bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-042: Xenopatients help in redefining medical therapeutic algorithms in high risk ovarian cancer
Authors: Erriquez Jessica , Olivero Martina , Mittica Gloria , Scalzo Maria Stella , Vaira Marco , De Simone Michele , Ponzone Riccardo , Katsaros Dionyssios , Aglietta Massimo , Calogero Raffaele , Di Renzo Maria Flavia , Valabrega Giorgio
Published: July 2016

Platinum-based chemotherapy is the recommended first-line treatment for high-grade serous (HGS) epithelial ovarian cancer (EOC). However, most patients relapse because of platinum refractory/resistant disease. We aimed at assessing whether other drugs, commonly used to treat relapsed HGS-EOC and poorly active in this clinical setting, might be more effective against chemotherapy-naïve cancers. We collected samples of advanced HGS-EOC from the same patients before and after neo-adjuvant platinum-based chemotherapy. Samples were propagated as Patient Derived Xenografts (PDXs) in immunocompromised mice (“xenopatients”). Xenopatients were treated with carboplatin, gemcitabine, pegylated liposomal doxorubicin (PLD) and trabectedin. One patient was studied who experienced clinically platinum-sensitivity first and then discordant responses of different tumor sites to platinum re-challenge. PDXs derived from this patient before chemotherapy showed responsiveness to carboplatin, trabectedin and gemcitabine. The PDXs from the same patient after chemotherapy did no longer respond to trabectedin and gemcitabine and showed a heterogeneous response to carboplatin. Expression profiling showed that loss of responsiveness to drugs of the post-chemotherapy PDXs was associated with the up-regulation of NR2F2 gene expression. A second patient with platinum refractory HGS-EOC responded poorly to PLD as second-line treatment. PDXs obtained from this patient's tumor before chemotherapy showed a complete response to PLD, which was lost in the post-chemotherapy PDXs. Response to PLD was associated with the over-expression of the TOP2A protein, which was lost in the post-chemotherapy PDXs. Thus, PDXs demonstrated that chemotherapy-naïve HGS-EOC might display susceptibility to agents not used commonly as first line treatment. These data suggest the importance of tailoring chemotherapy.

Citation Format: Jessica Erriquez, Martina Olivero, Gloria Mittica, Maria Stella Scalzo, Marco Vaira, Michele De Simone, Riccardo Ponzone, Dionyssios Katsaros, Massimo Aglietta, Raffaele Calogero, Maria Flavia Di Renzo, Giorgio Valabrega. Xenopatients help in redefining medical therapeutic algorithms in high risk ovarian 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 LB-042.
Abstract LB-058: Linker variation and structure-activity relationship analyses of salicylic, benzoic and hydroxamic acid-based small molecule Stat3 inhibitors
Authors: Yue Peibin , Lopez-Tapia Francisco , Brotherton-Pleiss Christine , Tius Marcus , Turkson James
Published: July 2016

Benzoic acid, salicylic acid and hydroxamic acid-based small molecules have been reported that inhibit Stat3 activation. However, the structural basis for their Stat3-inhibitory activities are not clearly defined. To investigate the molecular determinants for activity and to derive structurally optimized analogs of improved pharmacokinetic parameters and potency, we conducted a computer-aided molecule optimization program and synthesized new analogs of the previously reported lead Stat3 inhibitors, BP-1-102 (IC50, 6.8 ± 2.5), SH4-54 (IC50 4.4 ± 0.3), and SH5-07 (IC50 3.9 ± 0.6). All three leads are based on N-methylglycinamide scaffold with its two amine moieties condensed with three different functionalities. The three functionalities or the CH2 group of the glycolamide scaffold were separately modified to generate a series of compounds, some of which show improved potency compared to the corresponding parental compounds. Specifically, H012, H070 and H072 had improved inhibitory activity against Stat3 DNA-binding activity in vitro, with IC50 of 3.0 ± 0.9, 2.4 ± 0.4, and 2.2 ± 0.9 μM, respectively. The replacement of the pentafluorobenzene or the cyclohexylbenzene with other components was detrimental to activity and led to decreased potency of the new analogues, as did the substitution of the benzene of the pTyr-mimetic moiety with heterocyclic components containing nitrogen and oxygen elements. Pharmacokinetic study showed that analog H058 has improved permeability in Caco-2 bidirectional permeability studies, compared to the lower relative permeability of the lead agents. Ala-based analogs, such as H048 and H058, were low in microsomal stability, presumably due to the labile methyl groups of the Ala. Select active analogs inhibited constitutive Stat3 activation and the DNA-binding activity of Stat3 in cancer cells, and they further decreased the growth, colony formation, and migration of human cancer cells harboring persistently active Stat3. Studies together demonstrate a significant progress towards developing a more suitable Stat3 inhibitor with drug-like features. Additional efforts are focused on refining the physicochemical properties to further improve the activity and efficacy and ultimately furnish suitable Stat3-inhibiting candidates for clinical application.

