bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B181: The Chk1 inhibitor, SRA737, demonstrates chemical synthetic lethality with replication stress-inducing agents, including low-dose gemcitabine, in preclinical models of cancer
Authors: Hansen Ryan J. , Strouse Bryan , Anderes Kenna , Smith Gregg , Hassig Christian
Published: August 2018

Depending on genetic and other factors, tumor cells exhibit replication stress (RS) to various extents. Low levels of RS contribute to genomic instability, an enabling hallmark of cancer, whereas high levels may be cytotoxic. The DNA damage response (DDR) checkpoint kinase 1, Chk1, is essential for managing RS by stabilizing replication forks, abrogating origin firing, pausing the cell cycle, and fostering homologous recombination DNA repair. Consequently, tumor cells become highly reliant on Chk1 to manage RS and maintain proliferation and survival. Certain chemotherapeutic drugs, including nucleoside analogs, DNA cross-linkers, and topoisomerase inhibitors, are effective extrinsic inducers of RS. Here we demonstrate that the potent, selective oral Chk1 inhibitor, SRA737, strongly synergizes with a range of extrinsic inducers of RS to kill tumor cells in vitro and in vivo. We find that clinically used drugs including the DNA crosslinking agents mitomycin C and cisplatin, as well as the nucleoside analog gemcitabine, exacerbate cellular RS markers (e.g. γ-H2A.X and p-RPA) and synergize with SRA737 to increase tumor cell death in vitro. Combination indices (CI) at various doses across a cell line panel were 0.46, 0.54, and 0.25 for mitomycin C, cisplatin, and gemcitabine, respectively. Similar results were observed with both type I and type II topoisomerase inhibitors (SN-38 and etoposide). Notably, gemcitabine potentiates SRA737 in several bladder cancer cell lines including 5637, TCCSUP, and J82 cells, as well as in human primary bladder cancer patient 3D organoids. Consistent with the observed cytotoxicity, the combination of SRA737 and gemcitabine induces γ-H2A.X and p-RPA 3- to 5-fold over gemcitabine alone in tumor xenografts. Moreover, the combination inhibits tumor growth by 64% following administration of both agents at 100 mg/kg in a gemcitabine-insensitive patient-derived xenograft (PDX) model of bladder cancer. Importantly, SRA737 also synergizes with subtherapeutic doses of gemcitabine (40 mg/kg) in xenograft models of colorectal adenocarcinoma and osteosarcoma, indications where gemcitabine is not standard of care. These findings support the clinical development of SRA737 in combination with gemcitabine in bladder cancer, as well as the broader application of SRA737 in combination with extrinsic inducers of RS.

Citation Format: Ryan J. Hansen, Bryan Strouse, Kenna Anderes, Gregg Smith, Christian Hassig. The Chk1 inhibitor, SRA737, demonstrates chemical synthetic lethality with replication stress-inducing agents, including low-dose gemcitabine, in preclinical models of cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B181.
Abstract B189: New class of TOP1 inhibitors selective for HR deficiencies and SLFN11 expression
Authors: Marzi Laetitia , Pommier Yves G.
Published: August 2018

To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication-induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives are plagued by limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (the LMPs: LMP400, LMP776, and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2, and PALB2 are needed to repair DSB induced by TOP1 inhibitors. It has been shown that BRCA1 is a determinant of response to camptothecins in addition to olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives. To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival and cell cycle modifications after treatment with the LMPs. We found that SLFN11-negative cells are 10 times hypersensitive to LMPs compared to WT cells. Also, BRCA1-, BRCA2-, and PALB2-deficient cells are 3 to 5 times hypersensitive to the LMPs. Adding olaparib led to a greater cell death, especially in HR-deficient cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers.

Citation Format: Laetitia Marzi, Yves G. Pommier. New class of TOP1 inhibitors selective for HR deficiencies and SLFN11 expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B189.
Abstract B191: Synergistic anticancer activity of eribulin plus palbociclib in patient-derived xenograft (PDX) models of ER+/Her2- human breast cancer
Authors: Nomoto Kenichi , de Boisferon Marc H. , Littlefield Bruce A.
Published: August 2018

