DOCSLIB.ORG

Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-18-3374 CANCER RESEARCH | TRANSLATIONAL SCIENCE Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H. Diplas4, Song Liu5, Jianmei Liu2,3, Dingding Wang1, Yan Lu1, Qing Zhu6, Jiayu Wu1, Wenjia Wang1, Hai Yan4, Yi-Xin Zeng6, Xiaoyue Wang1, and Yuchen Jiao2,7 ABSTRACT ◥ The tumorsuppressor gene ATRX is frequently mutated ina variety also selectively inhibited the proliferation of patient-derived primary of tumors including gliomas and liver cancers, which are highly cell lines from gliomas with naturally occurring ATRX mutations, unresponsive to current therapies. Here, we performed a genome- indicating that the synthetic lethal relationship between WEE1 and wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to ATRX could be exploited in a broader spectrum of human tumors. As identify potential therapeutic targets specificforATRX-mutated WEE1 inhibitors have been investigated in several phase II clinical cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engi- trials, our discovery provides the basis for an easily clinically testable neered for ATRX loss, we identified 58genes, including the checkpoint therapeutic strategy specific for cancers deficient in ATRX. kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the Significance: ATRX-mutant cancer cells depend on WEE1, which growth of several ATRX-deficient HCC cell lines in vitro,aswellas provides a basis for therapeutically targeting WEE1 in ATRX-deficient xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was cancers. caused by accumulated DNA damage–induced apoptosis. AZD1775 SeerelatedcommentarybyCole,p.375 Introduction variety of human cancers, including pancreatic neuroendocrine tumors (PanNET), glioma, liver cancer, and neuroblastoma. Although Over 20 years ago, germline variations in ATRX, a member of the ATR-X syndrome-related germline variations are mainly missense in SWI/SNF chromatin remodeling superfamily, were found to underlie a the histone methylation recognition domain and the ATPase rare developmental disorder, X-linked mental retardation with alpha- domain (6), the cancer-associated somatic ATRX mutations are thalassemia (ATR-X syndrome; ref. 1). More recently, genomic predominantly truncating mutations, resulting in loss of ATRX studies (2–5) have revealed that ATRX is frequently mutated in a expression (7), indicating that ATRX functions as a tumor suppressor gene in these cancers. Although genomic sequencing has enabled the rapid discovery of 1 State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical the mutations driving the development of human cancer, significant Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, challenges remain regarding the effective translation of these data into Beijing, China. 2State Key Lab of Molecular Oncology, National Cancer Center/ National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy viable therapies. Most driver mutations are damaging the functions of of Medical Sciences and Peking Union Medical College, Beijing, China. 3Depart- suppressor genes and cannot serve as a direct therapeutic target. One ment of Hepatobiliary Surgery, National Cancer Center/National Clinical strategy for developing targeted therapies against such loss-of-function Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical mutations is “synthetic lethality.” Synthetic lethality refers to the 4 Sciences and Peking Union Medical College, Beijing, China. The Preston Robert relationship between 2 genes for which loss of both genes results in Tisch Brain Tumor Center at Duke, Pediatric Brain Tumor Foundation Institute at cell death but loss of either one alone does not. For example, poly Duke, and Department of Pathology, Duke University Medical Center, Durham, PARP1 BRCA1/2 North Carolina. 5Department of Central Laboratory, Peking Union Medical (ADP)-ribose polymerase 1 ( ) and are synthetic lethal College Hospital, Peking Union Medical College & Chinese Academy of Medical because loss of both genes abolishes 2 parallel DNA damage repair Sciences, Beijing, China. 6Department of Experimental Research, Sun Yat-sen pathways and causes cell apoptosis (8). University Cancer Center, State Key Laboratory of Oncology in Southern China, Motivated by the clinical success of PARP inhibitors in treatment of Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong BRCA1/2-deficient tumors, scientists have performed genome-wide 7 Province, China. Department of Clinical Laboratory, National Cancer Center/ screens in human cancer cells to identify synthetic lethal partners National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. relevant to other frequently mutated tumor suppressor genes. In such screens, genes essential for each cell line are first identified by high- Note: Supplementary data for this article are available at Cancer Research throughput perturbation of genes using RNA interference (RNAi) or Online (http://cancerres.aacrjournals.org/). CRISPR-Cas9 genome editing. Synthetic lethal interactions are then J. Liang, H. Zhao, B.H. Diplas, and S. Liu contributed equally to this article. inferred by comparing the gene dependency profiles across a panel of Corresponding Authors: Xiaoyue Wang, Institute of Basic Medical Sciences, either