Citation Format: Peibin Yue, Francisco Lopez-Tapia, Christine Brotherton-Pleiss, Marcus Tius, James Turkson. Linker variation and structure-activity relationship analyses of salicylic, benzoic and hydroxamic acid-based small molecule Stat3 inhibitors. [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 LB-058.
Abstract LB-062: Evaluation of drug conjugation sites in an anti-EGFR Centyrin
Authors: Cardoso Rosa , Goldberg Shalom , Lin Tricia , Spinka-Doms Tracy , Klein Donna , Jacobs Steven , Gilliland Gary , O’Neil Karyn
Published: July 2016

Centyrins are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with increased tissue penetration and simplified manufacturing. To identify optimal positions for chemical conjugation within the Centyrin scaffold, the crystal structure of an anti-EGFR Centyrin was solved and a library consisting of all possible single-cysteine variants was constructed and assessed for expression level in E. coli, efficiency of conjugation to maleimide derivatives, protein aggregation, thermostability, and EGFR binding. The best variants were conjugated to cytotoxic drugs and compared in a cell-killing assay. Three optimal positions were identified at which the Centyrin conjugated efficiently to maleimides; retained high expression, antigen binding, and biophysical properties; and showed increased cell-killing potency relative to the parent molecule.

Citation Format: Rosa Cardoso, Shalom Goldberg, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven Jacobs, Gary Gilliland, Karyn O’Neil. Evaluation of drug conjugation sites in an anti-EGFR Centyrin. [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 LB-062.
Abstract LB-065: Comparison of the serum proteome in individuals with cancers versus those without cancer, and its application to wellness
Authors: Zheng Haiyan , Zhao Caifeng , Roy Swapan , Soherwardy Amenah , Amin Ravish , Roy Devjit , Kuruc Matthew
Published: July 2016

For many diseases, pancreatic cancer for example, long term survival is critically dependent upon early detection. So various strategies for early detectable markers are being investigated. One such strategy is hypothesis driven, first by identifying a singular biomarker, usually derived from genomic analysis of the tumor type, and then determine its derivative protein concentration in blood. This has been challenging as many differentially regulated genes do not generate a differentially regulated protein, or one of sufficient concentration for current means of detection. An alternative biomarker strategy is purely data driven, as panels of proteins may be up and/or down regulated, or altered by post-translational modifications. These can be monitored and quantified to differences in diseased and normal/healthy individuals. In this study we adopt such a data driven strategy using a new product which combines Albumin depletion and on-bead digestion of the enriched serum proteome in a seamless process, called AlbuVoid™ LC-MS On-Bead. From these methods, we were able to compare labeled quantification of proteins from normal and disease state sera - for this case, breast, lung and pancreatic cancer at different clinically defined stages. The methods spectrally quantify over 250 total proteins, in a cost effective and reproducible manner. No offline peptide level fractionation prior to LC-MS was employed, lowering the LC-MS acquisition time 5-10x compared to common serum proteomic workflows. We describe these workflow advantages applied towards a “wellness” proteome algorithm combining knowledge and data surrounding individual normal and healthy proteomes, compared and contrasted to those with a clinically definable disease. From our initial tests, a candidate panel of 20 proteins are determined to be differentially expressed and such data suggests the feasibility for their use in the early detection of cancer. The critical parameters for success to our strategy are considered as well as some of the remaining challenges to clinical utility.

Citation Format: Haiyan Zheng, Caifeng Zhao, Swapan Roy, Amenah Soherwardy, Ravish Amin, Devjit Roy, Matthew Kuruc. Comparison of the serum proteome in individuals with cancers versus those without cancer, and its application to wellness. [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 LB-065.
Abstract LB-102: A pro-proliferative function of centriole duplication regulator, polo-like kinase-4 in human melanoma
Authors: Shabbir Maria , Nihal Minakshi , Singh Chandra K. , Longley B Jack , Ahmad Nihal
Published: July 2016

Malignant melanoma is one of the deadliest forms of skin cancer that remains resistant to existing therapeutic strategies. According to the SEER (Surveillance, Epidemiology, and End Results) program database of the National Cancer Institute, the 5-year survival rate for metastatic melanoma is a dismal 16%. Further, of the seven most common cancers in the USA, melanoma is the only neoplasm with constantly increasing incidence. Therefore, it is important to intensify our efforts to better-understand the genetics and biology of melanoma. This may lead to development of novel targets and biomarkers for management of this hard-to-treat neoplasm. This study was undertaken to determine the role and potential significance of polo-like kinase 4 (PLK4) in human melanoma. PLK4 is a serine/threonine kinase that has been shown to localize to centrioles and regulate centriole duplication during the cell cycle. While the potential role of PLK4 in cancer is beginning to be appreciated, there are evidence for its function as a tumor suppressor as well as tumor promoter. However, the role of PLK4 in melanoma is not known. Here, we employed in vitro and ex vivo experiments to determine the role of PLK4 in melanoma. Our data demonstrated that PLK4 was significantly overexpressed, both at mRNA and protein levels in human melanoma cells viz. A375, Hs294T, G361, 451Lu; when compared to the normal human epidermal melanocytes (NHEM). We also found a significant increase in PLK4 immunostaining in clinical melanoma tissues, compared to normal skin and nevus tissues. Further, treatment of melanoma cells with a selective small molecule PLK4 inhibitor, centrinone-B, was found to cause a significant decrease in the cell growth/viability and clonogenic survival in multiple melanoma cell lines. The observed anti-proliferative effects of centrinone-B were found to be accompanied with i) a G0/G1 phase arrest of cell cycle, and ii) induction of apoptosis, in human melanoma cells. Taken together, our study suggests a pro-proliferative role of PLK4 in human melanoma. Additional studies are ongoing in our laboratory to determine the functional significance of PLK4 in melanoma. We suggest that PLK4 should be further evaluated as a potential ‘druggable target’ and biomarker, for the management of melanoma.

