bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3438: PI3Kα/δ inhibitor, copanlisib is highly effective in ER-positive breast cancer
Authors: De Pradip , Aske Jennifer C. , Williams Casey , Dey Nandini , Leyland-Jones Brian
Published: August 2019

Purpose: Over 60% of ER+ breast cancer (BC) cases are associated with activation of the PI3K-AKT-mTORC1/C2 pathway. PIK3CA, the catalytic subunit of PI3K, is by far the most frequently mutated gene in luminal ER+ BC patients. It has long been known that hormone receptor and oncogene cooperation plays a key role in driving tumor development and that most hormone therapy-resistant BC depends on the PI3K pathway. Here, we present the results of a preclinical study of the PI3K α/δ (dominant) inhibitor copanlisib alone and in combination with letrozole in ER+ BC cell lines.

Method: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of copanlisib alone and in combination with letrozole were evaluated in a panel of ER +/PIK3CA mutated, and ER+/PTEN mutated breast cancer cell lines.

Results: 1) Copanlisib inhibited PI3K, mTOR and their downstream signaling molecules in ER+/PIK3CA or ER+/PTEN mutated BC cell lines; 2) interestingly, copanlisib also inhibited RAS-MAPK signaling in the earlier time points in ER+/PIK3CA mutated cells (T47D & MCF7) but not in ER+/PTEN mutated cells (CAMA1); 3) copanlisib caused a strong differential growth inhibition in ER+ BC cell lines as shown by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition was greater when copanlisib was combined with letrozole; 4) administration of copanlisib induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner; 5) unlike everolimus, copanlisib blocked HIF1α accumulation in hypoxic conditions and this blocking effect was reversed by prior treatment with the proteasome inhibitor carfilzomib, suggesting copalnisib enhanced proteasome-mediated HIF1α degradation; and 6) copanlisib also attenuated ER+ cell migration, an important phenotypic feature for metastasis, along with integrin-mediated RAC1 activation.

Conclusions: Copanlisib is highly effective (blocks proliferation, induces apoptosis, and inhibits PI3K and its downstream signaling targets) in ER+ BC cell lines (both PIK3CA and PTEN mutated). The addition of copanlisib to aromatase inhibitor might represent an improved treatment strategy for patients with hormone-resistant/relapse ER+ BC.

Citation Format: Pradip De, Jennifer C. Aske, Casey Williams, Nandini Dey, Brian Leyland-Jones. PI3Kα/δ inhibitor, copanlisib is highly effective in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3438.
Abstract 3441: A RPL39-SGK3 signaling pathway that may play a key role in breast cancer therapy resistance
Authors: Liu Dan , Dave Bhuvanesh
Published: August 2019

Breast cancer is the most common cancer in the United States, where 1 in 8 women are affected by it during their lifetime. In 2018, an estimated 266,120 new cases of invasive breast cancer are expected to be diagnosed in women in the U.S., along with 63,960 new cases of non-invasive (in situ) breast cancer. In order to identify novel targets and better understand the mechanism of survival of these cancers, we describe the mechanism of action of ribosomal protein like 39 (RPL39) as one potentially useful target for therapy resistance. RPL39 is over-expressed in breast cancer and regulated by nitric oxide signaling (NOS). In the present study we examine the detailed mechanism of action of RPL39 as it relates to treatment-resistant breast cancers and the role of SGK3 in modulating its signal transduction pathway. Methods and Results: Mass spectrometric analysis following immunoprecipitation of RPL39, in two breast cancer cell lines SUM159 and MDAMB231, indicated that proteins involved in translation, specifically the mTOR-dependent translational pathway, were part of the complex. In order to verify that the results we observed were due to targeting of the translational arm of mTOR signaling, we knocked down both the 4EBP-1 (translation) and S6K (cell size) arms of the mTOR pathway in three breast cancer cell lines (SUM159, MDAMB231 and MDAMB436). Analysis of these pathways by proliferation, apoptosis, sphere formation and western blotting in all three breast cancer cell lines demonstrated that the translation arm was significantly altered: self-renewal (p<0.05, Student's t test) and proliferation (p<0.05, student's t test) in a NOS signaling dependent manner. Additional bioinformatic analysis determined a potential role for SGK3 upstream of RPL39. SGK3 is an AGC kinase family member that is over-expressed in ER+ breast cancers. It functions downstream of PI 3-kinase and can activate the mTOR pathway. Conclusion: RPL39 gene may be modulated by the selective translational initiation complex which affects self-renewal and proliferation through SGK3 and NOS signaling. The significance of targeting SGK3 and RPL39 pathway allows a potentially novel therapeutic strategy for the treatment of breast cancer.

