bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2626A: A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo
Authors: Wang Piwen , Henning Susanne M. , Vadgama Jaydutt V.
Published: July 2016

Arctigenin is a novel anti-inflammatory lignan derived mainly from the seeds of Arctium lappa, an herb widely used in traditional Chinese medicine to treat inflammation related diseases such as cough, cold and swelling of throat. The present study was designed to investigate whether arctigenin is a potent inhibitor of prostate cancer using both in vitro and xenograft mouse models. The treatments with arctigenin at lower doses (< 2μM) for 48h dose-dependently inhibited the proliferation of two androgen-sensitive prostate cancer cell lines, LNCaP and LAPC-4, by 40-50% compared to control. There was no cytotoxicity observed in normal prostate epithelial PrEC cells with arctigenin doses up to 10μM. An intervention study was then conducted in severe combined immunodeficiency (SCID) mice to confirm the tumor-inhibitory effect of arctigenin in vivo. Male SCID mice (n = 10 per group) were inoculated with 5×10⁁5 LAPC-4 cells subcutaneously. One week later when tumors were palpable the intervention started. Mice were administered with arctigenin at 50mg/kg (LD) or 100mg/kg (HD) or vehicle control daily for 6 weeks by oral gavage. After 4 weeks of intervention the tumor growth was significantly inhibited by both of the arctigenin doses. After 6 weeks of intervention, the tumor volumes were reduced by 48% (LD arctigenin) and 67% (HD arctigenin) compared to control. There was no significant difference in food or water consumption or body weight among groups. An ELISA assay of growth factors demonstrates that arctigenin treatments significantly decreased the concentrations of VEGF, EGF, NGF-β, TNF-α, and FGF-β in tumor tissues compared to control. Western blot analysis revealed a significantly reduced expression of galectin-4 in tumor tissues by arctigenin treatment. Previous studies have shown that strong expression of galectin-4 can be induced in different types of cancer, associated with tumor growth and progression. A microRNA PCR array assay was performed to measure the tumor concentrations of 84 microRNAs involved in prostate cancer development and progression. A panel of microRNAs including miR-126 and miR-135 were identified to be responsive to arctigenin treatment. Further mechanistic investigation using tissue microarray assay is ongoing to measure the protein markers of proliferation and apoptosis in tumor tissues. This study provides a promising non-toxic agent to enhance chemoprevention of prostate cancer. These results warrant future clinical trial studies to confirm the anti-carcinogenic effect of arctigenin in humans.

Citation Format: Piwen Wang, Susanne M. Henning, Jaydutt V. Vadgama. A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2626A.
Abstract 2635: Methylation in benign prostate and risk of disease progression in men subsequently diagnosed with prostate cancer
Authors: Rybicki Benjamin A. , Rundle Andrew , Kryvenko Oleksandr , Chitale Dhananjay , Belinsky Steven , Tang Deliang
Published: July 2016

In DNA from prostate cancer tumors, methylation patterns in gene promoter regions can be a biomarker for disease progression. It remains unclear whether methylation patterns in benign prostate tissue—prior to malignant transformation—may provide similar prognostic information. To determine whether early methylation events predict prostate cancer outcomes, we evaluated histologically benign prostate specimens from 353 men who eventually developed prostate cancer and received “definitive” treatment (radical prostatectomy [58%] or radiation therapy [42%]). Cases were drawn from a large hospital-based cohort of men with benign prostate biopsy specimens collected between 1990 and 2002. Risk of disease progression associated with methylation was estimated using time-to-event analyses. Average follow-up was over 5 years; biochemical recurrence (BCR) occurred in 91 cases (26%). In White men, methylation of the APC gene was associated with increased risk of BCR, even after adjusting for standard clinical risk factors for prostate cancer progression (adjusted hazard ratio (aHR) = 2.26; 95%CI 1.23-4.16). APC methylation was most strongly associated with a significant increased risk of BCR in White men with low prostate specific antigen at cohort entry (HR = 3.66; 95%CI 1.51-8.85). In additional stratified analyses, we found that methylation of the RARB gene significantly increased risk of BCR in African American cases who demonstrated methylation of at least one of the other four genes under study (HR = 3.80; 95%CI 1.07-13.53). These findings may have implications in the early identification of aggressive prostate cancer as well as reducing unnecessary medical procedures and emotional distress for men who present with markers of indolent disease.

