bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1262: Type-2 cGMP-dependent protein kinase has a tumor-suppressive role in the colon epithelium
Authors: Islam Bianca N. , Sharman Sarah K. , Hou Yali , Ashby Justin , Wang Rui , Sridhar Subbaramiah , Kolhe Ravindra , Browning Darren D.
Published: August 2018

The cGMP level plays a central role in regulating homeostasis in the colon epithelium and is emerging as a potential target for colon cancer prevention. The signaling components downstream of cGMP, and the tumor suppressive mechanism remain poorly understood. The present study has examined the expression of cGMP-dependent protein kinase isoforms (PKG1, PKG2) in normal intestinal mucosa and in colon cancer from human specimens. Immunohistochemical analysis detected PKG1 in supportive cells in the lamina propria and in smooth-muscle tissue, but not in the epithelium of normal intestine and colon. In contrast, PKG2 was detected exclusively in the epithelium. In colon cancer, PKG1 was restricted to the stroma where it colocalized with vimentin, whereas PKG2 was only detected in the tumor epithelium where it colocalized with cytokeratin. This pattern of PKG isoform expression was similar in the mouse intestine and colon, where only PKG2 was detected in purified colonic crypts. PKG2 knockout (KO) mice were used interrogate possible anti-carcinogenic roles in the colon epithelium. When subjected to the AOM/DSS model of colon carcinogenesis, the PKG2 KO animals produced significantly more polyps than wild type controls (1.75-fold, p=0.0037). While the polyps from PKG2 KO animals were slightly smaller than those from wild type, the trend was not significant (p=0.13). Wild type and PKG2 KO mice responded equally to DSS treatment, but the PKG2-deficient animals exhibited crypt hyperplasia and increased apoptosis in luminal epithelium relative to wild type animals. Taken together these findings suggest that PKG2 has a tumor suppressive role in the colon epithelium, and that reducing the proliferative compartment may be part of the mechanism.

Citation Format: Bianca N. Islam, Sarah K. Sharman, Yali Hou, Justin Ashby, Rui Wang, Subbaramiah Sridhar, Ravindra Kolhe, Darren D. Browning. Type-2 cGMP-dependent protein kinase has a tumor-suppressive role in the colon epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1262.
Abstract 1279: Passenger mutation landscape in cancer genomes
Authors: Kumar Sushant , Warrel Jonathan , Mcgillivray Patrick , Meyerson William , Li Shantao , Salichos Leonidas , Harmanci Arif , Fundichely Alexander , Chan Calvin , Herrmann Carl , Nielsen Morten , Lochovsky Lucas , Zhang Yan , Li Xiaotong , Khurana Ekta , Getz Gad , Gerstein Mark
Published: August 2018

Cancer progression is an evolutionary process during which thousands of somatic variants are accumulated within an individual. In the classic view of cancer progression, a handful of driver variants are thought to give a positive selection advantage to the cancer cell. The remaining variants, termed passengers, represent the overwhelming majority of the variants in cancer genomes, and their functional consequences are poorly understood. Furthermore, the bulk of these passengers fall within noncoding regions of the genome, making these the main product of whole-genome sequencing of tumors. In this work, we explore the functional landscape of passenger variants in various cancer cohorts by leveraging extensive pan-cancer variant calls from ~2500 uniformly processed whole cancer genomes. More specifically, we integrate their annotations and predicted functional impact scores to quantify the overall burdening of various elements in cancer genomes. We show that disruption of genetic regulatory elements in the noncoding genome correlates with altered gene expression. Furthermore, we also show how overall functional burdening of various genomic elements correlate with patient survival time, and tumor clonality. Finally, we observe statistical signals consistent with the notion that aggregated subsets of passenger variants - particularly those we predict to be functionally impactful- might confer weak selective effects.

Citation Format: Sushant Kumar, Jonathan Warrel, Patrick Mcgillivray, William Meyerson, Shantao Li, Leonidas Salichos, Arif Harmanci, Alexander Fundichely, Calvin Chan, Carl Herrmann, Morten Nielsen, Lucas Lochovsky, Yan Zhang, Xiaotong Li, Ekta Khurana, Gad Getz, Mark Gerstein. Passenger mutation landscape in cancer genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1279.
Abstract 128: NKILA lncRNA promotes tumor immune evasion by mediating activation-induced cell death in tumor-specific CTLs
Authors: Song Erwei
Published: August 2018