independently derived cell lines with or without mutations at a Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing specific locus, or paired isogenic lines with engineered mutations. Such 100005, China. E-mail: [email protected]; and Yuchen Jiao, National screens have been used to identify potential pharmacologic targets that Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and enable selective killing of cancer cells with deficiencies in tumor Peking Union Medical College, Beijing 100021, China. Phone: 86-010- TP53 ARID1A 87788045; E-mail: [email protected] suppressors or (9, 10), or even with activation of “undruggable” oncogenes including RAS or MYC (11, 12). Cancer Res 2020;80:510–23 Among ATRX-mutant tumor types, hepatocellular carcinoma doi: 10.1158/0008-5472.CAN-18-3374 (HCC) is the 5th most common cancer in the world (13) and glioma Ó2019 American Association for Cancer Research. is the most common primary malignant brain tumor in adults. There AACRJournals.org | 510 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-18-3374 WEE1 Inhibitors Selectively Kill ATRX-Deficient Cancer Cells are currently no effective targeted therapies available for HCC or with DNAMAN multiple sequence alignment tools. The primers used glioma, making them among the most lethal tumors in the world. Here, for amplifying the flanked region of the target site were as follows: we performed a genome-wide pooled CRISPR-Cas9–based screen in 0 0 paired isogenic HCC cells with or without engineered mutations in ATRX_forward: 5 -CCGTGACTCAGATGGAATGGA-3 ATRX – WEE1 ATRX 0 0 . The G2 M checkpoint control kinase, , was one of the _reverse: 5 -GGTTACAGAGCCAGAACAGG-3 several candidate genes identified as potentially lethal in the context of ATRX deficiency. The lethality of WEE1 loss was further investigated Lentivirus production 7 2 with small molecule inhibitors of WEE1 through the treatment of Low passage HEK293T cells (4 Â 10 ) were seeded onto 500 cm engineered ATRX-mutant HCC cell lines and xenografts, as well as culture plates 1 day before transfection. When cells reached 80% to primary cell lines from naturally occurring gliomas. The results 90% confluence, a mixture containing the following library and support inhibition of WEE1 as the basis for the development of new packaging plasmids was transfected into HEK293T cells with Neofect: therapeutic strategies to treat multiple tumor types harboring ATRX GeCKO v2 library (63 mg; Addgene plasmid 1000000048), psPAX2 (45 mutations. mg; Addgene plasmid #12260), and pMD2.G (18 mg; Addgene plasmid #12259). Culture medium was changed 12 hours after transfection. The supernatant containing lentivirus was collected 60 hours post- Materials and Methods transfection, filtered through a PVDF filter membrane (0.45 mm; Ethics statement Millipore SteriCup 250 mL; Millipore) to remove cells, aliquoted, and All animal procedures were approved by and performed according stored at À80 C. to the animal ethical committee at the Cancer Hospital, Chinese Synthetic lethal screens Academy of Medical Sciences (Beijing, China). PLC/PRF/5 parental cells or PLC/PRF/5 ATRX KO cells (200 Â 106) Cell culture were transduced with lentivirus from the human GeCKO v2 library at PLC/PRF/5, HuH-7, and HeLa cell lines were obtained from the an MOI of 0.3 and an average of 500-fold coverage of the library. As the human GeCKO v2 library contains 123,411 sgRNAs, at least 6 Â National Infrastructure of Cell Line Resource (Beijing, China) and are 7 routinely authenticated via their short tandem repeat profile (latest 10 cells need to be infected to achieve 500-fold coverage. At 48 hours verification for PLC/PRF/5 and HeLa: April 2019; latest verification for postinfection, cells were selected in puromycin (2 mg/mL; Thermo
Load more

Recommended publications
  • Systematic Screening for Potential Therapeutic Targets in Osteosarcoma Through a Kinome-Wide CRISPR-Cas9 Library
    Cancer Biol Med 2020. doi: 10.20892/j.issn.2095-3941.2020.0162 ORIGINAL ARTICLE Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library Yuanzhong Wu*, Liwen Zhou*, Zifeng Wang, Xin Wang, Ruhua Zhang, Lisi Zheng, Tiebang Kang Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China ABSTRACT Objective: Osteosarcoma is the most common primary malignant bone tumor. However, the survival of patients with osteosarcoma has remained unchanged during the past 30 years, owing to a lack of efficient therapeutic targets. Methods: We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma. The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Python package. The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout. The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2: Genomics Analysis and Visualization Platform. Results: We identified 53 potential kinase targets in osteosarcoma. Among these targets, we analyzed 3 kinases, TRRAP, PKMYT1, and TP53RK, to validate their oncogenic functions in osteosarcoma. PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue, whereas TRRAP showed no significant difference. High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma. Conclusions: Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library, including target design, library construction, screening workflow, data analysis, and functional validation.
