bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C109: Discovery of new natural anti-tumoral compounds using a parallel target- and phenotypic-driven screening approach.
Authors: Feger Daniel , Weber Holger , Totzke Frank , Birkle Marianne , Siedentopf Oliver , Müller Melanie , Umber Sarah , Lin Wenhan , Proksch Peter , Ehlert Jan E. , Kubbutat Michael H.G.
Published: August 2014

In anticancer drug discovery a common strategy is to start with a target-specific screen followed later by characterization of hit compounds in phenotypic cell-based assays. One important advantage of this sequential screening procedure is the feasibility of high throughput compound testing. However, a significant disadvantage of the approach is the fact that due to the sequential nature of the approach, compounds showing promising phenotypic activities, but addressing so far unknown targets will be lost as false-negative compounds. Here we describe a medium throughput screening strategy, where we characterized 360 natural compounds isolated from marine organisms in a parallel target- and phenotypic-driven screening approach. As target-specific read out, we analyzed biochemically the inhibitory potential of the compounds against 16 recombinant protein kinases, known to be relevant targets in different kinds of human tumors. In parallel we determined the impact of the compounds on cell transformation, migration and angiogenesis by analyzing their behavior in soft agar growth, wound healing and three-dimensional endothelial cell sprouting. Furthermore, unspecific toxicity of the compounds was tested via a viability assay with human peripheral mononuclear cells from healthy donors. Examples of identified compounds showing differential inhibitory profiles in the different assays applied will be presented.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C109.

Citation Format: Daniel Feger, Holger Weber, Frank Totzke, Marianne Birkle, Oliver Siedentopf, Melanie Müller, Sarah Umber, Wenhan Lin, Peter Proksch, Jan E. Ehlert, Michael H.G. Kubbutat. Discovery of new natural anti-tumoral compounds using a parallel target- and phenotypic-driven screening approach. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C109.
Abstract C134: EN3356, a novel CYP17A inhibitor for the treatment of castration resistant prostate cancer.
Authors: Laping Nicholas J. , Sivanandhan Dhanalakshmi , Kethiri Raghava Reddy , Dewang Purushottam M. , Vijayendra Ajith , Zainuddin Mohd , Mullangi Ramesh , Rao Niranjan , Venkatesan Aranapakam M. , Reddy Sanjeeva , Gupta Sandeep , Smith Roger A.
Published: August 2014

Androgens are a major driver of prostate cancer growth. As prostate cancer progresses, it can escape standard androgen deprivation therapy by relying on extra-testicular CYP17 enzyme activity for androgen synthesis. EN3356 is a novel non-steroidal CYP17 inhibitor that reduces androgen synthesis and is designed to treat castration-resistant prostate cancer. EN3356 provides potent inhibition of CYP17 lyase activity in rat and human testicular microsomes, and in human adrenocortical carcinoma H295R cells it also inhibits testosterone synthesis potently. In contrast to abiraterone, EN3356 is selective for inhibiting testosterone synthesis over cortisol synthesis. This finding suggests that EN3356 may not require co-administration of prednisone to mitigate mineralocorticoid excess. In pre-clinical studies, EN3356 exhibited high oral bioavailability. EN3356 inhibited serum testosterone levels in the rat and caused a robust decrease of seminal vesicle and prostate weights following 14-days of oral administration. GLP safety studies were completed in two preclinical species and identified a high safety margin. A combination Phase I/II study in metastatic castration-resistant prostate cancer is currently under initiation in the US and is anticipated to start enrolling subjects in the fourth quarter of 2013. Based on the potent inhibitory activity of EN3356 against CYP17 lyase in vitro, selective inhibition of testosterone synthesis versus cortisol synthesis, and robust weight reduction of androgen sensitive organs in vivo, EN3356 has the potential to be a selective and efficacious therapy for the treatment of castration-resistant prostate cancer.

Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C134.

