AACR 2019 Proceedings: Abstracts 2749-5314

By CTI Meeting Technology

American Association for Cancer Research 2019 Proceedings: Abstracts 2749-5314 - Part B

• Language: English
• Publisher: CTI Meeting Technology
• Release date: Mar 28, 2019
• ISBN: 9780463372722

Book preview

CANCER CHEMISTRY

Cancer Chemical Biology and Therapeutics Development

#2749

Defining structure activity relationships for GPCR engagement and anti-cancer efficacy of imipridone small molecules.

Varun V. Prabhu,¹ Abed Rahman Kawakibi,¹ Neel S. Madhukar,² Lakshmi Anantharaman,³ Sean Deacon,³ Neil S. Charter,³ Mathew J. Garnett,⁴ Ultan McDermott,⁴ Cyril H. Benes,⁵ Wolfgang Oster,¹ Olivier Elemento,² Martin Stogniew,¹ Joshua E. Allen¹. ¹Oncoceutics, Inc, Philadelphia, PA; ²Weill Cornell Medicine, New York, NY; ³Eurofins DiscoverX, CA; ⁴Wellcome Trust Sanger Institute, United Kingdom; ⁵Massachusetts General Hospital, Boston, MA.

G protein-coupled receptors (GPCRs) represent the most widely exploited superfamily of drug targets for FDA-approved therapies for many diseases, however, these receptors are underexploited for oncology. ONC201 is a selective antagonist of GPCRs dopamine receptor D2 (DRD2) and DRD3 that has been shown to induce tumor regressions with a benign safety profile in high grade glioma patients. ONC201 (benzyl-2-methylbenzyl-imipridone) is the founding member of the imipridone class of small molecules that share a unique tri-heterocyclic core chemical structure. Imipridones share several chemical and biological properties that are desirable drug-like characteristics: oral administration, wide therapeutic window, chemical stability and blood brain barrier penetrance. In this study, we profiled a series of imipridones for GPCR engagement and anti-cancer efficacy. Several imipridones were screened against a large panel of human GPCRs using a β-arrestin recruitment assay. The imipridones tested resulted in GPCR agonist/antagonist activity (threshold set at >20% activity) that was heterogenous, but exclusive among Class A GPCRs that represent the largest class. Minor chemical modifications to the ONC201 chemical structure caused large shifts in agonist versus antagonist activity and selectivity for GPCRs. Specifically, switching the ONC201 imipridone core from an angular to a linear isomer resulted in loss of DRD2 antagonist activity and impaired inhibition of cancer cell viability, indicating the imipridone core structure is critical for GPCR engagement and anti-cancer effects. The addition of electron withdrawing groups (e.g. di- or tri-halogen substitution) to the methyl benzyl ring improved potency for GPCR engagement and anti-cancer effects, but not for the benzyl ring. Loss of the benzyl ring impaired anti-cancer effects. Among all of the GPCR hits identified, maximal variance in imipridone GPCR engagement was identified for DRD2/DRD3 antagonism and GPR132 agonism that were prioritized considering their known biological relevance in oncology. ONC206 (benzyl-2,4-difluoromethylbenzyl-imipridone) emerged as the most selective and potent antagonist for D2-like dopamine receptors that are overexpressed and critical for survival in several cancers. ONC212 (benzyl-4-trifluoromethylbenzyl-imipridone) was the most selective and potent agonist for tumor suppressor GPR132. Both compounds were tested in the GDSC panel of >1000 cancer cell lines and demonstrated broad spectrum nanomolar inhibition of cancer cell viability and a wide therapeutic window. GPCR target expression correlated with anti-cancer efficacy in the GDSC panel for both compounds, providing potential biomarkers of response. Thus, chemical derivatization of ONC201 has generated a class of novel GPCR-targeting agents with promising preclinical efficacy and safety profiles in oncology.

#2750

Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer.

Emma Adhikari, Shikha Mahajan, Harshani Lawrence, Yan Yang, Bin Fang, Eric Haura. Moffitt cancer center, Tampa, FL.

DUBs are involved in tumorigenesis and are of high interest as potential targets for cancer therapy. However, target discovery is problematic due to the absence of clear DUB activity within subsets of human cancers. Our goal was to profile DUBs and their activity in KRAS-mutated lung cancer cell lines using chemical biology strategies. Approximately 25% of patients with Lung adenocarcinoma have tumor associated KRAS mutations in non-small lung cancers (NSCLC), yet specific RAS inhibitors against KRAS-mutated lung cancer have not yet been successfully developed.

We used activity-based protein profiling (ABPP) to profile DUBs in 25 distinct human lung cancer cell lines harboring KRAS mutations. ABPP uses chemical probes that are active-site directed and covalently bind to a class of enzymes in complex proteome. Whole cell lysates were incubated with HA-UB-VME and HA-UB-PA probes and ABPP pull-down was performed. Enriched proteins were trypsin digested and peptides were analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). MAXQUANT software was used to quantify DUBs. We related activity of observed DUBs to effects on cell viability by examining publicly available RNAi (Project DRIVE) and CRISPR (PICKLES) databases.

A total of 50 DUBs were identified in our ABPP screen. Each cell line expressed at least 32 different DUBs and 22 DUBs were represented in all cell lines. The identified DUBs include 48% USP, 36% OTU, 8% JAMM and 8% UCH & MJD family. USP5, USP7, USP14, USP15, UCHL5, UCHL3, OTUB1, PRP8, PSMD7 and PSMD14 are the highly expressed DUBs. Functional enrichment approach and protein-protein interaction network reveal that 70% of the active DUBs are involved in cell cycle regulation. DDR, RNA splicing, TGF-beta receptor, NF-kB and Wnt signaling are some other enriched pathways. Of the 35 cell cycle DUBs, CRISPR screens identify 14 DUBs and DRIVE data identified 11 DUBS as having effects on cell viability. Combining the ABPP data, shRNA and CRISPR results, OTUD5, BRCC3, UCHL5, UFD1L, USP5, YY1, USP3, USP39, USP37, OTUB1, USP9X and COPS5 have activity in KRAS mutant lung cancer cell lines and demonstrate reduced cell viability with loss of function through CRISPR or RNAi.

We have identified and prioritized DUB targets in KRAS mutant lung cancer. Future studies will further validate DUB as targets and identify mechanisms of activity in KRAS mutant lung cancers, as well as examine DUB activity using ABPP in human lung cancer tumor tissues, including adenocarcinoma (KRAS mutant and wild-type) and squamous cell lung cancer.

#2751

Development of a specific Wee-1 inhibitor.

Mandy Watson, Tom Pesnot, Andrew Scott, Anthony Huxley, Gary Nelson, Montserrat Shelbourne, Jen Morton, Tilly Bingham. Concept Life Sciences, Manchester, United Kingdom.

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers. The resulting TP53-deficient cancer cells rely on WEE1 to trigger a G2/M arrest, which allows for DNA repair and survival. Inhibition of WEE1 has therefore emerged as an attractive therapeutic strategy to selectively sensitise TP53-deficient tumours to DNA damaging agents. The WEE1 kinase inhibitor adavosertib is the only agent undergoing evaluation in a range of clinical trials to validate this hypothesis. It has also been the tool compound of choice to interrogate WEE1 biology for over a decade.

