Synergistic Induction of Cell Death and Apoptosis by the Histone Deacetylase Inhibitor SAHA (Vorinostat) and the Demethylating Agent DAC in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2393-2393
Author(s):  
Edgar Jost ◽  
Claudia Schubert ◽  
Tim Bruemmendorf ◽  
Oliver Galm
Keyword(s):  
Cell Lines ◽  
Apoptotic Cell ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Rt Pcr ◽  
Advisory Committees ◽  
Dnmt Inhibitors ◽  
Induction Of Apoptosis ◽  
Rpmi 8226

Abstract Abstract 2393 Poster Board II-370 Introduction: Hypermethylation of CpG islands in the promoter region of genes is a well characterized epigenetic modification associated with transcriptional silencing of cancer related genes and plays a crucial role in carcinogenesis. In addition, acetylation of core histones is necessary for the maintenance of transcriptional activity of genes. DNA methylation and histone deacetylation are reversible and can be influenced by DNA methyltransferase (DNMT) inhibitors such as 5-aza-2`-deoxycytidine (DAC) or 5-azacytidine (AZA) and histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA), respectively. Clinical trials using a strategy based on the modification of epigenetic changes with DAC or AZA in combination with HDAC inhibitors have been promising and may help to generate new strategies in treatment of hematopoietic malignancies including multiple myeloma (MM). In MM however, only limited data are published about the possible synergistic effects between DNMT inhibitors and the highly potent pan-HDAC inhibitor SAHA. Material and Methods: To assess the in vitro effects of SAHA on the MM cell lines U266, LP-1, RPMI8226 or OPM-2 and the possible interactions with DNMT inhibitors, cells were first incubated with DAC in a final concentration of 0.1 or 0.2 mM for 72 hours. After exposure to DAC, cells were incubated for 72 or 96 hours with SAHA in a final concentration between 0.1 and 20 mM. The toxic effect of the treatment was assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing a 50 % killing of the cells compared to control cell survival was calculated from the dose-response curve (IC50). Induction of apoptosis was analysed by flow cytometry with annexin V-binding. In addition, the expression of the epigenetically silenced tumor suppressor genes SFRP-2 and DAB2 was determined by real time RT-PCR before and after exposure to DAC and SAHA. Results: In LP-1 and U-266 cells, no relevant enhancement in the cytotoxic effect of SAHA was observed after previous exposure to DAC. In contrast, in OPM-2 and RPMI-8226 cells, a significant increase in cytotoxicity of SAHA was observed, when the cells were first incubated with DAC with a decrease of the IC50 from 6.5 μM to 2.43 μM and 10.37 μM to 4.5 μM, respectively. We further analysed a possible synergism between SAHA and DAC for the induction of apoptosis by flow cytometry. After sequential exposure of the cells with DAC for 72 hours and with SAHA for 72 hours, no change in the apoptotic cell fraction was observed for the cell lines OPM-2 and RPMI-8226. However, for U-266 and LP-1, a significant increase in apoptotic cells was observed after incubation with SAHA, when the cells were previously exposed to DAC with a increase in the apoptotic cell fraction of 39.5 % to 55.4 % and 2.5 % to 14.4 %, respectively. By real-time RT-PCR, corresponding transcriptional silencing for SFRP-2 and DAB2 was demonstrated in untreated cells, and exposure of cell lines to DAC and SAHA resulted in reexpression. A synergism for the induction of reexpression of these genes was observed when cells were incubated with DAC and SAHA sequentially. Discussion: After treatment with SAHA, we observed a dose-dependent induction of cell death and apoptosis as assessed by MTT and annexin V assay, respectively. In the different MM cell lines, we observed a synergism between SAHA and DAC both for cytotoxic effects and the induction of apoptosis. A synergism was also observed for the reexpression of epigenetically silenced genes after exposure to DAC and SAHA. These in vitro data can be considered as a basis for further in vitro studies and preclinical models with SAHA in combination with demethylating agents such as DAC in order to improve treatment response and survival in MM patients. Disclosures: Jost: MSD: Research Funding. Bruemmendorf:Genzyme: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

A Novel SIRT1 Activator SIRT1720 Triggers In Vitro and In Vivo Cytotoxicity In Multiple Myeloma Via ATM-Dependent Mechanism

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3007-3007
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Teru Hideshima ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Advisory Committees ◽  
Rpmi 8226 ◽  
Sirt1 Activator

