Long-term Survival Assay Research Articles

Abstract 5943: Development of a SMART theranostic agent for precision pancreatic cancer therapeutics

Abstract Pancreatic cancer is predicted to emerge as the second leading cause of cancer-related deaths in the United States by 2030. Pancreatic ductal adenocarcinoma (PDAC) - the most common form of pancreatic cancer - is often diagnosed as metastatic disease and has demonstrated the least improvement in overall survival over the last three decades with incidence rising by 0.5% to 1.0% per year due in part to lack of effective and tumor-selective treatment strategies. One approach to reduce host toxicity is the development of novel molecules and innovative therapeutic approaches that are nontoxic to normal cells but can be activated only under tumor-specific conditions for lethality, which we coined as SMART (Selective Method of Activation for Response in Tumors) therapeutic agents. We utilized explicit properties and biomarkers present in PDAC tumors but not in normal tissues to develop a SMART DNA-damaging anti-cancer agent with both therapeutic and diagnostic (theranostic) activities. We evaluated the tumoricidal effect of our SMART agent using long-term survival assays in PDAC cell lines and 3D co-culture spheroids, as well as in tumor xenograft model studies in mice. We also tested the ability of our SMART agent to treat, detect/diagnose (via click chemistry), and identify critical resistance mechanisms (via proteomics) as a single agent or as a chemosensitizer with predictive tumor biomarkers for tumor-selective therapeutic response. We found that our SMART small molecule induces tumor-selective lethal effects as a monotherapy or in combination with DNA repair inhibitors or small molecules known to generate hydrogen peroxide (H2O2) in a tumor-selective manner for activation. Using “click” chemistry, we detected and quantified the incorporation of our SMART small molecule as a DNA-damaging agent via flow cytometry that correlated with therapeutic response, which is a feature that could facilitate the stratification, treatment monitoring, and management of patients with pancreatic cancers. We also found that our SMART agent is less toxic and more effective as a monotherapy than cisplatin in PDAC cell line-derived xenograft studies in mice. Overall, our novel SMART theranostic agent could provide a significant advancement as they are preferentially activated under tumor-specific conditions to induce tumor-selective therapeutic response. Furthermore, the diagnostic component our SMART agent could provide oncologists with essential information to make rapid clinical decisions on treatment strategies. Our SMART agent is also remarkably versatile as it can be utilized as a chemosensitizer or as a chemical probe to identify targetable vulnerabilities to potentiate its therapeutic effects at lower doses. Citation Format: Edward A. Motea, Jarrett J. Smith, Oluwasijibomi Ketiku. Development of a SMART theranostic agent for precision pancreatic cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5943.

MRE11A: a novel negative regulator of human DNA mismatch repair

BackgroundDNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3′–5′ exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear.MethodsInitially, short-term and long-term survival assays were used to measure the cells’ sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used.ResultsWe show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity.ConclusionsOur findings reveal that MRE11A is a negative regulator of human MMR.Graphical

Effect of Hybrid Compounds of Stilbene and Pentadienone on Inhibition of Tubulin Polymerization.

Tubulin polymerization inhibitors induce cancer cell death; therefore, they can be developed as chemotherapeutic agents. We hypothesized that hybrid compounds, including the trans-stilbene moiety contained in resveratrol and penta-1,4-dien-3-one contained in curcumin, could inhibit tubulin polymerization. Twenty-six hybrid stilbene and pentadienone compounds were designed and synthesized. The cytotoxicity of the hybrid compounds against MDA-MB-231 human breast cancer cells was determined using a clonogenic long-term survival assay. The relationship between cytotoxicity and structural properties was evaluated. Biological activities, including inhibition of tubulin polymerization and cell cycle progression, were investigated to select compounds with excellent anticancer properties. The molecular binding mode between the selected compound and the α, β-tubulin dimers was investigated. Twenty-six hybrid stilbene and pentadienone compounds were designed and synthesized. Among them, compound 13 exhibited the highest inhibitory effect on the clonogenicity of MDA-MB-231 cells. Compound 13 induced the destabilization of tubulins and inhibited cell cycle progression at the G2/M phase. Through in silico molecular docking analysis, compound 13 was predicted to bind to the colchicine binding site of α, β-tubulin. The stilbene and pentadienone hybrid compound 13 has a binding mode similar to that of colchicine. Compound 13 inhibited the clonogenicity of MDA-MB-231 cells through a mechanism that destabilizes tubulin polymerization, leading to cell cycle arrest at the G2/M phase.

