AACR 2016: Abstracts 2697-5293

Proceedings of the AACR

Volume 57 | April 2016

Part B: Abstracts 2697-5293

TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Bioinformatic Tools for Analysis and Mathematical Modeling of Clinical Samples

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Cycle Control and Checkpoints

DNA Double-Strand Break Repair Defects in Cancer: Therapeutic Strategies and Molecular Basis

DNA Methylation 1

Hypoxia and Oxidative Stress

Pharmacological Inhibitors of Cyclin-dependent Kinases

Senescence, Cell Death, and Unfolded Protein Response

Transcriptional Regulation and Gene Expression in Human Malignancies

Translational and Therapeutic Relevance of Perturbations of Gene Regulation in Malignancy

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Mechanisms of Drug Resistance 3

Monoclonal Antibodies and Antibody-Drug Conjugates

New Mechanisms of Anticancer Drug Action

Novel Targets and Pathways

Preclinical Radiotherapeutics

Small Molecule Inhibitors

CANCER CHEMISTRY:

Structural and Chemical Biology

CLINICAL RESEARCH:

Biomarkers for Gastrointestinal, Hematologic, and Uncommon Cancers

Circulating Biomarkers 2

Molecular Classification and Genomic Applications

Novel Approaches to Pediatric Cancers

IMMUNOLOGY:

Immune Checkpoints 2

Innate Immune System, Myeloid Cells, and Tumorigenesis

TUMOR BIOLOGY:

Antiangiogenic Therapy: Inhibitors and Resistance

Hematological Microenvironment

Microbiome in Cancer

Stemness Properties of Breast and Ovarian Cancer

Stemness Properties of Leukemias and Carcinomas

Tumor Angiogenesis: Host Interactions and the Tumor Microenvironment

Tumor Angiogenesis: Mediators and Mechanisms

EPIDEMIOLOGY:

Epidemiology of Cancer Prognosis and Survival

Exogenous Exposures and Cancer Risk

PREVENTION RESEARCH:

Behavioral and Social Science Studies across the Cancer Prevention Continuum

ENDOCRINOLOGY:

Clinical Endocrinology

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Death 1

Cell Death 2

Cell Death 3

Genetic Instability in Cancer: Molecular Basis and Tools

Genomic Technologies

Genomic Technologies and Analyses

Tumor Suppressor Genes and Pathways

Tumor Suppressors: TP53 Pathway

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Cell Death Pathways and DNA Repair

Gene and Vector-Based Therapy

Novel Antitumor DNA-Reactive Agents

Novel Targets

Oncogenes and Tumor Suppressor Genes as Therapeutic Targets

Targeting Protein Kinases, Death Pathways, and the Tumor Microenvironment

CANCER CHEMISTRY:

Proteomics and Mass Spectrometry

Therapeutics

CLINICAL RESEARCH:

Biomarkers for Breast Cancer

Circulating Biomarkers 3 / Immune Biomarkers

Novel Molecular Diagnostics and Imaging

IMMUNOLOGY:

Immune Modulation from Non-Immunotherapy: Preclinical

Mechanisms and Applications of Immune-based Therapies

TUMOR BIOLOGY:

Chemical and Viral Carcinogenesis

Host-Tumor Interactions

Imaging and Therapeutics of Metastasis

Immune Cell Activity

Mechanisms of Tumorigenesis in Animal Models of Cancer 2

Molecular and Cellular Imaging of Cancer 1

Molecular and Cellular Imaging of Cancer 2

New Cell Lines and 3D Models

EPIDEMIOLOGY:

Biomarkers of Endogenous and Exogenous Exposures

PREVENTION RESEARCH:

Diet and Cancer

CANCER CHEMISTRY:

Drug Design and Delivery

CLINICAL RESEARCH:

Clinical Qualification of Impactful NGS-based Biomarkers

EPIDEMIOLOGY:

Endogenous and Exogenous Factors in Cancer Epidemiology throughout the Life Course

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Identifying Targets and Combinations through Novel Approaches

IMMUNOLOGY:

Potentiating Immunotherapy Responses with Next Generation Agents and Combinatorial Partners

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Genomic Alterations and Their Functional Consequences

Oncogenic Cell Signaling: Mechanisms and Translational Insight

MULTIDISCIPLINARY:

New Cool Tools for Cancer Discovery

TUMOR BIOLOGY:

Therapeutic Studies in Cell Culture and Mouse Models

Tumor-supporting Microenvironment

Advocates Poster Session 2

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Signaling

DNA Methylation 2

Epigenetic Biomarkers and Therapies

Germline/Somatic Genomics and Personalized Medicine

Histone Modifications and Chromatin Dynamics

Protein Modification and Transcriptional Regulation

Receptors

Wnt, AKT, and Cell Survival Pathways

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Cellular Responses to Anticancer Drugs

Combination Therapies and Approaches to Sensitizing Cancer Cells to Drugs

Epigenetic Agents

HDAC, Methyltransferase Inhibitors, and Novel Anticancer Agents

Molecular Characterizations of Tumors and Translational Studies

Novel Chemotherapies

Targeted Therapy

CANCER CHEMISTRY:

Drug Design

IMMUNOLOGY:

Immune Modulating Agents 2

Immune Response Monitoring: Preclinical

CLINICAL RESEARCH:

Biomarkers for Gastrointestinal Cancer

Biomarkers for Genitourinary Cancers: Prostate

Immune Modulation from Non-Immunotherapy and Antibodies: Clinical

Predictive and Prognostic Biomarkers

TUMOR BIOLOGY:

Cell Adhesion and Tumor Treatment

Cellular and Molecular Dynamics of Cancer Migration and Invasion

Extracellular Microenvironment

Immunomodulation and Immunotherapy

Molecular Carcinogenesis

Therapeutic Studies in Patient-derived Xenografts

EPIDEMIOLOGY:

Description of Cancer Trends and Next-Generation Sequencing in Epidemiology

PREVENTION RESEARCH:

Chemoprevention Studies

BIOINFORMATICS AND SYSTEMS BIOLOGY:

New Bioinformatic Tools, Databases, Portals, and Data Resources

Tuesday, April 19, 2016

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Bioinformatic Tools for Analysis and Mathematical Modeling of Clinical Samples

#2697

Drug combinations for breast cancer based on synthetic lethality screens in yeast.

Michael Schwarz,¹ Erwin Tomasich,¹ Maximilian Marhold,¹ Andreas Heinzel,² Paul Perco,² Peter Horak,³ Bernd Mayer,² Michael Krainer¹. ¹Medical University of Vienna, Department for Internal Medicine I - Oncology, Vienna, Austria; ²emergentec biodevelopment GmBH, Vienna, Austria; ³SMZ Ost, Department for Medicine II - Oncology, Vienna, Austria.

Synthetic lethality describes an interdependent relationship between two genes, where the loss of either one alone can be compensated, while the simultaneous loss of both genes causes a non-viable phenotype. In recent years, first therapeutics based on this concept entered the clinic, most notably the PARP inhibitors for BRCA1/2-mutated cancers.

In the present study, we analyzed synthetic lethal interactions in yeast to identify new and potentially synergistic drug combinations for breast cancer therapy. We were able to confirm significantly enhanced cytotoxicity for predicted drug pairs in breast cancer cell lines in vitro.