Abstract Abstract 3007 Background and Rationale: SIRT1 belongs to the silent information regulator 2 (Sir2) family of proteins and functions as NAD+-dependent deacetylase. Previous studies showed that resveratrol, a polyphenolic SIRT1 activator, inhibits tumorigenesis in various solid tumor and hematologic malignancies, including human multiple myeloma (MM) cells. This notion led to the discovery and development of more potent and selective pharmacological activators of SIRT1 as potential anti-cancer therapeutics. In this context, a recent medicinal chemistry research using high-throughput screening, and mass spectrometry identified SRT1720, a small molecule activator of SIRT1 that is structurally distinct from resveratrol. Here, we examined the anti-tumor activity of SRT1720 in MM cells using in vitro and in vivo model systems. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, H929, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using trypan blue, MTT, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Results: We first confirmed the functional specificity of SRT1720 against SIRT1 using different experimental strategies. First, we utilized Fluor de Lys Deacetylase Assay to measure whether SRT1720 affects the SIRT1 deacetylase enzymatic activity. Treatment of MM.1R and RPMI-8226 MM cells with SRT1720 markedly increased the deacetylating activity; conversely, pre-treatment of cells with nicotinamide (NAM) - an inhibitor of SIRT1 – significantly blocked SRT1720-triggered deacetylating activity. Second, immunoblot analysis using antibodies specific against acetylated p53 (a known substrate of SIRT1) showed a marked decrease in acetylated state of p53 in SRT1720-treated MM cells. These findings in MM cells confirm SIRT1 as a selective target of SRT1720. We next examined the efficacyof SRT1720 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 3–7 uM) (P < 0.005; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for SRT1720. SRT1720-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, SRT1720 induced apoptosis in MM cells even in the presence of bone marrow stromal cells. Mechanistic studies showed that SRT1720-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of pATM, CHK2, endoplasmic reticulum stress molecules pEIF2, and BIP; as well as an increase in reactive oxygen species (ROS); 3) inhibition of MM cell growth and survival pathway via NF-kB; and 4) inhibition of VEGF-induced migration of MM cells and associated angiogenesis. Importantly, blockade of pATM using a biochemical inhibitor KU-5593 significantly attenuated SRT1720-induced MM cell death (P value < 0.002; n=2). These data suggest that SRT1720-induced MM cell apoptosis is predominantly mediated by an ATM-dependent apoptotic pathway. We next examined the in vivo efficacy of SRT1720 using a human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with SRT1720 (200 mg/kg, 5 days a week for three weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Finally, the combination of SRT1720 with bortezomib or dexamethasone triggered synergistic anti-MM activity. Conclusions: These preclinical studies provide the rationale for novel therapeutics targeting SIRT1 to improve patient outcome in MM. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Anti-Myeloma Activity of Enzymatically Activated Melphalan Prodrug J1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1838-1838
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Klaus Podar ◽  
Paul G Richardson ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Clinical Protocols ◽  
Advisory Committees ◽  
Pre Treatment ◽  
Rpmi 8226

