bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2319: The role of CDKN1A and CDKN2B in the reprogramming of AML cells toward cell cycle arrest
Authors: Roberts Michael , Crossland Grace , Forrester Jeffrey
Published: August 2018

AML is the most common acute leukemia in adults, showing rapid progression if left untreated. With current treatments, AML patients frequently achieve remission, but often experience relapse leading to the overall poor prognosis and high mortality of this cancer. We have employed human AML cell lines to examine the response of leukemia cells to phorbol esters. Phorbol esters mimic the action of diacyl glycerol (DAG), an activator of protein kinase C, which regulates several signal transduction pathways and cellular metabolic activities. In response to the phorbol ester PMA, AML cells undergo cell cycle arrest, differentiate into “normal” macrophage-like cells, and ultimately undergo programmed cell death. The transcriptome variations that accompany these changes have been explored by DNA microarray analysis and quantitative real-time PCR, revealing ~1250 genes that are differentially expressed as the cells transition from the cancer state to non-dividing, myeloid cells that initiate apoptosis. Two genes of great interest regarding cell cycle arrest are the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. We have shown that both of these genes are expressed at extremely low levels in AML cells, but are rapidly up-regulated during the PMA response, in keeping with their proposed function as tumor suppressor genes. We present here results from the transfection of expression plasmids containing the CDKN1A or CDKN2B genes into HL-60 cells, showing ~100 and ~1,000-fold overexpression, respectively. In addition, we have engineered a plasmid containing both genes and demonstrate their co-overexpression. RNA samples from cells transfected with CDKN1A, CDKN2B, and CDKN1A-CDKN2B have been subjected to RNA sequencing. RNA-seq analysis reveals changes in CDKN1A and CDKN2B-mediated gene expression, altering the AML transcriptome in ways that facilitate cell cycle arrest. Finally, we present results on the phenotypic and transcriptome changes of a stable HL-60 cell line containing an inducible CDKN2B gene after dox-induced overexpression, implicating CDKN2B as an important regulator of cell cycle progression in AML cells.

Citation Format: Michael Roberts, Grace Crossland, Jeffrey Forrester. The role of CDKN1A and CDKN2B in the reprogramming of AML cells toward cell cycle arrest [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 2319.
Abstract 2325: Protein homeostasis adaptation to Hsp70 inhibition in cancer
Authors: Sannino Sara , Guerriero Christopher J. , Sabnis Amit J. , Bivona Trever G. , Brodsky Jeffrey L.
Published: August 2018

Cancer cells experience acute stress conditions due to their increased proliferation rate and synthesis of misfolded proteins. However, by modulating the mechanisms that control protein-folding, cancer cells avoid cell death. Thus, it is perhaps not surprising that molecular chaperones, like Hsp70, are upregulated in cancer cells compared to their normal counterparts. These data suggest that these chaperones are potential targets for cancer therapy. Previous work in our lab demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that treatment of on RMS13 cell line with an Hsp70 inhibitor, known as MAL3-101, activates the PERK driven unfolded protein response that results in CHOP-dependent cell death (Sabinis et al., 2016). Nevertheless, the mechanism underlying how Hsp70 inhibition triggered apoptosis was poorly understood. By taking advantage of a MAL3-101-resistant cell line (RMS13-R), we have now determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying the increased demand on two protein degradation pathways upon inhibition of Hsp70. Specifically, autophagy related genes were upregulated, and increased conversion of LC3BI to LC3BII and accumulation of LC3BII was detected in RMS13-R cells. Further experiments demonstrated that autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components. Finally, we discovered that only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, which was apparent from an induction of apoptotic markers and cell death. These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, the unfolded protein response, and autophagy in rhabdomyosarcoma.

Citation Format: Sara Sannino, Christopher J. Guerriero, Amit J. Sabnis, Trever G. Bivona, Jeffrey L. Brodsky. Protein homeostasis adaptation to Hsp70 inhibition in 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 2325.
Abstract 2335: Disulfiram overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2/Akt signaling in HER2-positive breast cancer
Authors: Kim Yoon-Jae , Cho Youngkwan , Sung Daeil , Oh Eunhye , Cho Tae-Min , Jang Seojin , Kim Ji Young , Seo Jae Hong
Published: August 2018

Although trastuzumab has shown a significant clinical benefit for HER2-positive breast cancers, responses are limited by the frequent emergence of resistance. Trastuzumab resistance is a multi-factorial phenomenon thought to arise from the presence of cancer stem cells and interactions between truncated p95HER2 and HER family members. Disulfiram (DSF), an anti-alcoholism drug, is known to elicit cytotoxicity in numerous cancer cell types in the presence of copper (Cu). We sought to investigate the effect of DSF on apoptosis, HER2/Akt signaling, cancer stem-like properties and trastuzumab resistance in HER2-positive breast cancer cells in vitro and in vivo. DSF/Cu treatment induced apoptosis, associated with a significant downregulation of HER2, truncated p95HER2, phospho-HER2, HER3, phospho-HER3 and phospho-Akt levels in both trastuzumab-sensitive and -resistant cell lines. This was accompanied by the eradication of cancer stem-like populations, concomitant with the suppression of aldehyde dehydrogenase 1 (ALDH1) activity and mammosphere-forming ability. DSF administration also caused significant tumor suppression in trastuzumab-resistant xenografts, coinciding with the downregulation of BCSC-related markers and intracellular HER2 in vivo. These findings highlight the mechanisms of action of DSF in overcoming trastuzumab resistance in HER2-positive breast cancer.