First, a predictor built from publicly available yeast genetic interactions in the Data Repository of Yeast Genetic INteraction (DRYGIN) was used to predict potential synthetic lethal genetic interactions in human. Independently, a data set containing all pharmacological approaches, targeted or cytostatic, in breast cancer therapy was created. These drug combinations and their respective targets were then analyzed regarding their coverage of predicted synthetic lethal interactions. New drug combinations, previously unused in breast cancer therapy, were identified in silico by combining drugs already in use for breast cancer therapy (individually or in other combinations) in such a manner that their combination covers one or more potential synthetic lethal gene pairs. From this set of new drug combination and their synthetic lethal gene pairs we further pursued the predicted interdependencies between farnesyl diphosphate synthase (FDPS) and tubulin, beta 1 (TUBB1) and between FDPS and phosphoinositide-dependent kinase-1 (PDPK1) as well as prostaglandin-endoperoxide synthase 2 (PTGS2). Drugs targeting these genes are celecoxib (PDPK1, PTGS2), an anti-inflammatory drug, and zoledronic acid (FDPS), a bone degradation inhibitor, as well as the cytotoxic agent paclitaxel (TUBB1).

We performed cell viability and cytotoxicity assays to determine therapeutic effects of celecoxib, zoledronic acid and paclitaxel alone and in combination on selected breast cancer cell lines.

Our results showed statistically significant decreases in cell viability for the combinational treatment with zoledronic acid and paclitaxel as well as with the per se non-cytotoxic combination of zoledronic acid and celecoxib when compared to single agent treatment.

In conclusion, we present a bioinformatics approach to predict potentially synergistic gene interactions based on synthetic lethality found in yeast and a strategy for utilizing these interactions for identifying new potentially synergistic drug combinations.

#2698

Transcriptional profiling of human TAMs highlights differences in breast and endometrial tumour microenvironments.

Matina Fragkogianni,¹ Luca Cassetta,¹ Agnieszka Swierczak,¹ Lisa Wiechmann,² Harriet O. Smith,³ Andrew H. Sims,⁴ Hui Zhang,⁵ Lisa M. Coussens,⁶ J. W. Pollard¹. ¹MRC Centre for Reproductive Health, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; ²Northeast Medical Group, Yale New Haven Health, CT; ³Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, NY; ⁴CRUK Edinburgh Cancer Centre, Edinburgh, United Kingdom; ⁵Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, NY; ⁶Oregon Health & Science University, OR.

Background: Breast and endometrial cancers are two of the most frequently diagnosed reproductive cancers in women in the western world. Previously, research has focused on cancer cells, however increasing evidence shows that the tumour microenvironment plays an important role in cancer progression. Macrophages are almost entirely derived from circulating monocytes and are recruited into tumour sites. In murine studies, tumour-associated macrophages (TAMs) have been implicated in promoting angiogenesis, immune suppression and resistance to treatment. Their density has been associated with poor prognosis in breast cancer and decreased survival in endometrial cancer. However, little is known about the function of TAMs in humans. The aim of this study is to examine the transcriptional profiles of human TAMs in order to investigate their biological relevance and potential for therapeutic intervention.

Methods: RNA-sequencing was performed on purified normal macrophages and TAMs from breast tissue (4 breast cancer and 4 healthy breast) and endometrium tissue (5 endometrial cancer and 9 healthy endometrium). Statistical analysis using Limma was used to identify significantly differentially expressed genes (FDR< 0.05) with a minimum log2 fold change of 1.5. Gene set enrichment (GSEA) analysis and gene ontologies (GO) were employed for functional analysis and identification of important biological pathways.

Results: Transcriptome profiling revealed a significantly altered gene expression profile of TAMs when compared to normal macrophages. Furthermore, comparison of TAMs between breast and endometrial cancer also revealed differences suggesting that different tumour microenvironments induce different gene expression profiles in TAMs. Interestingly, comparison of normal macrophages between breast and endometrial tissue also revealed differences in gene expression suggesting tissue specificity for macrophages. Functional analysis of significant genes revealed similar biological pathways to those of murine studies suggesting that TAMs in humans may have similar functions. We were able to extract TAM-specific genes by comparing with publicly available datasets that could serve as markers for their identification. Finally, we identified a list of transmembrane receptors that could act as potential targets for targeted killing of TAMs.

Conclusion: This is the first study to carry out genome-wide profiling of TAMs in human breast and endometrial cancer. Expression profiles differed between TAMs and healthy patients, as well as between breast and endometrial cancer suggesting cancer specificity for TAMs and revealing not only potential TAM-specific markers, but also identifying possible cancer- and TAM-specific therapeutic targets.

#2699

A cancer treatment based on synergy between anti-angiogenic and immune cell therapies.

Luis F. Soto-Ortiz. East Los Angeles College, Monterey Park, CA.

A mathematical model integrating tumor angiogenesis and tumor-targeted cytotoxicity by immune cells was developed to identify the therapeutic window of two distinct modes to treat cancer: 1) an anti-angiogenesis treatment based on the monoclonal antibody bevacizumab that targets tumor vasculature, and 2) immunotherapy involving the injection of unlicensed dendritic cells to boost the anti-tumor adaptive response. The angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) contributes to the immunosuppressive tumor microenvironment, which is responsible for the short-lived therapeutic effect of cancer-targeted immunotherapy. The effect of immunosuppression on the width of the therapeutic window of each treatment was quantified. Experimental evidence has shown that neutralizing immunosuppressive cytokines results in an enhanced immune response against infections and chronic diseases. The model was used to determine treatment protocols involving the combination of anti-VEGF and unlicensed dendritic cell injections that enhance tumor regression. The model simulations predicted that the most effective method to treat tumors involves administering a series of biweekly anti-VEGF injections to disrupt angiogenic processes and limit tumor growth. The simulations also verified the hypothesis that reducing the concentration of the immunosuppressive factor VEGF prior to an injection of unlicensed dendritic cells enhances the cytotoxicity of CD8+ T cells and results in complete tumor elimination. Feasible treatment protocols for tumors that are diagnosed late and have grown to a relatively large size were identified.

#2700

Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors.

Andreas M. Hoff, Sen Zhao, Bjarne Johannessen, Rolf I. Skotheim. Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Norwegian Radium Hospital, Oslo, Norway.

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men. Embryonal carcinoma (EC) comprises a histological subgroup of TGCT that exhibits pluripotent characteristics similar to embryonic stem (ES) cells. The genetic drivers underlying malignant transformation of TGCT, including ECs, are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the formation of fusion genes or expression of aberrant transcript variants, we have previously performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. From this dataset, we identified and characterized eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. These were the first fusion genes to be shown in TGCT (Hoff et al., Cancer Research, in press).

To search for recurrence of the originally nominated TGCT specific fusion transcripts and other transcript variants in an external sample series, we have here parsed raw RNA sequencing data from 102 primary TGCTs obtained from The Cancer Genome Atlas (TCGA; unpublished). In this process, we have evaluated multiple fusion finder algorithms and approaches to determine the optimal method, on the basis of sensitivity and specificity, to query specific transcript variants and breakpoints in RNA sequencing datasets.

We were able to detect the recurrent fusion transcripts; RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and the transcript variant of ETV6, which was previously detected from our in-house RNA sequencing dataset from EC cell lines. We also demonstrate that expression of several fusion transcripts correlate significantly with the histological subtype. In conclusion, the novel fusion transcripts and ETV6 transcript variant are expressed from a high fraction of primary TGCTs with low differentiation grade, as detected across independent sample series.

