Functional effects of a novel BIM deletion polymorphism in mediating resistance to tyrosine kinase inhibitors in cancer

FUNCTIONAL EFFECTS OF A NOVEL BIM DELETION POLYMORPHISM IN MEDIATING RESISTANCE TO TYROSINE KINASE INHIBITORS IN CANCER JUAN WEN CHUN B.Sc. (Hons), NATIONAL UNIVERSITY OF SINGAPORE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ___________ ____________ Juan Wen Chun 25 November 2013 ACKNOWLEDGEMENTS I thank my PhD supervisor, Associate Professor Ong Sin Tiong, for accepting me as his graduate student and for giving me the wonderful opportunity to work on this project. Furthermore, I also thank him for giving me the freedom to develop my own ideas and for providing useful advice when I am in doubt. I thank my wife, Hui Ling, for all the love, patience and support that she has given me during the last four years. Hui Ling is also a scientist who has lots of research experience. I thank her for teaching me overlapping extension PCR, a technique that enabled me to generate mutations for analyzing cis-elements that regulate splicing of BIM. I thank my parents for their support during the last four years, and for being patient when I am frustrated over experiments that are not working. I thank Assistant Professor Xavier Roca for his invaluable advice on all the experiments pertaining to alternative splicing in this project. I also thank Xavier for piquing my interest to study the role of alternative splicing in human diseases. I thank all the members of my thesis advisory committee: Professor David Virshup, Professor Ruan Yijun and Dr Axel Hillmer, for their support and their advice in this project. I thank Professor Mariano Garcia-Blanco for his advice on the design of the WT and DEL minigenes to demonstrate that the BIM deletion polymorphism affects splicing of BIM. I thank all the current and the former members of the Tiong’s lab for all the joy, laughter, encouragement and advice that they have given me during the last four years. The time that I have spent in the lab is indeed a memorable one. These people are: King Pan, Sharon, Tun i Kiat, Aditi, Sheila, Siew Peng, John, Rauzan, Sathish, Galih, Angie, Sandy, Judy, Michael, Tuang Yeow and Vera. I thank all members of the Cancer and Stem Cell Biology department at Duke-NUS Graduate Medical School for the snippets of advice that they have given me during coffee breaks and the Research In Progress seminars. I thank Professor Hooi Shing Chuan, Associate Professor Maxey Chung, Associate Professor Vladimir Korzh, Guodong, Sandra, Cynthia and Cathleen for their guidance when I was an undergraduate student in NUS. Finally, I am grateful to all the patients who took part in this project. These important discoveries would not be made without their participation. ii TABLE OF CONTENTS Summary…………………………………………………………………………………… vi List of tables………………………………………………………………………………….viii List of figures and illustrations……………………………………………………………….ix List of symbols and abbreviations……………………………………………………………xii Chapter Introduction……………………………………………………………… 1.1 Tyrosine kinases and their signaling pathways…………………………… 1.2 The role of tyrosine kinases in cancer……………………………………… 1.3 Tyrosine kinase inhibitors as therapeutic agents in cancer………………… 1.4 Clinical resistance to tyrosine kinase inhibitors…………………………… 12 1.5 Molecular basis of resistance to tyrosine kinase inhibitors……………… . 13 1.6 Biomarkers that predict response to tyrosine kinase inhibitors……………. 18 1.7 The analysis of genome structural variations using next-generation sequencing of paired-end tags……………………………………………… 22 1.8 Chapter Aim of study……………………………………………………………… . 24 Functional effects of a novel BIM deletion polymorphism on gene expression………………………………………………………………… . 25 2.1 Introduction……………………………………………………………… 26 2.2 Identification of a 2,903-bp deletion polymorphism in the second intron of the BIM gene…………………………………………………………… 28 2.3 Effects of the BIM deletion polymorphism on gene expression…………… 32 2.4 Conclusion…………………………………………………………………. 40 Chapter Effects of aberrant splicing mediated by the BIM deletion polymorphism on resistance to tyrosine kinase inhibitors…………………………… . 42 3.1 Introduction……………………………………………………………… 43 iii 3.2 The BIM deletion polymorphism mediates resistance to tyrosine kinase inhibitors in chronic myelogenous leukemia……………………………. 43 3.3 The BIM deletion polymorphism mediates resistance to tyrosine kinase inhibitors in epidermal growth factor receptor-mutated non-small-cell lung cancer……………………………………………………………………. 60 3.4 Chapter Conclusion………………………………………………………………. 68 Identification of cis-acting RNA elements and trans-acting factors that regulate splicing of BIM via the BIM deletion polymorphism 69 4.1 Introduction…………………………………………………………… 70 4.2 Deletion and substitution analysis to identify cis-elements that regulate splicing of BIM exon 3…………………………………………………. 72 4.3 A 23-nt intronic splicing silencer is located at the 3’end of the BIM deletion polymorphism…………………………………………………. 80 4.4 Conclusion (Part 1)…………………………………………………… . 85 4.5 The role of PTBP1 in repressing the inclusion of BIM exon 3…………. 86 4.6 HnRNP H and hnRNP F not regulate splicing of BIM exon 3……… 90 4.7 The role of hnRNP C in repressing the inclusion of BIM exon 3………. 94 4.8 The identification of trans-acting factors that bind to the 23-nt intronic splicing silencer using RNA pull-down assay………………………… 97 4.9 Chapter 5.1 Conclusion (Part 2)…………………………………………………… 100 Discussion…………………………………………………………… . 