Citation Format: Yoon-Jae Kim, Youngkwan Cho, Daeil Sung, Eunhye Oh, Tae-Min Cho, Seojin Jang, Ji Young Kim, Jae Hong Seo. Disulfiram overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2/Akt signaling in HER2-positive breast 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 2335.
Abstract 2342: Digital gene expression of up to 96 targets in 96 samples for cell line screening with nCounter® PlexSet™
Authors: Ong Giang T. , Merritt Chris , Kharkia Anisha , Webster Philippa
Published: August 2018

Cell line screening studies require highly efficient protocols for studying many samples in parallel. We have developed a lyse-and-go protocol for digital gene expression profiling of 96-samples by 96-genes in parallel. This protocol is based on nCounter® PlexSet™ Reagents, which enable multiplexing of samples and gene targets. The NanoString® nCounter Analysis platform uses a novel molecular barcoding technology to measure multiplexed gene expression. The assay counts fluorescent barcodes hybridized to targets to provide precise digital data. This platform is used in a wide variety of research applications. The standard nCounter gene expression assay can be used to process 12 samples per run. We recognize an additional need for a higher sample-throughput assay that would enable researchers to quickly evaluate up to 96 multiplexed targets in samples processed in a 96-well plate-based format. The PlexSet Reagent protocol is simple and fast and can be divided into 3 steps: 1) lyse cells, 2) set-up hybridization with crude lysate, 3) initiate automated post-hybridization processing and digital counting. The data presented are crude cells lysates from various PBMC cell types, two suspension cell lines, and two adherent cell lines using the IO Lymphocyte Activity Panel to measure the gene expression of 5 housekeeper genes and 91 lymphocyte activity genes (96 genes in total). The data demonstrate that up to 96 high-quality, digital data points for gene expression in each of 96 samples can be generated from crude cell lysates in an efficient, high-throughput protocol and that the cell lysate data correlate well with purified total RNA.

Citation Format: Giang T. Ong, Chris Merritt, Anisha Kharkia, Philippa Webster. Digital gene expression of up to 96 targets in 96 samples for cell line screening with nCounter® PlexSet™ [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 2342.
Abstract 2346: ARDE: Detecting selectively expressed cancer driver mutations through integration of exome and transcriptome sequencing data
Authors: Wang Fang , Zhang Shaojun , Chen Yiwen , Hart Traver , Shaw Kenna , Meric-Bernstam Funda , Mills Gordon , Chen Ken
Published: August 2018

Integrating multi-omics data from cancer patient samples can potentially advance the discovery of cancer driver mutations (CDM) towards establishing a knowledgebase for cancer precision medicine. However, it remains unclear whether CDMs are selectively expressed at RNA levels unexpected from DNA copy numbers and whether identifying such selectivity will enable more accurate discovery of CDMs. To answer this question, we developed a novel statistical algorithm, ARDE (Allelic RNA DNA expression), which estimates tumor purity, allele-specific copy number and intratumor heterogeneity (ITH) from matched whole-exome and whole-transcriptome sequencing data, deconvolutes tumor allele-specific expression levels (ASEL) and uses a two-component mixture probability model to identify selectively expressed alleles (SEAs) in the tumor compartment of heterogeneous tumor tissues. We applied ARDE to analyze melanoma and breast samples from The Cancer Genome Atlas (TCGA). Tumor purities estimated by ARDE were highly correlated with estimation from pathology review, DNA methylation array, gene expression microarray and SNP array data. ITH scores can significantly stratify breast cancer subtypes and survival time of melanoma patients. The deconvoluted tumor ASEL showed consistently higher correlation with the averaged gene expression levels in tissue-matched single tumor cells than in normal cells. Higher proportions of somatic mutations are selectively expressed than germline. Top SEAs are highly enriched in deleterious and gain-of-function (GOF) mutations. Stratifying melanoma patients by the expression selectivity of BRAF V600E mutation resulted in a 10-fold improvement of significance in survival analysis. These results indicate that ARDE will likely be an effective, widely applicable approach for accurate discovery of CDMs from tumor tissue sequencing data. (See Table 1.)

