MERKEL CELL POLYOMAVIRUS POSITIVE MERKEL CELL CARCINOMA – FROM DIAGNOSIS TO THERAPY by Reety Arora Bachelors in Engineering, Panjab University, 2007 Submitted to the Graduate Faculty of University of Pittsburgh School of Medicine, Program in Integrative Molecular Biology, in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Reety Arora It was defended on May 4, 2012 and approved by Jeffrey L.Brodsky, Ph.D., Professor, Department of Biological Sciences James M. Pipas, Ph.D., Professor, Department of Biological Sciences Saleem A. Khan, Ph. D., Professor, Department of Microbiology and Molecular Genetics Thomas E. Smithgall, Ph. D., Professor, Department of Microbiology and Molecular Genetics Dissertation Advisor: Yuan Chang, MD, Professor, Department of Pathology ii Copyright © by Reety Arora 2012 iii MERKEL CELL POLYOMAVIRUS POSITIVE MERKEL CELL CARCINOMA - FROM DIAGNOSIS TO THERAPY Reety Arora University of Pittsburgh, 2012 Merkel cell polyomavirus (MCV) is one of the newer members of the polyomavirus family, recently discovered as clonally integrated into the genomes of a subset of Merkel cell carcinoma (MCC). MCV is the first polyomavirus that is widely accepted to cause a human cancer and its identification has resulted in a paradigm shift in the understanding of MCC biology. In the vast majority of the population, MCV is a harmless member of the normal human microbial flora, but can initiate an aggressive cancer if it integrates into the host genome and acquires a precise set of viral mutations that result in replication incompetence (in a susceptible host). This dissertation describes how the identification of a new viral cause of MCC was harnessed to develop new diagnostic assays and therapeutic options for MCC. To assess MCV infection and its association with MCC as well as other human diseases, a monoclonal antibody that specifically recognizes both endogenous and transfected MCV large T antigen was developed. Using this antibody, specific nuclear localization of MCV T antigen in MCC tumor cells was demonstrated. A quantitative PCR assay revealed that MCV is present in MCC at > 1 copy per tumor cell. These assays were used to survey non-MCC tissues including hematolymphoid malignancies, neuroendocrine tumors, and various dermatologic cancers. None showed association with MCV infection. Restricted expression of the MCV LT oncoprotein to MCC tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. To investigate treatment options for MCC two methods were used. An in vitro drug screen of 1360 chemotherapeutic and pharmacologically active compounds resulted in the iv identification of a proteasome inhibitor, bortezomib, as a potent but nonselective candidate. To rationally and specifically target MCV positive MCC, deep-sequence profiles of MCV positive MCC tumors were compared to MCV negative MCC tumors. Among 64 cell death related genes, a seven fold differential expression of survivin was observed in MCV positive MCC. MCV T antigen knock down in MCV positive cell lines decreased survivin mRNA and protein expression. Also, exogenously expressed MCV T antigen increased survivin protein in non-MCC primary cells in an RB protein binding dependent manner. A survivin inhibitor, YM155 initiated selective non- apoptotic MCV positive MCC cell death. YM155 was nontoxic and halted MCV positive MCC xenograft tumor growth in mice while bortezomib was inactive and caused significant toxicity in vivo. v TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................... XV 1.0 INTRODUCTION ....................................................................................................... 1 1.1 VIRUSES AND CANCER ..................................................................................... 2 1.1.1 Discovery of link between viruses and cancer ........................................... 2 1.1.2 Human tumor viruses ................................................................................ 2 1.1.3 Human immunodeficiency virus: an indirect carcinogen ........................... 8 1.2 POLYOMAVIRUSES ........................................................................................... 9 1.2.1 Introduction and phylogeny ...................................................................... 9 1.2.2 History and association with human cancer ............................................ 11 1.2.3 Genome organization .............................................................................. 13 1.2.4 Viral life cycle and general properties ..................................................... 17 1.2.5 Human polyomaviruses. .......................................................................... 20 1.3 SV40 T ANTIGENS- CLASSIC MODELS FOR STUDYING CANCER ........................ 23 1.3.1 Large T antigen (LT) ................................................................................. 24 1.3.1.1 Interaction with pRB family of proteins ........................................... 25 1.3.1.2 Interaction with p53 ........................................................................ 29 1.3.1.3 Other interactions ........................................................................... 30 vi 1.3.2 Small T antigen (ST) ................................................................................. 34 1.3.3 Insights into malignant progression from SV40 transgenic mouse models36 1.4 MERKEL CELL CARCINOMA ............................................................................. 38 1.4.1 Origin and pathology ............................................................................... 38 1.4.2 Risk factors and clinical features ............................................................. 40 1.4.3 Staging .................................................................................................... 40 1.4.4 Incidence ................................................................................................. 40 1.4.5 Molecular studies .................................................................................... 41 1.4.6 Treatment ............................................................................................... 41 1.5 MERKEL CELL POLYOMAVIRUS ....................................................................... 43 1.5.1 Discovery of MCV .................................................................................... 44 1.5.2 Genomic organization of MCV ................................................................. 45 1.5.3 Tumor specific signature truncation mutations ....................................... 47 1.5.4 T antigen dependency ............................................................................. 49 1.5.5 Transformation activity ........................................................................... 51 1.5.6 MCV seroprevalence ............................................................................... 51 1.5.7 Transmission ........................................................................................... 53 1.6 MCC CELL LINES ............................................................................................. 54 2.0 MERKEL CELL POLYOMAVIRUS T ANTIGEN EXPRESSION IN MCC AND OTHER CANCERS……… ....................................................................................................................... 56 2.1 MATERIALS AND METHODS ........................................................................... 59 vii 2.1.1 Human tissue samples ............................................................................. 59 2.1.2 Real time quantitative PCR ...................................................................... 61 2.1.3 Cell lines and transfection conditions ...................................................... 62 2.1.4 Plasmids .................................................................................................. 62 2.1.5 Generation of CM2B4 mAb ...................................................................... 63 2.1.6 Immunofluorescence and immunohistochemistry ................................... 63 2.1.7 Immunoblotting ...................................................................................... 64 2.2 RESULTS ......................................................................................................... 64 2.2.1 MCV T antigen expression in Merkel cell carcinoma tumors .................... 64 2.2.2 PBMC infection with MCV ....................................................................... 76 2.2.3 Survey of hematolymphoid malignancies for MCV infection ................... 76 2.2.4 Immunohistochemial analysis of combined primary cutaneous squamous and neuroendocrine carcinomas ............................................................................ 77 2.2.5 Immunohistochemical analysis of 26 primary pulmonary (small and large cell) neuroendocrine carcinomas ........................................................................... 77 2.2.6 MCV and chronic lymphocytic leukemia .................................................. 80 2.3 DISCUSSION ................................................................................................... 82 3.0 LITERATURE REVIEW OF MCV POSITIVITY IN MCC AND OTHER TISSUES ................. 86 3.1 MONOCLONAL INTEGRATION OF MCV IN MCC AT >1 COPY PER CELL ............ 86 3.2 MCV