Targeting the Insulin-Like Growth Factor-I Receptor (IGF-IR) in Multiple Myeloma Cells Using Selective IGF-IR Tyrosine Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 639-639 ◽  
Author(s):  
Thomas Stromberg ◽  
Simon Ekman ◽  
Leonard Girnita ◽  
Johan Lennartsson ◽  
Ulf Hellman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Rpmi 8226 ◽  
Β Chain ◽  
Tyrosine Phosphorylation Site ◽  
Rtk Inhibitors

Abstract In multiple myeloma (MM) emerging evidence suggest the IGF-1R as an important mediator of tumor cell survival and thus resistance to cytotoxic therapy. Since IGF-1R is not an absolute requirement for maintenance of normal cell homeostasis, interfering with IGF-1R signaling at the receptor tyrosine kinase (RTK) level represents an attractive strategy to improve anti-cancer treatment. However, most IGF-1 RTK inhibitors do not fully discriminate between the IGF-1 RTK and the insulin RTK, i.e. are diabetogenic. Recently, members of the cyclolignan family have been shown to selectively inhibit the phosphorylation of the IGF-1R β-chain without down regulating the RTK activity of the insulin R1. The effect of two of these compounds, picropodophyllin (PPP) and deoxypodophyllotoxin (DPPT), was studied in vitro using a panel of nine MM cell lines and primary tumor cells from MM patients, and in vivo using the 5TMM mouse MM model2. Both IGF-1 RTK inhibitors effectively inhibited growth in all MM cell lines providing increased apoptosis and cell cycle arrest in the G2/M-phase. Notably, the two drug-resistant subclones of the MM cell line RPMI 8226/Dox40 (doxorubicin) and RPMI 8226/LR5 (melphalan), were highly sensitive to the IGF-1 RTK inhibitors. In addition, PPP and DPPT showed inhibitory effects on primary MM cells cultured in vitro with bone marrow stromal cells. Inhibition of the IGF-1 RTK with PPP has been shown to be non-competitive with ATP suggesting interference with the IGF-1R at the substrate level1. To identify tyrosine phosphorylation site(s) on the IGF-1R β-chain potentially regulated by the IGF-1 RTK inhibitors, IGF-1R extracted from RPMI 8226 cells was analyzed by tryptic phosphopeptide mapping following 32P-labeling and treatment with PPP or DPPT. Cleaved phosphopeptides were separated by thin layer chromatography and analyzed by a modified radio-Edman degradation. The results show that the IGF-1 RTK inhibitors do not down regulate any specific tyrosine phosphorylation site of the IGF-1R in MM cells. In vitro kinase assay suggests that PPP/DPPT-induced inhibition of the IGF-1R instead is conducted via a general down regulation of the tyrosine kinase activity of the receptor. Extraction of tyrosine phosphorylated proteins followed by electrophoresis and mass spectrometric analysis revealed additional signaling molecules affected by treatment with the IGF-1 RTK inhibitors e.g. impaired tyrosine phosphorylation of CDK1/cdc2, a cell cycle associated protein known as a potent regulator of apoptosis. Taken together, we show that treatment of MM cells with selective IGF-1 RTK inhibitors decreases survival/proliferation and affects the function of crucial intracellular signaling proteins, thus emphasizing the pivotal role for IGF-1R signaling in MM.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

The Novel NFkB Inhibitor V1810 Induces Apoptosis and Cell Cycle Arrest in Multiple Myeloma Cells and Overcomes NFkB Mediated Drug Resistance.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1715-1715 ◽  
Author(s):  
Felix Meinel ◽  
Sonja Mandl-Weber ◽  
Philipp Baumann ◽  
Johann Leban ◽  
Ralf Schmidmaier
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Death ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cyclin D3 ◽  
Cycle Arrest ◽  
Rpmi 8226