Citation Format: Nicholas J. Laping, Dhanalakshmi Sivanandhan, Raghava Reddy Kethiri, Purushottam M. Dewang, Ajith Vijayendra, Mohd Zainuddin, Ramesh Mullangi, Niranjan Rao, Aranapakam M. Venkatesan, Sanjeeva Reddy, Sandeep Gupta, Roger A. Smith. EN3356, a novel CYP17A inhibitor for the treatment of castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C134.
Abstract C140: RIT1 mutations define a new genetic subset of lung adenocarcinoma.
Authors: Berger Alice , Imielinski Marcin , Duke Fujiko , Wala Jeremiah , Kaplan Nathan , Shi Geng-Xian , Andres Douglas , Meyerson Matthew
Published: August 2014

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF, and ERBB2, and translocations involving ALK, RET, and ROS1. Identification of these oncogenic events has transformed therapy of lung adenocarcinoma via application of therapies targeted towards specific genetic lesions in stratified patient populations. However, such mutations have been identified in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy may be involved in the remaining cases. We analyzed exome sequencing data from 413 lung adenocarcinomas and identified somatic mutations in the small GTPase gene RIT1 in ∼2% of samples. Mutations in RIT1 cluster in a hotspot near the switch II domain of the protein that is homologous to the Q61 region in RAS genes. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces activation of AKT and ERK signaling pathways and transforms NIH3T3 cells in vitro and in vivo. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C140.

Citation Format: Alice Berger, Marcin Imielinski, Fujiko Duke, Jeremiah Wala, Nathan Kaplan, Geng-Xian Shi, Douglas Andres, Matthew Meyerson. RIT1 mutations define a new genetic subset of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C140.
Abstract C151: Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells.
Authors: Tedeschi Philip M. , Markert Elke K. , Gounder Murugesan , Lin HongXia , Dolfi Sonia C. , Chan Leo Li-Ying , Qiu Jean , Hirshfield Kim M. , Boros Laszlo G. , Bertino Joseph R. , Oltvai Zoltan N. , Vazquez Alexei
Published: August 2014

Background: Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. Expression of genes in this pathway also correlate with sensitivity to the antifolate methotrexate (Vazquez A, et al, Cancer Res. 2013 Jan 15;73(2):478-82). Using a large-scale model of human cell metabolism we have predicted that serine synthesis can be routed to a pathway for ATP production. The pathway is composed by reactions from the serine synthesis, one carbon (folate) metabolism and the glycine cleavage system (SOG pathway) and its flux is predicted to increase at high proliferation rates.

Results: Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and its level of expression correlates with gene signatures of cell proliferation and Myc targets activation. To investigate the activity of the SOG pathway at the level of metabolic fluxes we estimated the metabolic fluxes of the NCI60 panel of tumor derived cell lines, using previously reported exchange fluxes and a flux balance model of cell metabolism. We show that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also find that the SOG pathway contributes significantly to the energy requirements of biosynthesis, the NADPH requirements of fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is subject to treatment with the antifolate methotrexate, we observe a decrease in the ATP levels, an inhibition of the proliferation rate and a decrease in the ribonucleotides and fatty acids synthesized from glucose.

Conclusions: Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C151.

Citation Format: Philip M. Tedeschi, Elke K. Markert, Murugesan Gounder, HongXia Lin, Sonia C. Dolfi, Leo Li-Ying Chan, Jean Qiu, Kim M. Hirshfield, Laszlo G. Boros, Joseph R. Bertino, Zoltan N. Oltvai, Alexei Vazquez. Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C151.
Abstract C152: Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism.
Authors: Issaq Sameer , Teicher Beverly , Monks Anne
Published: August 2014

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. A renewed interest in the metabolic properties of cancer cells, as well as the recognition of altered cellular metabolism as a critical hallmark of cancer, has led to an exploration of targeting metabolic dependencies of cancer cells as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human rhabdomyosarcoma and osteosarcoma cell lines in order to identify potential differences in sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Glycolytic and mitochondrial bioenergetic profiles were generated using Seahorse extracellular flux analysis to measure extracellular acidification rates and oxygen consumption rates, and significant differences in glycolytic and respiratory bioenergetics between rhabdomyosarcoma and osteosarcoma cell lines were identified. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely-used diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells versus embryonal rhabdomyosarcoma, osteosarcoma, and normal cells, whereas inhibition of mitochondrial respiration by oligomycin or metformin did not significantly affect viability. Combined inhibition of glycolysis and respiration decreased cell growth more effectively than either treatment alone in all sarcoma lines tested. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels in all sarcoma lines tested, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. Funded by NCI Contract No. HHSN261200800001E.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C152.

