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Targeting the Oncomicrorna Mir-155 in Acute Myeloid

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Targeting the Oncomicrorna Mir-155 in Acute Myeloid Targeting the oncomicroRNA miR-155 in Acute Myeloid Leukemia with the NEDD8-Activating Enzyme Inhibitor MLN4924 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jihane Khalife Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2015 Dissertation Committee: Ramiro Garzon, MD (advisor) Guido Marcucci, MD Michael Caligiuri, MD Robert Lee, PhD. Copyright by Jihane Khalife 2015 Abstract Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system characterized by maturation arrest and hyperproliferation of clonal myeloid precursors. Standard treatment for AML generally consists of cytarabine/anthracycline-based chemotherapy; however, only a minority of patients is cured with this approach. More intensive treatments, including allogeneic stem cell transplantation, albeit more effective, often result in increased toxicity and treatment-related mortality. Thus, novel therapies targeting are needed. microRNA-155 (miR-155) is a non-coding RNAs frequently deregulated in cancer and leukemia and its upregulation has been associated with more aggressive disease and chemoresistance. In AML, high expression of miR-155 independently predicts poor outcome in cytogenetically normal (CN) patients and is associated with high-risk FLT3 internal tandem duplication (ITD) mutations. Therapeutic silencing of this microRNA might represent a novel strategy to treat aggressive AML. Albeit targeting of miR-155 with anti-miR-based oligonucleotides may represent a more direct approach, these compounds are still in preclinical stage of development. Thus, we sought for an immediate strategy that would allow us to inhibit aberrant miR-155 expression in malignant blasts by pharmacological intervention. In AML, miR-155 is regulated by NF-κB, the activity of which is in part controlled by neddylation. We demonstrate that MLN4924, a neddylation inhibitor presently used in clinical trials, ii decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs survival on mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound already available in the clinic. Since high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients. iii This Thesis is dedicated to my wonderful cousin “Mano” whose soul is resting in peace. “You were my inspiration to search for a cure of a deadly disease. I hope that I could contribute even very slightly to help people who are still suffering. Our Journey isn’t over yet, this is just the beginning, you will keep being my inspiration, you will always be present in my mind and soul, Love you”. Jihane iv Acknowledgments I am first of all grateful to my advisor, Dr. Guido Marcucci, for his care, support, mentorship and his perseverance to always seek for our goals and not to give up. In his own words he said that the path of a scientist is hard work, perseverance without discouragement. I would like to thank my committee members, Drs. Michael Caligiuri, Ramiro Garzon and Bob Lee, for offering their support and guidance throughout the research progress. I would like to acknowledge Dr. Hanna Radomska for her great support and help, without her, this work wouldn’t have been accomplished. I would like to acknowledge former and present Dr. Marcucci‘s lab members, specifically Hongyan Wang, Xiaomeng Huang, Yue-Zhong Wu, John Curfman and Houda El Ashkar for their positive energy in trying to make the lab as harmonious as possible. A specific acknowledgment to Dr. Jason Mendler for his intuitive guidance in starting the project. Finally, I would like to thank my family and friends for their help and support as well as my colleague Christopher Walker for his encouragements and scientific advices. v Vita 2003....................................................B.S. in Biology, Saint Joseph University, Beirut, Lebanon 2006....................................................M.S. in Biology American University of Beirut, Beirut, Lebanon 2010 to present ................Graduate Research Associate, Molecular, Cellular and Developmental Biology, The Ohio State University, OH, US Publications Alachkar H, Santhanam R, Maharry K, Metzeler KH, Huang X, Kohlschmidt J, Mendler JH, Benito JM, Hickey C, Neviani P, Dorrance AM, Anghelina M, Khalife J, Tarighat SS, Volinia S, Whitman SP, Paschka P, Hoellerbauer P, Wu YZ, Han L, Bolon BN, Blum W, Mrózek K, Carroll AJ, Perrotti D, Andreeff M, Caligiuri MA, Konopleva M, Garzon R, Bloomfield CD, Marcucci G. SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome. J Clin Invest. 