Citation Format: Maria Shabbir, Minakshi Nihal, Chandra K. Singh, B Jack Longley, Nihal Ahmad. A pro-proliferative function of centriole duplication regulator, polo-like kinase-4 in human melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-102.
Abstract LB-103: Molecular exploitation of oncogenic stress in melanoma management
Authors: Kumar Raj , Ji Zhenyu , Miao Benchun , Njauw Ching-Ni Jenny , Tsao Hensin
Published: July 2016

Oncogenic B-RAF and N-RAS mutations are frequently reported in mitogen-activated protein kinase pathways of tumors. BRAF and NRAS mutations are found in more than 30% of all human tumors and 43% (88% V600E, 10% V600K) and 30% (48% Q61K, 40% Q61R) of malignant melanoma. These mutations were considered mutually exclusive except a few (0.7%) cases. BRAF and NRAS mutations are an important clinical marker and prognostic factor in metastatic melanoma. Recently developed BRAFV600E targeted therapies demonstrate significant clinical benefit but the targeted therapeutic options for NRAS-mutant melanomas are still under intense investigation. Importantly, BRAF and NRAS mutations are mutually exclusive, meaning that tumors are rarely found with these two mutations and are reported as lethal to the cancer cells. Hence, molecular behavior and interaction between BRAF and NRAS mutant proteins underline phenotypic complexity which is poorly understood. In the current study, we aimed to determine the translational significance of BRAF and NRAS mutations and their products in cell based studies with metastatic melanoma cell lines. Our lab sought to investigate the common molecular therapeutic target(s) of these genes, responsible for synthetic lethality. By using remote inducible expression system of BRAFV600E mutant gene in NRASQ61R mutant cell lines and vice versa. It is found that 25% (2/8) NRASQ61R mutant cell lines are sensitive to BRAFV600E and NRASQ61R mutation co-expression and have slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence, apoptosis and cell cycle arrest, more over rescued with BAP1 ectopic expression. While some NRAS mutant cell lines showed no lethal effect to BRAFV600E mutation expression which is well explained by human phospho-kinase array. These preliminary results suggest a complex interplay of tumor drivers (BRAF and NRAS) and passengers (X-factors) in cancer biology that need to be investigated in detail in melanoma.

Citation Format: Raj Kumar, Zhenyu Ji, Benchun Miao, Ching-Ni Jenny Njauw, Hensin Tsao. Molecular exploitation of oncogenic stress in melanoma management. [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 LB-103.
Abstract LB-104: Functional genomic screening identifies USP11 as a novel therapeutic target in breast cancer
Authors: O’Connor Darran , Dwane Lisa , O’Connor Aisling , Bernards Rene , Gallagher William
Published: July 2016

The estrogen receptor (ER) is the principal driver of growth and differentiation in breast cells and de-regulated receptor function is a key feature of almost 75% of breast cancers. Here, we investigated the role of de-ubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating the transcriptional activity of ER in breast cancer.

To identify DUBs involved in the regulation of ER transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all known human DUB genes (108 genes/432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter to estradiol (E2). In particular, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity (both in the presence and absence of E2), as demonstrated by a pronounced decrease in estrogen-response element (ERE) luciferase reporter activity. Subsequent validation of the screen using multiple individual hairpins and ZR-75-1 stable USP11 knockdown cells confirmed the suppression of ERE-reporter activity and further revealed a notable reduction in expression of the endogenous ER target genes TFF1 and PgR following USP11 knockdown. Growth assays and Western blot analysis also revealed a global decrease in cell survival, decreased ERK and AKT phosphorylation and increased sensitivity to DNA-damaging agents in USP11 knockdown cell lines.

In silico analysis of publically available breast cancer gene expression datasets revealed a highly significant association between high USP11 mRNA levels and poor prognosis. We observed a highly significant correlation between high expression of USP11 mRNA in ER positive patients and poor overall survival (OS)(HR 1.51, CI 1.07-2.14, p = 0.018) and distant metastasis-free survival (DMFS)(HR 1.35, CI 1.04-1.73, p = 0.023). This correlation was also significant in ER positive patients who had received endocrine therapy (OS, HR 3.4, CI 1.2-9.81, p = 0.014), DMFS, HR 2.16, CI 1.23-3.8, p = 0.0083). Tissue microarray validation using immunohistochemistry in an independent cohort (n = 144) confirmed USP11 as an independent marker of poor prognosis in ER+ breast cancer. These results suggest a role for USP11 in driving cellular growth and identifiy USP11 as a rationale and novel therapeutic target in breast cancer.