    [Show full text]
  • The Combination of the PARP Inhibitor Olaparib and the Wee1 Inhibitor AZD1775 As a New Therapeutic Option for Small Cell Lung Cancer
    Author Manuscript Published OnlineFirst on June 25, 2018; DOI: 10.1158/1078-0432.CCR-17-2805 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The combination of the PARP inhibitor olaparib and the Wee1 inhibitor AZD1775 as a new therapeutic option for small cell lung cancer Alice Lallo1,*, Kristopher K Frese1,*, Christopher J Morrow1, Robert Sloane1, Sakshi Gulati1, Maximillian W Schenk1, Francesca Trapani1, Nicole Simms1, Melanie Galvin1, Stewart Brown1, Cassandra L Hodgkinson1, Lynsey Priest1, Adina Hughes4, Zhongwu Lai5, Elaine Cadogan4, Garima Khandelwal3, Kathryn L Simpson1, Crispin Miller3, Fiona Blackhall2, Mark J O’Connor4,#, and Caroline Dive1,# 1 Clinical and Experimental Pharmacology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. 2 Institute of Cancer Sciences, University of Manchester, Manchester, United Kingdom. Christie NHS Foundation Trust, Manchester, M20 4BX, UK. 3 RNA Biology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. 4 Oncology Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, CB2 0RE, UK. 5 Oncology Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Waltham, 02451, USA. *These authors contributed equally to this work Running title: PARP and Wee1 inhibition in patient-derived models of SCLC C. Dive receives funding from the Cancer Research UK Manchester Institute core (C5759/A27412), the Cancer Research UK Manchester
    [Show full text]
  • Bioinformatics-Based Screening of Key Genes for Transformation of Liver
    Jiang et al. J Transl Med (2020) 18:40 https://doi.org/10.1186/s12967-020-02229-8 Journal of Translational Medicine RESEARCH Open Access Bioinformatics-based screening of key genes for transformation of liver cirrhosis to hepatocellular carcinoma Chen Hao Jiang1,2, Xin Yuan1,2, Jiang Fen Li1,2, Yu Fang Xie1,2, An Zhi Zhang1,2, Xue Li Wang1,2, Lan Yang1,2, Chun Xia Liu1,2, Wei Hua Liang1,2, Li Juan Pang1,2, Hong Zou1,2, Xiao Bin Cui1,2, Xi Hua Shen1,2, Yan Qi1,2, Jin Fang Jiang1,2, Wen Yi Gu4, Feng Li1,2,3 and Jian Ming Hu1,2* Abstract Background: Hepatocellular carcinoma (HCC) is the most common type of liver tumour, and is closely related to liver cirrhosis. Previous studies have focussed on the pathogenesis of liver cirrhosis developing into HCC, but the molecular mechanism remains unclear. The aims of the present study were to identify key genes related to the transformation of cirrhosis into HCC, and explore the associated molecular mechanisms. Methods: GSE89377, GSE17548, GSE63898 and GSE54236 mRNA microarray datasets from Gene Expression Omni- bus (GEO) were analysed to obtain diferentially expressed genes (DEGs) between HCC and liver cirrhosis tissues, and network analysis of protein–protein interactions (PPIs) was carried out. String and Cytoscape were used to analyse modules and identify hub genes, Kaplan–Meier Plotter and Oncomine databases were used to explore relationships between hub genes and disease occurrence, development and prognosis of HCC, and the molecular mechanism of the main hub gene was probed using Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis.
