CellTiter-Glo Luminescent Cell Viability Assay Research Articles

Abstract 2598: Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent B-lymphocyte neoplasm (Morton, Blood 2006). The activated B-cell (ABC) subtype is associated with poorer prognosis (Lenz, NEJM 2008). Over 20% of ABC-DLBCL cases have mutations in the CD79B or CD79A subunits of the B-cell receptor (BCR), generating chronically active BCR signaling (Staudt, ASH 2012). Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton's tyrosine kinase, an essential BCR pathway enzyme. Phase 2 trial results of single-agent ibrutinib in DLBCL showed efficacy in the ABC subtype, with an overall response rate of 40% and 60% in CD79B-mutant ABC-DLBCL (Wilson, ASH 2012). Identification of MYD88 L265P mutations in ABC-DLBCL revealed a link between Toll-like receptor (TLR) signaling and ABC-DLBCL (Ngo, Nature 2011). The findings that a TLR 7/8/9 antagonist selectively inhibits cell survival of MYD88 L265P-mutated ABC-DLBCL cells (Bhagat, AACR 2014) emphasize the importance of TLR signaling in ABC-DLBCL. We hypothesized that TLR-related kinases/pathways important to ABC-DLBCL may be unknown and tested for synergy between ibrutinib and inhibitors targeting these pathways in ABC-DLBCL cells. Methods: Combination effects of ibrutinib and TLR9 antagonists, a TAK1 inhibitor, or an endosomal TLR inhibitor were tested in ABC-DLBCL cell lines with MYD88 mutations. TMD-8, HBL-1, and OCI-LY10 cell lines were treated with inhibitors or antagonists alone or in combination with ibrutinib for 3 days; cell growth effects were determined by the CellTiter-Glo luminescent cell viability assay (Promega). The combination index (C.I.), a drug interactivity measurement, was calculated with CalcuSyn. Synergy scores were calculated by the Chalice Analyzer (Horizon CombinatoRx). Results: We identified synergistic inhibitory effects on ABC-DLBCL cell growth between ibrutinib and TLR9 antagonists as well as the TAK1 inhibitor. Different TLR9 antagonist combinations significantly enhanced ibrutinib killing effects in ABC-DLBCL cells in the presence or absence of class A, B, and C TLR9 agonists. Stimulation with TLR9 agonists increased resistance of ABC-DLBCL cells. Strong synergy was also identified between ibrutinib and an inhibitor of endocytic TLRs (TLR3, 7, 8, 9) with an average C.I. of 0.11 and synergy score of 4.22. Our assays demonstrated that an endosomal TLR inhibitor possesses similar ibrutinib synergistic effects as other TLR9 antagonists. Similar results were observed for inhibition of the downstream TLR signaling molecule TAK1. Conclusions: We provide evidence of strong synergistic effects between ibrutinib and inhibitors targeting TLR signaling. Survival of ABC-DLBCL cell lines with MYD88 mutations requires signals through both TLR and BCR. Combined, these results provide rationale for targeting both pathways simultaneously in MYD88-mutated ABC-DLBCL. Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Betty Chang. Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2598. doi:10.1158/1538-7445.AM2015-2598

Comparative Analysis of in Vitro Chemosensitivity to Four Proteasome Inhibitors in Human Myeloma Cell Lines

Proteasome inhibitors (PI) are effective chemotherapeutic agents in the treatment of multiple myeloma (MM), used alone or in combination with other anti-cancer agents, such as alkylating agents, topoisomerase inhibitors, corticosteroids, histone deacetylase inhibitors (HDACis) and immunomodulatory drugs (IMiDs). Bortezomib (Velcade/Bz) was the first PI to be approved by US-FDA for the treatment of relapsed and refractory MM. Other second generation PIs include carfilzomib (Kyprolis/Cz), ixazomib/Iz and oprozomib (Opz). Wide inter-individual variation in response to treatment with PIs is a major limitation in achieving consistent therapeutic effect in MM. Yet few studies have compared the efficacy of all four PIs in a range of myeloma subtypes.In our current study, we performed comprehensive in vitro chemosensitivity profiling of response to four (4) PIs (Bz, Cz, Ix and Opz) in a panel of forty-five (45) human myeloma cells lines (HMCLs) generated through the immortalization of primary multiple myeloma cells (MMCs) and representing the biological and genetic heterogeneity of MM with regards to chromosomal abnormalities, oncogene mutations (e.g. Ras), tumor suppressor variations (e.g. p53), cell surface phenotypes, or growth factor response. Cells were treated with increasing concentrations of Bz, Cz, Ix and Opz as single agents and cell viability assays were performed using CellTiter-Glo luminescent cell viability assay to generate survival curves and determine the half maximal inhibitory concentration (IC50) values by calculating the nonlinear regression using sigmoidal dose-response equation (variable slope). Our results in comparing the cellular responses to PI treatment among HMCLs showed wide range of variability in IC50 values identifying some lines which were highly sensitive and some lines relatively refractory to PI treatment. Pearson product-moment correlation (PPMC) test demonstrated statistically significant (adjusted p values < 0.001) positive correlation between IC50 values of the following drug pairs: Bz vs Opz (r = 0.82); and Ix vs Opz (r = 0.88); Bz vs Ix (r = 0.65); Cz vs Opz (r = 0.69) and Cz vs Ix (r = 0.63). Subgroup analysis revealed significant correlation between carfizomib IC50 and chromosome number (p < 0.05). Furthermore, it was interesting to note that although all 4 drugs belong to the same drug class (PI), not all cell lines responded the same across all PI treatments. This demonstrates tumor heterogeneity even in response to inhibitors of the same class, and further demonstrates tumors refractory to one PI may still respond to another. We are currently examining genetic characteristics that are associated with response among the four PIs, and analysis of these characteristics will be presented. DisclosuresNo relevant conflicts of interest to declare.