Activation-induced cell death (AICD) of T lymphocytes may be exploited by cancers to eliminate cytotoxic T cells (CTLs). However, the mechanisms underlying AICD in tumor specific CTLs remain unknown. Here, we demonstrated excessive apoptosis of tumor antigen-specific CTLs in breast and lung cancers. Interestingly, NKILA, an NFκB-interacting lncRNA, mediates AICD of CTLs by inhibiting NFκB activities after their activation. Mechanistically, NKILA promoter is closed for transcription by histone deacetylation in resting T cells, while calcium influx by TCR signaling activates calmodulin to remove the deacetylase from NKILA promoter and enhances its transcription. In vivo, administering CTLs with NKILA silencing to immunocompromised mice with breast cancer patient-derived xenografts (PDXs) effectively inhibits PDX growth by increasing CTL infiltration. Clinically, NKILA was overexpressed in the tumor-specific CTLs of breast and lung cancers, which was associated with less CTL infiltration in the tumors and shorter patient survival. Our findings present the first evidence that AICD in tumor-specific CTLs is crucial to cancer immune evasion, and targeting NKILA in CTLs emerges as a novel antitumor immunotherapy.

Citation Format: Erwei Song. NKILA lncRNA promotes tumor immune evasion by mediating activation-induced cell death in tumor-specific CTLs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 128.
Abstract 1285: Deep mutagenesis analysis on BRCA1 variants in high throughput DNA repair assays
Authors: Diabate Mariame , Adamovich Alexandrea , Banerjee Tapahsama , Freitas Michael , Parvin Jeffrey
Published: August 2018

Genetic screening of Hereditary Breast and Ovarian Cancer (HBOC)-associated genes can aid in prevention of the onset of cancer by increased surveillance or preventative surgery if the genetic information is clear. Often, the genetic information is ambiguous since missense substitutions may be common in aggregate but individually these missense variants occur too infrequently for genetic segregation to be conclusive and are known as Variants of Unknown Significance (VUS). One commonly screened HBOC gene is BRCA1, with mutations found in 40-45% of hereditary breast cancer cases. The BRCA1 protein has the critical tumor suppressive function mediating homology-directed repair (HDR), which is used for the repair of double-stranded breaks (DSB) in DNA. Clinical mutant databases, such as ClinVar, contain the clinical classification of BRCA1, but, unfortunately, in many of these databases, most of the variants are classified as VUS or have conflicting data. We suggest that laboratory-based functional analysis can augment the genetic and clinical data, enabling the understanding of whether a specific variant is benign or pathogenic and predisposing to breast and ovarian cancer. We have developed a high-throughput method for analyzing the function of individual missense variants of BRCA1 in the DNA repair process. The goal is to have functional analysis for every possible amino acid substitution in BRCA1, and this will be publicly available for geneticists to consider when evaluating the likelihood of cancer predisposition an individual with a VUS in BRCA1. We have completed analysis of the amino-terminal RING domain, and we will present new results for the carboxy-terminal BRCT domain. We develop a computational pipeline that starts with sequence data from DNA repair assays to obtain a score for each variant. This pipeline will include statistical programs to evaluate the confidence level for these results, which will then be compared to the information provided in the ClinVar database. In summary, this work describes a computational framework for deconvoluting sequencing results of a high-throughput approach to measure the functional impact of each possible missense variant in the BRCT domain of BRCA1.

Citation Format: Mariame Diabate, Alexandrea Adamovich, Tapahsama Banerjee, Michael Freitas, Jeffrey Parvin. Deep mutagenesis analysis on BRCA1 variants in high throughput DNA repair assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1285.
Abstract 1298: Mapping the comprehensive landscape of missense-mutation neoantigens across the human genome
Authors: Lee HoJoon , Greer Stephanie U. , Ji Hanlee P.
Published: August 2018

Specific somatic mutations in cancer genomes result in amino acid alterations and derivative novel immunogenic peptides called neoantigens. The MHC molecule mediates the immunologic presentation of these cancer mutation-derived peptides. Based on a limited number of polymorphisms, the configuration of the MHC molecule is one of the major factors in determining whether mutated peptides can elicit immune response or not. To provide insight in the full diversity of potential neoantigens across the entire human genome, we identified all possible amino acid substitutions and estimated their potential binding affinities to the 86 MHC genotypes. As a first step, we evaluated every possible nucleotide changes at every single position using exons and related alternative transcripts as defined by the Consensus Coding Sequence (CCDS) resource. We evaluated 159 million (M) possible nucleotide changes among ~31,000 autosomal transcripts from ~ 19,000 genes. Overall, this comprised a genome sequence “space” of 53 M nucleotides. Among this coding regions space, we evaluated 123 M candidate nonsynonmous mutations. Among these mutations, we identified short mutated peptides of nine amino acids in length across this mutation space. We estimated the binding affinity of all mutated peptides across 86 MHG polymorphisms. Based on the predicted binding affinity, 0.6% ~ 1.0 % were found to be potential strong binders to the one of MHC molecules while 1.7% ~ 2.5% of them are weak binder to the one of MHC molecules. HLA-A0201 had the high number of potential candidate neoantigens. The KRAS cancer had candidate epitopes that binds to >50 different MHC molecules include KRAS D12V, which is very hot spot, that make epitopes that binds to 30 MHC molecules. In addition, we mapped the frequencies of missense mutations observed in TCGA and COSMIC to these candidate neoantigens. For releasing the results of our study, we are developing the “Immunogenomic Explorer” available at http://genomeportal.stanford.edu/immune. This portal enables users to explore the potential immunogenic epitopes from genes of their interest such as KRAS. Users can search by HLA genotypes as well. This web interface allows researchers to address important questions regarding their immunotherapy or cancer vaccine.