    [Show full text]
  • NIH Public Access Author Manuscript J Theor Biol
    NIH Public Access Author Manuscript J Theor Biol. Author manuscript; available in PMC 2014 March 07. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: J Theor Biol. 2013 March 7; 320: . doi:10.1016/j.jtbi.2012.12.011. A predictive mathematical model of the DNA damage G2 checkpoint Kevin J. Kesselera, Michael L. Blinovb, Timothy C. Elstonc, William K. Kaufmanna, and Dennis A. Simpsona,* aDepartment of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, NC 27599-7255, USA bCenter for Cell Analysis and Modeling, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1507, USA cDepartment of Pharmacology, University of North Carolina at Chapel Hill,Chapel Hill, NC 27599-7260, USA Abstract A predictive mathematical model of the transition from the G2 phase in the cell cycle to mitosis (M) was constructed from the known interactions of the proteins that are thought to play significant roles in the G2 to M transition as well as the DNA damage- induced G2 checkpoint. The model simulates the accumulation of active cyclin B1/Cdk1 (MPF) complexes in the nucleus to activate mitosis, the inhibition of this process by DNA damage, and transport of component proteins between cytoplasm and nucleus. Interactions in the model are based on activities of individual phospho-epitopes and binding sites of proteins involved in G2/M. Because tracking phosphoforms leads to combinatorial explosion, we employ a rule-based approach using the BioNetGen software. The model was used to determine the effects of depletion or over-expression of selected proteins involved in the regulation of the G2 to M transition in the presence and absence of DNA damage.
    [Show full text]
  • Small-Molecule Binding Sites to Explore New Targets in the Cancer Proteome
    Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is © The Royal Society of Chemistry 2016 Small-molecule binding sites to explore new targets in the cancer proteome David Xu, Shadia I. Jalal, George W. Sledge Jr., and Samy O. Meroueh* Supplementary Text Druggable Binding Sites across all 10 Diseases. Using the previously established cutoffs, we identified genes that were overexpressed across multiple cancer types and featured druggable binding sites. We ranked these genes based on the total number of tumors that overexpressed the gene (Fig. S1). Using a simple PubMed query, we then counted the number of articles in which either the gene symbol or gene name was co-mentioned with the term ‘cancer’. Most of the most frequently occurring differentially-expressed genes correspond to proteins of well- established cancer targets. Among them are matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP12, which are implicated in tumor invasion and metastasis (1). There are several protein kinases, including TTK, AURKA, AURKB, and PLK1, that are involved in cell signaling and well-established oncology targets (2). Some genes among this list that have not been extensively studied nor targeted in cancer. These include the serine/threonine kinase PKMYT1 (MYT1) is a regulator of G2/M transition in the cell cycle, but lacks focused small molecule inhibitors that specifically target the kinase. Recent efforts in developing small molecule inhibitors involve repurposing of available kinase inhibitors to specifically target the kinase (3). A subunit of the GINS complex GINS2 (PSF2) is involved in cell proliferation and survival in cancer cell lines (4,5).
    [Show full text]
  • Ser 15 of WEE1B Is a Potential PKA Phosphorylation Target in G2/M Transition in One-Cell Stage Mouse Embryos
    MOLECULAR MEDICINE REPORTS 7: 1929-1937, 2013 Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos CHAO LIU1,2, YANCHUN LIU1, YANG LIU2, DIDI WU1, ZHIDONG LUAN2, ENHUA WANG3 and BINGZHI YU1,3 1Department of Biochemistry and Molecular Biology, China Medical University, Shenyang; 2Department of Developmental Biology, Liaoning Medical University, Jinzhou; 3Institute of Pathology and Pathopysiology, China Medical University, Shenyang, Liaoning, P.R. China Received December 17, 2012; Accepted March 28, 2013 DOI: 10.3892/mmr.2013.1437 Abstract. The WEE1 kinase family has been shown to be the Introduction major kinase family responsible for phosphorylating Tyr 15 on cyclin-dependent kinase 1 (CDK1). WEE1 homolog 2 (WEE2, M phase-promoting factor (MPF) is a universal regulator of M also known as WEE1B) was first identified in Xenopus laevis phase in the eukaryotic cell cycle (1,2). Its activation induces and more recently in humans and mice, and is responsible for entry into M phase and its subsequent inactivation is necessary phosphorylating the CDK1 inhibitory site and maintaining to exit from M phase. The activity of MPF in higher eukary- meiotic arrest in oocytes. However, the mechanism by which otic cells is primarily regulated by the formation of a complex WEE1B is regulated in one-cell stage mouse embryos remains between the catalytic cyclin-dependent kinase 1 (CDK1, to be elucidated. In the present study, we examined the role previously known as CDC2) subunit and the regulatory of WEE1B-Ser 15 in G2/M transition of one-cell stage mouse CCNB1 (previously known as cyclin B) subunit, and further embryos.