#2701

Combining whole-exome and RNA-Seq data improves the quality of PDX mutation profiles.

Manuel Landesfeind, Bruno Zeitouni, Anne-Lise Peille, Vincent Vuaroqueaux. Oncotest GmbH, Freiburg, Germany.

Patient-derived xenograft tumor models (PDX) are of increasing interest for anti-cancer agent testing due to their close resemblance to patient tumors. An accurate molecular characterization of the models is essential 1) to select the PDX that best fit the genetic requirements for a successful cancer therapy investigation and 2) to identify potential predictive biomarkers of response. In this study, we evaluated the quality of mutation profiles from whole-exome sequencing (WES) in terms of concordance with previously acquired mutation data in a large collection of PDX. Further, we analyzed the persistence of disclosed mutations at the transcript level with RNA-Seq.

From 339 PDX, DNA was extracted and enriched in exonic regions with Agilent SureSelect kits before Illumina HiSeq 2000 sequencing with a minimum expected average-of-coverage of 100X. Raw paired-end reads were analyzed by a PDX-specific bioinformatics pipeline to identify the human mutation profile. Sequenom Oncocarta and Sanger sequencing data acquired for 29 cancer genes in 272 PDX was used to evaluate the WES mutation profiles. In parallel, 92 PDX were profiled with RNA-Seq (100M sequencing reads required) and we investigated the expressed mutation profiles by comparing with mutations from WES data.

Among 502 point mutations found with classical methods, 95% were retrieved by WES analyses, revealing the very high sensitivity of the PDX-specific bioinformatics pipeline. 5% of mutations were missed because of a low coverage, particularly in the STK11 gene and in the KRAS gene of pancreatic models, possibly due to poor gene enrichment and high mouse stroma content, respectively. Deeper sequencing could potentially overcome this lack of coverage. Additionally, the WES analysis pipeline displays a high specificity, reporting only 1 additional mutation at gene positions covered with the classical methods. Finally, 507 mutations were detected by WES at positions not interrogated by classical methods emphasizing the necessity for next-generation sequencing (NGS) to obtain a comprehensive mutational spectrum. The number of mutations found using RNA-Seq data was on average two times lower and covered 15% of the mutations detected in WES. This was mainly due to the non-expression of genes or isoforms (40%), the mono-allelic expression of genes (30%), and low coverage data (15%). RNA-Seq analysis restricted to expressed genes represents a substantial complement to WES mutation data and enhances understanding of actual gene alterations in cancer cells.

This study demonstrated the high quality of mutation profiles obtained by WES and highlights the importance of integrating expression data to accurately predict the impact of a mutation at the protein level. An accurate molecular characterization of models is crucial for the selection of PDX with a specific genetic background for the evaluation of anticancer agents.

#2704

Radiotherapy and immunotherapy in cancer: A mathematical model.

Raphael Serre, Xavier Muracciole, Joseph Ciccolini, Sebastien Benzekry, Dominique Barbolosi. SMARTc Unit, Aix Marseille Université, INSERM, CRO2 UMR_S 911, Marseille, France.

The rise of immunotherapy is a major breakthrough in oncology. Recently, the combination of radiotherapy with the blockade of immune checkpoint inhibitors such as the PD1-PDL1 axis or the CTLA4 pathway has shown a synergistic potential: in addition to the direct cell kill induced by irradiation, radiotherapy unleashes neoantigens that can further induce an anti-tumoral immune response. However, this immune response can be inhibited by the immunosuppressive nature of the tumor micro-environment (TME). Hence, radiotherapy combined with immune check-point inhibitors is a promising solution and is the subject of preclinical and clinical research. However, defining the most efficient scheduling between radiotherapy and immunotherapy is a crucial issue that cannot be properly addressed solely by empirical trial-and-error practices. Consequently, developing mathematical models that can describe the synergy between immune checkpoint inhibitors and radiotherapy is critical. Hence, we have built a pharmocodynamic model of the combination of radiotherapy with inhibitors of the PD1-PDL1 axis and/or the CTLA4 pathway. We describe a mathematical representation of how a growing tumor first elicits and then inhibits an anti-tumoral immune response. This anti-tumoral immune response is described by a primary and a secondary response. The primary immune response appears first and is down-regulated by the PD1-PDL1 axis, while the secondary immune response happens next and is down-regulated by the CTLA4 pathway. We describe the effects of irradiation by a slightly modified version of the Linear-Quadratic model. In particular, we explain the biphasic relationship between the size of a tumor and its immunogenicity, as measured by the abscopal immune response. The ability of the model to forecast pharmacodynamic endpoints was retrospectively validated by reproducing results from experimental studies investigating radiotherapy and immune checkpoint inhibitors. This model clarifies the issue of synchronisation of immunotherapy with radiotherapy and it also explains why the CTLA4 blockade often occurs with a delay. The model also explains why a sustained response to immunotherapy may or may not happen after treatment discontinuation. We believe that this mathematical model could be further used as a simulation tool to help decision-makers determine the optimal scheduling between radiotherapy and immunotherapy and could be a building block for the in-silico design of optimized multimodal strategies.

#2705

A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations.

Alexander T. Pearson, Patrick Ingram, Shoumei Bai, Euisik Yoon, Trachette Jackson, Ronald J. Buckanovich. University of Michigan, Ann Arbor, MI.

We have developed a new mathematical algorithm and corresponding computer software that uses observations of single cell behavior to predict the growth of the cancer stem-like cell proportion in larger cancer cell groups. Cancer stem-like cells (CSCs) have been implicated in ovarian cancer tumor growth, chemotherapy resistance, and disease recurrence. Aldehyde dehydrogenase (ALDH) is a primary discriminator of CSCs in ovarian cancer as well as in other cancer types. Unfortunately, the rarity and ability of CSCs to rapidly differentiate makes them difficult to study in-vitro and in-vivo. Microfluidic capture devices now allow us to grow and evaluate single ovarian cancer cells in isolated culture. We deployed microfluidic devices to evaluate the different self-renewal and asymmetric division patterns of ALDH+ and ALDH(-) ovarian CSCs. We analyzed data gathered from arrays of single cell chamber observations and found that purified ALDH+ cells were more proliferative than ALDH(-) cells in both cell-line (n = 112, p < 0.001) and primary (n = 41, p = 0.008) ovarian cancer specimens. Importantly, ALDH+ cells could produce both ALDH+ and ALDH(-) cells, whereas ALDH- cells were only able to produce ALDH- cells. Based on this hierarchy-defining data, we developed an easy to use computer algorithm on the freely-available software R to predict cancer cell population growth in-vitro and in-vivo. In our algorithm, size changes of cell populations are simulated by drawing from observed events. We compared the predictions from our hybrid microfluidics chip and computational algorithm with validation experiments in-vivo and in-vitro for both cell line and primary ovarian cancer cells. We found a superb correlation between observed and predicted in-vitro total and CSC counts for both cell line and primary ovarian cancer cells (correlation r = 0.98, p < 0.0001). Furthermore, this approach appropriately predicted changes in cell growth in the presence of the CSC-promoting growth factor EGFL6 both in vitro and in vivo, over time frames of up to 28 days. The single cell division based sampling algorithm developed here allows for rapid and inexpensive means to predict in-vivo ovarian tumor growth based on microfluidic chip culture and can easily be adapted to evaluate new therapeutic options across other cancer subtypes.