Citation Format: HoJoon Lee, Stephanie U. Greer, Hanlee P. Ji. Mapping the comprehensive landscape of missense-mutation neoantigens across the human genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1298.
Abstract 1301: Using the evolutionary ages of genes to understand hallmark features of cancer
Authors: Goode David L. , Trigos Anna S. , Papenfuss Anthony T. , Pearson Richard B.
Published: August 2018

The evolutionary age of a gene is closely tied to its role in cancer. As organisms evolved into multicellular forms, new pathways emerged to control core cellular processes formed in unicellular ancestors such as cell division, DNA replication and energy metabolism. Tumors rely on many of these core processes, at the same time as they suppress key pathways linked to the emergence of multicellularity. This suggests the guiding principles behind many hallmarks of cancer could be better understood in an evolutionary context, by investigating interactions between unicellular and multicellular genes and how they are disrupted in cancer. We have found the evolutionary histories of genes are associated with their expression and regulation (Trigos et al., PNAS 2017), the selection and response to genetic alterations, as well as the rewiring of transcriptional networks across 7 tumor types. Our results reveal different transcriptional and mutational patterns in cancer between genes with different evolutionary ages, with selection for genetic alterations in early metazoan genes leading to the transcriptional rewiring of the regulation between unicellular and multicellular genes, driving carcinogenesis. Examples of how this approach can help prioritize genes for drug treatment and inform novel treatment strategies will be given.

Citation Format: David L. Goode, Anna S. Trigos, Anthony T. Papenfuss, Richard B. Pearson. Using the evolutionary ages of genes to understand hallmark features of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1301.
Abstract 1312: Predicting ligand-dependent tumors from multi-dimensional signaling features
Authors: Hass Helge , Masson Kristina , Wohlgemuth Sibylle , Paragas Violette , Allen John E. , Sevecka Mark , Pace Emily , Timmer Jens , Stelling Joerg , MacBeath Gavin , Schoeberl Birgit , Raue Andreas
Published: August 2018

Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors for growth factors that are frequently deregulated in cancer. Signaling through these receptors has been associated with increased cancer cell proliferation and resistance to cytotoxic therapies. To block this detrimental signaling, many companies are developing inhibitory antibodies against various RTKs. A key challenge in clinical studies is the optimal stratification of patients who may benefit from these therapies. For an RTK targeted antibody, the detection of the respective growth factor in the tumor microenvironment may be an important bio-marker. Beyond the physical presence of the growth factor, the decision whether a cancer cell will respond to growth factor-induced signals is governed by complex intra-cellular signaling networks. We compared different approaches to predict cellular responses and will highlight a hybrid approach that combines mechanistic modeling based on ordinary differential equations with a machine learning algorithm. The models are trained on in vitro drug response screens and then applied to predict response in patient samples. The mechanistic models are trained on quantitative data from signal transduction studies as well as RNAseq data for cellular characterization. Using the hybrid approach, a correlation between growth factor expression in the tumor microenvironment and its predicted response was identified. This supports the hypothesis of addiction of tumors to growth factors abundant in the tumor microenvironment, and might enable more robust patient stratification in the future.