Combination of Ibrutinib and BCL-2 or SYK Inhibitors in Ibrutinib Resistant ABC-Subtype of Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the US. DLBCL can progress quickly, and in advanced cases is inconsistently cured with current therapies. Ibrutinib, a first-in-class Bruton’s tyrosine kinase (BTK) inhibitor, is approved as a treatment for patients (pts) with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) who have had one prior treatment. The ABC subtype of DLBCL is considered especially high risk and characterized by chronic active B-cell receptor (BCR) signaling, which is blocked by ibrutinib. Recent phase II clinical trial results of ibrutinib as a single agent in DLBCL pts show an overall response rate of 41% in the ABC subtype (Wilson et al. ASH 2012). Responses of various cancers to single kinase targeted therapies are often limited by the cell’s ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway. It has been shown that a small number of CLL pts acquire resistance to ibrutinib through mutations in BTK and its substrate phospholipase C gamma 2 (PLCG2) in the B lymphoma cells following prolonged treatments (Woyach et al. NEJM 2014). Such mechanisms may be overcome by combinations of targeted agents. Through screening of wild-type and acquired ibrutinib-resistant ABC-DLBCL cell lines (e.g. expressing BTK C481S), we identify and report herein B-cell lymphoma-2 (BCL-2) and spleen tyrosine kinase (SYK) inhibitors that synergize with ibrutinib in vitro and in vivo.Methods: Gene expression was analyzed by RT-qPCR using TaqMan Gene Expression Master Mix. Human DLBCL cell lines were treated with drugs for 3 days and the effect on cell growth was determined by CellTiter-Glo luminescent cell viability assay. SCID mice were treated when the TMD8 tumors reached 100-150 mm3. Annexin-V-positive and PI-negative population was detected as apoptotic cells in tumor cells at sacrifice. Cell adhesion and migration assays were performed as previously described (Chang et al. Blood 2013). Analysis of clinical samples used for BCL-2 gene expression profiling was performed using Affymetrix microarrays on FFPE specimens from the phase 2 PCYC-1106 trial (NCT01325701) and a rank based statistic (RankProd) was used to determine the significance of gene expression changes.Results: DLBCL cell lines with higher BCL-2 expression were more sensitive to single agent ABT-199 than those with lower expression. Treatment of DLBCL cells with ibrutinib alone increased BCL-2 expression as well as their sensitivity to BCL-2 inhibitors. Combination treatment with BCL-2 inhibitors and ibrutinib completely inhibited tumor growth in murine models of ABC-DLBCL (Figure). Increased apoptotic cell populations were detected in the combination treated tumors compared to either treatment alone. Clinically, pretreatment tissue samples (n=28) from ABC-DLBCL pts who experienced objective responses to ibrutinib (CR+PR) had lower BCL-2 gene expression. A high BCL-2 mutation rate was observed in pts with poor response to ibrutinib (SD+PD). However, none of these mutations occurred in the BH3 or BH1 domains, both of which appear to interact with ABT-199 based on a 3-dimensional co-crystal structure of the inhibitor with BCL-2 (PDB code 4MAN) and further molecular simulation results. These findings suggest the potential benefit from combination therapy.SYK is another downstream mediator of BCR signaling. Pretreatment of DLBCL cells with SYK inhibitors (e.g. R406) increased their sensitivity to ibrutinib. Ibrutinib resistant B-lymphoma cells with either C481S BTK or R665W PLCG2 mutations were re-sensitized to ibrutinib in combination with BCL-2 or SYK inhibitors, inhibiting cell growth, IgM-induced calcium flux, cell adhesion or migration in mutant containing cells.Conclusions: Consistent with previous results from high-throughput combinatorial screenings of drugs interact favorably with ibrutinib (Mathews Griner, et al. PNAS 2013), we found BCL-2 and SYK may function in alternative survival pathways in DLBCL cells upon BTK inhibition. Human B lymphomas harboring ibrutinib-resistant C481S BTK or R665W PLCG2 may be re-sensitized by BCL-2 or SYK inhibitors, both of which provide a rationale for the design of combination therapies. [Display omitted] DisclosuresKuo:Pharmacyclics: Employment. Crowley:Pharmacyclics: Employment. Xue:Pharmacyclics: Employment. Schweighofer:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment. Hsieh:Pharmacyclics: Employment. Eckert:Pharmacyclics: Employment. Versele:Janssen Pharmaceutica: Employment. Chang:Pharmacyclics, Inc: Employment, Equity Ownership.