101 The BIM deletion polymorphism as a biomarker for TKI-resistance in kinase-driven cancers………………………………………………… 102 5.2 Other potential implications of the BIM deletion polymorphism in human diseases……………………………………………………………… . 104 iv 5.3 Alternative approaches to overcome TKI-resistance associated with the BIM deletion polymorphism…………………………………………………… 105 5.4 The role of polymorphisms in splicing-related diseases…………………. 107 5.5 Regulation of BIM exon splicing via the BIM deletion polymorphism . 108 Chapter Materials and Methods………………………………………………… 113 Ethics committee approval……………………………………………… . 114 Cell lines, culture and drugs………………………………………………. 114 Identification of structural variations by DNA-PET sequencing…………. 115 Genotyping individuals for the BIM deletion polymorphism…………… 115 Real-time RT-PCR……………………………………………………… . 117 Luciferase assay to determine enhancer activity………………………… 117 Minigene plasmids construction and assay for splicing changes…………. 118 Computational analysis to predict for cis-regulatory elements that regulate splicing……………………………………………………………………. 124 RT-PCR and sequencing of BIM splice variants…………………………. 124 Protein extraction and western blot………………………………………. 125 Trypan blue exclusion assay…………………………………………… . 126 BIM expression plasmids and siRNAs………………………………… . 126 Annexin V staining………………………………………………………. 127 Measurement of protein stability………………………………………… 127 Generating cell lines harboring the BIM deletion polymorphism using ZFNs…………………………………………………………………… . 128 ELISA-based DNA fragmentation assay……………………………… . 129 RNA pull-down assay and mass spectrometry analysis………………… 129 Statistical analysis………………………………………………………. 130 References 131 v SUMMARY The use of tyrosine kinase inhibitors (TKIs) to inhibit oncogenic kinases, such as BCR-ABL1 and EGFR, has led to remarkable responses in patients with chronic myelogenous leukemia (CML) and non-small-cell lung cancer with activating mutations in EGFR (EGFR NSCLC). Despite the high response rates, there are still patients who not respond adequately to TKIs. These findings suggest that there are additional genetic aberrations which could modulate a patient’s response to TKIs. Structural variations can be found in the cancer as well as in the normal human genome. However, their role in influencing responses to TKIs have not been well-established. Using paired-end DNA sequencing, we discovered a novel 2,903-bp deletion polymorphism in intron of the BIM gene. BIM is an apoptosis-inducing member of the BCL-2 family of proteins. Importantly, upregulation of BIM expression is required for sensitivity towards TKIs in CML and EGFR NSCLC. Using a minigene system, I demonstrated that the deletion favored the inclusion of exon over exon 4, an event that could impair the induction of apoptosis because the apoptosis-inducing BH3 domain is found only in exon 4. To study the role of this deletion in mediating TKI-resistance, we have identified and generated CML and EGFR NSCLC cell lines that contain the deletion polymorphism. Compared to non-deletion containing cells, cells harboring the deletion exhibited an increased exon 3- to exon 4-containing BIM transcripts and decreased induction of BH3-containing BIM proteins after exposure to TKIs. As a result, CML and EGFR NSCLC cells harboring the deletion are less sensitive to TKIs. We have also demonstrated that the BH3-mimetic, ABT737, can sensitize deletion-containing cells to TKI-induced apoptosis. Notably, individuals with CML and EGFR NSCLC harboring the deletion polymorphism experienced significantly poorer responses to TKIs than did individuals without the polymorphism. Mutation analysis of the 2,903-nt deleted fragment revealed that there are multiple redundant cis-acting elements that repress inclusion of exon 3. In addition, I have also demonstrated that a 322-nt sequence at the 3’end of the deletion contains regulatory elements that are sufficient but not necessary for repressing exon inclusion. Within this 322-nt vi 60. Ren, R. (2005). Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nature reviews. Cancer 5, 172-83. 61. Mahon, F. X., Rea, D., Guilhot, J., Guilhot, F., Huguet, F., Nicolini, F., Legros, L., Charbonnier, A., Guerci, A., Varet, B., Etienne, G., Reiffers, J. & Rousselot, P. (2010). Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least years: the prospective, multicentre Stop Imatinib (STIM) trial. The lancet oncology 11, 1029-35. 62. Bhatia, R., Holtz, M., Niu, N., Gray, R., Snyder, D. S., Sawyers, C. L., Arber, D. A., Slovak, M. L. & Forman, S. J. (2003). Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. Blood 101, 4701-7. 63. Mitsudomi, T., Morita, S., Yatabe, Y., Negoro, S., Okamoto, I., Tsurutani, J., Seto, T., Satouchi, M., Tada, H., Hirashima, T., Asami, K., Katakami, N., Takada, M., Yoshioka, H., Shibata, K., Kudoh, S., Shimizu, E., Saito, H., Toyooka, S., Nakagawa, K. & Fukuoka, M. (2010). Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase trial. The lancet oncology 11, 121-8. 64. Maemondo, M., Inoue, A., Kobayashi, K., Sugawara, S., Oizumi, S., Isobe, H., Gemma, A., Harada, M., Yoshizawa, H., Kinoshita, I., Fujita, Y., Okinaga, S., Hirano, H., Yoshimori, K., Harada, T., Ogura, T., Ando, M., Miyazawa, H., Tanaka, T., Saijo, Y., Hagiwara, K., Morita, S. & Nukiwa, T. (2010). Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. The New England journal of medicine 362, 2380-8. 65. Fukuoka, M., Yano, S., Giaccone, G., Tamura, T., Nakagawa, K., Douillard, J. Y., Nishiwaki, Y., Vansteenkiste, J., Kudoh, S., Rischin, D., Eek, R., Horai, T., Noda, K., Takata, I., Smit, E., Averbuch, S., Macleod, A., Feyereislova, A., Dong, R. P. & Baselga, J. (2003). Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL Trial) [corrected]. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, 2237-46. 66. Kris, M. G., Natale, R. B., Herbst, R. S., Lynch, T. J., Jr., Prager, D., Belani, C. P., Schiller, J. H., Kelly, K., Spiridonidis, H., Sandler, A., Albain, K. S., Cella, D., Wolf, M. K., Averbuch, S. D., Ochs, J. J. & Kay, A. C. (2003). Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA : the journal of the American Medical Association 290, 2149-58. 67. Quintas-Cardama, A. & Cortes, J. E. (2006). Chronic myeloid leukemia: diagnosis and treatment. Mayo Clinic proceedings. Mayo Clinic 81, 973-88. 68. Milojkovic, D. & Apperley, J. (2009). Mechanisms of Resistance to Imatinib and Second-Generation Tyrosine Inhibitors in Chronic Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 7519-7527. 136 69. Therasse, P., Arbuck, S. G., Eisenhauer, E. A., Wanders, J., Kaplan, R. S., Rubinstein, L., Verweij, J., Van Glabbeke, M., van Oosterom, A. T., Christian, M. C. & Gwyther, S. G. (2000). New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. Journal of the National Cancer Institute 92, 205-16. 70. Baccarani, M., Deininger, M. W., Rosti, G., Hochhaus, A., Soverini, S., Apperley, J. F., Cervantes, F., Clark, R. E., Cortes, J. E., Guilhot, F., Hjorth-Hansen, H., Hughes, T. P., Kantarjian, H. M., Kim, D. W., Larson, R. A., Lipton, J. H., Mahon, F. X., Martinelli, G., Mayer, J., Muller, M. C., Niederwieser, D., Pane, F., Radich, J. P., Rousselot, P., Saglio, G., Saussele, S., Schiffer, C., Silver, R., Simonsson, B., Steegmann, J. L., Goldman, J. M. & Hehlmann, R. (2013). European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood 122, 872-84. 71. Quintas-Cardama, A., Kantarjian, H. M. & Cortes, J. E. (2009). Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer control : journal of the Moffitt Cancer Center 16, 122-31. 72. Ou, S. H. (2012). Second-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs): a better mousetrap? A review of the clinical evidence. Critical reviews in oncology/hematology 83, 407-21. 73. Luzzatto, L. & Melo, J. V. (2002). Acquired resistance to imatinib mesylate: selection for pre-existing mutant cells. Blood 100, 1105. 74. Engelman, J. A. & Janne, P. A. (2008). Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 2895-9. 75. Kobayashi, S., Boggon, T. J., Dayaram, T., Janne, P. A., Kocher, O., Meyerson, M., Johnson, B. E., Eck, M. J., Tenen, D. G. & Halmos, B. (2005). EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. The New England journal of medicine 352, 786-92. 76. Weisberg, E., Manley, P. W., Breitenstein, W., Bruggen, J., Cowan-Jacob, S. W., Ray, A., Huntly, B., Fabbro, D., Fendrich, G., Hall-Meyers, E., Kung, A. L., Mestan, J., Daley, G. Q., Callahan, L., Catley, L., Cavazza, C., Azam, M., Neuberg, D., Wright, R. D., Gilliland, D. G. & Griffin, J. D. (2005). Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer cell 7, 129-41. 77. Lombardo, L. J., Lee, F. Y., Chen, P., Norris, D., Barrish, J. C., Behnia, K., Castaneda, S., Cornelius, L. A., Das, J., Doweyko, A. M., Fairchild, C., Hunt, J. T., Inigo, I., Johnston, K., Kamath, A., Kan, D., Klei, H., Marathe, P., Pang, S., Peterson, R., Pitt, S., Schieven, G. L., Schmidt, R. J., Tokarski, J., Wen, M. L., Wityak, J. & Borzilleri, R. M. (2004). Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. Journal of medicinal chemistry 47, 6658-61. 137 78. Kwak, E. L., Sordella, R., Bell, D. W., Godin-Heymann, N., Okimoto, R. A., Brannigan, B. W., Harris, P. L., Driscoll, D. R., Fidias, P., Lynch, T. J., Rabindran, S. K., McGinnis, J. P., Wissner, A., Sharma, S. V., Isselbacher, K. J., Settleman, J. & Haber, D. A. (2005). Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proceedings of the National Academy of Sciences of the United States of America 102, 7665-70. 79. Gorre, M. E., Mohammed, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P. N. & Sawyers, C. L. (2001). Clinical resistance to STI-571 cancer therapy caused by BCRABL gene mutation or amplification. Science 293, 876-80. 80. Neviani, P., Santhanam, R., Trotta, R., Notari, M., Blaser, B. W., Liu, S., Mao, H., Chang, J. S., Galietta, A., Uttam, A., Roy, D. C., Valtieri, M., Bruner-Klisovic, R., Caligiuri, M. A., Bloomfield, C. D., Marcucci, G. & Perrotti, D. (2005). The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer cell 8, 355-68. 81. Marega, M., Piazza, R. G., Pirola, A., Redaelli, S., Mogavero, A., Iacobucci, I., Meneghetti, I., Parma, M., Pogliani, E. M. & Gambacorti-Passerini, C. (2010). BCR and BCR-ABL regulation during myeloid differentiation in healthy donors and in chronic phase/blast crisis CML patients. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 24, 1445-9. 82. Chu, S., Holtz, M., Gupta, M. & Bhatia, R. (2004). BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells. Blood 103, 3167-74. 83. Abram, C. L. & Courtneidge, S. A. (2000). Src family tyrosine kinases and growth factor signaling. Experimental cell research 254, 1-13. 