Table 1. Top 10 significant positive-selectively expressed somatic mutations

MelanomaBreast cancerGenepSNVFunctionSIFTGenepSNVFunctionSIFTBRAFp. V600EGOFDAKT1p.E17KGOFDNRASp. Q61RGOFDPIK3CAp.H1047RGOFTCDKN2Ap. R114LGOFDERBB2p.L755SGOFDIDH1p. R132CSwitch of functionDTP53p.R273HGOFDEEF1A1p. H349YDGATA3p.M293KDRAC1p. P29SGOFDTP53p.P278TDNRASp. Q61KGOFDFOXA1p.S250FDSF3B1p. R625HGOFDTP53p. R175HGOFDNRASp. Q61LGOFDPIK3CAp.H1047LGOFTMAP2K1p. P124SGOFDERBB2p.V777LGOFT

Citation Format: Fang Wang, Shaojun Zhang, Yiwen Chen, Traver Hart, Kenna Shaw, Funda Meric-Bernstam, Gordon Mills, Ken Chen. ARDE: Detecting selectively expressed cancer driver mutations through integration of exome and transcriptome sequencing data [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 2346.
Abstract 2348: Low-cost and accurate human leukocyte antigen (HLA) class I typing of The Cancer Genome Atlas on the Seven Bridges Cancer Genomics Cloud
Authors: Malhotra Raunaq , Krasnitz Alexandar , Sethi Anurag , Lehnert Erik , Williams Elizabeth H. , Brandi Davis-Dusenbery N.
Published: August 2018

The Cancer Genome Atlas (TCGA)network has generated more than2.5petabytes of genomic dataover the last decade, with

petabyte-scale additions of dataexpected inthe coming years. Access and analysis of this informationonalocal compute

environment is challenging due tothe volume of dataand lack of sufficient computing resources at many research organizations.

The Cancer Genomics Cloud Pilot project from the National Cancer Institute (NCI)has helped indemocratizing access toTCGA

by co-localizing datawith computational resources onthe cloud. Funded as part of this project, the SevenBridges Cancer

Genomics Cloud (CGC)hosts nearly 5petabytes of public datafrom TCGA, the Simons Genome Diversity Project, the

Therapeutically Applicable Research toGenerate Effective Treatments (TARGET)initiative, The Cancer Imaging Archive (TCIA),

and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CGCprovides academic researchers with asecure,

scalable, cloud-based cancer research platform that includes collaborative tools for accessing, uploading, analyzing, and

visualizing data. The platform uses resource descriptionframeworks, dataharmonization, and metadatacurationtofacilitate

effective querying, and bioinformatics tools are implemented onthe CGCusing the CommonWorkflow Language (CWL), an

emerging standard for describing computational workflows, tosupport computational reproducibility. Since its launch in2016, the

CGChas enabled researchers from around the world tounderstand humangenetics and cancer biology through the analysis of

large public datasets and private datainacloud computing environment. Inthis poster, we present anexample of analysis of

TCGAdataonthe CGC. The OptiType tool for HumanLeukocyte Antigenclass I typing was used toprofile 8,872RNA-Seq

samples present inthe TCGAdataset. All samples were accurately processed within2days using arobust, fault-tolerant, and

cost-efficient CWL descriptionof OptiType that enabled analysis for less than50cents per sample onaverage. This case study

demonstrates how cloud computing resources canfacilitate the successful analysis of large cohorts of datausing custom

pipelines in a robust, scalable, and reproducible manner.

Citation Format: Raunaq Malhotra, Alexandar Krasnitz, Anurag Sethi, Erik Lehnert, Elizabeth H. Williams, Davis-Dusenbery N. Brandi. Low-cost and accurate human leukocyte antigen (HLA) class I typing of The Cancer Genome Atlas on the Seven Bridges Cancer Genomics Cloud [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 2348.
Abstract 2368: The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia
Authors: O'Leary Anna K. , Wei Min , Blackburn Jessica
Published: August 2018

Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers that is diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high-dose cytotoxic chemotherapies that ALL patients are given have long-term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large-scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. Upregulation of PRL3 is correlated progression of colorectal, breast, and gastric cancers, and is associated with poor patient prognosis. We have found that PRL3 is highly expressed in a subset of patient ALL samples, and I am examining the role of PRL3 in ALL progression. We have found that PRL3 expression enhances ALL proliferation, and promotes leukemia survival after cytotoxic chemotherapy treatment. Small-molecule inhibition of PRL3 induces apoptosis in ALL, and synergizes with common ALL chemotherapies. I am currently examining the localization of PRL3 and identifying signaling pathways that it may be involved with to promote ALL progression. If my project is successful, it will provide scientific data that support our hypothesis that PRL3 is related to acute lymphoblastic leukemia. If there is a correlation between PRL3 and ALL, PRL3 could be used as a more effective and less toxic treatment for patients with ALL.

Citation Format: Anna K. O'Leary, Min Wei, Jessica Blackburn. The phosphatase PRL3 as a novel therapeutic target in 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 2368.
Abstract 2369: AMPK regulation of apoptosis through OSBP
Authors: Ong Kai Li , Thurgood Trever , Christensen Michael , Malmrose Jonny , Ng Denise , Grose Julianne
Published: August 2018

Alterations in lipid metabolism have been frequently reported in tumor progression, with impaired PI(4,5)P2 homeostasis in the plasma membrane implicated in various cancers and metabolic disorders. Oxysterol binding protein (OSBP) and its related proteins (ORPs) constitute a large family of ubiquitously expressed, evolutionarily conserved lipid binding proteins that play critical roles in intracellular lipid trafficking. OSBP and ORPs associate with a wide range of cellular activities, however their functions and regulations are largely unclear. Emerging evidence has supported an essential role of OSBP in cell signaling pathways and cancer development. We recently identified yeast oxysterol binding protein Osh7 as a true substrate of yeast AMPK (Snf1). AMPK's involvement in cancer has been extensively studied in the past decade, and is an important mediator of cellular energy homeostasis. Despite many downstream targets identified, AMPK is not known to regulate oxysterol binding protein activities. We found that Snf1 may be responsible for the degradation of Osh7 and cells deficient in Osh7 and its homolog Osh6 display higher rates of apoptosis. Understanding the regulation of OSBP by AMPK may aid in better targeting AMPK for cancer treatment and prevention, at the same time provide a better understanding to the roles of OSBP in cancer development.

