bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4540: SAC abrogation by MPS1 kinase inhibition: preclinical proof of concept of a novel approach to tumor treatment
Authors: Mumberg Dominik , Siemeister Gerhard , Wengner Antje M. , Koppitz Marcus , Schulze Volker , Bader Benjamin , Prechtl Stefan , Kreft Bertolt , Ziegelbauer Karl
Published: May 2015

Cell cycle deregulation represents one of the classical hallmarks of cancer and consequently cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids). Targeted disruption of the cell cycle checkpoint offers a novel approach to cancer treatment: driving tumor cells into cell division despite DNA damage or unattached/misattached chromosomes resulting in a lethal degree of DNA damage or aneuploidy.

MPS1, a mitotic kinase that is overexpressed in several human cancers, has been shown to function as the key kinase which activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the identification and functional characterization of novel inhibitors of MPS1 from two structurally unrelated chemical classes. In biochemical assays, these molecules inhibited the kinase activity of MPS1 with IC50 values in the single digit nanomolar range and have shown an exquisite selectivity against a panel of kinases. In cellular assays the compounds were able to abrogate nocodazole-induced SAC, to reduce time in mitosis, to induce multinuclearity and apoptotic cell death, and to inhibit tumor cell proliferation with IC50 values in the low nanomolar range. In combination experiments MPS1 inhibitors showed cooperativity with low concentrations of paclitaxel. MPS1 inhibitors showed limited efficacy in monotherapy (T/C ca. 0.6) in tumor xenograft studies. However, when combined with paclitaxel dosed at the maximal tolerated dose, low doses of MPS1 inhibitor strongly improved efficacy over paclitaxel or MPS inhibitor monotreatment in intrinsically paclitaxel-insensitive xenograft models. The combination treatment was well tolerated. These results validate the concept of SAC abrogation preclinically and pave the way to a clinical proof of concept.

Citation Format: Dominik Mumberg, Gerhard Siemeister, Antje M. Wengner, Marcus Koppitz, Volker Schulze, Benjamin Bader, Stefan Prechtl, Bertolt Kreft, Karl Ziegelbauer. SAC abrogation by MPS1 kinase inhibition: preclinical proof of concept of a novel approach to tumor treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2014-4540
Abstract 4544: Induction of KLF4 by LOR-253 as an innovative therapeutic approach to induce apoptosis in acute myeloid leukemia
Authors: Lum Ronnie , Javadi Mojib , Cheng Tiffany , Peralta Robert , Cukier Howard , Lightfoot Jeff , Lee Yoon , Young Aiping , Rice William G.
Published: May 2015

LOR-253, a small molecule anticancer agent that acts through induction of the tumor suppressor Krüppel-like factor 4 (KLF4), has demonstrated antitumor activity as a single agent in a Phase I study in patients with advanced or metastatic solid tumors. Recently, the vast majority of patients with acute myeloid leukemia (AML) were shown to inappropriately express the embryonic CDX2 gene in bone marrow stem and progenitor cells, resulting in down-regulation of KLF4 expression as the leukemogenic event. Consequently, we examined the antitumor activity and mechanism of action for LOR-253 in AML cells and cells representing other hematological malignancies. Indeed, LOR-253 was found to inhibit proliferation of various human leukemia and lymphoma cell lines in vitro with low nM IC50 values. Further LOR-253 induced high levels of KLF4 mRNA expression in AML cells and a resultant significant increase in expression of p21, a cyclin-dependent kinase inhibitor that is transcriptionally regulated by KLF4. Consistent with these findings, in AML cells we found that LOR-253 induced G1/S cell cycle arrest and apoptosis, based on positive Annexin V staining, activated caspase-3, and increased BAX mRNA expression. Studies are underway to further characterize the pathway that mediates KLF4 induction by LOR-253, to characterize the effects of LOR-253 in combination with approved chemotherapies for AML, and to assess the efficacy of LOR-253 in animal models of AML. Induction of KLF4 represents a novel approach to the treatment of AML and other hematologic malignancies, and LOR-253 is the only clinical stage agent to act through this mechanism of action.

Citation Format: Ronnie Lum, Mojib Javadi, Tiffany Cheng, Robert Peralta, Howard Cukier, Jeff Lightfoot, Yoon Lee, Aiping Young, William G. Rice. Induction of KLF4 by LOR-253 as an innovative therapeutic approach to induce apoptosis in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2014-4544
Abstract 455: Increased copy number of Septin 9 (SEPT9) in women with high grade endometrial adenocarcinoma(EMCA): Is there a potential link to paclitaxel resistance
Authors: Zechmeister Jenna R. , Goldberg Gary L. , Montagna Cristina
Published: May 2015