84. Wu, J., Meng, F., Lu, H., Kong, L., Bornmann, W., Peng, Z., Talpaz, M. & Donato, N. J. (2008). Lyn regulates BCR-ABL and Gab2 tyrosine phosphorylation and c-Cbl protein stability in imatinib-resistant chronic myelogenous leukemia cells. Blood 111, 3821-9. 85. Donato, N. J., Wu, J. Y., Stapley, J., Gallick, G., Lin, H., Arlinghaus, R. & Talpaz, M. (2003). BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571. Blood 101, 690-8. 86. Ptasznik, A., Nakata, Y., Kalota, A., Emerson, S. G. & Gewirtz, A. M. (2004). Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells. Nature medicine 10, 1187-9. 87. Engelman, J. A., Zejnullahu, K., Mitsudomi, T., Song, Y., Hyland, C., Park, J. O., Lindeman, N., Gale, C. M., Zhao, X., Christensen, J., Kosaka, T., Holmes, A. J., Rogers, A. M., Cappuzzo, F., Mok, T., Lee, C., Johnson, B. E., Cantley, L. C. & Janne, P. A. (2007). MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039-43. 138 88. Swartz, M. A., Iida, N., Roberts, E. W., Sangaletti, S., Wong, M. H., Yull, F. E., Coussens, L. M. & DeClerck, Y. A. (2012). Tumor microenvironment complexity: emerging roles in cancer therapy. Cancer research 72, 2473-80. 89. Hockel, M. & Vaupel, P. (2001). Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. Journal of the National Cancer Institute 93, 266-76. 90. Semenza, G. L. (2003). Targeting HIF-1 for cancer therapy. Nature reviews. Cancer 3, 721-32. 91. Minakata, K., Takahashi, F., Nara, T., Hashimoto, M., Tajima, K., Murakami, A., Nurwidya, F., Yae, S., Koizumi, F., Moriyama, H., Seyama, K., Nishio, K. & Takahashi, K. (2012). Hypoxia induces gefitinib resistance in non-small-cell lung cancer with both mutant and wild-type epidermal growth factor receptors. Cancer science 103, 1946-54. 92. Grant, W. C. & Root, W. S. (1947). The relation of O2 in bone marrow blood to posthemorrhagic erythropoiesis. The American journal of physiology 150, 618-27. 93. Cipolleschi, M. G., Dello Sbarba, P. & Olivotto, M. (1993). The role of hypoxia in the maintenance of hematopoietic stem cells. Blood 82, 2031-7. 94. Giuntoli, S., Rovida, E., Barbetti, V., Cipolleschi, M. G., Olivotto, M. & Dello Sbarba, P. (2006). Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 20, 1291-3. 95. Giuntoli, S., Rovida, E., Gozzini, A., Barbetti, V., Cipolleschi, M. G., Olivotto, M. & Dello Sbarba, P. (2007). Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells. Stem cells 25, 1119-25. 96. Zhang, B., Li, M., McDonald, T., Holyoake, T. L., Moon, R. T., Campana, D., Shultz, L. & Bhatia, R. (2013). Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood 121, 1824-38. 97. Eechoute, K., Sparreboom, A., Burger, H., Franke, R. M., Schiavon, G., Verweij, J., Loos, W. J., Wiemer, E. A. & Mathijssen, R. H. (2011). Drug transporters and imatinib treatment: implications for clinical practice. Clinical cancer research : an official journal of the American Association for Cancer Research 17, 406-15. 98. Thomas, J., Wang, L., Clark, R. E. & Pirmohamed, M. (2004). Active transport of imatinib into and out of cells: implications for drug resistance. Blood 104, 3739-45. 99. Mahon, F. X., Deininger, M. W., Schultheis, B., Chabrol, J., Reiffers, J., Goldman, J. M. & Melo, J. V. (2000). Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 96, 1070-9. 139 100. Rumpold, H., Wolf, A. M., Gruenewald, K., Gastl, G., Gunsilius, E. & Wolf, D. (2005). RNAi-mediated knockdown of P-glycoprotein using a transposon-based vector system durably restores imatinib sensitivity in imatinib-resistant CML cell lines. Experimental hematology 33, 767-75. 101. Hegedus, C., Truta-Feles, K., Antalffy, G., Varady, G., Nemet, K., Ozvegy-Laczka, C., Keri, G., Orfi, L., Szakacs, G., Settleman, J., Varadi, A. & Sarkadi, B. (2012). Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance. Biochemical pharmacology 84, 260-7. 102. Elkind, N. B., Szentpetery, Z., Apati, A., Ozvegy-Laczka, C., Varady, G., Ujhelly, O., Szabo, K., Homolya, L., Varadi, A., Buday, L., Keri, G., Nemet, K. & Sarkadi, B. (2005). Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib). Cancer research 65, 1770-7. 103. Sokal, J. E., Cox, E. B., Baccarani, M., Tura, S., Gomez, G. A., Robertson, J. E., Tso, C. Y., Braun, T. J., Clarkson, B. D., Cervantes, F. & et al. (1984). Prognostic discrimination in "good-risk" chronic granulocytic leukemia. Blood 63, 789-99. 104. Baccarani, M., Cortes, J., Pane, F., Niederwieser, D., Saglio, G., Apperley, J., Cervantes, F., Deininger, M., Gratwohl, A., Guilhot, F., Hochhaus, A., Horowitz, M., Hughes, T., Kantarjian, H., Larson, R., Radich, J., Simonsson, B., Silver, R. T., Goldman, J. & Hehlmann, R. (2009). Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 6041-51. 105. McWeeney, S. K., Pemberton, L. C., Loriaux, M. M., Vartanian, K., Willis, S. G., Yochum, G., Wilmot, B., Turpaz, Y., Pillai, R., Druker, B. J., Snead, J. L., MacPartlin, M., O'Brien, S. G., Melo, J. V., Lange, T., Harrington, C. A. & Deininger, M. W. (2010). A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib. Blood 115, 315-25. 106. Lange, T., Gunther, C., Kohler, T., Krahl, R., Musiol, S., Leiblein, S., Al-Ali, H. K., van Hoomissen, I., Niederwieser, D. & Deininger, M. W. (2003). High levels of BAX, low levels of MRP-1, and high platelets are independent predictors of response to imatinib in myeloid blast crisis of CML. Blood 101, 2152-5. 