Citation Format: Kai Li Ong, Trever Thurgood, Michael Christensen, Jonny Malmrose, Denise Ng, Julianne Grose. AMPK regulation of apoptosis through OSBP [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 2369.
Abstract 2384: Preclinical evaluation of PD and efficacy of novel potent selective and orally bioavailable CDK12 covalent inhibitors in TNBC model
Authors: Poddutoori Ramulu , Rajagopalan Sujatha , Mukherjee Subhendu , Marappan Sivapriya , S Samiulla D , Sivakumar Sasirekha , Nayak Shilpa S. , V Ravindra M. , Murtuza Hadianawala , S Devaraja T. , Kondela Srinivas , Tgore Suraj , Dhudashiya Amit A. , B Charamanna K. , Antony Thomas , Daginakatte Girish , Giri Sanjeev , Chelur Shekar , Ramachandra Murali , Pandit Chetan , Samajdar Susanta
Published: August 2018

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes such as DNA damage response, splicing and pre-mRNA processing. In association with Cyclin K (CycK), CDK12 regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at Serine 2 in the C-terminal domain (CTD). Overexpression of CDK12 in various tumor types suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. Considering its critical role in transcription and RNA processing CDK12 is emerging as a potential therapeutic target for cancer. Multiple series of potent and selective CDK12 covalent inhibitors were identified by iterative medicinal chemistry efforts and SAR-based approaches. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumour activity. Very potent and highly selective CDK12 inhibitors have been identified from two distinct chemical series. The covalent mode of action for these biochemically potent compounds has been confirmed by CDK12 target engagement assay in the cellular context. These selective inhibitors showed significant anti-proliferative activity in TNBC and other cancer cell lines, which correlated with inhibition of pS2 (RNAP II), a bonafide CDK12 substrate and target engagement. In vivo target engagement, PD and efficacy data for optimized compounds with good oral bioavailability in a TNBC (HCC-70) xenograft model along with will be presented.

Citation Format: Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D S, Sasirekha Sivakumar, Shilpa S. Nayak, Ravindra M. V, Hadianawala Murtuza, Devaraja T. S, Srinivas Kondela, Suraj Tgore, Amit A. Dhudashiya, Charamanna K. B, Thomas Antony, Girish Daginakatte, Sanjeev Giri, Shekar Chelur, Murali Ramachandra, Chetan Pandit, Susanta Samajdar. Preclinical evaluation of PD and efficacy of novel potent selective and orally bioavailable CDK12 covalent inhibitors in TNBC model [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 2384.
Abstract 2387: The oncogenic role and therapeutic potential of PTPN11 in melanoma
Authors: Hill Kristen Suzanne , Roberts Evan , Marin Ellen , Wang Xue , Teer Jamie , Messina Jane , Wu Jerry , Kim Minjung
Published: August 2018

The RAS/RAF/MEK/ERK signaling pathway, which is intricately regulated by multiple proteins including PTPN11 (Tyrosine-Protein Phosphatase Non-Receptor Type 11, encoding SHP2), is frequently activated in melanoma. Although implicated as an oncogene in multiple cancer types, the oncogenic role of PTPN11 has not been established in melanoma. Recently, we preformed whole exome sequencing on tumors generated by a mouse model driven by loss of PTEN and CDKN2A (INK4A/ARF) and identified several conserved cross-species orthologous mutations in Kras, Erbb3, and Ptpn11. In this study, we addressed the functional roles of PTPN11 in melanoma tumorigenesis and tumor maintenance; as well as, PTPN11's effect on the RAS/RAF/MEK/ERK signaling pathway and its activation status in human melanoma.

PTPN11 can be activated by receptor tyrosine kinases (RTKs) or by point mutations. We observed activating phosphorylation on Tyr 542 of PTPN11 in 40% (n=15/38) of melanoma specimens and the majority of human melanoma cell lines (n=14), supporting frequent activation of PTPN11 in human melanoma even though the mutation rate is low (1~3%). PTPN11 knock-down suppressed ERK activation in NRAS mutant and BRAF/NRAS wild-type melanoma cells, but not in BRAF mutant cells. Moreover, we have shown that expression of active PTPN11E76K mutant drives soft-agar colony growth in vitro, tumor growth in nude mice, RAS/RAF/MEK/ERK activation, and resistance to MEK inhibition. Alternatively, knock-down of Ptpn11 reduces colony growth and ERK activation. We generated a tet-inducible, melanocyte-specific, PTPN11E76K transgenic mouse model in a PTEN and CDKN2A null background and observed melanoma formation. Implantation of melanoma cells derived from this model showed doxycycline dependent tumor growth in nude mice. Doxycycline withdrawal and subsequent extinction of PTPN11E76K caused regression of established tumors, supporting the tumor maintenance role of PTPN11. Subsequently, tumor tissue from this model underwent phosphor-tyrosine proteomic analysis to identify downstream effectors of PTPN11's protein tyrosine phosphatase activity. The proteins identified in this analysis are currently being confirmed. These data support the oncogenic roles of PTPN11 in melanoma by regulating RAS/RAF/MAPK pathway activation and the value of PTPN11 as a novel and actionable therapeutic target.