Septins are a unique family of GTPases that are important in the regulation of a variety of essential cellular functions. Specifically, SEPT9 has been demonstrated to be vital in normal development in a mouse model. When a full knockout is generated, the phenotype is embryonically lethal. SEPT9 has also been implicated in tumorigenesis and our group has identified SEPT9 as an oncogene which is amplified and overexpressed in breast cancer. We have demonstrated that in high grade breast carcinomas, the subtype with the worst clinical outcome, exhibit significant increases in SEPT9 copy number variation (CNV) when compared to other breast tumor grades. Therefore to explore a possible biomarker role of SEPT9 in other tumor types, we have investigated the CNV status of SEPT9 in EMCA using data from the publically available Cancer Genome Atlas (TCGA). Within the published dataset, 7.8% of patients have an increased CNV of the gene. The Kaplan-Meier survival curves between the 2 groups show a decreased overall survival in the EMCA patients who exhibit CNV (n=18) with a 5-year survival of 44% compared to 83% (p = 0.003) in the remaining EMCA patients without an alteration in CNV (n=214). Septins assemble into filaments that interact with the cytoskeleton. Published data on established cell lines with an overexpression of specific isoforms suggest an association between the overexpression and resistance to paclitaxel. Many high grade EMCA patients are treated with adjuvant paclitaxel-based regimens. We hypothesize that an increased CNV and overexpression of SEPT9 is associated with paclitaxel resistance on a molecular level and a worse clinical prognosis. To support our hypothesis, we will test established uterine papillary serous carcinoma cell lines (UPSC), an aggressive high grade EMCA, for CNV variation of SEPT9 and resistance to paclitaxel. In addition, tumor samples from patients with advanced stage (III-IV) UPSC will be investigated for an increased CNV of SEPT9.

Citation Format: Jenna R. Zechmeister, Gary L. Goldberg, Cristina Montagna. Increased copy number of Septin 9 (SEPT9) in women with high grade endometrial adenocarcinoma(EMCA): Is there a potential link to paclitaxel resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 455. doi:10.1158/1538-7445.AM2014-455
Abstract 4551: Emerging effects of Chinese herbal medicines on human cancer cells proliferation
Authors: Zhang Xufei , Li Hao , Espiritu Herschel Angela , Kim Anita , Zhong Li
Published: May 2015

Chinese herbal medicine is a major aspect of traditional Chinese medicine, which focuses on restoring a balance of energy, body, andspirit to maintain health rather than treating a particular disease or medical condition. Some of Chinese herbs have shown therapeutic effects in cancer patients. Yunnan Baiyao (White Medicine from Yunnan, YNBY) and Snow daisy are popular Chinese herbs for routine clinical usage. YNBY is a Chinese herbal medicinal powder used to stop bleeding, reduce swelling and improve circulation in traumatic injuries. While Snow daisy has effects on maintaining blood pressure and reducing coronary heart diseases and inflammation when consumed in form of tea. Although it has been hypothesized that both YNBY and Snow daisy flower tea (SDFT) may help to prevent tumor proliferation and even reduce tumor growth, yet no systematic study and analysis have been carried out. Therefore, in this study the effects of YNBY and SDFT on tumor cell proliferation were evaluated and the signal transduction pathways mediating the effects were also determined. We tested tumor cells (lung, breast and oral cancer cell lines) and normal human fibroblast cells with different YNBY and SDFT dilution in cultured medium. Then the proliferation ability of these treated cells was tested by MTT assay and visualized via Dil (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) staining. Subsequently, several apoptosis factors (Fas pathway related, i.e., Fas/CD95, Fas Ligand, Caspase-8) which may be secreted by YNBY and SDFT treated tumor cells were tested using ELISA and Western Blot. The results showed that YNBY and SDFT can significantly inhibit lung tumor cells proliferation when its concentration was above 0.4mg/mL and 2mg/mL, respectively (P<0.01). The data indicated that YNBY could induce lung tumor cells apoptosis through Fas related pathway. The expression of Fas/CD95 and caspase-8 in treated lung tumor cells was much higher than which in control groups (P<0.01) suggesting that YNBY and SDFT may provoke tumor cells death by inducing cell apoptosis through Fas pathway. YNBY and SDFT may constitute potential therapeutic effects for lung cancer patients.

Citation Format: Xufei Zhang, Hao Li, Herschel Angela Espiritu, Anita Kim, Li Zhong. Emerging effects of Chinese herbal medicines on human cancer cells proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2014-4551
Abstract 4557: Expression of p16 in colon cancer and cyclin D1 in gastric cancer predicts response to CDK4/6 inhibition in vitro
Authors: Wainberg Zev A. , Yufa Ann , Anghel Adrian , Rogers Amy M. , Manivong Tin , Adhami Shahriar , Hamidi Habib , Conklin Dylan , Finn Richard S. , Slamon Dennis J.
Published: May 2015

Background: Cyclin-dependent kinases (CDKs) play a significant role in regulating cell cycle progression through association with cyclins. CDK4 and CDK6 interact with cyclin D1 to mediate hyperphosphorylation of retinoblastoma (Rb) during early G1 phase. Palbociclib is a highly selective inhibitor of CDK4 and CDK6 which functions by blocking pRb phosphorylation resulting in G1 arrest in sensitive cell lines. We evaluated the effect of palbociclib in gastric and colon cancer cell lines to explore potential biomarkers of response and to guide patient selection in colon and gastric cancer.