107. Crossman, L. C., Druker, B. J., Deininger, M. W., Pirmohamed, M., Wang, L. & Clark, R. E. (2005). hOCT and resistance to imatinib. Blood 106, 1133-4; author reply 1134. 108. Wang, L., Giannoudis, A., Lane, S., Williamson, P., Pirmohamed, M. & Clark, R. E. (2008). Expression of the uptake drug transporter hOCT1 is an important clinical determinant of the response to imatinib in chronic myeloid leukemia. Clinical pharmacology and therapeutics 83, 258-64. 140 109. Herbst, R. S., Heymach, J. V. & Lippman, S. M. (2008). Lung cancer. The New England journal of medicine 359, 1367-80. 110. Subramanian, J. & Govindan, R. (2008). Molecular genetics of lung cancer in people who have never smoked. The lancet oncology 9, 676-82. 111. Pao, W., Wang, T. Y., Riely, G. J., Miller, V. A., Pan, Q., Ladanyi, M., Zakowski, M. F., Heelan, R. T., Kris, M. G. & Varmus, H. E. (2005). KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS medicine 2, e17. 112. Linardou, H., Dahabreh, I. J., Kanaloupiti, D., Siannis, F., Bafaloukos, D., Kosmidis, P., Papadimitriou, C. A. & Murray, S. (2008). Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. The lancet oncology 9, 962-72. 113. Bean, J., Brennan, C., Shih, J. Y., Riely, G., Viale, A., Wang, L., Chitale, D., Motoi, N., Szoke, J., Broderick, S., Balak, M., Chang, W. C., Yu, C. J., Gazdar, A., Pass, H., Rusch, V., Gerald, W., Huang, S. F., Yang, P. C., Miller, V., Ladanyi, M., Yang, C. H. & Pao, W. (2007). MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proceedings of the National Academy of Sciences of the United States of America 104, 20932-7. 114. Soh, S. & Ong, S. T. (2013). A novel BIM deletion polymorphism: implications and lessons for cancer targeted therapies. [Rinsho ketsueki] The Japanese journal of clinical hematology 54, 1714-9. 115. Wheeler, H. E., Maitland, M. L., Dolan, M. E., Cox, N. J. & Ratain, M. J. (2013). Cancer pharmacogenomics: strategies and challenges. Nature reviews. Genetics 14, 23-34. 116. Nelson, M. R., Wegmann, D., Ehm, M. G., Kessner, D., St Jean, P., Verzilli, C., Shen, J., Tang, Z., Bacanu, S. A., Fraser, D., Warren, L., Aponte, J., Zawistowski, M., Liu, X., Zhang, H., Zhang, Y., Li, J., Li, Y., Li, L., Woollard, P., Topp, S., Hall, M. D., Nangle, K., Wang, J., Abecasis, G., Cardon, L. R., Zollner, S., Whittaker, J. C., Chissoe, S. L., Novembre, J. & Mooser, V. (2012). An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people. Science 337, 100-4. 117. Dulucq, S., Bouchet, S., Turcq, B., Lippert, E., Etienne, G., Reiffers, J., Molimard, M., Krajinovic, M. & Mahon, F. X. (2008). Multidrug resistance gene (MDR1) polymorphisms are associated with major molecular responses to standard-dose imatinib in chronic myeloid leukemia. Blood 112, 2024-7. 118. Li, J., Cusatis, G., Brahmer, J., Sparreboom, A., Robey, R. W., Bates, S. E., Hidalgo, M. & Baker, S. D. (2007). Association of variant ABCG2 and the pharmacokinetics of epidermal growth factor receptor tyrosine kinase inhibitors in cancer patients. Cancer biology & therapy 6, 432-8. 141 119. Gebhardt, F., Zanker, K. S. & Brandt, B. (1999). Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1. The Journal of biological chemistry 274, 13176-80. 120. Ichihara, S., Toyooka, S., Fujiwara, Y., Hotta, K., Shigematsu, H., Tokumo, M., Soh, J., Asano, H., Ichimura, K., Aoe, K., Aoe, M., Kiura, K., Shimizu, K., Date, H. & Shimizu, N. (2007). The impact of epidermal growth factor receptor gene status on gefitinib-treated Japanese patients with non-small-cell lung cancer. International journal of cancer. Journal international du cancer 120, 1239-47. 121. Han, S. W., Jeon, Y. K., Lee, K. H., Keam, B., Hwang, P. G., Oh, D. Y., Lee, S. H., Kim, D. W., Im, S. A., Chung, D. H., Heo, D. S., Bang, Y. J. & Kim, T. Y. (2007). Intron CA dinucleotide repeat polymorphism and mutations of epidermal growth factor receptor and gefitinib responsiveness in non-small-cell lung cancer. Pharmacogenetics and genomics 17, 313-9. 122. Gregorc, V., Hidalgo, M., Spreafico, A., Cusatis, G., Ludovini, V., Ingersoll, R. G., Marsh, S., Steinberg, S. M., Vigano, M. G., Ghio, D., Villa, E., Sparreboom, A. & Baker, S. D. (2008). Germline polymorphisms in EGFR and survival in patients with lung cancer receiving gefitinib. Clinical pharmacology and therapeutics 83, 477-84. 123. Giovannetti, E., Zucali, P. A., Peters, G. J., Cortesi, F., D'Incecco, A., Smit, E. F., Falcone, A., Burgers, J. A., Santoro, A., Danesi, R., Giaccone, G. & Tibaldi, C. (2010). Association of polymorphisms in AKT1 and EGFR with clinical outcome and toxicity in non-small cell lung cancer patients treated with gefitinib. Molecular cancer therapeutics 9, 581-93. 124. Sadikovic, B., Al-Romaih, K., Squire, J. A. & Zielenska, M. (2008). Cause and consequences of genetic and epigenetic alterations in human cancer. Current genomics 9, 394-408. 125. Feuk, L., Carson, A. R. & Scherer, S. W. (2006). Structural variation in the human genome. Nature reviews. Genetics 7, 85-97. 126. Fullwood, M. J., Wei, C. L., Liu, E. T. & Ruan, Y. (2009). Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses. Genome research 19, 521-32. 127. Campbell, P. J., Stephens, P. J., Pleasance, E. D., O'Meara, S., Li, H., Santarius, T., Stebbings, L. A., Leroy, C., Edkins, S., Hardy, C., Teague, J. W., Menzies, A., Goodhead, I., Turner, D. J., Clee, C. M., Quail, M. A., Cox, A., Brown, C., Durbin, R., Hurles, M. E., Edwards, P. A., Bignell, G. R., Stratton, M. R. & Futreal, P. A. (2008). Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Nature genetics 40, 722-9. 128. Hillmer, A. M., Yao, F., Inaki, K., Lee, W. H., Ariyaratne, P. N., Teo, A. S., Woo, X. Y., Zhang, Z., Zhao, H., Ukil, L., Chen, J. P., Zhu, F., So, J. B., Salto-Tellez, M., Poh, W. T., Zawack, K. F., Nagarajan, N., Gao, S., Li, G., Kumar, V., Lim, H. P., Sia, Y. Y., Chan, C. S., Leong, S. T., Neo, S. C., Choi, P. S., Thoreau, H., Tan, P. B., Shahab, A., Ruan, X., Bergh, J., Hall, P., Cacheux-Rataboul, V., Wei, C. L., Yeoh, K. G., Sung, W. K., Bourque, G., Liu, E. T. & Ruan, Y. (2011). Comprehensive long-span 142 paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes. Genome research 21, 665-75. 129. Hanahan, D. & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646-74. 130. Adams, J. M. & Cory, S. (2007). The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26, 1324-37. 131. Delbridge, A. R., Valente, L. J. & Strasser, A. (2012). The role of the apoptotic machinery in tumor suppression. Cold Spring Harbor perspectives in biology 4. 132. Chipuk, J. E. & Green, D. R. (2008). How BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends in cell biology 18, 157-64. 133. Chipuk, J. E., Moldoveanu, T., Llambi, F., Parsons, M. J. & Green, D. R. (2010). The BCL-2 family reunion. Molecular cell 37, 299-310. 134. Kuroda, J., Puthalakath, H., Cragg, M. S., Kelly, P. N., Bouillet, P., Huang, D. C., Kimura, S., Ottmann, O. G., Druker, B. J., Villunger, A., Roberts, A. W. & Strasser, A. (2006). Bim and Bad mediate imatinib-induced killing of Bcr/Abl+ leukemic cells, and resistance due to their loss is overcome by a BH3 mimetic. Proceedings of the National Academy of Sciences of the United States of America 103, 14907-12. 135. Aichberger, K. J., Mayerhofer, M., Krauth, M. T., Vales, A., Kondo, R., Derdak, S., Pickl, W. F., Selzer, E., Deininger, M., Druker, B. J., Sillaber, C., Esterbauer, H. & Valent, P. (2005). Low-level expression of proapoptotic Bcl-2-interacting mediator in leukemic cells in patients with chronic myeloid leukemia: role of BCR/ABL, characterization of underlying signaling pathways, and reexpression by novel pharmacologic compounds. Cancer research 65, 9436-44. 136. Kuribara, R., Honda, H., Matsui, H., Shinjyo, T., Inukai, T., Sugita, K., Nakazawa, S., Hirai, H., Ozawa, K. & Inaba, T. (2004). Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors. Molecular and cellular biology 24, 6172-83. 137. Cragg, M. S., Kuroda, J., Puthalakath, H., Huang, D. C. & Strasser, A. (2007). Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics. PLoS medicine 4, 1681-89; discussion 1690. 138. Costa, D. B., Halmos, B., Kumar, A., Schumer, S. T., Huberman, M. S., Boggon, T. J., Tenen, D. G. & Kobayashi, S. (2007). BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS medicine 4, 1669-79; discussion 1680. 139. Luciano, F., Jacquel, A., Colosetti, P., Herrant, M., Cagnol, S., Pages, G. & Auberger, P. (2003). Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene 22, 6785-93. 143 140. Ng, K. P., Hillmer, A. M., Chuah, C. T., Juan, W. C., Ko, T. K., Teo, A. S., Ariyaratne, P. N., Takahashi, N., Sawada, K., Fei, Y., Soh, S., Lee, W. H., Huang, J. W., Allen, J. C., Jr., Woo, X. Y., Nagarajan, N., Kumar, V., Thalamuthu, A., Poh, W. T., Ang, A. L., Mya, H. T., How, G. F., Yang, L. Y., Koh, L. P., Chowbay, B., Chang, C. T., Nadarajan, V. S., Chng, W. J., Than, H., Lim, L. C., Goh, Y. T., Zhang, S., Poh, D., Tan, P., Seet, J. E., Ang, M. K., Chau, N. M., Ng, Q. S., Tan, D. S., Soda, M., Isobe, K., Nothen, M. M., Wong, T. Y., Shahab, A., Ruan, X., Cacheux-Rataboul, V., Sung, W. K., Tan, E. H., Yatabe, Y., Mano, H., Soo, R. A., Chin, T. M., Lim, W. T., Ruan, Y. & Ong, S. T. (2012). A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nature medicine 18, 521-8. 141. O'Connor, L., Strasser, A., O'Reilly, L. A., Hausmann, G., Adams, J. M., Cory, S. & Huang, D. C. (1998). Bim: a novel member of the Bcl-2 family that promotes apoptosis. The EMBO journal 17, 384-95. 142. U, M., Miyashita, T., Shikama, Y., Tadokoro, K. & Yamada, M. (2001). Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family. FEBS letters 509, 135-41. 143. Black, D. L. (2003). Mechanisms of alternative pre-messenger RNA splicing. Annual review of biochemistry 72, 291-336. 144. Carstens, R. P., McKeehan, W. L. & Garcia-Blanco, M. A. (1998). An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor pre-mRNA splicing. Molecular and cellular biology 18, 2205-17. 145. Kapustin, Y., Chan, E., Sarkar, R., Wong, F., Vorechovsky, I., Winston, R. M., Tatusova, T. & Dibb, N. J. (2011). Cryptic splice sites and split genes. Nucleic acids research 39, 5837-44. 146. Hastings, M. L., Resta, N., Traum, D., Stella, A., Guanti, G. & Krainer, A. R. (2005). An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites. Nature structural & molecular biology 12, 54-9. 147. Wang, G. S. & Cooper, T. A. (2007). Splicing in disease: disruption of the splicing code and the decoding machinery. Nature reviews. Genetics 8, 749-61. 148. Adachi, M., Zhao, X. & Imai, K. (2005). Nomenclature of dynein light chain-linked BH3-only protein Bim isoforms. Cell death and differentiation 12, 192-3. 149. Liu, Y., Li, H., Tanaka, K., Tsumaki, N. & Yamada, Y. (2000). Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene. The Journal of biological chemistry 275, 12712-8. 150. Brooks, A. R., Nagy, B. P., Taylor, S., Simonet, W. S., Taylor, J. M. & Levy-Wilson, B. (1994). Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice. Molecular and cellular biology 14, 2243-56. 144 151. Deininger, M. W., Goldman, J. M., Lydon, N. & Melo, J. V. (1997). The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 90, 3691-8. 152. Oltersdorf, T., Elmore, S. W., Shoemaker, A. R., Armstrong, R. C., Augeri, D. J., Belli, B. A., Bruncko, M., Deckwerth, T. L., Dinges, J., Hajduk, P. J., Joseph, M. K., Kitada, S., Korsmeyer, S. J., Kunzer, A. R., Letai, A., Li, C., Mitten, M. J., Nettesheim, D. G., Ng, S., Nimmer, P. M., O'Connor, J. M., Oleksijew, A., Petros, A. M., Reed, J. C., Shen, W., Tahir, S. K., Thompson, C. B., Tomaselli, K. J., Wang, B., Wendt, M. D., Zhang, H., Fesik, S. W. & Rosenberg, S. H. (2005). An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677-81. 153. Gazdar, A. F., Gao, B. & Minna, J. D. (2010). Lung cancer cell lines: Useless artifacts or invaluable tools for medical science? Lung cancer 68, 309-18. 154. Gazdar, A. F., Girard, L., Lockwood, W. W., Lam, W. L. & Minna, J. D. (2010). Lung cancer cell lines as tools for biomedical discovery and research. Journal of the National Cancer Institute 102, 1310-21. 155. Lu, Y., Liang, K., Li, X. & Fan, Z. (2007). Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor1alpha. Molecular cancer 6, 63. 156. Coldren, C. D., Helfrich, B. A., Witta, S. E., Sugita, M., Lapadat, R., Zeng, C., Baron, A., Franklin, W. A., Hirsch, F. R., Geraci, M. W. & Bunn, P. A., Jr. (2006). Baseline gene expression predicts sensitivity to gefitinib in non-small cell lung cancer cell lines. Molecular cancer research : MCR 4, 521-8. 157. Chen, M. & Manley, J. L. (2009). Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nature reviews. Molecular cell biology 10, 741-54. 158. McManus, C. J. & Graveley, B. R. (2011). RNA structure and the mechanisms of alternative splicing. Current opinion in genetics & development 21, 373-9. 159. Barbon, A. & Barlati, S. (2000). Genomic organization, proposed alternative splicing mechanisms, and RNA editing structure of GRIK1. Cytogenetics and cell genetics 88, 236-9. 160. Sheth, N., Roca, X., Hastings, M. L., Roeder, T., Krainer, A. R. & Sachidanandam, R. (2006). Comprehensive splice-site analysis using comparative genomics. Nucleic acids research 34, 3955-67. 161. Licatalosi, D. D. & Darnell, R. B. (2010). RNA processing and its regulation: global insights into biological networks. Nature reviews. Genetics 11, 75-87. 162. Zhou, Z. & Fu, X. D. (2013). Regulation of splicing by SR proteins and SR proteinspecific kinases. Chromosoma 122, 191-207. 145 163. Mauger, D. M., Lin, C. & Garcia-Blanco, M. A. (2008). hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor exon IIIc. Molecular and cellular biology 28, 5403-19. 164. Chou, M. Y., Rooke, N., Turck, C. W. & Black, D. L. (1999). hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Molecular and cellular biology 19, 69-77. 165. Crawford, J. B. & Patton, J. G. (2006). Activation of alpha-tropomyosin exon is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F. Molecular and cellular biology 26, 8791-802. 166. Chen, C. D., Kobayashi, R. & Helfman, D. M. (1999). Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes & development 13, 593-606. 167. Garneau, D., Revil, T., Fisette, J. F. & Chabot, B. (2005). Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x. The Journal of biological chemistry 280, 22641-50. 168. Han, K., Yeo, G., An, P., Burge, C. B. & Grabowski, P. J. (2005). A combinatorial code for splicing silencing: UAGG and GGGG motifs. PLoS biology 3, e158. 169. Sawicka, K., Bushell, M., Spriggs, K. A. & Willis, A. E. (2008). Polypyrimidinetract-binding protein: a multifunctional RNA-binding protein. Biochemical Society transactions 36, 641-7. 170. Yeowell, H. N., Walker, L. C., Mauger, D. M., Seth, P. & Garcia-Blanco, M. A. (2009). TIA nuclear proteins regulate the alternate splicing of lysyl hydroxylase 2. The Journal of investigative dermatology 129, 1402-11. 171. Paz, I., Akerman, M., Dror, I., Kosti, I. & Mandel-Gutfreund, Y. (2010). SFmap: a web server for motif analysis and prediction of splicing factor binding sites. Nucleic acids research 38, W281-5. 172. Zhang, X. H. & Chasin, L. A. (2004). Computational definition of sequence motifs governing constitutive exon splicing. Genes & development 18, 1241-50. 173. Stamm, S., Riethoven, J. J., Le Texier, V., Gopalakrishnan, C., Kumanduri, V., Tang, Y., Barbosa-Morais, N. L. & Thanaraj, T. A. (2006). ASD: a bioinformatics resource on alternative splicing. Nucleic acids research 34, D46-55. 174. Desmet, F. O., Hamroun, D., Lalande, M., Collod-Beroud, G., Claustres, M. & Beroud, C. (2009). Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic acids research 37, e67. 175. Piva, F., Giulietti, M., Nocchi, L. & Principato, G. (2009). SpliceAid: a database of experimental RNA target motifs bound by splicing proteins in humans. Bioinformatics 25, 1211-3. 146 176. Carstens, R. P., Wagner, E. J. & Garcia-Blanco, M. A. (2000). An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor through involvement of polypyrimidine tract binding protein. Molecular and cellular biology 20, 7388-400. 