However, recent reports show adavosertib has antiproliferative single agent activity, which is counter intuitive considering its postulated mode of action. Other studies suggest adavosertib exerts poor kinase selectivity, and inhibits PLK1 with similar potency as WEE1. Since PLK1 is a well-established mitotic regulator with biological functions throughout the cell cycle, its inhibition may contribute to the clinical efficacy and toxicity observed for adavosertib. Other reported WEE1 inhibitors also suffer from low selectivity and single agent toxicity, making then unsuitable tool compounds. There is a clear need to identify a selective Wee1 tool compound.

Using a structure-based drug design approach, we exploited subtle active site differences to identify novel, potent WEE1 inhibitors that display high selectivity over PLK1. These compounds allowed us to demonstrate the distinct effects of WEE1 and PLK1 inhibition on cellular toxicity, and confirm that adavosertib’s single agent efficacy is unlikely to be mediated solely through the inhibition of WEE1. Our ongoing studies aim to provide a WEE1 selective tool compound that more rigorously probes WEE1 biology, and eventually leads to the development of less toxic therapeutic agents.

#2752

Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound.

Wenting Liu,¹ Clement Lin,¹ Zong-wan Mao,² Danzhou Yang¹. ¹Purdue University, West Lafayette, IN; ²Sun Yat-Sen University, Guangzhou, China.

The human telomeric DNA, which consists of tandem repeat sequences of d(TTAGGG)n, plays an important role in cancers and cell aging. Previous studies in human cancer cells have demonstrated DNA G-quadruplex (G4) structure formation in telomeres. G-quadruplexes are four-stranded structures formed in guanine-rich sequences that are held together by guanine-guanine Hoogsteen hydrogen bonding, and G-quadruplex stabilization by small molecules induces tumor cell senescence and apoptosis by repressing telomerase activity and the DNA damage response pathway. Additionally, telomeric-overhang DNA can form biologically relevant higher-order DNA structures containing consecutive G-quadruplexes which provides additional binding sites for small molecules. Thus, telomeric DNA G-quadruplexes have attracted increasing interest as potential drug targets for cancer therapy. Here, we found that a novel platinum (II)-based tripod (Pt-tripod) specifically recognizes the hybrid-1 human telomeric G4, which is formed in the physiologically relevant K+ solution. Pt-tripod strongly represses telomerase activity and exhibits DNA-targeted photodynamic therapy anticancer activity. Using NMR, we show that Pt-tripod binds the hybrid-1 human telomeric G4 over other G-quadruplex structures and dsDNA. We determined solution structures of the 1:1 and the dimeric 4:2 Pt-tripod-hybrid-1 telomeric G4 complexes by NMR. Our 1:1 complex structure shows preferential binding of the Pt-tripod to the 5ʹ-end of hybrid-1 telomeric G4. At higher ligand ratio, Pt-tripod binds to the 3ʹ-end of the hybid-1 G4 and induces an unprecedented dimeric 4:2 structure interlocked by a non-canonical A:A pair at the 3ʹ-end. The specific binding of the Pt-tripod with the hybrid-1 G4 is achieved by unique interaction modes including the π-π stacking, hydrogen bonding, and electrostatic interactions with the loop and flanking segments. The non-planar tertiary amine conformation and three properly sized cationic platinum arms are critical for the specific and strong binding. Our structures provide significant insights into understanding the dynamic binding of small molecules with G-quadruplexes, and a structural basis for future rational anticancer drug design of non-planar small molecules targeting the human telomeric G4. Our studies also provide a potential handle to study the specific protein interactions and biological functions of hybrid-1 human telomeric G4.

#2753

The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy.

Jaafar N. Haidar,¹ Stephen Antonysamy,² Sneha Mathew,¹ Lan Wu,¹ Yi Zhang,¹ Margaret C. Kearins,² Leyi Shen,¹ J. Michael Sauder,² David Schaer,¹ Kyla E. Driscoll,¹ Michael Kalos¹. ¹Lilly Research Laboratories, Eli Lilly & Company, New York, NY; ²Lilly Biotechnology Center, Eli Lilly & Company, San Diego, CA.

The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells.

Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3.

#2754

Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor.

Ziqian Wang, Zhichao Zhang, Ting Song, Zongwei Guo. Dalian University of Technology, Dalian, China.

The identification of novel non-Bcl-2 BH3 receptors is still challenge owing to frequently weak, transient and reversible binding character of the PPIs mediated by them. Herein, we designed and synthesized an affinity-based probe (AfBP), S1b-probe, by introducing a photo-crosslinker to an artificial BH3 mimetic. Hsp70 proteins were identified as novel BH3 receptors in situ. The PPIs between Bcl-2 proteins and Hsp70 proteins were further validated by fluorescent polarization (FP), isothermal titration calorimetry (ITC) and Co-immunoprecipitation (Co-IP) experiments. Structural and functional analysis by photo-crosslinking/LC-MS/MS sequencing, ¹H-¹⁵N transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, single-turnover ATPase rates and denatured rhodanese aggregation measurement demonstrated that BimBH3 bind in the NBD domain of Hsp70 proteins to act as a co-chaperone that negatively regulates the ATPase activity and chaperone functions. S1g, derived from BH3 mimetics, was screened as a specific Hsp70 inhibitor in native physiological context by using S1b-probe.

#2755

Limited proteolysis coupled to mass spectrometry (LiP-MS), a novel drug target deconvolution strategy.

Nigel Beaton,¹ Roland Bruderer,¹ Kristina Beeler,¹ Nicholas Dupuis,¹ Ilaria Piazza,² Paola Picotti,² Lukas Reiter¹. ¹Biognosys AG, Schlieren, Switzerland; ²ETH Zurich, Zurich, Switzerland.

Background High attrition rates in target-centric drug development approaches, as well as a limited number of targets, have shifted the focus of drug development back towards phenotypic screening. In parallel, novel proteomics-based target deconvolution approaches to drug target identification have gained popularity. Limited proteolysis coupled with mass spectrometry (LiP-MS) is a new target deconvolution technique that exploits protein structural alterations driven by drug binding. A major advantage of LiP-MS is its unique focus on detection of peptides that report on ligand binding induced structural changes that are generated by a limited digestion and identified by proteomic analysis. Here we demonstrate the performance of LiP-MS using the protein phosphatase inhibitor Calyculin A, as well as two well known kinase inhibitors, selumetinib (SE) and staurosporine (ST), in HeLa cell lysate.

Materials and Methods Mechanically sheared HeLa cell lysate was incubated with compound at multiple concentrations. Next, a limited digest was performed using proteinase K. Finally, the limited digests were processed to peptides with trypsin for mass spectrometry analysis. A project-specific spectral library was generated using data-dependent acquisition (DDA) mass spectrometry and for quantitative analysis data-independent acquisition (DIA) data were recorded and analyzed using Spectronaut Pulsar X.

Results LiP-MS identifies several peptides for the phosphatase inhibitor Calyculin A, with IC50 values of 52 nM and 17 nM for PP1A and PP2A respectively. Additionally, a previously unknown target PP1B was also identified among the same family, although with a higher IC50 (74 nM). Through structural inspection of ligand-sensitive peptides we were able to map the drug’s binding site within the phosphatases and predict distal conformational changes, demonstrating that LiP-MS can be used to provide structural insights to ligand-protein binding. Similar dose response relationships were observed for both specific (SE) and broad (ST) kinase inhibitors. Amongst the top 200 identified target candidate peptides ranked by LiP score, GO enrichment analysis confirmed a highly significant 3-fold enrichment for kinase targets (p < 0.00002) in ST-treated lysate, while no such enrichment was observed for SE. However, in SE-treated lysate robust identification of multiple MEK1 peptides, one of the compound’s main targets, was observed. In the case of both kinase inhibitors LiP peptides could be successfully mapped to ATP binding sites, confirming the ability of LiP-MS to model drug-bound protein structure.