2014 124(4):1512-24. vi Mendler JH, Maharry K, Becker H, Eisfeld AK, Senter L, Mrózek K, Kohlschmidt J, Metzeler KH, Schwind S, Whitman SP, Khalife J, Caligiuri MA, Klisovic RB, Moore JO, Carter TH, Marcucci G, Bloomfield CD. In rare acute myeloid leukemia patients harboring both RUNX1 and NPM1 mutations, RUNX1 mutations are unusual in structure and present in the germline. Haematologica. 2013 98(8):e92-4. El-Ballouli A, Khnayzera, RS, Khalife JC et al., Diarylpyrenes vs. diaryltetrahydropyrenes: Crystal structures, fluorescence, and upconversion photochemistry. Journal of Photochemistry and Photobiology A: Chemistry 2013, 272: 49– 57. Mendler JH, Maharry K, Radmacher MD, Mrózek K, Becker H, Metzeler KH, Schwind S, Whitman SP, Khalife J, Kohlschmidt J, Nicolet D, Powell BL, Carter TH, Wetzler M, Moore JO, Kolitz JE, Baer MR, Carroll AJ, Larson RA, Caligiuri MA, Marcucci G, Bloomfield CD. RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and MicroRNA expression signatures. J Clin Oncol. 2012 30(25):3109-18. Harakeh S, Diab-Assaf M, Khalife JC, et al., Ascorbic acid induces apoptosis in adult T- cell leukemia. Anticancer Res. 2007 (1A):289-298. vii Harakeh, SM, Diab-Assaf, M, Niedzwiecki, A, Khalife, J et al., Apoptosis Induction by Epican Forte in HTLV-I-positive and -negative Malignant T-Cells. Leukemia Res. 30(7):869-881. Fields of Study Major Field: Molecular, Cellular and Developmental BiologyMolecular, Cellular and Developmental BiologyMolecular, Cellular and Developmental BiologyMolecular, Cellular and Developmental Biology viii Table of Contents Abstract ................................................................................................................... ………ii Dedication .......................................................................................................................... iv Acknowledgments................................................................................................................v Vita ..................................................................................................................................... vi List of Tables…………………………………………………………………………….xii List of Figures………………………………………………………………………......xiii Chapter 1: Background……………………………………………………………………1 1. Acute Myeloid Leukemia (AML)…………………………………………………....….1 1.1.Definition………………………………………………………………………...…....1 1.2.Demographics…………………………………………………………………..……..1 1.3.Etiology ………………………………………………………………………...……..2 1.4. Cytogenetic and Molecular Aberration in AML…………………………………..….3 1.5. Classifications……………………………………………………………………….14 1.6. Clinical Manifestation…………………………………………………………….…20 1.7. Treatment……………………………………………………………………………21 Chapter 2: MicroRNA 155 (miR-155) as a therapeutic target in AML ……………..…..26 2.1. miR-155 biogenesis and function…………………………………………………...26 ix 2.2. Role of miR-155 in cancer and inflammation……………………………………….34 2.3. How is miR-155 regulated?........................................................................................37 2.4. miR-155 as a therapeutic target in cancer…………………………………………...38 2.5. Significance of targeting miR-155 in AML ………………………………………...39 Chapter 3: The neddylation pathway and the importance of targeting it in AML………41 3.1. Description of the Neddylation Pathway……………………………………………41 3.2. The NEDD8 Activating Enzyme Inhibitor: MLN4924……………………………..44 3.2.1 Mechanism of action………………………………………………………...44 3.2.2. Role of MLN4924 in cancer………………………………………………..47 3.2.2.1. Preclinical studies………………………………………………47 3.2.2.2. Clinical Development……………………………………………49 3.3. Therapeutic benefit of using MLN4924 in AML…………………………………...50 Chapter 4: Inhibition of miR-155 by targeting neddylation in AML………………...…51 4.1. Introduction………………………………………………………………………….52 4.2. Materials and Methods……………………………………………………………...52 4.3. Results……………………………………………………………………………….61 4.3.1. Antileukemic effects of inhibiting miR-155 expression in AML cells………61 4.3.2. Treatment with MLN4924 downregulates miR-155 expression……………...71 4.3.3. MLN4924 disrupts binding of NF-κB to the miR-155 promoter………….…..80
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