Citation Format: Sameer Issaq, Beverly Teicher, Anne Monks. Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C152.
Abstract C171: Novel therapeutic strategies targeting the hedgehog signaling pathway and mTOR pathway in biliary tract cancer (BTC).
Authors: Zuo Mingxin , Rashid Asif , Churi Chaitanya , Hung Mien-Chie , Javle Milind
Published: August 2014

Background: BTC represents the second most common primary cancer of the hepatobiliary system and is associated with a poor prognosis. Activation of PI3K/mTOR and hedgehog pathways has been demonstrated in biliary cancer. Activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenesis through S6K1-mediated Gli phosphorylation. Recently, synergistic antitumor efficacy was demonstrated with the combination of hedgehog and mTOR inhibitors, particularly in cases with mTOR/S6K pathway in esophageal cancer (Cancer Cell 2012; 21(3):374-87).

Methods: Gene expression profiling was performed on 10 human biliary tract cancer cell lines (MMNK-1, Mz-ChA-1, Sk-ChA-1, KKU-M213, KKU-M214, KKU-M055, KKU-M139, M213L5H, and M213LOH) by real-time qRT-PCR. CellTiter-Glo® luminescent cell viability assay, colony formation, and stem cells morphology spheres were conducted to examine the effects of the combination of Rapamycin and Vismodegib on cell proliferation. Western-blot assay was used to examine the related protein expression. Fluorescence-activated cell sorting (FACS) was used to examine the effects of the combination on BTC stem cells.

Results: Based on Gli1 and S6K gene expression, the Mz-ChA-1 and Sk-ChA-1 cell lines were chosen to investigate the effects of the combination of Rapamycin and Vismodegib on BTC (Table 1). Combination of Rapamycin and Vismodegib exhibited the synergistic antiproliferative effect on Mz-ChA-1 cells. The combination of Rapamycin and Vismodegib inhibited the Mz-ChA-1 cell proliferation, colony formation and stem cell sphere formation. This combination therapy decreases the NANOG, OTC3/4, and E-Cadherin gene expression in Mz-ChA-1 stem cells. Furthermore, the drug combination can decrease the phospho-p70S6K and Gli1 protein and increase cleaved caspase-3 expression in Mz-ChA-1 cells. No such effect was observed in Sk-ChA-1 cells.Conclusions: Crosstalk between the mTOR pathway and HH pathway provides a new avenue for combination therapy in BTC with hedgehog and mTOR inhibitors.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C171.

Citation Format: Mingxin Zuo, Asif Rashid, Chaitanya Churi, Mien-Chie Hung, Milind Javle. Novel therapeutic strategies targeting the hedgehog signaling pathway and mTOR pathway in biliary tract cancer (BTC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C171.
Abstract C18: Antitumor activity of xanthohumol, an inhibitor of valosin-containing protein.
Authors: Shikata Yuki , Kanagaki Shuhei , Sasazawa Yukiko , Tashiro Etsu , Yoshimaru Tetsuro , Komatsu Masato , Katagiri Toyomasa , Imoto Masaya
Published: August 2014

Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate co-factor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation, and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II).On the other hand, it has been reported that the expression level of VCP is correlated with progression of cancer, and VCP-overexpressing cell lines showed the resistance to apoptosis. Therefore, in this study, we examined the antitumor effects of XN on several types of human tumor cells. First we tested the sensitivity to XN in 15 human tumor cell lines. Each cell was treated with XN for 48 hrs, and XN-induced apoptosis was quantitatively analyzed by flow cytometry. As a result, XN potently induced apoptosis in 6 cell lines (SW480, SW620, HCT116, A2058, A375 and SW48 cells) as judged from subG1 population, and another 9 tumor cell lines were resistant to XN. Next, we examined whether VCP is involved in XN-induced apoptosis. Although, these 15 cell lines showed similar expression levels of VCP, another VCP inhibitor, Eeyarestatin I, also induced apoptosis in XN-sensitive tumor cells (SW480 cells and HCT116 cells) but not in VCP-insensitive tumor cells (A431 cells and EC17 cells). These findings suggest that the several human tumor cell lines require VCP function for their survival, and VCP would be a potential chemotherapeutic target molecule for subset of tumor cells.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C18.