Histone deacetylase 6 activity is critical for the metastasis of Burkitt’s lymphoma cells

BackgroundBurkitt’s lymphoma is an aggressive malignancy with high risk of metastasis to extranodal sites, such as bone marrow and central nervous system. The prognosis of metastatic Burkitt’s lymphoma is poor. Here we sought to identify a role of histone deacetylase 6 (HDAC6) in the metastasis of Burkitt’s lymphoma cells.MethodsBurkitt’s lymphoma cells were pharmacologically treated with niltubacin, tubacin or sodium butyrate (NaB) or transfected with siRNAs to knock down the expression of HDAC6. Cell migration and invasion ability were measured by transwell assay, and cell cycle progression was analyzed by flow cytometry. Cell adhesion and proliferation was determined by CellTiter-Glo luminescent cell viability assay kit. Cell morphological alteration and microtubule stability were analyzed by immunofluorescence staining. Effect of niltubacin, tubacin and NaB on acetylated tubulin and siRNA efficacy were measured by western blotting.ResultsSuppression of histone deacetylase 6 activity significantly compromised the migration and invasion of Burkitt’s lymphoma cells, without affecting cell proliferation and cell cycle progression. Mechanistic study revealed that HDAC6 modulated chemokine induced cell shape elongation and cell adhesion probably through its action on microtubule dynamics.ConclusionsWe identified a critical role of HDAC6 in the metastasis of Burkitt’s lymphoma cells, suggesting that pharmacological inhibition of HDAC6 could be a promising strategy for the management of metastatic Burkitt’s lymphoma.

Abstract 2515: Synthesis of substituted chromone-2-phenylcarboxamides as anti-breast cancer agents

Abstract Breast cancer is one of the most commonly diagnosed cancers and causes the second leading cancer deaths in women. The overall survival rate of breast cancer patients has substantially increased during the last decades due to early tumor detection. Unfortunately, the recovery rate of advanced breast cancer by current available drug treatment is still unacceptably low. Although chemotherapy is the main stay of cancer treatment at present, most of the drugs cause general toxicity to any proliferating cells and tend to severely limit the therapeutic values of these drugs. In an attempt to overcome these problems, the synthesis of new anti-breast cancer agents with unique mechanisms of action was developed. Chromones are benzannulated oxygen containing heterocyclic compounds widely distributed in nature that have a -pyrone ring. The chemical core of chromone has been classifies as a privileged structures in drug discovery, due to its uses in a wide variety of pharmacologically active compounds such as anti cancer, anti-HIV, antibacterial and anti-inflammatory agents. Based on these considerations, we attempted the synthesis of novel substituted chromone-2-phenylcarboxamides as anti-breast cancer agents. The starting material chromones-2-carboxylic acid was obtained by the hydrolysis reaction of ethyl esters with the mixture of glacial acetic acid and concentrated HCl (2:1v/v) and the mixture was heated to reflux for 12 h. The acids were obtained in 70-80% yields. A suspension of substituted chromone-2-carboxylic acid in DMF was added to substituted anilines, in the presence of diphenylphosphorylazide and triethylamine. The reaction mixture was stirred at room temperature for 24 h, which furnished the target compounds i.e., substituted chromone-2-phenylcarboxamides. These compounds were evaluated for their cytotoxic effects on MCF-7 ER+ve breast cancer cells, MDA-MB-231 ER-ve breast cancer cell lines and Ishikawa cells using the CellTiter-Glo luminescent cell viability assay. N-(4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromene-2-carboxamide showed the most potent cytotoxicity with an IC50 values of 29.5 and 25.7 µM on MCF-7 and Ishikawa cell lines, respectively. This research was supported by National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Mohammad A. Ghaffari, Suresh Eyunni. Synthesis of substituted chromone-2-phenylcarboxamides as anti-breast cancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2515. doi:10.1158/1538-7445.AM2014-2515

Interactions Between Ataxia Telangiectasia Mutated Kinase Inhibition, Poly(ADP-ribose) Polymerase-1 Inhibition and BRCA1 Status in Breast Cancer Cells.