Abstract Abstract 1838 Background and Rationale: The alkylating drug melphalan is routinely used in clinical protocols for the treatment of multiple myeloma (MM). Importantly, clinical trials in MM have effectively utilized combination of melphalan with proteasome inhibitor bortezomib and prednisolone (VMP regimen) to reduce toxicity, overcome drug resistance and enhance cytotoxicity. These findings highlight the utility of conventional alkylating agent, and importantly, provide impetus to develop conventional agents based prodrugs with a potent cytotoxic activity. In this context, pharmacological screening of alkylating oligopeptides led to the identification of a novel melphalan-containing prodrug J1 (L-melphalanyl-p-L-fluoro phenylalanine ethyl ester) - a new molecular entity with a more distinct activity profile than melphalan (Gullbo J, et al., Anticancer Drugs 2003,14:617–24; Gullbo J, et al., Invest New Drugs 2004, 22:411–20; Wickstrom M, et al., Mol Cancer Ther 2007, 6:2409–17). J1 is rapidly incorporated into the tumor cells cytoplasm, followed by intracellular hydrolysis in part mediated by aminopeptidase N (APN), resulting in a 10-fold greater release of free intracellular melphalan than exposure to melphalan at the same molar concentration (Gullbo J, et al., J Drug Target 2003,11:355–63; Wickstrom et al., Biochem Pharmacol 2010, 79(9):1281-90). In vitro studies showed a greater cytotoxic potency of J1 versus melphalan against different human solid cancers; however, its effect in MM is undefined. In the present study, we examined the anti-tumor activity of J1 in MM cells using both in vitro and in vivo model systems. Methods and Models: We utilized MM.1S, MM.1R, RPMI-8226, melphalan-resistant derivative of RPMI-8226 (LR-5), KMS12BM, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability-, proliferation-, and apoptosis assays were performed using Trypan blue, MTT, thymidine incorporation, and Annexin V/Propidium iodide staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymologic assays. Statistical significance of data was determined using Student t test. Results: As aminopeptidase N (APN) has been shown to play a key role in conversion of J1 into melphalan in solid tumors, we first examined both expression and enzymatic activity of APN in MM cells. Immunoblot analysis showed a high expression of APN in various MM cell lines. Similarly, colorimetric analysis of APN enzymatic activity using the APN substrate L-alanine-4-nitro-anilide demonstrated elevated APN activity in MM cells. Moreover, pre-treatment of MM cells with APN inhibitor Bestatin showed a moderate, but significant blockade of J1-induced cytotoxicity in MM cells (P < 0.05; n=3). We next examined the effects of J1 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 0.5 – 1.0 uM; P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index forJ1. Of note, the IC50 range of melphalan for MM cell lines is 10–40 uM. J1-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by marked increase in Annexin V+ and PI-cell population (P < 0.001, n=3). Importantly, J1induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that J1-triggered apoptosis in MM cells is associated with 1) activation of caspase-7, caspase-8, caspase-9, caspase-3, and PARP; 2) induction of phospho-c-Jun and phospho-JNK, p53, and p21; 3) release of mitochondrial apoptogenic protein cytochrome-c; 4) inhibition of VEGF-induced migration of MM cells and angiogenesis; and 5) induction of DNA damage response, evidenced by increase in phospho-histone H2AX. Pre-treatment of MM cells with pan-caspase inhibitor Z-VAD-fmk attenuated J1-triggered MM cell apoptosis (P value < 0.001; n=3). Finally, treatment of tumor-bearing mice with J1 (3 mg/kg, twice a week for 2 weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Conclusions: Our study provides the rationale for clinical protocols evaluating J1, either alone or in combination, to improve patient outcome in MM. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Spira:Oncopeptide AB: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1852-1852 ◽  
Author(s):  
Malathi Kandarpa ◽  
Stephanie J Kraftson ◽  
Sean P Maxwell ◽  
Dilara McCauley ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Tumor Suppressors ◽  
Nuclear Export ◽  
Plasma Cells ◽  
Annexin V ◽  
Apoptosis Induction ◽  
Advisory Committees ◽  
Tumor Suppressor Proteins ◽  
Rpmi 8226

Abstract Abstract 1852 Background: CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012. Methods: To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1. Results: Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells. Conclusions: MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs. Disclosures: Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1361-1361 ◽  
Author(s):  
Daniel A Luedtke ◽  
Yongwei Su ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Combination Index ◽  
Survival Rates ◽  
Annexin V ◽  
Antileukemic Activity ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

scholarly journals Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 506 ◽  
Author(s):  
Tony Haykal ◽  
Peter Nasr ◽  
Mohammad H. Hodroj ◽  
Robin I. Taleb ◽  
Rita Sarkis ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Seed Extract ◽  
Leukemic Cells ◽  
Western Blots ◽  
Toxic Activity ◽  
Annona Cherimola

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.

scholarly journals Combination of CYC065, a Second Generation CDK2/9 Inhibitor, with Venetoclax or Standard Chemotherapies - a Novel Therapeutic Approach for Acute Myeloid Leukaemia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3938-3938
Author(s):  
Wittawat Chantkran ◽  
Daniella Zheleva ◽  
Sheelagh Frame ◽  
Ya-Ching Hsieh ◽  
Mhairi Copland
Keyword(s):  
Synergistic Effect ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Induction Of Apoptosis ◽  
Chemotherapy Agents