Citation Format: Kristen Suzanne Hill, Evan Roberts, Ellen Marin, Xue Wang, Jamie Teer, Jane Messina, Jerry Wu, Minjung Kim. The oncogenic role and therapeutic potential of PTPN11 in melanoma [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 2387.
Abstract 2394: Racial disparity in bladder cancer and identification of altered metabolism in African American compared to European American bladder cancer
Authors: Vantaku Venkata Rao , Donepudi Sri Ramya , Dorsey Tiffany , Putluri Vasanta , Ambati Chandrashekar , Wei Tang , Ittmann Michael M. , Terris Martha K. , Lerner Seth , Ambs Stefan , Lotan Yair , Sreekumar Arun , Putluri Nagireddy
Published: August 2018

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2018 Official Press Program. It will be posted online following its presentation.

Citation Format: Venkata Rao Vantaku, Sri Ramya Donepudi, Tiffany Dorsey, Vasanta Putluri, Chandrashekar Ambati, Tang Wei, Michael M. Ittmann, Martha K. Terris, Seth Lerner, Stefan Ambs, Yair Lotan, Arun Sreekumar, Nagireddy Putluri. Racial disparity in bladder cancer and identification of altered metabolism in African American compared to European American bladder 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 2394.
Abstract 2410: RIPK1-mediated induction of mitophagy compromises the viability of extracellular matrix-detached cells
Authors: Hawk Mark A. , Schafer Zachary T.
Published: August 2018

For cancer cells to survive during ECM-detachment, they must inhibit anoikis and rectify metabolic deficiencies that cause non-apoptotic cell death. Previous studies in ECM-detached cells have linked non-apoptotic cell death to ROS generation, although the mechanistic underpinnings of this link remain poorly defined. Here, we uncover a novel role for receptor-interacting protein kinase-1 (RIPK1) in the modulation of mitochondrial ROS levels and cell viability during ECM-detachment. We find that RIPK1 activation during ECM-detachment results in the induction of PINK1-dependent mitophagy through a mechanism requiring the mitochondrial phosphatase PGAM5. As a consequence of mitophagy induction, ECM-detached cells experience diminished IDH2-mediated NADPH production in the mitochondria and the subsequent elevation in mitochondrial ROS levels leads to non-apoptotic cell death. Furthermore, we find that antagonizing RIPK1 or PGAM5 enhances tumor formation in vivo. Intriguingly, upon mining a meta-analysis of published lung cancer patient microarray data sets, we find that low RIPK1 expression in lung cancer patients correlates with decreased overall and progression-free survival. Thus, RIPK1-mediated induction of mitophagy may be an efficacious target for novel therapeutics aimed at specifically eliminating ECM-detached cancer cells and deterring tumor progression.

Citation Format: Mark A. Hawk, Zachary T. Schafer. RIPK1-mediated induction of mitophagy compromises the viability of extracellular matrix-detached 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 2410.
Abstract 2416: CREB orchestrates metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer
Authors: Xu Jun , Jin Nan , Huang Min , Ding Jian , Geng Meiyu
Published: August 2018

Aberration of platelet-derived growth factor receptor A (PDGFRA) gene has been increasingly identified in multiple types of human cancers, including hematologic malignancies, gastrointestinal stromal tumor (GIST), medulloblastomas and gliomas. In particular, amplification of 4q12 that contains PDGFRA locus has been reported in 3% to 7% of lung adenocarcinomas and 8% to 10% of lung squamous cell carcinomas. However, it remains unclear how amplified PDGFRA drives malignancy of lung squamous cell cancer. To address this question, we used a lung squamous cell cancer cell line NCI-H1703, which has been previously reported to have 24-fold amplification of the 4q12 region. Either siRNA knockdown or small-molecule inhibition of PDGFRA significantly inhibited cell growth, suggesting that PDGFRA was required for driving cell proliferation. We further showed that both oxidative phosphorylation and aerobic glycolysis were significantly affected followed by interference of PDGFRA, by measuring of oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR). In particular, ATP generation level and maximum respiratory capacity, the key parameters of mitochondrial function, were significantly decreased in mitochondrial pressure testing. These observations suggested that oncogenic PDGFRA drove the metabolic reprogramming and promoted tumor malignancy in lung squamous cell cancer. To further explore the regulatory mechanism of the observed metabolic phenotype, we took the approach of genetic sequencing technology to analyze the transcriptome in PDGFRA-driven NCI-H1703 cells treated with or without PDGFRA inhibitor. It revealed that PDGFRA-driven transcriptional regulation of metabolic genes underpinned the specific metabolic phenotypes of mitochondrial function. In addition, analysis of transcriptome as well as a phosphorylated kinase array revealed that a transcription factor, cAMP response element-binding protein (CREB), regulated the metabolic gene expression in PDGFRA-addicted cells. Knockdown of CREB disrupted metabolic gene expression and the mitochondrial function, consistent with aforementioned findings of PDGFRA, suggesting that transcriptional regulation of metabolic genes was orchestrated by CREB. Together, we herein firstly provide the molecular link between oncogenic PDGFRA and metabolic vulnerability and reveal the important role of CREB in orchestrating metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer.

