bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2998: Molecular and cellular effects of individual and combinatorial silencing of HSP90 paralogs
Authors: Powers Marissa V. , Te Poele Robert H. , Murphy Ravindhi L. , de Billy Emmanuel , Clarke Paul A. , Workman Paul
Published: July 2016

HSP90 is an ATP-dependent molecular chaperone critical for the folding, stability and function of over 350 client proteins, including many that are oncogenic. Pharmacologic inhibition of the essential ATPase domain inhibits HSP90 function and produces a well-documented molecular and cellular response comprising depletion of client proteins, induction of heat shock proteins and reduction of tumor cell proliferation. There are four paralogs within the HSP90 family, HSP90α, HSP90β, GRP94 and TRAP1, and it is not currently clear what precise role each one plays in the molecular and therapeutic response to HSP90 inhibitors. Here we use an siRNA approach to selectively reduce the expression of each HSP90 paralog, alone or in combination, in an attempt to dissect the individual roles of each chaperone in the molecular and cellular response associated with pharmacologic HSP90 inhibition. In the human cancer models used we found that by simultaneously silencing HSP90α and HSP90β we could phenocopy aspects of pharmacologic HSP90 inhibition including reduction of cell proliferation, induction of HSP72 and other HSF-1 target genes and depletion of oncogenic client proteins. Through analysing representative HSP90 client proteins we observed interesting patterns of paralog-dependence that were related to their overall sensitivity to pharmacologic HSP90 inhibition. Our results reveal the importance of HSP90 paralogs in particular aspects of the molecular and cellular response to pharmacologic HSP90 inhibition.

Citation Format: Marissa V. Powers, Robert H. Te Poele, Ravindhi L. Murphy, Emmanuel de Billy, Paul A. Clarke, Paul Workman. Molecular and cellular effects of individual and combinatorial silencing of HSP90 paralogs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2998.
Abstract 3: Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes
Authors: Sweeney Shannon R. , Sentandreu Enrique , Tiziani Stefano
Published: July 2016

Despite the growing body of evidence that the tumor microenvironment protects leukemia cells from chemotherapeutic stresses (1-3), the effect of many extracellular metabolites remains largely unknown. To explore the influence of extracellular metabolites on different leukemia subtypes, cells were treated for 24 hours in vitro with either a supplemental amino acid or amino acid derivative. From this initial screening, a subset of metabolites were chosen for metabolomics analysis. Mass spectrometry (UPLC-MS/MS) was performed on intracellular fractions to identify metabolic differences that resulted from supplementation. Metabolite profiles were also compared between leukemia cell types, namely AML, pre-B cell ALL, and T cell ALL. Of the metabolites tested, lysine and 4-hydroxyphenylpyruvate, an intermediate of tyrosine and phenylalanine metabolism, had the greatest impact on global amino acid profiles. In AML and T cell ALL cell lines, intracellular glutamate, glutamine, proline, and aspartate were increased relative to their respective controls. These amino acids can enter the tricarboxylic acid (TCA) cycle as either α-ketoglutarate or oxaloacetate, suggesting a central role of the TCA cycle in both AML and T cell ALL metabolism. Interestingly, these metabolites were not significantly increased in pre-B cell ALL, signifying the inverse it true for pre-B cells. This observation provides metabolomics evidence that is consistent with a previous study that reported downregulated expression of TCA cycle related genes in pre-B cell ALL (4). Our findings indicate that uptake and metabolism of amino acids and their derivatives is distinct for different leukemia types. Moreover, supplementation with a single metabolite can result in global changes in intracellular metabolite profiles, suggesting an influence not only as an energy substrate, but on overall metabolic pathway activity. Specifically, we conclude that the TCA cycle is more active in AML and T cell ALL and can be modulated by changing the extracellular environment, while pre-B cells are less sensitive to amino acid modulation.

(1) Meads MB, et al. Clin Cancer Res 2008;14(9):2519-2526.

(2) Ayala F, et al. Leukemia 2009;23:2233-2241.

(3) Konopleva M, et al. Drug Resist Updat 2009;12:103-113.

(4) Boag JM, et al. Leukemia 2006;20:1731-1737.

Citation Format: Shannon R. Sweeney, Enrique Sentandreu, Stefano Tiziani. Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3.
Abstract 30: Kras mediates amino acid metabolism during nutrient stress
Authors: Gwinn Dana , Sweet-Cordero Alejandro
Published: July 2016

Kras is a small GTPase that functions in the transmission of extracellular mitogenic stimuli, and is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Previously, Kras has been demonstrated to play a role in tumor metabolism in certain cell types and cancers, but this has yet to be analyzed in the context of NSCLC. We have analyzed how Kras changes metabolic gene expression by expressing a doxycycline-inducible hairpin against Kras in 4 NSCLC cell lines. One of the most regulated metabolism genes is asparagine synthetase (Asns), which transfers the γ-amino group of glutamine to aspartate, yielding glutamate and asparagine. We find elevated levels of Asns in Kras mutant tumors, and that expression of Asns correlates with poor patient outcome. Knockdown of Asns in the absence of exogenous asparagine in the media results in inhibition of cell growth and induction of apoptosis that is rescued by addition of asparagine. Additionally, under conditions of low glutamine levels, knockdown of Asns sensitizes cells to apoptosis, and overexpression of Asns is protective under these conditions. We also find a role for the glutaminase-activity of Asns in glutamine-mediated anapleurosis of the TCA cycle. These observations suggest Asns may be an interesting therapeutic target for NSCLC tumors with mutations in Kras.