Methods: Panels of 17 gastric and 27 colon cancer cell lines were exposed in vitro to palbociclib over various concentrations to generate dose response curves. Analysis of variance (ANOVA) was used to identify differentially expressed genes between sensitive and resistant cell lines. Genes identified by ANOVA and effects of palbociclib on pRB were analyzed via western blot.

Results: Palbociclib was found to have potent anti-proliferative activity in the low nanomolar range (< 150 nM) for 14 of the 44 gastric and colon cancer cell lines tested. In gastric cancer, cyclin D1-amplified cells cells expressed greater sensitivity to the compound when compared to cyclin D1-negative cells. HER2 amplified cell lines were also statistically more sensitive than HER2 negative cells. Combination studies with palbociclib and trastuzumab demonstrated significant synergy in HER2 amplified gastric cancer models. Furthermore, cyclin E emerged as a biomarker for resistance to the compound in gastric cancers. Contrary to observations made in other cancers, expression levels of p16 (CDK4 inhibitor) and p21 (CDK2 inhibitor) in colon cancer indicated that p16 loss and p21 gain predict for resistance rather than sensitivity to CDK4 and CDK6 inhibition.

Conclusions: Palbociclib demonstrates anti-proliferative activity in several gastric and colon cancer cell lines. Molecular markers found to predict for sensitivity to this agent enhance patient selection for future clinical studies of palbociclib.

Citation Format: Zev A. Wainberg, Ann Yufa, Adrian Anghel, Amy M. Rogers, Tin Manivong, Shahriar Adhami, Habib Hamidi, Dylan Conklin, Richard S. Finn, Dennis J. Slamon. Expression of p16 in colon cancer and cyclin D1 in gastric cancer predicts response to CDK4/6 inhibition in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4557. doi:10.1158/1538-7445.AM2014-4557
Abstract 4565: Bet bromodomain inhibitors affects replication & cell cycle progression
Authors: Muralidharan Somsundar Veppil , Bhadury Joydeep , Green Lydia , Nilsson Lisa M. , Mclure Kevin G. , Nilsson Jonas A.
Published: May 2015

Myc family of transcription factors contribute to pathogenesis in most cancers and their expression projects a poor prognosis. Genetic and pharmacological inhibition of Myc and several Myc targets leads to apoptosis and tumor regression. Recently, inhibitors of BET Bromodomain proteins (BETi) were shown to have anti-tumor properties and this has been attributed to Myc down-regulation. In this study, we show that two structurally distinct BETi affects replication and cell cycle progression at low concentrations where the transcriptome remains largely unaltered. At these concentrations, S-phase progression is hindered, as assessed by thymidine incorporation and flow cytometry analyses. However, in a cell-free system replication is not impaired suggesting that BETi-mediated block of replication is linked to effects on chromatin. Furthermore, at higher concentration of BET, S-phase entry of cells is completely abrogated. Ectopic expression of Myc fails to rescue these phenotypes, suggesting a novel function of BET bromodomain proteins in replication and cell cycle regulation.

Citation Format: Somsundar Veppil Muralidharan, Joydeep Bhadury, Lydia Green, Lisa M. Nilsson, Kevin G. Mclure, Jonas A. Nilsson. Bet bromodomain inhibitors affects replication & cell cycle progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2014-4565
Abstract 4920: Luminol-based in situ photodynamic therapy for breast adenocarcinoma
Authors: Alshetaiwi Hamad S. , Shrestha Tej B. , Balivada Sivasai , Basel Matthew T. , Pyle Marla , Wang Hongwang , Bossmann Stefan H. , Troyer Deryl L.
Published: May 2015

Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT and can expand its use for metastatic tumors. Infiltration of tumor-activated polymorphonuclear neutrophils (PMNs) produces luminescence in the presence of luminol; this bioluminescence has been used for tumor detection in pre-clinical trials. Based on this rationale, we hypothesized that luminol-based bioluminescence can cause targeted PDT in breast adenocarcinoma tumors in the presence of the photosensitizer 5-aminolevulinic acid (ALA). To test this hypothesis, BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. After tumor formation, ALA and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the treatment hypothesis. Analysis of the data showed that treatment with a combination of luminol and ALA as well as treatment with luminol alone results in breast adenocarcinoma tumor growth attenuation. This study gives evidence for the antitumor activity of luminol on breast adenocarcinoma, possibly through PDT.