Objectives: Over the last 50 years, there has been a steady improvement in the treatment outcome of AML. However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. CYC065, a novel CDK inhibitor, has pre-clinical efficacy in AML. The survival of AML blasts is dependent on MCL-1 which is depleted following treatment with CYC065. The aims of this study are: i) to demonstrate target inhibition and characterise the mechanism of action of CYC065 in vitro; ii) to assess the effects of CYC065 on growth and survival of AML cells; and iii) to assess a synergistic effect of CYC065 in combination with other targeted or chemotherapy agents on growth and survival of AML cell lines and primary human AML cells. Methods: Western blotting was performed to elucidate the mechanism of action. CYC065 effects on gene expression were studied in three AML cell lines: OCI-AML3 (NPM1 and DNMT3A mutations), MOLM-13 (MLL-AF9) and MV4-11 (FLT3-ITD, MLL-AF4) by qRT-PCR using Fluidigm® Biomark technology. The efficacy of CYC065 was explored in the three cell lines in parallel with washout studies. A synergistic effect of CYC065 in combination with venetoclax, cytarabine, or azacitidine was explored in the three AML cell lines, seven primary AML and three normal control samples using a variety of assays measuring cell viability, apoptosis and proliferation. Results: Inhibition of CDK9 by 1µM CYC065 treatment for 4h and 24h was demonstrated in AML cell lines by a reduction in phosphorylation of pSer2 RNA polymerase II, leading to inhibition of transcription and loss of transcripts with short half-lives. As expected from the proposed mechanism of action, gene expression was generally suppressed following CYC065 treatment. Early events (4h) included decreases in cell cycle regulators including CDK7 and CDK9, E2F1, CDC25C and PPP1R10, pro-survival molecules, including MCL-1, BCL-2 and XIAP, and the MLL target genes, MEIS1 and RUNX1. Decreases in MCL-1 mRNA were confirmed at the protein level by Western blotting and preceded the induction of apoptosis (PARP cleavage). In functional assays, clinically relevant CYC065 concentrations of 0.75µM, 0.5µM and 1µM, which induce 50% apoptosis, were selected for the OCI-AML3, MOLM-13 and MV4-11 cell lines, respectively, to combine with 0.0018-0.9µM of venetoclax, 0.016-0.47µM of cytarabine, or 2-5.3µM of azacitidine using the drug combination ratio obtained from synergy assays using CompuSyn software. Preliminary results showed a synergistic activity in all cell lines. Washout studies showed a slight recovery of cell viability at low concentrations but not at 1μM CYC065. High diversity of genetic mutations was observed in the seven primary AML samples (see Table). In general, moderate to marked synergistic effects were observed in apoptosis and active caspase-3 assays when CYC065 in combination with the three partners when clinically achievable concentrations were used. The more complex the molecular genetic lesions or complexity of karyotype, the less efficacious the combination therapy. In proliferation assays, CYC065/venetoclax showed a slight synergistic effect, whereas, CYC065/cytarabine and CYC065/azacitidine combinations showed a marked synergistic effect in which an increase in a percentage of cell division arrest was observed. Conclusions: CYC065 pulse treatment effectively induced apoptosis in AML cell lines in vitro. Target inhibition was confirmed by analysis of gene and protein expression and was accompanied by induction of apoptosis and cell cycle arrest. A synergistic effect of CYC065 in combination with venetoclax, cytarabine, or azacitidine was seen in AML cell lines and primary AML cells. These results highlight the potential of CYC065 in combination with venetoclax or standard chemotherapy agents for the treatment of AML. Table Disclosures Zheleva: Cyclacel Ltd: Employment, Equity Ownership, Patents & Royalties. Copland:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Cyclacel: Research Funding.

Cyanidin 3-O-Glucoside Induces the Apoptosis in the Osteosarcoma Cells through Upregulation of the PPARγ and P21: An In Vitro Study

2020 ◽  
Vol 20 (9) ◽  
pp. 1087-1093
Author(s):  
Hesam A. Atashi ◽  
Hamid Z. Arani ◽  
Amirhossein Shekarriz ◽  
Hamidreza Nazari ◽  
Amirhossein Zabolian ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Mtt Assay ◽  
Malignant Tumors ◽  
Apoptotic Cell ◽  
Annexin V ◽  
In Vitro Study ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells

Background: Osteosarcoma (OS) is known as the malignant tumors in the bone. Cyanidin 3-OGlucoside (C3G) has a potential to induce the apoptotic cell death in different cancer cells; however, the mechanisms of action for C3G have not been clarified yet. Objective: In this study, the apoptotic effects of C3G on three different osteosarcoma cell lines including Saso-2, MG-63, and G-292 (clone A141B1) were investigated. Methods: The 24-hr IC50 of C3G for Saso-2, G-292, and MG-63 cells was evaluated by the MTT assay. Apoptosis induction in these cell lines after treatment with the C3G was approved by the Annexin V/PI flow cytometry. Changes at the mRNA expression level of PPARγ, P21, Bax, and Bcl-xl genes were investigated by real-time Polymerase Chain Reaction (PCR) technique, and P21 expression was further confirmed by the western blotting. Results: The MTT assay results demonstrated that the 24-hr IC50 of C3G was equal to 110μg/ml for Saso-2 and G-292 cells while it was about 140μg/ml for the MG-63 cells. The results of real-time PCR clearly showed that treatment of the cells with 24hrs IC50 of C3G caused the upregulation of PPARγ, P21, and Bax genes. Moreover, western blot analysis confirmed that P21 protein overexpressed endogenously after treatment of the cells with the C3G, and it was more upregulated in the MG-63 cells compared to the other cell lines. Conclusion: According to the findings of the study, the C3G is a novel anti-osteosarcoma agent with the ability to induce the apoptosis in different osteosarcoma cells through upregulation of the PPARγ and P21 genes.

Growth Inhibition and Synergistic Induction of Apoptosis By Synthetic Mir-125b-5p Mimics and Myc-Targeting Agents in Human Myeloma Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3019-3019
Author(s):  
Lavinia Biamonte ◽  
Cinzia Federico ◽  
Eugenio Morelli ◽  
Emanuela Leone ◽  
Maria Eugenia Gallo Cantafio ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Apoptotic Cell ◽  
Synergistic Effects ◽  
Annexin V ◽  
Transcriptional Level ◽  
Suppressor Activity

Abstract MicroRNAs (miRNAs), short non-coding RNAs which tune gene expression at post-transcriptional level, are recently emerging as key players in pathogenesis, progression and drug-resistance of multiple myeloma (MM). In this disease, they can act either with tumor-promoting or tumor-suppressing functions, depending on the nature of target mRNAs. Nowadays, effective strategies are available both to replace or to inhibit the expression of deregulated miRNAs, thus prompting the design of miRNA-based therapeutic strategies. We have recently demonstrated that miR-125b has tumor suppressor activity in MM and that enforced expression of synthetic miR-125b-5p mimics induces significant anti-MM activity in vitro and in vivo by targeting cell addiction to IRF4/cMyc pro-survival signaling. Moreover, we uncovered a functional feedback loop between cMyc and miR-125b in MM cells, whereas cMyc directly suppresses miR-125b transcription which, in turn, negatively regulates cMyc expression by targeting IRF4 mRNA. In the present study, we investigated the therapeutic potential of synthetic miR-125b-5p mimics combined with cMyc targeting agents, including the 10058-F4 small molecule inhibitor of cMyc-Max heterodimerization and the BET-bromodomain inhibitor JQ1, which is reported to inhibit cMyc transcription. At this aim, 3 MM cell lines (NCI-H929, SK-MM-1 and RPMI-8226) transfected with either miR-125b-5p mimics or scrambled oligonucleotides (miR-NC) were exposed to 10058-F4 (ranging from 10 to 100 μM) or JQ1 (ranging from 0,1 to 2μM) or DMSO. Effects on cell proliferation were then evaluated by CCK-8 assay at 24h, 48h and 72h time points, while the occurrence of apoptotic cell death was assessed by Annexin V flow-cytometry assay. Importantly, we found that enforced expression of miR-125b-5p mimics significantly and synergistically (synergistic index, SI >1) increases growth-inhibitory and pro-apoptotic activities of both 10058-F4 and JQ1. Similar results were observed in SK-MM-1 cells co-transfected with miR-125b-5p and cMyc siRNAs, while cMyc-defective U266 cells were not sensitized to either 10058-F4 nor JQ1 upon transfection with miR-125b-5p mimics. Furthermore, combinatorial treatments with JQ1 and miR-125b-5p mimics resulted in a stronger downregulation of cMyc protein, as compared to single molecules alone. Indeed, these results confirmed that impairment of cMyc activity/expression mediates the anti-MM synergistic effects between 10058-F4 or JQ1 and overexpression of miR-125b-5p by synthetic mimics. In conclusion, our data demonstrate a cMyc-mediated synergistic anti-MM activity of synthetic miR-125b-5p mimics with 10058-F4 or JQ1 cMyc targeting agents, providing the rationale for a more advanced preclinical investigations for the design of early clinical trials. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document