#2706

Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA.

Jun Koseki, Kenta Tsunekuni, Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Osaka University, Suita, Japan.

Trifluridine (FTD), known as main component of TAS-102, is beginning to be applied as anti-tumor agent due to the highly efficacious antitumor potency although there is little to distinguish FTD structure from thymidine structure. TAS-102 has first been used in Japan in clinical therapy as an oral agent. Some experimental results hypothesized that FTD pharmacological antitumor effect can be arisen by inhibition of thymidylate synthase and incorporation of FTD itself into DNA. One of them is the thermal denaturation experiments for DNA duplexes of containing some FTD or not, performed by J. C. Markley et al. Their experiments have shown that the DNA duplexes containing FTD are slightly less stable, with a melting temperature about three degrees lower than not containing duplexes. However, the change of thermodynamic structure and inter-nucleobase interaction changes are not yet known.

In this presentation, we have performed molecular dynamics (MD) simulations and ab initio molecular orbital (MO) calculations to analyze the differences of thermodynamics structure and inter-nucleobase interaction between containing and not containing FTD. From these results, we sought to qualitatively understand the effects of FTD. At the first step, we have estimated the stabilization structures of same two DNA duplexes (containing FTD and not) used by J. C. Markley, with energy minimization. Then, a large differences were shown between these structures. The hydrogen bond breaking is observed between FTD and complementary Adenine base while the not containing DNA duplex keeps having a stabilized structure. The behavior means that the essential difference of intra-DNA interactions was arisen. At the second step, MD simulations were performed to analyze the distribution of distances between FTD and complementary base (i.e. thermally available conformations). With some conformations from MD sampling, the MO calculations were performed to discuss the difference of intra-base interactions.

From our results, we found that thermodynamic conformational change of DNA duplex containing FTD occurs even if it is in internal body temperature, and the break hydrogen bonding between FTD and paired base was observed more frequently. In addition, strong interactions between FTD and nearby π-conjugate base were observed. These behaviors might be one of the factor of inducing unstabilization in DNA duplexes.

#2707

Quantitative analysis of the role of androgen receptor in radiation treatment for prostate cancer.

Mengdi Qian,¹ Alexandru Almasan,² Evren Gurkan-Cavusoglu¹. ¹Case Western Reserve University, Cleveland, OH; ²Cleveland Clinic, Cleveland, OH.

The purpose of this study is to quantitatively investigate the mechanism of androgen receptor (AR) and non-homologous end joining (NHEJ) interaction after and ionizing radiation (IR) and androgen deprivation therapy (ADT) treatment. In most clinical treatment approaches, a combination of ADT and IR is used in order to improve prostate patients’ overall survival probability. It is found that AR interacts with NHEJ pathway, a major double strand break (DSB) repair pathway. We have developed a mathematical model of the NHEJ pathway that is composed of a series of ordinary differential equations. The kinetic rate constants constitute the unknown parameters of the model and we have estimated these parameters using least square estimation. The data sets used in the parameter estimation were obtained from the literature and they were from both in vitro experiments as well as clinical data from prostate cancer patients. Both sets of data demonstrated the effects of AR on DSB repair by NHEJ. The in vitro data suggest that AR enhances DSB repair by inducing expression/activity of DNA-PKcs, which is a key protein in NHEJ. The clinical data show that the effect is through the first NHEJ protein complex that is recruited to DSB, Ku70/80. We have developed the models for IR treatment alone or in combination with ADT to determine which of these mechanisms can explain the behavior in both data sets simultaneously. We have carried out sensitivity analysis and identifiability tests on all the parameters from both models in order to determine the parameters that are reliably estimated. We have shown that when the rate constant for the initiation protein Ku70/80 is decreased to half in the IR+ADT case compared to the IR only case, the model outputs captured the observations in both experimental and clinical data. The rate constant for Ku70/80 is proved to be both sensitive and identifiable, indicating that the two-fold difference in the rate constants of IR only and IR+ADT cases is biologically relevant. Therefore, we conclude that when ADT is applied, AR significantly slows down the kinetic activity of Ku70/80, thus the DSB repair by NHEJ. The suppression of AR activity through ADT has the potential to enhance the IR treatment outcomes for the prostate cancer patients that are resistant to IR-only treatment.

#2708

Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma.

Abbas Shirinifard, Suresh Thiagarajan, Yogesh T. Patel, Abigail D. Davis, Megan O. Jacus, Stacy L. Throm, Jessica Roberts, Vinay Daryani, Clinton F. Stewart, András Sablauer. St. Jude Children's Research Hospital, Memphis, TN.

Resistance to chemotherapeutics and targeted therapies in pediatric solid tumors including neuroblastoma is a common cause of poor clinical outcome. These failures in part stem from shortcomings in understanding inter- and intra-tumor heterogeneities of drug penetration due to heterogeneities in blood perfusion. Herein we propose to develop an individualized 3D transport model of topotecan (TPT) for a patient-derived orthotopic xenograft model of pediatric NB5 neuroblastoma to account for inter- and intra-tumor heterogeneities in blood perfusion. The transport model uses a 3D reaction-diffusion equation to simulate diffusion of TPT from blood vessels into the tumor tissue and its flux in and out of intracellular space. Our transport model takes three types of inputs to predict TPT exposure maps defined over the volume of an individual tumor: a) plasma concentration-time profiles from an individualized physiologically-based pharmacokinetic (PBPK) model of TPT (separate cohort), b) 3D blood perfusion map of the individual tumor from contrast enhanced ultrasound (CEUS) using VisualSonics VEVO 2100 imaging system, and c) in vitro TPT cellular uptake and efflux kinetics from two-photon imaging. We use in vitro pharmacodynamics (PD) experiments with NB5 cells exposed to TPT to derive probabilistic PD-rules for drug effects (e.g., γ-H2AX response). Based on these rules and the exposure maps, we then compute probabilities of effects for the entire tumor volume. We will validate the predicted drug effect maps by comparing them to the observed effects measured by immunohistochemistry marker for γ-H2AX from the same tumor (location matched) using spatial correlation techniques.

#2709

Mathematical modeling for prediction of secondary BRCA1 and BRCA2 mutations in ovarian cancers with deleterious germline BRCA1 and BRCA2 mutations.

Dana-Adriana Botesteanu,¹ Doron Levy,¹ Jung-Min Lee². ¹Department of Mathematics and Center for Scientific Computation and Mathematical Modeling (CSCAMM), University of Maryland, College Park, MD; ²Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.

Background:

DNA damaging agents such as cisplatin or carboplatin and poly(ADP-ribose) polymerase inhibitors (PARPi) have clinical activity in germline BRCA1 and BRCA2 mutation-associated ovarian cancers. However, BRCA1 or BRCA2-mutated tumors often develop resistance to these drugs. Restoration of BRCA function due to secondary BRCA mutations has been recognized as one of the clinical resistance mechanisms. The aim of this mathematical analysis is to study the emergence and frequency of secondary BRCA mutations in a cohort of BRCA-deficient in silico ovarian cancer-positive cases.

Methods:

We develop a 2D stochastic cell-cycle specific mathematical model of BRCA-deficient ovarian cancer growth and progression prior to and post-treatment, which includes debulking surgery, platinum-based chemotherapy or PARPi. This is a continuum model based on a combination of ordinary differential equations using Gompertzian growth kinetics subject to random discrete-time jump processes. This approach enables us to reflect the inter-individual heterogeneity in disease progression and drug administration reported in published data. Calculations are performed for in silico cancer-positive growth curves in which the inception of the first ovarian malignant cell is assumed to have already occurred.