Citation Format: Dan Liu, Bhuvanesh Dave. A RPL39-SGK3 signaling pathway that may play a key role in breast cancer therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3441.
Abstract 3451: Interpreting gene lists from -omics experiments
Authors: Luna Augustin , Wong Jeffrey V. , Demir Emek , Rodchenkov Igor , Babur Özgün , Sander Chris , Bader Gary D.
Published: August 2019

Understanding the mechanisms responsible for a cellular behavior often begins with observations of genes and gene products. Depending on the type of experiment, the number of resulting genes can be small, but increasingly, researchers are faced with many thousands of measurements, as in the case of transcriptomic or protein-DNA binding observations. Here, we describe ways to pair experimental results consisting of one or more genes with analysis tools with the overall aim being to make results more biologically interpretable. In certain cases, experimental approaches such as screens for essential genes can generate one or a few ‘genes of interest’ and there is a desire to understand their relationship to one another as well as discover links to additional, interesting genes. To this end, ‘GeneMANIA’ is a web tool that accepts gene names and returns a network visualization of related genes based on similarity in expression, localization, protein domains and those involved in physical interactions. Likewise, ‘PCViz’ is a web tool that displays a network of interactions drawn from Pathway Commons, a web resource for pathway and interaction knowledge. In cases where experiments generate a lengthy list of genes, for instance, transcriptomic measurements, there is a desire to understand their relevance to a phenotype of interest. Pathway enrichment analysis methods aim to summarize gene lists as pathways, which have a closer link to cell function. An online ‘Guide’ by Pathway Commons includes workflows that illustrate how to chain together software tools to identify pathways from the corresponding gene-level data then organize and summarize the pathway-level results in an interactive visualization known as an Enrichment Map. For those wishing to drill-down to individual pathways, Pathway Commons offers a set of web apps, including ‘Search’ that enables users to query by keyword and visualize ranked search results. Ongoing development of web apps aims to enhance the accessibility to pathways and integrate support for analysis and visualization of experimental data. The full complement of data, tools and resources offered by Pathway Commons in support of pathway analysis are described.

Citation Format: Augustin Luna, Jeffrey V. Wong, Emek Demir, Igor Rodchenkov, Özgün Babur, Chris Sander, Gary D. Bader. Interpreting gene lists from -omics experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3451.
Abstract 3452: hnRNPUL1 regulation by ATR in DNA damage response
Authors: Zhang Hui , Head PamelaSara E. , Duong Duc M. , Seyfried Nicholas T. , Yu David S.
Published: August 2019

The DNA damage response (DDR) is a signaling network that recognizes damages to DNA and orchestrates a variety of DNA repair and cell cycle checkpoint pathways. The DDR is pivotal for cancer prevention. Ataxia Telangiectasia And Rad3-Related Protein (ATR), a protein kinase, functions as an essential transducer of the DDR signaling cascade. ATR primarily responds to single-stranded DNA (ssDNA) generated from double-strand break resection or at stalled replication forks. Depletion of ATR leads to cellular senescence and cancer related phenotypes. The mechanisms regarding how the ATR signal pathway is regulated, however, remain elusive. To discover novel DDR proteins regulated by ATR, we performed proteomic analysis to identify proteins that partner with ATR in response to DNA damage in cells. Our analysis revealed a network of proteins as potential ATR substrates, including Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1), a protein functions in RNA metabolism. We validated that ATR interacts with hnRNPUL1. In addition, hnRPUL1 is phosphorylated on SQ/TQ sites in response to IR. ATR depletion suppresses the IR induced hnRPUL1 phosphorylation. Our results identify hnRNPUL1 as a novel ATR substrate, critical for the DNA damage response and provide insight into how DDR and RNA metabolism might work synergically in maintaining genomic stability and preventing cancer. Additionally, hnRNPUL1 phosphorylation by ATR may be targeted as an adjunct to improve the efficacy of ionizing radiation for cancer therapy.