Conclusions This data demonstrates that LiP-MS can be used to effectively identify protein drug targets and characterize the binding properties, regardless of the specificity of the compound. These capabilities make LiP-MS a powerful target deconvolution and identification strategy.

#2756

A high-resolution G-quadruplex structure involved in cmyc oncogene regulation.

Jonathan Dickerhoff, Luying Chen, Guanhui Wu, Buket Onel, Clement Lin, Danzhou Yang. Purdue University, West Lafayette, IN.

Gene expression needs strict regulation in human cells to prevent tumorigenesis. Among other control mechanisms, guanine-rich sequences which are clustered in promoter regions of oncogenes can form four stranded nucleic acid structures, called G-quadruplexes (G4), and regulate their transcription. The cmyc protein is the most important cellular regulator for proliferation and is often overexpressed in cancer cells. The DNA G-quadruplex formed in the cmyc promoter is a transcriptional silencer element and stabilization of this G4 by small molecules suppresses cmyc transcription, making it a promising target for cancer therapy. High-resolution structures of the highly diverse G4s provide the opportunity to understand their molecular interactions with transcription factors and to design small molecules that specifically bind a particular G4 conformer. Characteristically, multiple G4s can be formed in gene promoter regions because their G-rich sequence generally contains more than the minimum of four G-stretches. While all reported cmyc G4s share the same core with parallel oriented strands, their overall shape is individualized by distinct combinations of varying flanking and loop motifs. In this study, we present a new high-resolution NMR structure of a G4 found to form in the cmyc promoter region. The determined cmyc G4 has a 1:6:1 loop-size arrangement and its conformation is largely defined by its long central loop of 6 nt. We show that this G4 structure is the preferred target of nucleolin protein in comparison to other G4s formed in the cmyc promoter, demonstrating how the polymorphism of a G-rich sequence can modulate the interaction with transcription factors and other regulators. Our NMR structure shows that, despite the length of the 6-nt loop motif and the associated increased flexibility, the folding converges well due to a base pairing between the central loop and the 5’-flanking. Thus, the long loop is pulled towards the 5’-end while analogous loop-flanking interactions are prevented at the opposite 3’-site. In conclusion, the presented new addition to the ensemble of possible cmyc promoter G4 conformations extends the folding landscape for this promoter region, in particular due to the long central loop. This may shed light on how proteins such as nucleolin can target specific G4 and shift the equilibrium of folding to fine-tune gene expression. Finally, this new structure is another promising target for cancer drug development.

#2757

Discovery and characterization of covalent Pin1 inhibitors targeted to an active site cysteine.

Benika Pinch,¹ Zainab Doctor,² Christopher M. Browne,² Hyuk-Soo Seo,³ Behnam Nabet,³ Shingo Kozono,⁴ Xiaolan Lian,⁴ Daniel Zaidman,⁵ Dina Daitchman,⁵ Nir London,⁵ Lu Gong,⁴ Theresa Manz,³ Yujin Chun,⁶ Li Tan,⁷ Jarrod Marto,³ Stephen Buratowski,⁶ Sirano Dhe-Paganon,³ Xiao Zhou,⁴ Kun Ping Lu,⁴ Nathanael S. Gray². ¹Harvard University, Cambridge, MA; ²Dana-Farber Cancer Institute; Harvard Medical School, Boston, MA; ³Dana-Farber Cancer Institute, Boston, MA; ⁴Beth Israel Deaconess Medical Center; Harvard Medical School, Boston, MA; ⁵Weizmann Institute of Science, Rehovot, Israel; ⁶Harvard Medical School, Boston, MA; ⁷Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China.

Proline-directed phosphorylation at serine or threonine residues (pSer/Thr-Pro) regulates numerous cellular processes, including the cell cycle, transcription, and differentiation. Deregulation of such signaling networks is a hallmark of transformation and oncogenesis. Pin1, a peptidyl-prolyl isomerase, regulates the function and stability of phosphoproteins by catalyzing the cis/trans isomerization of pSer/Thr-Pro motifs. Pin1 is frequently overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC), and Pin1 is required for activated Ras to induce tumorigenesis. While mutations in KRAS are observed in 90-95% of human PDAC cases, it has historically proven very challenging to develop small molecules that inhibit mutant Ras function. Consequently, drug discovery efforts have turned to targets required for Ras-mediated transformation, such as Pin1. However, existing Pin1 inhibitors lack the potency, selectivity, and/or cell permeability to serve as informative cellular probes. We report a highly potent, cell-permeable Pin1 inhibitor that covalently targets Cys113, a conserved cysteine residue in the Pin1 active site. Through iterative rounds of synthesis and characterization, we developed inhibitor 1b. With a Ki of 15 nM as measured in biochemical binding and isomerase inhibition assays, 1b is currently the most potent Pin1 inhibitor available. Furthermore, in a chemoproteomic study using Covalent Inhibitor Target Site Identification (CITe-Id) to quantify the dose-dependent covalent labeling of 1b to individual cysteines across the proteome, Pin1 Cys113 was the only identified target, highlighting the pronounced selectivity of 1b for Pin1. We show that treatment with 1b diminishes viability of human PDAC cell lines, which can be fully rescued in corresponding Pin1 knockout cells generated using CRISPR/Cas9, showing that this phenotype is on-target. In parallel to inhibitor development, we used CRISPR/Cas9 GFP-dropout screens to further validate the dependence of these cell lines on Pin1. Genetic disruption of Pin1 led to antiproliferative effects, confirming the results of 1b treatment. We also employed the degradation tag (dTAG) approach to assess the effects of rapid and selective targeted Pin1 degradation through generation of FKBP12F36V-Pin1, Pin1-/- human PDAC cell lines. Treatment with a small molecule FKBP12F36V-degrader led to rapid ubiquitination and degradation of FKBP12F36V-Pin1, enabling comparisons of targeted inhibition and Pin1 degradation. Through the development of a selective Pin1 inhibitor coupled with genetic approaches and the chemical-genetic dTAG strategy, we demonstrate that Pin1 inhibition represents a tractable strategy in PDAC.

#2758

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

Chun-Jung Chen,¹ Li-Jin Hsu². ¹National Synchrotron Radiation Research Center, Hsinchu, Taiwan; ²National Cheng Kung University, Tainan, Taiwan.

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers, including hepatocellular carcinoma, pancreatic cancer, oral cancer, breast cancer and non-small cell lung cancer. However, the functional mechanisms of human HDGF had been puzzling because of a lack of essential knowledge of its intact structure in complex with DNA. We present the first crystal structures of the human HDGF PWWP domain (residues 1 - 100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

#2759

Chemoproteomic profiling of the oncometabolite fumarate.

Sarah E. Bergholtz,¹ Chloe A. Briney,¹ Rhushikesh A. Kulkarni,¹ David Wei,² Daniel W. Bak,³ Jonathan H. Shrimp,¹ Eranthie Weerapana,³ W. Marston Linehan,² Jordan L. Meier¹. ¹National Cancer Institute, Frederick, MD; ²National Cancer Institute, Bethesda, MD; ³Boston College, Chestnut Hill, MA.