Eribulin is a synthetic analog of the marine sponge natural product halichondrin B. Its clinical formulation is currently approved in numerous countries for treating certain patients with advanced breast cancer or advanced liposarcoma. Eribulin’s anticancer mechanisms include relatively fast antimitotic effects that lead to cell death by apoptosis, and slower complex effects on the tumor microenvironment that include increased vascular perfusion, reduced hypoxia, and changes in phenotype associated with decreased migration and invasiveness in vitro. Such changes in tumor phenotype led us to ask whether eribulin could be successfully combined with inhibitors of cyclin dependent kinases 4 and 6 (cdk 4/6), which work at the G1/S cell cycle checkpoint where phenotypic changes are typically regulated. It is now recognized that patient-derived xenograft (PDX) tumor models more faithfully recapitulate human tumor biology and drug responsiveness than standard xenograft models derived from established human cancer cell lines. Accordingly, in the current study, we used two ER+/PR+/Her2- PDX models developed from patients with luminal B breast cancers, OD-BRE-0192 and OD-BRE-0745, to test in vivo combinations of eribulin and the cdk 4/6 inhibitor palbociclib. Eribulin and palbociclib dose levels were chosen that led to only minimal effects when given alone, and were administered on Q7Dx3 and Q1Dx5[x3] schedules, respectively, with palbociclib withheld the day before and the day of weekly eribulin dosing to avoid cell cycle-based antagonism that could occur between mitotic and G1/S blockers. In the OD-BRE-0745 model only, a fourth cycle of eribulin followed by 5 days of palbociclib was administered starting 13 days after the third eribulin dose. Under these conditions, combining eribulin and palbociclib led to markedly superior anticancer activity in both models (minimum T/C values of 29% and 41%) compared to either agent alone (T/C: 55-67% and 88-98%, respectively). These preclinical PDX results support clinical exploration of eribulin and palbociclib combinations for appropriate patients with ER+/Her2- breast cancers.

Citation Format: Kenichi Nomoto, Marc H. de Boisferon, Bruce A. Littlefield. Synergistic anticancer activity of eribulin plus palbociclib in patient-derived xenograft (PDX) models of ER+/Her2- human breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B191.
Abstract CN01-02: Chromosomal instability as a driver of tumor metastasis
Authors: Bakhoum Samuel F.
Published: August 2018

Chromosomal instability (CIN) is a hallmark of cancer, and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of rupture-prone micronuclei that expose their DNA content to the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. Using single-cell RNA sequencing, we uncover a CIN-induced transcriptional switch from a proliferative and metabolically active state to a mesenchymal phenotype associated with inflammatory pathways, offering an opportunity to target chromosome segregation errors for therapeutic benefit.

Citation Format: Samuel F. Bakhoum. Chromosomal instability as a driver of tumor metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr CN01-02.
Abstract LB-A09: Predicting PARPi sensitivity in patient derived ex vivo 3D tumor cultures
Authors: Basten Sander , Herpers Bram , Yan Kuan , Vader Willemijn , Price Leo
Published: August 2018

Ex vivo culture of patient derived tumor material has broad applications including functional testing to support treatment decisions, as well as testing drugs in development. Because average therapeutic response rates are low, there is a need for clinical tools to predict treatment response. Such tools may also reduce overtreatment and provide alternatives when treatment options are exhausted. Here we present data for 3D tumor cultures focusing on standard chemotherapy in combinations with targeted therapy, namely PARPi.

By embedding and optimizing growth for small amounts of heterogeneous tumor material in extracellular matrix protein hydrogels, we have generated 3D tumor cultures from various cancer indications. Initiated from both fresh and cryopreserved material, tumor cultures are seeded into 384 well plates and exposed to standard-of-care therapies, targeted therapies and drug combinations. An automated high content screening platform measures cell and tissue morphology, and reports responses such as tumor cell killing, growth arrest and local invasion. Depending on the tumor type and drug mechanism of action, morphological features are selected as standard read-outs for the response.

We will present results of proof-of-concept experiments that aim to compare drug sensitivity of tumor cultures with treatment responses in the clinic. Tumor material of gastric, endometrial, cervical, and ovarian cancer patients was used to generate 3D cultures, which were subsequently tested for drug sensitivity. Patient-dependent responses are identified for treatment schedules including platinum-based drugs, taxanes, anthracyclines and 5-FU. In addition, responses to drugs that are not (yet) considered standard of care (PARP inhibitors) were measured.

In conclusion, our technology can determine drug sensitivities of 3D cultured tumor tissues. This allows patient-specific treatment responses to standard-of-care drugs to be determined. In addition, this material can be used to test experimental drugs currently in development and in clinical studies. Ongoing proof-of-concept trials will reveal the correlation of our in vitro test with treatment responses in the clinic.

Keywords; in vitro diagnostic; organoids; tumor cultures; drug sensitivity testing.