Citation Format: Hui Zhang, PamelaSara E. Head, Duc M. Duong, Nicholas T. Seyfried, David S. Yu. hnRNPUL1 regulation by ATR in DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3452.
Abstract 3460: MPRIP-ALK,a novel ALK rearrangement that responds to ALK inhibitor in non-small-cell lung cancer
Authors: Fang Wenfeng , Gan Jiadi , Lu Feng , Deng Yangyang , Chen Liang , Yang Yunpeng , Zhang Li
Published: August 2019

Oncogenic rearrangements of the anaplastic lymphoma receptor tyrosine kinase (ALK gene) have recently been described in 3% to 5% of lung adenocarcinomas. Kinds of fusion partners of ALK have been reported in non-small-cell lung cancer (NSCLC), with echinoderm microtubule-associated protein-like 4 (EML4)-ALKbeing the most prevalent one. Several tyrosine kinase inhibitors have been approved for treating patients with advanced-stage ALK-positive, NSCLC by Food and Drugs Administration (FDA).

Comprehensive genomic profiling performed by means of next-generation sequencing assay demonstrated a novel myosin phosphatase-Rho-interacting protein gene (MPRIP)-ALK fusion in our case, in which a translocation involving chromosomes 2 and 9 has taken place. The rearrangement was verified by reverse transcription reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. ALK immunohistochemistry demonstrated strong staining in patient’s tumor sample as extracted from formalin-fixed paraffin-embedded (FFPE) sections.

We expressed MPRIP-ALK cDNA construct in non-cancer cell line Ba/F3 which showed IL-3-independent growth compared the growth stop of cells carrying empty vector, moreover, hyper-activation of the protein ALK, ERK, AKT were detected in Ba/F3 cells with MPRIP-ALK over-expressed. Furthermore, the colonies and activations of ALK downstream signaling pathways could be inhibited by crizotinib. The patient received standard chemotherapy on the first-line. She experienced progressive disease after 7 months, and commenced on crizotinib (250mg, twice per day) from March 2018. The patient showed significant improvement in cough, dorsalgia, and polypnea within one week. Partial response was gained after one month treatment and confirmed at the third month. She still remains on the crizotinib treatment without disease relapse up to now. In this study, we report a novel ALK rearrangement in a patient with NSCLC by RNA-seq. Our in vivo and in vitro studies have shown that the novel MPRIP-ALK fusion is a driver gene and can be targetable by crizotinib.

Citation Format: Wenfeng Fang, Jiadi Gan, Feng Lu, Yangyang Deng, Liang Chen, Yunpeng Yang, Li Zhang. MPRIP-ALK,a novel ALK rearrangement that responds to ALK inhibitor in non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3460.
Abstract 3466: The oncogenic roles of ANXA1 in gastrointestinal tract cancer
Authors: Saito Motonobu
Published: August 2019

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein that is associated with anti-inflammatory effects, regulation of cellular proliferation, and apoptosis and, therefore, ANXA1 is associated with cancer development and metastasis. We have previously reported that ANXA1 was upregulated in gastrointestinal cancer and associated with venous invasion (P=0.023) and lymph node metastasis (P=0.042). We also performed in vitro cell experiments and revealed that elevated ANXA1 expression was induced resistance to 5-FU in the colon cancer cells. So next, we have evaluated the association between ANXA1 and cancer related genes, including p53 by immunohistochemical (IHC) staining in two independent gastric cancer cohorts (n=200 and n=220). The percentage of positive ANXA1 was significantly higher in p53 positive tumors compared with p53 negative tumors (P=0.023 and P=0.022, respectively). We also evaluated the association with ARID1A, the chromatin remodeler AT-Rich interactive 1A, that is frequently mutated in gastric cancer and is considered as a candidate therapeutic target by synthetic lethality. Although it was reported that the expression of ANXA1 was upregulated in ARID1A mutated breast cancer cases, significant association between ANXA1 and ARID1A expressions was not found in gastric cancer. We next performed IHC staining for ANXA1 and p53 in three human gastric cancer cell lines (MKN7, NUGU4, KATOIII). Positive staining for both ANXA1 and p53 was detected in cells with TP53 missense mutation. On the other hand, weak positive staining for ANXA1 and negative staining for p53 was found in NUGC4 cells with TP53 wild-type and KATOIII cells with TP53 truncating mutation. These results suggested that ANXA1 expression may be induced by aberrant p53 protein, resulting cancer progression and resistance to 5-FU. Our results also provide a possible strategy to overcome 5-FU resistance by modulating ANXA1 expression.

Note: This abstract was not presented at the meeting.