Background:Cells harboring BRCA1/BRCA2 mutations are hypersensitive to inhibition of poly(ADP-ribose) polymerase-1 (PARP-1). We recently showed that interference with PARP-1 activity by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells but not BRCA1-deficient SKBr-3 cells. These unexpected observations prompted speculation that other PARP-1 inhibitor(s) may be more cytotoxic towards SKBr-3 cells. In addition, interference with the DNA damage signaling pathway via (for instance) Ataxia telangiectasia mutated (ATM) kinase inhibition may induce synthetic lethality in DNA repair-deficient breast cancer cells and pharmacological interference with ATM activity may sensitize breast cancer cells to PARP-1 inactivation.Methods:We determined drug cytotoxicity in human MCF-7 and SKBr-3 breast cancer cells using the CellTiterGLO Luminescent cell viability assay and a Tecan multi-label, multitask plate counter to measure generated luminescence. Changes in cell cycle progression were monitored by flow cytometric measurement of DNA content in cells stained with propidium iodide.Results:Unlike NU1025, AZD2461, a new PARP-1 inhibitor, markedly reduced the numbers of living MCF-7 and SKBr-3 cells. ATM kinase inhibition (CP466722) was also cytotoxic for both MCF-7 and SKBr-3 cells. Furthermore, AZD2461 enhanced the cytotoxicity of CP466722 in both cell lines by inducing apoptosis, and concurrent inhibition of ATM and PARP-1 reduced cell proliferation more strongly than either single treatment.Conclusions:Our data show that inhibition of PARP-1 by AZD2461 is synthetically lethal for NU1025-resistant MCF-7 and SKBr-3 breast cancer cells. They also indicate that DNA damage signaling is essential for survival of both SKBr-3 and MCF-7 cells, especially after inactivation of PARP-1.

Brucine inhibits the proliferation of human lung cancer cell line PC-9 via arresting cell cycle

背景与目的已有的研究表明：Cyclin D1和Cyclin E是细胞周期中重要的正性调控因子，其高表达与肿瘤的增殖密切相关。本研究旨在探讨马钱子碱（Brucine）对人肺癌细胞株PC-9增殖的影响，及其与Cyclin D1和Cyclin E表达的影响。方法将PC-9细胞分为4组：空白对照组、DMSO对照组（2‰）、150 μM Brucine组、300 μM Brucine组。CellTiter-Glo发光法、平板克隆形成实验观察该药对PC-9细胞增殖的影响，流式细胞仪检测细胞周期，qRT-PCR检测细胞周期相关基因Cyclin D1、Cyclin E mRNA的表达，Western blot检测细胞周期相关基因Cyclin D1、Cyclin E蛋白的表达。结果与对照组比较，CellTiter-Glo发光法、平板克隆形成实验结果显示：Brucine可以抑制人肺癌细胞株PC-9的增殖，并呈时间-剂量依赖性（P < 0.01）；流式结果显示对细胞周期的影响主要是阻滞PC-9细胞于G0/G1期；qRT-PCR结果显示Cyclin D1、Cyclin E mRNA的表达下调；Western blot结果显示Brucine使Cyclin D1、Cyclin E的表达降低。结论Brucine能明显抑制人肺癌细胞株PC-9的增殖，机制主要与其通过下调Cyclin D1、Cyclin E表达，进而阻滞细胞周期有关。

Inhibition of Activin A Ameliorates Skeletal Muscle Injury and Rescues Contractile Properties by Inducing Efficient Remodeling in Female Mice

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Leonurusoleanolides E–J, Minor Spirocyclic Triterpenoids from Leonurus japonicus Fruits

Six new (leonurusoleanolides E-J, 1-6) and five known (7-11) nortriterpenoids were isolated and characterized from the dried fruits of Leonurus japonicus. They all contain a distinctive 19(18→17)-abeo-28-noroleanane-type spirocylclic skeleton with a trans or a cis acyl substituent at C-3 or C-23. Similar to the previously known leonurusoleanolides A/B (7/8) and C/D (9/10), compounds 1/2 and 3/4 were also found to exist as equilibrium mixtures of trans and cis isomers. The isolated pure compounds and mixtures were evaluated for their cytotoxicity against a small panel of human cancer cell lines (BGC-823 and KE-97 gastric carcinoma, Huh-7 hepatocarcinoma, Jurkat T cell lymphoblasts, and MCF-7 breast adenocarcinoma) using the CellTiter-Glo luminescent cell viability assay method. Among them, (2α,3β,17R*,18β)-3-O-(trans-caffeoyl)-19(18→17)-abeo-28-norolean-12-ene-2,18,23-triol (leonurusoleanolide J, 6) showed the most potent cytotoxic activity, with IC50 values less than 10 μM.

ONO-4059, A Novel Bruton's Tyrosine Kinase (Btk) Inhibitor: Synergistic Activity In Combination With Chemotherapy In a ABC-DLBCL Cell Line

