Diverse Human Cancer Cell Lines Research Articles

Structure‐Activity Analysis of Bisphenol‐Z Derivatives for Anticancer Activity

Much of the research surrounding bisphenol derivatives focuses on their use as polymers and plasticizers for industry. Research has demonstrated that bisphenol‐A (BPA) adversely affects humans via endocrine disruption through hormonal modulation. Despite the recent explosion in BPA research, little effort has been devoted to researching the biological activities of other bisphenol derivatives, including bisphenol‐Z (BPZ). Initial screening of BPZ revealed moderate in vitro anticancer activity against four diverse human cancer cell lines (A375 malignant melanoma, A‐172 glioblastoma, HT‐29 colorectal adenocarcinoma, and MCF7 breast adenocarcinoma) with IC50 values of approximately 65 μM following 24 hour treatments. In an attempt to enhance the activity of BPZ, seven analogs with varying degrees of polarity, lipophilicity, and sterics on the cyclohexane ring were synthesized. Screening of these analogs revealed that polar substitutions resulted in a loss of activity, while a simple methyl substitution had similar activity to BPZ. Anticancer activity was enhanced relative to BPZ following substitution with an ethyl, isopropyl, or N‐palmitoyl group at the 4‐ position. The most active compounds from this structure‐activity study were the N‐palmitoyl (LS4) and isopropyl (LS6) derivatives with 24 hour IC50 values ranging from 25 to 53 μM and 33 to 42 μM respectively.Given the increased activity of LS4 and LS6, additional studies were performed including a trypan blue exclusion assay and a Caspase‐Glo® 3/7 assay. The results of these studies revealed exclusion of trypan blue at supra‐IC50 concentrations six hours after treatments, suggestive of a non‐necrotic mechanism of death. To determine if caspase‐dependent apoptosis was involved, we performed a Caspase‐Glo® 3/7 assay. Functional screening of executioner caspase‐3 and ‐7 revealed significant increases in activity following a 12 hour treatment with LS4 in all cells lines. This effect was least evident in the MCF7 cells, potentially due to a mutation wherein these cells lack expression of caspase‐3. In contrast, LS6 failed to activate executioner caspases‐3 and ‐7 in these same cell lines.In conclusion, this structure‐activity study revealed that nonpolar substitution at the 4‐ position of BPZ enhanced anticancer activity. Additionally, membrane integrity remained intact following exposure to LS4 and LS6. While LS4 activated executioner caspases‐3/7, LS6 failed to activate this hallmark pathway of apoptotic death. Further investigations are ongoing to elucidate the mechanisms of cell death, identify putative cellular targets, and determine potential selectivity against normal human cell lines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure‐Activity Relationship of Novel Compounds Based on Tamoxifen with Proposed Sigma‐2 Receptor Activity

Sigma‐2 receptors are overexpressed in several different types of human cancer cells and are regarded as potential therapeutic targets. Ligands for sigma‐2 receptors have been shown to elicit anticancer properties including growth arrest and apoptosis. While much research has been devoted to designing sigma‐2 receptor ligands for therapeutic benefits, none have stood up to rigorous testing and clinical trials needed for approval in human patients. In an attempt to provide more literature on the potential usefulness of sigma‐2 receptor ligands for anticancer activity, we synthesized a series of analogs based upon the known sigma‐2 receptor ligand tamoxifen that lack estrogen receptor binding properties.We previously reported on the first molecule in this series, SP‐1, with IC50 activity ranging from 30–60 μM against several dissimilar cell lines. To further elucidate structurally important regions of the molecule and ascertain a structure‐activity relationship (SAR) for anticancer activity, six additional molecules were synthesized and tested. These molecules included modifications to both the terminal phenyl and nitrogen moieties of SP‐1 (N,N‐dimethyl‐2‐(4‐(1‐(p‐tolyl) vinyl)phenoxy)ethan‐1‐amine). The XTT cell viability assay was then used to screen these compounds for anticancer activity in four diverse human cancer cell lines including A‐172 human glioblastoma, A375 human malignant melanoma, HT‐29 human colorectal adenocarcinoma, and MCF7 human breast adenocarcinoma. Interestingly, hydration of the ethylene moiety attached to the carbon linker between the two phenyl rings markedly reduced anticancer activity of SP‐2, SP‐3, SP‐7 and SP‐8. Substantial anticancer activity was not observed at concentrations less than 90 μM regardless of structural modifications made to the periphery of these molecules. However, SP‐5 and SP‐6, which conserved the nonpolar central linker present in SP‐1, demonstrated moderately potent anticancer activity against the four cancer cell lines. SP‐5, which only differs from SP‐1 by the addition of one carbon to the peripheral phenyl ring, was found to have nearly the same potency as SP‐1. However, SP‐6, which includes the addition of a morpholine ring to the periphery of the molecule, demonstrated slightly less potent activity than that of SP‐1 and SP‐5. However IC50 values were still found to range from 45–90 μM in all four cells lines. While the changes made to these analogs failed to improve the efficacy of our parent molecule SP‐1, the information regarding the SAR will help guide the synthesis of further analogs. Additional studies investigating the mechanisms of death and putative targets are ongoing for these lead molecules.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