Citation Format: Yuki Shikata, Shuhei Kanagaki, Yukiko Sasazawa, Etsu Tashiro, Tetsuro Yoshimaru, Masato Komatsu, Toyomasa Katagiri, Masaya Imoto. Antitumor activity of xanthohumol, an inhibitor of valosin-containing protein. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C18.
Abstract C183: Targeted disruption of the BCL9/b-catenin complex inhibits oncogenic WNT signaling.
Authors: Carrasco Ruben D. , Walensky Loren
Published: August 2014

Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers yet the development of targeted therapies to disrupt the pathway has remained a formidable challenge due to the toxicities associated with disrupting the pathway's homeostatic functions and the large protein interaction surfaces involved. BCL9 is an important co-activator for β-catenin-mediated transcription and is highly expressed in tumors but not in the cells of origin, presenting an opportunity to selectively block pathologic β-catenin activity. BCL9 drives β-catenin signaling through a direct binding interaction mediated by its α-helical homology domain-2. We report the development of a Stabilized Alpha-Helix of BCL9 (SAH-BCL9) that targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and elicits mechanism-based anti-tumor responses. The clinical translation potential of this approach is underscored by the capacity of SAH-BCL9 to effectively suppress the growth, angiogenesis, invasion, and metastasis of Wnt-dependent colorectal cancer and multiple myeloma in vivo.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C183.

Citation Format: Ruben D. Carrasco, Loren Walensky. Targeted disruption of the BCL9/b-catenin complex inhibits oncogenic WNT signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C183.
Abstract C199: Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules.
Authors: Guo Huifang , Gao Meng , Lu Yiling , Liang Jiyong , Lorenzi Philip L. , Bai Shanshan , Hawke David H. , Li Jie , Dogruluk Turgut , Scott Kenneth L. , Jonasch Eric , Mills Gordon B. , Ding Zhiyong
Published: August 2014

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated due to the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. While basal AKT2 phosphorylation was sensitive to inhibition of PI3K, basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C199.

Citation Format: Huifang Guo, Meng Gao, Yiling Lu, Jiyong Liang, Philip L. Lorenzi, Shanshan Bai, David H. Hawke, Jie Li, Turgut Dogruluk, Kenneth L. Scott, Eric Jonasch, Gordon B. Mills, Zhiyong Ding. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C199.
Abstract C201: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics.
Authors: Chen Li , Dastur Anahita , Benes Cyril
Published: August 2014

The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a novel gain-of-function forward genetic approach for identifying potential mechanisms of resistance. This system uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activates nearby gene expression. By combining this with high-throughput sequencing, we have performed genome-wide screens for paclitaxel resistance mechanism in a variety of cancer cell lines. Validating this approach, we identified the multidrug transporter ABCB1 as the major contributor to resistance. In addition, we identified several potential novel resistance genes. Therefore we have developed a powerful new pipeline tool to systematically discover common and tumor specific pathways of resistance in human cancers. This cost-effective approach can be readily applied to different cell lines. Moreover, its ability to identify gene interactions and to probe complex genetic context provides a significant advantage over RNAi- or cDNA-based screens.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C201.

Citation Format: Li Chen, Anahita Dastur, Cyril Benes. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C201.
Abstract C202: Probing EGFR, HER2 and c-Met protein-protein interactions using an antibody array.
Authors: Knowles Jacob , Capodilupo Stephen , Noring Richard , Campos-González Roberto , Herrera Julio , Gechtman Zev
Published: August 2014