Citation Format: Helge Hass, Kristina Masson, Sibylle Wohlgemuth, Violette Paragas, John E. Allen, Mark Sevecka, Emily Pace, Jens Timmer, Joerg Stelling, Gavin MacBeath, Birgit Schoeberl, Andreas Raue. Predicting ligand-dependent tumors from multi-dimensional signaling features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1312.
Abstract 1320: An integrative and interactive colorectal cancer biomarker database
Authors: Zhang Xueli , Sun Xiao-Feng , Cao Yang , Ye Benchen , Peng Qiliang , Liu Xingyun , Shen Bairong , Zhang Hong
Published: August 2018

We created a colorectal cancer (CRC) biomarker database (CBD) with credible content as a powerful and time-saving tool to provide better and accurate information concerning CRC biomarkers. CRC biomarker database (http://sysbio.suda.edu.cn/CBD/) was established based on 870 identified CRC biomarkers and their relevant information from 1115 original articles in PubMed published from 1986 to 2017. In this version of the CBD, CRC biomarker data was collected, sorted, displayed, and analysed. The CBD with the credible contents as a powerful and time-saving tool can provide more comprehensive and accurate information for further CRC biomarker researches. The CBD was constructed under MySQL server. HEML, PHP and JavaScript languages have been used to implement the web interface. The Apache was selected as HTTP server. All of these web operations were implemented under the Windows system. The CBD was concluded the multiple individual biomarker information and categorised into the biological category, source and application of biomarkers; the experiment methods, results, authors and publication resources; the research region, the patient's age, gender, race, the number of tumours, tumour location, and stage. The key words for searching in PubMed are: (((biomarker[Title/Abstract] OR marker[Title/Abstract]) OR indicator[Title/Abstract]) OR predictor[Title/Abstract]) AND ((colorectal cancer[Title/Abstract] OR rectal cancer[Title/Abstract]) OR bowel cancer[Title/Abstract]). The CBD can provide a professional platform to researchers who are interested in CRC research to communicate, exchange their research ideas and further design high quality research in CRC. They can submit their new findings to our database via th submission page, and communicate with us in the CBD.

Citation Format: Xueli Zhang, Xiao-Feng Sun, Yang Cao, Benchen Ye, Qiliang Peng, Xingyun Liu, Bairong Shen, Hong Zhang. An integrative and interactive colorectal cancer biomarker database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1320.
Abstract 1329: Combination treatment with CB-839 and romidepsin induces apoptosis and suppresses cell viability in preclinical models of chondrosarcoma
Authors: Sheikh Tahir N. , Patwardhan Parag P. , Schwartz Gary K.
Published: August 2018

Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Several studies have reported that mTORC1 stimulates glutamine metabolism and increased mitogenic signaling through activation of the mTORC pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of certain malignancies. In the present study, we hypothesized that concurrent inhibition of glutamine metabolism and histone acetylation could result in decreased viability of chondrosarcoma cells. To test this, we used a panel of chondrosarcoma cell lines including IDH wild type (CH2879), IDH1 mutant (JJ012) and IDH2 mutant (SW1353) cell line. Results from our in vitro proliferation assay showed that combination of sub-IC50 concentrations of the CB-839 (Glutaminase inhibitor) and Romidepsin (HDAC inhibitor) resulted in decreased cell viability of all the three chondrosarcoma cell lines when compared to either of the two drugs alone. Western blot analysis showed induction of cleaved Poly-ADP Ribose Polymerase (PARP) as well as cleaved caspase 3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 and Phospho-p44/42 MAPK such as ERK1/2. Combination treatment also resulted in increased induction of histone acetylation (AcH3). Cell cycle analysis showed induction of sub-G1 population phase in combination treatment. Previous reports have shown that CpG methylation is a critical event in transcriptional repression of hyper methylated genes in cancer. To this end, we show that combination treatment with CB-839 and Romidepsin results in down regulation of (H3K9) methylation. Taken together, our data strongly suggests that combination treatment with CB-839 (Glutaminase inhibitor) and Romidepsin (HDAC inhibitor) is a novel treatment approach and merits evaluation in the treatment of chondrosarcoma.

Citation Format: Tahir N. Sheikh, Parag P. Patwardhan, Gary K. Schwartz. Combination treatment with CB-839 and romidepsin induces apoptosis and suppresses cell viability in preclinical models of chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1329.
Abstract 1342: Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells
Authors: Biel Thomas , Rao Ashutosh
Published: August 2018

Mitochondria-targeted drugs are designed to accumulate in the mitochondrion to modulate metabolism. Triphenylphosphonium (TPP) conjugated agents are a class of mitochondria-targeted drugs that induce mitochondrial dysfunction in cancer cells. Mitochondrial dysfunction is known factor that facilitates the onset of mitophagy. However, the activation of mitophagy to remove drug-induced mitochondrial damage is unknown. Here, we subjected breast MDA-MB-231 cancer cells and MCF-12A noncancerous cells to a series of mitochondria-targeted redox-active agents (MTA) that contain TPP to investigate the onset and activation of autophagy, mitophagy and mitochondrial dysfunction. Collectively, we identified three different MTAs from the series that sequentially damage the mitochondria, enhance autophagic flux, and activate mitophagy selectively in MDA-MB-231 cells. Furthermore, we generated stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines to establish a mitophagy flux assay for screening new agents. FACS analysis of mt-mKeima, a pH-sensitive fluorophore, revealed MTAs induced lysosomal dependent mitochondrial degradation and the presence of bafilomycin, a lysosomal inhibitor, suppressed MTA-induced mitophagy. To confirm MTAs induced mitochondrial autophagy, we identified mitochondria located within autophagosomes using confocal microscopy, an endogenous autophagy-related protein complex by coimmunoprecipitation and mitochondrial PINK1 accumulation using immunoblot. These in vitro studies were translated into an in vivo rat SST-2 xenograph breast cancer model to demonstrate that mitochondrial PINK1 accumulates in rat tumor tissue following MitoQ treatment. These data suggest that mitochondria-targeted agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A noncancerous cells.

Citation Format: Thomas Biel, Ashutosh Rao. Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1342.
Abstract 1357: Functional characterization of FANCD2 in esophageal squamous cell carcinoma
Authors: Lei Lisa Chan , Yu Valen Zhuoyou , Ning Lvwen , Ko Josephine Mun-Yee , Wang Li Dong , Lung Maria Li
Published: August 2018

Introduction:

Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value.

Methods:

We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability.

Results:

The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome.

Conclusion:

These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance.

Acknowledgement:

We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative

Research Fund (C7031.15G to M.L.L.).

Citation Format: Lisa Chan Lei, Valen Zhuoyou Yu, Lvwen Ning, Josephine Mun-Yee Ko, Li Dong Wang, Maria Li Lung. Functional characterization of FANCD2 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1357.
Abstract 1383: A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML
Authors: Bhogal Balpreet , Weir Barbara , Crescenzo Ramona , Kwon Min Chul , Philippar Ulrike , Attar Ricardo , Cowley Glenn , Pocalyko David
Published: August 2018

DNA methylation is an epigenetic mechanism that regulates gene expression during many stages of development, including genomic imprinting, stem cell regulation, and X-chromosome inactivation. Moreover, aberrant DNA methylation patterns, characterized by genome-wide hypomethylation and promoter-specific hypermethylation, are a prominent feature of cancer. Methylation of DNA at the 5-position of cytosines is mediated by the DNA methyltransferase (DNMT) protein family, which regulates both maintenance methylation (DNMT1) and de novo methylation (DNMT3A and DNMT3B). Loss-of-function mutations of DNMT3A have been identified in hematological malignancies including acute myeloid leukemia (AML), where DNMT3A is mutated in approximately 25% of known cases.

Published reports suggest the existence of a synthetic lethal interaction between DNMT3A and DNMT1/3B. To further study this potential genetic interaction, we are performing a CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that are synthetic lethal with DNMT3A. We generated a lentiviral library containing 777 and 421 single guide RNAs (sgRNAs) that tile the coding region of DNMT1 and DNMT3B, respectively and performed viability screens in AML cell lines that are either wild-type or mutant for DNMT3A. This screen was designed to identify in-frame alterations within functional domains that lead to effects on cell viability. Next generation sequencing of sgRNAs identified three functional domains of DNMT1 which, when mutated, leads to decreases in cell viability. Current efforts are focused on verifying the essentiality of these functional domains using CRISPR-Cas9-based approaches as well as mutagenesis by integrated tiles (MITE)-seq analyses.

Citation Format: Balpreet Bhogal, Barbara Weir, Ramona Crescenzo, Min Chul Kwon, Ulrike Philippar, Ricardo Attar, Glenn Cowley, David Pocalyko. A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1383.
Abstract 1385: Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells
Authors: Donovan Micah G. , Selmin Ornella I. , Romagnolo Donato F.
Published: August 2018

The aryl hydrocarbon receptor (AhR) is a driver of breast cancer progression and its overexpression is often observed in triple negative breast cancer (TNBC). Activation of AhR results in epigenetic silencing of the breast cancer 1 gene (BRCA1), a necessary factor in the transcriptional activation of estrogen receptor-alpha (ERα). The purpose of this study was to examine the capacity for galangin (GAL; 3,5,7-trihydroxy-2-phenylchromen-4-one), a natural AhR antagonist, to mimic the effects of synthetic AhR antagonists alpha-naphthoflavone (αNF, 2-phenylbenzo[h]chromen-4-one) and 3'-methoxy-4'-nitroflavone (MNF; 2-(3-methoxy-4-nitrophenyl)chromen-4-one) in regards to counteracting epigenetic silencing of BRCA1 and restoring ERα protein expression. We utilized TNBC cells with hypermethylated BRCA1 and constitutively active AhR (HCC38 cells) for these experiments. After 72h in phenol-red free media, cells were treated for up to 72h against a range of GAL, αNF, and MNF concentrations in the presence or absence of estradiol (E2). Transcript levels of AhR and its downstream target genes CYP1A1 and CYP1B1 were determined by real-time polymerase chain reaction (RT-PCR). Protein levels of BRCA1, ERα, and AhR were determined by Western blot. Treatment with GAL results in stabilization of BRCA1 and ERα protein levels, similarly to αNF and MNF. To determine potential mechanisms of these observations, we are currently conducting experiments to analyze changes in BRCA1 promoter methylation using methylation-specific RT-PCR (MSP) and DNA methyltransferase 1 (DNMT1) protein levels by Western blot. These data suggest the naturally occurring flavanoid GAL may be an effective compound for modulating epigenetic repression of BRCA1 and restoring ERα levels, which may provide options for anti-hormonal therapy in ERα-negative breast cancers.

Citation Format: Micah G. Donovan, Ornella I. Selmin, Donato F. Romagnolo. Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1385.
Abstract 1405: PAX2 induces tubular structure in ovarian cancer cells
Authors: Alwosaibai Kholoud
Published: August 2018

In adult tissues, PAX2 protein is expressed in normal oviductal epithelial cells (OVE) but not in normal ovarian surface epithelial cells (OSE). Recent studies reported that PAX2 is expressed in serous ovarian carcinoma cases but the role of PAX2 in enhancing ovarian cancer is unrevealed yet. The aim of this study is to understand the biological consequences after Pax2 overexpression in mouse ovarian surface epithelial (MOSE) cells. We found that Pax2 overexpression in MOSE cells induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells.

Citation Format: Kholoud Alwosaibai. PAX2 induces tubular structure in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1405.
Abstract 1411: Proteogenomic analysis of the syngeneic mouse gastric stem cell-like cancer cell line
Authors: Yang Hanna , Kim Hark Kyun
Published: August 2018

Gastric cancer is the third main cause of cancer deaths worldwide. To understand molecular mechanism of gastric cancer incidence and metastasis, we generated NCC-S1M lacking Smad4, Trp53 and Cdh1, which is the unique syngeneic gastric cancer cell line transplant model in the scientific society. NCC-S1M showed cancer stem cell-like features and strong tumor-initiating potential. Using proteogenomic analysis of NCC-S1M, we elucidated the specific signaling pathways that control gastric cancer stem cell-like properties and immune checkpoint. Cd274, Ccne1 and Il1rl1 were overexpressed and their protein levels were higher than normal gastric tissues. Cd274 encodes PD-L1, which is one of the important immune checkpoint proteins. The growth rate of NCC-S1M allograft was reduced after treatment with anti-PD-1. When Smad4 was ectopically expressed in NCC-S1M, Cd274 expression was decreased. In addition, TGF-β treatment induced reduction in Cd274 expression of Smad4 overexpressed NCC-S1M cells even more comparing with normal Smad4 overexpressed NCC-S1M. The knockdown of Il1rl1 in NCC-S1M reduced tumorigenicity and in vivo chemoresistance. We demonstrated that NCC-S1M focally amplified Ccne1 gene through array comparative genomic hybridization. Ccne1 knocked down NCC-S1M showed reduced metastatic potential. The proteogenomic characterization of NCC-S1M explains that Smad4 is important for PD-L1 immune evasion and that Il1rl1 has a significant role in cancer stem cell-like properties. Furthermore, it could imply that Ccne1 regulates metastasis.

Citation Format: Hanna Yang, Hark Kyun Kim. Proteogenomic analysis of the syngeneic mouse gastric stem cell-like cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1411.
Abstract 1419: Dissection of single-cell transcriptional and translational regulation by digital mRNA and protein quantification
Authors: Lam Gretchen , Shum Eleen , Chang Christina , Shah Hemi , Jensen Devon , Ghadiali James , Martin Jody , Rosenfeld David , Fan Christina H.
Published: August 2018

The immune system consists of complex gene regulatory networks that allow a rapid transition of different cellular states during an immune response. Cell-surface marker analysis using flow cytometry or single cell RNA-seq has allowed characterization of immune subpopulations and a greater understanding of the complexity of immune cells. However, restrictions on protein-only or RNA-only analysis can greatly limit the understanding of how genes are regulated in cells. For example, many cell surface markers - such as CD4 in T cells - has thousands of protein copies per cell using antigen density calculations, yet is fueled by a small number of mRNA transcripts per CD4+ T cell. Moreover, conventional whole-transcriptome analysis of mRNA can further mute the expression detection of CD4 mRNA in T cells due to the abundance of housekeeping ribosomal genes. To bridge the understanding of protein and mRNA expression differences, we used Ab-Seq on BD RhapsodyTM platform to provide digital quantification of both protein and mRNA expression level in single cells. An oligo-conjugated antibody panel against immune-relevant cell-surface markers was created and used for this multi-omic gene expression profiling effort. This approach is coupled with mRNA analysis using the BD Rhapsody Immune Response Panel, a targeted method of RNA-seq that allows a higher sensitivity of immune markers than conventional whole transcriptome assays. We studied the dynamics of early T cell activation in vitro to understand this response on transcriptional, post-transcriptional, and translational levels. Different time points following anti-CD3 and anti-CD28 treatment were collected and multiplexed on to BD Rhapsody cartridge for single cell capture and analysis. Using Ab-Seq on BD Rhapsody, we were able to detect the difference in mRNA and protein levels of crucial markers, allowing us to dissect the intricate gene regulatory pathways during an immune response in a single cell level.

Citation Format: Gretchen Lam, Eleen Shum, Christina Chang, Hemi Shah, Devon Jensen, James Ghadiali, Jody Martin, David Rosenfeld, Christina H. Fan. Dissection of single-cell transcriptional and translational regulation by digital mRNA and protein quantification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1419.
Abstract 1468: ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator
Authors: Jahn Arne , Rane Grishma , Paszkowski-Rogacz Maciej , Sayols Sergi , Bluhm Alina , Han Chung-Ting , Draškovič Irena , Londoño Arturo , Kumar Alan Prem , Buchholz Frank , Butter Falk , Kappei Dennis
Published: August 2018

Telomeres consist of repetitive TTAGGG repeats and together with the shelterin complex protect the ends of linear chromosomes from DNA repair mechanisms. Due to telomere shortening with every cell division proliferating cells need to ensure telomere maintenance by telomerase or in about 15% of cancer cells via the recombination based alternative lengthening of telomeres. Recently ZBTB48 was identified as a novel telomere-associated factor throughout the vertebrate lineage. Here we show that ZBTB48 binds directly to telomeres in vitro and in vivo, independently of the telomere maintenance mechanism. Loss of the protein leads to longer telomeres indicating a role as negative regulator of telomere length. Furthermore RNAseq, ChIPseq and quantitative expression proteomics demonstrate a function as transcriptional activator for a small set of target genes, including mitochondrial fission process 1 (MTFP1). This regulatory role links its telomeric function to mitochondrial metabolism and might play a role in cancer development. In summary ZBTB48 is a novel telomere binding protein which regulates telomere length and transcriptional activity.

Citation Format: Arne Jahn, Grishma Rane, Maciej Paszkowski-Rogacz, Sergi Sayols, Alina Bluhm, Chung-Ting Han, Irena Draškovič, Arturo Londoño, Alan Prem Kumar, Frank Buchholz, Falk Butter, Dennis Kappei. ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1468.
Abstract 1482: SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL)
Authors: Vukadin Lana , Park Eun young , Kim Jung Hyun , Ahn Erin Eun Young
Published: August 2018

Individuals with Down syndrome (DS) are highly predisposed to develop hematopoietic malignancies, especially acute megakaryoblastic leukemia (DS-AMKL). Generally, genes located on chromosome 21 tend to be increased in Down syndrome patients, but how these individual genes contribute to development of DS-AMKL is still unclear. SON, a gene located on chromosome 21, is upregulated in Down syndrome patients, and it has been reported that DS-AMKL cells have higher level of SON expression. The SON protein functions as a splicing factor and a transcription regulator in leukemia but the role of SON upregulation in DS-AMKL pathogenesis is still not fully elucidated. In addition, runt-related transcription factor 1 (RUNX1) is also located on chromosome 21, but its expression level is downregulated in DS-AMKL patients despite the increased gene dosage. We show that SON represses RUNX1 expression and blocks terminal megakaryocytic differentiation in DS-AMKL. Knockdown of SON in DS-AMKL cell lines leads to morphologic changes and increased cell adhesion. Moreover, cell surface marker analyses demonstrate that the depletion of SON induces increased CD41+ CD61+ populations as well as polyploidy cells, indicating megakaryocytic differentiation. Consistent with these results, SON deletion causes upregulation of RUNX1, which is a critical factor in the megakaryocytic differentiation. Furthermore, our data show that knockdown of SON increases several megakaryocytic transcription factors and decreases erythropoietic transcription factors, facilitating terminal megakaryocytic differentiation of leukemic blasts. Mechanistically, SON binds to the proximal promoter region as well as two intronic enhancer regions upstream of the proximal promoter at the RUNX1 gene locus, and suppresses transcription of RUNX1 by lowering the level of H3K4me3. Taken together, we propose that the increased genomic dosage of SON in Down syndrome impairs terminal differentiation of megakaryo-erythroblasts into mature megakaryocytes through binding to the RUNX1 promoter/enhancer to repress its transcription, which contributes to the higher incidence of AMKL in Down syndrome patients. Our results reveal a novel connection of SON to DS-AMKL pathogenesis and provide clinical importance of SON upregulation in DS. These findings suggest that SON could be a promising therapeutic target for DS-AMKL patients.

Citation Format: Lana Vukadin, Eun young Park, Jung Hyun Kim, Erin Eun Young Ahn. SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1482.
Abstract 1483: Targeting oncogenic enhancer activity with a small-molecule CBP/P300 bromodomain inhibitor
Authors: Raisner Ryan , Bainer Russell , Chan Emily , Garcia Jesse , Merchant Mark , Gascoigne Karen
Published: August 2018

Transcriptional networks are often dramatically altered in cancer, this dysregulation occurring at both the genetic and epigenetic level. Recently much attention has been given to the role of enhancer-driven gene expression in cancer, with the observation that numerous key oncogenes are driven by super-enhancers. This opens up the possibility for therapeutic intervention with small molecules that modulate enhancer activity, leading to control of expression of otherwise un-drugable oncogenes. CBP/P300 are histone acetyl transferases (HATS) that are critical mediators of enhancer activity. Here we use a novel small molecule inhibitor of the CBP/P300 bromodomain to specifically probe the role of these HATS at enhancers, and more broadly evaluate the consequences of enhancer disruption on cancer cell growth in vitro and in vivo.

Citation Format: Ryan Raisner, Russell Bainer, Emily Chan, Jesse Garcia, Mark Merchant, Karen Gascoigne. Targeting oncogenic enhancer activity with a small-molecule CBP/P300 bromodomain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1483.
Abstract 1491: Expression of osteopontin splicing isoforms in childhood B-cell precursor acute lymphoblastic leukemia
Authors: Bastos Ana Clara Santos da Fonseca , Blunck Caroline Barbieri Blunck , Ferreira Luciana Bueno , Pombo-de-Oliveira Maria do Socorro , Emerenciano Mariana , Gimba Etel R.
Published: August 2018

Osteopontin (OPN) suffers alternative splicing generating three OPN splicing isoforms (OPN-SI), named OPNa, OPNb and OPNc. However, OPN-SI roles in hematological malignancies (HMs) are still under investigation. Detection of specific gene rearrangements allows the identification of relevant prognostic subgroups of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Patients harbouring the ETV6-RUNX1 rearrangement are classified as a good prognostic subgroup, while those with the KMT2A-AFF1 fusion are included into poor prognostic subgroup. This work aimed to analyze the expression levels of OPN-SI in BCP-ALL cell lines and patient samples. We tested 4 BCP-ALL cell lines: 207, ALL-PO, RS4;11 and REH. Patient samples included 28 samples from patients harbouring the ETV6-RUNX1 fusion and 26 samples presenting the KMT2A-AFF1 rearrangement. We found that these three OPN-SIs are expressed in all BCP-ALL cell lines and in 70% of both patient prognostic subgroups tested. Both 207 and REH cell lines displayed higher OPNc and OPNb levels than OPNa. We found that OPNc is expressed in higher levels than OPNb and OPNa in unfavorable prognostic subgroup and is also overexpressed in relation to OPNc levels in relation to the ETV6-RUNX1 fusion the comparative subgroup. Interestingly, we found that higher OPNc transcriptional levels are associated with poor prognostic features, such as central nervous system (CNS), initial white blood cell counting and high risk of relapse. In conclusion, we found that the three tested OPN-SI are expressed in most tested BCP-ALL cell lines and BCP-ALL human samples. Since higher OPNc levels are associated to prognostic features, our data provide early evidence that these OPN-SIs could specifically contribute to step BCP-ALL progression. Further work should investigate their putative applications as additional risk-stratification and prognostic markers for BCP-ALL, as well as, their roles on modulating HM progression.

Citation Format: Ana Clara Santos da Fonseca Bastos, Caroline Barbieri Blunck Blunck, Luciana Bueno Ferreira, Maria do Socorro Pombo-de-Oliveira, Mariana Emerenciano, Etel R. Gimba. Expression of osteopontin splicing isoforms in childhood B-cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1491.