Citation Format: Benjamin A. Rybicki, Andrew Rundle, Oleksandr Kryvenko, Dhananjay Chitale, Steven Belinsky, Deliang Tang. Methylation in benign prostate and risk of disease progression in men subsequently diagnosed with prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2635.
Abstract 2657: Target specificity of epigenetic therapy in cancer
Authors: Sato Takahiro , Cesaroni Matteo , Panjarian Shoghag , Tran Anthony , Madzo Jozef , Okamoto Yasuyuki , Zhang Hanghang , Chen Xiaowei , Jelinek Jaroslav , Issa Jean-Pierre J.
Published: July 2016

A major question facing the use of epigenetic therapies in cancer is specificity in modulating gene expression. In addition, combined targeting of DNA and histone methylation remains largely unexplored despite the promising synergistic effects observed from combining DNA methyltransferase inhibitors with HDAC inhibitors. To address these questions, we performed RNA-seq, DNA methylation analysis and ChIP-seq (H3K4me2, H3K9me2, and H3K27me3) to study the effects of inhibitors of DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343) in three different cancer models (colon cancer, breast cancer, and leukemia). In colon cancer cells (YB5), DAC affected 3% of the transcriptome and 93% of the effect was gene upregulation. DAC had a greater effect on genes expressed in normal tissues and silenced in cancer (443 genes) compared to genes that do not change in cancer (194 genes). 90% of DAC targets genes showed no promoter DNA methylation in normal colon but gained methylation in cancer. Depsi changed the expression of 35% of the transcriptome and showed little specificity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 had limited effects on their own (<1.5% of the transcriptome), but UNC0638 and GSK343 preferentially targeted genes with H3K9me2 or H3K27me3, respectively. DAC combined with histone methylation inhibitors led to synergistic gene upregulation while still maintaining specificity for DNA methylated and silenced genes. These synergistic genes had limited overlap, indicating the possibility to target distinct sets of genes based on different epigenetic therapy combinations. IPA analysis demonstrated that these genes are enriched in cancer pathways, and consistent with this analysis, the combination therapies were able to decrease cancer cell proliferation more effectively than monotherapy. Broadly similar results were seen with genome wide studies in both breast cancer (MCF7) and leukemia (HL-60) cells. These results demonstrate that DNA methyltransferase inhibitors preferentially target cancer relevant genes, and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining specificity.

Citation Format: Takahiro Sato, Matteo Cesaroni, Shoghag Panjarian, Anthony Tran, Jozef Madzo, Yasuyuki Okamoto, Hanghang Zhang, Xiaowei Chen, Jaroslav Jelinek, Jean-Pierre J. Issa. Target specificity of epigenetic therapy in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2657.
Abstract 2661: The genomic landscape and clinical relevance of A-to-I RNA editing in human cancers
Authors: Liang Han
Published: July 2016

Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of >6000 patient samples of 20 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions. We experimentally demonstrated the functional effects of several RNA editing events and provide the evidence that RNA editing could selectively affect drug sensitivity. These results highlight RNA editing as an exciting theme for investigating cancer mechanisms, biomarkers and treatments.

Citation Format: Han Liang. The genomic landscape and clinical relevance of A-to-I RNA editing in human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2661.
Abstract 2672: Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures
Authors: Graham Nicholas A. , Minasyan Aspram , Christofk Heather R. , Mellinghoff Ingo K. , Graeber Thomas G.
Published: July 2016

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including FDG-avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A cross-species comparison of CNA identified 21 conserved altered DNA regions containing 13 enzymes in the glycolysis and pentose phosphate pathways in addition to known cancer driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution.