Citation Format: Hamad S. Alshetaiwi, Tej B. Shrestha, Sivasai Balivada, Matthew T. Basel, Marla Pyle, Hongwang Wang, Stefan H. Bossmann, Deryl L. Troyer. Luminol-based in situ photodynamic therapy for breast adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4920. doi:10.1158/1538-7445.AM2014-4920
Abstract 4992: Zkscan3 facilitates invasion of colorectal cancer associated with ceacam5
Authors: Roh Seon A. , Kim Chan Wook , Tak Ka Hee , Kim Jin Cheon
Published: May 2015

Purpose: ZKSCAN3 is over-expressed in invasive colonic tumor cells and regulate the expression of several genes favoring tumor progression including integrin β4. We evaluated the role of ZKSCAN3 in invasive signaling pathway with stage 4 colorectal cancer (CRC).

Materials and methods: 119 metastatic CRC (mCRC) patients who palliatively or curatively resected and 331 sporadic CRC patients who curatively resected from January 2004 to December 2009 were used to identify correlations between single-nucleotide polymorphism (SNP) rs733743 and clinically prognostic parameters. To validation of prior correlation, the genotypes of SNP identified by pyrosequencing using 81 patients (33 with mCRC and 48 CRC patients) and CRC cell lines. We then performed immunohistochemistry (IHC) and Western blot on 36 mCRC patients and 49 CRC patients. Up-regulated putative down-stream targets included genes contributing tumor aggressiveness and invasiveness; VEGF, cyclin D2, Akt, phospho-Akt, integrin β4, KRAS, CEACAM5, and integrin α5β1. To test biological utility assay, ZKSCAN3 mRNA was knocked down by ZKSCAN3 specific siRNA in metastatic CRC cells (LOVO) and ZKSCAN3 cDNA was overexpressed in the low-ZKSCAN3 expressed CRC cells (HCT116).

Results: Wild-type alleles (GG) of ZKSCAN3 rs733743 was related with male dominant, family history of malignancy, high CEA concentration, and mCRC. The wild-type alleles of ZKSCAN3 rs733743 using tissue sample was related with lymphvascular invasion. ZKSCAN3 IHC positive tissue was related with lymphovascular invasion. ZKSCAN expression was higher in LOVO and SW620 cell lines which were derived from metastatic site. ZKSCAN3 high groups were related with CEA, integrin β4, VEGF, and AKT and ZKSCAN3 low groups were related with RAS, VEGF, AKT, and PAKT in metastatic tumor tissue. Therefore, ZKSCAN3 seemed to be related with CEA and integrin β4 to metastasis. Additional CEA expression was observed a significant decrease in ZKSCAN3 knockdowned LOVO cell (1.0 vs 0.89, P= 0.046) and invasiveness of the ZKSCAN3 overexpressed HCT116 cells was higher in CEA coated filters (75.6±6.0 vs 91.3±4.5, P=0.023).

Conclusion: ZKSCAN3 is related with colorectal tumor progression and invasion. ZKSCAN3 overexpression tumor may facilitate metastasis of colorectal cancer associated with CEACAM5.

Citation Format: Seon Ae Roh, Chan Wook Kim, Ka Hee Tak, Jin Cheon Kim. Zkscan3 facilitates invasion of colorectal cancer associated with ceacam5. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4992. doi:10.1158/1538-7445.AM2014-4992
Abstract 5116: Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells
Authors: Jane Esther P. , Premkumar Daniel R. , Morales Alejandro , Foster Kimberly A. , Pollack Ian
Published: May 2015

Identification of therapeutic strategies that might enhance the efficacy of Bcl-2 inhibitor ABT-737 is of great interest in many cancers including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with glioma, as PI3K/Akt inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. We assessed the combination of the PI3K inhibitor BKM120 and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8, 3, PARP and cell death irrespective of PTEN status. The enhanced lethality observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, activation of Bax and Bak caused by these inhibitors was the key factor for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony forming ability, suggesting the potential use of BKM120 as a promising agent to improve the anti-cancer activities of ABT-737.

Citation Format: Esther P. Jane, Daniel R. Premkumar, Alejandro Morales, Kimberly A. Foster, Ian Pollack. Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2014-5116
Abstract 5131: Cadmium induces cytotoxicity in human bronchial epithelial cell in a p53-independent, nuclear factor NF-kappaB-dependent manner
Authors: Du Ji-Ying , Chen De-Ju , Xu Yan-Ming , Lau Andy T. Y.
Published: May 2015

Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well known carcinogens. Here, we report that in CdCl2-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can up-regulate the protein levels of eukaryotic translation initiation factor 5A-1 (eIF-5A-1) and redox-sensitive transcription factor NF-κB p65 in a time- and dose-dependent manner. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF-5A-1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF-5A-1 in Cd cytotoxicity. As eIF-5A-1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF-5A-1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF-5A-1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF-5A-1 promoter region of Cd-treated cells. Taken together, our findings report for the first time a regulatory mechanism for the pro-apoptotic protein eIF-5A-1 in which its level is controlled in an NF-κB-dependent manner.

