Multitarget inhibition of drug-resistant multiple myeloma cell lines by dimethyl-celecoxib (DMC), a non–COX-2 inhibitory analog of celecoxib

2,5-dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib that lacks COX-2 inhibitory function. We and others have demonstrated that DMC, despite its inability to block COX-2, is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. In this current study, we investigated whether DMC would also be able to inhibit the growth of highly drug-resistant tumor cell variants. We focused on human multiple myeloma (MM) cells, as patients with MM frequently develop drug-resistant disease and ultimately succumb to death. Here we show that DMC (and celecoxib) inhibits the proliferation of various multiple myeloma cell lines, including several (multi) drug-resistant variants. Growth inhibition in drug-sensitive and drug-resistant cells is mediated via multiple effects, which include diminished signal transducer and activator of transcription 3 (STAT-3) and mitogen-activated protein (MAP) kinase kinase (MEK) activity, reduced expression of survivin and various cyclins, and is followed by apoptotic cell death. Thus, our study demonstrates that inhibition of proliferation and induction of apoptosis by DMC (and celecoxib) can be accomplished even in highly drug-resistant multiple myeloma cells, and that this effect is achieved via the blockage of multiple targets that are critical for multiple myeloma cell growth and survival.

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Melphalan and XPO1 Inhibitor Combination Therapy for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2084.2084 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2084-2084 ◽

Cited By ~ 1

Author(s):

Joel G Turner ◽

Jana L Dawson ◽

Steven Grant ◽

Kenneth H. Shain ◽

Yun Dai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

De Novo ◽

Ex Vivo ◽

Myeloma Cell ◽

Drug Resistant ◽

Cell Viability Assay ◽

Myeloma Cells ◽

Multiple Myeloma Cell

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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In silico Prediction Followed By in Vitro validation Identifies the Survivin Inhibitor YM155 As a Potent Secondary Drug Against Pi-Resistant Myeloma

Blood ◽

10.1182/blood-2019-131923 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5543-5543

Author(s):

Harish Kumar ◽

Ujjal Kumar Mukherjee ◽

Li Chen ◽

Amit Kumar Mitra

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Cell Lines ◽

In Silico ◽

Proteasome Inhibitors ◽

Standard Of Care ◽

Myeloma Cell ◽

Drug Resistant ◽

Multiple Myeloma Cell

Drug resistance is a major cause of concern in cancer chemotherapy that not only reduces efficacy but also causes toxicity through overdosing. Multiple myeloma (MM) is the second-most common haematological malignancy in the USA that remains a challenging disease to cure due to the presence of significant complexity and heterogeneity at molecular level with high treatment cost. Proteasome inhibitor, the standard of care drug, has proven to remarkably improve myeloma patient outcomes but is associated with dose limiting toxicities and drug resistance. Therefore, new combination treatment regimens are urgently needed for the treatment of PI-resistant MM that can lower the required dose of standard-of-care drug without compromising on treatment efficacy. We have created an optimization-regularization based computational prediction method called secDrug that uses large-scale pharmacogenomics databases to identify novel secondary drugs against PI-resistance when used as single agent or in combination. We applied secDrug to PIs-resistant cell lines (>100) in the Genomics of Drug Sensitivity in Cancer (GDSC) database and predicted the top drugs that can be best combined with PIs to overcome PI-resistance in MM. These include: Survivin inhibitor (YM155), Nicotinamide phospho ribosyl transferase or Nampt inhibitor (FK866), PIKfyve inhibitor (YM201636), Raf inhibitor (PLX-4720), Bcl2 inhibitor (Navitoclax),HSP90 inhibitor (17-AAG), AKT inhibitor (KIN00102), HDAC inhibitors (Panobinostat, SAHA), S6K1-specific inhibitor (PF-4708671), and the neddylation inhibitor (MLN4924). To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib and >10 clonally derived acquired resistant cells with the top predicted best secondary drugs. Next, we treated three pairs of parental PI-sensitive (P) and clonally-derived PI-resistant (R) cell lines from PI-sensitive (P) HMCLs representing acquired PI-response with the top predicted drug combinations. Our preliminary in vitro data confirmed our in silico predictions of secondary drugs based on secDrug analysis. Among these, YM155 and YM155 + Ixazomib showed strikingly very high in vitro cytotoxicity against PI- resistant MM cell lines (Figure 1). The results, based on Chou-Talalay's combination index (CI) theorem, show that this combination works highly synergistically where the IC50 of ixazomib in MM cells was significantly reduced in presence of YM155. Similar results were obtained for the other top predicted combination regimens. Our findings provide a strong case for combining YM155 with ixazomib to enhance sensitivity or overcome resistance to ixazomib and thereby improve patient outcome. Further, this study provides additional support towards using secDrug for predicting secondary drugs in cancers resistant to the standard-of-care therapy. Currently, we are exploring the molecular mechanisms behind the synergism between proteasome inhibitors and the top drugs identified by our algorithm as candidates for secondary combination therapies using next-gen transcriptomics and single-cell analysis approaches. Earlier studies have shown that YM155 potentially induces selective cell death in human pluripotent stem cells. Further, the presence of cancer stem cells sub population/ MM-CSCs in drug-resistant tumors have been shown to significantly contribute to the development of PI-resistance in MM. Hence, we propose that YM155 reverses PI-resistance by reducing the stem cell load in multiple myeloma cell line model system. Therefore, our future plan is to assess the effects of YM155 on MM-cancer stem cells like subclones/ sub population/ MM-CSCs including CD19- CD138- quiescent stem cells, ALDH+ and side populations/SP) in drug-resistant tumors. Overall, our in vitro validation results corroborated with our in silico prediction of secondary drugs. Disclosures No relevant conflicts of interest to declare.

