bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 572: Genome-scale CRISPR/Cas9 screen identifies factors required for sensitivity to pyrimidine nucleoside analogs
Authors: SARR Awa , Bré Jennifer , Mullen Peter , Blagden Sarah , Um In Hwa , Harrison David J. , Reynolds Paul A.
Published: August 2018

NUC-1031 (NuCana plc), is a phosphoramidate transformation of gemcitabine, which bypasses cancer cell resistance mechanisms to gemcitabine. This compound showed promising results in clinical studies (phase I/II), demonstrating tumor reduction and durable disease control in patients who were refractory to or had relapsed on gemcitabine. Here, we adopted an unbiased approach to uncover genes and pathways involved in gemcitabine and NUC-1031 resistance. We performed a genome-wide knockdown screen using CRISPR/Cas9 technology in the pancreatic cancer cell line MiaPaCa2. Potential candidates from the screen were validated by gene inactivation using individual or combined single guide (sg)RNA targeting sequences. The effect of gene knockdown on NUC-1031 and gemcitabine resistance was assessed by monitoring cell survival after drug treatment using Celigo cytometry to assess cell number. EC50 values were determined using the resulting survival curves. This screen generated a list of 4 candidate genes potentially involved in NUC-1031 resistance. Two candidates were investigated further: DCK and DCTPP1, both involved in pyrimidine metabolism, especially in the maintenance of the dCMP/dCTP pool. DCK knockdown induced significant resistance to gemcitabine (EC50 350 times higher than in control cells) and a degree of resistance to NUC-1031 (EC50 7 times higher than in control cells). A less robust effect was observed when DCTPP1 was inactivated. DCTPP1 knockdown induced a small decrease in sensitivity to NUC-1031 (EC50 1.5 times higher than in control cells).

These results highlight the potential role of dCMP/dCTP pool regulation by DCK and DCTPP1 in sensitivity to pyrimidine nucleoside analogs and may provide a useful biomarker for patient selection.

Citation Format: Awa SARR, Jennifer Bré, Peter Mullen, Sarah Blagden, In Hwa Um, David J. Harrison, Paul A. Reynolds. Genome-scale CRISPR/Cas9 screen identifies factors required for sensitivity to pyrimidine nucleoside analogs [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 572.
Abstract 5737: Genomic drivers of cancer are enriched and mutually exclusive within non-T cell-inflamed tumors
Authors: Olson Brian , Bao Riyue , Fessler Jessica , Luke Jason , Gajewski Thomas F. , Patnaik Akash
Published: August 2018

While immunotherapy has seen dramatic successes in a variety of malignancies, the majority of patients fail to respond. In particular, less than 10% of patients with metastatic, castrate-resistant prostate cancer (mCRPC) respond to immunotherapy. Based on previous work demonstrating robust clinical activity of immunotherapies in patients with a T cell-inflamed tumor microenvironment, we investigated whether non-T cell-inflamed cancers show evidence for distinct genomic alterations that might mediate immune escape. RNA-seq gene expression data were analyzed from two prostate cancer cohorts, The Cancer Genome Atlas (TCGA) of primary tumors (n=437) and a Stand Up to Cancer (SU2C) mCRPC cohort (n=43). These datasets were interrogated for common molecular alterations and their association with non-T cell-inflamed and T cell-inflamed cancers, focusing on copy number deletions of PTEN, RB1/CDKN2A, and activating mutations in CTNNB1 (β-catenin). Similar approaches were used to analyze 30 solid tumor types from TCGA (n=9555). Our analysis identified that 94% of primary prostate cancers harbor a non-T cell-inflamed tumor microenvironment, with only 6% of patients having a T cell-inflamed gene signature. Similar results were found in mCRPC patients from the SU2C cohort. Higher frequencies of PTEN loss and CTNNB1 activation, as well as a significant enrichment of RB1/CDKN2A deletion, were found in non-T cell-inflamed primary and metastatic prostate tumors. Moreover, we observed a direct correlation between the expression of PTEN/RB1 and CD8A expression within the tumor microenvironment. Strikingly, analysis of 30 human cancers revealed that loss of PTEN and/or RB1/CDKN2A correlates with T-cell exclusion across multiple malignancies. When combined with β-catenin pathway activation, we found that these alterations are mutually exclusive, with only 1% of non-T cell-inflamed cancers sharing all three mechanisms, 24% sharing two out of the three, and 75% of the samples harboring only one mechanism. Taken together, these data reveal that deregulated PTEN/PI3K, Rb and Wnt-β-catenin signaling networks are enriched in a mutually exclusive manner within non-T cell-inflamed cancers, suggesting that such cancers evolutionarily co-opt nonredundant signaling pathways to achieve a common goal of immune evasion. Targeting these pathways pharmacologically may provide an opportunity to enhance responsiveness to immunotherapy across multiple malignancies.

