Biology of Type 2 Phosphatidylinositol-5- Phosphate 4-Kinase The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Shim, Hyeseok. 2015. Biology of Type 2 Phosphatidylinositol-5- Phosphate 4-Kinase. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845419 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 Biology of type 2 phosphatidylinositol-5-phosphate 4-kinase A dissertation presented by Hyeseok Shim to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts August 2015 © 2015 Hyeseok Shim All rights reserved Professor Lewis C. Cantley Hyeseok Shim Biology of type 2 phosphatidylinositol-5-phosphate 4-kinase Abstract Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three genes, PIP4K2A, PIP4K2B and PIP4K2C that encode the enzymes PI5P4Kα, PI5P4Kβ and PI5P4Kγ respectively. Studies in mice showed that PI5P4Kβ is a negative regulator of insulin signaling (Lamia et al., 2004) and that co-deletion of Pip4k2b and Trp53 resulted in synthetic embryonic lethality (Emerling et al., 2013). Also, deletion of two alleles of Pip4k2a and one allele of Pip4k2b suppressed the appearance of tumors in Trp53-/- mice. These studies suggest that drugs targeting PI5P4Kα and β could be effective therapies for treating insulin resistance, type 2 diabetes and TP53 mutant cancers. While less is known about PI5P4Kγ, several genome-wide association studies have revealed a SNP in front of the PIP4K2C at an autoimmunity susceptibility loci (Raychaudhuri et al., 2008). To evaluate the role of PI5P4Kγ, I generated Pip4k2c-/- mice and found an inflammatory phenotype with increased tissue immune infiltrates and pro-inflammatory cytokines, correlating with increased helper T cells and decreased regulatory T cells. Also, Pip4k2c-/- mice exhibited upregulated mammalian target of rapamycin complex 1 (mTORC1) signaling in tissues and rapamycin treatment reduced the inflammation of these mice. These studies support the concept that the SNP identified at the PIP4K2C iii locus in human patients with autoimmunity contributes to disease by reducing expression of PI5P4Kγ and indicates that inhibition of mTORC1 would be beneficial to these patients. Finally, in collaboration with Dr. Nathanael Gray’s laboratory we identified small molecules that covalently react with PI5P4Ks and thereby cause irreversible inhibition. These compounds, PIP4Kin1 and PIP4Kin2 mimicked the effect of shRNA mediated knockdown or knockout of PI5P4Kα and PI5P4Kβ, and impaired the growth of several TP53 mutant cancer cell lines, with little effect on most TP53 wild type cell lines. Utilizing the xenograft tumor model with BT474 (TP53 mutant) and MCF7 (TP53 wild type) cells, we showed that daily treatment of the mice with PIP4Kin2 inhibited the growth of the BT474 tumors but not the MCF7 tumors, without causing any obvious toxicity. These results further validate PI5P4Kα and PI5P4Kβ as targets for treating TP53 mutant cancers. iv Table of Contents Title i Abstract iii Table of Contents v Acknowledgements vi List of Tables viii List of Figures ix List of Abbreviations xii Chapter 1: Introduction 1 Chapter 2: Deletion of a Novel Phosphatidylinositol Kinase results in 14 Hyperactivation of the Immune System Chapter 3: Identification of PI5P4K inhibitors 46 Chapter 4: Summary and Future Directions 85 Appendix A: Depletion of a Putatively Druggable Class of 93 Phosphatidylinositol Kinases Inhibits Growth of p53-Null Tumors Bibliography 137 Supplemental Materials 143 v Acknowledgments First of all, I am deeply grateful to my advisor, Dr. Lewis C. Cantley, for mentoring my graduate study. His unlimited support, guidance and confidence encouraged me to move forward and complete all the steps required for achieving a Ph.D degree. I would like to thank my thesis committee, Drs. Karen Cichowski, John Blenis and Alex Toker for giving valuable comments and suggestions on my thesis. I thank Drs. Nathanael Gray and Tinghu Zhang for collaborating with me on developing PI5P4K inhibitors and Drs. Vijay Kuchroo and Chuan Wu on analyzing T cells in Pip4k2c -/- mice. I thank Drs. Sirano Dhe-Paganon and Hyuk- Soo Seo for crystalizing PI5P4Kβ with PIP4Kin2 and Drs. Scott Ficarro and Jarrod Marto for identifying modified residues of PI5P4K using mass spectrometry. I thank members of Cantley laboratory. I thank Jihye Yun and Kaitlyn Bosch for helping me on rapamycin treatment of Pip4k2c -/- mice, Yuxiang Zheng for performing HPLC to measure phospholipids and Gina DeNicola for measuring ROS in cells. I give my thanks to Shivan Ramsamooj for culturing tissues and performing western blots and Zhiwei Yang and Rayman Choo-Wing for mouse genotyping. I