Evaluation of 3,5-Diphenyl-2-Pyrazolines for Antimitotic Activity by Inhibition of Tubulin Polymerization

Chalcones have a skeleton of diphenyls connected via an α,β-unsaturated carbonyl group. Many chalcone derivatives have been shown to interact with tubulin. Based on previous reports, chalcones derived by substitution of a carbonyl group with 2-pyrazoline can be expected to inhibit tubulin polymerization. Therefore, 3,5-diphenyl-2-pyrazolines were prepared to investigate their ability to inhibit tubulin polymerization. The clonogenic long-term survival assay showed that derivative 4, 5-(3,5-dimethoxyphenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, was the most effective at inhibiting the clonogenicity of HCT116 human colon cancer cells. Derivative 4 induced G2/M cell cycle arrest. In addition, derivative 4 caused dispersed microtubules, a disorganized spherical arrangement of chromosomes, and inhibition of mitotic spindle formation. The binding mode between tubulin and derivative 4 was elucidated by in silico molecular docking. Derivative 4 was superimposed with colchicine and entered the colchicine-binding site well. These results suggest that derivative 4 inhibits tubulin polymerization by binding to the colchicine binding site of tubulin, thus preventing mitotic spindle formation during mitosis in HCT116 colon cancer cells. We propose that derivative 4 could be used as a promising antimitotic chemotherapeutic agent.

The BRD9/7 Inhibitor TP-472 Blocks Melanoma Tumor Growth by Suppressing ECM-Mediated Oncogenic Signaling and Inducing Apoptosis.

Simple SummaryMelanoma is an aggressive form of skin cancer and the leading cause of skin cancer-related deaths. Current therapies, including those targeting oncogenic pathways and immunotherapies, provide therapeutic benefits to only a subset of melanoma patients. Therefore, more options for therapeutic interventions are needed. Epigenetic alterations play an important role in tumor development and progression. In this study, we identified that TP-472 a small molecule inhibitor of BRD7/9 blocks melanoma tumor growth in cell cultures and in mouse models of melanoma growth. Further studies revealed that TP-472 downregulates cancer-promoting signaling pathways and induces cell death. Thus, this study identifies TP-472 as a potentially useful therapeutic agent for melanoma therapy.Melanoma accounts for the majority of all skin cancer-related deaths and only 1/3rd of melanoma patients with distal metastasis survive beyond five years. However, current therapies including BRAF/MEK targeted therapies or immunotherapies only benefit a subset of melanoma patients due to the emergence of intrinsic or extrinsic resistance mechanisms. Effective treatment of melanoma will thus require new and more effective therapeutic agents. Towards the goal of identifying new therapeutic agents, we conducted an unbiased, druggable epigenetic drug screen using a library of 32 epigenetic inhibitors obtained from the Structural Genome Consortium that targets proteins encoding for epigenetic regulators. This chemical genetic screening identified TP-472, which targets bromodomain-7/9, as the strongest inhibitor of melanoma growth in both short- and long-term survival assays and in mouse models of melanoma tumor growth. Mechanistically, using a transcriptome-wide mRNA sequencing profile we identified TP-472 treatment downregulates genes encoding various extracellular matrix (ECM) proteins, including integrins, collagens, and fibronectins. Reactome-based functional pathway analyses revealed that many of the ECM proteins are involved in extracellular matrix interactions required for cancer cell growth and proliferation. TP-472 treatment also upregulated several pro-apoptotic genes that can inhibit melanoma growth. Collectively, our results identify BRD7/9 inhibitor TP-472 as a potentially useful therapeutic agent for melanoma therapy.