Citation Format: Ji-Ying Du, De-Ju Chen, Yan-Ming Xu, Andy T. Y. Lau. Cadmium induces cytotoxicity in human bronchial epithelial cell in a p53-independent, nuclear factor NF-kappaB-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2014-5131
Abstract 5170: Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in colorectal cancer
Authors: Mamlouk Soulafa , Childs Liam , Redmer Thorben , Durek Pawel , Schumacher Dirk , Bläker Hendrik , Aust Daniela , Winterfeld Moritz von , Heinemann Volker , Modest Dominik , Möhr Kerstin , Morkel Markus , Schäfer Reinhold , Sers Christine
Published: May 2015

Complex sets of driver mutations critically control clinical course and treatment response in colorectal cancer (CRC). It is expected that multiple mutations in different signaling pathways interact to establish an individual cancer cell phenotype. Furthermore, mutational patterns can evolve under selective pressure, for instance during tumor metastasis or targeted therapy. Yet only few mutations, such as these in KRAS, NRAS and BRAF, are routinely assessed in the clinic today. With the introduction of next generation sequencing (NGS) many more genes can be simultaneously sequenced, allowing detailed analyses of mutational patterns.

We have assembled the CRC5.2 NGS panel for application with Ion Torrent PGM covering 100 frequently mutated genes with 784 amplicons (covering 21,000 COSMIC mutations), going well beyond the scope of commercially available cancer panels. In particular, exon coverage was optimized to embrace all genomic information related to drug sensitivity, using several drug and signaling network databases.

The German Consortium for Translational Cancer Research (DKTK) comprises the top cancer centers in Germany. As a part of this group, we employ our custom CRC5.2 panel for the analysis of mutational patterns in CRC. We address several key questions relevant for current diagnostics and treatment in colorectal cancer. In retrospective and prospective clinical studies, we assess mutational heterogeneity in primary tumors, synchronous and metachronous metastases of individual patients. In-depth NGS mutational analyses, transcriptome analysis and methylation studies will identify (epi-)genetic alterations occurring as a consequence of selective pressure during tumor evolution e.g. during metastasis, chemotherapy or targeted tumor therapy. Thus, we will uncover new links between cancer-specific mutations, clinical course and treatment response as well as novel mechanisms of secondary resistance. In addition we aim to identify novel predictive and prognostic mutational markers for the benefit of future CRC patients

Citation Format: Soulafa Mamlouk, Liam Childs, Thorben Redmer, Pawel Durek, Dirk Schumacher, Hendrik Bläker, Daniela Aust, Moritz von Winterfeld, Volker Heinemann, Dominik Modest, Kerstin Möhr, Markus Morkel, Reinhold Schäfer, Christine Sers. Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2014-5170
Abstract 5492: Effect of GnRH analogues and pharmacoperone IN3 treatment in survival of prostatic cancer cells to radiation
Authors: Sanchez Catherine A. , Salgado Apolo , Castellon Enrique A.
Published: May 2015

Prostate cancer (PCa) is the second cause of cancer related death in men. Radiotherapy of localized tumours is highly effective and reduces the risk of urinary incontinence and impotence compared with radical surgery. Radiotherapy is more effective when associated with GnRH analogues (as leuprolide), which has been related with decreased circulating androgens due to hypophysis-testis axis blockade. We propose that GnRH analogues increase the cytotoxic effect of radiotherapy in PCa cells, effect that can be potentiated with IN3, a pharmacoperone that increases GnRH receptor (GnRHR) expression in cell membrane.

To demonstrate our hypothesis, we stablished primary cell cultures from PCa samples from patients of our Institutional Hospital (n=12). Cells were treated during 24 hr with 1 ug/ml IN3 (Merck Sharp & Dohme Corp., USA,) and then with different concentrations (20-80 ng/ml) of leuprolide (Sigma-Aldrich Co., USA) for 24 hr. Later, cells were irradiated at 3 Gy and we evaluated cell survival at 24 hr (through MTT), changes in cell cycle at 24 hr and apoptosis at 6 hr (through FACS). GnRHR expression in cell membrane after 24 hr of IN3 treatment was evaluated trough fluorescence microscopy. Results were evaluated using Anova and Kruskal-Wallis tests, followed by post-test of Dunn. P <0.05.

As we previously described, we observed that leuprolide decreased PCa cell survival. When cells were pre-treated with IN3, GnRHR expression in cell membrane increased in about 60% and the effect of leuprolide treatment was higher. When cells were irradiated, cell survival decreased in 10%, this effect was higher when cells were previously treated with leuprolide (15%) and IN3/leuprolide (20%). This was related with an increase of 10% in apoptosis and cell cycle arrest in G2. The effect was dependant of leuprolide dose. At the dose used, IN3 had no effect in cell survival.