One-Pot Synthesis of Ginsenoside Rh2 and Bioactive Unnatural Ginsenoside by Coupling Promiscuous Glycosyltransferase from Bacillus subtilis 168 to Sucrose Synthase.

Ginsenosides, the major effective ingredients of Panax ginseng, exhibit various biological properties. UDP-glycosyltransferase (UGT)-mediated glycosylation is the last biosynthetic step of ginsenosides and contributes to their immense structural and functional diversity. In this study, UGT Bs-YjiC from Bacillus subtilis 168 was demonstrated to transfer a glucosyl moiety to the free C3-OH and C12-OH of protopanaxadiol (PPD) and PPD-type ginsenosides to synthesize natural and unnatural ginsenosides. In vitro assays showed that unnatural ginsenoside F12 (3- O-β-d-glucopyranosyl-12- O-β-d-glucopyranosyl-20( S)-protopanaxadiol) exhibited remarkable activity against diverse human cancer cell lines. A one-pot reaction by coupling Bs-YjiC to sucrose synthase (SuSy) was performed to regenerate UDP-glucose from sucrose and UDP. With PPD as the aglycon, an unprecedented high yield of ginsenosides F12 (3.98 g L-1) and Rh2 (0.20 g L-1) was obtained by optimizing the conversion conditions. This study provides an efficient approach for the biosynthesis of ginsenosides using a UGT-SuSy cascade reaction.

Defining a Cancer Dependency Map

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one suchpredictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Binding of the anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study

BI-2536 is a potent Polo-like kinase inhibitor which induces apoptosis in diverse human cancer cell lines. The binding affinity of BI-2536 for human serum albumin (HSA) protein may define its pharmacokinetic and pharmacodynamic profile. We have studied the binding of BI-2536 to HSA by means of different spectroscopic techniques and docking calculations. We have experimentally observed that the affinity of BI-2536 for HSA is higher than that of other common HSA binding drugs. Therefore, it can be postulated that the drug dose should be increased to achieve a certain concentration of free drug in plasma, although BI-2536 could also reach tumour tissues by uptaking HSA/BI-2536 complex. Only a single binding site on HSA has been observed for BI-2536 which seems to correspond to the subdomain IIA pocket. The formation of the HSA/BI-2536 complex is a spontaneous and entropy-driven process that does not cause a significant change of the secondary structure of the protein. Its endothermic character could be related to proton release. Thermodynamic analysis showed that the main protein-drug interactions are of the van der Waals type although the presence of amide and ether groups in BI-2536 could also allow H-bonding with some residues in the subdomain IIA pocket.