Dysregulated metabolism is a feature of many diseases, including cancer. In addition to providing energy and building blocks to growing cells, altered metabolism can assist in tumor growth by producing oncometabolites. For example, in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) inactivating mutations in fumarate hydratase (FH) leads to accumulation of millimolar levels of an oncometabolite, fumarate. At such high concentrations, fumarate can spontaneously react with protein cysteine residues leading to a post-translational modification (PTM) called cysteine succination. This PTM can directly stimulate tumorigenic signaling, however, current methods to study this PTM are limited. Our lab has developed chemical proteomic methods to investigate proteome-wide targets of cysteine succination. Using these methods, we have identified hundreds of targets of fumarate in normal kidney cells as well as HLRCC cells. One interesting target identified that has implications in cancer progression is SMARCC1, a core subunit of a nucleosome remodeling complex known as the SWI/SNF complex. The SWI/SNF complex is a well-known tumor suppressor and mutation of this complex leads to cancer development. We have demonstrated that excess fumarate levels result in hyper-modification of SMARCC1 and decrease in SWI/SNF complex stability. This instability also results in an increase in EZH2 expression as well as activity, and an increase in H3K27Me3. Our current efforts are aimed at understanding how fumarate affects the function of this complex in vivo and exploring the potential of using EZH2 inhibitors in the treatment of HLRCC.

#2760

Photoinducible detection of the oncometabolite fumarate.

Chloe Briney,¹ Sarah Bergholtz,¹ Rhushikesh Kulkarni,¹ Daniel Crooks,² Chandrasekhar Mushti,³ Stephen Lockett,¹ Rolf Swenson,³ W. Marston Linehan,² Jordan Meier¹. ¹National Institutes of Health, Frederick, MD; ²National Institutes of Health, Bethesda, MD; ³National Institutes of Health, Rockville, MD.

Dysregulated metabolism is an important marker of many disease states, including cancer. For example, in hereditary leiomyomatosis and renal cell cancer (HLRCC), inactivating mutations in fumarate hydratase (FH) lead to accumulation of high levels of fumarate, a so-called oncometabolite. Substantial evidence indicates that fumarate stimulates various oncogenic signaling pathways, necessitating sensitive methods to detect the oncometabolite in order to more rapidly diagnose HLRCC as well as to identify new disease settings in which fumarate may play a signaling role. Here, we report development of novel photoactivatable, fluorogenic chemical probes for detection and profiling of fumarate in biological systems. These chemical probes, diaryl tetrazoles, are by themselves inert towards fumarate. However, upon irradiation with UV light, they release nitrileimines that can form fluorescent cycloadducts with fumarate. We have demonstrated that diaryl tetrazoles can sensitively detect FH activity as well as low micromolar levels of fumarate in complex biological samples. We have also shown that diaryl tetrazoles can be used to monitor changes in intracellular fumarate levels in biological samples by live-cell imaging and flow cytometry. Moreover, these compounds are capable of visualizing differences between patient-derived primary HLRCC tumors lacking FH activity and the adjacent normal kidney, highlighting their potential utility in clinical diagnostics. By offering new insights into fumarate reactivity, our studies provide the chemical basis for novel approaches to therapy and diagnosis in cancers driven by oncometabolite accumulation.

#2761

Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies.

Annaleen Vermeulen, Zaklina Strezoska, Sarah M. Dickerson, Maren M. Gross, Eldon T. Chou, Elena Maksimova, Matthew R. Perkett, Shawn McClelland, Anja V. Smith. Dharmacon, a Horizon Discovery Company, Lafayette, CO.

Functional gene analysis studies have been empowered by development of CRISPR-Cas9 gene knockout tools, however the CRISPR-Cas9 system has also been adapted for inhibition or activation of gene transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains to produce an RNA-guided transcription factor for either inhibition (CRISPRi) or activation (CRISPRa) of target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based gene expression, because genes are upregulated from their native promoter and endogenous genomic context. The majority of CRISPRa research performed to date has utilized single guide RNA (sgRNA) expressed from a DNA vector, primarily in the context of pooled lentiviral screening approaches. Here we describe the development of CRISPRa synthetic guide RNAs for the use in arrayed screening, so that we combine this next-generation transcriptional activation method with the ability to support more complex assays in a one-gene-per well format. Considerations for arrayed activation screens will be shown. The combination of gain-of-function from CRISPRa with loss-of-function using RNAi or canonical CRISPR-Cas9 for gene knockout allows for robust characterization of gene mechanisms and pathways.

#2762

Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment.

Qiongling Wang, Kevin F. Staveley-O’Carroll, Guangfu Li. University of Missouri, Columbia, MO.

Background and objective: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of pancreas with an overall 5-year survival rate of less than 5%. Surgical operation is an curable treatment, however, only 10-20% of patients with PDAC are respectable at diagnosis. Taking advantage of the progression in nanoparticle study, we developed a novel peptide-formed nanoparticle (PF-Nano) and investigate its therapeutic effect in PDAC treatment.

Methods: Phage display peptide library was used to screen a high-affinity tumor-homing peptide (THP) for pancreatic cancer (PC) cells. Ligation of THP with cell penetrating peptide was used to prepare a tandem peptide, which automatically formed a nanoparticle. We used this PF-Nano as a vehicle to load and deliver siRNA. Dynamic light scattering (DLS) was employed to define nanoparticle size; and fluorescence imaging was applied to assess PF-Nano siRNA delivery efficiency. In vitro, we used MTT assay and colony formation to evaluate cell growth; wound healing assay to detect cell migration, and flow cytometry to evaluate cell cycle and apoptosis. Therapeutic effect was evaluated by measuring final tumor weight in the mice receiving different treatments. qPCR and Western Blot (WB) were used to detect gene expression in mRNA and protein level. Immunoprecipitation in combination with mass spectrometry and immunofluorescence were used to identify THP’s ligands.

Results: The screened peptide has a high-affinity with PC cells. DLS identified evenly distributed PF-Nano with a diameter of approximately 230nm. Detection of fluorescence signal from labeled siRNAs in PC cells and tumor tissue suggested that PF-Nano owned the capacity to successfully deliver siRNA in vitro and in vivo. In vitro treatment with PF-Nano significantly suppressed PC cell proliferation, colony formation, and cell migration. Also it caused cycle arrest in S phase (DNA synthesis) and increase in cell apoptosis. Treatment of tumor-bearing mice with PF-Nano by intraperitoneal injection markedly suppressed tumor growth, evidenced by the reduction of tumor weight to 25% relevant to that in control mice without treatment. Immunoprecipitation and mass spectrometry revealed that protein phosphatase 1 catalytic subunit gamma (PP1C) was a potential binding target of the specific THP. Mechanistically, PF-Nano treatment strongly suppressed RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation,

suggesting PP1-AKT signal pathway may be responsible for peptide effect in pancreatic tumor.

Conclusion: The established PF-Nano is able to specifically deliver siRNAs to PC tumors and therapeutically suppresses PC tumor growth.

#2763

Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface.

Hyunbum Jang, Ruth Nussinov. National Cancer Institute at Frederick, Frederick, MD.

Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1’s kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

#2764

Small activating RNA induced expression of VHL gene in renal cell carcinoma.