177. Chan, R. C. & Black, D. L. (1997). The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream. Molecular and cellular biology 17, 4667-76. 178. Mulligan, G. J., Guo, W., Wormsley, S. & Helfman, D. M. (1992). Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of betatropomyosin pre-mRNA. The Journal of biological chemistry 267, 25480-7. 179. Xue, Y., Zhou, Y., Wu, T., Zhu, T., Ji, X., Kwon, Y. S., Zhang, C., Yeo, G., Black, D. L., Sun, H., Fu, X. D. & Zhang, Y. (2009). Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Molecular cell 36, 996-1006. 180. Hubner, A., Barrett, T., Flavell, R. A. & Davis, R. J. (2008). Multisite phosphorylation regulates Bim stability and apoptotic activity. Molecular cell 30, 415-25. 181. Wiggins, C. M., Johnson, M. & Cook, S. J. (2010). Refining the minimal sequence required for ERK1/2-dependent poly-ubiquitination and proteasome-dependent turnover of BIM. Cellular signalling 22, 801-8. 182. Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M. & Ule, J. (2010). iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nature structural & molecular biology 17, 909-15. 183. Tanizaki, J., Okamoto, I., Fumita, S., Okamoto, W., Nishio, K. & Nakagawa, K. (2011). Roles of BIM induction and survivin downregulation in lapatinib-induced apoptosis in breast cancer cells with HER2 amplification. Oncogene 30, 4097-106. 184. Gordon, P. M. & Fisher, D. E. (2010). Role for the proapoptotic factor BIM in mediating imatinib-induced apoptosis in a c-KIT-dependent gastrointestinal stromal tumor cell line. The Journal of biological chemistry 285, 14109-14. 185. Au, W. Y., Caguioa, P. B., Chuah, C., Hsu, S. C., Jootar, S., Kim, D. W., Kweon, I. Y., O'Neil, W. M., Saikia, T. K. & Wang, J. (2009). Chronic myeloid leukemia in Asia. International journal of hematology 89, 14-23. 186. Hentschel, J., Rubio, I., Eberhart, M., Hipler, C., Schiefner, J., Schubert, K., Loncarevic, I. F., Wittig, U., Baniahmad, A. & von Eggeling, F. (2011). BCR-ABLand Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML. International journal of oncology 39, 585-91. 187. Pellegrini, M., Belz, G., Bouillet, P. & Strasser, A. (2003). Shutdown of an acute T cell immune response to viral infection is mediated by the proapoptotic Bcl-2 147 homology 3-only protein Bim. Proceedings of the National Academy of Sciences of the United States of America 100, 14175-80. 188. Bouillet, P., Purton, J. F., Godfrey, D. I., Zhang, L. C., Coultas, L., Puthalakath, H., Pellegrini, M., Cory, S., Adams, J. M. & Strasser, A. (2002). BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature 415, 922-6. 189. Egle, A., Harris, A. W., Bouillet, P. & Cory, S. (2004). Bim is a suppressor of Mycinduced mouse B cell leukemia. Proceedings of the National Academy of Sciences of the United States of America 101, 6164-9. 190. Cragg, M. S., Harris, C., Strasser, A. & Scott, C. L. (2009). Unleashing the power of inhibitors of oncogenic kinases through BH3 mimetics. Nature reviews. Cancer 9, 321-6. 191. Kuroda, J., Kimura, S., Andreeff, M., Ashihara, E., Kamitsuji, Y., Yokota, A., Kawata, E., Takeuchi, M., Tanaka, R., Murotani, Y., Matsumoto, Y., Tanaka, H., Strasser, A., Taniwaki, M. & Maekawa, T. (2008). ABT-737 is a useful component of combinatory chemotherapies for chronic myeloid leukaemias with diverse drugresistance mechanisms. British journal of haematology 140, 181-90. 192. Wei, J., Stebbins, J. L., Kitada, S., Dash, R., Placzek, W., Rega, M. F., Wu, B., Cellitti, J., Zhai, D., Yang, L., Dahl, R., Fisher, P. B., Reed, J. C. & Pellecchia, M. (2010). BI-97C1, an optically pure Apogossypol derivative as pan-active inhibitor of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins. Journal of medicinal chemistry 53, 4166-76. 193. Goff, D. J., Recart, A. C., Sadarangani, A., Chun, H. J., Barrett, C. L., Krajewska, M., Leu, H., Low-Marchelli, J., Ma, W., Shih, A. Y., Wei, J., Zhai, D., Geron, I., Pu, M., Bao, L., Chuang, R., Balaian, L., Gotlib, J., Minden, M., Martinelli, G., Rusert, J., Dao, K. H., Shazand, K., Wentworth, P., Smith, K. M., Jamieson, C. A., Morris, S. R., Messer, K., Goldstein, L. S., Hudson, T. J., Marra, M., Frazer, K. A., Pellecchia, M., Reed, J. C. & Jamieson, C. H. (2013). A Pan-BCL2 inhibitor renders bonemarrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell stem cell 12, 316-28. 194. Youle, R. J. & Strasser, A. (2008). The BCL-2 protein family: opposing activities that mediate cell death. Nature reviews. Molecular cell biology 9, 47-59. 195. Nakagawa, T., Takeuchi, S., Yamada, T., Ebi, H., Sano, T., Nanjo, S., Ishikawa, D., Sato, M., Hasegawa, Y., Sekido, Y. & Yano, S. (2013). EGFR-TKI resistance due to BIM polymorphism can be circumvented in combination with HDAC inhibition. Cancer research 73, 2428-34. 196. Emery, A. E. (2002). The muscular dystrophies. Lancet 359, 687-95. 197. Aartsma-Rus, A., Fokkema, I., Verschuuren, J., Ginjaar, I., van Deutekom, J., van Ommen, G. J. & den Dunnen, J. T. (2009). Theoretic applicability of antisensemediated exon skipping for Duchenne muscular dystrophy mutations. Human mutation 30, 293-9. 148 198. Muntoni, F., Torelli, S. & Ferlini, A. (2003). Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet neurology 2, 731-40. 199. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L., Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J., Wells, D. J., Dickson, G., Wood, M. J., Wilton, S. D., Straub, V., Kole, R., Shrewsbury, S. B., Sewry, C., