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Molecular Targeting and Enhancing Anticancer Efficacy of Oncolytic HSV-1 to Midkine Expressing Tumors

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Molecular Targeting and Enhancing Anticancer Efficacy of Oncolytic HSV-1 to Midkine Expressing Tumors University of Cincinnati Date: 12/20/2010 I, Arturo R Maldonado , hereby submit this original work as part of the requirements for the degree of Doctor of Philosophy in Developmental Biology. It is entitled: Molecular Targeting and Enhancing Anticancer Efficacy of Oncolytic HSV-1 to Midkine Expressing Tumors Student's name: Arturo R Maldonado This work and its defense approved by: Committee chair: Jeffrey Whitsett Committee member: Timothy Crombleholme, MD Committee member: Dan Wiginton, PhD Committee member: Rhonda Cardin, PhD Committee member: Tim Cripe 1297 Last Printed:1/11/2011 Document Of Defense Form Molecular Targeting and Enhancing Anticancer Efficacy of Oncolytic HSV-1 to Midkine Expressing Tumors A dissertation submitted to the Graduate School of the University of Cincinnati College of Medicine in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (PH.D.) in the Division of Molecular & Developmental Biology 2010 By Arturo Rafael Maldonado B.A., University of Miami, Coral Gables, Florida June 1993 M.D., New Jersey Medical School, Newark, New Jersey June 1999 Committee Chair: Jeffrey A. Whitsett, M.D. Advisor: Timothy M. Crombleholme, M.D. Timothy P. Cripe, M.D. Ph.D. Dan Wiginton, Ph.D. Rhonda D. Cardin, Ph.D. ABSTRACT Since 1999, cancer has surpassed heart disease as the number one cause of death in the US for people under the age of 85. Malignant Peripheral Nerve Sheath Tumor (MPNST), a common malignancy in patients with Neurofibromatosis, and colorectal cancer are midkine- producing tumors with high mortality rates. In vitro and preclinical xenograft models of MPNST were utilized in this dissertation to study the role of midkine (MDK), a tumor-specific gene over- expressed in these tumors and to test the efficacy of a MDK-transcriptionally targeted oncolytic HSV-1 (oHSV). The overall goals of this dissertation were to 1) discover mechanistic insights into the role of MDK in cancer using a feature-enabled bioinformatics approach and to 2) exploit MDK biology to increase the efficacy of oncolytic HSV-1 towards MPNST tumors without compromising safety. To search for mechanistic insights into the role of MDK in cancer, A Pearson correlation of genes that co-express with MDK in 121 microarrays of MPNST and colorectal cancers was performed. A cluster of 348 genes that were co-regulated with MDK were identified. MDK co- expressed genes significantly enriched in functional categories such as DNA replication and cell cycle regulation. Out of 348 genes, 66 are transcription complex genes, many of which have been associated with cancer or shown to be cancer causal genes, such as HMGB1, CREB1, and YY1. To assess upstream regulation of MDK co-expressed genes, a cis-element promoter analysis was performed and predicted enrichment of 4 elements: HLF, NFIL3, MEF2A, and FOXD3, a possible cis-regulatory module. Validation and functional studies are needed to confirm these hypotheses. oHSV has been engineered to be safe by deletion of viral genes necessary for virus replication and killing in normal cells. To target midkine-producing tumors, oHSV-MDK-34.5 iii was engineered for this dissertation which contains the HSV-1 neurovirulence γ134.5 transgene driven by the MDK promoter. The HSV neurovirulence protein ICP34.5 has been shown to increase virus replication and infectivity by preventing the shutdown of host cell protein translation. oHSV-MDK-34.5 targeted MDK-producing MPNST with increased replication and oncolysis yet retains attenuation in normal non-MDK producing tissues. MDK promoter activity and transgene biological activity was confirmed in human MPNST (S462, STS26T) and Ewing sarcoma cells (A673). In vitro replication and cytotoxicity in human fibroblasts and STS26T cells by plaque and MTT assays showed that oHSV-MDK-34.5 increased replication and cytotoxicity compared to the Luciferase-containing control virus, oHSV-MDK-Luc. In contrast, no significant difference in cytotoxicity was detected between these viruses in normal human fibroblasts. oHSV-MDK-34.5 impaired in vivo STS26T tumor growth and increased median survival of tumor-bearing nude mice. No evidence of extratumoral spread of oHSV-MDK-34.5 was found in nude mouse organs 14 days after tumor injection. The novel MDK-targeted oncolytic virus engineered in this dissertation, oHSV-MDK-34.5, successfully impaired human MPNST tumor growth and prolonged the survival of MPNST tumor-bearing mice without compromising tumor-specificity and safety. This oHSV is a new addition to the armamentarium of oncolytic viruses being studied for personalized anti-cancer therapy. iv ©2011 Arturo Rafael Maldonado ALL RIGHTS RESERVED v RESEARCH GRANTS & AWARDS The work in this dissertation was supported by a research award from the National Cancer Institute, grants from the NIH, and a grant from the Shriners Hospitals for Children. NIH NCI National Research Service Award F31 CA132613-01 (Arturo Maldonado PI) R01 DK074055 (Timothy Crombleholme PI) R01 DK072446 (Timothy Crombleholme PI) R01 CA114004 (Timothy Cripe PI) R21 CA133663 (Timothy Cripe PI) Shriners Hospitals for Children Grant 8901 (Timothy Crombleholme PI) vi ACKNOWLEDGEMENTS Most special thanks to my thesis committee members and mentors Drs. Timothy Crombleholme, Timothy Cripe, Jeffrey Whitsett, Dan Wiginton, Rhonda Cardin, Bruce Aronow, Anil Jegga and Punam Malik for your insight, instruction and leadership. Thanks to the students of the University of Cincinnati Graduate Programs in Molecular and Developmental Biology, Cell and Cancer Biology, and Neuroscience: especially Hongyan Zhu, Karunyakanth (KK) Mandapaka, David Hahn, Diva Jonatan and Tom Lu for friendship, scientific discussion and support. Thanks to the members of the Crombleholme, Cripe and Malik laboratories. Special thanks to the Crombleholme laboratory: Chuck Klanke, Jose Fernando Solis Vuletin, Shuichi Katayama, Sundeep Keswani, Foong Lim, Mounira Habli, Louis Le and Stephanie Lang. Also thanks to the Cripe laboratory: Mark Currier, Pin-Yi Wang, William Baird and Jennifer Leddon. Also special thanks to the Malik laboratory: Drs. Tomoyasu Higashimoto and Ping Xia. Very special thanks to Drs. Helen Jones and Yoni Mahller, for the excellent scientific discussions, encouragement, guidance, and friendship. My warmest and deepest gratitude goes to Drs. Timothy Crombleholme and Timothy Cripe for their unbridled support, enthusiasm and mentorship. Without their assistance, this work would not have been possible. Thanks to Paul Steele, M.D., director of the Cincinnati Children’s clinical laboratory for advice and technical assistance. Very special thanks to Jessica and Betsy Honig for scientific discussions and manuscript assistance. Also, thanks to my collaborators Drs. Yoshinaga Saeki, E. Antonio Chiocca, Balveen Kaur (The Ohio State University), Robert Cohen (University of Cincinnati), Ian Mohr (SUNY at Stony Brook). Thanks to Michael Burhans, John Shannon vii (Cincinnati Children’s Research Foundation), and Kazunari Yokoyama (RIKEN Institute, Ibaraki, Japan) for kindly providing advice and reagents. My warmest and special thanks to Katrina and my family for unwavering love and support. viii TABLE OF CONTENTS ABSTRACT .................................................................................................................................. iii RESEARCH GRANTS & AWARDS ........................................................................................ vi ACKNOWLEDGEMENTS ....................................................................................................... vii LIST OF FIGURES AND TABLES .......................................................................................... xii CHAPTER 1: General Introduction ........................................................................................... 1 Overall background and clinical significance .......................................................................................... 1 A brief history of chemotherapy and the advent of molecular therapy for cancer ................................... 2 Oncolytic Viruses: Cancer killing viruses as an emerging treatment for cancer ...................................... 4 The era of recombinant viral oncolytics: increasing oncolytic efficacy and biosafety ............................. 6 Virus-mediated cancer cell lysis ............................................................................................................... 7 Transcriptional targeting: an approach to increasing oncolytic efficacy and to personalized predictive oncologic medicine ................................................................................................................................... 7 Advantages of Oncolytic HSV (oHSV) for cancer treatment .................................................................. 8 The research problem: improving oncolytic HSV-1 efficacy without compromising biosafety .............. 9 The oHSV constructed for transcriptional targeting: oHSV-MDK-34.5 .................................................. 9 The role of the HSV-1 protein ICP34.5 in evading viral host defense ................................................... 10 Developmental and cancer-specific MDK expression ........................................................................... 11 Transcriptional regulation and systems biological analyses of the MDK promoter in cancer ............... 12 Transcriptional targeting using the MDK promoter ..............................................................................
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