Citation Format: Darran O’Connor, Lisa Dwane, Aisling O’Connor, Rene Bernards, William Gallagher. Functional genomic screening identifies USP11 as a novel therapeutic target in 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 LB-104.
Abstract LB-105: High-throughput target identification using CRISPR/Cas9
Authors: Revankar Chetana M. , Braun Julia , Wong LaiYee , Wetter Justin , Yang Jian-Ping , Ravinder Namritha , Chesnut Jon , Piper David
Published: July 2016

One of the major challenges of the drug discovery process is the identification of novel, validated targets, whose pharmacological modulation may yield the desired therapeutic outcomes. The use of CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease and guide RNA can potentially impact the entire drug discovery process from generation of models based on clinical findings through to the target identification. The ability of CRISPR/Cas9, to efficiently and precisely edit a cell's DNA and introduce a complete genetic knockout, while minimizing off-target effects, offers an improved approach to target identification. Moreover, the ability to scale this approach through the generation of genome wide CRISPR libraries, coupled with the use of lentiviral delivery methods enables high-throughput (HTP) loss-of-function screens to be performed rapidly and identify genes whose activity is important for the specific endpoint being measured. For example, in this study, we have used CellSensor® cell lines targeting various signaling pathways (AP-1, c-Fos and NF-kB) to identify key targets critical to these pathways. Transcription factors like AP-1, c-Fos and NF-kB have shown to play an important role in cancer initiation and progression. Using the key signaling molecules along these pathways as controls we have data that supports HTP target identification. Furthermore, we will present our results from screening of a subset of a CRISPR library targeting 160 different kinases. Eventually, we plan to screen the entire CRISPR library targeting 750 kinases with 4 gRNA per gene against each of these signaling pathways. Similar screenings can be performed using other functional assay formats, like the cell health assays or high content imaging. These screenings can provide a wealth of data on the normal functioning of a target and in turn, should yield better validated targets for progression into full drug discovery. Ultimately, the hits from the HTP screenings can be followed up by using CRISPR technology to generate animal knockout models that would support translating the screen to the pre-clinical trials. This in turn could provide better correlation to the clinical setting and thereby reduce candidate drug attrition.

Citation Format: Chetana M. Revankar, Julia Braun, LaiYee Wong, Justin Wetter, Jian-Ping Yang, Namritha Ravinder, Jon Chesnut, David Piper. High-throughput target identification using CRISPR/Cas9. [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 LB-105.
Abstract LB-115: Mechanisms of acquired resistance to allosteric versus ATP-competitive AKT inhibition
Authors: Zimmerman Kristin M. , Lee Brian , Chung Wei-Jen , Gnad Florian , Lin Eva , Martin Scott , Lin Kui
Published: July 2016

The PI3K/AKT signaling pathway mediates a variety of cellular processes including metabolism, glucose uptake, angiogenesis, growth, survival and proliferation. Increased activity of this pathway is observed in many cancer types and is associated with tumor invasiveness, survival, and proliferation. Various regulators of this pathway are being investigated as therapeutic targets in oncology, including the AKT kinases. Two distinct classes of AKT inhibitors (AKTi) are being evaluated as therapeutic agents in the clinic: allosteric inhibitors (e.g. MK-2206) and ATP-competitive inhibitors (e.g. Ipatasertib/GDC-0068). In an effort to elucidate pathway alterations in response to these inhibitors and, ultimately, to improve therapeutic outcome, we are currently evaluating mechanisms of acquired resistance to both AKT inhibitor classes. The establishment of clonal cell populations resistant to either MK-2206 or GDC-0068 and subsequent characterization of alterations in these lines revealed various genetic and non-genetic variations associated with AKTi resistance. Here, we will present findings on biological mechanisms that contribute to acquired AKTi resistance in prostate and ovarian cancer cell line models.

Citation Format: Kristin M. Zimmerman, Brian Lee, Wei-Jen Chung, Florian Gnad, Eva Lin, Scott Martin, Kui Lin. Mechanisms of acquired resistance to allosteric versus ATP-competitive AKT inhibition. [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 LB-115.
Abstract LB-124: Preliminary anti-proliferative effect of ethanolic extracts of Xylopia aethiopica on prostate and breast cancer cell lines
Authors: Iweala Emeka E.J , Bankole Eunice W.
Published: July 2016