Citation Format: Nicholas A. Graham, Aspram Minasyan, Heather R. Christofk, Ingo K. Mellinghoff, Thomas G. Graeber. Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2672.
Abstract 2676: Epithelial-mesenchymal transition does not require transcriptional repression of the epithelial program in vivo
Authors: Aiello Nicole M. , Balli David , Norgard Robert , Li Jinyang , Sahmoud Amine , Stanger Ben Z.
Published: July 2016

Epithelial-mesenchymal transition (EMT), the process by which an epithelial cell loses its adhesion to neighboring cells and acquires a motile, fibroblast-like phenotype, is critical for many aspects of embryogenesis and has been strongly implicated in tumor cell invasion and metastasis. EMT is thought to be primarily regulated at the transcriptional level through repression of epithelial genes by transcription factors such as Snail, Zeb and Twist. To date, the mechanisms driving EMT have been parsed out almost exclusively in vitro under pre-defined conditions (ie, using TGFβ as an EMT inducer), but it remains to be seen whether the machinery that regulates in vitro EMT is physiologically relevant. Using a lineage labeled mouse model of pancreatic ductal adenocarcinoma (PDAC), we isolated tumor cells that have spontaneously undergone EMT to interrogate the molecular mechanisms driving this process in vivo. By comparing the transcriptomes of epithelial (E-cadherin+) and mesenchymal (E-cadherin-) tumor cells, we found that for a majority of PDAC tumors, EMT does not require transcriptional repression of the epithelial program. These cells were fully capable of upregulating a mesenchymal program, as evidenced by the increased expression of collagens, SPARC, MMP2, PDPN, and PRRX1; however, despite the loss of E-cadherin protein, we detected no change in transcript levels of E-cadherin or other epithelial genes after EMT. Our results suggest that in vivo, cells that undergo EMT retain epithelial gene transcription, which may confer the ability to rapidly oscillate between epithelial and mesenchymal states.

Citation Format: Nicole M. Aiello, David Balli, Robert Norgard, Jinyang Li, Amine Sahmoud, Ben Z. Stanger. Epithelial-mesenchymal transition does not require transcriptional repression of the epithelial program in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2676.
Abstract 2696: Four new brain tumor entities emerge from molecular classification of CNS-PNETs
Authors: Sturm Dominik , Orr Brent , Toprak Umut H. , Hovestadt Volker , Jones David T.W. , Capper David , Lichter Peter , Korshunov Andrey , Pfister Stefan M. , Ellison David W. , Kool Marcel
Published: July 2016

CNS-primitive neuroectodermal tumors (CNS PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Histological diagnosis is complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other CNS tumors. To investigate this we have analyzed 323 fresh-frozen or paraffin-embedded institutionally diagnosed CNS-PNETs using DNA methylation and expression arrays. Data were compared to 211 reference cases of other pediatric and adult brain tumors representing more than 20 well-known entities.

DNA methylation and gene expression patterns showed that a significant proportion of CNS PNETs display molecular profiles indistinguishable from those of various other well defined CNS tumor entities. Hallmark genetic alterations for each of these well-defined entities, such as amplification of 19q13.42, mutations in IDH1 or H3F3A, mutations/deletions of the SMARCB1 locus, or RELA fusions, were frequently detected among the reclassified cases. From the remaining fraction of CNS PNETs we have identified four new CNS tumor entities, each associated with a unique recurrent genetic alteration and distinct histopathological and clinical features. Based on these findings we designated these four new entities as “CNS neuroblastoma with FOXR2 activation (CNS NB FOXR2)”, “CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT CIC)”, “CNS high grade neuroepithelial tumor with MN1 alteration (CNS HGNET MN1)”, and “CNS high grade neuroepithelial tumor with BCOR alteration (CNS HGNET BCOR)”.

Our findings reinforce the importance of incorporating molecular information into the next revision of the WHO classification of CNS tumors, and warrant a replacement of the term “CNS PNET” with biologically specific designations. Our study provides an innovative framework for improving diagnostic accuracy and prognostication in malignant CNS tumors. The approach is amenable to retrospective analyses of patients treated with current regimens and will facilitate the design of more meaningful clinical trials for patients with malignant brain tumors.

Citation Format: Dominik Sturm, Brent Orr, Umut H. Toprak, Volker Hovestadt, David T.W. Jones, David Capper, Peter Lichter, Andrey Korshunov, Stefan M. Pfister, David W. Ellison, Marcel Kool. Four new brain tumor entities emerge from molecular classification of CNS-PNETs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2696.
Abstract 27: Understanding mechanisms of nutrient homeostasis: Amino acid availability and regulation of V-ATPase activity
Authors: Stransky Laura A. , Forgac Michael
Published: July 2016