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Simvastatin Induces Death of Multiple Myeloma Cell Lines

Journal of Investigative Medicine ◽

10.1136/jim-52-05-34 ◽

2004 ◽

Vol 52 (5) ◽

pp. 335-344 ◽

Cited By ~ 11

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.

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Ruthenium(II)-arene complexes with dibenzoylmethane induce apoptotic cell death in multiple myeloma cell lines

Inorganica Chimica Acta ◽

10.1016/j.ica.2016.04.031 ◽

2017 ◽

Vol 454 ◽

pp. 139-148 ◽

Cited By ~ 19

Author(s):

Riccardo Pettinari ◽

Fabio Marchetti ◽

Agnese Petrini ◽

Claudio Pettinari ◽

Giulio Lupidi ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Apoptotic Cell ◽

Myeloma Cell ◽

Apoptotic Cell Death ◽

Arene Complexes ◽

Multiple Myeloma Cell

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Antitumor Effects and Mechanisms of Curcumin On Human Multiple Myeloma Cell Lines.

Blood ◽

10.1182/blood.v114.22.4896.4896 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4896-4896

Author(s):

Qingxian Bai ◽

Qifa Liu

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Synergistic Effect ◽

Cell Lines ◽

Myeloma Cell ◽

Dependent Manner ◽

Antitumor Effects ◽

Low Concentration ◽

Multiple Myeloma Cell ◽

Human Multiple Myeloma

Abstract Abstract 4896 BackgroundF Multiple myeloma(MM) is a malignant plasma disease, which is characterized as high relapse rate and high resistance to chemotherapy. Curcumin is a polyphenol derived from the rhizome of Curcuma spp. It possesses diverse pharmacologic actions, such as antitumor, anti-inflammatory,anti- oxidation properties .Curcumin has the property of inhibit multiple tumor cell lines, in which included multiple myeloma cell. The real mechanism is not completely clear yet. We explored the mechanisms of curcumin on human multiple myeloma cell lines (RPMI8226 and H929), and investigated whether the combination of curcumin and adriamycin(Adr) has a synergistic effect. MethodsF The effect of curcumin on proliferation of RPMI8226 and H929 was observed with MTT assay. The synergetic effect of curcumin and Adr was analyzed by median-effect principle. Cell cycle distribution and apoptosis were studied with flow cytometry. Expression of surviving, bcl-2, bax mRNA was detected by RT-PCR. ResultsF Curcumin could inhibit the proliferation of RPMI8226 and H929 cells in a time- and dose-dependent manner. The IC50 values for RPMI8226 and H929 cell line were 12.15 μmol/L,17.24μmol/L respectively. The combination of curcumin and Adr showed synergistic effect even at low concentration of Adr. Apoptotic ratio of treated cells was significantly higher than untreated controls (36.9% vs 10.6%, p<0.05). Cells treated with curcumin showed cell cycle arrest at G2/M phase. Curcumin upregulated expression of survivin, bcl-2, while bax mRNA was significantly downregulated. ConclusionF Curcumin could suppress the proliferation of multiple myeloma cells and induce apoptosis. Adr combining with curcumin can show synergistic effect at low concentration of Adr. The mechanism of curcumin's antitumous effect might be related to down-regulation of surviving, bcl-2 mRNA and up-regulation of bax mRNA. Disclosures No relevant conflicts of interest to declare.

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Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽

10.1182/blood.v122.21.3493.3493 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3493-3493

Author(s):

Ahmad-Samer Samer Al-Homsi ◽

Zhongbin Lai ◽

Tara Sabrina Roy ◽

Niholas Kouttab

Keyword(s):

Multiple Myeloma ◽

T Cell ◽

Cell Lines ◽

Mechanism Of Action ◽

Peripheral Blood ◽

Research Funding ◽

Myeloma Cell ◽

Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

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