Jong Soon Kang,¹ Moo RIm Kang,² Ki Hwan Park¹. ¹Korea Research Inst. of Biosci. Biotech., Cheongju, Republic of Korea; ²Ractigen Therapeutics, Nantong, China.

Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences in a mechanism termed RNA activation (RNAa). In the present study, we designed four candidate dsRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these dsRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Consistent with previous studies on RNAa of other genes, single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, resulting in inhibition of cell growth and induction of apoptosis. Targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.

TUMOR BIOLOGY

Expression Profiling and Biomarkers of Tumor Progression and Metastasis

#2765

Exosomal miRNAs as circulating biomarkers for prediction of metastasis in stage II/III gastric cancer.

Xin Liu,¹ Kent-Man Chu². ¹Univ. of Hong Kong Faculty of Medicine, Hong Kong, Hong Kong; ²Univ. of Hong Kong Faculty of Medicine, Queen Mary Hospital, Hong Kong, Hong Kong.

Background: Metastasis is the fatal character of cancer, leading to mortality of gastric cancer. In recent years, exosomes have been reported as extracellular vesicles secreted by cells. They contain various biological molecules including miRNAs from the original cells. Exosomal miRNAs are significant for pre-metastatic niche formation, as they can change the microenvironment and make it favorable for future metastasis. Therefore, the aim of this study was to identify dysregulated exosomal miRNAs as circulating biomarkers for prediction of metastasis of gastric cancer.

Materials and Methods: Pre-treatment serum samples of 36 metastatic patients and 55 non-metastatic patients with stage II/III gastric cancer were collected. Exosomes were extracted and validated by western blot, transmission electron microscope and nanoparticle tracking analysis. MiRCURY LNA™ miRNA miRNome PCR Array (containing 752 miRNAs) was performed in three pairs of serum exosomes. Each pair samples were with the same stage, race, gender and similar age. These three pairs of samples represented stage II, IIIA or IIIB gastric cancer, respectively. A panel of commonly dysregulated exosomal miRNAs was released from the PCR Array. Expressions of exosomal miRNAs were validated in the remaining 33 metastatic and 52 non-metastatic patients by RT-qPCR, as well as in gastric cancer cell culture medium and cell lines.

Results: By comparing the expressions of exosomal miRNAs between the metastatic patients and matched non-metastatic ones, 13 upregulated and 6 downregulated miRNAs were released after normalization with miR-16-5p and miR-93-5p. Among these miRNAs, 7 of them were selected for further validation according to their fold changes, p-values and association with cancer development. MiR-379-5p and miR-410-3p were validated to be significantly upregulated in metastatic patients (P<0.01). Sensitivity, specificity and AUC were 67.31%, 69.70% and 0.6894 for miR-379-5p, and 65.38%, 66.67% and 0.6719 for miR-410-3p. Higher expression of serum exosomal miR-379-5p or miR-410-3p showed shorter overall survival of the patients (P<0.05). It was also found that miR-379-5p and miR-410-3p expressed significantly higher in gastric cancer cell culture medium comparing with gastric cancer cells.

Conclusions: Exosomal miRNAs are dysregulated in the serum of metastatic gastric cancer patients before they are clinically diagnosed. Upregulation of serum exosomal miR-379-5p and/or miR-410-3p are promising circulating biomarkers for prediction of metastasis in stage II/III gastric cancer.

#2766

Amplification-associated upregulation of genes involved in oxidative phosphorylation for disseminated prostate cancer cells.

Chun-Lin Lin, Tan Xi, Chia-Nung Hung, Pawel A. Osmulski, Chih-Wei Chou, Meizhen Chen, Chiou-Miin Wang, Kohzoh Mitsuya, Nameer Kirma, Maria E. Gaczynska, Chun-Liang Chen, Tim H.-M. Huang. University of Texas Health San Antonio, San Antonio, TX.

Purpose: Little is known about adaptive selection of tumor cells transiting from in situ proliferation to distant colonization through blood circulation. This study used genomic, transcriptomic, and biophysical analyses at single-cell levels to explore biological and physical properties of circulating tumor cells (CTCs) undergoing hemodynamic stress. The aims are trying to understand how CTCs strive to survive in the bloodstream and to develop strategies for therapeutic intervention.

Experimental Design: We compared genomic profiles of CTCs in blood and primary tumor cells shed in urine of prostate cancer patients to identify amplified regions preferentially retained in CTCs. Single-cell RNA-seq was used to confirm amplification-associated overexpression of genes in CTCs.

Results: Among 261 recurrently amplified genomic regions in the analysis, 70 were found predominantly shown in CTCs relative to primary tumor cells. In line with the results at the genomic level, the transcriptomic data of CTCs demonstrate a great amount of cells showing high expression levels in the oxidative phosphorylation (OXPHOS) pathway compared to other hallmark gene sets. The finding suggests that tumor cells with these pre-existing genomic alterations were adaptively selected for transcription reprogramming during blood circulation. Specifically, amplified genes associated with OXPHOS were exploited by CTCs for alternative fuels. As to the upstream of this transcription event, we found the expression of MEN1, encoding menin known to form a transcription factor complex with the mixed-lineage leukemia protein (MLL) in prostate cancer cells, was positively correlated with 11 of the 14 OXPHOS loci in the Cancer Genomic Atlas prostate cancer cohort. In vitro assay showed that MEN1 knockdown by shRNA resulted in attenuation of both mRNA and protein expression of OXPHOS loci in PC-3 cells. Taken together pre-existing amplification of OXPHOS loci can be used as a transcription apparatus by menin for metabolic reprogramming of tumor cells in response to harsh microenvironments in the bloodstream.

Conclusions: Single-cell profiling identified signaling pathways that are crucial for CTCs to survive in the bloodstream. The finding suggests that a metabolic shift from Warburg to OXPHOS metabolism can be associated with a hybrid mesenchymal-epithelial phenotype of CTCs. Moreover, the study demonstrates the feasibility of routinely conducting single-cell analysis of exfoliated tumor cells for minimally invasive monitoring of disease progression and treatment response in prostate cancer patients

#2767

Expression of defensin-associated genes may be correlated with lymph node metastasis of early stage tongue cancer.

Doh Young Lee. Seoul National University, Seoul, Republic of Korea.

OBJECTIVES: Appropriate patient selection for elective neck dissection in N0 tongue cancer has been controversial, while there has been no genetic consideration so far. We aimed to analyze genetic difference between early and late cervical lymph node metastasis in oral tongue cancer patients using the Seoul National University Hospital (SNUH) cancer cohort and cancer genome atlas (TCGA) data, and to suggest genetic background for choosing eligible patient for elective neck dissection. METHODS: A total of 35 cases with RNAseq in SNUH cancer cohort with tongue cancer were also enrolled in this study. To investigate gene expression in early cervical lymph node metastasis, genomic data of following 2 groups was compared; 1) N0 group: T1/2 and N0, 2) N+ group: T1/2 and N+. Differentially expressed genes (DEGs) were extracted using R and limma package in bioconductor program. Gene ontology and pathway enrichment analysis were performed using DAVID online tool. Immune cell infiltration was analyzed using CIBERSORT online program. Additionally, 70 cases with matched RNAseq data of primary tumor and clinical TCGA data were analyzed to validate the role of DEGs. RESULTS: N0 and N+ groups showed no difference in age, gender, size and thickness of tumor, and nearest resection margin. In addition, there was no significant difference in 22 types of immune cell infiltration. A total of 51 DEGs were identified, and 14 genes were significantly upregulated and 37 genes were significantly downregulated (p<0.01, fold-change >2). Significant genes were PLA2G4D, NELL2, PCOLCE2, PI3, and PLA2G3. On KEGG pathway analysis, calcium, muscle contraction, and arachidonic acid metabolism-related pathway was significantly correlated. The most significant difference was found in 6 genes as follows; DEFB4A, SPRR2B, DEFB103B, SPRR2G, DEFB4B, FAM25A.DEFB which was the most significantly difference in expression was associated with ‘antibiotic’, ‘antimicrobial’, ‘beta defensing type’, defense reponse to bacterium’, and ‘defensin’. In TCGA data, DEFB4A and DEFB103B were more highly expressed in N0 group than N+ group, although it did not attain significant significance. CONCLUSIONS: Defensin (DEFB4A, DEFB103B, DEFB4B) may be a novel biomarker for early regional metastasis in T1/2 tongue cancer. Considering that defensin can be detected in saliva, simple saliva test for prediction of metastasis can be developed in the future.