We concluded that leuprolide sensitizes cells from PCa to radiation, and this effect was increased with pretreatment with IN3 due, at least in part, for the increase of GnRHR expression in cell membrane, a receptor that decreases its expression during PCa progression. In clinical practice, leuprolide has direct effect over PCa cells, decreasing cancer cell survival and improving the cytotoxic effect of radiation. The utility of IN3 as a potential adjuvant therapy needs to be addressed.

Citation Format: Catherine A. Sanchez, Apolo Salgado, Enrique A. Castellon. Effect of GnRH analogues and pharmacoperone IN3 treatment in survival of prostatic cancer cells to radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5492. doi:10.1158/1538-7445.AM2014-5492
Abstract 5510: ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines
Authors: Foster Kimberly A. , Premkumar Daniel R. , Jane Esther P. , Morales Alejandro , Pollack Ian F.
Published: May 2015

We have previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apopotsis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACi) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that cotreatment of glioma with a BH3-mimetic and HDACi may synergistically produce cell death. We assessed the combination of the Bcl-2 family inhibitor ABT-737 and the HDACi vorinostat in established and primary cultured glioma cells. We found that combined treatment with these agents led to significant cellular death when compared to either drug as a single agent and demonstrated activation of the caspase cascade. The enhanced apoptosis observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. We observed that the upregulation of Noxa, truncation of Bid, and the activation of Bax caused by this combination were important factors for synergistic cell death. We demonstrated that Noxa functioned to occupy Mcl-1 in cells exposed to the combination, yet, contrary to studies in other malignancies, this combination in glioma occurred without the release of proapoptotic proteins Bim and Bak from Mcl-1. We showed this combination to be less effective in PTEN-deficient glioma cells. Both genetic manipulation of PTEN status and pharmacologic inhibition of PI3 kinase sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as the Bcl-2 family, and regulating epigenetic aberrancies, such as histone regulation, may induce cell death. Moreover, we reiterate that therapies to target the PI3-kinase/Akt/mTor pathway may be additionally necessary in cancers lacking PTEN functionality.

Citation Format: Kimberly A. Foster, Daniel R. Premkumar, Esther P. Jane, Alejandro Morales, Ian F. Pollack. ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2014-5510
Abstract 5517: PTC596-induced Bmi1 hyper-phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion
Authors: Kim Min Jung , Cao Liangxian , Sheedy Josephine , Risher Nicole , Dumble Melissa , Lee Chang-Sun , Sydorenko Nadiya , Baiazitov Ramil , Du Wu , Moon Young-Choon , Weetall Marla L. , Colacino Joseph , Davis Thomas W.
Published: May 2015

The Polycomb group (PcG) transcription repressor BMI1 is highly expressed in human cancers and is required for the clonogenic self-renewal and tumorigenesis of human cancer cells including those in hematological cancer and neuroblastoma. PTC596 is efficacious in vivo across a range of xenograft tumor models, including models of glioblastoma, fibrosarcoma and leukemia as well as orthotopic models of GBM. With EC50 values of 30-200 nM in a variety of tumor cell lines, PTC596 selectively reduces the level of functional BMI1 protein resulting in the depletion of the tumor stem cell fraction. PTC596 induces the hyper-phosphorylation of BMI1 leading to its degradation and the reduction of polycomb repressive complex 1 (PRC1) activity. Mechanistic studies suggest that PTC596 inhibits APC/CCDC20 activity resulting in the persistent activation of CDK1 and CDK2 which mediate the hyperphosphorylation of BMI1. Studies are ongoing to elucidate the mechanism of PTC596 inhibition of APC/CCDC20 and its preferential depletion of the tumor stem cell fraction.

Citation Format: Min Jung Kim, Liangxian Cao, Josephine Sheedy, Nicole Risher, Melissa Dumble, Chang-Sun Lee, Nadiya Sydorenko, Ramil Baiazitov, Wu Du, Young-Choon Moon, Marla L. Weetall, Joseph Colacino, Thomas W. Davis. PTC596-induced Bmi1 hyper-phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2014-5517
Abstract 5521: TTFields reduce cancer cell clonogenic potential through abnormal chromosome segregation during mitosis
Authors: Schneiderman Rosa S. , Giladi Moshe , Porat Yaara , Munster Mijal , Kirson Eilon D. , Weinberg Uri , Palti Yoram
Published: May 2015

TTFields cause mitotic arrest and cell death by the disruption of spindle microtubule arrangement and interference with cytokinesis. Recently it was reported that TTFields application results in reduced clonogenicity of cells surviving treatment. We hypothesized that TTFields can impair chromosome segregation during mitosis thus leading to a long term effect on surviving cancer cells.