Citation Format: Sander Basten, Bram Herpers, Kuan Yan, Willemijn Vader, Leo Price. Predicting PARPi sensitivity in patient derived ex vivo 3D tumor cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A09.
Abstract LB-A17: Identification and Characterization of Cyclic Peptide Inhibitors for Ras/PI3K and Ras/Raf Protein Complexes
Authors: Ismail Mohamed S. , Downward Julian
Published: August 2018

Ras oncogenic mutations are present in almost 25% of human cancers - indicative of their involvement in important signalling pathways that regulate many cellular functions such as: cell growth, proliferation, differentiation and survival. PI3K and Raf Kinases control two of the main effector signalling pathways that are activated by Ras proteins. There have been long efforts in discovering drugs for Ras mutants however these drugs either lacked strong binding affinity or specificity to Ras mutants. Here, in collaboration with Ali Tavassoli’s lab at Southampton University, we apply a new approach for discovering inhibitors that can block the Ras/Raf and Ras/PI3K interactions. The technique gives a great advantage of the possibility to discover inhibitors against Ras, Raf or PI3K proteins at the same time, rather than following the traditional route of screening drugs against Ras in isolation. In addition, because the effector-binding site of Ras is negatively charged and both of the binding sites of Raf and PI3K with Ras are positively charged - this opens up the opportunity for both negatively and positively charged inhibitors to compete with both interactions. The technique we are applying combines two methodologies: The “split-intein cyclisation of peptides and proteins” (SICLOPPS), for producing random cyclic peptides in vivo; and a bacterial reverse two-hybrid system (RTHS), can be used for high throughput screening (HTS). SICLOPPS has the capacity to produce millions of random cyclic peptides, which significantly increases the chances of finding suitable inhibitors, to interrupt protein complexes. Alongside this the RTHS provides a time efficient and effective technique to screen such a high number of peptides. We have developed a robotic HTS process that allows the screening of thousands of peptides per day. Initial characterisation of positive peptides that disrupt the interaction of the Ras/p110 α complex is currently in progress.

Citation Format: Mohamed S. Ismail, Julian Downward. Identification and Characterization of Cyclic Peptide Inhibitors for Ras/PI3K and Ras/Raf Protein Complexes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A17.
Abstract LB-A19: Targeting ERBB2 pathway in ovarian cancer
Authors: Kovtun Irina V. , Piyan Zhang , Harris Faye R. , Hou Xiaonan , Weroha Saravut J. , Vasmatzis George
Published: August 2018

Ovarian cancer (OC) is most lethal gynecologic malignancy. About 75% of OC patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in OC are reported to vary and responses to HER2 inhibitors have been feeble.

Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using whole-genome mate-pair next generation sequencing protocol we profiled a set of serous OC to identify copy number changes and gene fusions. Specific focus was on DNA alterations which were therapeutically targetable. The efficiency of anti-Her2 therapy was tested in three different PDX lines with alterations at genes of ERBB pathway and various levels of HER2 protein expression. Tumor in one line harbored a duplication of ERBB2 and high expression of HER2 protein. Second PDX model had a fusion at NRG1 gene, a ligand for HER4 and HER4 receptors, that was highly expressed on protein level. Third line harbored an amplification of NRG3 gene, a known ligand for HER4 receptor. Treatment responses to pertzumab/trastuzumab were compared in each PDX line to standard carboplatin and paclitaxel combination treatment. In all three PDX models HER2-targeted therapy resulted in significant inhibition of tumor growth compared to untreated control. However, the responses in each were inferior to chemotherapy, even in chemo-resistant case. There was an extra benefit when chemotherapy and HER-2 targeted therapy were administered together, as a significant regression of tumor was observed after 6 weeks of treatment using this regiment compared to chemotherapy alone. Post-treatment analysis of the tissues, nevertheless, revealed inhibition of ERBB2 pathway on the level of phosphorylation and expression of downstream targets. In conclusion, targeting of presumably activated ERBB2 pathway alone in OC results in a modest treatment benefit. A combination therapy including both chemo drugs and Her2 inhibitors provides far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to Her2 targeted therapy.

Citation Format: Irina V. Kovtun, Zhang Piyan, Faye R. Harris, Xiaonan Hou, Saravut J. Weroha, George Vasmatzis. Targeting ERBB2 pathway in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A19.
Abstract PL05-01: Personal regulome navigation
Authors: Chang Howard Y.
Published: August 2018

In every human cell, two meters of DNA are packed into a ~10-micron nucleus. The genome is extensively compacted, except for the active regulatory DNA elements that remain accessible. We have developed an Assay of Transpose Accessible Chromatin by sequencing (ATAC-seq) as a facile and sensitive method to map the landscape of gene regulation. The landscape of accessible chromatin from primary cells and tissues is uniquely informative for cell types, cell states, and the molecular basis of genetic variants associated with human diseases. Single-cell chromatin accessibility reveals DNA elements and trans-acting factors that mediate cell-to-cell variation, which directly impacts cancer heterogeneity. Real-time monitoring of gene regulation landscape in patients can provide prognostic prediction for response to cancer therapy. New technologies that incorporate 3-dimensional chromosome conformation, in situ imaging, and single-cell multi-omics provide new levels of precision and insights into human diseases.