Citation Format: Motonobu Saito. The oncogenic roles of ANXA1 in gastrointestinal tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3466.
Abstract 3469: CYP1B1 induces cancer progression through regulation of TRAIL pathway and uPA-uPAR system
Authors: Kwon Yeo-Jung , Chun Young-Jin
Published: August 2019

Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor-specific hyper-expression. To explore the role of CYP1B1 on progression of human cancer cells, we studied the effects of CYP1B1 in MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 significantly induced invasion of cancer cells while strongly inhibited cancer cell apoptosis. Suppression of CYP1B1 by si/shRNA or treatment with TMS, a specific inhibitor for CYP1B1, markedly increased the protein expression level along with the level of release from cells of TNF-related apoptosis inducing ligand (TRAIL), a death ligand for cancer cells, whereas significantly decreased the expression of urokinase-type plasminogen activator receptor (uPAR) as well as uPA. We also found that CYP1B1 showed to inhibit the expression of key factors in TRAIL-induced apoptotic pathway and promote the inducers in uPA-uPAR metastasis system including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down-regulated p53 expression through MDM2 activation and nutlin-3a, an inhibitor for MDM2, blocked the promoting effects of CYP1B1 inducer, DMBA, on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2-p53 system. Furthermore, we found that CYP1B1 suppresses TRAIL-related apoptosis through not only inhibition of TRAIL expression but also induction of DNA methylation on the promoter region of DR4, a receptor for TRAIL. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPA-uPAR pathway which is one of the targets of p53 signaling and suppresses cancer cell apoptosis through inhibition of TRAIL-induced apoptosis by suppression of TRAIL release as well as post-translational regulation on DR4.

Citation Format: Yeo-Jung Kwon, Young-Jin Chun. CYP1B1 induces cancer progression through regulation of TRAIL pathway and uPA-uPAR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3469.
Abstract 3470: The antagonistic functional duality of p21WAF1/CIP1 in cells derived from multicellular tumor spheroids
Authors: Das Viswanath , Annadurai Narendran , Holub Dušan , Hajdúch Marián
Published: August 2019

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a key mediator of p53-dependent cell cycle arrest after DNA damage, in addition to p53-independent mechanisms. Being one of the major transcriptional targets of p53 and due to its anti-cell proliferative activity, p21 was considered initially as a potent tumor suppressor. However, emerging studies now show the anti-apoptotic and pro-cell proliferative effects of p21, highlighting the oncogenic role of p21 in cancer. In particular, p21 results in genomic instability and the development of aggressive and chemo-resistant traits in a subset of highly proliferating tumor cells through p53-independent pathways. The cellular localization of p21 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Herein, using a combination of gene knockout, cytotoxicity assay, immunofluorescence, immunoprecipitation, and mass spectrometry techniques, we study the consequence of nuclear and cytoplasmic localization of p21 on cell survival and drug response in cells derived from multicellular spheroids of multiple human cancer cell lines. Our study shows that cells derived from multicellular spheroids show an increased induction of p21 despite the reversal from three-dimensional to two-dimensional cultures. The p21-overexpressing, highly proliferative cells are significantly resistant to a number of standard anti-cancer agents. Further analysis shows that the majority of p21 in this subset of spheroid-derived cells is localized in the cytoplasm and forms a potential 'anti-apoptosome'-like complex with mitochondrial apoptosis-associated proteins. These findings add to the shifting paradigm on the oncogenic role p21 due to cellular mislocalization in tumor cells.

Citation Format: Viswanath Das, Narendran Annadurai, Dušan Holub, Marián Hajdúch. The antagonistic functional duality of p21WAF1/CIP1 in cells derived from multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3470.
Abstract 348: ABI-009 (nab-Sirolimus) improves tumor accumulation and antitumor activity over oral mTOR inhibitors
Authors: Hou Shihe , Schmid Anita , Desai Neil
Published: August 2019

Background: mTOR pathway has been implicated in cell survival and proliferation and is an attractive target for cancer therapy. ABI-009 (nab-sirolimus) is an injectable nanoparticle form of human albumin-bound sirolimus with a mean particle size of approximately 100 nm developed with a proprietary nanoparticle albumin-bound (nab®) technology. ABI-009 is currently in phase 1 and 2 clinical studies for the treatment of multiple cancer types, including bladder cancer, soft-tissue sarcomas, neuroendocrine tumors, colorectal cancer, glioblastoma, and various childhood cancers, and a registrational phase 2 study for malignant perivascular epithelioid cell carcinoma (PEComa). In nonclinical and clinical studies, ABI-009 demonstrated a PK profile distinct from oral mTOR inhibitors. This study compared the antitumor activity, tissue penetration, and pharmacodynamics of ABI-009 with equal doses of oral mTOR inhibitors at clinically relevant doses in a mouse tumor xenograft model.

