Phenotypic Screening of Parthenolide Derivatives Reveals The

Phenotypic Screening of Parthenolide Derivatives Reveals The

PHENOTYPIC SCREENING OF PARTHENOLIDE DERIVATIVES REVEALS THE CHEMOPROTECTIVE ROLE OF GALECTIN-1 IN ACUTE MYELOID LEUKEMIA by JESSICA NICOLE PONDER B.S. Truman State University, 2010 M.S. University of Colorado, 2013 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Toxicology Program 2018 This thesis for the Doctor of Philosophy degree by Jessica Nicole Ponder has been approved for the Toxicology Program by David Ross, Chair Craig Jordan, Advisor Peter Crooks Brad Bendiak Kristopher Fritz Date: May 18, 2018 ii Ponder, Jessica Nicole Ph.D., Toxicology Phenotypic Screening of Parthenolide Derivatives Reveals the Chemoprotective Role of Galectin-1 in Acute Myeloid Leukemia Thesis directed by Professor of Medicine Craig T. Jordan ABSTRACT Despite decades of investigation, the diagnosis of acute myeloid leukemia still carries with it a bleak prognosis, primarily as a result of the failure of clinical chemotherapy to target the leukemic stem cell population. In order to overcome this obstacle a library of derivatives of the small molecule parthenolide, a leukemic stem cell targeting natural product, were screened against acute myeloid leukemia (AML). Using the M9-ENL cell line as a high-throughput screening model for leukemic stem cells, over four hundred novel parthenolide derivatives were screened by flow cytometry for apoptotic activity. Using this method, more than forty compounds with improved efficacy against primary AML were identified, but the most remarkable compounds were those that were dimers of melampomagnolide B (MMB dimers), which had virtually no measurable toxicity to healthy blood stem and progenitor cells. Despite their dramatic improvement in therapeutic index, these derivatives were only weakly associated with the leukemic stem cell targeting mechanism of the parent compound. Pharmacological investigation utilizing a chemoproteomic approach reveals that, unlike parthenolide, these bivalent MMB dimers potently and rapidly induce the depletion of nuclear galectin-1, a chemoprotective protein that is found to be overexpressed by more than two orders of magnitude in leukemic stem cells at diagnosis and relapse. In primary AML, nuclear monomeric galectin-1 is depleted within fifteen minutes of exposure to a 4 µM dose of MMB dimer JVM 3-88A. This discovery is significant both because galectin-1 has not previously been iii demonstrated to play a role in acute myeloid leukemia, and because to date, no clinical inhibitors of this emerging cancer target yet exist. Future investigations will seek to further understand the mechanism by which galectin-1 protects leukemic stem cells from apoptosis and how these compounds are able to alter its nuclear localization. The form and content of this abstract are approved. I recommend its publication. Approved: Craig T. Jordan iv Jai Guru Deva v ACKNOWLEDGEMENTS The author would like to acknowledge Shanshan Pei, Nabilah Khan, Mohd Minhajuddin, and Biniam Adane of the Jordan lab for their invaluable contributions to this work, intellectually and materially. Venumadhav Janganati, Bommagani Shobanbabu, and Narsimha Reddy Penthala, led by Peter Crooks at University of Arkansas for Medical Science, deserve the clear majority of credit for synthesis of the small molecules screened, as well as labeled small molecules used for chemoproteomic target identification. A few compounds synthesized by Suresh Kuarm Bowroju and Soma Shekar Dachavaram are also included and compound initials (i.e. JVM, BS, PNR, BSK, or DSS) indicate the identity of the organic chemist for each molecule. From the very first chapter and throughout the body of the text, the author hopes to have made clear to the reader the significance of the contributions of these synthetic chemists to this project. Additional credit goes to Monika Dzieciatkowska, of the University of Colorado AMC Proteomic Mass Spectrometry Core and Michael Becker of the University of Rochester. Specific experimental credits are embedded within the text where appropriate. Additionally, this work was made possible by the financial contributions of the National Institutes of Health R01CA158275. Additional funds were provided through the generous contribution of the UC Denver Anschutz Medical Campus Graduate School, in no small part due to recommendations by former Dean Barry Shur and professors Brad Bendiak and Vasilis Vasiliou. The author is grateful for this financial support and the endorsement implied, without which this work would surely not have been possible. Ultimately, it would be an indefensible oversight to neglect the leukemia patients and clinical research team responsible for maintaining the primary sample tissue bank used in this project. Without the willingness of patients and the dedication of physicians, nurses, and other vi clinical and research staff to provide these samples and care for them, in many cases long after the donor has deceased, as researchers we would not have the ability to truly understand this tragic disease and how to fight it, no matter how advanced our technological resources. So, it is with humble but sincere gratitude that the author would like to recognize the patients as well as the clinical and research staff who provide this priceless resource. Only by partnering together will we ultimately be able to develop a cure for cancer. vii TABLE OF CONTENTS CHAPTER I. ACUTE MYELOID LEUKEMIA: CHALLENGES AND OPPORTUNITIES ......................... 1 Introduction ................................................................................................................................. 1 The Challenge of Acute Myeloid Leukemia ............................................................................... 4 Clinical acute myeloid leukemia ............................................................................................. 4 Leukemic stem cells ................................................................................................................ 6 The Opportunity of Parthenolide................................................................................................. 8 Summary ................................................................................................................................... 10 II. HIGH-THROUGHPUT SCREENING OF PARTHENOLIDE DERIVATIVES ................... 11 High-Throughput Screening Platform ....................................................................................... 11 High-Throughput Screening Results ......................................................................................... 12 Dose-response curves and structures of derivatives .............................................................. 12 Quantitative results ................................................................................................................ 14 Discussion ................................................................................................................................. 14 Quantitative structure-activity relationships .......................................................................... 14 Simple combinations ......................................................................................................... 17 Trans-annular cyclized (TAC) derivatives ........................................................................ 18 C13-substituted (C13) derivatives .................................................................................... 18 C14-substituted (C14) derivatives .................................................................................... 18 Dimer derivatives .............................................................................................................. 26 viii Conclusions ............................................................................................................................... 29 Inactive modifications ........................................................................................................... 29 Potent modifications .............................................................................................................. 30 Methods ..................................................................................................................................... 30 Library handling and storage ................................................................................................. 30 M9-ENL cell culture .............................................................................................................. 30 Apoptosis assay ..................................................................................................................... 31 Quantitative results ................................................................................................................ 31 III. DISCOVERY OF AML-SELECTIVE MMB DIMERS ........................................................ 33 Therapeutic Index Screening ..................................................................................................... 33 LSC-Selective Mechanism of Action ........................................................................................ 34 Therapeutic Index Screening Results ........................................................................................ 35 Primary acute myeloid leukemia ........................................................................................... 35 Determination of therapeutic

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