Bcl-2-ASO. Most important, however, pretreatment of myeloma cells expressing high levels of Bcl-2 with Bcl-2-ASO increased the extent of DEX-, TAX-, and Ad-p53–induced apoptosis from 10%-20% to 70%-90%. Increased apoptosis was accompanied by additional decrease in Bcl-2 protein. Similar results for down-regulation of Bcl-2 and apoptosis were obtained with freshly isolated myeloma cells. These data support development of clinical trials with combinations of Bcl-2-ASO and DEX, TAX, or Ad-p53 in the treatment of refractory myeloma patients.
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Preudhomme, Claude, Thierry Facon, Marc Zandecki, et al. "Rare occurrence of P53 gene mutations in multiple myeloma." British Journal of Haematology 81, no. 3 (1992): 440–43. http://dx.doi.org/10.1111/j.1365-2141.1992.tb08253.x.
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Ortega, Manoela M., Helen N. Honma, Lair Zambon, et al. "GSTM1 and codon 72 P53 polymorphism in multiple myeloma." Annals of Hematology 86, no. 11 (2007): 815–19. http://dx.doi.org/10.1007/s00277-007-0347-x.
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