Citation Format: Dana Gwinn, Alejandro Sweet-Cordero. Kras mediates amino acid metabolism during nutrient stress. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 30.
Abstract 3009: Targeting G-protein coupled receptor-related pathway in mucinous tumors
Authors: Dilly Ashok K. , Lee Yong J. , Zeh Herbert J. , Bartlett David L. , Choudry. Mohammad Haroon A.
Published: July 2016

Introduction: Pseudomyxoma peritonei (PMP) is an indolent malignancy characterized by excessive production of Mucin 2 (MUC2; a gel-forming secreted mucin). We have previously demonstrated mucinous tumor growth inhibition in a patient-derived murine xenograft model of PMP using the cyclooxygenase-2 (COX-2) inhibitor celecoxib. We evaluated the downstream mechanism for celecoxib-mediated MUC2-inhibition.

Methods: In vitro and in vivo studies were conducted using MUC2-secreting LS174T cells, PMP explant tissue and a unique intraperitoneal patient-derived murine xenograft model of PMP (PDX model of PMP).

Results: LS174T cells treated with COX-2 inhibitor celecoxib (5-20 μM) for 24 hours demonstrated a significant reduction in MUC2 protein expression measured by flow cytometric analysis. Similarly, in the PDX model of PMP chronic oral therapy with celecoxib (20 mg/kg/d) for 28 days significantly inhibited MUC2 protein expression in harvested tumor tissue. In the LS174T cells, PGE2 (a COX-2 product) induced MUC2 mRNA (real-time PCR) and protein expression (flow cytometry) and this was inhibited by the G-protein coupled-EP4 receptor inhibitor AH23848 as well as siRNA for CREB (cAMP response element-binding protein). Using chromatin immunoprecipitation assay in LS174T cells, we found that treatment with celecoxib or protein kinase A inhibitor (fragment 6-22 amide) significantly decreased CREB-transcription factor binding to the MUC2 promoter.

Conclusions: These data suggest that celecoxib inhibits MUC2 expression via the G-protein coupled receptor/ cAMP/ Protein kinase A/ CREB pathway. Since activating G-protein (GNAS) mutations are commonly found in PMP and may be responsible for its unique mucinous phenotype, our data provide a rationale for targeting this pathway to control mucin production in this malignancy.

Citation Format: Ashok K. Dilly, Yong J. Lee, Herbert J. Zeh, David L. Bartlett, Mohammad Haroon A. Choudry. Targeting G-protein coupled receptor-related pathway in mucinous tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3009.
Abstract 3014: ONC201 anti-cancer effects against solid tumors are mediated through eIF2α kinases HRI and PKR but are PERK-independent
Authors: Kline Christina Leah B. , El-Deiry Wafik S.
Published: July 2016

ONC201 is a first-in-class small molecule inducer of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. We recently reported that ONC201-mediated induction of the TRAIL pathway is largely promoted by a preceding early activation of the integrated stress response pathway (ISR) (Kline et al., Sci. Sig., in press, 2015). ONC201 engages the ISR to exert its broad-spectrum anti-cancer effects through pro-apoptotic and anti-proliferative activities. ONC201 upregulates ATF4, the key indicator of ISR activation, in a manner that is dependent on two eIF2α kinases: heme-regulated inhibitor (HRI) and double-stranded RNA activated protein kinase (PKR). Of note, the activation of ATF4 and downstream cell death signaling by ONC201 is PERK-independent. We now further elucidate how the novel dual engagement of HRI and PKR by ONC201 contributes to the anti-cancer effects of ONC201. ONC201 treatment results in early phosphorylation of eIF2α in a manner that is dependent on HRI. On the other hand, we have shown that ONC201-induced downregulation of cyclin D1 is dependent, at least in part, on PKR, but not on eIF2 α phosphorylation per se. This occurs potentially via PKR-mediated ubiquitination and subsequent proteasome degradation of cyclin D1. Treating cells with ONC201 and the proteasome inhibitor MG132 prevents ONC201-induced decrease of cyclin D1. Despite distinctions in their regulatory protein domains, HRI and PKR have both been shown to respond to oxidative stress. Treating cells with ONC201 in the presence of the antioxidant N-acetylcysteine abrogated ATF4 induction. Our findings document a unique activity of ONC201 on protein translation, via activation of HRI and PKR, as well as effects on protein stability through the proteasome leading to anti-cancer effects.