Results:

Our modeling approach generates an empirical cumulative distribution of disease-free survival times prior to recurrence or disease progression, given the heterogeneous cancer progression illustrated by our in silico cohort. Our preliminary computational results indicate that an initially rare resistant cell clone carrying a secondary BRCA mutation pre-exists to treatment in circa 90% of the simulated progression curves. For the remaining 10% of the simulated curves, it is more likely that the secondary mutations are acquired during chemotherapy or PARPi via increased mutation rates, possibly conferred by the increasing amount of damaged DNA lesions caused by the cancer treatment.

Conclusions:

Our mathematical model provides some insight on the dynamics of secondary BRCA mutations that cannot be accessed through existing genomic analysis methods, as current DNA sequencing depth thresholds cannot detect the presence of rare tumor cell clones carrying such mutations. We predict that a large proportion of initially resistant cell clones with restored BRCA function more likely originate prior to rather than post-treatment. If such clones get selected for by subsequent platinum-based chemotherapy or PARPi in a Darwinian fashion, our model generates an empirical distribution of disease-free survival times prior to recurrence. This may have direct implications on the possible duration of subsequent treatment response in our simulated ovarian cancer-positive population.

#2710

Model evolution technique as a novel concept for characterization of tumor heterogeneity in dynamic contrast enhanced MRI studies.

Hassan Bagher-Ebadian,¹ Azimeh NV Dehkordi,² Rasha Alamgharibi,³ David Nathanson,¹ Hamid Soltanian-Zadeh,¹ Arbab S. Ali,⁴ Stephen Brown,¹ Meser M. Ali,¹ Tom Mikkelsen,¹ James R. Ewing¹. ¹Henry Ford Hospital, Detroit, MI; ²Shahid Beheshti University, Tehran, Islamic Republic of Iran; ³Oakland University, Rochester, MI; ⁴Georgia Regents University, Augusta, GA.

Introduction: Many studies have shown that tumor vascular network and the assortment of tumorous cells inside and on the periphery of solid tumors are spatially heterogeneous. Variation in cell packing density (VCPD), hypoxia, acidosis, and elevated interstitial fluid pressure (IFP) are main characteristic features of solid tumors. Elevated IFP and VCPD in solid tumors can be generally relevant to the pathological structures at the cellular level that is fundamental to understanding the chance of response to treatment and recurrence. Therefore, non-invasive quantification of tumor heterogeneity for the same types of tumors can play an important role in diagnosis and treatment planning.

Hypothesis: In this pilot study, using Nested Model (NM) selection technique, Model Evolution (ME) concept is framed and introduced to quantify the evolutions of 3 different physiologically NM that are derived from standard Tofts model, throughout the course of Dynamic Contrast Enhanced (DCE) Magnetic Resonance Imaging (MRI) experiment. Using ME technique for pharmacokinetic (PK) modeling and DCE-MRI data analysis, a heterogeneity measure is formulated and introduced based on the evolutionary profile of the estimated extra-cellular extra-vascular (ve) volume. We hypothesized that the ME profiles in the course of DCE-MRI experiment, highly depend on the inward diffusion and outward convection of contrast agent concentration and contain abundant information for describing the compartmentalization and heterogeneity levels of solid tumors.

Material and Methods: 24 athymic Nude rats with U251n rat tumor model of cerebral tumor were studied. Look-Locker T1 mapping and DCE-MRI experiments (Dual Gradient Echo, 150 image sets at 4.0 sec intervals over 10 min: matrix = 128x64, five 2.0 mm slices, NE= 2, NA=1, TE/TE/TR = 2.0/4.0/40ms with bolus intravenous injection of the Magnevist at 0.25 mmol/kg) acquired at 7T field strengths. In each animal, in-vivo measurement of tumor IFP was done right after the DCE-MRI experiment using a wick-in-needle technique. The ME technique was applied on DCE-MR data of 24 U251n rat tumors to characterize the heterogeneity of each tumor and then the results were compared to their known in vivo measure of IFPs.

Results and Conclusions: Results of this pilot study clearly attest that the evolutionary profile of ve can be used to characterize the heterogeneity level of solid tumors. The ME results imply that as the slop of the evolutionary profile increases, the IFP of tumor increases. Also, the latency of the profile during the course of MR experiment can reliably explain the tumor compartmentalization and their elevated IFP. This pilot study confirms that the ME concept can make a paradigm shift in non-invasive quantification of tumor heterogeneity from DCE-MRI studies

#2711

Spatiotemporal progression patterns in metastatic lung cancer treated with bevacizumab.

Jeremy M. Mason, Paul K. Newton, Gino K. In, Sonia Lin, Peter Kuhn, Jorge J. Nieva. University of Southern California, Los Angeles, CA.

We describe a Markov chain mathematical model of lung cancer progression based on a longitudinal dataset of 722 lung cancer patients. Specifically, we investigate the patterns of metastatic spread in lung cancer and how the VEGF inhibitor, bevacizumab, alters these metastatic patterns. Stochastic models were used to simulate metastatic spread by means of random walk processes on directed graphs. We created spatiotemporal diagrams of cancer progression to analyze the differences between patients treated and not treated with bevacizumab. Patients with squamous lung cancer or brain metastases at baseline were ineligible for bevacizumab and excluded from analysis. Our results demonstrated that not only does bevacizumab extend survival, but it also alters patterns of metastatic spread. Patients treated with bevacizumab were characterized by greater heterogeneity in their pathways of metastatic progression, compared to those patients not treated with bevacizumab, which showed more homogeneity in their pathways. Furthermore, we quantify this spread and characterize metastatic sites as ‘spreaders’ and ‘sponges’ based on their probability of spreading the disease.

#2712

Joint somatic mutation and germline variant identification and scoring from tumor molecular profiling and ct-DNA monitoring of cancer patients by high-throughput sequencing.

Francisco M. De La Vega,¹ Ryan T. Koehler,¹ Yannick Pouliot,¹ Yosr Bouhlal,¹ Austin So,¹ Federico Goodsaid,¹ Sean Irvine,² Len Trigg,² Lincoln Nadauld³. ¹TOMA Biosciences, Foster City, CA; ²Real Time Genomics, Hamilton, New Zealand; ³InterMountain Healthcare, Saint George, UT.

Cancer tumor profiling by targeted resequencing of actionable cancer genes is rapidly becoming the standard approach for selecting targeted therapies and clinical trials in refractory cancer patients. In this clinical scenario, a tumor sample is obtained from an FFPE block and sequenced by targeted next-generation sequencing (NGS) to uncover actionable somatic mutations in relevant cancer genes. Some of the challenges that arise in analyzing tumor-derived NGS data include distinguishing between somatic and germline variants in the absence of normal tissue data, recognizing pathogenic germline variants, and identifying sequencing errors (which occur at about 0.5% rate). Additional challenges arise when considering other clinical applications of NGS such as sequencing cell-free tumor DNA (cf-DNA) from plasma samples to monitor disease response or disease recurrence. Here we present a principled approach to identify both single-nucleotide and small insertion/deletion somatic mutations and germline variants from NGS data of tumor tissue that leverages the allelic fraction patterns in tumors and prior information from external databases through the use of a Bayesian Network algorithm. Our approach allows us to score each putative mutation or variant with respect to its probability of belonging to each variant class, versus classification as a sequencing error. The method enables the joint calling of related samples form the same patient, such as cases where a cf-DNA sample and primary tumor sample are both profiled improving sensitivity and specificity. We validated our method by analyzing data obtained with the TOMA OS-Seq targeted sequencing RUO assay for 98 cancer genes from a mixture of well-known genomes, patient case triads (where normal, tumor and cf-DNA are available), and a retrospective analysis of tumor patient data that underwent clinical tumor profiling for therapy selection.

