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VALIDATION of the PRE-B CELL RECEPTOR AS a THERAPEUTIC TARGET in B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA Michael F

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VALIDATION of the PRE-B CELL RECEPTOR AS a THERAPEUTIC TARGET in B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA Michael F University of New Mexico UNM Digital Repository Biomedical Sciences ETDs Electronic Theses and Dissertations Spring 4-7-2017 VALIDATION OF THE PRE-B CELL RECEPTOR AS A THERAPEUTIC TARGET IN B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA Michael F. Erasmus University of New Mexico Medical School Follow this and additional works at: https://digitalrepository.unm.edu/biom_etds Part of the Bioinformatics Commons, Biophysics Commons, Biotechnology Commons, Immunology of Infectious Disease Commons, Medical Biotechnology Commons, Medical Cell Biology Commons, Medical Immunology Commons, Molecular Biology Commons, and the Structural Biology Commons Recommended Citation Erasmus, Michael F.. "VALIDATION OF THE PRE-B CELL RECEPTOR AS A THERAPEUTIC TARGET IN B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA." (2017). https://digitalrepository.unm.edu/biom_etds/162 This Thesis is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Biomedical Sciences ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. i M. FRANK ERASMUS Candidate BIOMEDICAL SCIENCES - PATHOLOGY Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: BRIDGET S. WILSON, Ph.D. , Chairperson DIANE S. LIDKE, Ph.D. GNANA S. GNANAKARAN, Ph.D. STUART S. WINTER, M.D. ii VALIDATION OF THE PRE-B CELL RECEPTOR AS A THERAPEUTIC TARGET IN B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA by M. FRANK ERASMUS B.S. Biological Sciences, California Polytechnic, USA, 2008 M.S. Molecular & Cell Biology, San Diego State Univ., USA, 2012 DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biomedical Sciences The University of New Mexico Albuquerque, New Mexico May 2017 iii DEDICATION To my amazing wife and love of my life, Evan Feldman, for her relentless support and patience through the ups-and-downs of this process. I could not have been able to get through this without her commitment and belief in my ability to achieve this degree. To my loving mother-in-law and father-in-law, Lori Brody and Fabrizio Giovannini, for their unconditional love and support. And I dedicate this dissertation to my grandfather-in-law Edmond Brody for the reminding me of the importance of hard work and positive thinking as the means to a happy life. iv ACKNOWLEDGEMENTS To my incredible mentor, Dr. Bridget Wilson. Her dedication and drive throughout this process was truly inspirational. Thank you for continually pushing me through every step of the way. I truly appreciate all of your constructive criticism that fostered my skills as a scientist. There were many memorable moments over the years and I could not have asked for a better mentor. It is an honor to have been your graduate student. I knew from the get-go this was a very intriguing idea. Thank you for taking me on for the ride. To my dissertation committee. Dr. Diane Lidke, for her guidance on a large portion of this project. I have always been intrigued by your incredible work, so it was a true inspiration to learn from you over the years. All of your advice has been essential to the progress of these projects. Thank you for being a great mentor and critical leader. Dr. Gnanakaran, it was a tremendous privilege to work with you up at Los Alamos and to be exposed to all of the exciting research that you are doing. I am very appreciative for always keeping me in the loop to fascinating discussions and cutting-edge science. The ability to engage with you and your postdocs was very enlightening. I took a lot away from this experience, especially over the past year. I look forward to continuing to incorporate theory into my work due to this exhilarating experience. Dr. Winter, I cannot thank you enough for all of our talks and your support through the years. The ‘B Cells Gone Bad’ group was essential in keeping the clinical component a central feature of this project. I am very grateful to have had the opportunity to work with you over the past four years. From this experience I will always be reminded to keep a translational component in mind. v VALIDATION OF THE PRE-B CELL RECEPTOR AS A THERAPEUTIC TARGET IN B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA by M. FRANK ERASMUS B.S., Biological Sciences, California Polytechnic St. University, USA, 2008 M.S., Molecular & Cell Biology, San Diego St. University, USA, 2012 Ph.D., Biomedical Sciences, University of New Mexico, USA, 2017 ABSTRACT This dissertation is built upon the fundamental idea that the pre-B cell receptor (pre-BCR) is important to leukemia cell survival and a logical therapeutic target in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The pre-BCR is expressed early at a specific stage during B cell development where it plays a central role in survival of healthy B lymphocytes. This receptor is composed of the membrane heavy chain (mIgµ) associated with surrogate light chain components, 5 and VpreB. Through the use of advanced imaging modalities, in particular two-color single particle tracking (SPT), we showed that pre-BCRs formed transient, homotypic interactions. These receptors displayed correlated motion at short separation distances that is consistent with the formation of dimers. These encounters were sufficient to generate an autonomous signal that helped to maintain leukemia blast survival. Incubation of the BCP-ALL cells with galectin-1, which can bind both 5 and glycans, led to aggregate formation that drastically slowed down the diffusion of pre-BCRs. This correlated to a downregulation of pre-BCR signaling through proximal Syk activation. Blocking galectin-1’s lectin-binding capability with saturating concentrations of -lactose partially restored diffusion to “tonic” levels. The introduction of this sugar also impacted the downstream signaling profile. Due to these differential vi diffusion and signaling profiles observed in experimental procedures we turned computational biology to gain additional structural insight. Molecular dynamics (MD) simulations supported the idea that the pre-BCR can allosterically control galectin-1’s binding capacity at its distal carbohydrate-binding site (CBS). We generated a monovalent Fab against the dimer interface of the pre-BCR using phage display technologies and showed that it not only could disrupt dimerization but also disrupted downstream signaling. Due to recent clinical advances in chimeric antigen receptor (CAR) T cell therapy, we reformatted our Fab into a scFv, the binding region of the CAR, using genetic cloning techniques. Through the use of computer-aided design we successfully generated a scFv with high affinity to the VpreB target. This antibody binding fragment was cloned into a lentiviral vector containing the CD28 co-stimulatory domain and immunoreceptor tyrosine-based activation motif (ITAM)-containing TCR-CD3. After efficient transduction of primary CD8+ T cells, we have shown efficient in vitro efficacy against BCP-ALL cells. vii Table of Contents CHAPTER 1: INTRODUCTION ................................................................. 1 Model system: B cell precursor acute lymphoblastic leukemia .............................................. 1 The pre-BCR checkpoint in B cell development ....................................................................... 2 Technical Approach I: Single Particle Tracking......................................................................... 5 Insights into pre-BCR Homotypic Interactions through Quantitative Imaging ........................ 8 Galectin, a pre-BCR crosslinking agent .................................................................................. 10 Technical Approach II: Molecular Dynamics .......................................................................... 12 MD Simulations Reveal Allosteric Control of Galectin-1 by the pre-BCR .............................. 16 Small molecule and antibody-based therapeutics for leukemia ........................................... 17 The design and promise of CAR T cell therapy ...................................................... 17 Chapter 2: PRE-BCR DIMER – SPT......................................................... 22 ABSTRACT ............................................................................................................................... 22 INTRODUCTION ...................................................................................................................... 24 MATERIALS AND METHODS ................................................................................................... 26 RESULTS ................................................................................................................................. 32 DISCUSSION ............................................................................................................................ 56 CHAPTER 3: GALECTIN-1 - MD ............................................................ 63 ABSTRACT ............................................................................................................................... 63 INTRODUCTION ...................................................................................................................... 65 METHODS ............................................................................................................................... 67 RESULTS ................................................................................................................................
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