Citation Format: Howard Y. Chang. Personal regulome navigation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr PL05-01.
Abstract 1000: PD-L1 binding peptide identified by phage peptide display inhibits PD-1/PD-L1 interaction and activates T cells
Authors: Gurung Smriti , Khan Fatima , Lee Soo-Woong , Yoon Jaewon , Lee Byungheon
Published: August 2018

Immune suppressive mechanism within the tumor microenvironment inhibits antitumor T cell function, leading to evasion of tumor from immune attack. One of the mechanisms is the up-regulation of programmed death-ligand 1 (PD-L1) expression on tumor cells which binds to programmed cell death protein 1 (PD-1) on activated T cells and inhibits T cell functions. Inhibiting the PD-L1/PD-1 interaction could reverse the tumor microenvironment and enhance the endogenous antitumor immune response. To identify a novel peptide that selectively binds to PD-L1, we screened a phage-displayed peptide library against cells transiently expressing PD-L1 at high levels. After multiple rounds of screening, selected phage clones were sequenced. Of these, two phage clones showed more selective binding to cells expressing PD-L1 compared to a control clone. The peptides displayed on the clones were synthesized for further study and named as PD-L1Pep-1 (9mer) and PD-L1Pep-2 (7mer). Fluorescein-conjugated PD-L1Pep-1 and PD-L1Pep-2 selectively bound to cells highly expressing PD-L1, whereas only negligible binding to cells expressing PD-L1 at low levels. The cell binding of the PD-L1-binding peptides was enhanced after treatment of cells with interferon-γ which is a potent inducer of PD-L1 in many types of cancer cells. FACS analysis showed that the pre-treatment of cells with an anti-PD-L1 antibody competed and reduced the cell binding of the PD-L1-binding peptides, suggesting the antibody and peptides bind to the same epitope on PD-L1. Co-culture experiments of CT26 mouse colon tumor cells with primary T cells isolated from a mouse spleen showed that, similar to the anti-PD-L1 antibody, the PD-L1-binding peptides increased the secretion of Interleukin-2 and interferon-γ by the T cells, indicating that the PD-L1-binding peptides can reactivate the T cells that were suppressed by the co-cultured tumor cells. In-vivo fluorescence imaging of BALB/c mice bearing subcutaneously transplanted CT26 mouse colon tumor cells showed tumor targeting ability of the PD-L1-binding peptides to PD-L1-expressing tumor. These findings suggest that PD-L1Pep-1 and PD-L1Pep-2 can inhibit PD-L1/PD-1 interaction and activate T cells in vivo and hold a potential as a cancer immunotherapeutic. Keywords: Colon tumor, immunotherapy, PD-L1, peptide, phage display

Citation Format: Smriti Gurung, Fatima Khan, Soo-Woong Lee, Jaewon Yoon, Byungheon Lee. PD-L1 binding peptide identified by phage peptide display inhibits PD-1/PD-L1 interaction and activates T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1000.
Abstract 1010: Establishing lobular-like and interlobular-like fibroblast cell lines from human breast reduction mammoplasties
Authors: Morsing Mikkel , Jafari Abbas , Kassem Moustapha , Petersen Ole William , Rønnov-Jessen Lone
Published: August 2018

Introduction: The importance of epithelial-stromal interactions for tissue homeostasis and maintenance is well documented. We have recently provided evidence that two distinct fibroblast lineages in the human breast stroma, lobular and interlobular, respectively, can be prospectively isolated and stably propagated in serial-passage subculture. The aim of the present study was to generate immortalized and stable cell lines derived from lobular and interlobular fibroblasts with preserved lineage identity and functionality. The availability of such cell lines will allow for an as of yet unprecedented detailed characterization of mammary fibroblast lineages including their specific interactions with normal and malignant breast epithelium.

Results: Lobular- and interlobular-derived fibroblast cell strains in passage 8 were immortalized by retroviral introduction of human telomerase reverse transcriptase (hTERT) followed by neomycin selection. At abstract submission deadline, lobular and interlobular hTERT expressing fibroblasts had reached passage 56 corresponding to 60 and 100 population doublings, respectively, while mock-transfected controls had seized proliferating in passage 21. Lineage restriction of immortalized cells was maintained as lobular fibroblasts express higher levels of CD105 than interlobular fibroblasts. Also, lobular fibroblasts are able to undergo adipogenic and osteogenic differentiation upon relevant induction in culture, but fail to generate bone in an in vivo ectopic bone formation model, thus functionally distinguishing lobular fibroblasts from bone marrow-derived mesenchymal/skeletal stem cells. Upon co-culture with primary breast luminal progenitors, lobular fibroblasts support epithelial growth and branching morphogenesis, including correct apical-basal polarization.

Conclusion: Lobular and interlobular fibroblasts have been successfully immortalized while preserving both lineage restriction and functional characteristics of their primary counterparts. With 80% of breast cancers arising in lobular epithelium, these established fibroblast models may prove instrumental to unravel such focal susceptibility to breast cancer development.