PurposeBruton's tyrosine kinase (Btk) is a key regulator of the B-cell receptor (BCR) signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. Recent studies indicate that targeting Btk is effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC50 in the sub-nmol/L range. The activated B-cell-like (ABC) sub-type of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and a substantial unmet need still exists. The standard treatment with Rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) for ABC-DLBCL in particular being associated with a poorer outcome. The combination of ONO-4059 with the Bcl-2 inhibitor, ABT-263 has demonstrated greater apoptosis in ABC-DLBCL cell lines than that seen with single agent treatment (Ryohei K. et al., EHA 2013 meeting). To explore rational combinations, ONO-4059 combined with chemotherapy drugs was evaluated in a ABC-DLBCL cell line. MethodsABC-DLBCL cell line (TMD-8) was incubated with ONO-4059 monotherapy (1-1000 nmol/L) and in combination with various agents that target various pathways thought to be involved in DLBCL. Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay. Apoptosis was determined by AnnexinV-FITC/7AAD (AnnV+/7AAD-) staining. ResultsONO-4059 inhibited the TMD-8 cell growth and Btk phosphorylation in a concentration-dependent manner, with an IC50 of 3.59 for 72 hr and 23.9 nmol/L for 4 hr respectively. Furthermore, the decrease in Btk phosphorylation subsequently down-regulated Erk phosphorylation. At the 48 hr time-point, 7% of apoptosis occurred in TMD-8 cells at 10 nmol/L, an earlier time-point than that observed for dead cell. After the combination of 10 nmol/L of ONO-4059 with 25 nmol/L of doxorubicin, 500 nmol/L of etoposide, 1.5 nmol/L of vincristine and 1000 nmol/L of dexamethasone, the increased apoptosis ratio was observed, 25, 20, 17 and 29%, respectively. Synergy was observed when ONO-4059 was combined with the CHOP regimen. ConclusionRecent studies indicate that targeting Btk induces BIM expression, the pro-apoptotic protein, following inhibition of Erk phoshphorylation. Our results demonstrate that treatment with ONO-4059 in combination with chemotherapy results in a synergistic effect, inducing apoptosis and is more effective compared with respective monotherapies. ONO-4059 is currently being developed in a Phase I clinical trial for the treatment of B-cell malignancies. Additional combination studies are underway using the TMD-8 xenograft model, testing ONO-4059 in combination with various agents. Disclosures:Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Yoshizawa:Ono Pharmaceutical CO., Ltd: Employment. Birkett:Ono Pharma UK: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

HZ08 reverse the aneuploidy-induced cisplatin-resistance in Gastric cancer by modulating the p53 pathway

We evaluated the influence of DNA aneuploidy on chemotherapy-resistance in human Gastric cancer cell MKN45; we also evaluated the reversal effects of HZ08 on these cells and then preliminary investigated the possible involved pathway. We made use of a pair of human Gastric cancer cell dip-MKN45 (diploid MKN45) and aneu-MKN45 (aneuploid MKN45). Growth inhibition in response to chemotherapeutic drugs was evaluated by CellTiter-Glo Luminescent Cell Viability assay and clone formation assay. Flow cytometry and immuno-assay were applied to evaluate apoptosis and the expression of relative signaling molecules. MKN45 xenograft was generated to evaluate in vivo action. Aneu-MKN45 developed a resistance to cisplatin which could be reversed by HZ08; Flow cytometry and western-blot indicates that HZ08-combination could induce apoptosis and increase the expression of apoptosis-related biomarkers on aneu-MKN45; in vivo study also reflect the same correlation between aneuploidy and cisplatin-resistance, which could be antagonized by HZ08 combination; When investigating the involved pathway, in anue-MKN45, the expression of molecules in p53 pathway was decreased; HZ08 could increase the expression of p53 down-stream molecules as well as elevate the activity of p53, while inhibiting Mdm2, the major negative regulator of p53; p53 inhibitor Pifithrin-α could completely abrogate HZ08's synergism effects, and mimic cisplatin-resistance on dip-MKN45.Lower p53 pathway expression that attenuates cisplatin-induced apoptosis might be at least partly the reason of cisplatin-resistance occurred in aneuploid MKN45 both in vitro and in vivo; Combination of HZ08 could sensitize cisplatin-induced apoptosis through the activation of the p53 pathway, therefore represented a synergism effect on aneuploid MKN45 cells.

Abstract 2229: Synthesis of heterocyclic compounds as anticancer agents.

Abstract Cancer is a very complex disease, linked with different initiating causes, cofactors and promoters, and several types of cellular damages. There is a strong correlation between chronic inflammatory conditions in a particular organ and cancer specific to that organ. The longer the inflammation persists, the higher the risk of associated carcinogenesis. Drug discovery process has changed dramatically over the past decade as there is an increasing demand to obtain more drug candidates and decrease attrition during drug development. Chromone scaffold [(4H)-1-benzopyran-4-one] has been recognized as a pharmacophore of a large number of bioactive molecules of either natural or synthetic origin. Until now, numerous biological effects such as anti-inflammatory, antitumoral, and antimicrobial activities especially in popular medicine, have been attributed to this benzo-γ-pyrone nucleus. Compounds of this type also possess inhibition properties towards different enzymes such as oxidoreductases, kinases, tyrosinases, lipoxygenases and cyclooxygenases. Chemotherapeutic agents like tetrahydropyridine (THP) analogs exhibit diverse biological activities like analgesic and anti-inflammatory effects. Our earlier research results indicated that some of the THP derivatives showed COX-2 inhibition and anti-inflammatory activities on rat paw edema assay studies. Incorporation of THP moiety might enhance biological activity of the chromone derivatives. Herein, we report the synthesis of substituted N-(5,6-dihydropyridin-1-(2H)-yl)-4-oxo-4H-chromene-2-carboxamide as anticancer and anti-inflammatory agents. The starting material 4-oxo-4H-chromene-2-carbonyl chloride was obtained by the reaction of 4-oxo-4H-chromene-2-carboxylic acid, dissolved in 1,2-dichloroethane and SOCl2 containing few drops of DMF under reflux for 4h. The crude acid chloride obtained by removing the excess of SOCl2 and solvent under vaccum, was used immediately for next step. Reaction of the acid chloride with substituted N-amino pyridinium salt in anhydrous tetrahydrofuran gave stable ylides. This was followed by reduction with sodim borohydride in absolute ethanol, which furnished the target compound. These compounds were evaluated for their cytotoxic effects on MCF-7 ER positive breast cancer cells, MDA-MB ER receptor negative breast cancer cells, and Ishikawa cell line using the CellTiter-Glo (CTG) luminescent cell viability assay. N-(4-tert-butyl-5,6-dihydropyridin-1(2H)-yl)-6-chloro-7-methyl-4-oxo-4H-chromene-2-carboxamide showed the cytotoxicity with an IC50 values of 73.28 μM on MDA-MB-231 cell lines. This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Mohammad A. Ghaffari, Suresh Eyunni, Nelly Mateeva, Bereket Mochona. Synthesis of heterocyclic compounds as anticancer agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2229. doi:10.1158/1538-7445.AM2013-2229