Abstract Multiple Myeloma (MM) is a fatal malignancy characterised by the accumulation and expansion of antibody producing plasma cells in the bone marrow. Evidence is increasing that nuclear factor kappa B (NFκB) is a promising target for new anti-myeloma therapies. In this study, we assessed the in vitro activity of V1810, a novel NFκB inhibitor. V1810 potently induces cell death in all tested MM cell lines (OPM-2, U266, NCI-H929, RPMI-8226) with an IC50 ranging between 5μM to 10μM as well as in primary MM cells from patients. Cell death induced by V1810 clearly shows biological features of apoptosis such as DNA fragmentation and caspase 3 cleavage. In OPM2, U266 and RPMI-8226 cells induction of apoptosis is accompanied by cell cycle arrest. Western blots revealed downregulation of cyclin D1 (U266) or cyclin D2 (OPM2, NCI-H929, RPMI-8226) respectively, but not cyclin D3. Consistent with the downregulation of cyclin D1/2, retinoblastoma protein was found to be hypophosphorylated. Considering that cyclin D1 and D2 are known to be NFκB target genes, this is in line with our finding that V1810 inhibits baseline NFκB activity in MM cells (36% relative reduction). Importantly, V1810 also abrogates NFκB activation induced by genotoxic drugs like melphalan and doxorubicin. Accordingly, V1810 and melphalan synergistically decrease MM cell viability. Taken together, V1810 induces apoptosis and cell cycle arrest in MM cells by inhibition of NFκB and overcomes NFκB mediated drug resistance to melphalan. The maximum tolerable dose (MTD) of V1810 in BalbC mice was 10mg/kg i.v. and plasma concentrations of 9.5μM are achievable in NRMI mice after 5mg/kg V1810 i.v., which corresponds well to the used in vitro concentrations. This study strongly supports the further development of NFkB inhibitors in MM, especially in combination with genotoxic drugs.

scholarly journals A single tyrosine phosphorylation site in cortactin is important for filopodia formation in neuronal growth cones

2019 ◽  
Vol 30 (15) ◽  
pp. 1817-1833 ◽  
Author(s):  
Yuan Ren ◽  
Yingpei He ◽  
Sherlene Brown ◽  
Erica Zbornik ◽  
Michael J. Mlodzianoski ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Leading Edge ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Src Tyrosine Kinase ◽  
Actin Organization ◽  
Neuronal Growth Cones ◽  
Tyrosine Phosphorylation Site

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin–independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

scholarly journals In Vitro JAK1/2 Inhibition, in Association with Bortezomib and Lenalidomide, Is Comparable with Bortezomib, Lenalidomide and Dexametasone: An Alternative for Multiple Myeloma Patients with JAK2 overexpression?

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5663-5663
Author(s):  
Mariana Bleker de Oliveira ◽  
Veruska Lia Fook Alves ◽  
Angela Isabel Eugenio ◽  
Rodrigo Carlini Fernando ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
U266 Cell ◽  
Late Apoptosis ◽  
Rpmi 8226 ◽  
Number Of Cells ◽  
Stat Pathway

Abstract Background: Despite the absence of JAK1 and JAK2 mutations in multiple myeloma (MM), high levels of IL-6 constitutively activate the JAK/STAT pathway promoting survival and proliferation of tumor cells. Therefore, pharmacological inhibition of JAK proteins can be a potentially therapeutic strategy for myeloma treatment. Aims: 1) to identify expression of JAK1 and JAK2 in MM cell lines and in recently diagnosed MM patients; 2) to perform functional in vitro studies in MM cell lines treated with JAK/STAT pathway inhibitor (ruxolitinib), associated with drugs currently used in MM first line treatment (bortezomib, lenalidomide and dexamethasone), with and without co-culture with normal stromal cells; 3) to evaluate global gene expression of JAK/STAT pathway in cell lines treated with ruxolitinib to elucidate its mechanism of action in MM. Methods: JAK1 and JAK2 expression were analyzed in four cell lines (RPMI-8226, U266, SKO-007 and SKM-M2) and in bone marrow samples from 30 MM patients and 3 healthy controls by real time PCR. After IC50 calculation, drugs concentrations were: bortezomib (B) 10 nM for both RPMI-8226 and U266 cell lines; ruxolitinib (R) 30 µM for RPMI-8226 and 40 µM for U266 cell lines; lenalidomide (L) 10 µM for both cell lines; and dexamethasone (D) 1 µM for both cell lines. Apoptosis and cell cycle were evaluated by flow cytometry. PCR array for 92 JAK/STAT pathway related genes (Taqman® Array Human JAK/STAT Pathway, Applied Biosystems, Foster City, CA, USA) was performed in RPMI-8226 and U266 wild type and B+R treated cell lines, in duplicates. Results: Among the four cell lines, U266 presented the highest expression of JAK1 and JAK2 genes. JAK1 was overexpressed in 27% and JAK2 in 57% of 30 MM patients (considering at least 2-fold increase). After B+R treatment, RPMI-8226 showed increased number of cells in SubG0 phase (p<0.001) with reduction of cells in S (p<0.01) and G2/M (p<0.001) phases. In U266 cell line, there is a slight increase of cells in SubG0 phase (p<0.05). Also, after B+R treatment, both RPMI-8226 and U266 presented 50% of cells in late apoptosis, which was accompanied by reduction of expression levels of BCL-2 and BCL-XL anti-apoptotic genes. The expression profile of JAK/STAT pathway after B+R treatment showed that many JAK/STAT, Ras/Raf/MAPK and PI3K/Akt/mTOR pathways genes lost their expression, mainly in RPMI-8226, with insignificant changes in U266 expression pattern. Co-culture of RPMI-8226 with normal stromal cell line HS5 protected tumor cells from apoptosis, as the number of cells in late apoptosis decreased from 50% to 32% (p<0.001). The addition of immunomodulatory drug lenalidomide to the schedule (B+R+L) increased tumor cell death from 32% to 73% in co-culture (p<0.001). Despite the impressive results, B+R+L schedule was equivalent to currently used treatment for standard risk MM patients B+L+D (67% of cell death, p>0.05), in co-culture. Conclusion: B+R combination induced cell cycle arrest and apoptosis in U266 and RPMI-8226. The new drug combination B+R+L has in vitro results comparable with B+L+D and presents an alternative for MM treatment of almost 60% of cases bearing JAK2 overexpression. Our results support future studies using JAK inhibitors as an alternative for MM treatment in a Precision Medicine approach. Financial support: FAPESP 2010/17668-6 and CNPq. Disclosures No relevant conflicts of interest to declare.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