Xylopia aethiopica (African Pepper), a plant found throughout West Africa, has both nutritional and medicinal uses. The aim of this research was to study the preliminary anti-proliferative effects of ethanolic extracts of Xylopia aethiopica on prostate and breast cancer cell lines. Dried X. aethiopica fruits were extracted with 70% ethanol and tested against prostate(LNCaP) and breast cancer (MCF7 and MDA-MB231) cells’ viability in vitro using the MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) assay. The IC50 values for the extract were determined through linear regression using GraphPad Prism 6 software. The data obtained were expressed as mean ± SEM. Statistical analysis was carried out by one-way ANOVA followed by Dunnet's test (α = 0.05) to compare experimental means with controls. The data generated indicates that X. aethiopica fruits extract showed a dose-dependent anti-proliferative activity against LNCaP, MDA-MB231and MCF7 cancer cell lines after treatment for 48 and 72 hours. The extract induced a 16.96% and 93.5% inhibition on MDA-MB231 cells, 29.76% and 94.03% on the MCF7 cells and a 9.15% and 94.61% inhibition on the LNCaP cells at the lowest (1μg/ml) and highest (100μg/ml) dose respectively after 48 hours. The extract induced a 12.26% and 91.8% cell growth inhibition on the MDA-MB231 cells, 3.35% and 87.36% growth inhibition on the MCF7 cells and 2.28% and 92.42% cell growth inhibition on the LNCaP cells at the lowest (1μg/ml) and highest (100μg/ml) dose respectively after 72 hours. The IC50 was estimated to be 3.408μg/ml, 2.064μg/ml, 3.371μg/ml for MDA-MB231, MCF7 and LNCaP cells respectively. The data generated from this research indicates the anti-proliferative effects of ethanolic extract of X. aethiopica fruits on breast and prostate cancer cells. This can be seen from the inhibition of cell growth by this extract thus highlighting its potential as a therapy against breast and prostate cancer.

Citation Format: Emeka E.J Iweala, Eunice W. Bankole. Preliminary anti-proliferative effect of ethanolic extracts of Xylopia aethiopica on prostate and breast cancer cell lines. [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 LB-124.
Abstract LB-129: Epigenetic regulators to predict docetaxel sensitivity; a guide for treatment choice
Authors: Gurard-Levin Zachary A. , Wilson Laurence OW , Pancaldi Vera , Postel-Vinay Sophie , Sousa Fabricio G. , Reyes Cecile , Marangoni Elisabetta , Gentien David , Valencia Alfonso , Pommier Yves , Cottu Paul , Almouzni Genevieve
Published: July 2016

The limited capacity to predict the response of a given patient to cancer therapy is a major hurdle in cancer management. To date, many standard-of-care chemotherapy agents act by directly binding or modifying DNA or indirectly during processes that rely on chromatin architecture, as exemplified with docetaxel. Therefore, alterations in chromatin organization may impact resistance mechanisms for these drugs. Here, we explore the mis-regulation of chromatin regulators—factors involved in the establishment and maintenance of functional chromatin domains—as a tool to predict the response to docetaxel. For a series of chromatin regulators, we exploit Affymetrix microarray and NanoString gene expression data generated from breast cancer patient derived xenograft (PDX) models, colorectal cancer organoids, and breast cancer patient samples treated with docetaxel. Random Forest classification reveals a limited set of chromatin regulators that readily distinguish docetaxel responders from non-responders. Since the candidate predictive factors feature key subunits of the chromatin remodeler SWI/SNF complex, we then explore the behavior of SWI/SNF subunits in the response to docetaxel in the large NCI-60 dataset and in cultured cells. All together, our results showcase the predictive value of chromatin regulators and reveal that the SWI/SNF chromatin remodeler associates tightly with docetaxel response in several settings. Our findings open avenues for applications as biomarkers, but also as therapeutic targets to sensitize patients towards docetaxel. These approaches offer a means to potentially combat drug resistance.

Citation Format: Zachary A. Gurard-Levin, Laurence OW Wilson, Vera Pancaldi, Sophie Postel-Vinay, Fabricio G. Sousa, Cecile Reyes, Elisabetta Marangoni, David Gentien, Alfonso Valencia, Yves Pommier, Paul Cottu, Genevieve Almouzni. Epigenetic regulators to predict docetaxel sensitivity; a guide for treatment choice. [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 LB-129.
Abstract LB-137: IGF2BP1 and miR-34a genetic instability found in deadly neuroblastoma
Authors: Bell Jessica L. , Hüttelmaier Stefan
Published: July 2016