Cellular homeostasis requires accurate sensing of nutrient levels and subsequent regulation of biosynthetic and catabolic cellular processes. In cancer cells, metabolism is frequently disregulated to meet the increased demand for cellular building blocks to enable growth and proliferation. Thorough understanding of how homeostatic processes are regulated is necessary to determine how these processes could be manipulated to stop tumor growth. This study aims to understand amino acid homeostasis, with particular focus on the regulation of lysosomal activity, which allows protein breakdown and release of free amino acids. We have identified a novel mechanism of amino acid homeostasis whereby amino acid starvation leads to increased activity of the Vacuolar-ATPase (V-ATPase), as indicated by measurement of V-ATPase-dependent FITC-dextran fluorescence quenching in isolated lysosomes. The V-ATPase is the proton pump responsible for acidifying intracellular compartments, which is necessary for lysosomal function and protein degradation. This increased activity is due to an increase in the amount of fully assembled holoenzymes, as detected by cell fractionation and Western blotting to assess peripheral subunit association with membranes. Assembly of V-ATPase holoenzymes is a tightly controlled process. We describe the function of these changes with respect to lysosomal protein turnover and mTORC1 activation, in which the V-ATPase plays a critical role. We have also sought to characterize the signaling events that transmit the amino acid starvation signal and controls V-ATPase activity in response to changes in amino acid availability in normal and cancer cells that rely on lysosomal function. These changes do not depend on PI3K or mTORC1 activity, both of which have been linked to changes in V-ATPase activity and assembly in other response to other stimuli, such as glucose and growth factor stimulation and are highly activated across cancers. Together, these results shed light on a poorly understood aspect of cellular biology and novel mechanisms of amino acid homeostasis.

Citation Format: Laura A. Stransky, Michael Forgac. Understanding mechanisms of nutrient homeostasis: Amino acid availability and regulation of V-ATPase activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 27.
Abstract 2708: Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma
Authors: Shirinifard Abbas , Thiagarajan Suresh , Patel Yogesh T. , Davis Abigail D. , Jacus Megan O. , Throm Stacy L. , Roberts Jessica , Daryani Vinay , Stewart Clinton F. , Sablauer. András
Published: July 2016

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.

Citation Format: 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. Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2708.
Abstract 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
Authors: De La Vega Francisco M. , Koehler Ryan T. , Pouliot Yannick , Bouhlal Yosr , So Austin , Goodsaid Federico , Irvine Sean , Trigg Len , Nadauld Lincoln
Published: July 2016

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.

Citation Format: Francisco M. De La Vega, Ryan T. Koehler, Yannick Pouliot, Yosr Bouhlal, Austin So, Federico Goodsaid, Sean Irvine, Len Trigg, Lincoln Nadauld. Joint somatic mutation and germline variant identification and scoring from tumor molecular profiling and ct-DNA monitoring of cancer patients by high-throughput sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2712.
Abstract 2716: Lipid mediated “nutrients sensing” checkpoint in G1 phase of the cell cycle
Authors: Patel Deven , Foster David
Published: July 2016

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.

Citation Format: Deven Patel, David Foster. Lipid mediated “nutrients sensing” checkpoint in G1 phase of the cell cycle. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2716.
Abstract 2718: Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity
Authors: Hitchcock Marion , Siemeister Gerhard , Briem Hans , Fernandez-Montalvan Amaury Ernest , Holton Simon , Mengel Anne , Mönning Ursula , Brands Michael , Ziegelbauer Karl , Mumberg Dominik , Nussbaum. Franz von
Published: July 2016

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.

Citation Format: 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. Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2718.
Abstract 2721: Discovery and development of orally available subnanomolar potent checkpoint kinase 1 inhibitors as potential anticancer therapies
Authors: Vo Alex C. , Taylor Janelle , Rosler Robert , Leviten Dina , Sierra Teresa , Boyce Richard , Boyle Robert , Peterson Scott , Klucher Kevin
Published: July 2016

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

Citation Format: Alex C. Vo, Janelle Taylor, Robert Rosler, Dina Leviten, Teresa Sierra, Richard Boyce, Robert Boyle, Scott Peterson, Kevin Klucher. Discovery and development of orally available subnanomolar potent checkpoint kinase 1 inhibitors as potential anticancer therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2721.
Abstract 2722: Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy
Authors: Sturgill Emma G. , Ohi Ryoma
Published: July 2016

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.

