The Pennsylvania State University

The Pennsylvania State University

The Pennsylvania State University The Graduate School Department of Molecular Medicine IDENTIFICATION OF SOMATIC MUTATIONS IN LGL LEUKEMIA THROUGH WHOLE GENOME SEQUENCING AND CORRELATION OF STAT3 Y640F MUTATION WITH TREAMENT RESPONSE TO METHOTREXATE. A Dissertation in Molecular Medicine by Thomas Lynn Olson 2013 Thomas Lynn Olson Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2013 The dissertation of Thomas L. Olson was reviewed and approved* by the following: Thomas P. Loughran Jr., Physician Director of Penn State Cancer Institute Professor of Medicine Dissertation Advisor Rosalyn B. Irby Associate Professor of Medicine Chair of Committee Richard J. Courtney Professor Emeritus Chair and Distinguished Educator of Microbiology and Immunology Thomas E. Spratt Associate Professor of Biochemistry and Microbiology Director, IBIOS-Chemical Biology Option Director, Biochemistry and Molecular Biology Program Bruce A. Stanley Director, Section of Research Resources Director, Proteomics and Mass Spectrometry Facility Co-Director, Translational Technologies, Penn State Clinical and Translational Sciences Institute Director, Basic Science Cores, Penn State Hershey Cancer Institute Special Member Charles Lang Vice Chair, Distinguished Professor of Cellular and Molecular Physiology, and Surgery. Director of Molecular Medicine Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT The rare disorder, large granular lymphocyte (LGL) leukemia is characterized by a clonal expansion of cytotoxic cells. Normal LGL are critical to the removal of virally-infected or tumor cells from the body. These cells normally expand when there is an immune challenge and then rapidly undergo apoptosis when the antigen is cleared, but leukemic LGLs are resistant to apoptosis. Serological evidence indicates that LGL leukemia may be driven by chronic infection of an unknown retrovirus. Patients with LGL leukemia frequently experience autoimmune conditions such as rheumatoid arthritis and also experience cytopenias requiring medical intervention. The study of LGL leukemia is therefore important both from a patient standpoint and as a model of normal LGL. We have recently discovered and characterized multiple somatic mutations in exon 20 and 21 of STAT3 from exome sequencing of an LGL leukemia patient and confirmed this mutation to be present in both Natural Killer and T cell leukemias. A small percentage was also found to have mutations in STAT5B, with some associated with an aggressive phenotype. Concurrently, we have undertaken whole genome sequencing to determine what other mutations may contribute to LGL leukemia and to expand our knowledge of STAT3 activation. Given the antigen driven nature of LGL leukemia, it is unclear what mutations are necessary for their lack of apoptosis and whether these will resemble those seen in other leukemias. The current treatment of choice for LGL leukemia is immunosuppressive methotrexate. Firstly, we present the first large prospective trial of immunosuppressive therapy in LGL leukemia. In this trial we identify a correlation between a particular Y640F mutation in STAT3 and a favorable response to first line treatment with methotrexate. We measure and report 27 serum cytokines collected for this trial, none of which were a priori predictive of treatment response. Through microarray analysis we identified a gene expression signature indicative of response. Interestingly, this gene signature does not appear to be driven by STAT3 mutation in iv all samples. We propose that there are other methods of STAT activation in these samples and advance a model of how the Y640F mutation enforces response. Secondly, we collaboratively sequenced 6 paired, leukemic/saliva genomes from three LGL patients at a high rate of coverage. Two patient genomes contain a direct STAT3 mutation. In the third patient we observe a mutation in the 3' UTR of the IL6R that could activate JAK- STAT signaling if it is shown to regulate translation. Mutations in the histone modifiers, MLL2, MLL3 and NCOR1 are supported in the sequence reads of all three genomes. Alterations in this pathway would be a new observation for LGL leukemia. We identify and catalog numerous other mutations which may give insight into LGL biology that could allow us to better understand how symptoms arise and differ between patients and why current treatments may fail. Lastly, we present data identifying important cis-regulatory modules in LGL leukemia gathered through ChIP-Seq of genomic regions occupied by the master regulator TBET. We report evidence of binding in the promoter region of numerous constituents of lytic granules which may indicate these are important downstream effectors of TBET. Overlap between the patients used for genome work above and ChIP-Seq is allowing us to determine whether mutations can have an effect on TBET binding and subsequent transcription of nearby genes. As we collect the footprints of additional factors, this holds the promise of elucidating the potential impact of the tens of thousands of non-coding mutations observed in these samples. In summary, we present the results of large collaborative efforts to sequence mutations in LGL leukemia and determine their effect on LGL biology. We advance the knowledge of STAT3 mutations in LGL biology while identifying and stratifying other mutations for the future study of their potential impacts. Bioinformatic methods of analysis were focused on publicly available resources such as Galaxy for which new tools, file formats, and workflows were developed. This allows easy reproduction of methods by other groups and facilitates visualization and future analysis by our own. v TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... ix Abbreviations ........................................................................................................................... x Acknowledgements .................................................................................................................. xiii Chapter 1 Introduction to LGL Leukemia ............................................................................... 1 Clinical Entity .................................................................................................................. 1 Diagnosis and Symptoms ................................................................................................. 1 Treatment ......................................................................................................................... 3 Natural Born Killers ......................................................................................................... 3 LGL Are Resistant to Apoptosis ...................................................................................... 4 LGL Are Terminal Effector Memory Cells. .................................................................... 5 Viral Antigen.................................................................................................................... 6 Survival Network of LGL Leukemia ............................................................................... 8 STAT3 Dysregulation and Somatic Mutation .................................................................. 10 Chapter 2 STAT3 Y640F Mutation Predicts Response to Immunosuppressive Therapy of LGL Leukemia: Results of a Prospective Multicenter Phase II Study of Initial Treatment with Methotrexate by the Eastern Cooperative Oncology Group (E5998) ..... 14 Introduction ...................................................................................................................... 14 Methods ............................................................................................................................ 15 Eligibility Criteria .................................................................................................... 15 Objectives ................................................................................................................. 16 Study Design ............................................................................................................ 16 Response Criteria ..................................................................................................... 17 Statistical Methods ................................................................................................... 18 Array Data ................................................................................................................ 18 Cytokine Studies ...................................................................................................... 20 STAT3 Mutational Analysis. ................................................................................... 21 STAT3 Mutagenesis ................................................................................................. 22 STAT3 Reporter Assay ............................................................................................ 22 Western Blot ............................................................................................................. 22 Results .............................................................................................................................. 23 Demographics .........................................................................................................

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    158 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us