Citation Format: Mikkel Morsing, Abbas Jafari, Moustapha Kassem, Ole William Petersen, Lone Rønnov-Jessen. Establishing lobular-like and interlobular-like fibroblast cell lines from human breast reduction mammoplasties [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 1010.
Abstract 1041: XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data
Authors: Krijgsman Oscar , Kluin Roelof JC , Kemper Kristel , Kuilman Thomas , Ruiter Julian R. de , Iyer Vivek , Forment Josep V. , Cornelissen-Steijger Paulien , Rink Iris de , Brugge Petra ter , Song Ji-Ying , Klarenbeek Sjoerd , McDermott Ultan , Jonkers Jos , Velds Arno , Adams David J. , Peeper Daniel S.
Published: August 2018

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analysis of their RNA and DNA profiles is challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin can result both in the generation of false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. Therefore, we developed the open-source R-package XenofilteR, which separates mouse from human sequence reads based on the number of discordant base pairs between each read and the reference genomes. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human whole genome and whole exome DNA sequence data. This analysis revealed that XenofilteR removes >99.9% of sequence reads of mouse origin while retaining sequence reads of human origin. The filtering allowed for mutation analysis of PDX samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic mutations present in the original tumor. These findings were further validated in breast cancer and melanoma PDX samples, confirming the retrieval of accurate variant allele frequencies and somatic mutations. In conclusion, XenofilteR accurately dissects sequence reads from mouse and human origin in PDX sequence data, thereby outperforming currently available tools.

Citation Format: Oscar Krijgsman, Roelof JC Kluin, Kristel Kemper, Thomas Kuilman, Julian R. de Ruiter, Vivek Iyer, Josep V. Forment, Paulien Cornelissen-Steijger, Iris de Rink, Petra ter Brugge, Ji-Ying Song, Sjoerd Klarenbeek, Ultan McDermott, Jos Jonkers, Arno Velds, David J. Adams, Daniel S. Peeper. XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1041.
Abstract 105: CADM1 is a TWIST1-regulated suppressor of melanoma invasion and survival
Authors: Hartsough Edward J. , Weiss Michele B. , Heilman Shea A. , Purwin Timothy J. , Kugel Curtis H. , Rosenbaum Sheera R. , Erkes Dan A. , Tiago Manoela , Chervoneva Inna , Aplin Andrew E.
Published: August 2018

Metastatic melanoma is the deadliest form of skin cancer; however, patients diagnosed and treated prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion of melanoma cells but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Modulation of CADM1 resulted in significant effects on the migration and invasion of melanoma cells. In addition, elevated CADM1 elicited cell death in non-adherent conditions, an effect that could not be rescued with a pan-caspase inhibitor. Analyses suggest that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Furthermore, clinical data from TCGA indicates that CADM1 expression is correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be a biomarker for improved survival of melanoma patients.

Citation Format: Edward J. Hartsough, Michele B. Weiss, Shea A. Heilman, Timothy J. Purwin, Curtis H. Kugel, Sheera R. Rosenbaum, Dan A. Erkes, Manoela Tiago, Inna Chervoneva, Andrew E. Aplin. CADM1 is a TWIST1-regulated suppressor of melanoma invasion and survival [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 105.
Abstract 1051: Mutation detection in patient-derived xenografts by WES and RNAseq
Authors: Qian Wubin , Li Henry Q.X. , Guo Sheng
Published: August 2018

Patient derived xenograft (PDX) is widely used in preclinical cancer research for drug efficacy evaluation, biomarker discovery and validation, drug mechanism and resistant mechanism study, tumor biology studies and so on. Many PDX libraries are now available to researchers1-4. A good library is composed of PDXs with high-quality characterization for genomics, pharmacology, histology, and clinical information associated with the originating patient tumors. Various technologies are used for genomic profiling of PDX models. For example, RNAseq is used for transcriptomic profiling to obtain data on gene expression, RNA-level mutations, gene fusion; whole exome sequencing (WES) is used to infer DNA-level mutations and gene copy numbers. In this study, we compared mutations inferred by RNAseq and WES for 600 PDX models with both datasets.

We found that about 50% of mutations detected by WES were uncovered by RNAseq. The other half are missing because these mutation alleles were covered by insufficient reads (<5) or the mutations are located in poly-N regions, which make it hard to call mutations. By contrast, 99% of the RNAseq-based mutations were detected by WES. It is not surprising to see such difference. WES amplifies exons at DNA level, and is only affected by experimental variations such as hybridization efficacy. RNAseq yields more reads for genes with higher expression, therefore, mutations in genes with no or low expression cannot be reliably detected. Our results also indicate that there is limited RNA editing. We also examined mutation ratios and found good agreement between WES and RNAseq. Pearson's correlation coefficient is 0.91 and 0.81 between the two mutation ratios when homologous mutations are included and excluded, respectively.