Citation Format: Christina Leah B. Kline, Wafik S. El-Deiry. ONC201 anti-cancer effects against solid tumors are mediated through eIF2α kinases HRI and PKR but are PERK-independent. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3014.
Abstract 303: Compensatory up-regulation of NEK9 contributes to resistance to PLK1 inhibition in triple-negative breast cancer cells
Authors: Wong Nelson K. Y. , Mitchell Devon , Khan Mohamed K.
Published: July 2016

Patients with triple-negative breast cancers (TNBCs) have very poor prognosis in terms of relapse and overall survival. There is currently no targeted therapy for this heterogeneous group of patients and it presents a pressing unmet medical need. Recent data suggest that Polo-like kinase 1 (PLK1) is a plausible target for treating TNBCs, and many small-molecule inhibitors are currently under development for targeting this kinase. Since clinical data have shown that single-agent treatment often leads to resistance and relapse of the cancer, it is essential to identify potential resistance mechanisms of TNBCs to PLK1 inhibition such that combination therapy can be rationally designed to increase treatment efficacy.

We have selected ten individual cell clones from a TNBC cell line, MDA-MB-231, through long-term incubation of the cells with increasing concentrations of a PLK1 inhibitor BI 2536. We hypothesized that increased expression and/or activities of other kinases can compensate for the PLK1 inhibition. To identify these kinases, we screened the expression levels of >500 human kinases in the resistant clones in comparison to the parental cells, using the Nanostring platform, and generated a list of candidate kinases. Among the candidates, NEK9, a downstream effector of PLK1, was confirmed to be over-expressed in all resistant cell clones through Western blotting. MDA-MB-231 parental cells were then infected with a lentiviral over-expression vector that contains NEK9 and GFP. When a mixed population of NEK9-over-expressing cells (GFP+) and parental cells (GFP-) were incubated in the presence of BI 2536, increased proportion of GFP+ cells and increased mean GFP fluorescence intensity was observed over time through flow cytometry. Increase of NEK9 expression was also confirmed with Western blotting in the mixed cell culture experiment, indicating the selection of the NEK9-over-expressing cells in the presence of BI 2536.

In conclusion, we have constructed a list of over-expressed kinase candidates that may contribute to the resistance to PLK1 inhibition. We further identify that over-expression of NEK9 contributes to the resistance phenotype. Our data prompt the need to further study the biology around NEK9 and suggest the possibility of combination therapy through inhibition of PLK1 and NEK9 in treating TNBCs.

Citation Format: Nelson K. Y. Wong, Devon Mitchell, Mohamed K. Khan. Compensatory up-regulation of NEK9 contributes to resistance to PLK1 inhibition in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 303.
Abstract 3030: CT179: A novel small molecule Olig2 inhibitor for the treatment of glioblastoma
Authors: Alton Gordon , Beaton Graham , Knowles Susan , Stein Greg , Mukthavaam Rajesh , Kesari Santosh
Published: July 2016

Glioblastoma (GBM) is an orphan disease with a high unmet medical need. Despite optimal treatment, the median survival is only 12 to 15 months in GBM patients of which only 4% achieve 5-year survival. OLIG2 is a transcription factor that is almost exclusively expressed in the CNS that has been linked to the tumorigenesis of GBM cells and their resistance to radiotherapy. Importantly OLIG2 is a key transcription factor that maintains the stemness of the glial stem cells, drives proliferation and invasion and recurrent tumor growth. Inhibitors of this pathway are therefore of interest as potential treatments for this unmet need.

CT-179 is a small molecule inhibitor that was initially designed to prevent homodimerization of Olig2. CT-179 is highly cytotoxic to OLIG2 expressing GBM cells with a high therapeutic index for non-OLIG2 expressing cell types. This inhibitor demonstrates functional inhibition of the OLIG2 pathway through specific reduction in nuclear Olig2 protein levels in treated cells. A cell pathway analysis indicates that CT-179 impacts GBM proliferation through cell cycle arrest and induction of apoptosis. Secondarily an immunosuppression mechanism-of-action may also be relevant. CT-179 penetrates the CNS to attain high brain concentrations at levels far in excess of its cytotoxicity to GBM cells. Efficacy studies reveal dose dependent reductions of tumor growth which is enhanced with radiotherapy. CT-179 demonstrates impressive extension of survival in orthotopic human PDX GBM. Safety studies indicate that there is a large therapeutic window. The compound has a long half-life, low metabolism and has excellent formulation properties and PK supporting once per day oral dosing. CT-179 show promise as both a cytotoxic agent and as potential radiosensitizer for use in the treatment of GBM.

Citation Format: Gordon Alton, Graham Beaton, Susan Knowles, Greg Stein, Rajesh Mukthavaam, Santosh Kesari. CT179: A novel small molecule Olig2 inhibitor for the treatment of glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3030.
Abstract 3039: Lurbinectedin specifically targets transcription in cancer cells, triggering DNA breaks and degradation of phosphorylated Pol II
Authors: Santamaria-Nunez Gema , Genes-Robles Carlos M. , Martínez-Leal Juan F. , Galmarini Carlos M. , Egly Jean Marc
Published: July 2016