2019 ◽  
Vol 19 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Mariana B. de Oliveira ◽  
Luiz F.G. Sanson ◽  
Angela I.P. Eugenio ◽  
Rebecca S.S. Barbosa-Dantas ◽  
Gisele W.B. Colleoni
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Treatment Options ◽  
Compensatory Mechanism ◽  
Protein Homeostasis ◽  
Protein Response ◽  
Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

scholarly journals Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

2021 ◽  
Author(s):  
Yu-bo Zhou ◽  
Yang-ming Zhang ◽  
Hong-hui Huang ◽  
Li-jing Shen ◽  
Xiao-feng Han ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Targets ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Preclinical Study ◽  
Parp Cleavage ◽  
Rpmi 8226 ◽  
Ongoing Phase

AbstractHDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2745-2754 ◽  
Author(s):  
Alistair J. Standish ◽  
Jonathan J. Whittall ◽  
Renato Morona
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Capsular Polysaccharide ◽  
Regulatory Mechanism ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Regulatory Proteins ◽  
Amidase Activity ◽  
The Past

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

scholarly journals Immunotherapy of Multiple Myeloma With a Monoclonal Antibody Directed Against a Plasma Cell-Specific Antigen, HM1.24

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3179-3186 ◽  
Author(s):  
Shuji Ozaki ◽  
Masaaki Kosaka ◽  
Shingo Wakatsuki ◽  
Masahiro Abe ◽  
Yasuo Koishihara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Plasma Cell ◽  
Severe Combined Immunodeficiency ◽  
Specific Antigen ◽  
Chemotherapeutic Agents ◽  
Scid Mice ◽  
Dependent Manner ◽  
Rpmi 8226

Abstract Multiple myeloma remains an incurable malignancy because of marked resistance of tumor cells to conventional chemotherapeutic agents. Alternative strategies are needed to solve these problems. To develop a new strategy, we have generated a monoclonal antibody (MoAb), which detects a human plasma cell-specific antigen, HM1.24. In this report, we evaluated the in vivo antitumor effect of unconjugated anti-HM1.24 MoAb on human myeloma xenografts implanted into severe combined immunodeficiency (SCID) mice. Two models of disseminated or localized tumors were established in SCID mice by either intravenous or subcutaneous injection of human myeloma cell lines, ARH-77 and RPMI 8226. When mice were treated with a single intraperitoneal injection of anti-HM1.24 MoAb 1 day after tumor inoculation, the development of disseminated myeloma was completely inhibited. In mice bearing advanced tumors, multiple injections of anti-HM1.24 MoAb reduced the tumor size and significantly prolonged survival, including tumor cure, in a dose-dependent manner. The proliferation of cultured human myeloma cells was inhibited in vitro by anti-HM1.24 IgG-mediated complement-dependent cytotoxicity, but not by the antibody alone. Moreover, spleen cells from SCID mice mediated antibody-dependent cell cytotoxicity against RPMI 8226 cells. These results indicate that anti-HM1.24 MoAb can be used for immunotherapy of multiple myeloma and related plasma cell dyscrasias.

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