Overall, our study shows that RNAseq data is reliable to infer mutations in highly expressed genes and can be readily used for such when WES data is not available.

References

1. Budinská E, Popovici V. The EurOPDX consortium: Sharing patient tumor-derived xenografts for collaborative multicentric preclinical trials. Molecular cancer therapeutics 2014;12:A8-A

2. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nature medicine 2015;21:1318-25

3. Ledford, H. (2016). Us cancer institute to overhaul tumour cell lines. Nature, 530.

4. Guo, S., Qian, W., Cai, J., Zhang, L., Wery, J. P., & Li, Q. (2016). Molecular pathology of patient tumors, patient derived xenografts and cancer cell lines. Cancer Research, 76(16), 4619.

Citation Format: Wubin Qian, Henry Q.X. Li, Sheng Guo. Mutation detection in patient-derived xenografts by WES and RNAseq [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 1051.
Abstract 1058: CRLF1 promotes proliferation and metastasis in papillary thyroid carcinoma via activating MAPK/ERK and PI3K/AKT pathway
Authors: ZHONG QIAN , Yu Shitong , Huang Xiaoming , Zeng Musheng
Published: August 2018

Papillary thyroid cancer (PTC) is the most common cancer in endocrine system which accounts for the majority component of thyroid cancer during the past decades. PTCs usually are indolent and curable with 5-year survival rate over 95%. However, some of PTCs will dedifferentiate and become aggressive with a poor prognosis. Current established biomarkers may not fully explain the mechanisms of the development of PTC. Therefore, it is still urgent to find novel biomarkers of PTC to better predict patients' prognosis and lead to the personalized therapies for PTC patients. By analyzing The Cancer Genome Altas (TCGA) database, we identified Cytokine Receptor Like Factor-1(CRLF1) as a potential oncogene. We identified CRLF1 is highly expressed in papillary thyroid carcinoma (PTC) compared with normal thyroid tissues both at mRNA and protein level. High CRLF1 level was associated with aggressive clinicopathological features and worse disease-free survival rate. By loss-of-function and gain-of-function assays, we found that CRLF1 not only boosts capacity of cell migration and invasion in vitro, but promotes tumor growth both in vitro and in vivo. Besides, CRLF1 can induce epithelial-mesenchymal transitions. Overexpressing CRLF1 could activate ERK1/2 and AKT pathways. The oncogenic effects induced by CRLF1 were suppressed by treating the cells with the MEK inhibitor U0126 or the AKT inhibitor MK-2206. These results suggest an oncogenic role CRLF1 plays in PTC tumorigenesis by regulating MAPK/ERK and PI3K/AKT signaling pathways. These results suggest that CRLF1 is a potential biomarker in PTC patients and it may be a valuable therapeutic target for PTC in the future.

Citation Format: QIAN ZHONG, Shitong Yu, Xiaoming Huang, Musheng Zeng. CRLF1 promotes proliferation and metastasis in papillary thyroid carcinoma via activating MAPK/ERK and PI3K/AKT pathway [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 1058.
Abstract 1062: Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer
Authors: Tian Zhongxian , Jin Honglei , Li Jingxia , Huang Chao , Huang Chuanshu
Published: August 2018

Our previous studies have demonstrated that X-linked inhibitor of apoptosis protein (XIAP) plays a regulatory role in cancer invasion via mediation of RhoGDIα protein SUMOylation at Ser138 in colorectal cancer HCT116 cells. However, the alteration/association of XIAP in invasion, progression, and lung metastasis of bladder cancer (BC) as well as the molecular mechanisms leading to this XIAP alteration during BC development have not been explored yet. Herein, we found that XIAP was overexpressed in human high grade BCs, high metastatic human BC T24T cells, and in mouse invasive BCs. Mechanistic studies indicated that XIAP overexpression in the highly metastatic T24T cells was due to increased mRNA stability of XIAP in comparison to the parental non-metastatic T24 cells. Results from XIAP mRNA stability regulation revealed that miR-200c was profoundly downregulated in T24T cells and in highly invasive BCs, and that such miR-200c downregulation was mediated by CREB inactivation. Furthermore, miR-200c downregulation reduced its binding to the 3'-UTR region of XIAP mRNA, and resulted in its mRNA stabilization, protein overexpression, and invasive capabilities in T24T cells. Moreover, our results from XIAP-specific shRNA and miR-200c ectopic expression showed that suppression of XIAP profoundly attenuated lung metastasis of T24T cells in nude mice. In conclusion, our findings demonstrate the molecular basis leading to XIAP overexpression and the crucial role of XIAP in BC invasion in vitro and lung metastasis in nude mice.