Chromosomal 17q21-ter gain in neuroblastoma is both a common and clinically significant occurrence. The oncogenic, insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. We have previously reported that in 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein levels are significantly higher in stage 4 compared with stage 1 tumors. Moreover, clinical and experimental data pointed to a strong association of IGF2BP1 with the MYCN oncogene. From the late 1990s onwards, 17q gain and 1p deletions (either alone or both) have been speculated as essential precursors to the MYCN amplification. As the tumor suppressive, miR-34a is located within 1p and is known to directly target MYCN and putatively IGF2BP1 mRNAs (Targetminer website), in this study we analyzed the co-occurrence of IGF2BP1 gain, miR-34a loss and MYCN amplification) in 69 neuroblastomas. qPCR was utilized to assess copy number. Changes were found for IGF2BP1 (58 tumors), miR-34a (15) and MYCN (12). miR-34a was never completed deleted. Interestingly, in all (12/12) MYCN amplified tumors, miR-34a was partially lost. Whilst, IGF2BP1 was gained in 9/12 MYCN amplified tumors. IGF2BP1 and miR-34a were aberrant together only twice with the absence of MYCN amplification. Of the 17 patients that died, all but one had at least an IGF2BP1 or miR-34a change in copy number. This stage 4 patent also had low MYCN protein levels, comparable to the stage 1 patients, indicating an aggressive tumor not involving the MYCN pathway. may A second larger cohort is certainly required to confirm these important results. IGF2BP1 and miR-34a locus have aberrant copy numbers in the majority of neuroblastoma, most strikingly, in the patients that died. IGF2BP1 and miR-34a may just be representative DNA of the larger locus changes and not be the causative event in affected neuroblastoma, but because of their known roles in cancer, the above results are intriguing. Although further experimental data is required, we speculate that either IGF2BP1 or mir-34a oncogenic changes are required for the clonal expansion of MYCN amplified tumor cells in neuroblastoma, and that these factors are crucial in the ability for an increase in MYCN transcription to be oncogenic in neuroblastoma.

Citation Format: Jessica L. Bell, Stefan Hüttelmaier. IGF2BP1 and miR-34a genetic instability found in deadly neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-137.
Abstract LB-147: Simultaneous profiling of mRNA and protein in single human induced pluripotent stem cells
Authors: Liu Benjamin , Dakshinamoorthy Gajalakshmi , Gong Haibiao , Ooi Aik , Li Nianzhen , Unger Marc , Holcomb Ilona , Ramakrishnan Ramesh
Published: July 2016

The cancer stem cell theory proposes that a discrete, small number of “stem-cell-like” cancer cells are primarily responsible for driving cancer. Thus, future cancer treatments are likely to be developed by targeting these cancer stem cells. One approach to modeling cancer therapy utilizes induced pluripotent stem cell (iPSC) technology. While gene expression studies have measured differentiation propensities between different iPSC clones and compared protein coding gene profiles between iPSCs and embryonic stem cells (ESCs), relatively little is known about the expression characteristics of individual RNA transcripts and their corresponding protein within each single iPSC. In addition, studies of the dynamic interaction between RNA and protein during the cell differentiation stage remains rare. Here we present an integrated assay that simultaneously quantifies mRNAs and proteins of interest within single iPSCs. The C1™ system was utilized for individual cell capture, cell lysis, DNA template generation for protein detection and cDNA synthesis. Final products from each iPSC were quantified on the 96.96 Dynamic Array™ IFC on the Biomark™ HD system. PCR data were analyzed using the Singular™ Analysis Toolset. For each run, up to 5,000 data points for 52 protein targets and 2,880 data points for 30 RNA targets, in addition to control and reference assays, were simultaneously generated and analyzed in less than two days. Combined RNA and protein signatures of each iPSC were identified. By comparing these results to those obtained from non-induced fibroblast cells, we demonstrate that this method is capable of obtaining expression levels of both RNA and protein associated with pluripotency marker genes at a single-cell level. More interestingly, the profiling data also indicates changes of some key members (EGFR, TGFB1, EPCAM, etc.) of cancer related common signaling pathways. The current system significantly controls lab-specific differences in reprogramming processes, progenitor cells or cell culture and handling conditions. We believe this powerful method makes it a suitable approach of choice for providing new insights into cell differentiation, reprogramming and tumor progression, as well as cancer therapeutic targets research.

Citation Format: Benjamin Liu, Gajalakshmi Dakshinamoorthy, Haibiao Gong, Aik Ooi, Nianzhen Li, Marc Unger, Ilona Holcomb, Ramesh Ramakrishnan. Simultaneous profiling of mRNA and protein in single human induced pluripotent stem 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 LB-147.
Abstract LB-148: DDX5 promotes ARF gene expression and apoptosis induced by deregulated E2F1
Authors: Kurayoshi Kenta , Ohtani Kiyoshi
Published: July 2016

The transcription factor E2F, the main target of the tumor suppressor pRB, plays central roles not only in cell proliferation but also in tumor suppression. E2F promotes cell proliferation by activating growth-related genes in response to normal growth stimulation. On the other hand, E2F contributes to tumor suppression by activating pro-apoptotic and growth-suppressive genes such as ARF and TAp73 when deregulated from pRB upon dysfunction of pRB, a major oncogenic change. ARF is an upstream activator of the tumor suppressor p53, one of the two major tumor suppressors in addition to pRB. TAp73 is a member of the p53 family and activates p53-responsive genes. Importantly, physiological E2F activity induced by growth stimulation does not activate the latter genes, underscoring importance of deregulated E2F activity in tumor suppression upon oncogenic changes. However, the regulatory mechanism of deregulated E2F activity is not known in detail.