Development of ROC-325: A Novel Small Molecule Inhibitor of Autophagy with Promising Anti-Leukemic Activity

Autophagy contributes to therapeutic resistance and malignant progression by generating alternative metabolic fuel to maintain cell survival under stress conditions including those imposed by hypoxia, radiation, chemotherapy, and targeted agents. The FDA approved anti-malarial drug hydroxychloroquine (HCQ) inhibits autophagy through the disruption of lysosomal function. Robust efforts to repurpose HCQ for cancer therapy based on these properties stimulated numerous clinical trials where HCQ was combined with an array of other anticancer regimens and ultimately produced modest clinical activity. However, it was unclear if the maximum tolerated dose of HCQ was sufficient to completely inhibit autophagy in tumors. New autophagy inhibitors with increased potency and more favorable therapeutic indices are clearly needed to rigorously investigate the potential benefit of this approach.Polyvalent molecules can yield nonlinear, multifold potency against their respective targets compared to their corresponding monomers. We generated a series of novel dimeric compounds containing modified core elements of HCQ, CQ and the anti-schistosomal drug lucanthone with the goal of developing new autophagy inhibitors with superior potency, tolerability, and anticancer efficacy. Initial screens that tested for drug-induced increased expression of p62, a protein that is specifically turned over by autophagy, and anticancer activity identified ROC-325 as a lead agent. The structure of ROC-325 was confirmed by NMR and MS analyses. Direct comparison of the in vitro activity of ROC-325 and HCQ in 13 different genetically and histologically diverse human cancer cell lines demonstrated that ROC-325 was approximately 10-fold more potent than HCQ based on IC50 analyses. Acute myeloid leukemia (AML) was selected as a primary malignancy for intensive investigation based on the high sensitivity of FLT3-ITD+ MV4-11 cells to this agent in preliminary studies. Transmission electron microscopy analyses demonstrated that treatment with ROC-325 triggered all of the hallmark features of autophagy inhibition including the accumulation of autophagosomes with undegraded cargo, an increase in lysosomal membrane permeability, deacidification of lysosomes, and elevated LC3B, p62, and cathepsin D expression. Bafilomycin A1 clamp experiments showed that ROC-325 potently inhibited autophagic flux. Genetic impairment of two different genes that are essential for functional autophagy, ATG5 and ATG7, using lentiviral shRNA approaches significantly diminished the anticancer effects of ROC-325, thus indicating that autophagy inhibition is a key component of its anticancer mechanism of action.RNA sequencing and gene level analyses demonstrated that treatment with ROC-325 (1 µM) in MV4-11 cells altered the levels of autophagy-dependent degradation pathways while preserving protein synthesis through the upregulation of post-translational ribosomal, methylation, and splicing components.In vitro treatment of a panel of human AML cell lines and normal human bone marrow progenitors demonstrated that ROC-325 diminished AML cell viability (IC50 range 0.7-2.2 µM), antagonized clonogenic survival, and induced apoptosis in a manner that was therapeutically selective. Analysis of primary blasts from patients with AML showed that its activity was not significantly affected by adverse cytogenetics or multi-drug resistance due to relapsed/refractory clinical status. Oral administration of 50 mg/kg ROC-325 (QDx5) to mice bearing disseminated MV4-11 human AML xenografts significantly increased lifespan (P<0.05) and enhanced the efficacy of azacitidine (5 mg/kg IV, 2X per week). ROC-325 was well tolerated and no notable toxicities were observed other than a modest, non-significant reversible reduction in mean body weight. Immunohistochemical analysis of specimens collected from animals treated with ROC-325 demonstrated significant increases in the autophagic markers LC3B and p62 and apoptotic blasts. Our data demonstrate that ROC-325 is a novel orally available autophagy inhibitor that is well tolerated, significantly more potent than HCQ, and has promising anti-AML activity. These findings support further investigation of the safety and efficacy of ROC-325 as a novel agent for the treatment of AML and other disorders where lysosomal activity contributes to disease pathogenesis. DisclosuresKelly:Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau.

Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity.