Citation Format: Zhongxian Tian, Honglei Jin, Jingxia Li, Chao Huang, Chuanshu Huang. Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1062.
Abstract 1066: L1CAM as a pivotal mediator for disease progression and prognostic biomarker in gastric cancer patients
Authors: Okugawa Yoshinaga , Toiyama Yuji , Mohri Yasuhiko , Yamamoto Akira , Shigemori Tsunehiko , Yin Chengzeng , Narumi Aya , Ide Shozo , Kitajima Takahito , Yasuda Hiromi , Fujikawa Hiroyuki , Hiro Junichiro , Yoshiyama Shigeyuki , Ohi Masaki , Tanaka Koji , Araki Toshimitsu , Kusunoki Masato
Published: August 2018

Background/Aims: Although significant progress has been made in the diagnosis and treatment of Gastric cancer (GC) patients, GC is the second leading cause of cancer-related deaths worldwide. Identification of critical factors driving the progression of metastasis and new prognostic biomarkers are urgently required to improve both early detection of recurrence and prognosis of patients with advanced GC. L1 cell adhesion molecule (L1CAM) has been observed to be highly expressed in several types of tumors, which may play a vital role in tumor progression. The present study investigated the clinical significance of L1CAM, in the development and progression of gastric cancer.

Experimental Design: At first, the effects of L1CAM alteration were investigated using proliferation, migration, invasion, and anoikis and xenograft model. Next, we analyzed L1CAM expression in surgical tissue speciemens from 131 gastric cancer patients (Cohort 1) by quantitative real-time reverse transcription-PCR and 309 patients (Cohort 2) by immunohistochemistry.

Results: Knockdown of L1CAM using small interfering RNA in GC cell lines resulted in reduced cell proliferation, invasion, migration, and anoikis in gastric cancer cells. Furthermore, Knockdown of L1CAM inhibited metastasis in a xenograft model. Interestingly, L1CAM expression in cancer tissues was significantly higher in patients with lymph node metastasis and distant metastasis. The expression of L1CAM was significantly associated with poor prognosis. Furthermore, L1CAM emerged as an independent prognostic predictor and a predictor of lymph node metastasis in GC patients.

Conclusions: Our data highlight that L1CAM appear to play an important role in disease development and L1CAM expression may serve as a novel predictor for the identification of patients who are at high risk for tumor recurrence and strict surveillance in curative GC patients.

Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Yasuhiko Mohri, Akira Yamamoto, Tsunehiko Shigemori, Chengzeng Yin, Aya Narumi, Shozo Ide, Takahito Kitajima, Hiromi Yasuda, Hiroyuki Fujikawa, Junichiro Hiro, Shigeyuki Yoshiyama, Masaki Ohi, Koji Tanaka, Toshimitsu Araki, Masato Kusunoki. L1CAM as a pivotal mediator for disease progression and prognostic biomarker in gastric cancer patients [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 1066.
Abstract 107: Interaction of BIR domain of XIAP with Sp1/E2F1 activates MMP2 and bladder cancer cell invasion by inhibiting src protein translation
Authors: Hua Xiaohui , Xu Jiheng , Jin Honglei , Huang Chuanshu
Published: August 2018

X-linked inhibitor of apoptosis protein (XIAP) is important in the regulation of cancer cell biological behaviors. However, little is known about the molecular mechanism by which the XIAP domains promote aggressive tumor behavior in human bladder cancer (BC). Herein, we discovered that ectopic expression of the BIR domains of XIAP rescued the MMP2 activation and human BC invasion in human BC cells with XIAP deletion. Interestingly, Src expression was upregulated in XIAP/BIR domain-deficient cells, whereas restoration of BIR domain in XIAP-deletion cells recused Src expression. Moreover, knockdown of Src reversed the defects of MMP2 activation and BC invasion in XIAP-deficient BC cells, revealing that Src acts as a XIAP downstream negative regulator for MMP2 activation and BC invasion. Mechanistic studies revealed that the regulatory effect of the BIR domains of XIAP on Src expression was due to directly inhibition of its protein translation by upregulation of miR-203 resulting from activation of the transcription factors Sp1 and E2F1, while the BIR domains of XIAP was found to interact with both Sp1 and E2F1 to activate these transcription factors. Overall, our studies demonstrate that BIR domain of XIAP is able to bind to Sp1 and E2F1 leading to their transcriptional activation, resulting their targeted miR-203 transcription, consequently directly targets the 3'-UTR of src mRNA and therefore attenuating SRC protein translation, and activating MMP2 and BC invasion.

