Inhibition of Metabolic Enzymes As Differentiation Therapy in Acute Myeloid Leukemia
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Inhibition of Metabolic Enzymes as Differentiation Therapy in Acute Myeloid Leukemia The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37945013 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Inhibition of Metabolic Enzymes as Differentiation Therapy in Acute Myeloid Leukemia A dissertation presented by Jason Michael Law to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts October, 2016 © 2016 Jason Michael Law All rights reserved Dissertation Advisor: Professor Stuart L. Schreiber Jason Michael Law Inhibition of Metabolic Enzymes as Differentiation Therapy in Acute Myeloid Leukemia Abstract Acute myeloid leukemia (AML) is among the deadliest of cancers: it carries a 27% 5-year survival rate and is responsible for over 10,000 deaths annually in the United States alone. Despite the clear medical need, new therapeutics effective in most AML patients have not been developed in forty years, and cytotoxic chemotherapy remains the typical treatment. Fortunately, advances in the understanding of AML biology have the potential to lead to new treatments. A hallmark of AML is the differentiation block: cancer cells are halted at an early phase of hematopoietic development in which they are programmed to proliferate rapidly. In a rare subset of AML—acute promyelocytic leukemia—a genetically targeted therapy is able to overcome the differentiation block, leading to complete remissions in over 90% of patients. In this work, I describe two projects, utilizing different strategies, aimed at the development of differentiation therapy agents effective in AML more broadly. First, I describe how frequent mutations of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) found in AML tumors inspired me to design and run a target-based compound screen with the aim of developing a chemical probe selective for the mutant allele of IDH1. The result of this effort is BRD2879, a validated inhibitor of the IDH1-R132H mutant enzyme. The compound is active in cells and possesses a structure markedly different from other known inhibitors. Second, I describe a phenotypic, cell-based screen for AML differentiation, and the target identification and validation experiments leading to the iii surprising discovery that inhibition of dihydroorotate dehydrogenase (DHODH)—another metabolic enzyme—reliably induces differentiation across many models of AML. I also describe my efforts to uncover a biochemical rationale for why DHODH inhibition leads to differentiation. Together, the outcomes of the two projects suggest a profound relationship between a cell’s metabolic state and its differentiation state, with the implication that modulation of a cell’s metabolism might be exploited to cause therapeutically beneficial differentiation in diseases like AML. iv Table of Contents Acknowledgements .............................................................................................................................. viii List of Abbreviations .............................................................................................................................. xii Chapter 1 – Approaches to Differentiation Therapy in Acute Myeloid Leukemia 1 1.1 – Treatment of acute myeloid leukemia is an unmet medical need ................................................ 2 1.2 – AML is a disease of impaired differentiation ................................................................................. 2 1.3 – Differentiation therapy is highly effective in APL .......................................................................... 3 1.4 – Targeted vs. phenotypic screening ................................................................................................ 5 1.5 – Many AML tumors harbor mutations in IDH1 or IDH2 .................................................................. 6 1.6 – Most AML tumors are driven by overexpression of HoxA9 .......................................................... 9 1.7 – Two approaches to differentiation therapy ................................................................................ 10 1.8 – References ................................................................................................................................... 11 Chapter 2 – Development of a Biologically Relevant Assay for IDH1 Inhibition 16 2.1 – The importance of a biologically relevant assay ......................................................................... 17 2.2 – Assay design for IDH1-R132H inhibition ...................................................................................... 18 2.3 – The enzymatic assay functions most easily with Mn2+ as a cofactor .......................................... 20 2.4 – Screening identifies BRD5667 as an IDH1-R132H inhibitor ......................................................... 21 2.5 – Synthesis and testing of analogs defines structure-activity relationships for BRD5667 ............. 22 2.6 – BRD5667 and its analogs work poorly in cells ............................................................................. 24 2.7 – Compound activity depends on which metal cofactor is used .................................................... 26 2.8 – Mg2+ is superior to Mn2+ as a cofactor in the IDH1 enzymatic assay........................................... 28 2.9 – All assay conditions were optimized before the final round of screening .................................. 30 2.10 – Experimental methods: biology and biochemistry .................................................................... 31 2.11 – Experimental methods: chemistry, with compound characterization ...................................... 34 2.12 – References ................................................................................................................................. 43 v Chapter 3 – Development of BRD2879, a Cell-Active Inhibitor of Mutant IDH1 44 3.1 – High-throughput screening identifies BRD2879 as a promising lead .......................................... 45 3.2 – Resynthesis of BRD2879 and exploration of structure-activity relationships ............................. 48 3.3 – Validation of BRD2879 specificity through biophysical and cell-based assays ........................... 52 3.4 – Prospects for the use of BRD2879 as a probe compound ........................................................... 57 3.5 – Experimental methods: biology and biochemistry ...................................................................... 57 3.6 – Experimental methods: chemistry, with compound characterization ........................................ 62 3.7 – References ................................................................................................................................... 67 Chapter 4 – Discovery of DHODH as a Target for AML Differentiation 68 4.1 – The role of phenotypic screening in development of differentiation therapy ........................... 69 4.2 – Lysozyme-GFP-ER-HoxA9 cells establish a screenable model for AML differentiation ............... 70 4.3 – A high-throughput screen identifies small-molecule inducers of AML differentiation ............... 73 4.4 – Analysis of resistant cell lines identifies DHODH as the target ................................................... 74 4.5 – Dihydroorotate dehydrogenase is a key metabolic enzyme ....................................................... 76 4.6 – Targeting of DHODH confirmed by in vitro enzyme inhibition assay .......................................... 77 4.7 – Medicinal chemistry leads to ML390, a more potent differentiating agent ............................... 78 4.8 – X-ray crystallography of DHODH with ML390 defines binding ................................................... 82 4.9 – Brequinar, another DHODH inhibitor, is suitable for in vivo studies ........................................... 85 4.10 – Brequinar demonstrates anti-leukemia activity and differentiation in vivo ............................. 86 4.11 – Differentiation is caused by lack of pyrimidine metabolites ..................................................... 88 4.12 – The mechanism of myeloid differentiation in response to uridine deprivation ....................... 90 4.13 – The differentiation effects of DHODH inhibitors are stronger than those of standard chemotherapy ........................................................................................................................... 95 4.14 – The history of DHODH as a therapeutic target .......................................................................... 98 4.15 – The potential of brequinar as differentiation therapy in AML .................................................. 99 4.16 – Experimental methods ............................................................................................................ 101 4.17 – References .............................................................................................................................. 102 vi Chapter 5 – Lessons on Probe Development and the Link Between Metabolism and Differentiation in AML 107 5.1 – Unifying themes and lessons