Enhancing radiation response by a second-generation TRAIL receptor agonist using a new in vitro organoid model system

BackgroundFor many cancer types, including colorectal carcinoma (CRC), combined modality treatments have shown to improve outcome, but are frequently associated with significant toxicity, illustrating the need for new therapeutic approaches. Based on preclinical data, TRAIL receptor agonists appeared to be promising agents for cancer therapy especially in combination with DNA damaging regimens. Here, we present the combination of the second-generation TRAIL receptor agonist APG-880 with radiation in a new and clinically relevant 3D model system. MethodsTo investigate the effect of APG-880 in combination with radiation we performed short-term cytotoxicity and long-term clonogenic survival assays in established CRC cell lines, and in tumor organoids derived from colon cancer patients. ResultsAPG-880 is a potent inducer of apoptosis in CRC cell lines and in patient-derived CRC organoids. Furthermore, a supra-additive effect on cytotoxicity was found when APG-880 and radiation were combined simultaneously, with combination indices around 0.7. Lastly, in the long-term survival assays, we demonstrated a radiosensitizing effect of APG-880 with dose enhancement factors between 1.3 and 1.5. ConclusionsIn a new, clinically relevant CRC-organoid model system we demonstrated a more than additive combined effect between the second-generation TRAIL receptor agonist APG-880 and radiation.

Synthesis, Single Crystal X-Ray Structure, Hirshfeld Surface Analysis, DFT Computations, Docking Studies on Aurora Kinases and an Anticancer Property of 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-6-methoxy-4H-chromen-4-one

The isoflavone compound 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-methoxy-4H-chromen-4-one (6) was prepared and structurally characterized using NMR, mass spectrum and X-ray crystallography. Compound 6, C18H14O5, was crystallized in the monoclinic space group P21/n with the cell parameters; a = 7.1869(4) Å, b = 10.2764(6) Å, c = 19.6771(10) Å, β = 99.442(2)°, V = 1433.57(14) Å3, Z = 4. In the title compound, the chromenone ring system is slightly twisted from planarity and the dihedral angle formed between the plane of the chromenone ring and benzene ring is 47.75°. Several intermolecular hydrogen bonds make the crystal stabilized in the three-dimensional structure, which was confirmed by Hirshfeld surface analysis. Density functional theory (DFT) calculations at the B3LYP/6-311++G(d,p) level were carried out and the calculated geometric parameters were compared with the experimental results. A frontier molecular orbital calculation was performed to reveal that the energy values of highest occupied molecular orbital (HOMO) and lowest un-occupied molecular orbital (LUMO) were −5.8223 eV and −1.8447 eV, and the HOMO–LUMO energy gap was 3.9783 eV. A clonogenic long-term survival assay of compound 6 against HCT116 human colon cancer cells showed an anti-cancer ability, with GI50 value of 24.9 μM. Docking experiments within the active sites of aurora kinase A and B were carried out to explain the anti-cancer property of compound 6.

Effect of serotonin modulation on dystrophin-deficient zebrafish.

ABSTRACTDuchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the dystrophin gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.

Design, synthesis, and biological evaluation of polyphenols with 4,6-diphenylpyrimidin-2-amine derivatives for inhibition of Aurora kinase A