Citation Format: Brian Olson, Riyue Bao, Jessica Fessler, Jason Luke, Thomas F. Gajewski, Akash Patnaik. Genomic drivers of cancer are enriched and mutually exclusive within non-T cell-inflamed tumors [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 5737.
Abstract 574: BRAF
V600E mutation and loss of CDX2 synergize in serrated colorectal tumorigenesis
Authors: Sakamoto Naoya , Feng Ying , Hattori Takuya , Shimoda Masayuki , Ishikawa Akira , Honma Ririno , Hinoi Takao , Egi Hiroyuki , Ohdan Hideki , Okada Yasunori , Fearon Eric , Yasui Wataru
Published: August 2018

While many CRCs arise from adenomatous polyps through what we call “adenoma-carcinoma sequence,” it has been estimated that up to 20% of CRCs likely evolve from an alternative pathway, so-called ‘‘serrated pathway.' However, only 8-10% of CRCs display definitive serrated morphology at diagnosis. We previously examined expression of differentiation and molecular markers that are most likely to be involved in serrated tumor development by using 36 serrated CRCs and found CDX2 loss or BRAF mutations often together. We then generated a mouse model that can be induced concurrent biallelic inactivation of Cdx2 (Cdx2-/-) and expression of mutant BRAFV600E in adult mouse colon epithelium, and the Cdx2-/-/BrafV600E tumors showed a quite similar phenotype to that of CDX2-negative serrated CRCs. Through the validation of global gene expression profile including the mouse models and human CRCs, we focused on ADAM28 as a candidate because ADAM28 was significantly upregulated in Cdx2-/-/BrafV600E tumor and CRCs with low CDX2 expression and BRAFV600E mutation. We examined ADAM28 expression using 12 CRC cases with BRAFV600E mutation (8 of 12 displayed serrated feature) and 12 CRC cases with KRAS codon 12/13 mutation and found that ADAM28 was specifically upregulated in BRAF mutant CRC cases. Among 10 CRC cell lines (DLD-1, Lovo, SW48, SW480, SW837, SW1116, Caco2, RKO, HT-29, HCT-116), only HT-29, which has many phenotypic and genetic similarities to serrated CRC, showed a robust expression of membranous type ADAM28, and 9 of 10 CRC cells, except for RKO, showed weak to modest expression of active-form ADAM28. Further in-depth studies are ongoing to clarify the solid evidences, especially the utility of ADAM28 as a diagnostic marker for serrated CRCs and the direct effect of ADAM28 onto cell growth of CRC cells using ADAM28-specific antibody. We present a novel transgenic model of human serrated CRC to highlight the suitability of centering on CDX2 loss and BRAFV600E in the pathogenesis. This model has a great potential to figure out the mechanisms underlying in serrated tumor progression and identify the candidates of new biomarkers and therapeutic targets of CRCs with serrated morphology.

Citation Format: Naoya Sakamoto, Ying Feng, Takuya Hattori, Masayuki Shimoda, Akira Ishikawa, Ririno Honma, Takao Hinoi, Hiroyuki Egi, Hideki Ohdan, Yasunori Okada, Eric Fearon, Wataru Yasui. BRAFV600E mutation and loss of CDX2 synergize in serrated colorectal tumorigenesis [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 574.
Abstract 5755: Cancer immunity contributes to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers
Authors: Jung Hyunchul , Choi Jung Kyoon , Lee Eunjung Alice
Published: August 2018

Long interspersed nuclear element-1 (L1) retrotransposons are normally suppressed in somatic tissues, mainly by DNA methylation and antiviral defense. However, L1s can be de-suppressed in cancers to act as insertional mutagens and cause genomic instability by creating DNA double-strand breaks and chromosomal rearrangements. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, our pan-gastrointestinal cancer genome analyses for stomach, colorectal, and esophageal tumors identified multiple correlates of L1 activity. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. Potential L1 expression suppressors such as TP53 and DNMT1, a DNA methyltransferase, were inactivated in tumors with frequent L1 insertions. Importantly, tumors with high immune activity, for example, due to viral infection or high tumor-antigen load, tended to carry a low number of L1 insertions in their genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1. Our analysis of the transcriptional effects of intragenic retrotransposon insertions demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found a splicing-disrupting L1 insertion in an exon of MOV10, a key L1 suppressor, which caused exon skipping with evidence of nonsense-mediated decay in a tumor with a high L1 insertion load. Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition, particularly in gastrointestinal cancers.

Citation Format: Hyunchul Jung, Jung Kyoon Choi, Eunjung Alice Lee. Cancer immunity contributes to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers [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 5755.
Abstract 5763: Large-scale use of knockout validation to confirm antibody specificity to key oncology targets
Authors: Heaton Sam , Sassi Monica , Solache Alejandra , Hamilton Bruce , Dave Sambhav
Published: August 2018

Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. As a result, experimental irreproducibility leads to wasted resources and compromises the advancement of science. In response to this, Abcam has introduced knockout (KO) validation to help produce target-specific antibodies. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line. This results in a frameshift and a complete loss of gene expression. These KO cell lines provide a true negative control for antibody validation as a lack of signal confirms that the antibody binds to its intended target. As a result, both antibody specificity and reproducibility on a large scale can be addressed. We have KO-validated over a thousand antibodies, including several that are relevant to immuno-oncology research. Examples of such targets are the ERK1/2 pathway, which regulates cell survival, proliferation and cell death. Inhibition of this pathway is an attractive target for the treatment of malignant tumors with increased ERK1/2 activity. Similarly, Ki67 expression is strongly associated with tumor cell proliferation and can be used as a marker for tumor aggressiveness. Finally, overexpression of vimentin in tumors correlates with increased growth and invasion, leading to poor prognosis. It is imperative that reproducible data can be produced on these targets to further our understanding of tumor biology and patient prognosis. Here we present the data for antibodies to these key targets that have been validated in both flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. The antibodies shown gave signal in wild-type cells but showed complete loss of signal when tested in knockout cell lines. By providing researchers with reliable and specific antibodies that work first time we hope to minimize wasted resources and improve reproducibility.