Citation Format: Emma G. Sturgill, Ryoma Ohi. Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2722.
Abstract 2730: Association of TERT promoter mutations with telomerase expression in melanoma
Authors: Lee Seungjae , Opresko Patricia , Pappo Alberto , Kirkwood John , Bahrami Armita
Published: July 2016

Telomerase reverse transcriptase (TERT) promoter mutations occur at a high frequency in melanoma, but the functional consequences of these mutations in melanoma remain to be clarified. In a large study of melanoma samples by The Cancer Genome Atlas network, mRNA expression analysis using RNA sequencing data showed that only TERT promoter mutations at C228T were associated with high TERT expression levels. The effect of hotspot C250 or C242T/C243T tandem mutations on telomerase expression has not yet been determined. To assess telomerase expression levels in melanomas harboring C250 or C242T/C243T mutations, we used real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in a sample set of pediatric melanocytic tumors for which the methylation status and the mutational profile of the TERT promoter were known. We previously showed that in a subset of melanoma, TERT expression is mediated epigenetically by promoter hypermethylation rather than by promoter point mutations. Formalin-fixed paraffin-embedded (FFPE) tissues from 10 metastatic melanomas, of which 8 harbored a hotspot TERT promoter mutation (5 C250T; 2 C228; 1 C242T/C243T) and 2 carried a hypermethylated wild-type TERT promoter were examined. For controls, 9 atypical Spitz tumors with a wild-type unmethylated TERT promoter were also studied. Total RNA was isolated from FFPE tumor tissues and converted to cDNA. TERT expression levels were determined by RT-qPCR by using the TaqMan® Gene Expression Assay and normalized with GAPDH as the endogenous control. In the atypical Spitz tumor samples, TERT mRNA expression was undetectable or negligible. In contrast, TERT expression in all melanoma samples was elevated, ranging from 3- to 71-fold (median, 25-fold) when compared to an atypical Spitz tumor as a reference. Our data support that in melanoma, TERT promoter hotspot C250 and C242T/C243T mutations, similar to C228T mutations, correlate with TERT overexpression and that C250 and C242T/C243T mutations likely contribute biologically to tumorigenesis in melanoma.

Citation Format: Seungjae Lee, Patricia Opresko, Alberto Pappo, John Kirkwood, Armita Bahrami. Association of TERT promoter mutations with telomerase expression in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2730.
Abstract 2736: Regulation of cytokinesis by polo-like kinase 4
Authors: Press Michael F. , Xie Bin , Davenport Simon , Zhou Yu , O’Brien Neil , Palazzolo Michael , Mak Tak , Brugge Joan , Slamon Dennis J.
Published: July 2016

Introduction. The serine/threonine mitotic kinase, polo-like kinase 4 (PLK4), is known to play a critical role in centrosome duplication in preparation for cell division. Based on our preliminary observations with a PLK4 kinase inhibitor, we investigated the possibility that PLK4 may also play a role in regulation of cytokinesis.

Experimental Procedures. Immunofluorescence was used to localize PLK4 and phospho-PLK4 in cultured human breast, ovarian, and colorectal cancer cell lines throughout the cell cycle without and with CFI-400945 PLK4 inhibitor and MG-115 protease inhibitor. Flow cytometry and videomicroscopy were used to analyze the consequences of PLK4 inhibition on cytokinesis.

Results. Using immunofluorescence, PLK4 was localized to centrosomes; however, we also found that phospho-PLK4 was cleaved and distributed to kinetochores (metaphase and anaphase), cleavage furrow (telophase), and middle body (cytokinesis) during cell division in colorectal, ovarian, and breast cancer cells. Distribution of phospho-PLK4 to the cleavage furrow and middle body raised the possibility that this kinase plays a functional role in cytokinesis. Using either CFI-400945 PLK4 kinase inhibitor or the MG-115 protease inhibitor, we found that PLK4 accumulated in centrosomes with inhibition of translocation of PLK4 to the middle body. This change in subcellular distribution of PLK4 was associated with generation of large, multi-nucleated tumor cells or tumor cells with polyploidy. Videomicroscopy confirmed that treatment with CFI-400945 PLK4 inhibitor was associated with prevention of cellular abscission in treated cells.

Conclusions. These observations demonstrate a role for phospho-PLK4 in facilitation of cytokinesis. A regulatory role for PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.

