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Lkb1 Loss Induces Characteristic Pathway Activation in Human LKB1 LOSS INDUCES CHARACTERISTIC PATHWAY ACTIVATION IN HUMAN TUMORS AND CONFERS SENSITIVITY TO MEK INHIBITION ASSOCIATED WITH ATTENUATED PI3K-AKT-FOXO3 SIGNALING By JACOB KAUFMAN Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cancer Biology December, 2013 Nashville, Tennessee Approved: Professor William Pao Professor Alissa M. Weaver Professor Ethan Lee Professor Pierre P. Massion Professor David P. Carbone This work is dedicated, with love, to my amazing wife, Elizabeth and To our three wonderful daughters, Claire, Caroline, and Mary ii!! ACKNOWLEDGMENTS This work would not have been possible without the financial support from Vanderbilt’s Medical Scientist Training Program and the Strategic Partnering to Evaluate Cancer Signatures grant NCI U01CA114771. It is a privilege to have had such resources available to fund my training and education. I am deeply grateful for the incredible support of my mentor, David Carbone. Dr. Carbone has shared not only his inexhaustible knowledge of biology and cancer, but also his dedication to improving the lives of others, and his sincere compassion both for patients and colleagues. Working with him has given me a model of what being a physician scientist should be, and I hope to emulate his character and compassion throughout my career. This work could not have been completed without the help of my co-authors, especially Joe Amann and Paul Park, as well as Horton Li, Rajesh Arasada and Yu Shyr. I have also benefited immeasurably from many faculty members with whom I’ve had the honor of working and interacting, especially Yu Shyr, Pierre Massion and David Johnson, and my committee members William Pao, Alissa Weaver, and Ethan Lee. Thank you all for everything you have done for me. Most of all, I am forever indebted to my family for their constant love, support, and encouragement. My parents have supported me in more ways than I can count, and throughout my life have been models of goodness, compassion, love, generosity, and good doctoring. My three wonderful daughters bring a smile to my face every day. I am so blessed by each of them. And to my amazing wife, Elizabeth, you mean more to me than anything. Thank you, from the bottom of my heart, for all the help, love, support, iii! ! and friendship that you have sustained me with through our years, and for everything you are to me. I love you, E. iv! ! TABLE OF CONTENTS Page DEDICATION .......................................................................................................................ii ACKNOWLEDGEMENTS ...................................................................................................iii LIST OF FIGURES ...............................................................................................................viii LIST OF TABLES .................................................................................................................ix Chapter I. INTRODUCTION .......................................................................................................1 Discovery of the LKB1 Tumor Suppressor ....................................................................1 Structure, function, and regulation of LKB1 ...................................................................2 LKB1 regulation of AMPK .............................................................................................7 LKB1 and the mTOR pathway ........................................................................................10 Regulation of other processes by LKB1 ..........................................................................12 Somatic LKB1 loss in human tumors ..............................................................................14 Mouse models of LKB1 loss ..........................................................................................17 Murine model of LKB1/KRAS mutant lung cancer ........................................................20 Gene expression analysis .................................................................................................23 II. GENE EXPRESSION SIGNATURE OF LKB1 LOSS .............................................29 Introduction ......................................................................................................................29 Methods and materials .....................................................................................................31 Results ..............................................................................................................................36 LKB1 loss results in consistent gene expression changes in human tumors .............36 A 16-Gene LKB1-loss classifier accurately predicts mutational and non-mutational loss of LKB1 in resected human lung Adenocarcinomas .........................................................................................41 The LKB1-loss classifier accurately predicts mutational and non-mutational loss of LKB1 in NSCLC cell lines .......................................................................50 The LKB1-loss classifier is associated with LKB1 mutations in cell lines derived from non-lung primary cancers ...............................................51 Association of LKB1-loss signature with loss of LKB1 in resected, non-lung primary tumors .....................................................................................54 Development of an assay for LKB1-loss score suitable for analysis of clinical samples using the nanoString n-Counter assay ...................................58 Discussion ........................................................................................................................61 v!! III. INTERPRETING THE BIOLOGICAL SIGNIFICANCE OF THE LKB1 SIGNATURE .........................................................................................................................65 Introduction ......................................................................................................................65 Methods and materials .....................................................................................................67 Results ..............................................................................................................................73 Genes associated with LKB1 loss correspond to particular tumor phenotypes and transcription factors ...................................................................73 Activation of the NRF2 pathway is common among LKB1-deficient tumors ..........74 NRF2 association with LKB1 is partially explained by deletion events affecting LKB1 and KEAP1 ................................................................................78 Gene set enrichment analysis of the mitochondria/mTOR cluster and the down-regulated cluster .........................................................................................80 Gene set enrichment analysis of the LKB1-loss cluster identifies association with FOXO3, FOXA2, and CREB Transcription Factors ...................................83 Association between LKB1 loss and prevalence of other mutations .........................85 Clinical phenotypes of LKB1-deficient lung adenocarcinomas ................................88 Protein, microRNA, and copy number alterations associated with LKB1 status ......90 Wild-type LKB1 decreases the expression of the LKB1-associated signature genes .....................................................................................................93 Discussion ........................................................................................................................96 IV. LKB1 LOSS IS ASSOCIATED WITH SENSITIVITY TO MEK INHIBITION AND ALTERATIONS IN PI3K-AKT-FOXO3 SIGNALING .............................................105 Introduction ......................................................................................................................105 Methods and materials .....................................................................................................108 Results ..............................................................................................................................110 LKB1-deficient cell lines show increased susceptibility to MEK inhibition ............110 Restoring LKB1 expression in LKB1-mutant cell lines induces resistance to MEK inhibitors. ...................................................................................................113 The expression of the 16-gene signature is independent of MEK/ERK signaling ....114 Restoring LKB1 to cell lines in vitro induces phosphorylation of AKT and FOXO3 ..........................................................................................................116 PI3K/AKT signaling is attenuated in resected human tumors with LKB1 loss ........117 Discussion ........................................................................................................................120 V. CONCLUSION ..........................................................................................................125 Appendix A. DATA SOURCES AND STATISTICAL COMPARISONS ....................................135 B. INITIAL GENES IN FOUR TRANSCRIPTIONAL CLUSTERS ..........................139 C. TOP 200 GENES ASSOCIATED WITH EACH CLUSTER ...................................140 vi! ! REFERENCES ......................................................................................................................143
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