    [Show full text]
  • Biomarkers, Master Regulators and Genomic Fabric Remodeling in a Case of Papillary Thyroid Carcinoma
    G C A T T A C G G C A T genes Article Biomarkers, Master Regulators and Genomic Fabric Remodeling in a Case of Papillary Thyroid Carcinoma Dumitru A. Iacobas Personalized Genomics Laboratory, CRI Center for Computational Systems Biology, Roy G Perry College of Engineering, Prairie View A&M University, Prairie View, TX 77446, USA; [email protected]; Tel.: +1-936-261-9926 Received: 1 August 2020; Accepted: 1 September 2020; Published: 2 September 2020 Abstract: Publicly available (own) transcriptomic data have been analyzed to quantify the alteration in functional pathways in thyroid cancer, establish the gene hierarchy, identify potential gene targets and predict the effects of their manipulation. The expression data have been generated by profiling one case of papillary thyroid carcinoma (PTC) and genetically manipulated BCPAP (papillary) and 8505C (anaplastic) human thyroid cancer cell lines. The study used the genomic fabric paradigm that considers the transcriptome as a multi-dimensional mathematical object based on the three independent characteristics that can be derived for each gene from the expression data. We found remarkable remodeling of the thyroid hormone synthesis, cell cycle, oxidative phosphorylation and apoptosis pathways. Serine peptidase inhibitor, Kunitz type, 2 (SPINT2) was identified as the Gene Master Regulator of the investigated PTC. The substantial increase in the expression synergism of SPINT2 with apoptosis genes in the cancer nodule with respect to the surrounding normal tissue (NOR) suggests that SPINT2 experimental overexpression may force the PTC cells into apoptosis with a negligible effect on the NOR cells. The predictive value of the expression coordination for the expression regulation was validated with data from 8505C and BCPAP cell lines before and after lentiviral transfection with DDX19B.
    [Show full text]
  • Activation of Diverse Signalling Pathways by Oncogenic PIK3CA Mutations
    ARTICLE Received 14 Feb 2014 | Accepted 12 Aug 2014 | Published 23 Sep 2014 DOI: 10.1038/ncomms5961 Activation of diverse signalling pathways by oncogenic PIK3CA mutations Xinyan Wu1, Santosh Renuse2,3, Nandini A. Sahasrabuddhe2,4, Muhammad Saddiq Zahari1, Raghothama Chaerkady1, Min-Sik Kim1, Raja S. Nirujogi2, Morassa Mohseni1, Praveen Kumar2,4, Rajesh Raju2, Jun Zhong1, Jian Yang5, Johnathan Neiswinger6, Jun-Seop Jeong6, Robert Newman6, Maureen A. Powers7, Babu Lal Somani2, Edward Gabrielson8, Saraswati Sukumar9, Vered Stearns9, Jiang Qian10, Heng Zhu6, Bert Vogelstein5, Ben Ho Park9 & Akhilesh Pandey1,8,9 The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets. 1 McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 527, Baltimore, Maryland 21205, USA.
    [Show full text]
  • Supplementary Information
    Osa et al Supplementary Information Clinical implications of monitoring nivolumab immunokinetics in previously treated non– small cell lung cancer patients Akio Osa, Takeshi Uenami, Shohei Koyama, Kosuke Fujimoto, Daisuke Okuzaki, Takayuki Takimoto, Haruhiko Hirata, Yukihiro Yano, Soichiro Yokota, Yuhei Kinehara, Yujiro Naito, Tomoyuki Otsuka, Masaki Kanazu, Muneyoshi Kuroyama, Masanari Hamaguchi, Taro Koba, Yu Futami, Mikako Ishijima, Yasuhiko Suga, Yuki Akazawa, Hirotomo Machiyama, Kota Iwahori, Hyota Takamatsu, Izumi Nagatomo, Yoshito Takeda, Hiroshi Kida, Esra A. Akbay, Peter S. Hammerman, Kwok-kin Wong, Glenn Dranoff, Masahide Mori, Takashi Kijima, Atsushi Kumanogoh Supplemental Figures 1 – 8 1 Osa et al Supplemental Figure 1. The frequency of nivolumab-bound T cells was maintained in patients who continued treatment. Nivolumab binding in CD8 and CD4 T cells was analyzed at two follow-up points, as indicated, in fresh peripheral blood from three representative cases from protocol 1 that continued treatment. 2 Osa et al Supplemental Figure 2. Long-term follow-up of nivolumab binding to T cells from fresh whole blood. Nivolumab binding was followed up in fresh peripheral blood from an additional case, Pt.7. 3 Osa et al Supplemental Figure 3. Long-term duration of nivolumab binding is due to sustained circulation of residual nivolumab in plasma. (A) PBMCs acquired from Pt.8 and 9 at pretreatment (pre PBMCs) and after a single dose (post 1 PBMCs) were cultured in regular medium without nivolumab (top and middle). Pre PBMCs were also incubated with 10 µg/ml nivolumab in vitro before the cultures were started (bottom). Nivolumab binding status was monitored at the indicated time points.