The receptor tyrosine kinase (RTK) family comprises important disease drivers and validated drug targets. Cooperation between certain RTKs is often achieved through physical interactions and is thought be a contributing factor to some cases of tumor resistance to targeted therapies. Epidermal Growth Factor Receptor (EGFR), HER2/ErbB2, and c-Met are prominent examples of disease driver RTKs that cooperate and form heterodimers. Current methods to detect RTK protein-protein interactions lack proper throughput, are not quantitative, or rely on highly engineered systems and cumbersome workflows. We have developed a planar surface antibody array-based sandwich assay that enables the detection and relative quantification of interactions between EGFR, HER2, and c-Met. The antibody-based assay revealed co-existing combinatorial receptor interactions that were unique to each cell type tested. The assay also pointed to an existence of distinct topological configurations of EGFR-c-Met and EGFR-HER2 heterodimers, unveiling previously not described nuances of these interactions. Lastly, the array-based assay allowed monitoring the disruption of receptor complexes using small molecule inhibitors and revealed differential sensitivity of EGFR-HER2 and EGFR-c-Met complexes to a small molecule EGFR kinase inhibitor, gefitinib. The antibody array sandwich assay described here allows utilizing larger panel of affinity reagents and can be applied to broader range of cases with various topological requirements, thus offering an advantage over technologies that have strict proximity requirement. This novel protein interaction array enables parallel detection of RTK protein-protein interactions, allowing a more systematic monitoring of response to activators or inhibitors.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C202.

Citation Format: Jacob Knowles, Stephen Capodilupo, Richard Noring, Roberto Campos-González, Julio Herrera, Zev Gechtman. Probing EGFR, HER2 and c-Met protein-protein interactions using an antibody array. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C202.
Abstract C213: Protein interaction analysis of mll-aff4 and mll-fel oncogenic fusion proteins using data mining approach.
Authors: Hovhannisyan Grant Henry , Marqaryan Ashot Vardan
Published: August 2014

Mixed-lineage leukemia (MLL) is a transcriptional co-activator that plays an essential role in early development and hematopoiesis. MLL H3K4 methyltransferase activity mediates chromatin remodeling associated with epigenetic regulation of transcription activation. Multiple chromosomal translocations of MLL gene lead to its fusion to numerous partner genes and production of chimeric proteins, which are enrolled in progression of similar phenotypes of leukemia [5]. However, protein interaction models of different MLL fusions are still debated. To assess the pattern of protein interactions of MLL chimeras, we have performed protein-protein interaction (PPI) network analysis for two MLL fusion proteins, MLL-FEL - t(4;11)(q21;q23) and MLL-AFF4 - ins(5;11)(q31;q13q23), which are involved in acute leukemia progression [2]. Protein interaction data was extracted from data mining software GPS-Prot v. 2.0 [4]. PPI network visualization and network parameters evaluation were performed using Cytoscape v. 2.8 [7]. Cluster analysis was made by MCODE plug-in. The statistical significance of cluster extraction was assessed as described [3]. Comparative experimental interaction data taken from previously published papers were manually discarded from our analysis. Our results show that each of the components of studied fusion proteins in native form compose highly interconnected distinct regions. MLL PPI network analysis reveals a clique cluster with cluster density equal to 1, indicating that MLL interacters are united in protein macromolecular complex, which is in a good accordance with previously published experimental data [6]. Worth of mention, that FEL and AFF4 proteins form the same clique clusters, which also is in a good congruence with experimentally obtained data [1]. Therefore, we assume that the MLL-AFF4 and MLL-FEL fusion proteins generate the identical PPI network, which underlie the manifestation of similar malignancy. Further corroboration of this result will confirm the assumption that different fusions can bring to the same disease network, which could possibly has its impact in leukemia therapeutical approaches. To obtain more detailed pattern of protein interactions of MLL-caused leukemias further network analysis is required.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C213.

Citation Format: Grant Henry Hovhannisyan, Ashot Vardan Marqaryan. Protein interaction analysis of mll-aff4 and mll-fel oncogenic fusion proteins using data mining approach. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C213.
Abstract C221: A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene.
Authors: Min Chengyin , Konieczkowski David J. , Kwon Christine , Linja Marika , Vasudevan Krishna , Weir Barbara , Goetz Eva , Garraway Levi
Published: August 2014