#2769

A squalene epoxidase-cholesterolaxis drives colorectal cancer progression and metastatic dissemination.

Soo Young Jun,¹ Andrew J. Brown,² Ji-Yong Yoon,¹ Jeong-Ju Lee,¹ Ngee Kiat Chua,³ Jin OK Yang,¹ Ju-Sik Min,¹ Insu Jang,¹ Su-Jin Jeon,¹ Min-Hyuk Choi,¹ Tae-Ik Choi,⁴ Cheol-Hee Kim,⁴ Nam-Soon Kim¹. ¹Korea Research Inst. of Biosci. and Biotech., Daejeon, Republic of Korea; ²School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, Australia; ³School of Biotechnology and Biomolecular Sciences, University of NEw South Wales, Sydney, Australia; ⁴Department of Biology, Chungnam National University, Daejeon, Republic of Korea.

Links between cholesterol and cancer are well-documented, but the mechanisms remain unclear. Squalene epoxidase (SQLE), a key enzyme in cholesterol biosynthesis degraded by excess cholesterol, is suggested as a proto-oncogene. Paradoxically, we found reduced SQLE in aggressive colorectal cancer (CRC); low SQLE being associated with a shortened survival of CRC patients. This was confirmed in a spontaneous CRC metastasis mouse model where SL-15 reduction, by either a high-cholesterol regimen or genetic knockdown, strikingly promotes CRC aggressiveness through the production of migratory cancer stem cells. Experiments in CRC cell-lines demonstrated that SQLE reduction helps overcome constraints for malignant transformation. Specifically, we uncovered a surprising interaction of SQLE with GSK3β and p53. Depletion of SQLE disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. Our findings provide mechanistic insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC progression and potentially a biomarker for risk assessment.

#2770

Spatio-temporal genetic heterogeneity and clonal evolution in advanced papillary thyroid carcinomas and matched distant metastases.

Sara Gil-Bernabe, Noa Feas Rodríguez, Miriam Vega Herrero, Jose Javier Estébanez García, Ginesa M. Garcia-Rostan. Institute of Molecular Biology and Genetics (IBGM), Valladolid, Spain.

Papillary Thyroid Carcinoma (PTC) represents 65-80% of all thyroid cancers. Though the vast majority of PTCs are indolent tumors, around 5-15% behave aggressively, developing blood-borne metastases, which cause patient´s death. The molecular mechanisms underlying metastatic spread are poorly understood. Little it is known about the contribution of intratumor molecular heterogeneity to distant metastases (DM). Dynamic changes in mutation distribution through space and time have not been in deep characterized. In this study, by genotyping 13 cases of matched primary tumors (PrT) and DMs, we sought to determine the prevalence of mutations in genes that have been associated with tumor progression and aggressiveness in follicular thyroid carcinogenesis (TERTp, MED12, BRAF, NRAS, KRAS, HRAS, PIK3CA). To asses the contribution of intratumor heterogeneity and clonal evolution to DMs, 54 tumor areas, including different areas across space and time within the PrTs and the DMs were characterized. Mutational analysis was approached by means of PCR and SSCP or direct sequencing. Twelve cases (92%) were mutated in at least one of the genes screened [TERTp=9 cases (69%), BRAF=7 cases (54%), KRAS=3 cases (23%), NRAS=3 cases (23%), HRAS=2 cases (15.4%) and PIK3CA=2 cases (15.4%)]. No mutations were seen at MED12. Among the mutated cases 67% exhibited more than 1 gene activated. Three mutated genes co-existed in 62.5% of the cases with concomitant mutations [3 cases (60%) TERTp+RAS+BRAF and 2 cases (40%) TERTp+BRAF+PIK3CA]. Concurrent activation of TERTp+RAS or TERTp+BRAF was seen in 5 cases each event (62.5%). Simultaneous activation of BRAF and RAS was found in 4 cases (50%). In all the cases in which more than 1 area of DM, across space and time, was analyzed, the mutations at TERTp, KRAS and HRAS resulted clonal. De novo mutations at DM, not present in the PrT, were seen in 3 cases mutated at TERTp, 2 cases mutated at NRAS, 1 case mutated at KRAS. Among the mutated cases, in which more than one area of PrT was analyzed, clonality was seen in 80% of the cases mutated at TERTp and subclonality was seen in 80% of the cases mutated at BRAF. The activation of RAS within the PrT tend to be a clonal event. Conclusions: The number of mutational events in PTC with DM is strikingly higher than in in PTC without DM. While TERTp and RAS mutations tend to be clonal within the PrT and the DMs, BRAF mutations tend to be subconal. TERTp and RAS mutations may appear the novo at DM. PTC with DM display a much higher rate of genetic heterogeneity (67%). The coexistence of mutations in different genes is in agreement with the hypothesis that tumor progression relies on progressive accumulation of genetic alterations. MED12 does not play a role in aggressive papillary thyroid carcinomas. Concurrent mutations at TERTp, and different PI3K/AKt and MAPK pathway genes are common in poorly differentiated and anaplastic carcinomas.

#2771

Identification of novel genes associated with metastasis from TCGA transcriptomic data.

Takahiko Koyama, Laxmi Parida. IBM T.J. Watson Research Center, Yorktown Heights, NY.

Introduction: Many cancer patients suffer from metastasis in bone, brain, liver and lung. However, understanding of metastasis in molecular level is quite limited. To improve survival rate and quality of life, preventing metastasis is crucial. In this study, we analyzed transcriptomic data with tumor node metastasis (TNM) classification from The Cancer Genome Atlas (TCGA) to identify genes associated with metastasis.

Method: TCGA samples in 20 solid tumor types with both transcriptome and TNM annotation data are used. To obtain the best cut point separating cohorts of high and low gene expression, Hothorn and Lausen’s method is used. A contingency table is created with expression separated by the the cut point and presence of staging T, N, M for each gene. Association between the staging category and the gene was calculated with Fisher’s exact test.

Results: In total, 13,220 distinct genes including miRNAs are associated with either lymph node or distant metastasis for some cancer types with a loose criteria of p-value < 1.0e-3. Among them, we identified 1,357 genes with p-value < 1.0e-8. ADAMTS12, AURKB, BUB1, C17orf53, CCNA2, CEP55, EPR1, KIF4A, KIF11, KRT80, MELK, MKI67, NCAPG, NEIL3, PBK, PLK1, RRM2, TPX2, miR139, miR30a, miR375, and miR379 appear most commonly in lymph node metastasis for many cancer types. Likewise, NOP56, PKD1L2, miR150, miR365, miR9, miR149, miR210, miR425, miR675, and miR937 are most commonly associated genes in distant metastasis. Many of these genes have been already reported in association with metastasis.