Citation Format: Sam Heaton, Monica Sassi, Alejandra Solache, Bruce Hamilton, Sambhav Dave. Large-scale use of knockout validation to confirm antibody specificity to key oncology targets [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 5763.
Abstract 5766: Multifunctional biologics for targeted T-cell therapy based on in vivo matured fully human VH domains
Authors: Mcguinness Brian , Legg James , Bland-Ward Phil , Pack Peter
Published: August 2018

Crescendo Biologics has created a proprietary transgenic mouse devoid of any antibody light chains from which it generates highly diverse fully human VH domain (‘Humabody') building blocks. In vivo maturation optimizes Humabody potency and develops superior biophysical properties. Their small size (13kDa) and high stability permits Humabody assembly into an almost limitless array of multifunctional formats optimally configured for therapeutic efficacy. These small versatile single VH domain building blocks permit the rapid exploration of a vast range of 3D format space to identify optimal therapeutic solutions. This fully modular plug & play approach lacks the constraints of traditional mAbs and enables a radical rethink of how a multifunctional molecule can be designed and assembled to deliver enhanced therapeutic benefit. Such molecules are capable of target engagement that is unachievable using regular mAbs. Using Humabody VH addressing key targets in the immune-oncology space, Crescendo has exemplified the potential for optimally configured molecules to deliver enhanced efficacy both in vitro and in vivo. Examples include: CB307, a unique T-cell engaging format with a novel mode of action. CB201, a biparatopic antagonist which ‘handcuffs' PD1: one domain binding and blocking PD1 from engaging with its ligands, the other binding a nonblocking epitope. With a binding affinity of 175pM and a unique ability to localise rapidly to PD-1 positive T-cells, CB201 demonstrates significantly enhanced in vivo efficacy over IgG-based PD-1 antagonists in a PDX tumour model in NSG mice. CB108, a half-life extended paratopic Humabody binding PSMA has demonstrated superior tumour targeting with enhanced internalisation and highly efficient accumulation in target-positive tumours with up to 18 percent ID per g recorded in a 24hr imaging study. These data support the progression of highly differentiated therapeutics into the clinic.

Citation Format: Brian Mcguinness, James Legg, Phil Bland-Ward, Peter Pack. Multifunctional biologics for targeted T-cell therapy based on in vivo matured fully human VH domains [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 5766.
Abstract 5772: Engineered lectins to treat cancer
Authors: Murarasu Thomas , Johannes Ludger
Published: August 2018

Targeted delivery of cytotoxic compounds to tumors has become one of the most dynamic areas of research and development in the cancer therapy field. In this context, glycosphingolipids (GSLs) represent a promising class of tumor markers. Indeed, cancer patients have altered GSL expression patterns on their tumoral cells compared to healthy tissue (Krengel et al., 2014). In a recent prioritization of cancer antigens by the National Cancer Institute at Rockville (USA), 4 out of 75 were GSLs (GD2, GD3, fucosyl-GM1, and GM3), with GD2 arriving already at the 12th position (Cheever et al., 2009). However, GSLs are only rarely addressed in the context of tumor targeting, obtaining GSL specific antibodies being a notorious difficult task. An exception is the anti-GD2 antibody, dinutuximab, which entered the market for the treatment of high-risk neuroblastoma patients, which is unfortunately associated to dose-limiting acute toxicity (Yu et al., 2010). This project aims at developing a novel class of therapeutic products for the delivery of therapeutic compounds by targeting tumor-specific GSLs. Our products are based on a naturally occurring GSL-binding lectin with intrinsic tumor targeting capacity. We will rely on a proprietary technology to derive products which are tuned to transport cytotoxic compounds specifically into cancer cells. As a proof of concept, we apply our technology to a tumor-specific GSL, GD2, to generate a therapeutic formulation against neuroblastoma, one of the most frequent cancer in children under 5 years old. Our technology has truly groundbreaking potential as, in principle, it can apply to any GSL to specifically target several common tumors.