Oncogenic mutations in BRAF are found in 50-70% of malignant melanomas and in ∼8% of all cancers. Activating BRAF mutations confer exquisite dependency on RAF/MEK/ERK signaling and RAF and MEK inhibitors achieved striking clinical response in the majority of patients with BRAFV600E-mutant melanoma. However, nearly all patients progress to therapeutic resistance and there are limited treatment options for patients with relapsed tumors. Identification of genes that are essential only in the presence of BRAFV600E mutation may inform alternative as well as combinatorial therapeutic approaches. Here, we used systematic RNA interference to discover synthetic lethal interactions in melanoma cells carrying BRAFV600E mutation. In addition, Project Achilles at the Broad Institute reported systematic loss-of-function studies in more than 100 human cancer cell lines. We intersected the list of genes that are synthetic lethal with BRAFV600E mutation in our melanoma study and that of Project Achilles. As a result, we have identified novel “druggable” targets for cells driven by BRAFV600E mutation. Our extensive in vitro and in vivo validation has confirmed and established key synthetic lethal interactions with BRAFV600E mutation. One major mechanism of acquired resistance to RAF and MEK inhibition is reactivation of MEK/ERK pathway. Importantly, we demonstrated that these synthetic lethal genes are also essential in the context of acquired resistance to RAF and MEK inhibition. Together, our study has identified novel targets for the treatment of oncogenic BRAF-driven melanoma and suggests targeting these genes as novel therapeutic strategies to overcome acquired resistance to RAF and MEK inhibition.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C221.

Citation Format: Chengyin Min, David J. Konieczkowski, Christine Kwon, Marika Linja, Krishna Vasudevan, Barbara Weir, Eva Goetz, Levi Garraway. A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C221.
Abstract C228: A retrospective study of patients with non-small cell lung cancer who have Life Technologies Pervenio™ Lung RS test performed.
Authors: Singh Jaswinder , Hasan Raza , Feeback Jennifer
Published: August 2014

With lung cancer causing the highest mortality in cancer patients, an accurate method to stratify lung cancer patients has been needed. This stratification would allow physicians to tailor their management. Furthermore, even with resection of early-stage NSCLC, there is a 35-50% recurrence, considerably poor when compared to other solid tumors. A new 14-gene PCR assay was created to stratify the risk of patients based on their lung cancer. The Pervenio™ Lung RS Test was established to discriminate the low vs intermediate vs high risk patients in order to provide information regarding mortality rates to guide management. Using this clinically proven PCR assay in a site-specific study, results mirrored the original study. The patients who were stratified as high-risk with the Pervenio™ Lung RS Test, were the same patients who had recurrence and death. Amongst the high-risk group, one patient expired and three remained alive. Furthermore, of the three living patients, two patients had progressive disease with deteriorating conditions and recurrence. This new assay successfully has proven to accurately stratify patients, in regards to survival and recurrence. This stratification will allow physician to alter their management and monitoring. Comprehension of the projected disease course is useful in planning a course of management, whether it may be curative versus palliative.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C228.

Citation Format: Jaswinder Singh, Raza Hasan, Jennifer Feeback. A retrospective study of patients with non-small cell lung cancer who have Life Technologies Pervenio™ Lung RS test performed. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C228.
Abstract C23: Functional characterization of viable circulating tumor cells using nanowells.
Authors: Yao Xiaosai , Choudhury Atish D. , Yamanaka Yvonne J. , Hahn William C. , Wittrup K Dane , Love J Christopher
Published: August 2014

Current characterizations of circulating tumor cells (CTCs) are limited to enumeration, genotyping, and gene expression. Because these assays each require fixation or lysis, it has not been possible to evaluate several important characteristics of live CTCs at the single-cell level. We have interrogated viable, single CTCs using arrays of subnanoliter wells (nanowells), performing functional measurements of viability, migration, and secretion of soluble factors.

We developed a process using nanowells to isolate and characterize viable CTCs from whole blood. Each poly(dimethylsiloxane) (PDMS) array comprises 84,762 cubic wells of 275 pL each. CTCs were enriched from whole blood by negative selection against CD45 and loaded onto the array to settle into the wells by limiting serial dilution. Because CTCs are rare, the loading biased the occupancy of the wells to single CTCs or CTCs in preformed clusters, allowing comparisons among individual CTCs. The defined address of each well allowed spatiotemporal tracking of single CTCs, and subsequent registration of data for functional measures on each CTC. Using this approach, we made three types of measurements on viable CTCs isolated from prostate cancer patients: (1) immediate and short-term viability of CTCs, (2) migration and invasion potential of CTCs, and (3) secretion of soluble factors.