Methods: Athymic mice bearing subcutaneous UMUC3 bladder cancer xenografts were treated with saline, ABI-009 (IV, 7.5 mg/kg, 2x/wk), sirolimus and everolimus (PO, 3 mg/kg/day, 5 days/wk). Body weights, tumor measurements, and clinical signs were recorded 3x/wk for up to 5 wks. Tumor and blood sirolimus concentrations were also obtained (tumor: D1 1h and 24h, D4 1h, D7; blood: D4 1h, D7). Markers for mTOR pathway activation (pS6, p4EBP1) will also be analyzed.

Results: Overall, all treatments were well tolerated with no significant body weight loss in any group. ABI-009 IV resulted in significantly higher sirolimus concentrations in the tumor compared with oral mTOR inhibitors at all time points tested. Tumor AUC with ABI-009 IV over 1 week were significantly higher vs equal weekly dose of oral sirolimus (43 fold) and everolimus (12 fold) (p < 0.0001). The enhanced tumor drug delivery by ABI-009 corresponded with significantly greater tumor growth inhibition (TGI: ABI-009 69.6% vs oral sirolimus 24.3%, p < 0.00001; vs oral everolimus 36.2%, p = 0.0023) and longer animal survival (median OS: ABI-009 not reached vs oral sirolimus 21 days, p < 0.05; vs oral everolimus 19 days, p < 0.05). mTOR pathway inhibition in tissues is being assessed.

Conclusions: Compared with equal weekly dose of oral mTOR inhibitors, ABI-009 at clinically relevant dose of administered IV demonstrated significantly greater tumor drug delivery, stronger antitumor activity, and prolonged animal survival. These advantages observed in this nonclinical study support the clinical investigation of ABI-009 in numerous oncology indications.

Citation Format: Shihe Hou, Anita Schmid, Neil Desai. ABI-009 (nab-Sirolimus) improves tumor accumulation and antitumor activity over oral mTOR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 348.
Abstract 3494: Centrobin is involved in the cellular response to DNA damage
Authors: Ryu Na-mi , Jung Jin Ki , Jang Seok Won , Kim Jung Min
Published: August 2019

DNA-repair proteins have been found to localize to the centrosomes and defects in these proteins cause centrosome over-duplication. Centrobin is a centriole-associated protein that is required for centriole duplication. In the present study, we investigated a possible role of centrobin in DNA damage response. Centrobin was phosphorylated in response to UV radiation and its phosphorylated form was detected only in the detergent / DNase 1-resistant fraction. UV-induced phosphorylation of centrobin was significantly decreased by ATR inhibition, but not by ATM inhibition. In addition, centrobin-depleted cells showed reduced survival rate after UV exposure compared with control cells. Moreover, centrobin knockdown in DR-GFP/U2OS cells had a significant reduction in DNA repair efficiency of homologous recombination (HR) and this defect was recovered by expression of wild-type centrobin, but not by a mutant form of centrobin. Taken together, these results suggest that centrobin may play a critical role in DDR and that ATR dependent phosphorylation of centrobin may be at least partially responsible for this function.

Citation Format: Na-mi Ryu, Jin Ki Jung, Seok Won Jang, Jung Min Kim. Centrobin is involved in the cellular response to DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3494.
Abstract 3496: Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors
Authors: Ratnaparkhe Manasi , Wong John K. , Wei Pei-Chi , Hlevnjak Mario , Kolb Thorsten , Simovic Milena , Haag Daniel , Paul Yashna , Devens Frauke , Northcott Paul , Jones David T. , Kool Marcel , Jauch Anna , Pastorczak Agata , Mlynarski Wojciech , Korshunov Andrey , Kumar Rajiv , Downing Susanna M. , Pfister Stefan M. , Zapatka Marc , McKinnon Peter J. , Alt Frederick W. , Lichter Peter , Ernst Aurelie
Published: August 2019

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous recombination repair or non-homologous end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