Abstract 3816: DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma.

Abstract Background: Neuroblastoma remains responsible for a disproportionately high number of pediatric cancer deaths, necessitating novel treatment approaches. Our lab has previously reported DNA variations in the BRCA1-associated RING domain 1 (BARD1) gene to be significantly associated with high-risk neuroblastoma in an ongoing genome-wide association study (Capasso Nat Gen 2009). The expression of an oncogenic isoform, BARD1β, correlated with this disease associated common variation, and isoform specific depletion showed differential growth inhibition in a risk haplotype dependent manner (Bosse Can Res 2012). Furthermore, this isoform was shown to bind to and stabilized aurora kinase A and B in neuroblastoma cells, suggesting a mechanism for its oncogenicity and a potential therapeutic strategy. Methods: Short interfering RNAs (siRNAs) targeting AURKA, AURKB and BARD1β were tested individually in a panel of neuroblastoma cell lines (n=8) stratified by risk haplotype. Cell growth was measured by real time cell electronic sensing (RT-CES), and mRNA knockdown was confirmed by quantitative PCR. The pharmacologic inhibitors MLN8237, a selective aurora kinase A inhibitor; AZD1152, a selective aurora kinase B inhibitor; and CCT137690, a pan-aurora kinase A and B inhibitor were tested in vitro in an expanded neuroblastoma cell line panel (n=10), and cell viability was measured with a CellTiter-Glo(R) Luminescent Cell Viability Assay. Results: AURKB knockdown resulted in a trend of greater growth inhibition in neuroblastoma cell lines harboring a BARD1 risk haplotype, directly correlating with growth inhibition following BARD1β knockdown (R2=0.57, p=0.03). AURKA depletion showed significant growth inhibition (mean inhibition=45%, p&amp;lt;0.0001), but no correlation with BARD1 genotype was observed. Aurora A kinase inhibition with MLN8237 resulted in significant cytotoxicity (IC50 30 -750nM), and further analysis of published studies using MLN8237 in vivo on murine xenograft models of neuroblastoma showed that the cell lines with the BARD1 risk haplotype had significantly greater tumor reduction with this single agent therapy. AZD1152 and CCT137690 were less potent inhibitors of growth (IC50 30nM-10mM and 250nM-3mM, respectively). Conclusion: Taken together, these data support a model of BARD1 acting in a pathway involving the aurora kinase proteins in neuroblastoma cells, suggesting that BARD1 genotypes have the potential to be a biomarker of aurora kinase inhibitor activity, but the associations are complex and unlikely to be binary predictors of sensitivity or insensitivity. These data support the continued investigation of MLN8237 in clinical trials for high-risk neuroblastoma and further exploration of the mechanisms by which DNA variation at BARD1 influences neuroblastoma oncogenicity and therapeutic vulnerabilities. Citation Format: Vanessa M. Pineros, Kris R. Bosse, Lori S. Hart, Sharon J. Diskin, JulieAnn Rader, John M. Maris. DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2013-3816

Antiplasmodial Activity and Cytotoxicity of 10β-Aminoquinolinylethylethers of Artemisinin

Each year roughly 800 000 people die of malaria, with 95% being African children. The shortcomings of the current drugs and the emergence of P. falciparum resistance to the artemisinin class of compounds warrant the search for new classes or derivatives. In search for such compounds, a series of 10β-amino-quinolinylethylethers of artemisinin, previously synthesized from this laboratory were screened for antimalarial activity against both the chloroquine-susceptible 3D7 and -resistant K1 strains of P. falciparum. Their cytotoxicity was also assessed against HEK 293 and HepG2 cell lines.The parasitic and mammalian cells were incubated with compounds at various concentrations for 72 h. The antimalarial activity was determined using SYBR Green I-based fluorescence. For cytotoxicity determination, cells were grown to confluence and CellTiter-Glo luminescent cell viability assay was used.All derivatives proved to be active against both strains with good selectivity towards the parasitic cells. The derivative 11 featuring 2 artemisinin moieties and an aminoethylpiperazine linker was the most active of all. It possessed 17- and 166-fold more potency than artemether against 3D7 (EC50: 9.5 vs. 166 nM) and K1 (10.9 vs. 1723.3 nM), respectively, while was found to be as potent as artesunate against both strains.Derivative 11 stands as a good candidate to be further investigated primarily in vitro in comparison with an equimolar combination of chloroquine (CQ) and artemisinin to ascertain its advantages, if any, over the combination.