We found a rare subset of CTCs to possess malignant traits indicative of metastatic potential in late-stage, progressing prostate cancer patients. These CTCs were resistant to anoikis after being in the circulation as they stained Calcein AM+/Annexin V−, and remained viable for at least a week in culture. They were invasive in their epithelial state when embedded in Matrigel. They could also secrete proteases capable of cleaving peptide substrates. However, we did not observe such metastatic potential in every CTC, pointing to the presence of heterogeneity within CTCs and suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients.

By interrogating the functional behavior of individual CTCs, we identified a rare subset of CTCs with phenotypes consistent with more efficient metastasis. The nanowell technology holds the potential to further investigate the cellular characteristics of rare tumor cells and design therapies to counter metastasis.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C23.

Citation Format: Xiaosai Yao, Atish D. Choudhury, Yvonne J. Yamanaka, William C. Hahn, K Dane Wittrup, J Christopher Love. Functional characterization of viable circulating tumor cells using nanowells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C23.
Abstract C233: Activation of p53 by the NEDD8 activating enzyme inhibitor MLN4924 is independent of Cdt1 and involves the cullin-RING ligase CRL4-DTL.
Authors: Blank Jonathan L. , Liu Xiaozhen J. , Bernard Hugues , Liao Hua , Cosmopoulos Katherine , Lightcap Eric S.
Published: August 2014

NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of E3 ubiquitin ligases termed the cullin-RING ligases (CRLs) through conjugation of cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrates targets them for degradation by the 26S proteasome. NAE thereby regulates the stability of many proteins including those required for cancer cell growth and survival. MLN4924 is an investigational small molecule inhibitor of NAE with 2-3 orders of magnitude selectivity over the related E1’s ubiquitin-activating enzyme (UAE) and sumo-activating enzymes (SAE; Ref. 1). Recently, a genome-wide siRNA screen in the A375 melanoma cell line identified key DNA damage response genes affecting sensitivity of cells to MLN4924, many of which were associated with the p53 pathway which activates a p21-dependent intra-S-phase checkpoint (2). A comparison of the effects of TP53 RNAi in A375 cells to genetic deletion of TP53 in HCT116 cells (HD 104-001 cells, Horizon Discovery Ltd) suggests that p21 induces a cell cycle arrest within 24 h, whereas analysis of the p53-regulated transcriptional signature indicates that proapoptotic proteins are up-regulated at later times. Surprisingly, stabilization of p53 by MLN4924 appears independent of the replication licensing factor Cdt1, ATR activation, or p53 neddylation, but rather involves the E3 ligase complex Cul4A/B-Ddb1-DTL. Furthermore, Nutlin-3induced ubiquitination of p53 could be blocked by MLN4924, suggesting that Mdm2 and DTL may cooperate in the ubiquitination of p53.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C233.

Citation Format: Jonathan L. Blank, Xiaozhen J. Liu, Hugues Bernard, Hua Liao, Katherine Cosmopoulos, Eric S. Lightcap. Activation of p53 by the NEDD8 activating enzyme inhibitor MLN4924 is independent of Cdt1 and involves the cullin-RING ligase CRL4-DTL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C233.
Abstract C235: An investigation of the p53 ubiquitin-proteasome system using a novel non-steady-state enzyme kinetic model.
Authors: Talwai Prem M.
Published: August 2014