Citation Format: Thomas Murarasu, Ludger Johannes. Engineered lectins to treat 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 5772.
Abstract 5773: Sequencing and cloning IDH1 R132H-targeted monoclonal T cell receptors from CD4+ T cells facilitated by opto-electric-positioning technology
Authors: Smith Duane , Watchmaker Payal , Stadler Guido , Marks Natalie , Bronevetsky Yelena , Chapman Keviin , Okada Hideho
Published: August 2018

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are enzymes that catalyze the conversion of isocitrate to α-ketoglutarate, while reducing NADH to NADPH. More than 80% of low-grade gliomas and secondary glioblastomas harbor point mutations in IDH. The R132H mutation is the most common mutation in IDH1, and vaccination with the mutant peptide elicited an MHC class II-specific antitumor response, suggesting mutant IDH1 as a potential target for immunotherapy (1). Discovering monoclonal T cell receptors (TCRs) that specifically recognize the R132H mutation is a key but challenging step towards viable immunotherapy for glioma/glioblastomas. Using opto-electric-positioning (OEP) technology in a chip-based, NanoPen culture environment, we have successfully identified and isolated a monoclonal T cell that specifically recognizes the R132H mutant IDH1 peptide. We cultured and identified monoclonal CD4+ T cells on chip, stimulated by monocyte-derived dendritic cells, pulsed with R132H IDH1 peptide. Activated, CD4+ T cells were selectively exported from the chip and their T-cell receptors (TCRs) were subsequently deep sequenced. The most likely pairs of alpha and beta TCR chains were cloned into mammalian expression vectors for functional validation in vitro, producing a TCR that specifically binds mutant IDH1 tetramer. Taken together, we show that the combined platform of the OEP and nanofluidic device provides a powerful solution to help identify cell type-specific, monoclonal T cells that can be exported and sequenced to reveal distinct TCRs, which in turn can be cloned, expressed and validated for future immunotherapies.

Reference: 1. Schumacher et al., Nature 2014;512:324-27.

Citation Format: Duane Smith, Payal Watchmaker, Guido Stadler, Natalie Marks, Yelena Bronevetsky, Keviin Chapman, Hideho Okada. Sequencing and cloning IDH1 R132H-targeted monoclonal T cell receptors from CD4+ T cells facilitated by opto-electric-positioning technology [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 5773.
Abstract 5778: Preclinical evaluation of TQB3616, a highly potent and selective small-molecule CDK4/6 inhibitor
Authors: Zhaobing Xu , Lihong Hu , Yingchun Liu , Ding Charles Z. , Zhu Xiaoyu , Chen Chen , Zhu Yusong , Wu Jiahu , Wu Dongdong , Chen Shuhui , Zhang Xiquan , Yang Ling , Tian Xin
Published: August 2018

Objective: The G1 restriction point is critical for regulating the cell cycle and is controlled by the retinoblastoma protein (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). Selective CDK4/6 inhibitors, such as palbociclib and ribociclib have been approved by US FDA in combination with letrozole to treat patients with HR+/Her2- breast cancer, and selective CDK4/6 inhibitor LY2835219 is in Phase 3 trials for the same and related indications. Here, we disclose TQB3616 for the first time, a novel selective, potent, oral inhibitor of CDK4/6.

Method: The anti-proliferative activity of TQB3616 was evaluated in a panel of 18 Rb-proficient cell lines in a 72-hour viability assay. Daily oral administration was used to evaluate in vivo antitumor activity of TQB3616 in the MCF-7 cell-derived xenograft (CDX) breast cancer model. Effect on CDK4/6-cyclin D1-Rb signaling was assessed by western blot analysis of downstream effector protein p-Rb. In vivo antitumor activity of TQB3616 was also tested in a patient-derived xenograft (PDX) lung cancer model LU-01-0393.

Result: TQB3616 displayed potent anti-proliferative activity in 8 of 18 Rb-proficient cancer cell lines (>50% inhibition at 0.3 µM). Inhibition of p-Rb in the tumor samples was verified by western blot analysis. TQB3616 showed in vivo antitumor activity in the MCF-7 CDX model with TGI of 60% @7.5 mpk and 93% @15 mpk, better than palbociclib (TGI = 52% @20 mpk, 80% @40 mpk) and LY2835219 (TGI = 45% @7.5 mpk, 76% @15 mpk). TQB3616 also showed good in vivo antitumor activity in the LU-01-0393 lung cancer PDX model with 65% of TGI @35 mpk.

Conclusion: TQB3616 is a potent and selective CDK4/6 inhibitor and shows excellent antitumor activity in preclinical tumor models. These results warrant TQB3616 going to the clinic for exploration of its potential for treating HR+/Her2-/Rb+ breast cancer as well as other Rb+ cancer types.

Citation Format: Xu Zhaobing, Hu Lihong, Liu Yingchun, Charles Z. Ding, Xiaoyu Zhu, Chen Chen, Yusong Zhu, Jiahu Wu, Dongdong Wu, Shuhui Chen, Xiquan Zhang, Ling Yang, Xin Tian. Preclinical evaluation of TQB3616, a highly potent and selective small-molecule CDK4/6 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5778.
Abstract 5782: Selumetinib-based therapy in uveal melanoma patient-derived xenografts
Authors: Decaudin Didier , Botty Rania EL , Diallo Béré , Massonnet Gerald , Fleury Justine , Naguez Adnan , Dubois Marine , Davies Emma J. , Smith Aaron , Wilson Joanne , Howes Colin , Smith Paul , Cassoux Nathalie , Mariani Pascale , Piperno-Neumann Sophie , Roman Sergio Roman , Nemati Fariba
Published: August 2018

Purpose: The prognosis of metastatic uveal melanoma (UM) patients remains one of the worst observed in human cancers. The identification of frequently mutated GNAQ/GNA11 and the activation of MAPK signaling in UM raise the possibility of using efficient targeted therapies such as MEK inhibition. The aim of our study was to evaluate drug combinations that might enhance the efficacy of the MEK inhibitor, selumetinib (AZD6244, ARRY-142886), using patient-derived xenografts.