Cancer cells are characterized for their avid demand for active transcription, which reaches the level of a real addiction in solid tumors as small cell lung cancer (SCLC) or triple-negative breast cancer. Pharmacological modulation of transcription may thus provide a therapeutic approach to treat tumor types that depend on deregulated transcription for the maintenance of their oncogenic state. Lurbinectedin, currently under evaluation in a Phase III clinical trial for platinum-resistant ovarian cancer patients, and with very promising activity in combination with doxorubicin in SCLC, inhibits active transcription. Here we demonstrate that, after binding to specific DNA triplets highly represented in the CG-rich region surrounding the promoter of genes, this drug induces a rapid degradation of RNA Polymerase II (Pol II). Our results show that the hyperphosphorylated form of Pol II, already engaged in transcription elongation and likely blocked during this process by the lurbinectedin-DNA adduct, is then specifically submitted to the ubiquitin/proteasome degradation process, which finally removes the majority of the Pol II protein pool in treated cells. Disappearance of Pol II is followed by the formation of DNA breaks, process in which the nucleotide excision repair (NER) machinery, specifically the endonuclease XPF, has an important role. Pol II degradation and subsequent DNA damage were not only abrogated by inhibitors of CDK7 and CDK9 cyclin dependent kinases (DRB and flavopyridol), ubiquitin ligation (PYR-41), or proteasome activity (MG132), but also correlated with the antiproliferative activity of lurbinectedin in different cancer cell line models. In summary, lurbinectedin exemplifies a prototype drug for targeting transcriptional dependency in tumor cells and, thus, it could represent a new therapeutic alternative for solid tumors with this addiction.

Citation Format: Gema Santamaria-Nunez, Carlos M. Genes-Robles, Juan F. Martínez-Leal, Carlos M. Galmarini, Jean Marc Egly. Lurbinectedin specifically targets transcription in cancer cells, triggering DNA breaks and degradation of phosphorylated Pol II. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3039.
Abstract 3049: Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis
Authors: Chung Eun Joo , Valle Luca F. , White Ayla O. , Citrin Deborah E.
Published: July 2016

Introduction: Ionizing radiation (IR) is commonly used in the treatment of thoracic malignancies. Exposure of adjacent normal lung may result in lung injury and fibrosis. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in parynchymal depletion and precedes pulmonary fibrosis. Arachidonate 12-lipoxygenase (12-LOX) oxidizes arachidonic acid to form 12-Hydroxyeicosatetraeonic acid (12-HETE), a pro-inflammatory mediator. Increased expression of ALOX-12 has previously been associated with aging. We hypothesized that mice deficient in ALOX-12 would be resistant to IR induced fibrosis and senescence.

Methods: C57/Bl6J (WT) and Alox12 homozygous knockout (Alox12-KO) mice (n>3 per group) were exposed to thoracic IR (0Gy, 5Gy, 17.5Gy, 5×5Gy, or 6×5Gy). Fibrotic foci were identified with aniline blue staining of fixed lung sections. Levels of 12-LOX mRNA and protein were assessed in WT lungs after IR with microarray, quantitative PCR, and western blotting. In vitro studies with primary murine pneumocyte cultures and A549 cells included staining for senescence associated β-galactosidase activity and western blotting for NOX4, p21, and PML following IR and 12-HETE treatment.

Results: A significant increase in 12-LOX mRNA and protein was observed in murine lungs exposed to fibrogenic doses of IR (17.5 Gy, 5×5 Gy and 5×6 Gy) compared to low dose IR (5 Gy) or controls (0 Gy). 12-LOX deficiency significantly reduced fibrotic foci in murine lungs receiving fibrogenic doses of thoracic IR (6×5 Gy) at 18 weeks. Treatment of murine primary pneumocytes with 12-HETE (1 and 150 nM) increased the rate of pneumocyte senescence (2.1 fold). Treatment of murine primary pneumocytes with 12-HETE increased the expression of NOX4 (a mediator of superoxide generation), p21, and PML (senescence markers), paralleling increases observed after IR. Similarly, human type II pneumocyte A549 cells exhibited increased 12-LOX expression in β-galactosidase-stained senescent cells at 3 days after IR.

Conclusion: These studies identify 12-LOX as a critical mediator in radiation lung fibrosis and type II pneumocyte senescence. 12-LOX may serve as a novel therapeutic target in mitigating IR-induced lung fibrosis.

Citation Format: Eun Joo Chung, Luca F. Valle, Ayla O. White, Deborah E. Citrin. Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3049.
Abstract 3054: CD47 signaling regulates a DNA damage response pathway by suppressing the expression of Schlafen-11 (SLFN11)
Authors: Schwartz Anthony L. , Kaur Sukhbir , Tang Sai-Wen , Pommier Yves , Roberts David D.
Published: July 2016