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Cycle Control and Checkpoints

#2713

Combination of PLK1 and PI3K inhibitors shows strong synergy in anaplastic thyroid cancer.

Emrullah Yilmaz,¹ Arturo Orlacchio,² Antonio Di Cristofano². ¹Montefiore Medical Center, Bronx, NY; ²Albert Einstein College of Medicine, Bronx, NY.

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer with median of 6 months of survival from diagnosis. Molecular alterations are well defined recently. p53 mutations, and activation of PI3K, RAS and BRAF are among the most common alterations. PLK1 overexpression has also been identified in ATC.

We have previously shown that PLK1 inhibitors are effective in ATC cell lines. However PLK1 inhibition in cell lines with PI3K activation resulted in escaping growth arrest and subsequent mitotic catastrophe through mitotic slippage. Since the PI3K activation is common in ATC, there is risk of generating polyploid, genetically unstable cell populations by targeting these tumors with a PLK1 inhibitor. So we tested the effect of combining PLK1 and PI3K inhibitors in ATC cell lines.

We combined PLK1 inhibitor BI6727 and PI3K inhibitor BKM120 and measured the cell viability using Alamar Blue. Combination of these two drugs resulted in significant synergy in mouse derived PTEN deleted ATC cell lines. We observed the same synergy in the human ATC cell lines with PIK3CA mutations.

BI6727 showed significant G2/M arrest in the cell lines. The combination two drugs enhanced the G2/M arrest. In a low dose that the single drugs showed no inhibition in cell cycle, combination resulted in G2/M arrest.

We treated the cells with a dual PI3K and PLK1 inhibitor Rigosertib. Although we have seen some sensitivity of the cells with PIK3CA mutation, the cell lines with PTEN were strongly resistant to Rigosertib.

Our results show that combination of PLK1 and PI3K inhibitors is an effective treatment for ATC cells with PI3K activation. This combination results in cell cycle arrest suggesting the role of using combination therapies in this aggressive cancer.

#2714

Inhibition of human telomerase by new purine analogs.

Wilnelly M. Hernandez-Sanchez,¹ Diane Baus,¹ Anthony J. Berdis,² Derek J. Taylor¹. ¹Case Western Reserve University, Cleveland, OH; ²Cleveland State University, Cleveland, OH.

Telomeres are the end caps of linear chromosomes that help to ensure genomic stability. Because of the inability of DNA polymerases to synthesize the extreme ends of the lagging strand, telomeres get slightly shorter each time a cell divides. When telomeres get extremely short, a state of senescence is triggered which halts cell division. Some cells that require an extended lifespan (e.g. embryonic cells, germ lines and stem cells) use an enzyme called telomerase to maintain telomeres at a length that is sufficient to escape senescence. Whereas telomerase is undetectable, in adult somatic tissue, it is upregulated in 85-90% of all metastatic tumors; enabling cancer cells to divide indefinitely. Due to this unique property of cancer cells, targeting telomerase is a viable chemotherapeutic approach. Nucleotide analogs have been shown to inhibit telomerase activity (e.g. azidothymidine or AZT), albeit modestly, to promote telomere shortening in cancer cell lines. Also, most of these nucleotide analogs are limited by requirement of high dosage (e.g. The IC50 of AZT varies from 0.25 to 1.35 mM). Therefore, while promising, there is a need to develop other nucleotide analogs that are more potent in inhibiting telomerase activity. We have screened a series of new purines analogs using a direct in vitro telomerase assay. These nucleotide analogs were developed to have different chemical modifications around the indole group of the nucleobase. We have identified two purine analogs (5-MeCITP and 6-NITP) that inhibit telomerase activity in vitro. 5-MeCITP was measured to have an inhibition constant (ki) of ~45μM, which is significantly efficient. Future efforts will include evaluation of the lead nucleoside analogs in telomerase positive cancer cell lines to determine their effect on telomere length and cell survival.

#2715

Cell cycle regulated mitophagy: a key step at the G1/S metabolic checkpoint.

Daniela Annibali. KU Leuven, Leuven, Belgium.

How tumor cells coordinate their high demand for biosynthetic processes with nutrient availability, proliferation rate and oxidative stress tolerance is still not completely understood. We recently showed that highly proliferating cells, whether normal or tumorigenic, display different metabolic requirements throughout the cell cycle, in term of glucose and glutamine utilization. The role of autophagy in cancer cells is still debated, but recent evidence pointed to its involvement in the regulation of cellular bioenergetics and nutrients utilization. To investigate autophagy regulation during cancer cells proliferation and to characterize in detail the metabolic behaviour associated with tumor cell proliferation in the different phases of the cell cycle, we used synchronized HeLaS3, a human epithelial carcinoma cell line. Treatment of the cells with a double thymidine block (DTB) results in an arrest at the G1/S boundary and the possibility to study their progression through a complete division cycle. Here we show that in the highly proliferating HeLaS3 cancer cells basal autophagy is a cell cycle regulated phenomenon, responsible for mitochondria quality control and that it plays an essential role at the G1/S checkpoint. Both general autophagy genetic inhibition and mitophagy pharmacological inhibition with Mdivi-1 during cell cycle progression lead the cells to enter faster in S phase. However, probably due to the high levels of oxidative stress dictated by their proliferation rate, autophagy-incompetent cells start to accumulate insulted and less functional mitochondria. Autophagy is likely to be involved in the induction of resistance mechanisms to treatments in different types cancers. Because of this, there is an increased interest in the possibility to use inhibitors of the autophagic pathways as adjuvants for current anti-tumor therapies. Further investigations of the mechanisms here described will be of importance to elucidate whether drugs which inhibit autophagy may be a viable and effective approach in combination with chemotherapy for the treatment of highly proliferating cancers.

#2716

Lipid mediated nutrients sensing checkpoint in G1 phase of the cell cycle.

Deven Patel,¹ David Foster². ¹The Graduate Center of CUNY, New York, NY; ²The Hunter College of CUNY, New York, NY.

One of the hallmarks of cancers is dysregulation of G1 phase cell cycle progression- where cells decide their fate whether they should divide, arrest or undergo apoptosis. There is a growth factor-dependent Restriction point (R), where cells commit to replicate its genome or to enter a state of quiescence. Unicellular organisms only respond to the nutrients, a point known as START. In mammalian system, we have shown equivalent to START, Cell Growth checkpoints, where it senses Essential Amino Acids (EAAs), a Conditionally EAA glutamine (Q), and at last a checkpoint regulated by mTOR, which we think it senses nutrients and is the final arbitrator of the G1-S phase progression. mTOR- mammalian/mechanistic target of rapamycin integrates the growth factor signaling with nutrient sensing signaling and known to be involved in protein translation and proliferation. Another key nutrient needed for cell growth is lipids which is required as building blocks for plasma membrane synthesis and signaling molecules. Phosphatidic acid has been shown to be required for the activity of mTORC1 and mTORC2. In this study, we investigated the involvement of lipids in the cell cycle progression and how the signals are integrated. Upon lipid deprivation, normal fibroblasts cells arrest in G1-phase of the cell cycle. There is a distinct and distinguishable lipid sensing checkpoint from EAAs and Q. Temporally it is different from other nutrient sensing checkpoints in the late G1-phase of the cell cycle. In summary, there is a series of nutrient sensing checkpoints late in the G1-phase followed by mTOR Growth checkpoint. Dysregulation of these checkpoints in cancer cells may present better opportunity to target them with different therapeutic approach.