Citation Format: Jun Xu, Nan Jin, Min Huang, Jian Ding, Meiyu Geng. CREB orchestrates metabolic reprogramming in PDGFRA-addicted lung squamous cell 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 2416.
Abstract 2417: eIF4E phosphorylation licenses Myc-driven metabolic and stress reprogramming
Authors: Ruan Hang , Li Xiangyun , Leibowitz Brian , Tong Jingshan , Lu Xinghua , Sonenberg Nahum , Xu Xiang , Zhang Lin , Yu Jian
Published: August 2018

eIF4E, Eukaryotic Translation Initiation Factor 4E, regulates the rate limiting cap-binding step in mRNA translation. Oncogenic pathways converge to increase eIF4E expression, as well as phosphorylation of eIF4E (S209) and its inhibitor 4EBPs, which are believed to facilitate the translation of “weak'' mRNAs including cell type-specific and oncogenic targets. eIF4E phosphorylation, but not eIF4E, is dispensable for normal development. Pharmacologic inhibition of either p-eIF4E or p-4EBP has limited antitumor efficacy. Our current study aims to better define the role of p-eIF4E in colon cancer. We find that p-eIF4E, not eIF4E transcript or protein is elevated in human colon tumors and precursors. Knockin (KI) of phosphorylation-defective eIF4E mutant allele (4ES209A) in human colon cancer cells strongly suppresses Myc translation and cell growth under stress or in mice, while minimally impacting Cap binding or global translation. Surprisingly, p-eIF4E is found to be required to maintain p-4EBP1 levels, dissociate eIF4E from 4E-BP1, and stimulate glutamine metabolism via integrated stress response (ISR). Further, p-eIF4E and p-4EBP1 are highly elevated in the polyps in APC min/+ mice compared to adjacent normal intestinal crypts, and 4ES209A KI significantly inhibits intestinal polyposis and glutamine metabolism gene signatures. Finally, p-eIF4E renders CRC cells addicted to glutamine and glutaminolysis by preventing Myc- and ISR-driven metabolic crisis and cell death. In summary, we demonstrate that eIF4E phosphorylation is central to oncogenic translation, and is required to license Myc-driven metabolic adaptation and addiction via ISR-dependent transcriptional programs in colon cancer. These novel findings help reconcile the long-standing and context-dependent roles of ISR in cancer cell growth, death, plasticity and heterogeneity, and provide new ways for its therapeutic exploitation.

Citation Format: Hang Ruan, Xiangyun Li, Brian Leibowitz, Jingshan Tong, Xinghua Lu, Nahum Sonenberg, Xiang Xu, Lin Zhang, Jian Yu. eIF4E phosphorylation licenses Myc-driven metabolic and stress reprogramming [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 2417.
Abstract 2430: Targeting Nrf2 in esophageal adenocarcinoma sensitizes cancer cells to cisplatin treatment
Authors: Peng Dunfa , Hu Tianling , Zhu Shoumin , El-Rifai Wael
Published: August 2018

Background: The incidence rate of the esophageal adenocarcinoma (EAC) has increased rapidly among men since 1976 in the USA. EAC is considered to originate from premalignant Barrett's esophagus (BE) which results from chronic gastroesophageal reflux disease (GERD). It has been reported that oxidative stress is steadily increased in GERD-BE-EAC progression cascade. NFE2-related factor 2 (Nrf2) is a major regulator of cellular anti-oxidant properties that maintains cell viability and cellular homeostasis.

Methods and Results: Using Western blot analysis, we detected overexpression of Nrf2 protein in several EAC cell lines, as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines. Nrf2 overexpression was also confirmed in primary EAC samples, using immunohistochemistry on a tissue microarray that contained 76 EACs. Knockdown of Nrf2 by siRNA or CRISPR/CAS9 in EAC cells (FLO1 and OE33), that have constitutive high levels of Nrf2, enhanced the sensitivity of cancer cells to cisplatin (CDDP). We confirmed these results by using a Nrf2 specific inhibitor (Brusatol). Further studies demonstrated that dysfunction of Nrf2, either through knockdown of Nrf2 expression or using its specific inhibitor, depleted several Nrf2 target genes such as GR, HO-1, GSTM2, GPX3 and GPX4, which have anti-oxidative properties, leading to a significant increase in cellular ROS levels. The excessive ROS levels resulted in an increase in oxidative DNA damage (measured by 8-oxoguanine) and double strand breaks (measured by p-H2AX) which were further increased by treatment with CDDP with subsequent increase in cancer cell death.