Citation Format: Manasi Ratnaparkhe, John K. Wong, Pei-Chi Wei, Mario Hlevnjak, Thorsten Kolb, Milena Simovic, Daniel Haag, Yashna Paul, Frauke Devens, Paul Northcott, David T. Jones, Marcel Kool, Anna Jauch, Agata Pastorczak, Wojciech Mlynarski, Andrey Korshunov, Rajiv Kumar, Susanna M. Downing, Stefan M. Pfister, Marc Zapatka, Peter J. McKinnon, Frederick W. Alt, Peter Lichter, Aurelie Ernst. Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3496.
Abstract 350: Investigating the mechanisms of cellular uptake and metabolism of ICT2588, an MT-MMP-activated prodrug
Authors: Barnieh Francis Mprah , Race Amanda D. , Shnyder Steven D. , Loadman Paul M. , Falconer Robert A.
Published: August 2019

ICT2588 is a non-toxic MT1-MMP-activated peptide prodrug of the potent vascular disrupting agent (VDA) azademethylcolchicine [1]. ICT2588 produces significant tumor-selective delivery (≥10-fold) of the toxic and potent azademethylcolchicine, as compared to the administration of azademethylcolchicine alone, in murine models of human colorectal, breast, lung and prostate cancers. In addition, ICT2588 exhibited reduced potential for toxicity, and cardiotoxicity in particular (a phenomenon associated with most VDAs) as a result of its metabolic stability in plasma and normal tissues [2]. ICT2588 is progressing towards clinical evaluation in the UK in 2019. In this study we aim to further investigate the cellular uptake and metabolism of ICT2588 and related compounds in MT1-MMP positive (HT1080) and MT1-MMP negative (MCF-7) cells in vitro. ICT2588 and related compounds were synthesized in-house and purified by preparative HPLC in excellent yield and purity. Activation of ICT2588 by recombinant MT1-MMP and its preferential metabolism in MT1-MMP positive tumor xenograft homogenates has been shown previously [1]. We are currently studying the mechanism of cellular uptake and metabolism of these peptide prodrugs by incubating these compounds in HT1080 and MCF-7 cell cultures. Cellular concentrations of ICT2588 and all metabolites identified were determined using RP-HPLC, mass spectrometry and immunofluorescent microscopy. We describe the first data on the cellular mechanism of uptake and metabolism of ICT2588 and related peptide prodrugs in cancer cells. Our findings have important implications in the future design of effective peptide-based tumor-targeted prodrugs. Reference: [1] Atkinson, J.M et al “Development of a tumor-targeted vascular disrupting agent activated by Membrane-type Matrix Metalloproteinases (MT-MMPs).” Cancer Research, 2010, 70, 6902-12. [2] Gill, J.H et al “Tumor-targeted prodrug ICT2588 demonstrates therapeutic activity against solid tumors and reduced potential for cardiovascular toxicity”. Mol Pharm. 2014,11:1294-300.

Citation Format: Francis Mprah Barnieh, Amanda D. Race, Steven D. Shnyder, Paul M. Loadman, Robert A. Falconer. Investigating the mechanisms of cellular uptake and metabolism of ICT2588, an MT-MMP-activated prodrug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 350.
Abstract 3501: Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer
Authors: Gupta Vijayalaxmi G. , Fernandez Yoskaly Lazo , Roby Katherine , Pathak Harsh , Hirst Jeff , Wilson Andrew J. , Godwin Andrew , Khabele Dineo
Published: August 2019

Introduction: Ovarian cancer is a rare but often fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising new drugs that are most effective in patients with tumors with altered homologous recombination (HR) DNA repair genes, particularly BRCA1/2. To expand their clinical utility, PARPi are being tested in combination with other drugs. Our group has published that histone deacetylase inhibitors (HDACi) sensitize HR proficient BRCA wild-type ovarian cancer cells to PARPi. We have developed an investigator-initiated phase 1/2 clinical trial of the PARPi olaparib combined with the HDACi entinostat in patients with recurrent, HR proficient/BRCA wild-type ovarian cancer.

Objective: To test the effects of a novel combination of olaparib and entinostat in preclinical models of BRCA wild-type ovarian cancer.

Methods: HR proficient BRCA wild-type SKOV3 and ID8 ovarian cells were treated with Entinostat followed by Olaparib alone or the combination of Entinostat and Olaparib. Treated and control cells were analyzed for cell proliferation by sulforhodamine B assays, clonogenicity, and DNA damage by Comet assays. SKOV-3-luciferase cells were injected intraperitoneally into NOD-SCID mice. Mice were randomized into 4 treatment groups and treated with vehicle, entinostat, olaparib, or the combination. The mice were monitored for toxicity and by bioluminescence imaging (BLI). At sacrifice, tumor burden was quantified.