1674P - Identification of Phosphorylated Ribosomal Protein S6 as a Potential Predictor of Hepatocellular Carcinoma Response to Sorafenib by Pathway-Based Phosphoprotein Profiling

ABSTRACT Backgroud Sorafenib is a multi-kinase inhibitor that has been proved to be effective for the treatment of advanced hepatocellular carcinoma (HCC). However, only a small proportion of patients receiving sorafenib obtain the anticipated therapeutic benefits. It would therefore be desirable to clarify the precise molecular mechanisms that confer resistance to sorafenib and identify a predictive biomarker for unresponsiveness to sorafenib. Methods We constructed reverse-phase protein microarrays (RPPAs) onto which whole lysates of 95 cell lines derived from various tumors, including 23 HCCs, were plotted and examined for phosphorylation expression of 183 proteins serving as nodes in 120 signaling pathways registered in the NCI-Nature curated pathway (NCI-Nature_Curated.bp2.owl 2011.12.19). The sensitivity (IC50 value) of the 23 HCC cell lines to sofafenib was quantified using the CellTiter-Glo Luminescent Cell Viability Assay kit. Results Use of fluorescence dye significantly expanded the sensitivity and dynamic range of the RPPAs. We found that the relative level of phosphorylation of ribosomal protein S6 (p-rpS6) at the Ser235/236 residues showed the highest correlation with sensitivity to sorafenib among the 183 signaling nodes (P = 0.0044, Spearman correlation analysis), followed by phosphorylation of the same protein at the Ser240/244 residues (P = 0.0070). Immunoblot analysis confirmed that the sorafenib-resistant cell lines had the highest level of rpS6 phosphorylation. We found that in some HCC cell lines the mitogen-activated protein kinase (MAPK) pathway was activated downstream of RAF kinase and showed sensitivity to MEK (CI-1040) and RSK (SL0101) inhibitors. Conclusions To achieve personalization of molecular therapies, precise mapping of activated signaling pathways in individual patients is essential. RPPA requires only a small amount of protein and is ideal for application to clinical settings. We are collecting tumor biopsy samples from HCC patients to verify the present findings, and plan to present the data at the meeting. Disclosure All authors have declared no conflicts of interest.

Abstract 2021: ONO-WG-307, a novel, potent and selective inhibitor of Bruton's tyrosine kinase (Btk), results in sustained inhibition of the ERK, AKT and PKD signaling pathways

Abstract Purpose: Btk is a key regulator of B cell receptors (BCR) which play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk may be effective in the treatment of B-cell lymphoma. Response between cell survival and BCR signaling is implicated in different types of B-cell lymphoma. Therefore, we evaluated the inhibitory effect of ONO-WG-307 on Btk-dependent signal transduction by large-scale and quantitative phosphoproteome analysis to a depth of more than 10,000 phosphorylation sites. Methods: Two tumor cell lines (sensitive and non-sensitive) were treated with ONO-WG-307. After 72h, cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay. P-Btk was used as a marker for Btk activity determined by Western blot and Flow Cytometry. Quantitative phosphoproteomics were enabled by differential SILAC labeling of lymphoma cells. After 1 hr incubation with ONO-WG-307, the total cellular proteins were digested and phosphopeptides were enriched by a combination of strong cation exchange and immobilized metal affinity chromatography. Site-specific phosphorylations were identified and by LC-MS analysis on a LTQ-Orbitrap-Velos mass spectrometer followed by bioinformatic processing. Results: Btk is potently and selectively inhibited by ONO-WG-307 in an in vitro Btk kinase assay, with an IC50 in the subnanomolar range whereas IC50 values for other tyrosine kinases (Lck, Lyn and Fyn) were above 1 µM. The concentration required for growth inhibition for the sensitive cells was 3.59 nmol/L (IC50), whereas considerably higher concentrations were required to suppress growth of non-sensitive cells. Surprisingly, inhibition of cellular Btk and ERK phosphorylation by ONO-WG-307 levels of P-Btk and P-ERK were similar in both sensitive and non-sensitive cells. However, our quantitative phosphoproteome studies revealed selective suppression of Akt-mediated signaling and cellular protein kinase D activity in sensitive compared to non-sensitive cells.Conclusion: These results suggest that the selective ONO-WG-307 inhibition of “responder” cell growth might be due to a critical role of Btk-mediated signaling through Akt and protein kinase D. These data shed new light on the cellular mode-of-action of Btk inhibition and support the potential clinical utility of ONO-WG-307 in B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2021. doi:1538-7445.AM2012-2021

Abstract 5385: Effect of lenalidomide on antileukemia activity in vitro against adult T-cell leukemia (ATL)