Conclusion: These results indicate that constitutive overexpression of Nrf2 in EAC cells protects tumor cells from high level of ROS and favors tumor cell survival and drug resistance. Targeting Nrf2 through its specific inhibitor may have a therapeutic window in EAC. Additional studies are ongoing to determine the molecular mechanisms underlying NRF2 overexpression in EACs.

Citation Format: Dunfa Peng, Tianling Hu, Shoumin Zhu, Wael El-Rifai. Targeting Nrf2 in esophageal adenocarcinoma sensitizes cancer cells to cisplatin treatment [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 2430.
Abstract 2438: Reducing plate edge effect by controlling cell handling conditions for in vitro tumor hypoxia assays
Authors: Henn Alicia , Darou Shannon , Yerden Randy
Published: August 2018

To reduce the impact of high variability in the edge wells of 96-well plates, the "edge effect", researchers often fill edge wells with buffer to try to insulate the rest of the plate. This removes over a third of the wells from the assay, adding cost, handling time, and space problems to in vitro assays. This is of particular concern for researchers working with cells in low oxygen, as handling more plates increases the time that cells are out of optimum conditions, risking HIF-1a modulation. For this project, we tested the idea that controlling the entire cell handling space to conditions that match the incubator, including the working space floor temperature and gas levels, may help reduce the edge effect seen in 96-well plates. The null hypothesis was that cell handling conditions would make no difference in the variability of cell density in 96-well plates loaded in traditional room air conditions compared to plates loaded in controlled conditions matching the incubator. To address cell loading specifically, A549 cells were harvested from cell culture flasks in traditional cell handling conditions (room temp, 20% O2, 0% CO2), then split. Half of the cells were transferred to an Xvivo System for controlled cell handling conditions. The cells in both conditions were transferred to media pre-conditioned to hypoxic incubator conditions and then plated in 96-well plates. Temperature mapping was performed with 16 thermal probes simultaneously in replicate plates loaded with medium only. The cells were incubated for 24 hours, then stained for cell density, and read on a plate reader. Cell viability was greater than 90% (n=4 plates/condition/trial x 4 trials). We found that edge well temperatures were more stable in plates handled in unbroken optimal conditions and edge effect on cell density was greatly reduced. In room temperature conditions, columns 1 and 12 cooled most rapidly. We concluded that full-time control of environmental conditions allowed for use of the whole plate with reduced edge effect. This helps reduce cost, time, number of plates and the risks to cells during cell handling.

Citation Format: Alicia Henn, Shannon Darou, Randy Yerden. Reducing plate edge effect by controlling cell handling conditions for in vitro tumor hypoxia 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 2438.
Abstract 2441: Effects of hypoxia on Src family members' activity in prostate cancer cells
Authors: Dai Yao , Siemann Dietmar
Published: August 2018

Metastasis is the major reason of disease progression and poor prognosis in prostate cancer. Src family kinases (SFKs), including c-Src, Lyn and Fyn, are non-receptor tyrosine kinases that have been shown to play an essential role in castration resistance and metastasis in prostate cancer. Hypoxia, a typical hallmark of solid malignancies, is able to promote metastasis-associated functions. However, whether SFK members are upregulated under hypoxia is unclear. The current project will detect the impact of hypoxia on cell functions and molecular features in prostate cancer cells. For hypoxic exposure, prostate cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h). To test cell migration, “wound-healing” assay was used. Cell invasion was detected by seeding cells into Matrigel-coated transwell chamber and cells on the bottom membranes were counted after treatment. Clonogenic assay was performed to test cell survival. At the molecular level, SFKs' phosphorylation in cancer cells and patient tissues was detected by Western blotting, and gene knockdown was accomplished by siRNA transfection. To detect the effects of Src inhibitors, cells were treated with saracatinib for indicated time periods. Phosphorylation of all three typical SFKs, e.g., c-Src, Lyn and Fyn, were highly expressed in prostate cancer patient tissues. At the cellular level, while short term hypoxic exposure (2-6 h) induced greater effects than prolonged hypoxia (24 h) in PC-3ML, these cellular functions were increased with 24 h hypoxic exposure in C4-2B cells. Further, hypoxia enhanced SFK phosphorylation in the pattern that was consistent with cell functions in both cell lines. Knockdown SRC, but not LYN or FYN, abolished hypoxia-induced invasion and p-SFK expression. Lastly, SFK inhibitor saracatinib showed stronger inhibition on functional behaviors facilitated under hypoxia than normal conditions. Our data show that hypoxia is able to enhance metastatic phenotypes by activating SFKs in prostate cancer cells. Interestingly, c-Src may be the most important signaling molecule for hypoxia-mediated behaviors. More importantly, SFK inhibitors are able to impair hypoxia-induced cell functions, suggesting their therapeutic potential by suppressing tumor metastasis that is driven under hypoxia in prostate cancer.