Results: In cell culture assays, entinostat significantly enhanced the sensitivity of SKOV3 and ID8 cells to Olaparib. Entinostat combined with olaparib led to synergistic reductions in cell proliferation and clonogenicity in SKOV-3 and ID8 ovarian cancer cells, compared to each drug alone. Evidence of DNA damage was enhanced by the drug combination. In the SKOV3-luc xenograft mouse model, a significant reduction in tumor burden in the group of mice treated with the combination of entinostat and olaparib, compared to each drug alone. There was no significant toxicity with drug treatment.

Conclusion: Entinostat sensitizes ovarian cancer cells to Olaparib in vitro and in vivo. Experiments in ID-luciferase and patient-derived xenograft models are ongoing. These preclinical models are powerful tools that will be used to discover markers and mechanisms of sensitivity and resistance to inform the parallel clinical trial.

Citation Format: Vijayalaxmi G. Gupta, Yoskaly Lazo Fernandez, Katherine Roby, Harsh Pathak, Jeff Hirst, Andrew J. Wilson, Andrew Godwin, Dineo Khabele. Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3501.
Abstract 3515: Functional antigen presentation is required to interpret the tumor mutation burden (TMB) test
Authors: Cerqueira Gustavo C. , Keefer Laurel A. , Nichol Donna , Meyer Julie R. , Dracopoli Nicholas C.
Published: August 2019

Therapeutic response to checkpoint inhibitors requires a prior, suppressed immune response that is released by a monoclonal antibody blocking the interaction of the checkpoint receptors with their cognate ligands. The presence of this suppressed immune response can be detected by a variety of tests including tumor mutation burden (TMB), tumor infiltrating leukocytes, T-cell receptor clonality, circulating interferons and cytokines, and upregulation of the expression of the checkpoint ligands. Today, TMB has proven to be the most predictive biomarker of response to immunotherapy.

TMB is a surrogate marker of immune response. The test works by sampling a small region of the cancer genome to estimate the number of mutations/Mb of the cancer exome. A high TMB score is associated with better response to immunotherapy because it carries more somatic mutations and a higher chance of presenting an immunogenic neoepitope that will lead to CD-8+ T-cell mediated destruction. Restriction or loss of antigen presentation caused by mutations and loss of heterozygosity (LOH) of the beta-2-microglobulin (B2M) and HLA Class I genes has been shown to be a common means of evading CD-8+ T-Cell destruction. Consequently, the outcome for high TMB tumors will be dependent on their ability to present antigens, so that the mutation count will not matter in cells that are unable to present antigens in their HLA Class I/B2M complex.

We propose that TMB should be considered together with the ability of the tumor to present these putative neoantigens. To test this hypothesis, we used the PGDx elio™ Tissue Complete test (507 genes in 1.3 Mb) to measure TMB and antigen presentation in the same assay. We tested 212 cancer patients and showed that in FFPET samples with >20% tumor content, we could detect LOH of the MHC Class I and B2M genes with >90% accuracy compared to whole exome data from the same sample. These data confirmed our hypothesis that it was possible to measure TMB and evaluate the genes involved in antigen presentation in the same in silico analysis of a 507 gene next-generation sequencing (NGS) panel. We are currently using this combined analysis to measure TMB and somatic alterations of the antigen presentation complex in several different cancers and to test the hypothesis that adding neoantigen presentation capability to TMB scores will significantly improve prediction of response to a variety of immunotherapies.

Citation Format: Gustavo C. Cerqueira, Laurel A. Keefer, Donna Nichol, Julie R. Meyer, Nicholas C. Dracopoli. Functional antigen presentation is required to interpret the tumor mutation burden (TMB) test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3515.
Abstract 3519: High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma
Authors: Wu Liang , Wang Yuzhou , Jiang Miaomiao , Liu Shiping
Published: August 2019