A2780 human ovarian cancer cells were treated with TTFields for 24, 48 and 72 hours and the cytotoxic effect and clonogenic potential were determined. Abnormal mitotic figures were analyzed using fluorescence microscopy and cell karyotyping was performed using Giemsa staining and SKY analysis.

TTFields application led to 36, 52 and 43% cell growth inhibition after 24, 48 and 72 hours, respectively. Clonogenic assays revealed that only 12.9% of cells treated with TTFields for 72 hours maintained their ability to form colonies after 2 weeks. Immunofluorescence images revealed a significant increase in the number of abnormal mitotic figures amongst treated cells. Karyotyping analysis following 24 and 48 hours of TTFields application revealed 38.5% and 53.8% abnormal segregation events, respectively. After 24 hours of treatment, the proportion of cells with reduced and increased chromosomes number was equal, however, following 48 hours of treatment the population with a reduced chromosome number disappeared.

These results demonstrate that in addition to their cytotoxic effect leading to mitotic death, TTFields also lead to abnormal chromosome segregation. The surviving aneuploid cells are impaired, providing a possible explanation for the reduced clonogenic potential reported in cancer cells treated with TTFields.

Citation Format: Rosa S. Schneiderman, Moshe Giladi, Yaara Porat, Mijal Munster, Eilon D. Kirson, Uri Weinberg, Yoram Palti. TTFields reduce cancer cell clonogenic potential through abnormal chromosome segregation during mitosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5521. doi:10.1158/1538-7445.AM2014-5521
Abstract 5523: Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments
Authors: Dastur Anahita , Benes Cyril
Published: May 2015

The serine-threonine protein kinase, BRAF, is a frequently mutated oncogene, found in cancers of the skin, thyroid and large intestine. It is especially prevalent in malignant melanoma, where it occurs in 40-60% of the cases. The most common BRAF mutation is the valine to glutamate substitution at position 600 of the kinase (V600E). This substitution, seen in ninety percent of the BRAF cases, leads to constitutive activation of the kinase, and the downstream MEK-ERK pathway. Appropriately, several drugs have been designed to inhibit BRAF, with a broad range of outcomes in clinical trials. Unfortunately, all patients, including those showing complete responses, develop resistance to single agent therapy. Our goal is to come up with effective drug combinations that would delay, or even prevent, the cancer from becoming resistant to treatment.

We used a panel of 43 melanoma lines, comprising of 28 BRAF mutants, 12 NRAS mutants and 3 lines that were wild type for these two genes. Besides BRAF and NRAS, we have the mutational status of 65 other commonly mutated cancer genes for all the lines. We assembled a set of 25 drugs targeting a wide range of proteins, including PI3K/mTOR inhibitors, the apoptosis machinery, as well as some epigenetic modulators. The melanoma lines were treated with these drugs, either (i) alone or (ii) in combination with the B-RAF inhibitor vemurafenib or (iii) in combination with the MEK inhibitor Selumetinib, for 72 hrs over a nine point, 256-fold concentration range. Viability and apoptosis were measured using cell titer glo (Promega).

The results from this pilot study are discussed here.

Note: This abstract was not presented at the meeting.

Citation Format: Anahita Dastur, Cyril Benes. Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5523. doi:10.1158/1538-7445.AM2014-5523
Abstract 5524: 5-aza-2′-deoxycytidine, a DNA demethylating agent, inhibits metastatic melanoma invasiveness
Authors: Etiévant Chantal , Desjobert Cécile , Carrier Arnaud , Delmas Audrey , Tost Jorg , Favre Gilles , Riond Joelle , Arimondo Paola
Published: May 2015

Metastatic melanomas are the deadliest form of skin cancer and are very aggressive tumors showing highly invasive properties and a rapid chemoresistance to standard treatment (Dacarbazine) and to specific BRAF-V600E kinase inhibitors (Vemurafenib). Thus, targeting these tumors remains a major concern for novel therapeutic proposals.

Abnormal patterns of DNA methylation, an epigenetic modification that cells use to control gene expression, have been described in these tumors. These epigenetic modifications participate in melanoma formation and maintenance. The aim of our project is to characterize the DNA methylation changes that occur in the most aggressive form of melanoma and to reverse these changes by using clinically active DNA methyltransferase inhibitors (5-aza-2′-deoxycytidine, 5-aza-dC). To date a limited number of datasets have been published depicting the effects of inhibitors of DNA methylation on invasive capacities of metastatic melanoma cells. The work presented here, focuses on the study of the effects of DNA methylation inhibition on metastatic melanoma invasiveness.

We have set up an original method to quantify DNA methylation by FACS and shown that non cytotoxic nanomolar 5-aza-dC concentrations were able to demethylate DNA of WM-266-4 metastatic melanoma cells. Then using an in vitro 3D spheroids cell invasion assay and fluorescent microscopy to measure invasion capacities of metastatic cell lines, we showed that 5-aza-dC was able to inhibit invasion of WM-266-4 cells at these non-cytotoxic demethylating concentrations.