Conclusions: This study created comprehensive list of metastasis associated genes for the 20 solid tumors in TCGA. They would be useful as diagnostic and prognostic biomarkers; however, we found therapeutically relevant genes such as PLK1, AURKB, MET, MELK, CDK1, BUB1, and PBK. Some adhesion molecules associated with a particular cancer type might explain its organ destination preference. Among distant metastatic miRNAs discovered, metastasis promoting miRNAs in high abundance might be engaged in homing as exosomal miRNAs by reprogramming destination niche in favor of migrating tumor cells. Further work to reveal metastasis mechanism and to discover some pharmaceutical agents to prevent metastasis will be conducted.

#2772

Recurrent alterations of the TenascinC in highly aggressive neuroendocrine sub-type of prostate cancer.

Prachi Mishra,¹ Michael Kebish,² Jennifer Cullen,¹ Amina Ali,¹ Alagasamy Srinivasan,¹ Inger Rosner,¹ David McLeod,¹ Leonardo Rodrigues,² Viatcheslav Akmaev,² Rangaprasad Sarangarajan,² Shiv Srivastava,¹ Niven Narain,² Albert Dobi¹. ¹Center for Prostate Disease Research, Uniformed Service University of Health Sciences, Rockville, MD; ²BERG Health, Farmingham, MA.

Introduction: Early detection and prognosis of prostate cancer (CaP) is challenging due to its wide spectrum of biological features. Multi-analytic prognostic marker panels have increased sensitivity than single analytes among the biopsy tests and require advanced bioinformatics platform. We aimed to develop a panel of serum biomarkers using multi-omics approach and further characterized their involvement in prostate cancer progression. In a collaborative study between CPDR, Department of Surgery, USU and Berg Health, serum samples (N=385) were examined by multi-omics (proteomics, lipidomics and metabolomics) and Tenascin C (TNC) was identified as one of the promising markers for disease progression in combination with three other analytes, Apolipoprotein AIV, 1-Methyladenosine and a phosphatidic acid (PA 18:0-22:0). The combination of two clinical features, pathological measurement of T-Stage and Gleason score, along with the four molecular analytes further increased the AUC to 0.89 with a NPV of 0.96 and an odds ratio of 12.4. TNC, an extracellular matrix protein, is poorly studied in prostate cancer, though it is expressed in several cancer tissues such as the breast, lung, colon, and the gastrointestinal tract.

Methods: Publically available prostate cancer databases (cBioportal Version 1.17.1, http://www.cbioportal.org/index.do) were queried for TNC, and known driver oncogenes in prostate cancer, ERG, AR and MYC.

Results: Publically available prostate cancer databases (cBioPortal) demonstrated alterations (either amplifications or mutations) in TNC in 10 out of 16 datasets. Query of TNC along with the driver oncogenes of prostate tumorigenesis, including ERG, MYC and AR, in the aggressive neuroendocrine prostate cancer (NEPC) tumor whole genome sequencing datasets (N=77) demonstrating significant alterations (predominantly amplifications) in 74% of patient samples. CaP genomic levels TNC (30%) was significantly co-amplified (P < 0.001) with ERG (27%), AR (56%) and MYC (53%). TNC protein expression was detected in all examined prostate cancer cell lines VCaP, LNCaP, PC3 and DU145.

Conclusion: Genomic alterations of TNC was thus associated with major oncogenic drivers of CaP, such as ERG, AR and MYC in NEPC genomic datasets. Multi-analyte serum biomarkers offers new opportunities with potential impact on primary treatment and surveillance strategies. Functional involvement of the analytes in disease progression to address their mechanistic link with major CaP oncogenic pathways will be further investigated.

#2773

Prognostic value of KRASP53PIK3CA in non-small cell lung cancer patients.

Yiping Han. Shanghai Changhai Hospital, Shanghai, China.

We explored the relationship of KRAS, PIK3CA, and TP53 mutations with the clinical features and survival prognosis in 50 non-small cell lung cancer (NSCLC) patients. The most common concurrent single gene mutation was TP53, followed by KRAS and PIK3CA. Coexisting mutations were found in 17 patients. KRAS, PIK3CA, and TP53 mutations were related to Ca19-9 expression, invasive growth, vacuolar signs, and margin lobulation in chest CT imaging. The incidence of distant metastasis (bone and adrenal) with KRAS and TP53 mutations was greater than that of local metastasis (pleura). Patients the wild-type gene had longer progression-free survival than those with KRAS, TP53, KRAS/TP53, or PIK3CA/TP53 mutations. Patients with KRAS/TP53 or PIK3CA/TP53 mutations had shorter progression free survival than those with a single KRAS or TP53 mutation. KRAS, PIK3CA, and TP53 mutations were associated with distant metastases and poor prognosis. NSCLC patients should receive routine KRAS, PIK3CA, and TP53 gene sequencing to determine mutations for the analysis of clinical characteristics and prognosis.

#2774

Pain signals oral cavity cancer metastasis.

Aditi Bhattacharya,¹ Malvin N. Janal,¹ Hyesung Kim,¹ Susanna Wang,¹ Angie K. Wu,¹ Mari Hagiwara,² Alexander R. Kerr,¹ Brian L. Schmidt,¹ Donna G. Albertson¹. ¹NYU College of Dentistry, New York, NY; ²NYU School of Medicine, New York, NY.

Metastasis to the cervical (neck) lymph nodes is the primary determinant of survival for patients diagnosed with oral cavity cancer. Imaging (MRI, PET, CT) and clinical examination lack sensitivity to accurately detect cervical metastasis. Therefore most clinically node negative patients receive surgery to remove the cervical lymph nodes (elective neck dissection) at the time of surgical tumor removal. Half of these patients do not benefit from this additional surgery, highlighting the need for improved preoperative assessment in predicting metastatic risk. Oral cancer patients suffer severe chronic and mechanically-induced pain at the site of the cancer. We asked whether patient reported pain measured prior to surgery by a validated instrument, the University of California San Francisco Oral Cancer Pain Questionnaire (UCSFOCPQ), was an indicator of risk for lymph node metastasis. Seventy-two oral cancer patients were consented and enrolled, patients rated their pain using the UCSFOCPQ. Clinical and pathological characteristics of the patient cohorts were collected from pathology reports and medical records. Inclusion criteria included diagnosis of an oral squamous cell carcinoma, planned curative resection of the cancer and completion of the UCSFOCPQ within six weeks of surgery. Exclusion criteria included prior chemo- or radiation therapy for cancer, failure to complete or understand the UCSFOCPQ and less than one year post surgery follow-up. Sixty-six patients met the inclusion/exclusion criteria (35 node negative and 31 node positive). Patients with metastasis reported higher pain scores prior to surgery. Low pain score and depth of invasion each predicted low metastatic risk with high sensitivity and negative predictive values. Amongst patients who were clinically staged N0 by current standard of care imaging and physical exam prior to surgery, low pain scores correctly identified patients determined by pathology to be N0. Conclusions: Low pain scores assessed prior to surgery could identify patients at low risk for metastases who would not benefit from neck dissection. Pain score might be added to the standard of care preoperative assessments and decision making processes for determining whether to recommend an elective neck dissection.