also thank Brooke Emerling, Atsuo Sasaki, Hui Liu, Edouard Mullarky, Jared Johnson, Florian Karreth, Jonathan Yang and Diana Wang for all their helpful suggestions and support during my PhD study. vi Finally, I would like to thank my parents, Hyunho Shim and Dahye Lee for giving me encouragement and financial help. I thank Pom and Kumdong for staying with me. Above all, I am truly indebted to my husband, Young Kwon for his endless support and encouragement in both life and science. vii List of Tables Table 3.1 Metabolic pathways affect by PIP4Kin2 57-58 viii List of Figures Figure 1.1 Compartmentalization of phosphoinositide pathways 2-3 Figure 1.2 Working Model for the role of PI5P4Kγ in immune regulation 10 Figure 2.1 Generation of Pip4k2c-/- mice 19 Figure 2.2 Validation of Pip4k2c-/- mice 20 Figure 2.3 Immune cell infiltration is increased in tissues of Pip4k2c-/- 22 mice Figure 2.4 Immune cell infiltration in the liver of Pip4k2c-/- mice 23 Figure 2.5 Immune infiltrates in liver tissue of Pip4k2c-/- mice are mostly 23 T cells and B cells Figure 2.6 Pro-inflammatory cytokines are increased in Pip4k2c-/- mice 24 Figure 2.7 T cell derived IFNγ and IL-17 levels are elevated in Pip4k2c- 25 /- mice Figure 2.8 Plasma IgG3 levels are elevated in Pip4k2c-/- mice 26 Figure 2.9 Pip4k2c-/- mice exhibit an increase in CD44+ active T cells 27 and a decrease in CD62L+ naïve T cells Figure 2.10 CD4+ and CD8+ T cells are elevated in the spleen of 27 Pip4k2c-/- mice Figure 2.11 T cells from Pip4k2c-/- mouse have enhanced growth rates 28 Figure 2.12 Regulatory T cells are suppressed in Pip4k2c-/- mice 28 Figure 2.13 Signaling downstream of mTORC1 is upregulated in various 30 ix tissues of Pip4k2c-/- mice Figure 2.14 Signaling downstream of mTORC1 is upregulated in the 31 spleen of Pip4k2c-/- mice Figure 2.15 Rapamycin reduces mTORC1 signaling in Pip4k2c-/- mice 33 Figure 2.16 Changes in plasma cytokine levels after treatment with 34 rapamycin Figure 2.17 Changes in immune cell infiltration after treatment with 35 rapamycin Figure 2.18 Model for the role of PI5P4Kγ in regulation of immunity 36 Figure 3.1 Structures of PIP4Kin1 and PIP4Kin2 50 Figure 3.2 PIP4Kin1 and PIP4Kin2 inhibit the kinase activity of 51 PI5P4Ks Figure 3.3 PIP4Kin1 and PIP4Kin2 inhibit proliferation of BT474 cells 52 but not MCF7 cells Figure 3.4 PIP4Kin1 and PIP4Kin2 inhibit proliferation of BT474 cells 53 even after washout Figure 3.5 Reducing the temperature of the culture media to 32 oC, 55 where mutant p53 in BT474 cells is partially functional, partially rescues PIP4Kin1/2 mediated inhibition of cell growth Figure 3.6 PIP4Kin1 enhances Insulin-dependent activation of Akt 56 Figure 3.7 A biotin-PIP4Kin1 pull down assay indicates that PIP4Kin1 60 x and PIP4Kin2 are covalent inhibitors of the PI5P4K enzymes Figure 3.8 Mapping PI5P4Kβ cysteine residues targeted by PIP4Kin1 62 Figure 3.9 Co-crystal of PI5P4Kβ and PIP4Kin2 63-65 Figure 3.10 The half-life of PIP4Kin2 in serum following a single i.v. 66 injection is 9.63 hours Figure 3.11 Maximum tolerance dose test with PIP4Kin2 67 Figure 3.12 PIP4Kin2 has significant antitumor activity in an orthotopic 69-70 BT474 xenograft model Figure 3.13 In vivo target engagement of PIP4Kin2 72 Figure 4.1 Overexpressing PI5P4Kβ mutant (C307S C318S) partially 89-90 rescues the impaired growth of BT474 cells by PIP4Kin2 xi List of Abbreviations PI5P4K Type 2 phosphatidylinositol-5-phosphate 4-kinase mTOR Mammalian target of rapamycin mTORC1 Mammalian target of rapamycin complex 1 p53 Tumor protein p53 Akt Protein kinase B PI3K Phosphoinositide 3-kinase PIKFYVE FYVE finger-containing phosphoinositide 5-kinase PI4P5K Type 1 phosphatidylinositol-4-phosphate 5-kinase p70-S6K p70- ribosomal protein S6 kinase SREBP1 Sterol regulatory element-binding protein 1 PI-5-P Phosphatidylinositol-5-phosphate PI-4-P Phosphatidylinositol-4-phosphate PI-4,5-P2 Phosphatidylinositol-4,5-bisphosphate SNP Single nucleotide polymorphism Th Helper T cell Treg Regulatory T cell i.v. Intravenous p.o. Oral gavage i.p. Intraperitoneal xii Chapter 1. Introduction In 1997, Rameh et al. discovered that a family of enzymes that was thought to be phosphatidylinositol-4-phosphate 5-kinases (PI4P5Ks) were actually phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks). In other words, rather than producing phosphatidylinositol-4,5-P2 (PI-4,5-P2) from phosphatidylinositol-4- phosphate (PI-4-P), as had been thought for the previous 8 years, these enzymes were utilizing an impurity in bovine brain-derived PI-4-P, phosphatidylinositol-5- phosphate (PI-5-P) as a substrate to generate PI-4,5-P2 (Figure 1.1).