Germ line-specific genes are activated in somatic cells during tumorigenesis, and are accordingly referred to as cancer germline genes. Such genes that act on piRNA (Piwi-interacting RNA) processing play an important role in the progression of cancer cells. Here, we show that the spermatogenic transposon silencer maelstrom (Mael), a piRNA-processing factor, is required for malignant transformation and survival of cancer cells. A specific Mael isoform was distinctively overexpressed in diverse human cancer cell lines and its depletion resulted in cancer-specific cell death, characterized by apoptosis and senescence, accompanied by an increase in reactive oxygen-species and DNA damage. These biochemical changes and death phenotypes induced by Mael depletion were dependent on ATM. Interestingly Mael was essential for Myc/Ras-induced transformation, and its overexpression inhibited Ras-induced senescence. In addition, Mael repressed retrotransposon activity in cancer cells. These results suggest that Mael depletion induces ATM-dependent DNA damage, consequently leading to cell death specifically in cancer cells. Moreover, Mael possesses oncogenic potential that can protect against genetic instability.

Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.

Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine.

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.

Role of ATF5 in the invasive potential of diverse human cancer cell lines

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-α2 and integrin-β1 expression and that the depletion of integrin-α2 or integrin-β1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-α2 and integrin-β1 in several human cancer cell lines.

Synthesis and biological evaluation of a novel artesunate–podophyllotoxin conjugate as anticancer agent

A novel conjugate of artesunate-podophyllotoxin was prepared and evaluated for its cytotoxicity against diverse normal and multidrug resistance human cancer cell lines by CCK-8 assay. The conjugate exhibited good cytotoxicity on all the cell lines with IC50 values of 0.453±0.156-3.011±0.272μM and reduced the resistant factor. The conjugate was further found to disrupt the microtubule network and induce G2/M cell cycle arrest in multidrug resistance K562/ADR cells. Meanwhile, Hoechst staining analysis suggested that conjugate induced cell death by apoptosis. Furthermore, conjugate could downregulate the levels of P-glycoprotein (P-gp) in P-gp overexpressing K562/ADR cells.

Pro-Oxidant Activity of Amine-Pyridine-Based Iron Complexes Efficiently Kills Cancer and Cancer Stem-Like Cells.

Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds 1, 2 and 5 were cytotoxic in the low micromolar range against a panel of molecularly diverse human cancer cell lines. Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.

Antiproliferative activity of Origanum compactum extract on lung cancer and hepatoma cells

Natural products have been shown to present interesting biological and pharmacological activities and are used as cancer preventive and therapeutic agents. Plants have historically been used in treating cancer and are recognized for their ability to produce secondary metabolites. Origanum compactum Benth. (Lamiaceae) is a well-known Moroccan plant with cancer-related ethnobotanical use. Previously, we demonstrated that ethyl acetate extract of O. compactum had antiproliferative potential on human breast tumor MCF-7 cells. The purpose of this study was to investigate if the antiproliferative effect of this extract was similar for diverse human cancer cell lines such as A549 lung cancer and SMMC-7721 hepatoma cells. Furthermore, this study essentially focused on the intrinsic apoptotic signaling pathway. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide on A549 and SMMC-7721 cells. The characterization of the mechanisms involved in this effect was determined by lactate dehydrogenase test, apoptosis assays and protein expression analyses. Our present work has shown that this extract remarkably inhibited proliferation of A549 (IC50: 198 ± 12 μg/ml) and SMMC-7721 (IC50: 266 ± 14 μg/ml) cells. The characterization of antiproliferative activity demonstrated that this extract was an apoptosis inducer in both cell lines tested. The results of protein expression analyses have shown in A549 cells that this extract activated caspase signaling triggered by the modulation of Bcl-2 family proteins. These results suggest that these natural extract-induced effects may have novel therapeutic applications for the treatment of various cancer types.