Several 4,6-diarylpyrimidin-2-amine derivatives show anticancer properties. However, their mode of action is not fully characterized. To develop potent anticancer chemotherapeutic agents, we designed and synthesized 25 4,6-diphenylpyrimidin-2-amine derivatives containing a guanidine moiety. Clonogenic long-term survival assays were performed to screen anticancer compounds. To derive the structural conditions showing good cytotoxicities against cancer cells, quantitative structure-activity relationships (QSAR) were calculated. Biological activities were determined by flow cytometry for cell cycle analysis and by immunoblot analysis for the detection of Aurora kinase A (AURKA) activity. Because 2-(2-Amino-6-(2,4-dimethoxyphenyl)pyrimidin-4-yl) phenol (derivative 12) selectively inhibited AURKA activity from the kinome assay, in silico docking experiments were performed to elucidate the molecular binding mode between derivative 12 and AURKA. The pharmacophores were derived based on the QSAR calculations. Derivative 12 inhibited AURKA activity and reduced phosphorylation of AURKA at Thr283 in HCT116 human colon cancer cells. Derivative 12 caused the accumulation of the G2/M phase of the cell cycle and triggered the cleavages of caspase-3, caspase -7, and poly(ADP-ribose) polymerase. The binding energies of 30 apo-AURKA - derivative 12 complexes obtained from in silico docking ranged from -16.72 to -11.63kcal/mol. Derivative 12 is an AURKA inhibitor, which reduces clonogenicity, arrests the cell cycle at the G2/M phase, and induces caspase-mediated apoptotic cell death in HCT116 human colon cancer cells. In silico docking demonstrated that derivative 12 binds to AURKA well. The structure-activity relationship calculations showed hydrophobic substituents and 1-naphthalenyl group at the R2 position increased the activity. The existence of an H-bond acceptor at C-2 of the R1 position increased the activity, too. Graphical abstract Derivative 12 inhibits Aurora kinase A activity and causes the G2/M phase arrest of the cell cycle.

Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones

A moderate elevation in reactive oxygen species (ROS) levels can generally be controlled in normal cells, but may lead to death of cancer cells as the ROS level in cancer cells is already elevated. Therefore, a ROS-generating compound can act as a selective chemotherapeutic agent for cancer cells that does not affect normal cells. In our previous study, a compound containing a Michael acceptor was selectively cytotoxic to cancer cells without affecting normal cells; therefore, we designed and synthesized 26 compounds containing a Michael acceptor. Their cytotoxicities against HCT116 human colon cancer cell lines were measured by using a clonogenic long-term survival assay. To derive the structural conditions required to obtain stronger cytotoxicity against cancer cells, the relationships between the half-maximal cell growth inhibitory concentration values of the synthesized compounds and their physicochemical properties were evaluated by Comparative Molecular Field Analysis and Comparative Molecular Similarity Indices Analysis. It was confirmed that the compound with the best half-maximal cell growth inhibitory concentration triggered apoptosis through ROS generation, which then led to stimulation of the caspase pathway.

Clonogenic long-term survival assay of HCT 116 colorectal cancer cells after treatment with the synthesized diphenyl imidazoline derivatives

Fourteen diphenyl imidazoline derivatives were designed, synthesized, and identified using NMR spectroscopy and high-resolution mass spectrometry. Their cytotoxicities in HCT 116 colorectal cancer cell lines were measured using a clonogenic long-term survival assay and the half-maximal cell growth inhibitory concentration (GI50) values were in the range 3.1–58.4 μM. As the anticancer effects of diphenyl imidazolines were reported to be caused by the inhibition of mouse double minute 2 homolog (MDM2), the inhibitory effects of the most potent derivative on MDM2 were assessed through Western blotting analysis. In silico docking experiments revealed the binding mode between this derivative and MDM2.

Relation between structures of naphthalenylchalcone derivatives and their cytotoxic effects on HCT116 human colon cancer cells

To find potent chemotherapeutic agents, cytotoxic effects of 42 synthetic chalcone derivatives bearing naphthyl groups on HCT116 human colon cancer cell lines were tested using the clonogenic long-term survival assay. The relationships between their half-maximal cell growth inhibitory concentrations (GI50) and structural properties were obtained using comparative molecular field analysis and comparative molecular similarity indices analysis. The structural conditions that showed maximum cytotoxic effects on the colon cancer cells were determined. In addition, a derivative, (E)-1-(2-hydroxy-4,5-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one, showing the best GI50 value, was assessed for stimulating reactive oxygen species (ROS) production. While its treatment on non-tumorigenic epithelial MCF-12A cell line did not affect the intracellular ROS levels, its treatment on MDA-MB-231 human breast cancer cell line showed ROS accumulation. These findings demonstrate that naphthalenylchalcones can be developed as potent chemotherapeutic agents.

Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of chalcone

To identify new potent chemotherapeutic agents, we synthesized compounds with 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC) skeletons and evaluated their cytotoxicities using a clonogenic long-term survival assay. Their half-maximal cell growth inhibitory concentrations ranged from a few hundred nanomolars to a few micromolars. Further biological experiments including flow cytometry and western blotting analysis were performed with the derivative showing the best cytotoxicity. To identify a target protein of the selected compound, an in vitro kinase assay was carried out, which revealed that aurora kinases A and B were inhibited by the test compound, and this was confirmed using western blot analysis. The molecular binding mode between the selected compound and the kinases was elucidated using in silico docking. The structural conditions required for good cytotoxicity were identified based on the quantitative relationships between the physicochemical properties of the derivatives and their cytotoxicities.

Colorectal anticancer activities of polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamides

To develop potent chemotherapeutic agents for treating colorectal cancers, polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamide derivatives were designed. Twenty-two novel derivatives were synthesized and their cytotoxicities were measured using a clonogenic long-term survival assay. Of these derivatives, 3-(1-hydroxynaphthalen-2-yl)-N-(3-methoxyphenyl)-5-(4-methoxyphenyl)-pyrazoline-1-carbothioamide (NPC 15) exhibited the best half-maximal cell growth inhibitory concentrations (196.35nM). To explain its cytotoxicity, further biological experiments were performed. Treatment with NPC 15 inhibited cell cycle progression and triggered apoptosis through the caspase-mediated pathway. Its inhibitory effects on several kinases participating in the cell cycle were investigated using an in vitro kinase assay. Its half-maximal inhibitory concentrations for aurora kinases A and B were 105.03μM and 8.53μM, respectively. Further analysis showed that NPC 15 decreased phosphorylation of aurora kinases A, B, and C and phosphorylation of histone H3, a substrate of aurora kinases A and B. Its molecular binding mode for aurora kinase B was elucidated using in silico docking. In summary, polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamides could be potent chemotherapeutic agents.

NQO1-Mediated Tumor-Selective Lethality and Radiosensitization for Head and Neck Cancer.

Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNC). However, the 5-year overall survival rate for locally advanced HNCs is approximately 50% and better therapeutic efficacy is needed. quinone oxidoreductase 1 (NQO1) is overexpressed in many cancers, and β-lapachone (β-lap), a unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) that synergize with IR to kill by programmed necrosis. β-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of IR. Immunohistochemical staining and Western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs expressed elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by β-lap using long-term survival assays. The combination of nontoxic β-lap doses and IR significantly enhanced NQO1-dependent tumor cell lethality, increased ROS, TUNEL-positive cells, DNA damage, NAD(+), and ATP consumption, and resulted in significant antitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating β-lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to β-lap-induced radiosensitization. Mol Cancer Ther; 15(7); 1757-67. ©2016 AACR.

Abstract B19: Genome-wide integrated epigenomics identifies FZD-X as novel modulator for platinum sensitivity in high-grade serous ovarian cancer