Materials: Five UM PDXs were used in this study, i.e., MP34, MP46, MP55, MP77, and MM26, all mutated for GNAQ or GNA11. Selumetinib was administered alone or combined with chemotherapies (dacarbazine and docetaxel) or targeted therapies: the ERK inhibitor, AZ6197 and the mTORC1/2 inhibitor, vistusertib (AZD2014). All targeted therapies were administered orally five days/week at the following doses: selumetinib at 25 mg/kg/day BID, AZ6197 at 50 mg/kg QD, AZD2014 at 15mg/kg QD. Dacarbazine was administered at 40 mg/kg for 5 consecutive days every 28 days and docetaxel at 15 mg/kg weekly. Both were administered intraperitoneally.

Results: The combination of selumetinib and dacarbazine did not show improvement of antitumor efficacy in MP34, MM26 and MP55.The results showed a slight increase of overall response rate when selumetinib was combined with docetaxel, in comparison to the treatment with selumetinib or docetaxel alone. Importantly, we observed a significant antitumor effect when selumetinib was used in combination with AZ6197 or AZD2014 with respect to the monotherapies. Preliminary pharmacodynamics studies showed variation of the expression of p-ERK and also p-AKT and/or p-S6 when UM PDX were treated with AZ6197 or AZD2014 alone or in combination with selumetinib.

Conclusion: Our data suggest that ERK and mTORC1/2 targeting could represent promising therapeutic approaches for the treatment of metastatic UM patients.

Citation Format: Didier Decaudin, Rania EL Botty, Béré Diallo, Gerald Massonnet, Justine Fleury, Adnan Naguez, Marine Dubois, Emma J. Davies, Aaron Smith, Joanne Wilson, Colin Howes, Paul Smith, Nathalie Cassoux, Pascale Mariani, Sophie Piperno-Neumann, Sergio Roman Roman, Fariba Nemati. Selumetinib-based therapy in uveal melanoma patient-derived xenografts [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 5782.
Abstract 5783: Strong antitumor activity of ASN007, an oral ERK1/2 inhibitor, in PDX tumor models with MAP kinase pathway alterations including KRAS mutations
Authors: Reddy Sanjeeva P. , Rao Niranjan S. , Smith Roger A. , Thompson Scott K. , Toker Sarper
Published: August 2018

The RAS/RAF/MEK pathway is frequently dysregulated in a broad range of cancers through mutations in several targets such as KRAS, NRAS, HRAS, BRAF, NF1 and receptor tyrosine kinases. Although MEK and BRAF inhibitors improve the outcome for patients with BRAF mutant melanoma and lung cancers, the duration of response is limited due to reactivation of the pathway. The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and BRAF kinases, serve as a key node in this crucial signaling pathway. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. Here, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1 and Elk1 in various cell lines with low nM IC50 values. ASN007 showed potent anti-proliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more robust activity against RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a daily oral dosing regimen, ASN007 demonstrated strong tumor growth inhibition in BRAF, KRAS and NRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 showed strong efficacy using various intermittent dosing regimens. It also showed a strong anti-tumor activity in colorectal PDX models with various KRAS mutations. Furthermore, ASN007 showed regression of tumor growth in a melanoma PDX model that was resistant to both MEK and BRAF inhibitors. In addition to the single agent activity, greater efficacy was observed when ASN007 was combined with immunotherapeutic agents such as an anti-PD-1 antibody or an IDO-1 inhibitor. ASN007 does not exhibit significant inhibition of CYP enzymes or the hERG channel. A Phase 1 trial with ASN007 in patients with BRAF and K-, H- and N-RAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is being planned.

Citation Format: Sanjeeva P. Reddy, Niranjan S. Rao, Roger A. Smith, Scott K. Thompson, Sarper Toker. Strong antitumor activity of ASN007, an oral ERK1/2 inhibitor, in PDX tumor models with MAP kinase pathway alterations including KRAS mutations [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 5783.
Abstract 5784: A novel ERK inhibitor in the treatment of uveal melanoma
Authors: Musi Elgilda , Ambrosini Grazia , Munck Joanne , Schwartz Gary K.
Published: August 2018