Citation Format: Xiaohui Hua, Jiheng Xu, Honglei Jin, Chuanshu Huang. Interaction of BIR domain of XIAP with Sp1/E2F1 activates MMP2 and bladder cancer cell invasion by inhibiting src protein translation [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 107.
Abstract 1072: Potential biomarkers correlated with poor survival rate of melanoma patients
Authors: Morais Alice S. , Monteiro Ana C. , Andrade Fernando , Goedert Lucas , Espreáfico Enilza , Schneider-Stock Regine , Fujiota André , Jasiulionis Miriam G.
Published: August 2018

Malignant melanoma is an extremely aggressive tumor, responsible for 90% of skin cancer mortality. Widespread metastasis is the main cause of death in melanoma patients. Therefore, the identification of molecules related to the degree of melanoma malignancy could be used as prognostic biomarkers. In the present study, using a murine model of melanoma progression, different genes, such as Snai1, Chd1, Gata4, Id4, Cbs, Vav1, and Itga4, were first identified as presenting aberrant DNA methylation in their promoters along melanoma progression by Methylight analysis. The gene expression pattern and epigenetic regulation were validated in these murine cell lines by RT-qPCR and 5AzaCdR treatment, respectively. The genes Angpt2 and Fgf1 were also identified as differentially expressed along tumor progression in this murine model. The expression profile of these genes was also evaluated in primary human melanocytes and RGP (radial growth phase), VGP (vertical growth phase) and metastatic melanoma cell lines. Clinical and gene expression data from 460 patients were obtained from TCGA (The Cancer Genome Atlas). Using an univariate test, patients presenting tumors with low levels of Chd1, Vav1, Id4 and Fgf1, and high levels of Snai1 and Angpt2 were shown to exhibit poor overall survival rate. Indeed, the multivariate analysis using Cox regression model identified high Snai1 (HR=1.217; 95% CI 1.075-1.377; p=0.002) and Angpt2 (HR=1.186; 95% CI 1.059-1.329; p=0.003) expression and low Chd1 (HR=0.757; 95% CI 0.575-0.996; p=0.047), Fgf1 (HR=0.858; 95% CI 0.780-0.944; p=0.002) and Vav1 (HR=0.859; 95% CI 0.764-0.967; p=0.011) expression as independent prognostic factors for the overall survival rate of patients with melanoma. Also using a multivariate analysis, the low levels of Id4 promoter methylation (HR=0.689; 95% CI 0.531-0.891; p=0.045) was shown to be an independent predictive factor for poor survival rate, although no correlation was found between Id4 expression and survival rate. In conclusion, the upregulation of Snai1 and Angpt2, and the downregulation of Chd1, Vav1 and Fgf1 are associated with unfavorable survival outcome, indicating they might be valuable prognostic biomarkers for melanoma. Supported by CNPq and FAPESP.

Citation Format: Alice S. Morais, Ana C. Monteiro, Fernando Andrade, Lucas Goedert, Enilza Espreáfico, Regine Schneider-Stock, André Fujiota, Miriam G. Jasiulionis. Potential biomarkers correlated with poor survival rate of melanoma patients [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 1072.
Abstract 1078: Identification of peptides binding to mesenchymal subtype breast cancer from phage display peptide library
Authors: Jones Kelvin M. , Samant Rajeev , Singh Shree , Bedi Deepa
Published: August 2018

Most available therapeutic drugs efficiently treat tumors; however, absence of specificity has deleterious effects on patients. Identifying a tumor ablation method that has specificity with minimal to no side effects is needed. Phage display allows for expression of peptide and protein libraries on the surface of phage with a diversity of 109 peptides of different permutations, which leads to the selection of peptides and proteins, with high affinity and specificity. Phage display can be used as a tool to identify specific targets using a pIII phage library for identification of cell surface markers. To do so, we performed an initial depletion assay to isolate specific phages that express an affinity to human cells. We isolated a phage diversity of 10-2 from 109. Subsets of this population were used to identify phage and breast cancer cell interaction in vitro using an ELISA assay. Briefly, phages were exposed to epithelial and mesenchymal breast cancer cells and fibroblast cells. We have identified a phage profile that selectively targets tumor cells based on their transition from epithelial to mesenchymal. Interestingly, PE-26, a phage subset, indicated a greater affinity for more mesenchymal-like tumor cells. Moreover, we identified a 5-fold increase in the binding affinity for MDA-MB-231 and MCF-7 (overexpressed with TGF-β) in comparison to the control fibroblast cells. Additionally, PE-21 demonstrates a 6-fold increased affinity for breast cancer cells when contrasted with our control fibroblast cells. Our data provide further support that the identified phage profiles exhibit a preferential affinity for more mesenchymal-like tumor cells, which are known to be aggressive. This suggests their potential chemotherapeutic capability. Our future endeavors include further validation of selected phages and development of peptides to be employed as imaging agents to study EMT in vivo and as diagnostics to detect circulating tumor cells.

Citation Format: Kelvin M. Jones, Rajeev Samant, Shree Singh, Deepa Bedi. Identification of peptides binding to mesenchymal subtype breast cancer from phage display peptide library [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 1078.