Abstract 5427: Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors

Abstract Vascular endothelial growth factor (VEGF) is a key tumor angiogenesis factor that is overexpressed by many tumors; the Fas/CD95 death receptor induces apoptosis when activated by its ligand FasL. Agents that inhibit VEGF such as bevacizumab, or activate Fas receptor have been intensively developed as cancer therapeutics. Bevacizumab only modestly improves overall survival or has no effect on overall survival in most cancer patients. More effective VEGF-targeted agents are needed. We conceived of a new strategy that goes beyond VEGF inhibition and converts VEGF to act as a tumor cell death factor. Our aim is to co-opt overexpressed VEGF within the tumor microenvironment to initiate apoptosis via Fas death receptors expressed on tumor cells and/or stromal cells. We created a recombinant homotrimeric fusion protein designated R1FasL that combines the VEGF-binding domain of human VEGFR1 with the trimerization and death receptor-binding domains of human FasL ligand. R1FasL was produced in CHO cells and purified by FLAG antibody affinity chromatography to yield a 135 kD homotrimer. In vitro studies confirmed that VEGF binds to and crosslinks the R1FasL fusion protein to form higher-order complexes that efficiently aggregate and activate Fas death receptors to induce apoptosis. Jurkat cells were killed by R1FasL only when VEGF was added. R1FasL induced apoptosis in diverse human cancer cell lines in vitro, including renal cell, glioblastoma and breast cancers. When VEGF was sequestered by a VEGFR1-Fc trap protein, R1FasL did not induce apoptosis, demonstrating that R1FasL used the VEGF secreted by the cancer cells to kill them. We previously demonstrated that a single intravenous dose of R1FasL induced widespread tumor apoptosis in a xenograft model of human renal cell carcinoma. We have now tested R1FasL in an intracranial xenograft model of human glioblastoma. Nude mice bearing U87 intracranial xenografts were treated with a single intratumoral injection of R1FasL (0.025 to 0.9 mg/kg in 10 microliters). Twenty-four hours later tumors were collected and analyzed for evidence of apoptosis by caspase-3 immunohistochemistry. Mice treated with a single intratumoral dose of R1FasL at 0.1 mg/kg demonstrated significant apoptosis within the tumor but not in surrounding brain. In a safety study, intracranial injection of R1FasL (0.1 or 0.8 mg/kg) into normal brains of nude mice did not cause increased weight loss or death versus PBS injection. In a subsequent survival study, mice treated with a single intratumoral dose of R1FasL (0.8 mg/kg) survived significantly longer than PBS-treated mice (39.5 versus 30 days, p=0.018). Non-invasive imaging demonstrated a median 59% reduction in tumor size 48 hours after R1FasL injection. By converting VEGF to act as a Fas death receptor ligand, the R1FasL fusion protein may be an effective agent to simultaneously target VEGF and death receptors to selectively induce apoptosis in the tumor microenvironment. Citation Format: Timothy Quinn, YJ Lu, Raquel Santos, Tomoko Ozawa, David James, Mary C. Nakamura. Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors. [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 5427. doi:10.1158/1538-7445.AM2014-5427

Abstract 3154: RSK2-mediated phosphorylation of stathmin promotes microtubule polymerization, providing a pro-invasive advantage to metastatic cancer cells

Abstract Metastasis is responsible for over 90% of cancer related deaths. Breast cancer, lung cancer, and head and neck cancer commonly metastasize to vital organs resulting in poor clinical outcomes. However, the molecular processes underlying such cellular changes remain elusive. Therefore, identifying genes that induce cancer cell signaling changes and understanding how these changes control pro-metastatic mechanisms is necessary to develop molecular therapies for metastasis and to improve clinical prognosis for late-stage cancer patients. Protein kinases have been implicated in mediating pro-metastatic signaling in human cancers. We previously demonstrated that a serine/threonine kinase, p90 ribosomal S6 kinase 2 (RSK2) promotes cancer cell invasion and migration, anoikis resistance, and tumor metastasis in human cancers, including head and neck cancer, breast cancer, and lung cancer. Here, we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. SiRNA-mediated knockdown of RSK2 disrupted microtubules in diverse metastatic human cancer cell lines including lung cancer A549, breast cancer SKBR3, and head and neck cancer 212LN cells. Stathmin (STMN) is a microtubule destabilizing protein, which is an essential regulator of microtubule polymerization. We found that stable knockdown of RSK2 resulted in decreased phosphorylation of STMN at Serine 16 in the aforementioned metastatic cancer cell lines. Moreover, we found that RSK2 directly phosphorylated STMN at Ser16 in an in vitro RSK2 kinase assay using recombinant RSK2 and STMN proteins. We observed that phosphorylation of STMN by RSK2 resulted in reduced STMN-mediated microtubule depolymerization in vitro, while stable expression of phospho-deficient STMN S16A mutant but not STMN wild type (WT) led to decreased invasive potential of metastatic cancer cells. These data together suggest STMN as a potential novel RSK2 substrate/effector, which may contribute to RSK2-mediated pro-metastatic signaling. Our finding demonstrates that RSK2 signals through STMN to regulate microtubule polymerization and cancer cell invasion via direct phosphorylation of STMN at Serine 16, providing a pro-invasive and metastatic advantage to cancer cells. Therefore, the RSK2-STMN pathway represents a promising therapeutic target in the clinical prognosis and treatment of metastatic human cancers. Citation Format: Gina Alesi, Dan Li, Lingtao Jin, Georgia Z. Chen, Dong M. Shin, Fadlo Khuri, Sumin Kang. RSK2-mediated phosphorylation of stathmin promotes microtubule polymerization, providing a pro-invasive advantage to metastatic cancer cells. [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 3154. doi:10.1158/1538-7445.AM2014-3154