Overzealous MDM2-mediated ubiquitination of p53 characterizes and sustains over 50% of all human cancers. Successful targeted cancer therapy hinges on a thorough understanding of the ubiquitination process. Unfortunately, current steady-state enzyme kinetic models fail to accurately describe the ubiquitin-proteasome system, due to the assumption that the enzyme (E3 ligase) is expressed at infinitesimally smaller concentrations than the substrates (ubiquitin-conjugated E2 and target protein). This limitation of the quasi-steady state assumption prohibits the use of steady-state models when analyzing the cancerous consequences of extreme ubiquitin-ligase overexpression. This paper derives a novel non-steady-state mathematical model of reversible ordered ternary complex enzyme kinetics, which can be used to simulate the behavioral response of the ubiquitin-proteasome system to specific variations in the cellular concentrations of p53, MDM2, and ubiquitin-conjugated E2D3. Computer simulations of the model were used to study the effects of E2D3-Ub and MDM2 concentrations on the rates of p53 ubiquitination at different temperatures. In each process, it was observed that the ubiquitin-ligase MDM2 accelerates the carcinogenic ubiquitination process, while ubiquitin-conjugated E2 inhibits it. It was also discovered that E2D3-Ub is more effective inhibitor of overzealous p53 ubiquitination when present at higher concentrations. However, it was also observed that high concentrations of p53 hinder the ability of E2D3-Ub to decelerate the reaction. The mathematical model successfully reproduced the experimentally observed p53-MDM2 interaction. The derived model therefore suggests MDM2 as a prospective target for cancer therapy. In addition, the findings of this project propose recombinant E2D3-Ub as a new promising protein-based anticancer drug for targeting overzealous p53 ubiquitination. The derived model can suggest new therapeutic solutions for decreasing the harmful effects of many human cancers, bacterial infections, and inflammatory diseases (such as rheumatoid arthritis) characterized by an overzealous ubiquitin-proteasome system. Finally, computational simulation of this novel model provides a safe, fast, and cost-effective preliminary alternative to expensive in vitro experimentation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C235.

Citation Format: Prem M. Talwai. An investigation of the p53 ubiquitin-proteasome system using a novel non-steady-state enzyme kinetic model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C235.
Abstract C240: The synthetic parasite-derived peptide GK1 increases survival in a preclinical mouse melanoma model.
Authors: Vera Aguilera Jesus , Perez Torres Armando , Vera Aguilera Carlos , Monterverde Suarez Diego , Moreno Aguilera Eduardo , Hernaiz Leonardo Juan Carlos , Estrada Barcenas Daniel
Published: August 2014

Purpose: The therapeutic efficacy of a synthetic parasite-derived peptide GK1, an immune response booster, was evaluated in a mouse melanoma model. This melanoma model correlates with human stage IIb melanoma, which is treated with wide surgical excision, a parallel study employing a surgical treatment was carried out as an instructive goal.

Experimental Design: C57BL/6 mice were injected subcutaneously in the flank with 2×105 B16-F10 murine melanoma cells. When the tumors reached 20 mm3, mice were separated into two different groups: the GK1 group, treated weekly with peritumoral injections of GK1 (10 μg/100 μl of sterile saline solution); and the control group, treated weekly with an antiseptic peritumoral injection of 100 μl of sterile saline solution without further intervention. All mice were monitored daily for clinical appearance, tumor size, and survival. Surgical treatment was performed in parallel when the tumor size was 20 mm3 (group A), 500 mm3 (group B) and greater than 500 mm3 (group C).

Results: The GK1 peptide effectively increased the mean survival time by 9.05 days, corresponding to an increase of 42.58%, and significantly delayed tumor growth from day 3 to 12 of treatment. In addition, tumor necrosis was significantly increased (P<0.05) in the treated mice. The overall survival rates obtained with surgical treatment at 6 months were 83.33% for group A, 40% for group B and 0% for group C, with significant differences (P< 0.05) among the groups.

Conclusions: The GK1 peptide demonstrated therapeutic properties in a mouse melanoma model, as treatment resulted in a significant increase in the mean survival time of the treated animals (42.58%). The potential for GK1 to be used as a primary or adjuvant component of chemotherapeutic cocktails for the treatment of experimental and human cancers remains to be determined, and surgical removal remains as a challenge for any new experimental treatment of melanoma in mouse models.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C240.

Citation Format: Jesus Vera Aguilera, Armando Perez Torres, Carlos Vera Aguilera, Diego Monterverde Suarez, Eduardo Moreno Aguilera, Juan Carlos Hernaiz Leonardo, Daniel Estrada Barcenas. The synthetic parasite-derived peptide GK1 increases survival in a preclinical mouse melanoma model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C240.