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Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention

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Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 12-2012 Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention Nathan Ihle Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons, Molecular Biology Commons, and the Systems Biology Commons Recommended Citation Ihle, Nathan, "Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention" (2012). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 314. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/314 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas M. D. Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY By Nathan Ihle, B.S. Houston, Texas December, 2012 Dedication To my darling wife and children who have loved and supported me in and despite all the decisions I have made. Mena Abdelmelek, a great help in the preparation of this work and Garth Powis, a great mentor in science and life. iii Acknowledgements All the present and past members of the Powis Lab who have taught me so much through the years. My committee for great insights and input into this work and the hardworking scientists and physicians of MD Anderson. I perhaps owe the greatest acknowledgement to those patients who sacrificed time or comfort to provide samples to perform these studies and made this sacrifice so that future sufferers of cancer may have long healthy lives. iv Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention By Nathan Ihle B.S. Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated by mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure shows that different amino acids substituted at the codon 12 position influences how KRas interacts with its effecters. In the absence of a direct inhibitor of mutant KRas several agents have recently entered clinical trials alone and in combination directly targeting two of the common downstream effecter pathways of KRas, namely the Mapk pathway and the Akt pathway. These inhibitors were evaluated for efficacy against different KRAS activating mutations. An isogenic panel of colorectal cells with wild type KRas replaced with KRas G12C, G12D, or G12V at the endogenous loci differed in sensitivity to Mek and Akt inhibition. In contrast, screening was performed in a broad panel of lung cell lines alone and no correlation was seen between types of activating KRAS mutation due to concurrent oncogenic lesions. To find a new method to inhibit KRAS driven tumors, siRNA screens were performed in isogenic lines with and without active KRas. The knockdown of CNKSR1 (CNK1) showed selective growth inhibition in cells with an oncogenic KRAS. The deletion of CNK1 reduces expression of mitotic cell cycle proteins and arrests cells with active KRas in the G1 phase of the cell cycle similar to the deletion of an activated KRas regardless of activating substitution. v CNK1 has a PH domain responsible for localizing it to membrane lipids making KRas potentially amenable to inhibition with small molecules. The work has identified a series of small molecules capable of binding to this PH domain and inhibiting CNK1 facilitated KRas signaling. vi Table of contents Approval page .......................................................................................................................... i Title page .................................................................................................................................................. ii Dedication ................................................................................................................................................ iii Acknowledgements ................................................................................................................................ iv Abstract ..................................................................................................................................................... v Table of contents .................................................................................................................................... vii List of illustrations .................................................................................................................................. xii List of tables ........................................................................................................................................... xvi List of abbreviations ............................................................................................................................. xvii Chapter 1: Introduction ............................................................................................................. 1 1.1 Ras background and function ........................................................................................................ 1 1.1.1 The Ras activation cycle .............................................................................................................. 2 1.1.2 Ras localization .............................................................................................................................. 5 1.1.3 KRAS in development ................................................................................................................... 7 1.2 KRas signal transduction ................................................................................................................ 8 1.3 KRAS in cancer ............................................................................................................................... 11 1.3.1 Activating KRAS mutations ........................................................................................................ 11 vii 1.3.2 KRAS as a driver of tumorigenesis .......................................................................................... 12 1.3.3 KRAS in cancer therapy ............................................................................................................. 13 Chapter 2: Materials and methods .......................................................................................... 15 2.1 BATTLE clinical trial ....................................................................................................................... 15 2.2 Patient microarray data .................................................................................................................. 15 2.3 Cells and culture conditions ......................................................................................................... 17 2.4 Preparation of protein lysates and reverse-phase protein array (RPPA) .............................. 17 2.5 Immunoblots and immunoprecipitations ..................................................................................... 18 2.6 Viral vector construction ................................................................................................................ 19 2.7 Soft agar growth assays ............................................................................................................... 19 2.8 Molecular modeling ........................................................................................................................ 19 2.9 Screening of compounds against isogenic mutant KRAS lines .............................................. 20 2.10 Screening of compounds against NSCLC cell line panel ....................................................... 20 2.11 siRNA screening ........................................................................................................................... 20 2.12 Individual siRNA and plasmid transfection ..............................................................................
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