Schlafen-11 (SLFN11) expression was recently shown to determine the sensitivity of human cancer cells to DNA damaging agents including topoisomerase inhibitors, DNA alkylating agents, and DNA synthesis inhibitors. High expression of SLFN11 in cancer cell lines results in increased sensitivity to these agents, whereas depletion of SLFN11 induces cellular protection. In addition, high expression of SHFN11 is associated with greater overall survival than low SLFN11 expression in ovarian patients treated with cisplatin. These observations suggest that SLFN11 may be an integral part of a critical DNA repair pathway. However, the pathways that regulate SLFN11 expression have not been reported. CD47 is an integrin-associated protein widely expressed on mammalian cells and frequently elevated in cancers. Its ligand, thrombospondin-1 (TSP1) induces CD47 signaling that regulates some growth factor receptors, cell fate, viability, and responses to cellular stresses such as radiation and chemotherapy. Our laboratory has previously shown that CD47 deficiency results in protection of nontransformed cells, whereas high CD47 expression sensitizes the same cells to ionizing radiation and DNA-damaging chemotherapy. We identified SLFN11 expression as a potential target of CD47 signaling by microarray analysis of a CD47-deficient mutant cell line and found that CD47 expression also correlates with that of SLFN11 in some human cancers. Thus, we sought to investigate the possible regulation of SLFN11 by CD47 by treating various cell lines with the function-modifying CD47 antibody B6H12 or its physiological ligands TSP1 and signal regulatory protein-α (SIRPα). qRT-PCR analysis revealed significant decreases in SLFN11 expression at 3 and 6 hours post-B6H12 treatment. Furthermore, treatment with TSP1 significantly decreased SHFN11 mRNA expression at the same time points, but SIRPα-Fc did not. Conversely, transient re-expression of CD47 in deficient cells elevated SLFN11 expression. These data demonstrate that pharmacological and physiological modulation of CD47 signaling acutely alters SLFN11 expression and suggests a potential mechanism by which CD47 modulates cellular sensitivity to DNA damaging chemotherapy agents. SLFN11 may in turn be a functional target and useful biomarker for the therapeutic CD47 antibodies currently in clinical trials.

Citation Format: Anthony L. Schwartz, Sukhbir Kaur, Sai-Wen Tang, Yves Pommier, David D. Roberts. CD47 signaling regulates a DNA damage response pathway by suppressing the expression of Schlafen-11 (SLFN11). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3054.
Abstract 3057: Synthetic novel Psammaplin A derivatives enhance radiation lethality in human cancer cells
Authors: Kim Jin Ho , Wee Chan Woo , Kim Hak Jae , Suh Soo Youn , Ma Eun Sook , Shin Boom Soo , Kim Il Han
Published: July 2016

DNA methyltransferase (DNMT) inhibitors show not only anticancer effects but radiosensitization activity. Psammaplin A (PsA), a known DNMT inhibitor, enhances radiation lethality of human cancer cells, but the chemical instability hampers in vivo application of PsA. The purpose of the present study was to synthesize novel radiosensitizers using PsA as the backbone structure. Eight novel PsA-based derivatives (MA2, MA3, MA5, MA6, MA7, MA8, MA9 and MA5M) were synthesized. They were tested for in vitro cytotoxicity and radiosensitizing effects in A549 (lung cancer) and U373MG (glioblastoma) cells using a clonogenic assay. A paired ratio t-test was used to test a statistical significance of sensitizer enhancement ratios of the compounds. All 8 PsA derivatives inhibited in vitro cell survival with 50% inhibitory concentrations ranging 16-150 μM (A549) and 15-50 μM (U373MG). MA7, MA9 and MA5M significantly enhanced radiation lethality in A549 cells, while compounds MA2, MA3 and MA7 showed a significant radiosensitization in U373MG cells (p<0.05). MA3 and MA6 marginally radiosensitized A549 and U373MG cells, respectively (p<0.10). For further investigation, we selected MA3 and MA7, because only these two compounds showed radiosensitization in both A549 and U373MG cell lines. A DNMT activity assay showed that MA3 inhibited DNMT1 activity, but that MA7 showed no inhibitory effect on DNMT1. Pharmacokinetic parameters were determined in mice by a validated LC-MS/MS assay method using a non-compartmental analysis. The average degradation half-life of MA3 and MA7 was under 10 minutes in mouse serum. In the present study, we synthesized two novel PsA-derived compounds that enhanced radiation lethality in two human cancer cell lines. However, they lacked chemical stability to allow for in vivo experiments. Based on the present study, a further study is necessary to improve bioavailability of PsA-derived radiosensitizers. [Supported by 2013M2A2A7043683]

Citation Format: Jin Ho Kim, Chan Woo Wee, Hak Jae Kim, Soo Youn Suh, Eun Sook Ma, Boom Soo Shin, Il Han Kim. Synthetic novel Psammaplin A derivatives enhance radiation lethality in human cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3057.
Abstract 3059: Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry
Authors: Warder Scott E. , McLoughlin Shaun M. , Hansen T. Matthew , Richardson Paul L. , Wilcox Denise M. , Addo Sadiya N. , Tang Hua , Sun Chaohong , Petros Andrew M. , Panchal Sanjay C. , Park Chang H. , Duggan M. Shannon , Patterson Melanie J. , Buchanan F. Greg , Cheng Dong , Davis Heather M. , Calderwood David J. , Elmore Steven W. , Shen Yu
Published: July 2016