#2717

Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma.

Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Ogan Abaan, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sean R. Davis, Sven Bilke, Paul S. Meltzer. NCI-CCR, Bethesda, MD.

The unlimited proliferative capacity of cancer cells is closely linked to maintenance of their telomeres, which shorten with each cell division in normal cells. Cancer cells are able to maintain telomere length by multiple mechanisms, including activation of telomerase and the recombination based alternative lengthening of telomeres (ALT) pathway. ALT is prevalent in osteosarcoma, with approximately 50% of osteosarcoma cases using ALT for telomere maintenance. Mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 correlate with activation of the ALT pathway in several tumor systems. While loss of ATRX is a frequent event in osteosarcoma tumors, alterations of DAXX have not been reported. We characterized the telomere maintenance mechanisms utilized by 11 osteosarcoma cell lines. Of these, 45% (5/11) were ALT positive and 45% (5/11) were telomerase positive. One cell line possessed features of both telomere maintenance mechanisms. Among ALT positive osteosarcoma cell lines, we observed frequent loss of ATRX expression (4/5) and a previously unreported translocation resulting in disruption of DAXX. The translocation abolishes recruitment of DAXX to nuclear PML bodies and prevents normal DAXX function. By reintroducing full length DAXX, we were able to suppress telomere maintenance by ALT as evidenced by multiple assays including loss of C-circles and ALT-associated PML bodies, thus demonstrating that continued DAXX deficiency is necessary for maintenance of the ALT mechanism. Suppression of ALT by DAXX reintroduction did not result in compensatory activation of telomerase. This first demonstration of ALT suppression by DAXX supports a mechanistic connection between loss of the ATRX/DAXX chromatin remodeling complex and telomere maintenance by ALT. Understanding this relationship may uncover vulnerabilities specific to ALT tumors that could potentially lead to the development of targeted therapies for diverse cancers that depend on the ALT pathway.

#2718

Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity.

Marion Hitchcock, Gerhard Siemeister, Hans Briem, Amaury Ernest Fernandez-Montalvan, Simon Holton, Anne Mengel, Ursula Mönning, Michael Brands, Karl Ziegelbauer, Dominik Mumberg, Franz von Nussbaum. Bayer Pharma AG, Berlin, Germany.

BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase.

Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of DNA damage and misattached chromosomes.

Here, we report the characterization of a novel benzylpyrazole lead-structure series inhibiting BUB1 exemplified by BAY-320, a novel, first-in-class small molecule inhibitor of BUB1 kinase. This structure class was initially discovered as a single hit in a high-throughput screen, and resulted in a lead compound by chemical optimization. Benzylpyrazole BAY-320 is highly selective for BUB1 with single digit nanomolar biochemical potency and single-digit micromolar cellular potency (HeLa proliferation assay). Synergistic effects can be observed when BUB1 inhibitor BAY-320 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylpyrazoles allowed for a better understanding the binding mode for rational property design. Further data on structure-activity relationship including pharmacokinetic, drug metabolism and the synthesis of BAY-320 and analogues will be presented.

These results validate the benzylpyrazoles as novel selective BUB1 inhibitors and BUB1 as a promising approach for cancer treatment.

#2719

ADA3, a cell cycle regulator, regulates chromosome segregation.

Shashank Srivastava, Shakur Mohibi, June Wang-France, Sameer Mirza, Xiangshan Zhao, Hamid Band, Vimla Bnad. University of Nebraska Medical Center, Omaha, NE.

The RNA polymerase II mediated transcription requires the higher degree structure of chromatin to be relieved so that general transcription machinery can access the DNA. The acetylation of DNA bound histones at specific loci is the major epigenetic modification by which opening of chromatin is achieved. HATs (Histone Acetyl Transferase) are the enzymes that catalyze the acetylation of histones and require association with mediator proteins for their function. One such mediator protein is ADA3 (Alteration/Deficiency in Activation 3), which was initially discovered as a component of multi-protein complex that contains either GCN5 (General Control Non-repressed 5) or PCAF (p300/CBP Associated Factor) as HAT and subsequent studies showed that ADA3 associates with another HAT, p300. As a HAT interacting protein, ADA3 enhances the acetylation of histones as well as non-histone proteins, such as p53.

We have recently shown that ADA3 is a cell cycle regulatory protein and is important for both G1 to S phase transition as well as mitosis. Conditional deletion of Ada3 from Ada3FL/FL MEFs (Mouse Embryonic Fibroblasts) causes cell cycle arrest and severe mitotic defects. To further explore the mechanism of ADA3 mediated mitosis, we performed ChIP-seq analyses and found that ADA3 bound to higher order repeat region of the centromere across most of the chromosomes. Further studies showed ADA3 interaction with centromere was mediated by a centromeric protein CENP-B. More importantly, siRNA mediated knockdown of ADA3 decreases the occupancy of CENP-B at centromere and causes chromosomal segregation defects during mitosis. These studies demonstrate a novel function of ADA3 in cell cycle regulation. Our current studies are focused on defining the exact mechanism of recruitment of ADA3 complex to CENP-B to regulate mitosis and its effect on genomic stability.

#2721

Discovery and development of orally available subnanomolar potent checkpoint kinase 1 inhibitors as potential anticancer therapies.

Alex C. Vo,¹ Janelle Taylor,¹ Robert Rosler,¹ Dina Leviten,¹ Teresa Sierra,¹ Richard Boyce,² Robert Boyle,² Scott Peterson,¹ Kevin Klucher¹. ¹Oncothyreon, Seattle, WA; ²Sentinel Oncology, Cambridge, United Kingdom.

Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division by arresting progression in the S & G2 phases of the cell cycle in response to genotoxic stress. Pharmacological inhibition of Chk1 selectively uncouples the completion of DNA replication from G2/M phase transition in tumor cells lacking parallel cell cycle checkpoint controls, resulting in mitotic catastrophe and cell death. These properties make Chk1 inhibition a unique therapeutic approach as a single agent or as a means to enhance the efficacy of DNA-targeted chemotherapeutic drugs.