Abstract Background: Lenalidomide is an FDA approved agent for del 5q myelodysplastic syndromes and in combination with dexamethasone for previously treated multiple myeloma. Recently, phase II clinical trial has shown its anti-tumor activity against recurrent and refractory T-cell lymphomas. However, the mechanism underlining its effect is poorly understood. Adult T-cell leukemia (ATL) is a mature T cell malignancy caused by Human T-cell leukemia virus type I (HTLV-I) that is endemic in southwestern Japan, the Caribbean islands and Central Africa, and it still remains as one of the most difficult malignancies to be cured. We have evaluated the anti-ATL activity of lenalidomide in vitro using HTLV-I positive cell lines. Methods: The effect of lenalidomide (10-3-102µM) and Fas antibody (25-400 ng/mL) was assessed on the proliferation of HTLV-I positive cell lines by CellTiter-Glo Luminescent Cell Viability Assay. The cytotoxic effect against HTLV-I positive cell lines of lenalidomide on IL-2 primed peripheral blood mononuclear cells (PBMNCs) as well as sorted CD56 positive and CD3 negative cells from IL-2 primed PBMNCs from healthy volunteers was studied by 51Cr releasing assay. Results: Lenalidomide alone did not inhibit cellular proliferation. Even 6 day incubation did not inhibit or stimulate proliferation of HTLV-1 positive cell lines. Cytotoxic activity of IL-2 primed PBMNCs from healthy volunteers against HTLV-I positive cell lines was enhanced by lenalidomide. Sorted NK cells have also shown enhanced cytotoxic activities on HTLV-1 positive cells. That effect was observed regardless of Tax or cellular FLICE-inhibitory protein (cFLIP) expression. Therefore, even Fas resistant HTLV-1 positive cell lines were succumbed to death by enhanced cytotoxicity by lenalidomide, whereas lenalidomide did not interact Fas inducing apoptotic pathways. Conclusions: These results have shown that lenalidomide enhances cytotoxic effects against HTLV-I positive cells, without direct anti-proliferative effect on those cell lines. That effect was mediated through CD56 positive and CD3 negative cells stimulated by lenalidomide. Further study is now on-going to elucidate the mechanism of those actions in an in vivo setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5385. doi:1538-7445.AM2012-5385

Abstract 3903: Synthesis of substituted N-{4-[(2-hydroxyethyl)sulfanyl]-3,6-dihydropyridin-1(2h)-yl} benzamide/benzenesulfonamide as antiinflamatory and anticancer agents

Abstract Breast cancer is the most common type of cancer among women in the industrialized world, accounting for nearly one of every three cancers diagnosed. There is a strong association between chronic inflammatory conditions in a particular organ and cancer specific to that organ. The longer the inflammation persists, the higher the risk of associated carcinogenesis. There is abundant epidemiological and experimental evidence that NSAID's might inhibit tumor development in a number of organs. COX-2 expression may be a fundamental step in breast cancer pathogenesis acting through prostaglandin-dependent mechanisms. Tetrahydropyridine (THP) analogs are compounds of diverse biological activities like analgesics, anti-inflammatory and chemotherapeutic agents. Our earlier research results indicated that some of the THP derivatives showed COX-2 inhibition and anti-inflammatory activities on rat paw edema assay studies. There has been remarkable effort in developing new classes of compounds to minimize the side effects of the NSAIDs and increase the selectivity of COX-2 inhibitors. We now report the synthesis of substituted N-{4-[(2-hydroxyethyl)sulfanyl]-3,6-dihydropyridin-1(2H)-yl} benzamide/benzenesulfonamide as anti-inflammatory and anticancer agents to continue the development of new classes of anti-inflammatory and anticancer agents. The starting material ethyl (pyridin-4-ylsulfanyl)acetate was obtained by the reaction of (4-pyridylthio)acetic acid dissolved in ethanol and several drops of 98% H2SO4 and the mixture was heated to reflux for 12 h. Mesitylene sulfonyl chloride was added with stirring to a solution of ethylacetohydroxymate and triethylamine in dimethylformamide at 0o C. Hydrolysis of this compound with the mixture of p-dioxane-70% perchloric acid and allowing them to react for 45 min gave a white solid of O-mesitylene sulfonyl hydroxylamine (MSH). Ethyl (pyridin-4-ylsulfanyl)acetate was reacted with MSH in dichloromethane to produce 1-amino-4-(2-ethoxy-2-oxoethylthio)pyridinium mesitylenesulfonate. Reaction of the amino salt with substituted benzoyl chloride/benzenesulfonyl chloride in anhydrous tetrahydrofuran gave stable benzoyl/benzene sulfonyl imino ylides. This was followed by reduction with sodium borohydride in absolute ethanol, which furnished the target THP compounds. These compounds were evaluated for their cytotoxic effects on MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cell line, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay. Two compounds showed potent anticancer activities using these cell lines. This research was supported by NIH/ RCMI Grant # G12 RR 03020 and Pharmaceutical Research Center NIH/NCRR Grant 1 C06 RR12512-01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3903. doi:1538-7445.AM2012-3903

Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.