As part of a multi-year technology integration strategy to identify unprecedented targets, AbbVie has committed to building broad-endpoint profiling assays to enable phenotypic screening campaigns and compound prioritization. Early on, phenotypic cell-based screening employing a panel of protein-fragment complementation assays (PCAs) identified A-1107604 as a hit with potent activity against a subset of endpoints. While its activity profile had some commonalities with known anti-cancer agents, the overall profile of PCAs that were significantly and concomitantly modulated represented a unique signature. Further analysis revealed A-1107604 to have potent and selective activity in a panel of human tumor cell lines. Inhibitor affinity capture from cellular lysates coupled with mass spectrometry identified the BET family of proteins as the putative cellular targets of A-1107604. Binding was localized to the bromodomain of the target proteins using affinity capture-protease digestion and was confirmed by thermal shift assay, solution

binding and X-ray crystallography. This binding was found to be highly selective when A-1107604 was counter-screened against a 150-member kinase panel and an 80-member receptor panel. To correlate target affinity with cellular efficacy, a series of analogs were prepared with affinities spanning 3 orders magnitude. Affinity for BRD4, a BET family member, strongly correlated with efficacy in human tumor cell lines. Finally, A-1107604 was evaluated in human tumor xenograft models where it demonstrated significant tumor growth inhibition. This discovery effort laid the foundation for our BET inhibitor program. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the

publication.

Citation Format: Scott E. Warder, Shaun M. McLoughlin, T. Matthew Hansen, Paul L. Richardson, Denise M. Wilcox, Sadiya N. Addo, Hua Tang, Chaohong Sun, Andrew M. Petros, Sanjay C. Panchal, Chang H. Park, M. Shannon Duggan, Melanie J. Patterson, F. Greg Buchanan, Dong Cheng, Heather M. Davis, David J. Calderwood, Steven W. Elmore, Yu Shen. Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3059.
Abstract 306: Analysis of differentially expressed exosomal genes from ibrutinib resistant diffuse large B cell lymphoma
Authors: Jain Neeraj , Sehgal Lalit , Khashab Tamer , Samaniego Felipe
Published: July 2016

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Selective pressure provided by chemotherapy causes aberrant gene expression and selection of pre-existing population of chemo-resistant cells. These chemo-resistant cells can also transmit chemo-resistance to chemo-sensitive cells through exosomal transfer of molecules (miRNA, mRNA and proteins). In this study, we have analysed differentially expressed mRNA and miRNA from syngeneic ibrutinib (an irreversible BTK inhibitor, FDA approved) resistant DLBCL cells and from their isolated exosomes. Five DLBCL-GCB and five ABC subtype were analysed for their sensitivity with ibrutinib. We found three ABC subtype and one GCB subtype (OCL-LY1) were sensitive to ibrutinib. We have generated syngeneic ibrutinib drug resistant model from sensitive cells (at least 10 fold increase in IC50 value) by continuous culturing them in the presence of ibrutinib with gradual increase in drug concentration in each passage. Ibrutinib sensitive DLBCL cells when cultured with the syngeneic resistant cells in transwell apparatus, acquired resistance to ibrutinib. We also analysed the sensitivity of ibrutinib resistant cells with other conventional drugs for non-Hodgkin lymphomas. We further tested the expression of drug resistant genes in syngeneic resistant cells by real time PCR analysis. Later, we isolated the total RNA and miRNA species from DLBCL ibrutinib sensitive, their syngeneic resistant cells and from respective exosomes and analysed for differential expression by micro array analyses. Thus in conclusion, our study demonstrates that chemo-resistance can be transferred from resistant cells to sensitive cells via exosomal horizontal transmission of miRNA and mRNA in diffuse large B cell lymphoma cells. Moreover, our study identified potential targets that can be used to overcome chemo-resistance in diffuse large B cell lymphoma.

Citation Format: Neeraj Jain, Lalit Sehgal, Tamer Khashab, Felipe Samaniego. Analysis of differentially expressed exosomal genes from ibrutinib resistant diffuse large B cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 306.
Abstract 307: GPR120/FFAR4 activation by fatty acid 16:4(n-3) plays a key role in resistance to chemotherapy
Authors: Houthuijzen Julia , Oosterom Ilse , Hudson Brian , Hirasawa Akira , Daenen Laura , McLean Chelsea , Hansen Steffen , van Jaarsveld Marijn , Peeper Daniel , Jafari Sadatmand sahar , Roodhart Jeanine , van de Lest Chris , Ulven Trond , Ishihara Kenji , Milligan Graeme , Voest Emile
Published: July 2016

Although chemotherapy is designed to eradicate tumor cells it also has a significant impact on normal tissues. These host-responses can have large effects on the efficacy of treatment and overall survival. Fatty acids are increasingly recognized to play important signaling roles. 12-S-HHT and 16:4(n-3) are two platinum-induced fatty acids (PIFAs) that induce systemic resistance to a broad range of DNA-damaging chemotherapeutics. PIFAs exert their chemoprotective effect on tumor cells via an indirect mechanism involving splenic F4/80+/CD11blow macrophages. Here we identified GPR120 on mouse and human splenic macrophages as the relevant receptor for 16:4(n-3). GPR120 (FFAR4) is a free fatty acid receptor that binds medium- to long-chain fatty acids and omega-3 fatty acids and is involved in anti-inflammatory response and metabolic control. Although both GPR40 (FFAR1) and GPR120 can be activated by 16:4(n-3) in vitro, only inhibition or genetic loss of GPR120 was able to block 16:4(n-3)-mediated chemoresistance in vivo. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in splenic macrophages resulting in the production and secretion of resistance-inducing lysophosphatidylcholine 24:1 (LPC(24:1)). Taken together, we identified a novel function for GPR120. Activation by 16:4(n-3) leads to enhanced cPLA2 activity and production of LPC(24:1) resulting in chemotherapy resistance.