Citation Format: Michael F. Press, Bin Xie, Simon Davenport, Yu Zhou, Neil O’Brien, Michael Palazzolo, Tak Mak, Joan Brugge, Dennis J. Slamon. Regulation of cytokinesis by polo-like kinase 4. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2736.
Abstract 2740: Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications
Authors: Dann Stephen , Yuan Jing , Chionis John , Liu Chaoting , Xie Tao , Lee Nathan V. , Chen Enhong , Wei Ping , Rejto Paul A. , Shields David J. , VanArsdale Todd
Published: July 2016

Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (IbranceTM), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications.

Citation Format: Stephen Dann, Jing Yuan, John Chionis, Chaoting Liu, Tao Xie, Nathan V. Lee, Enhong Chen, Ping Wei, Paul A. Rejto, David J. Shields, Todd VanArsdale. Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2740.
Abstract 2741: A novel branched DNA-based multiplex method for telomere analysis at the single-cell level by flow cytometry
Authors: Lai Steve , Sias Leslie , Funatake Castle
Published: July 2016

Telomeres are protective structures at the ends of chromosomes, containing multiple repeats of the repetitive sequence (TTAGGG) in vertebrates. It undergoes progressive shortening during normal aging. Abnormalities in the regulation of telomere length are implicated in various diseases including cancers. Current methods for telomere analysis are tedious and lack the flexibility to allow identification of unique cell subsets in a heterogeneous sample. Here we describe a novel flow cytometric method for analyzing relative telomere length at the single-cell level based on the branched DNA technology. This method, named PrimeFlow Assay can be combined with antibody staining and mRNA detection enabling simultaneous analysis of telomere length as well as protein and RNA for a comprehensive view in any specific cell subset. With this method, we investigated various cell lines and primary cells of human and mouse origin. Our results indicate that transformed cell lines possess longer telomeres than primary cells. In human peripheral blood mononuclear cells, we observed that lymphocytes had longer telomeres compared to monocytes, consistent with previously published results by another flow cytometric method. These data demonstrate that the PrimeFlow assay is a valuable method for high-throughput analysis of relative telomere length that may be combined with the analysis of protein and gene targets and, thus, provides data at the single-cell level for a more complete understanding of sample heterogeneity.

Citation Format: Steve Lai, Leslie Sias, Castle Funatake. A novel branched DNA-based multiplex method for telomere analysis at the single-cell level by flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2741.
Abstract 2748: Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10
Authors: Turchick Audrey L. , Glazer Peter M.
Published: July 2016

There is growing evidence that many solid tumors have defective DNA repair pathways and cell cycle checkpoints. This has spurred development of therapeutics to specifically target cancer cells with defective DNA repair, but few have been successful outright due to problems such as general toxicity. One novel anti-cancer agent of interest to us is 3E10, a unique cell-penetrating, anti-DNA autoantibody. This molecule is non-toxic to normal cells on its own but has deleterious effects on cells with DNA repair deficiencies. Specifically, 3E10 has been shown to be synthetically lethal in BRCA2-deficient human cancer cells, to be synergistic with radiation and other commonly used DNA damaging therapies, and to reduce the efficiency of Rad51 mediated strand exchange and thus homologous recombination (HR). The goal of this project is to better understand how 3E10 can be used as a new paradigm for cancer treatment. New evidence suggests that 3E10 inhibits Exo1 exonuclease activity. Additionally, preliminary data shows that 3E10 physically interacts with Rad51, ultimately inhibiting Rad51 nuclear localization and foci formation. Additionally, a point mutation in 3E10 that confers increased DNA binding mediates increased cell death in BRCA2-deficient human cancer cells. Furthermore, studies investigating the potential therapeutic effect of 3E10 in patient derived primary melanoma cell lines show that 3E10 is synthetically lethal with PTEN deficiency. This data suggests that 3E10 inhibits HR by binding to DNA at double strand breaks and occupying sites normally processed by core repair machinery, as well as by physically interacting with Rad51. Overall, 3E10 holds great promise as a novel therapeutic agent for various cancers, including PTEN deficient melanomas.

Reference:

Hansen, J. et al. Targeting cancer with a lupus autoantibody. Science Translational Medicine 4, (2012)

Citation Format: Audrey L. Turchick, Peter M. Glazer. Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2748.