In this report, we describe our progress in the development of orally bioavailable, potent and selective Chk1 inhibitors derived from an aminopyrazole chemical scaffold. Starting with the lead compound, ONT-2409 (IC50 of <0.1 nM against the Chk1 enzyme), a series of Chk1 inhibitors have been developed. Lead candidates have been generated with potent biochemical IC50s against Chk1 (IC50s =0.1-0.3 nM). In a panel of 125 protein kinases the top candidates were shown to be highly selective for Chk1, with little appreciable activity against Chk2 or other kinases involved in cell cycle control/DNA damage response. The lead candidates have potent single agent activity against a panel of diverse cancer cell lines (EC50s=0.03-0.4 µM) and show synergistic enhancement in the activity of clinically relevant DNA targeted chemotherapeutics. Mechanistically, the lead candidates abrogate gemcitabine-induced cell cycle arrest and induce apoptotic cell death. Biomarker studies show the lead candidates inhibit gemcitabine-induced auto-phosphorylation of Chk1 at S296 and reduce phosphorylation of CDK1 at Y15. Compared with ONT-2409, the optimized candidates display improved cell permeability/efflux ratios, superior intrinsic microsomal and hepatic half-lives, and have excellent oral bioavailability in mice and rats. Lead candidate Chk1 inhibitors have also demonstrated potent potentiation of DNA targeted chemotherapies and single agent activity in xenograft tumor models

#2722

Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy.

Emma G. Sturgill, Ryoma Ohi. Vanderbilt University, Nashville, TN.

Mitosis serves as a powerful opportunity for therapeutic intervention in the treatment of neoplastic diseases like cancer. Anti-mitotic pharmacological agents tame the proliferative capacity of tumor cells by interfering with proper function of the mitotic spindle. Several next generation anti-mitotics target kinesins involved in assembling the mitotic spindle. Of these mitotic kinesins, the kinesin-5 Eg5 (also known as KSP) is perhaps the most compelling because of its primacy during spindle assembly in animal cells. Indeed, Eg5-inhibitors (K5Is) induce a potent and lethal mitotic arrest in cell culture and murine xenografts; however, K5Is failed to induce tumor regression during Phase II trials. This devastating clinical performance has halted further development of K5I monotherapies and motivated investigation into the underlying reason(s) for K5I inefficacy.

We previously showed that tumor cells in culture are capable of acquiring K5I resistance by employing parallel spindle assembly pathways. By studying a limited number of K5I-resistant cell lines, we showed that resistance involved the kinesin-12 Kif15; however, the generality of this finding was unclear. In this study we capitalize on the latest methodologies in targeted genome editing to study mechanisms of K5I resistance on a broader cell population level. We find that cells require Kif15 in all cases of K5I resistance, and that rescue is sensitive to the timing and extent of Kif15 expression. We propose that K5Is steer cells into an evolutionary bottleneck that requires Kif15, and that adaptive changes occur during passage through the bottleneck that enable populations to recover and diversify. These findings prompt a search for pharmacological inhibitors of Kif15 to test in combination with K5Is as an anti-mitotic chemotherapeutic regimen.

#2723

The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia.

Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. University of Bologna, Bologna, Italy.

Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL.

Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.

#2724

Collaborative regulation of cell cycle progression by Chk1 and E2F1 in small cell lung cancer cells.

Wei-Hsun Hsu, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Yu-Wen Zhang, Giuseppe Giaccone. Georgetown University, Washington, DC.

Introduction

Small cell lung cancer (SCLC) harbors very frequent mutations in p53 and Rb, key cell cycle regulators in normal cells. In absence of such tumor suppressors, SCLC cells rely on Chk1 for cell cycle arrest in the event of DNA damage. High expression of E2F1 and Chk1 have been found in SCLCs, which is associated with poor prognosis in various solid tumors. In human SCLC cell lines, Chk1 inhibition sensitizes chemotherapy agents. The interaction between Chk1 and E2F1 is understudied. Here, we report a collaborative role of Chk1 and E2F1 in the cell cycle regulation in SCLC cells.

Methods

Three Rb-mutated human SCLC cell lines (GLC4, NCI-H128, and NCI-H209) were used. GLC4 carries mutant p53; H209 and H128 are wild type. Chk1 inhibition was achieved by either siRNA knockdown or LY2940930; E2F1 was knocked down by siRNA. Ectopic overexpression of Chk1 or E2F1 was achieved using Lonza electroporation kit. Cell viability was measured by Cell-Titer Glo assay. Cell cycle analysis was assayed by PI staining and FACS. Western blotting was used to evaluate caspase activation and other signaling proteins.

Results

Chk1 inhibition by siRNA knockdown or LY2940930 treatment enhanced cisplatin cytotoxicity in SCLC cell lines, regardless of p53 status. Also, a significant decrease of E2F1 protein was observed following Chk1 inhibition in these cells. Knockdown of E2F1 by siRNA enhanced the cytotoxic activity of cisplatin in the GLC4 and H209 cells. In GLC4 cells, Chk1 siRNA combined with cisplatin treatment caused activation of caspase-2 and caspase-3 as well as the cleavage of BID. Interestingly, knocking down caspase-2 reversed caspase-3 activation resulting from the combination of Chk1 siRNA and cisplatin, and restored the level of E2F1 protein. In contrast, ectopic overexpression of Chk1 resulted in an increase of E2F1, while increase of Chk1 expression was also noticeable upon E2F1 overexpression. It has been shown that Chk1 inhibition can abrogate cell cycle arrest and increase phospho-histone H3 expression (mitotic marker). We found that E2F1-overexpressing GLC4 and H128 cells displayed higher levels of phospho-histone H3 when treated with LY2940930, compared to that of Mock-transfected controls. Furthermore, exogenous E2F1 overexpression significantly reduced the IC50 of cisplatin (1331nM vs. 3698nM; p=0.0028 by paired t-test) in GLC4 cells, but not that of LY2940930 (14nM vs. 19nM; p=0.25).

Conclusions

Either downregulation of Chk1 or overexpression of E2F1 leads to cell cycle progression, resulting in more cell death when combined with DNA damaging agents. Decrease of E2F1 by Chk1 inhibition might represent a mechanism of self-protection to avoid excessive mitosis after DNA damage, and this regulation is likely involving caspase-2. Further exploration of Chk1 and E2F1 regulatory mechanisms is warranted in SCLC.

#2725

Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524.

Anna P. Baron,¹ Conrad von Schubert,¹ Fabien Cubizolles,¹ Gerhard Siemeister,² Marion Hitchcock,² Anne Mengel,² Jens Schröder,² Amaury Fernández-Montalván,² Martin Lange,² Franz von Nussbaum,² Dominik Mumberg,² Erich Nigg¹. ¹Biozentrum, University of Basel, Basel, Switzerland; ²Bayer Pharma AG, Berlin, Germany.

The maintenance of correct chromosome number (euploidy) during cell division is ensured by a highly conserved surveillance mechanism termed ‘spindle assembly checkpoint’ which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. The mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) was reported to contribute to both chromosome congression and checkpoint function, yet the role of BUB1 catalytic activity in these processes remains a matter of debate.

To differentiate between catalytic and non-catalytic functions of BUB1 we compared phenotypes provoked by BUB1 protein depletion with specific BUB1 kinase inhibition using two novel small molecule inhibitors of BUB1, termed BAY-320 and BAY-524.

BAY-320 and BAY-524 were highly potent and selective ATP-competitive inhibitors of BUB1 kinase activity with IC50 values in the single digit nanomolar range (at 10 micromolar ATP concentration). By monitoring phosphorylation of Thr120 in histone H2A, we showed that both compounds acted as potent BUB1 kinase inhibitors both biochemically and in human cells. We found that BUB1 inhibition substantially altered the chromosomal association of Shugoshin and the chromosomal passenger complex without major effects on global Aurora B function. Consequently, inhibition of BUB1 kinase clearly impaired chromosome arm resolution but, in stark contrast to depletion of BUB1 protein, only had a minor effect