A convergent synthesis of alkyne–azide cycloaddition derivatives of 4-α,β-2-propyne podophyllotoxin depicting potent cytotoxic activity

A facile synthetic approach to construct the O-propargyl derivatives of 4α and 4β-(1,2,3-triazol-4-yl)-podophyllotoxin (9a–k & 10a–k) and 4′-Demethyl-4′-4β-(1,2,3-triazol-4-yl)-epipodophyllotoxin (12a–d) were synthesized by means of click chemistry. The chemical structures were confirmed by 1H, 13C, 2D NMR and HRMS spectral analysis and their cytotoxicities were measured against diverse human cancer cell lines viz. PC-3, PANC-1, COLO-205 and A-549 by MTT assay. Some of the compounds were found more potent than the parent molecule Podophyllotoxin, like; 9a &10a, 9h &10h, 9k &10k, 10d, 8 and 12a. The most potent molecule discovered was compound 9k that exhibited the highest cytotoxicity on all the four cancer cell lines with IC50 values of 3.8–22 nM. The compound further found to induce apoptosis and strongly hindered the motility of aggressive prostate cancer PC-3 cells.

The Effects of Combination Treatment Using Phenoxodiol and Docetaxel, and Phenoxodiol and Secreted Frizzled-related Protein 4 on Prostate Cancer Cell Lines

Although much progress has been made for the treatment of prostate cancer, patients with advanced prostate cancer still have a poor 5 year survival rate. Current practices for hormone-refractory/castrate resistant, metastatic prostate cancer involve the use of taxanes. Docetaxel, in particular, is being incorporated in numerous current clinical trials either as a single or combination agent against androgen-independent prostate cancer. Combination therapies have the potential to increase the effectiveness of drug treatments while simultaneously increasing quality of life by reducing side effects, lowering effective dosage rates, or by increasing effectiveness of one compound once combined with another. Using three diverse human prostate cancer cell lines, LNCap, DU145, and PC3, we studied the effect of the novel prostate cancer drug phenoxodiol in combination with docetaxel by utilizing isobolograms, and found that docetaxelinduced cell death was attenuated by co-treatment or pre-treatment of cells with phenoxodiol. This attenuation is associated with the prevention of cells from entering the G2/M phase of the cell cycle where docetaxel is functional in damaging the spindle fibers, and potentially due to p21WAF1 mediated cell survival after docetaxel treatment. We also investigated the use of the Wnt signaling pathway antagonist secreted frizzled-related protein 4 (sFRP4) to increase the effectiveness of phenoxodiol treatment. We found that, through stabilization of the GSK3β molecule, sFRP4 induces degradation of active β-catenin, which causes an increased sensitivity to isoflavone cytotoxic induction by increasing p21WAF1 expression and decreasing expression of c-Myc, Cyclin-D1, and other potent oncogenes. Phenoxodiol induces significant cytotoxicity when combined with a Wnt/β-catenin receptor blocker such as sFRP4. This promotes the concept that combination therapy of a Wnt inhibitor with phenoxodiol might increase the effectiveness of phenoxodiol and give a subset population of prostate cancer sufferers a more effective treatment regime.