#2775

Investigating myeloid-derived suppressor cells (MDSC) gene expression in predicting outcome in stage matched colorectal cancer patients.

Saleh G. Heneidi, Chance Bloomer, Pankaj Ahluwalia, Ashis Mondal, Ravindra Kolhe. Augusta University, Augusta, GA.

Colorectal cancer (CRC) has emerged as the third most commonly diagnosed cancer in males and second most common in females. Although surgery, chemotherapy, and emerging immunotherapies have reduced mortality rates, tools that can assist in tailoring individualized treatments are needed. MDSC are immune-suppressive cells that interfere with the functioning of T cells and immune-inflammatory pathways. An extensive literature search identified 8 MDSC-centric genes which were tested for their prognostic significance in CRC. These genes; CEBPB, IL10, NOS2, RORC, S100A8, SOCS1, SOCS3, and TGFB1, along with their expression in tumor microenvironments could give new insights when correlated with clinical-pathologic features of colon cancer patients. The aim of the study is to quantify MDSC gene signatures in colorectal cancer patients. Under an IRB approved protocol, a total of 750 colon cancer patients at the Medical College of Georgia with 5 years of follow-up were initially selected. A total of 88 patients fit in our inclusion criteria, on the basis of survival duration after diagnosis. The FFPE blocks were acquired and patients were stratified on the basis of overall survival in two groups, with higher (>5 years) and lower survival (<1 year), as well as AJCC staging (I to IV), grade, gender and age. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation. SOCS1 (p = 0.00*) was expressed in higher levels in patients with high survival (>5 years). Patients with low expression of TGFB1 survived longer compared to high expression group (p = 0.02*). On race comparison, CEBPA (p = 0.01*) showed significant expression in Caucasian population while NOS2 (p = 0.01*) expressed at higher levels in African Americans. These findings points to the clinical significance of MDSC based genes and tested the utility of a MDSC-related gene expression based prognostic biomarker panel for CRC patients.

#2776

Mitochondrial calcium uniporter in pancreatic cancer metastasis and metabolic stress resistance.

Xiuchao Wang,¹ Shengchen Lin,¹ Jianwei Sun,¹ Jiaxin Kang,¹ Jihui Hao,² Shengyu Yang¹. ¹Penn State College of Medicine, Hershey, PA; ²Tianjin Cancer Hospital, Tianjin, China.

Despite recent advances in cancer diagnosis and therapeutic modalities, the prognosis for pancreatic ductal carcinoma (PDAC) remains very poor. Most PDAC patients succumb to local and distant metastasis soon after the initial diagnosis. Therefore understanding molecular mechanisms underlying PDAC metastasis is crucial to the development of more effective PDAC therapies. Here we examined the role of mitochondrial calcium uniporter (MCU), a mitochondrial inner membrane calcium channel responsible for mitochondrial Ca2+ uptake, in pancreatic cancer metastasis and progression. We discovered that MCU is overexpressed in PDAC when compared to adjacent pancreatic tissues. MCU overexpression in PDAC patients is significantly associated with PDAC lymph node metastasis, histologic grade and pTNM stage. We also found that higher levels of MCU in PDAC patients was associated with worse overall survival (OS, P<0.01) and relapse-free survival (RFS, P<0.05) after surgical resection of tumors. MCU knockdown inhibits the PDAC cell motility, invasiveness, metabolic stress resistance and chemoresistance in cell culture models and PDAC metastasis in an orthotopic xenograft mouse model. Mechanistically, MCU controls the activation of the Keap1-Nrf2 signaling pathway through promoting mitochondrial ROS production and the oxidation of mitochondrion-recruited Keap1. Our data support that MCU regulates cancer cell metabolic stress resistance and oxidative stress response to drive PDAC metastasis and progression. Inhibiting MCU expression or blocking mitochondrial calcium influx may be an attractive approach for treating metastatic PDAC.

#2777

LOXL4-expressing neutrophils are found in colorectal cancer liver metastases resistant to anti-angiogenic therapy.

Vincent Palmieri,¹ Anthoula Lazaris,² Stephanie Petrillo,² Hussam Alamri,¹ Abdellatif Amri,² Woong-Yang Park,³ Zu-hua Gao,¹ Peter Metrakos¹. ¹McGill University Health Centre, Montreal, Quebec, Canada; ²Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada; ³Samsung Genome Institute, Seoul, Republic of Korea.

Three different histopathological growth patterns (HGP) have been identified in colorectal cancer liver metastases (CRCLM) resected from patients: the desmoplastic HGP, the pushing HGP, and the replacement HGP. Evidence suggests that the predominant growth pattern with which a CRCLM presents has clinically relevant prognostic implications. We have shown that patients with replacement HGP lesions who received bevacizumab plus chemotherapy had a worse pathological response and five-year overall survival than those with desmoplastic HGP lesions receiving the same treatment. We have also demonstrated that CRCLM with the replacement HGP promote vessel co-option for vascularization, rather than sprouting angiogenesis as seen in desmoplastic HGP metastases. These findings point to the growth patterns in CRCLM possibly serving as predictive biomarkers of response to anti-angiogenic therapy, when no such biomarker has been validated to date. However, HGP scoring is performed on resected liver metastatic tissue, implying that preoperative treatment precedes growth pattern assessment. Therefore, surrogate markers for the HGPs that can be appraised prior to surgery would be helpful to inform clinical decision-making about whether a patient with CRCLM may benefit from anti-angiogenic treatment.

This project aims to identify genes that are differentially expressed between CRCLM presenting with either the replacement or desmoplastic HGP. RNA sequencing (RNA-Seq) was used to compare the transcriptional profiles of these distinct CRCLM. A gene expression signature was generated from the RNA-Seq data to include genes that were upregulated in the replacement HGP liver metastases. Immunostaining of select genes from this signature was performed to validate these findings on human tumor tissue.

Initial analysis of our RNA-Seq data identified 525 genes that are differentially expressed between the replacement and desmoplastic HGP CRCLM, of which 53 genes met the criteria for the gene expression signature. Pathway analysis of these genes identified pathways involved in the immune system and extracellular matrix (ECM) to be upregulated in the replacement HGP lesions. Subsequent immunostaining revealed the expression pattern of lysyl oxidase like-4 (LOXL4) protein to be distinct between the HGPs - significantly greater quantities of LOXL4-expressing neutrophils were detected in the replacement HGP tumor microenvironment.

Characterizing differences in gene expression between replacement and desmoplastic HGP CRCLM is expected to provide some insight into the mechanisms responsible for generating these distinct growth patterns. Our findings suggest that further investigations into the role of tumor-associated leukocytes and ECM remodelling may ultimately lead to the development of biomarkers and new therapeutic targets for replacement HGP CRCLM.

#2778

ADD3-loss in glioblastoma is associated with increased angiogenesis and malignancy.

Mei-Yee Kiang, Gilberto Ka-Kit Leung. The University of Hong Kong, Hong Kong, Hong Kong.

Introduction: Adducin 3 (ADD3) is one of the major functional component in the cytoskeleton. Dysregulation of ADD3 has been implicated in various diseases including cancer and participate in different signaling transductions. However, little is known about the functional properties of ADD3 in cancer, its role in cancer biology remains unclear and controversial. We aim