We found that deletion of N-terminal domain of E2F1 dramatically reduced its ability to activate ARF and TAp73 promoter, suggesting that E2F1 interacts with a factor(s) through its N-terminal domain to activate the tumor suppressor genes. Hence we explored novel E2F1-interacting factors using co-immunoprecipitation and LC/MS. Among the candidates, we found DEAD box 5 (DDX5) that functions not only RNA helicase but also transcriptional coactivator. DDX5 is reported to function as a coactivator for p53. Furthermore, E2F1 activity is regulated in similar manners as p53 such as by Chk1 and PRMT5. These observations suggest that DDX5 enhances not only p53 activity but also deregulated E2F1 activity. We thus examined effect of DDX5 on deregulated E2F1 activity induced by overexpression of E2F1.

DDX5 enhanced E2F1 activation of ARF promoter depending on the integrity of N-terminal domain of E2F1. DDX5 mutant lacking RNA helicase activity retained the ability to enhance E2F1 activation of ARF promoter, indicating that the enhancement is independent of helicase activity. Furthermore, DDX5 enhanced endogenous ARF gene expression and apoptosis induced by E2F1 without affecting E2F1 expression level. Finally, knock down of DDX5 reduced E2F1-mediated activation of ARF promoter and ARF gene expression. These results suggest that DDX5 upregulates deregulated E2F1 activity and contributes to E2F1-induced apoptosis to protect cells from tumorigenesis.

Citation Format: Kenta Kurayoshi, Kiyoshi Ohtani. DDX5 promotes ARF gene expression and apoptosis induced by deregulated E2F1. [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 LB-148.
Abstract LB-149: LYAR promotes cell proliferation by repressing CHAC1 expression in neuroblastoma
Authors: Sun Yuting , Atmadibrata Bernard , Liu Bing , Liu Tao
Published: July 2016

Introduction: Neuroblastoma is a cancer of the sympathetic nervous system occurring mostly in infants and young children. Despite considerable progress in the treatment of neuroblastoma, 5-year survival rates have remained less than 50% for the majority of children with high-risk disease. Amplification of the MYCN oncogene and consequent overexpression of the N-Myc oncoprotein is a major prognostic indicator in neuroblastoma, which is strongly associated with aggressive tumour behaviour and poor patient outcome. N-Myc oncoprotein induces neuroblastoma by modulating gene and protein expression and consequently causing cell proliferation. LYAR (Ly-1 antibody reactive clone), a novel nucleolar protein with zinc finger DNA-binding motifs, is mostly involved in cell growth regulation in many cancers.

Results: Here we showed that N-Myc up-regulated LYAR expression in neuroblastoma cells. Conversely, LYAR up-regulated N-Myc expression. Affymetrix gene array studies revealed that the gene most significantly repressed by LYAR siRNAs was ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). Consistent with this finding, RT-PCR and immunoblot confirmed that LYAR siRNAs increased CHAC1 gene and protein expression. Alamar blue assays and cell cycle analyses demonstrated that LYAR induced MYCN-amplified neuroblastoma cell proliferation in a CHAC1-depedent manner. Additionally, high levels of LYAR gene expression in human neuroblastoma tissues were associated with MYCN gene expression and predicted poor patient survival independent of patient age and disease stage.

Conclusions: Our data demonstrate LYAR as a potential target in neuroblastoma oncogenesis, and provide critical evidence to support the application of LYAR inhibitors for the therapy of neuroblastoma patients.

Citation Format: Yuting Sun, Bernard Atmadibrata, Bing Liu, Tao Liu. LYAR promotes cell proliferation by repressing CHAC1 expression in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-149.
Abstract LB-157: Chromatin alterations induce C-MYC and BRD4 downregulation in pediatric sarcoma
Authors: Da Costa Elodie , Armaos Gregory , Jacques-Ricard Simon , Beaudry Annie , Raynal. Noël
Published: July 2016

Sarcoma accounts for 15% of all pediatric cancers diagnosed every year. Refractory patients have a poor prognosis with a 5-year survival rate of 10%, demonstrating the need for development of new therapeutic approaches. Epigenetic events, including DNA methylation and post-translational histone modifications (such as histone acetylation), are responsible for silencing tumor suppressor genes and overexpressing oncogenes. In sarcomas, a better understanding of epigenetic alterations is needed to develop new epigenetic targeted therapies. Several epigenetics drugs have demonstrated anticancer activity against sarcoma cell lines but the identification of a specific target is still missing. We demonstrated that targeting histone acetylation may represent a specific

epigenetic mark in pediatric sarcomas. We identified that proscillaridin A, a newly defined epigenetic drug, has epigenetic and anticancer effects on osteosarcoma (U2OS) and rhabdomyosarcoma (RD) cell lines. Our findings show a decrease of histone 3 acetylation on specific lysine residues after a 48h-treatment with proscillaridin A at 5 nM, a clinically relevant dose. These effects correlate with downregulation of histone acetylation associated proteins such as bromodomain BRD4 and oncogene C-MYC. A 48h-treatment with proscillaridin A produces a G2/M block and a reduction in the clonogenic potential of both U2OS and RD cells with IC50 value of approximately 6 nM. Interestingly, remaining colonies of RD cells fully lost their abilities to growth after drug removal, suggesting a long-term epigenetic reprograming. We demonstrated that proscillaridin A epigenetic effects occurred through calcium signaling since its mechanism of action may be dependent on calcium-calmodulin kinase activity.These promising results illustrate the epigenetic and anticancer potential of proscillaridin A against pediatric sarcoma. Additional experiments are currently ongoing in order to further characterize the mechanism of action of proscillaridin A in vitro and in vivo.