Inhibition Of RNA Polymerase I Transcription By CX-5461 As a Therapeutic Strategy For The Cancer-Specific Activation Of p53 In Highly Refractory Haematological Malignancies

Malignant transformation is commonly associated with dysregulated ribosome biogenesis and for more than 100 years, pathologists have utilized the increase in size and number of nucleoli, the site of ribosome biogenesis, as a marker for aggressive malignancies (Hein et al., Trends in Molecular Medicine, 2013). Recently we demonstrated that hyperactivated RNA Polymerase I (Pol I) transcription, a rate limiting step in ribosome biogenesis, can be specifically targeted by the small molecule inhibitor, CX-5461 (Drygin et al., Cancer Research, 2011; Bywater et al. Cancer Cell, 2012). When evaluated for its anti-proliferative activity against a panel of genetically diverse human cancer cell lines, those derived from p53 wild type hematological malignancies were the most sensitive (Drygin et al., Cancer Research, 2011). Evaluation of whether Pol I transcription inhibition could serve as therapeutic strategy in vivo revealed that CX-5461 administration significantly prolongs the overall survival of Em-Myc lymphoma bearing mice. This survival advantage was associated with rapid activation of the ribosomal protein (Rp)-MDM2-p53 nucleolar stress response (Deisenroth et al., Oncogene, 2010) and subsequent induction of tumour cell-specific p53-dependent apoptosis; importantly the wild type B-cell population was maintained and did not activate p53 pathways (Bywater et al. Cancer Cell, 2012).To explore the therapeutic potential of Pol I transcription inhibition via CX-5461 in hematological malignancies refractory to standard chemotherapy, we employed mouse models of highly aggressive MLL-driven Acute Myeloid Leukemia (AML) (MLL/AF9 + Nras and MLL/ENL + Nras) (Zuber et al., Genes Dev, 2009) and V*κ-Myc-driven Multiple Myeloma (MM) (Chesi et al. Cancer Cell, 2009). CX-5461 administration significantly increased the overall survival time of AML-bearing mice (MLL/ENL Nras median 17 days for vehicle vs 36 days for drug, P<0.0001 (Figure 1); MLL/AF9 Nras median 15 days for vehicle vs 23 days for drug, P 0.0009) without severe adverse effects. The extended survival of MLL-driven AML bearing mice was associated with acute activation of p53, induction of cell death and an aberrant G2/M cell cycle progression, which consequently delayed tumour progression as monitored by bioluminescence-imaging and white blood cell counts (WBC). In contrast to CX-5461, standard-therapy with a regime containing Cytarabine/Doxorubicin (5:3) failed to induce p53 in MLL/ENL Nras AML-bearing mice, resulting in a significantly lower survival advantage as compared to animals treated with CX-5461 therapy. [Display omitted] In ongoing experiments with MM-bearing mice, chronic CX-5461 administration robustly reduced the secretion of serum monoclonal Ig, as detected by serum protein electrophoresis (SPEP), and significantly prolonged the overall survival time (Figure 2) (V*κ-Myc median >160 days for drug vs, 103.5 days for vehicle, ongoing) demonstrating that CX-5461 exhibits potent anti-tumour activity in MM model. Analysis of the molecular mechanism responsible for the therapeutic benefit of CX-5461 in MM is ongoing but appears to involve both p53 mediated apoptosis and aberrant cell cycle progression. [Display omitted] These data demonstrate that hyperactivated Pol I transcription can be successfully targeted through small molecule inhibitors to treat models of human AML and MM that are refractory to standard therapy. Based on these and previously published results (Bywater et al., Cancer Cell, 2012), we have recently initiated a first-in-human, phase I, dose escalation clinical trial of this first-in-class drug, CX-5461 in patients with advanced hematological malignancies. The toxicity, response, pharmacokinetic and co-relative data from this trial will offer valuable insights as to whether inhibition of ribosome synthesis might offer a new non-genotoxic therapeutic approach in the fight against cancers, which are highly refractory to standard therapies. Disclosures:O'Brien:Senhwa Biosciences, Inc: Employment.