Citation Format: Yao Dai, Dietmar Siemann. Effects of hypoxia on Src family members' activity in prostate 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 2441.
Abstract 2451: Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27
Authors: ZHANG YUNA YUAN , Yan Xu Guang , Farrelly Margaret , Yari Hamed , Feng Yuchen , La Ting , Tabatabaee Hessam , Jin Lei , Zhang Xu Dong
Published: August 2018

Evading apoptosis and limitless replicative potential are two hallmarks of malignant cells. Here we report that melanoma cells surviving the induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77 have even higher proliferative potential, and that this is associated with the increased expression of the long noncoding RNA (lncRNA) OVAAL which competes with p27 mRNA for binding to polypyrimidine tract binding protein 1 (PTBP1) and thus represses p27 mRNA translation. Prolonged exposure to TRAIL or Mcl-1 resulted in stable melanoma cell populations with increased proliferation activity. Comparative RNA-seq analysis identified OVAAL as the most significantly upregulated lncRNA in melanoma cells resistance to TRAIL and Mcl-1. ShRNA knockdown of OVVAL reversed the increased proliferative potential of resistant cells, and also inhibited proliferation in a subset of parental melanoma cell lines that constitutively expressed relatively high levels of OVAAL. RNA pulldown followed by Mass Spectrometry analysis revealed that among proteins that were physically associated with OVAAL was a protein called PTBP1 that bound to and enhanced the activity of the internal ribosomal entry site (IRES) in the 5' untranslated region (5'-UTR) of p27 mRNA. Indeed, OVAAL knockdown resulted in an increase in the association between PTBP1 and p27 IRES leading to upregulation of the p27 protein, whereas overexpression of OVAAL caused reduction in binding of PTBP1 to the IRES of p27. Furthermore, co-knockdown of PTBP1 abolished upregulation of p27 triggered by OVAAL knockdown. These results suggest that OVAAL competes with p27 mRNA for binding to PTBP1 and thus inhibits p27 expression and promotes cell proliferation. This was substantiated by binding assays using in vitro-synthesized biotin-labelled OVAAL, p27 IRES and purified PTBP1 in a cell-free system. Our results showed that the increased c-Myc activity was responsible for upregulation of OVAAL in melanoma cells selected for resistance to apoptosis induced by TRAIL or UMI-77. Collectively, these results identify the expression of OVAAL as an important mechanism promoting melanoma cell proliferation, with potential implications for targeting OVAAL in combination with apoptosis-inducing therapeutics in the treatment of melanoma.

Citation Format: YUNA YUAN ZHANG, Xu Guang Yan, Margaret Farrelly, Hamed Yari, Yuchen Feng, Ting La, Hessam Tabatabaee, Lei Jin, Xu Dong Zhang. Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27 [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 2451.
Abstract 2456: The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer
Authors: Baumann Bethany , Gao Shang , Lugli Giovanni , Nonn Larisa
Published: August 2018

Low serum vitamin D is a risk factor for aggressive prostate cancer. piRNAs and their interacting partners, PIWI-like (PIWIL) proteins, are aberrantly expressed in many cancers, but few studies have focused on piRNAs in the prostate. We used laser-capture microdissected (LCM) human prostate epithelium from frozen samples collected for a clinical trial of high dose vitamin D to examine the small non-coding RNA population by next-generation sequencing. The 8 patients for this study were selected for having either high (avg=48.7pg/ml) or low (avg=21.4pg/ml) prostatic vitamin D (1,25(OH)2D) levels. VDR ChIP-seq was performed as well, using human primary epithelial cells treated for 3hr with EtOH or 50nM vitamin D. In LCM-collected prostate epithelium unexpectedly piRNAs represented ~20% of the mapped reads. Of all the small RNA species investigated, piRNAs were the most affected by vitamin D status, increasing in abundance in tissues wish high vitamin D (p=0.036, t-test). The LCM data were compared to small-RNA sequencing data from benign prostate available from The Cancer Genome Atlas (TCGA), which represent whole prostate tissue and may contain stromal bias. The LCM-collected samples had a distinct, more consistent profile of small RNAs than TCGA samples. Two unambiguous piRNAs that were highly expressed in LCM data, but not in TCGA data, were verified as epithelial-specific in prostate by in situ hybridization. Several piRNA-biogenesis genes lie within 100kb of VDR ChIP peaks, including TDRKH, which was found to be vitamin D-regulated experimentally. The expression of piRNA-interacting PIWIL proteins was assessed by RT-qPCR, immunoblot and immunofluorescence on a tissue microarray containing paired benign and cancer tissues (N=23). Of the 4 human PIWIL proteins, PIWIL1, 2 and 4 were present in normal prostate epithelial cells by PCR. PIWIL1 protein was also detected in PrE and RWPE-1 benign cells, but PIWIL1 was not detected in the PC3 prostate cancer cell line. By immunofluorescence, PIWIL1 protein in prostate epithelium significantly decreased in prostate cancer (p=0.016, paired t-test). In conclusion, vitamin D status associated with the expression of piRNA in prostate epithelium in clinical trial samples. PIWIL1 protein levels were lower in prostate cancer, suggesting a pro-differentiation role for the PIWI pathway that may be affected in vitamin D-deficient patients.

Citation Format: Bethany Baumann, Shang Gao, Giovanni Lugli, Larisa Nonn. The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in 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 2456.