Citation Format: Elodie Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Noël Raynal. Chromatin alterations induce C-MYC and BRD4 downregulation in pediatric sarcoma. [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 LB-157.
Abstract LB-162: BRCA2, and not BRCA1, dependent crosstalk between replication stress suppression and nucleotide excision repair - Clinical implications
Authors: Pathania Shailja , Reed Rachel , Duan Haohui , Culhane Aedin , Garber Judy
Published: July 2016

BRCA2, a breast and ovarian tumor suppressor, is required for replication stress suppression through its role in repair of stalled replication forks. Here we report that inefficient resolution of stalled replication forks in absence of BRCA2 results in persistent accumulation of mostly un-phosphorylated RPA32 (at S33, T21 and S4/S8) at stalled replication forks. Furthermore, this ‘persistent’ accumulation of RPA32 coated ssDNA not only results in fork collapse, as marked by increased accumulation of both 53BP1 and Mre11 at the site of stalling, but also leads to suppression of nucleotide excision repair (NER), especially in S phase population. No such persistent RPA32 coated ssDNA is observed in BRCA1 depleted cells, suggesting that this intermediate is exclusive to BRCA2 deficiency. Furthermore, generation of this intermediate was not dependent on Mre11 implying that persistent accumulation of RPA32 coated ssDNA was not a result of excessive DNA processing by Mre11 exonuclease in absence of BRCA2. This is the first report of differences between BRCA1 and BRCA2 role at stalled replication forks. In this report, we also show that BRCA2 heterozygous primary mammary epithelial cells, and not BRCA1 heterozygous cells, are haploinsufficient for nucleotide excision repair. This would imply higher predisposition to skin lesions, including melanoma in BRCA2 mutation carriers. Risk for melanoma is indeed higher in BRCA2 mutation carriers compared to BRCA1 mutation carriers. We further confirmed that there exists a difference in mutational signature in whole genome sequences derived from BRCA1 and BRCA2 tumors, in line with the kind of differences expected if there is an increased NER defect in BRCA2 mutated cells when compared to BRCA1 mutated cells.

Our findings provide first such DNA repair mechanism based evidence that could potentially explain the differences in BRCA1 and BRCA2 mutation driven risk for melanoma. This mechanistic difference between BRCA1 and BRCA2 response at stalled replication forks, and subsequently a crosstalk with nucleotide excision repair, might also contribute towards other clinical differences observed in BRCA1 and BRCA2 mutation driven breast cancer.

Citation Format: Shailja Pathania, Rachel Reed, Haohui Duan, Aedin Culhane, Judy Garber. BRCA2, and not BRCA1, dependent crosstalk between replication stress suppression and nucleotide excision repair - Clinical implications. [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 LB-162.
Abstract LB-165: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma
Authors: Powers John T. , Tsanov Kaloyan , Roels Frederik , Spina Catherine , Ebright Richard , Seligson Marc , de Soysa Yvanka , Cahan Patrick , Pearson Daniel , Theißen Jessica , Tu Ho-Chou , LaPier Grace , Osborne Jihan , Ross Samantha , Collins James , Berthold Frank , Daley George
Published: July 2016

The let-7 microRNA family are known tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma, a neural crest derived tumor, is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7 target. The let-7 biogenesis inhibitor LIN28B has recently been implicated as a critical regulator of MYCN, but through CRISPR-mediated gene disruption we show that LIN28B is dispensable for both MYCN protein expression and growth of MYCN-amplified neuroblastoma cell lines despite robust de-repression of let-7, prompting us to explore additional mechanisms for let-7 disruption. Consequently, we have found a novel non-coding role for amplified MYCN mRNA as a potent let-7 sponge that through exceptionally high expression defines a sub-class of self-sponging amplified-competing-endogenous-RNA (aceRNA) and reconciling the dispensability of LIN28B in neuroblastoma cell lines. Furthermore, by analyzing a large cohort of tumor samples from patients, we observe frequent genomic loss of let-7 that inversely associates with MYCN-amplification, providing a functional explanation for the known MYCN-amplification-independent pattern of chromosome 3p and 11q loss, which harbor let-7g and let-7a2, respectively. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN-amplified tumors marks decreased survival, further underscoring its importance. The inverse selective relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis.

Citation Format: John T. Powers, Kaloyan Tsanov, Frederik Roels, Catherine Spina, Richard Ebright, Marc Seligson, Yvanka de Soysa, Patrick Cahan, Daniel Pearson, Jessica Theißen, Ho-Chou Tu, Grace LaPier, Jihan Osborne, Samantha Ross, James Collins, Frank Berthold, George Daley. Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-165.