Uveal melanoma is one of most common intraocular malignancies of the adult eye. GNAQ/ GNA11 genetic alterations are present in 85% of diagnosed cases resulting in activation of MAPK pathway. Recently, MEK inhibitors, such as selumetinib have been used to treat GNAQ/GNA11-mutant uveal melanoma patients. However, selumetinib has shown modest effect compared to chemotherapy. As ERK is the primary downstream effector of the MAPK pathway, it is hypothesized that ERK inhibitors have the potential to improve clinical activity in these patient populations. We examined the effects of a selective and potent ERK inhibitor (Compound A), on several uveal melanoma cell lines including 4 GNAQ cell lines (92.1, Mel202, Omm1.3, MP46), 2 GNA11 cell lines (MP41, Omm1), 2 BRAF cell lines (OCM1A, OCM1) and a Wild Type (Mel290) cell line. The uveal melanoma cell lines were sensitive to ERK inhibition by Compound A, with IC50s concentrations determined to be between 50 to 250 nM. Compound A was more effective at inhibiting growth than the MEK inhibitor selumetinib at nanomolar concentrations. Compound A at 250 nM inhibited pERK1/2 within at least 2 hours of drug exposure. By 24 hours inhibition of pRB and suppression of cyclin D1 was detected in all the cell lines tested except the Wild type cell line Mel290. Cell cycle analysis of Compound A dosed at 250 nM for 24 hours showed significant G1 arrest (85-90% G1). These in vitro results support further evaluation of ERK inhibition in the treatment of uveal melanoma.

Citation Format: Elgilda Musi, Grazia Ambrosini, Joanne Munck, Gary K. Schwartz. A novel ERK inhibitor in the treatment of uveal 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 5784.
Abstract 5788: Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT
Authors: Li Gang , Hu Lihong , Mao Weifeng , Li Dongfang , Ding Charles Z. , Chen Shuhui
Published: August 2018

Objective: AKT is a component of the PI3K/AKT/mTOR (PAM) pathway, which plays an important part in cell proliferation, growth and survival, activated in many cancers and has been validated as a therapeutic target in the clinic. Inhibition of AKT activity can block the PAM pathway and proliferation of various types of tumor cells with loss of tumor suppressor PTEN, mutational activation of p110a catalytic subunit of PI3K and amplification of the gene encoding either AKT or PI3K. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of HZB0071, a small molecule AKT kinase inhibitor, in preclinical models of solid cancer with AKT overexpression.

Method: Kinase inhibiting activity of HZB0071 was determined with AKT1, 2, 3 kinase assays. Cellular anti-proliferative activity was evaluated with AKT overexpressed LNcap cell line and DU145 cell line without AKT overexpression. The antitumor activity of HZB0071 was evaluate in vivo and the synergistic effect combined with Paclitaxel in AKT highly expressed patient-derived mouse xenograft (PDX) models of Gastric Cancer ST-02-0013 and cell-derived mouse xenograft (CDX) models of Breast Cancer BT474.

Result: HZB0071 displayed potent kinase inhibiting activity for AKT1, 2, 3 with IC50 1.3±0.41 nM, 50.3±13.4 nM and 90.4±5.84 nM, respectively. HZB0071 inhibited cell proliferation in AKT overexpressed LNcap cells with IC50 67±7 nM. In contrast, HZB0071 showed much weaker anti-proliferative activity in DU145 cells with IC50 >10,000 nM. HZB0071 showed antitumor efficacy in AKT highly expressed gastric cancer PDX model ST-02-0013 (TGI = 62% @30 mpk, QD as single agent; TGI = 61% @15 mpk + Paclitaxel @10 mpk, QD, in combination with Paclitaxel), as well as in the breast cancer BT474 CDX model (TGI = 88% @30 mpk + Paclitaxel @15 mpk, QD).

Conclusion: We have identified a novel potent AKT inhibitor HZB0071. Preclinical studies shows antitumor efficacy of HZB0071 in AKT overexpressed solid cancer models. HZB0071 represents a promising clinical candidate for treating solid cancers with high AKT expression.

Citation Format: Gang Li, Lihong Hu, Weifeng Mao, Dongfang Li, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT [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 5788.
Abstract 5799: Characterization and profiling of CB-103, a novel small-molecule protein-protein interaction inhibitor targeting the NOTCH transcription complex
Authors: Lehal Rajwinder , Urech Charlotte , Vigolo Michele , Murone Maximilien , Radtke Freddy
Published: August 2018

NOTCH signalling is a key development pathway whose aberrant activation is known to play a role in multiple human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such over expression of ligands/receptors, GOF mutations in NOTCH receptors, including protein stabilizing mutations in the PEST domain of NOTCH, chromosomal translocations, or loss-of-function mutations in the E3 ubiquitin ligase FBXW7 and other negative regulators of the pathway (SPEN, NUMB). Activation of signalling due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). However, in human tumors where NOTCH signalling is constitutively activated due to chromosomal translocations in the NOTCH receptors (NOTCH1 and 2), none of the above-mentioned strategies will be effective. Moreover, due to on-target and off-target toxicities associated with blocking antibodies and GSIs, these anti-NOTCH agents failed to advance in clinical trials, although some of them showed signs of clinical efficacy. Given the role of NOTCH signalling in human tumors, there is a need to identify novel targets in the NOTCH pathway and develop new and more selective anti-NOTCH agents. To inhibit pan-NOTCH signalling in human tumors independently of the mechanisms of NOTCH activation, and in the most downstream part of the pathway, we have previously reported the discovery of a new class of small molecules able to target the NOTCH transcription complex enabling the specific inhibition of NOTCH target gene expression (e.g. cMYC, HES1, DTX1, CCND1). These small molecules act as protein-protein interaction inhibitors, and thereby compromise the assembly of functional NOTCH transcription complex.