Abstract B16: Inhibition of RNA Polymerase I transcription by CX-5461 as a therapeutic strategy for the cancer-specific activation of p53 in highly refractory haematological malignancies

Abstract Malignant transformation is commonly associated with dysregulated ribosome biogenesis and for more than 100 years, pathologist have utilized the increase in size and number of nucleoli, the site of ribosome biogenesis as a marker for aggressive malignancies. Recently we demonstrated that hyperactivated RNA Polymerase I (Pol I) transcription, a rate limiting step in ribosome biogenesis, can be specifically targeted by the small molecule inhibitor, CX-5461. When evaluated for its anti-proliferative activity against a panel of genetically diverse human cancer cell lines, those derived from p53 wild type haemotological malignancies were the most sensitive (Drygin et al., Cancer Research, 2011). Evaluation of whether Pol I transcription inhibition could serve as therapeutic strategy in vivo revealed that CX-5461 administration significantly prolongs the overall survival of Eμ-Myc lymphoma bearing mice (Bywater et al., Cancer Cell, 2012). This survival advantage was associated with rapid activation of the ribosomal protein (Rp)-MDM2-p53 nucleolar stress response (Deisenroth et al., Oncogene, 2010) and subsequent induction of tumour-specific p53-dependent apoptosis (Bywater et al. Cancer Cell, 2012). New data and future directions: To further explore the therapeutic potential of Pol I transcription inhibition in haemotological cancers that are refractory to standard therapy, we employed mouse models of MLL-driven Acute Myeloid Leukemia (AML) (MLL/AF9 + Nras and MLL/ENL + Nras) (Zuber et al., Genes Dev, 2009) and V*κ-Myc-driven Multiple Myeloma (MM) (Chesi et al. Cancer Cell, 2009). CX-5461 administration significantly increased the overall survival time of AML-bearing mice (MLL/ENL Nras 17 days for vehicle vs 36 days for drug, P&amp;lt;0.0001; MLL/AF9 Nras 15 days for vehicle vs 23 days for drug, P 0.0009))without severe adverse effects. The extended survival of MLL-driven AML bearing mice was associated with acute activation of p53, an aberrant G2/M cell cycle progression and consequently a delay in tumour progression as monitored by bioluminescence-imaging. In contrast to CX-5461, treatment of AML -bearing mice with Cytarabine/Doxorubicin, a regime used as standard-therapy in the clinic failed to induce p53 in MLL-driven AML resulting in a significantly less survival time than for CX-5461. In ongoing experiments with MM-bearing mice, chronic CX-5461 administration robustly reduced the secretion of serum monoclonal Ig, as detected by serum protein electrophoresis (SPEP), and significantly prolonged survival time demonstrating that CX-5461 possess potent anti-tumour activity in this highly refractory MM model. Analysis of the molecular mechanism responsible for the therapeutic benefit of CX-5461 in MM is ongoing but appears to involve both p53 mediated apoptosis and aberrant cell cycle progression. Conclusion: These data demonstrate that hyperactivated Pol I transcription can be successfully targeted through small molecule inhibitors to therapeutically treat models of human AML and MM that are refractory to standard therapy.. Based on these and published results (Bywater et al., Cancer Cell 2012) and a favorable toxicology profile, we have launched a first-in-human clinical trial of this first in class drug, CX-5461 in patients with hematological malignancies. The outcomes of this trial will offer valuable insights as to whether inhibition of ribosome synthesis might offer a new non-genotoxic therapy approach in the fight against cancers, which are highly refractory to standard-therapies. Citation Format: Nadine Hein, Denis Drygin, Sean O'Brien, Carleen Cullinane, Geoff Matthews, Marta Chesi, Leif Bergsagel, Johannes Zuber, Scott Lowe, Ricky Johnstone, Rick Pearson, Grant McArthur, Ross Hannan. Inhibition of RNA Polymerase I transcription by CX-5461 as a therapeutic strategy for the cancer-specific activation of p53 in highly refractory haematological malignancies. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B16.

Abstract 1692: SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines.

Abstract SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments. Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [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 1692. doi:10.1158/1538-7445.AM2013-1692