Citation Format: Julia Houthuijzen, Ilse Oosterom, Brian Hudson, Akira Hirasawa, Laura Daenen, Chelsea McLean, Steffen Hansen, Marijn van Jaarsveld, Daniel Peeper, sahar Jafari Sadatmand, Jeanine Roodhart, Chris van de Lest, Trond Ulven, Kenji Ishihara, Graeme Milligan, Emile Voest. GPR120/FFAR4 activation by fatty acid 16:4(n-3) plays a key role in resistance to chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 307.
Abstract 3071: Proteasome subunit composition contributes to unusually poor sensitivity of H727 cells to carfilzomib
Authors: Lee Min Jae , Kim Kyung Bo
Published: July 2016

Background: While proteasome inhibitors (PIs) have significantly improved the survival of multiple myeloma (MM) patients, significant subsets of MM patients do not respond to the PI- based therapy. Currently, mechanisms of de novo PI resistance in these patients remain completely unknown. In this study, we aim to identify previously unexplored mechanisms of de novo carfilzomib (Cfz) resistance using H727 cancer cells with poor sensitivity to carfilzomib.

Results: Our carfilzomib sensitivity investigation in a panel of 16 cancer cell lines revealed that H727 lung cancer cells are markedly resistant to Cfz compared to other cell lines. H727 cells displayed no detectable P-glycoprotein (P-gp) expression and subsequently were not sensitized by reversin 121 (a P-gp inhibitor) to Cfz, ruling out the involvement of P-gp in the resistance of H727 cells to Cfz. Interestingly, we found that H727 cells are killed by PYR-41 (ubiquitin activating enzyme E1 inhibitor) and WP-1130 (deubiquitinase inhibitor) as effectively as Cfz- sensitive H23 cells, suggesting that ubiquitin-proteasome pathway is essential in H727 cells as in H23 cells. In addition, knock-down of proteasome α7 subunit, a component required for the assembly of a proteasome α-ring and thereby a 20S proteasome complex, resulted in an almost complete cell death, indicating that the proteasome is indispensable for the survival of H727 cells. Intriguingly, despite their poor sensitivity to Cfz, H727 cells were highly sensitive to other structurally distinguished PIs such as bortezomib, epoxomicin, and UK-101. Finally, exposure of H727 cells to interferon-γ (IFN-γ), which has been widely used as a biochemical tool to switch cellular proteasome catalytic subunit composition to the one containing immunoproteasome subunits, sensitized H727 cells to Cfz (Changing IC50 values from 1,355 nM to 185 nM). On the contrary, IFN-γ pretreatment did not affect the sensitivity of H23 cells to Cfz.

Conclusion: We report that H727 cells have a poor sensitivity to carfilzomib (or de novo resistance to Cfz), and that previously reported resistance mechanism involving P-gp upregulation does not play a role. Given our data presented here, we conclude that de novo Cfz resistance of H727 cells may be attributed to distinguished proteasome subunit composition. We believe that our investigation using cancer cell line models may provide important insights into understanding the mechanisms of de novo PI resistance in clinic.

Citation Format: Min Jae Lee, Kyung Bo Kim. Proteasome subunit composition contributes to unusually poor sensitivity of H727 cells to carfilzomib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3071.
Abstract 3077: Potent inhibition of bromodomain-containing BET family with ABBV-075 induces robust antitumor efficacy in preclinical models of breast cancer and exhibits in vitro synergy with doxorubicin
Authors: Faivre Emily J. , Lin Xiaoyu , Wilcox Denise M. , Huang Xiaoli , Sarthy Aparna , Magoc Terry , Albert Daniel H. , Fang Guowei , Rosenberg Saul H. , McDaniel Keith F. , Kati Warren M. , Shen Yu
Published: July 2016

Breast cancer diagnosisis highly prevalent in the US, with an estimated 231,840 new cases occurring in 2015. Targeted therapies, such as tamoxifen or Trastuzumab (herceptin) are available to woman whose tumors are estrogen receptor (ER) positive or HER2 positive, respectively. However, women with triple negative breast cancer or relapsed metastatic disease have limited treatment options. ABBV-075 is a novel BET family bromodomain inhibitor currently under phase I clinical investigation for a wide spectrum of cancer indications. Here we show that ABBV-075 exhibits broad anti-proliferative activity across cell lines representing ER positive, HER2+, and triple negative breast cancer. Notably, ABBV-075 induced G1 arrest and growth inhibition of breast cancer cell lines regardless of ER or RB status. This result suggests that ABBV-075 may provide therapeutic benefit to a broader patient population relative to the recently approved cdk4/6 inhibitor, palbociclib. The G1 arrest mechanism of ABBV-075 was efficacious in breast cancer xenograft studies, where significant tumor growth inhibition was observed. Additionally, ABBV-075 exhibited synergy in ER positive cell lines with doxorubicin, a TOPO2 inhibitor and DNA intercalating agent prescribed to ER positive patients refractory to hormone therapies. Intriguingly, the mechanism by which ABBV-075 and doxorubicin interact was independent of the TOP2-trapping activity of doxorubicin and instead required DNA intercalation. Together, these results demonstrate the potential of ABBV-075 as a treatment option across the different subtypes of breast cancer and in combination with doxorubicin in patients with ER positive disease