Here we present further in vitro and in vivo characterization of the lead molecule CB-103. The anti-cancer activity of CB-103 was extensively profiled in several human cancer cell lines representing NOTCH positive solid tumors, leukemias and lymphomas. Moreover, CB-103 responsiveness of these cell lines correlates with a downregulation of the NOTCH signature following treatment with CB-103. Specifically, we will present data outlining the in vivo pharmacokinetic and pharmacodynamic properties of CB-103.

Citation Format: Rajwinder Lehal, Charlotte Urech, Michele Vigolo, Maximilien Murone, Freddy Radtke. Characterization and profiling of CB-103, a novel small-molecule protein-protein interaction inhibitor targeting the NOTCH transcription complex [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 5799.
Abstract 5809: Synergistic effect of alectinib and everolimus on anaplastic large cell lymphoma cells via ALK-mTOR pathway inhibition
Authors: Kim Dongchan , Park Hyejoo , Koh Youngil , Yoon Sung-Soo
Published: August 2018

Purpose Anaplastic lymphoma kinase (ALK) fusion is associated with various kinds of cancer, including anaplastic large cell lymphoma (ALCL). We investigated the efficacy of combination between ALK inhibitor and mammalian target of rapamycin (mTOR) inhibitor in ALK-positive ALCL cells.

Methods & Results ALK-positive cancer cell lines were treated with ALK and/or mTOR inhibitors and assessed for cell viability. Treatment of ALK-positive ALCL cell lines with the combination resulted in a synergistic effect in Karpas299 cells but not in SU-DHL-1 cells. Viability of Karpas299 cell was reduced to 26.4% (p<0.001) by alectinib-everolimus combination treatment compared to the single treatment groups (alectinib: 59.5%, everolimus: 51.6%); the Combination Index (CI) value of 0.316 indicated a synergistic effect of the combination treatment. Moreover, changes in intracellular signaling were analyzed by western blot. Combination treatment augmented increase of ALK-mTOR de-phosphorylation, and cleavage of poly ADP ribose polymerase (PARP) molecules. Combination treatment significantly increased the numbers of Karpas299 cells in subG1 (p = 0.0096 everolimus), and G0/G1 (p = 0.0018 alectinib; 0.0013 everolimus) cell cycle arrest compared to single treatment groups. We investigated the biological differences between Karpas299 and SU-DHL-1 cell lines using RNA sequencing data obtained from Sequence Read Archive (SRA). The RNA expression pattern of the ALK isoforms ALK-201 and 203 was different between the two cell lines. These data indicated that the relatively high expression of ALK-203 in SU-DHL1 cells could explain the lack of synergism in response to the combination treatment observed.

Conclusions Combination treatment with alectinib and everolimus synergistically reduced cell survival, augmented inhibition of cellular signaling, and increased cell cycle arrest in Karpas299 but not in SU-DHL1 cells. These differences might be attributable to differential expression of ALK isoforms between the two cell lines.

Citation Format: Dongchan Kim, Hyejoo Park, Youngil Koh, Sung-Soo Yoon. Synergistic effect of alectinib and everolimus on anaplastic large cell lymphoma cells via ALK-mTOR pathway inhibition [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 5809.
Abstract 5837: Acquired resistance to anticancer drug of high glucose-exposed HepG2
Authors: Kishimoto Shuichi , Fukushima Shoji
Published: August 2018

Reports show that diabetic patients with cancer tend to show resistance to anticancer drug treatment and have poor prognosis compared to non-diabetic patients. We observed that HepG2-HG, which is a derivative of the hepatocellular carcinoma cell line HepG2 exposed continuously to high glucose, shows high resistance to etoposide. In this study, we aimed to understand the involvement of excretion transporter and apoptosis regulatory factor in anti-cancer drug resistance in HepG2-HG. HepG2 was cultured in minimum essential medium (MEM) (with 5.5 mM glucose), and HepG2-HG was cultured in MEM supplemented with up to 25 mM glucose. Sensitivity to anticancer drugs was evaluated by the SRB assay. mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on Rotor-Gene Q using the KAPA SYBR FAST qPCR kit. Proteins extracted using the Ultra-RIPA kit were electrophoresed, transferred to a membrane by the iBlot 2 gel transfer device, reacted with antibodies, and evaluated for expression using enhanced chemiluminescence (ECL) prime reagent. IC50 values of etoposide in HepG2 and HepG2-HG were 0.59 µM and 17.5 µM, respectively. Comparison of the expression of the excretion transporter showed that the expression of p-glycoprotein (p-gp) was markedly enhanced in HepG2-HG compared to that in HepG2. Continuous contact of HepG2-HG with high glucose induced the expression of the glucose transporter GLUT1, which may provide a survival mechanism for cancer cells via escape from hypoxia-induced apoptosis. Furthermore, the expression of phospho-p70S6K was high in HepG2-HG, suggesting an increase in the activity of the mammalian target of rapamycin (mTOR). Therefore, we suggest that increased p-gp expression and mTOR activity in HepG2-HG provided resistance to etoposide by decreasing intracellular drug levels and suppressing the apoptotic mechanism. Estimation of the contribution of both mechanisms to etoposide resistance of HepG2-HG requires further examination.