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing DNA amplification and library preparation methods are expensive and introduce coverage bias during sample pre-amplification. Based on a massively parallel 5184-microwell chip, we developed a high-throughput unbiased single-cell DNA library preparation approach up to 1800 cells for copy number variation (CNV) detection, which provides a comprehensive, scalable solution for revealing genome heterogeneity. Compared with existing methods, it is more efficient and cost-effective to construct a barcoded-single-cell library without pre-amplification in 2 days. Besides, it has significantly lower Median Absolute Pairwise Difference (MAPD:0.17±0.004) values which are used to evaluate the amplification biases and noises, than using MDA (multiple displacement amplification) method (MAPD: 0.79±0.23) or MALBAC (multiple annealing and looping-based amplification cycles) approach (MAPD : 0.24±0.002). We further successfully applied this approach to obtain single-cell CNV information of more than 500 cells from a tumor sample of hepatocellular carcinoma in one run. Using the unsupervised clustering, we identified at least two clonal subpopulations with a multitude of non-integer alterations across the genome. Overall, this is an unbiased, effective, high-throughput and low cost approach to figure out tumor heterogeneity and understand clonal evolution in tumor at the single-cell level.

Citation Format: Liang Wu, Yuzhou Wang, Miaomiao Jiang, Shiping Liu. High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3519.
Abstract 3523: A high-throughput screening strategy for the identification of novel lymphoproliferative elements
Authors: Jadin Laurence , Shaban Hiba , Kundu Anirban , Schreiber Gregory , Willis Scooter , Haerizadeh Farzad , Onuffer James , Frost Gregory
Published: August 2019

Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy.

Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation.

Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model.

Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future.

Citation Format: Laurence Jadin, Hiba Shaban, Anirban Kundu, Gregory Schreiber, Scooter Willis, Farzad Haerizadeh, James Onuffer, Gregory Frost. A high-throughput screening strategy for the identification of novel lymphoproliferative elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3523.
Abstract 3524: Whole-exome sequencing of esophageal squamous cell carcinoma identifies recurrent mutations in Chinese subjects
Authors: Liu Meng , Feng Lin , An Hai yin
Published: August 2019

Background and Aims:

Epidemiological and etiological studies have shown that environmental and genetic factors play vital roles in esophageal carcinogenesis. However the genetic risk factors of esophageal squamous cell carcinoma (ESCC) still remain unclear. Here, we aimed to identify the recurrent mutations of ESCC in Chinese Uygur subjects from the China Xinjiang high-risk region by using a whole-exome sequencing method.

Materials and Methods:

We collected 50 Chinese Uygur patients with ESCC tumors and matched blood samples and adjacent normal oesophageal tissues (≥5cm from tumour site) at Xinjiang tumor hospital in China. Informed consent was obtained from each participant, and this study was approved by the Institutional Review Board of the Xinjiang tumor hospital. All the individuals underwent oesophagectomy and received no chemotherapy or radiotherapy before surgery. Whole-exome sequencing analysis (100bp) of DNA samples from 50 ESCC was performed on the Illumina HiSeq X Ten System.

Results:

We obtained 353,56 SNVs and 1157 indels in the exonic regions of 50 ESCC samples. Besides, we found the predominant mutations were the C>T and C>A transitions at TpCpW trinucleotide sites. And Three ESCC signatures were derived, which are highly similar to validated Signatrue-1, Signature-5 and Signature-13 in Cosmic. We identified four significantly mutated genes including TP53, CDKN2A, LGALS3 and C16orf3 (q<0.5). Moreover, the aberrant pathways including the receptor tyrosine kinases (RTK/RAS/ phosphoinositide-3 kinase (PI3K)), NOTCH, WNT, Hippo, TP53 and cell cycle were identified.

Conclusions:

We provided mutated genes and disrupted pathways in ESCCs. These findings might be used to specific treatments for esophageal squamous cell carcinoma in Chinese Uygur subjects.

Acknowledgements: This work was supported by the Research Fund of Key Laboratory of Xinjiang oncology (No. 2017D04006) and Outstanding Youth Science and technology training project fund of Xinjiang (No. 2017Q058).

Citation Format: Meng Liu, Lin Feng, Hai yin An. Whole-exome sequencing of esophageal squamous cell carcinoma identifies recurrent mutations in Chinese subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3524.
Abstract 3529: Sample quality control of cell-free DNA
Authors: Graf Eva
Published: August 2019

Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on pre-analytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns.

Citation Format: Eva Graf. Sample quality control of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3529.
Abstract 3532: Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow
Authors: Pendleton Kathryn E. , Liu Yang L. , Lin Lifeng , Lee Lucie S. , Liu Jeffery , Liu Guoying , Liu Zhitong
Published: August 2019

Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB.

Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics’ variant calling algorithm.

Using the CleanPlex technology, this prototype TMB panel exhibits >95% uniformity at 0.2X mean, limited GC bias, and >94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours.

Citation Format: Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3532.