Academic literature on the topic 'P73, multiple myeloma'

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.

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 <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.

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 > 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.

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.

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.

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.

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.

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.

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.