Citation Format: Shuichi Kishimoto, Shoji Fukushima. Acquired resistance to anticancer drug of high glucose-exposed HepG2 [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 5837.
Abstract 5840: Inhibition of cystathionine-β-synthase (CBS) sensitizes colon cancer cells to 5-FU via increasing apoptosis and inhibiting cellular bioenergetics
Authors: Chen Shanwen , Wang Pengyuan , Pan Yisheng , Liu Yucun
Published: August 2018

Increased expression of cystathionine-β-synthase (CBS) has been validated in multiple kinds of tumor tissues, including colorectal cancer. Endogenous hydrogen sulfide (H2S), produced by CBS, was able to promote the proliferation and migration of colon cancer cells by inhibiting apoptosis and injuries caused by noxious environmental factors, including chemotherapeutic agents. Recently, overexpression of CBS has been reported to play a pivotal role in tumorigenesis of colon cancer cells, rendering the normal colon epithelial cell line acquiring the features of malignant cells. However, the potential role of CBS in the development of chemoresistance of colon cancer cells has not be illustrated. In the present study, overexpression of CBS in tumor tissues was further validated by realtime PCR and the effect of inhibition of both the activity and expression of CBS on the sensitivity to 5-fluorouracil (5-FU) was investigated in colon cancer cell lines. Inhibition of the activity of CBS by CBS inhibitor aminooxyacetate (AOAA) significantly increased the sensitivity of colon cancer cell lines to 5-FU, featured by decreased IC50 and colony forming efficiency. However, utilizing a slowly releasing H2S donor GYY4137, the present study suggested that exogenous hydrogen sulfide was able to increase the resistance to 5-FU in both colon cancer cell lines. What's intriguing was that, 5-FU, at clinically relevant concentrations, significantly inhibited the expression of CBS. Co-treatment of AOAA significantly increased the expression of Bax and decreased the expression of Bcl-2, indicating the involvement of increased apoptosis. AOAA also predisposed the colon cancer cells to the inhibition of cellular energetics induced by 5-FU, featured by the further reduced level of cellular ATP. These results suggested that inhibition of CBS promoted the sensitivity of colon cancer cells to 5-FU via increasing apoptosis and inhibiting cellular bioenergetics. Furthermore, inhibition of CBS expression by shRNA sensitizes the 5-FU resistant colon cancer cell line HCT-8/5-FU to treatment with 5-FU. The increased expression of CBS and the consequently increased level of endogenous H2S might be a potential therapeutic target for chemoresistance in colon cancer.

Citation Format: Shanwen Chen, Pengyuan Wang, Yisheng Pan, Yucun Liu. Inhibition of cystathionine-β-synthase (CBS) sensitizes colon cancer cells to 5-FU via increasing apoptosis and inhibiting cellular bioenergetics [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 5840.
Abstract 5856: Identification and validation of Nrf2 inhibitors in esophageal squamous cell carcinoma
Authors: Weir Seth , Chen Xiaoxin , Paiboonrungruan Chorlada
Published: August 2018

Background: The Nrf2/Keap1 pathway is critical for human cells to respond to oxidative stress. In basal conditions the Keap1 homodimer is bound to Nrf2 in the cytoplasm. When stressed, Keap1 releases Nrf2 which translocates into the nucleus, binds the antioxidant response element, and activate antioxidative genes. Somatic mutations of Nrf2 or Keap1 gene can cause constitutive activation of this pathway, and thus results in chemoradioresistance and poor survival in patients with esophageal squamous cell carcinoma (ESCC). Our goal is to identify chemical Nrf2 inhibitors for targeted therapy of Nrf2high ESCC in the future.

Methods: Possible Nrf2 inhibitors were identified using high throughput screening of 1,280 compounds in the Prestwick library, and 35,000 compounds in the Asinex library with Nqo1-EGFP H1299 cells stimulated by an Nrf2 activator (CDDO). Nrf2 activity was monitored via Incucyte by measuring the total normalized green fluorescence divided by the total normalized red fluorescence (tagged nuclear cherry marker). The red channel assessed cell viability and cytotoxicity of compounds. Inhibitors were identified if they met a threshold of >50% inhibition. Lead compounds will be further validated in vitro with KYSE 450 (Nrf2low+Keap1 knockdown) and KYSE 70 (Nrf2high) by examining their effects on expression of nuclear Nrf2 and its downstream target gene (Nqo1). These compounds will also be validated in vivo using Sox2CreER;Nrf2Ki/Ki mice.

Results: We successfully established the in vitro assay with Nqo1-EGFP H1299 cells for high throughput screening of Nrf2 inhibitors. Eight lead compounds were identified from the Prestwick library and validated as

Nrf2 inhibitors via a dose-response experiment (IC50<1μM). Screening of the Asinex library and further validations are still ongoing.

Conclusion: We have identified Nrf2 inhibitors through high-throughput screening and will test them in vitro and finally in vivo.

Citation Format: Seth Weir, Xiaoxin Chen, Chorlada Paiboonrungruan. Identification and validation of Nrf2 inhibitors in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5856.