Citation Format: Chantal Etiévant, Cécile Desjobert, Arnaud Carrier, Audrey Delmas, Jorg Tost, Gilles Favre, Joelle Riond, Paola Arimondo. 5-aza-2′-deoxycytidine, a DNA demethylating agent, inhibits metastatic melanoma invasiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2014-5524
Abstract 5535: Gcn5 modulates the cellular response to oxidative stress induced by histone deacetylase inhibitors
Authors: Gaupel Ann-Christin , Begley Thomas J. , Tenniswood Martin P.
Published: May 2015

Aberrant histone post-translational modifications including acetylation are common in human cancers and result in dysregulation of gene expression. Our laboratory has focused on studying the effects of hydroxamic acid based HDAC inhibitors (HDACis) including TSA, SAHA and CG-1521.

We have employed a whole genome yeast deletion library screen to gain insight into the mechanism of the HDAC inhibitor CG-1521 and identify potential combinatorial drug targets. Gene ontology enrichment analysis shows that sensitive strains are highly enriched in processes regulating chromatin remodeling and transcription. Deletions of components of the SAGA HAT complex are highly overrepresented in the sensitive strains, including the catalytic subunit Gcn5. The gcn5 deletion strain displays increased sensitivity to CG-1521 induced cell death compared to the wild type strain, suggesting that the SAGA complex is required to reduce the effect of CG-1521 on cell growth. Gene expression analysis indicates that CG-1521 elicits an oxidative stress response in both the wild type and the gcn5 deletion strain. Gcn5 deletion substantially increases the level of oxidative stress induced by CG-1521 as measured by flow cytometry.

In the colorectal cancer cell line HT29, treatment with CG-1521 is accompanied by an increase in oxidative stress and co-treatment with the Gcn5 specific inhibitor MB-3 or RNAi-mediated knockdown of the human Gcn5 homologs, Gcn5 and PCAF, enhances the growth-inhibitory effect of CG-1521.

These studies have shown that disruption of the SAGA HAT complex with MB-3 or RNAi-mediated knockdown sensitizes tumor cells to CG-1521. Taken together, our work suggests that the combination of selective HAT and HDAC inhibitors may be an effective treatment of cancer.

Citation Format: Ann-Christin Gaupel, Thomas J. Begley, Martin P. Tenniswood. Gcn5 modulates the cellular response to oxidative stress induced by histone deacetylase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5535. doi:10.1158/1538-7445.AM2014-5535
Abstract 5547: A genetic variant near KLK3 gene is associated with aggressive prostate cancer and can stratify Gleason score 7 patients
Authors: Gu Jian , He Yonggang , Strom Sara S. , Logothetis Christopher J. , Kim Jeri , Wu Xifeng
Published: May 2015

Purpose: Gleason score (GS) 7 prostate cancer is a heterogeneous disease with significant differences in aggressiveness and prognosis. We sought to identify genetic biomarkers that may predict the aggressiveness of GS 7 diseases. Experimental Design: We genotyped 72 prostate cancer susceptibility single nucleotide polymorphisms (SNPs) identified in genome-wide association studies in 1827 white men with histologically confirmed prostate adenocarcinoma. SNPs associated with disease aggressiveness were identified by comparing high-aggressive (GS ≥8) and low-aggressive (GS ≤6) cases. The significant SNPs were then tested to see whether they could further stratify GS 7 prostate cancer. Results: Three SNPs_rs2735839, rs10486567, and rs103294_were associated with biopsy-proven high-aggressive (GS ≥8) prostate cancer (P < 0.05). Rs2735839 and rs10486567 were also associated with high D'Amico risk groups. Furthermore, the frequency of the variant allele (A) at rs2735839 was significantly higher in patients with biopsy-proven GS 4+3 disease than in those with GS 3+4 disease (P = 0.003). In multivariate logistic regression analysis, patients carrying the A allele at rs2735839 exhibited a 1.85-fold (95% 1.31-2.61) increased risk of being GS 4+3 compared to those with GS 3+4. Rs2735839 is located 600 base pair downstream of KLK3 gene (encoding PSA) on 19q13.33 and has been shown to modulate PSA level, providing strong biological plausibility for its association with prostate cancer aggressiveness. Conclusions: We showed the association of the rs2735839 with high-aggressive prostate cancer (GS ≥8). Moreover, we reported for the first time that rs2735839 can stratify GS 7 patients, which would be clinically important for more accurately assessing the clinical behavior of the intermediate-grade prostate cancer and for tailoring personalized treatment and post-treatment management.

Citation Format: Jian Gu, Yonggang He, Sara S. Strom, Christopher J. Logothetis, Jeri Kim, Xifeng Wu. A genetic variant near KLK3 gene is associated with aggressive prostate cancer and can stratify Gleason score 7 patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5547. doi:10.1158/1538-7445.AM2014-5547