Citation Format: Emily J. Faivre, Xiaoyu Lin, Denise M. Wilcox, Xiaoli Huang, Aparna Sarthy, Terry Magoc, Daniel H. Albert, Guowei Fang, Saul H. Rosenberg, Keith F. McDaniel, Warren M. Kati, Yu Shen. Potent inhibition of bromodomain-containing BET family with ABBV-075 induces robust antitumor efficacy in preclinical models of breast cancer and exhibits in vitro synergy with doxorubicin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3077.
Abstract 3078: GSK-3 inhibitor 9ING41 potentiates the antitumor effects of CPT-11 in human breast cancer
Authors: Ugolkov Andrey , Gaisina Irina , White Kevin , Kozikowski Alan , O’Halloran Thomas , Mazar Andrew
Published: July 2016

Glycogen Synthase Kinase-3beta (GSK-3beta), a serine/threonine protein kinase, has been demonstrated to be potential therapeutic target in human breast cancer. Our objective was to evaluate novel GSK-3 inhibitors alone and in combination with chemotherapeutic drugs for the targeted therapy of breast cancer. Using a short-term treatment, we found that the GSK-3 inhibitor 9ING41 significantly potentiates the antitumor effects of CPT-11, cisplatin and 5-FU to inhibit breast cancer cell growth. We established hormone-refractory BC-1 and BC-2 patient-derived xenograft (PDX) tumor models from metastatic pleural effusions that were obtained from breast cancer patients. We considered BC-1 and BC-2 PDX tumors as chemoresistant, because metastatic disease progressed in both cases in despite of the fact that patients received many cycles of chemotherapy. Although we found that BC-1 and BC-2 PDX tumors express estrogen receptor (ER), both PDX tumors carried activating mutations in ESR1. These mutations confer ligand-independent activity of the ER in tamoxifen-resistant breast tumors. We found that BC-1 and BC-2 PDX tumors grew without estradiol supplementation in immunodeficient mice, which suggests a hormone-refractory nature of these tumors. We found that GSK-3 inhibitor 9ING41 potentiates the effects of chemotherapeutic drug CPT-11, and leads to the regression of BC-1 and BC-2 breast PDX tumors. Our results demonstrate inhibition of GSK-3 as a promising therapeutic approach to overcome breast cancer chemoresistance, and identify GSK-3 inhibitor 9ING41 as a drug candidate for the targeted therapy of human breast cancer.

Citation Format: Andrey Ugolkov, Irina Gaisina, Kevin White, Alan Kozikowski, Thomas O’Halloran, Andrew Mazar. GSK-3 inhibitor 9ING41 potentiates the antitumor effects of CPT-11 in human breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3078.
Abstract 3094: Identification of mimotopes of the epithelial mucin-1 (MUC1) in non-small cell lung cancer
Authors: Qiu Yu , Jianfei Wang , Ruoqiong Cao , Yi Zhang , Yangyang Sun , Zhong Li
Published: July 2016

Background

Mimotope analysis has been widely used in mapping epitopes for drug target, and also used in the development of new diagnostics, therapeutics and vaccines. The epithelial mucin-1 (MUC1) is a glycoprotein in human endometrium and has been found highly expressed in tumor tissues of NSCLC patients. Mimotopes of MUC1 has great potentials to be immunotherapy targets for NSCLC. In this study, we use phage display technology to screen a T7 NSCLC phage library to identify mimotope peptides of MUC1 proteins.

Method

A T7 NSCLC phage library was screened by 3-round of biopanning with anti-MUC1 monoclonal antibody acting as a molecular target. Forty positive clones were picked for ELISA confirmation using anti-MUC1 antibody. In addition, these clones were then amplified and coated with ELISA plates. Forty NSCLC patient and 40 control serum samples were used to test the ELISA plates. Statistical analysis was preformed, and the most significant clones were picked and PCR amplified for sequencing.

Result

Two peptide sequences, AAPDFRP and SAPDDRP, were identified and the competitive inhibitory rates of MUC1 were more than 50%. The intensity between MUC1 monoclonal antibody and the positive clones of these two sequences had positive correlation. In addition, in serum binding specificity detection, these two amino acid sequences showed higher tumor binding specificity with lung cancer patients serum than that of healthy human serum(P<0.05).

Conclusion

Peptides of AAPDFRP and SAPDDRP showed a high homology in the mimic epitopes of the epithelial mucin-1 (MUC1) glycoprotein in NSCLC. These may be potential immunotherapy targets which could also act as candidates of tumor vaccines

Citation Format: Yu Qiu, Wang Jianfei, Cao Ruoqiong, Zhang Yi, Sun Yangyang, Li Zhong. Identification of mimotopes of the epithelial mucin-1 (MUC1) in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3094.