Inhibition of Monocarboxylate Transporter 1 by AZD3965 as a therapeutic approach in oncology Richard A Noble Thesis submitted in partial fulfilment of the requirements of the regulation for Doctor of Philosophy Newcastle University Faculty of Medical Science Graduate School Northern Institute for Cancer Research September 2016 Abstract Many tumours display a metabolic phenotype distinct from non‐malignant cells, with an increased reliance on glycolysis. This results in a greater production of lactate even under aerobic conditions. Lactate efflux is facilitated by monocarboxylate transporters 1‐4 (MCT1‐4) and is essential to maintain energy homeostasis. A sub‐group of cancers express only MCT1 and are therefore exclusively reliant on this transporter to export lactate. Inhibition of MCT1 has been proposed as a therapeutic approach to prevent lactate export in tumour cells with low MCT4. In this thesis, inhibition of monocarboxylate transporter 1 (MCT1) was investigated using the oral MCT1 inhibitor AZD3965 which is currently undergoing phase I clinical development. Low MCT4 expression was found to be a common characteristic of Burkitt lymphoma (BL) and Diffuse Large B‐cell Lymphoma (DLBCL) in immortalised cell lines and patient samples. In cell line models AZD3956 treatment caused a rapid accumulation of intracellular lactate and altered cellular metabolite profiles consistent with feedback inhibition of glycolysis including an increase in TCA cycle intermediates. A substantial growth inhibitory response was observed in vitro in BL and DLBCL cell lines and also in an in vivo model of BL following daily oral AZD3965 treatment. The combination with a mitochondrial Complex I inhibitor, BAY 87‐2243, triggered significant lymphoma cell death in vitro and also reduced disease burden in vivo. This work supports the use of AZD3965 in the treatment of lymphoma patients who have become refractory to standard therapy but also highlights the potential need for combination strategies to optimally target the altered tumour metabolic phenotype. i ii Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor Steve Wedge for his continuous support of my research, for his patience allowing me to pursue my own ideas, his motivation and insight. Also, my industrial supervisor Susan Critchlow (AstraZeneca) for supporting my placement at Alderley Park which was a great experience and has contributed substantially to this work, and my progress review panel Chris Bacon and Ross Maxwell for their invaluable feedback throughout. I would like to acknowledge all the collaborators who have contributed to the project especially Natalie Bell who was key to generating in vivo data. I’d also like to thank Noel Edwards who introduced me to the lab and trained me in a number of techniques used in this thesis. I’m very grateful to the other members of the lab and the Drug Discovery team who have provided technical advice, camaraderie, a stimulating and fun environment to work in, and cake. A special mention to my friends and family outside the lab especially to my parents who have always given me their fullest support. Finally, I would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca for funding this CASE studentship and to the British Association for Cancer Research (BACR) for funding a travel grant to present some of this work. iii iv Contents Abstract ........................................................................................................................................ i Acknowledgements ................................................................................................................... iii List of Figures and Tables ........................................................................................................... xi List of Abbreviations ................................................................................................................ xiv Chapter 1. Introduction .......................................................................................................... 1 1.1. Cellular metabolism ..................................................................................................... 1 1.1.1. Glycolysis .............................................................................................................. 1 1.1.2. Oxidative phosphorylation ................................................................................... 3 1.2. Reprogramming energy metabolism ‐ An emerging hallmark of cancer ..................... 6 1.2.1. The Warburg effect ............................................................................................... 6 1.2.2. Regulation of cellular metabolism in cancer ........................................................ 8 1.3. Therapeutic targeting of tumour metabolism ........................................................... 11 1.3.1. Nucleotide biosynthesis ...................................................................................... 13 1.3.2. Amino acid metabolism ...................................................................................... 13 1.3.3. Oxidative phosphorylation ................................................................................. 14 1.3.4. Glycolysis ............................................................................................................ 14 1.4. Monocarboxylate transporter 1 ................................................................................. 16 1.4.1. Structure ............................................................................................................. 16 1.4.2. Function of monocarboxylate transporters 1‐4 ................................................. 16 1.4.3. Mechanism of lactate transport ......................................................................... 18 1.4.4. Regulation ........................................................................................................... 18 1.5. MCT1 as a therapeutic target in cancer ..................................................................... 20 1.5.1. Differential expression of MCTs in cancer .......................................................... 20 1.5.2. Lactate shuttle hypothesis .................................................................................. 21 1.5.3. MCT1 inhibition in in vitro and in vivo cancer models ....................................... 21 v 1.6. AZD3965: A potent, selective orally‐bioavailable MCT1 inhibitor ............................ 36 1.6.1. Development ...................................................................................................... 36 1.6.2. Selectivity ........................................................................................................... 36 1.6.3. Structure ............................................................................................................ 37 1.6.4. Mode of binding ................................................................................................. 39 1.6.5. Safety .................................................................................................................. 39 1.7. Project objectives ...................................................................................................... 40 Chapter 2. Materials and methods ...................................................................................... 42 2.1. Preparation of AZD3965 and BAY 87‐2243 ................................................................ 43 2.2. Cell culture ................................................................................................................. 43 2.2.1. Subculturing cells ............................................................................................... 45 2.2.2. Cryopreservation ................................................................................................ 45 2.3. Protein extraction and quantitation .......................................................................... 45 2.3.1. Protein extraction .............................................................................................. 45 2.3.2. Protein quantitation – BCA protein assay .......................................................... 45 2.4. SDS‐Page and Western Blot ....................................................................................... 46 2.4.1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) ......... 46 2.4.2. Western Blot and ECL detection ........................................................................ 46 2.5. In vitro and ex vivo lactate quantification ................................................................. 48 2.5.1. Background principles ........................................................................................ 48 2.5.2. In vitro sample preparation ................................................................................ 48 2.5.3. Ex vivo sample preparation ................................................................................ 49 2.5.4. Assay................................................................................................................... 49 2.5.5. Data analysis ...................................................................................................... 50 2.6. Cell viability assays .................................................................................................... 50 2.6.1. XTT assay ...........................................................................................................