Abstract The major hindrance for successful treatment of advanced stage ovarian cancer patients stems from platinum-based chemotherapy resistance. In cancer, the epigenetic alterations like DNA methylation, play a crucial role in regulation of genes related to response towards chemotherapies. Identifying these epigenetically regulated key genes, which modulate platinum response, may improve patient selection, response to therapy, and find novel targeted strategies to overcome platinum resistance. Here we applied integrated epigenomics approach to identify novel genes associated with high grade serous ovarian cancer platinum response. We performed next generation sequencing on methylation-enriched genomic DNA of ovarian cancer patient material from responders (≥18 months progression free survival, n=8) and non-responders (≤6 months progression free survival, n=10) to platinum chemoresponse. Expression data of same patients was also integrated to identify differentially methylated and expressed genes. These genes were validated further on external patients cohort using bisulfite-pyrosequencing and other publically available datasets. Furthermore, candidate genes were functionally tested on ovarian cancer cell lines panel using several in vitro assays. Based on integrated epigenomics analysis, we identified Frizzled receptor (FzdX) as an epigenetically regulated gene, significantly unmethylated (p<0.0005) and highly expressed (p<0.003) in non-responder group of ovarian cancer patients. Further, FzdX was successfully validated by bisulfite-pyrosequencing on independent external patient cohort (responder: n=21 and non-responder: n=31). Concordant hypermethylation and corresponding gene silencing of FzdX was observed in a large panel of ovarian cancer cell lines (n=11). Additionally, demethylating agents caused reduction in methylation and high expression of FzdX in ovarian cancer cell lines, confirming FzdX as an epigenetically regulated gene. Targeted genetic inhibition of FzdX using siRNAs in ovarian cancer cell lines caused significant sensitization towards cisplatin treatment in short and long-term survival assays. FzdX silencing made ovarian cancer cells more apoptosis prone as showed by apoptotic acridine orange staining and high expression of cleaved PARP and caspase 3. Furthermore, siRNAs mediated silencing of FzdX in ovarian cancer cell lines led to reduction in their migratory and invasion potential as compared to control counter parts in various functional assays. Conclusively, our integrative epigenomics analysis revealed FzdX as novel epigenetic modulator for platinum-sensitivity in ovarian cancer patients, which might be exploited for predictive and therapeutic approaches. This study has been granted by the Dutch Cancer Society, KWF (RUG 2010-4833). Citation Format: Tushar Tomar, Nicolette G. Alkema, Gert Jan Meersma, Tim De Meyer, Wim van Criekinge, Harry G. Klip, Ate GJ van der Zee, Steven de Jong, G. Bea A. Wisman. Genome-wide integrated epigenomics identifies FZD-X as novel modulator for platinum sensitivity in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B19.

Synthetic Naphthoflavonoids Showing Inhibitory Effects on Clonogenicity against Cisplatin-Resistant A2780/Cis Human Ovarian Cancer Cells

Detecting early-stage ovarian cancer, the fourth leading cause of cancerrelated deaths in women, is difficult, and the development of novel chemotherapeutic agents is required. Since several flavonoids show anticancer activities against ovarian cancer, 35 naphthylated flavonoids including 3’,4’-naphthochalcones, 5’,6’-naphthochalcone, 7,8-nap-hthoflavones, 5,6-naphthoflavanones, 5,6-naphthoflavones, 2,3-naphthochalcone, N-phenylpyrazolyl-5’,6’-naphthocha-lcones, carbothioamidepyrazolyl-5’,6’-naphthochalcones, pyrazolyl-3’,4’-naphthochalcone, and carbothioamidepyrazolyl-3’,4’-naphthochalcone were designed and synthesized. To explore the anticancer effects of these compounds, clonogenic long-term survival assays were applied on human cisplatin-resistant A2780/Cis ovarian cancer cells. Relationships between the structural properties of 35 naphthylated flavonoids and their clonogenicities were explored using comparative molecular field analysis and hologram quantitative structure– activity relationships. As a result, several structural features that increased cell growth inhibitory activity were identified, and a compound satisfying these conditions, 5-(2,3-dimethoxyphenyl)-3-(1-hydroxynaphthalen-2-yl)-N-phenyl-4,5-dihydro-1Hpyrazole- 1-carbothioamide, was designed and synthesized. This novel compound´s half-maximal cell growth inhibitory concentration was lower than those of the 35 flavonoid derivatives tested here. Therefore, the structural features observed in this report can be used to design and develop potent chemotherapeutic agents to treat ovarian cancer cells. Keywords: Clonogenicity, CoMFA, flavonoids, HQSAR, ovarian cancer.

Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases

Due to toxicity problems, various plant-derived compounds have been screened to find the chemotherapeutic agents. As anticancer therapeutic agents, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. Chromenones show anticancer activities too. Therefore, hybrids of chalcone and chromenone may be potent chemotherapeutic agents. We prepared 16 synthetic chromenylchalcones and applied a clonogenic long-term survival assay method for them on HCT116 human colorectal cancer cell lines. One of chromenylchalcones tested here, chromenylchalcone 11, showed IC50 of 93.1nM which can be competed with the IC50 values of well-known flavonoids such as catechin gallate and epicatechin gallate. Further biological experiments including cell cycle analysis, apoptosis assay, Western blot analysis, and immunofluorescent microscopy were carried out for this compound. In addition, in vitro kinases binding assay performed to explain its molecular mechanism demonstrated the compound inhibited aurora kinases. The binding modes between chromenylchalcone 11 and aurora kinases were elucidated using in silico docking experiments. These findings could be used for designing cancer therapeutic or preventive plant-derived chromenylchalcone agents.

Cell Death of Streptococcus mutans Induced by a Quorum-Sensing Peptide Occurs via a Conserved Streptococcal Autolysin

Streptococcus mutans, a member of the human indigenous oral microbiome, produces a quorum-sensing peptide called the competence-stimulating peptide (CSP) pheromone. We previously demonstrated that S. mutans expresses its CSP pheromone under specific stresses and responds to high levels of CSP by inducing cell death in a fraction of the bacterial population. Streptococci lack the classical SOS response, and the induction of the SigX regulon has been proposed to act as a general stress response in Gram-positive bacteria. We show here that inactivation of SigX abolished the CSP-induced cell death phenotype. Among SigX-regulated genes, SMU.836 (now named lytF(Sm)), encoding a conserved streptococcal protein, is a functional peptidoglycan hydrolase involved in CSP-induced cell lysis. We also demonstrated that LytF(Sm) is most likely a self-acting autolysin, since LytF(Sm) produced by attacker cells cannot trigger CSP-induced lysis of LytF(Sm)-deficient target cells present in the same environment. Electron microscopy revealed important morphological changes accompanying autolysis of CSP-induced wild-type cultures that were absent in the LytF(Sm)-deficient mutant. The LytF(Sm) promoter was activated in the physiological context of elevated concentrations of the CSP pheromone under stress conditions, such as exposure to heat, hydrogen peroxide, and acid. In a long-term survival assay, the viability of a mutant deficient in LytF(Sm) autolysin was significantly lower than that observed for the wild-type strain. The results of this study suggest that cell death of S. mutans induced by its quorum-sensing CSP pheromone may represent a kind of altruistic act that provides a way for the species to survive environmental stresses at the expense of some of its cells.

Abstract B201: Identification of miRNA modulators to PARP inhibitor response.

Abstract Many pre-clinical and clinical studies have shown that inhibitors of the DNA repair protein PARP can have potentiating effects when combined with DNA damaging treatment modalities such as IR and temozolomide. Moreover, PARP inhibitors can be used as an efficient single agent in treatment of tumors carrying homologous recombination (HR) gene defects such as BRCA1 and BRCA2. Genetic screens have previously been used to identify additional genetic deficiencies, which render tumor cells sensitive to PARP inhibitors. As yet, the contribution of microRNAs (miRNAs) to PARP inhibitor sensitivity has not been comprehensively studied. In this study our aim was to identify miRNA determinants of PARP inhibitor sensitivity and resistance, and in doing so potentially refine the eventual clinical use of these agents. The human genome encodes over 1000 well annotated miRNAs. We used a high throughput method of miRNA mimetic screening to identify novel modulators of PARP inhibitor response. The assay involved transfecting cells with a library of approximately 900 miRNA mimics in a 384-well plate format. 48 hours after transfection half of the cells were treated with PARP inhibitor Olaparib and the other half with the drug vehicle. We identified several miRNA mimetics that modulate the response to Olaprib. Hits were validated by long-term survival assays as well as by analyses of DNA damage foci. We used several strategies to determine the target genes of our hits. We performed westerns, qRT-PCR, luciferase reporter assays and immunoprecipitation assays and demonstrated several miRNAs involved in regulating the HR repair pathway. Genes that modulate the response to PARP inhibitors are often implicitly involved in controlling DNA repair by HR and thus frequently found to be mutated in cancers. Our future work will focus upon whether the miRNAs identified in our screen drive HR dysfunction in human tumors and could be used as biomarkers to direct the use of therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B201.