    [Show full text]
  • Investigations on the Human Myt1 Kinase: Substrate Studies, Assay Development and Inhibitor Screening Alexander Rohe
    Investigations on the Human Myt1 Kinase: Substrate Studies, Assay Development and Inhibitor Screening Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I - Biowissenschaften Martin-Luther-Universität Halle-Wittenberg von Alexander Rohe geboren am 06.11.1985 in Saarbrücken Gutachter: 1. Prof. Dr. W. Sippl 2. Prof. Dr. M. Schutkowski 3. Prof. Dr. M. Jung Halle (Saale), 28.August 2013 Tag der Verteidigung: 15. Januar 2014 II III Durchgeführt am Institut für Pharmazie, Abteilung Medizinische Chemie, Martin-Luther-Universität Halle-Wittenberg Gutachter: Prof. Dr. W. Sippl, Martin-Luther-Universität Halle-Wittenberg Zweitgutachter: Prof. Dr. M. Schutkowski, Martin-Luther-Universität Halle-Wittenberg Drittgutachter: Prof. Dr. M. Jung, Albert-Ludwigs-Universität Freiburg i. Br. IV Table of Contents V Table of Contents Table of Contents .................................................................................................... V Abbreviations and Symbols .................................................................................. IX List of Figures ...................................................................................................... XV List of Tables..................................................................................................... XVII 1. Introduction ...................................................................................................... 1 2. Theoretical Background ..................................................................................
    [Show full text]
  • Brain-Restricted Mtor Inhibition with Binary Pharmacology 2 3 Ziyang Zhang1, Qiwen Fan2,3, Xujun Luo2,3, Kevin J
    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Brain-Restricted mTOR Inhibition with Binary Pharmacology 2 3 Ziyang Zhang1, Qiwen Fan2,3, Xujun Luo2,3, Kevin J. Lou1, William A. Weiss2,3,4,5, Kevan 4 M. Shokat1,* 5 6 1 Department of Cellular and Molecular Pharmacology and Howard Hughes Medical 7 Institute, University of California, San Francisco, California 8 2 Helen Diller Family Comprehensive Cancer Center, San Francisco, California 9 3 Department of Neurology, University of California, San Francisco, California 10 4 Department of Pediatrics, University of California, San Francisco, California 11 5 Department of Neurological Surgery, University of California, San Francisco, California 12 * Corresponding author. [email protected] (K.M.S.). 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 13 Abstract 14 On-target-off-tissue drug engagement is an important source of adverse effects 15 that constrains the therapeutic window of drug candidates. In diseases of the central 16 nervous system, drugs with brain-restricted pharmacology are highly desirable.
    [Show full text]
  • Defining the KRAS-Regulated Kinome in KRAS-Mutant Pancreatic Cancer
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441678; this version posted April 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Submitted Manuscript: Confidential Defining the KRAS-regulated kinome in KRAS-mutant pancreatic cancer J. Nathaniel Diehl1, Jennifer E. Klomp2, Kayla R. Snare2, Devon R. Blake3, Priya S. Hibshman2,4, Zane D. Kaiser2, Thomas S.K. Gilbert3,5, Elisa Baldelli6, Mariaelena Pierobon6, Björn Papke2, Runying Yang2, Richard G. Hodge2, Naim U. Rashid2,7, Emanuel F. Petricoin III6, Laura E. Herring3,5, Lee M. Graves2,3,5, Adrienne D. Cox2,3,4,8, Channing J. Der1,2,3,4* 1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 3Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 4Cell Biology and Physiology Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 5UNC Michael Hooker Proteomics Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 6Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, USA; 7Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 8Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. *Corresponding author: Email: [email protected] Running title: Defining the KRAS-regulated kinome 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441678; this version posted April 30, 2021.
    [Show full text]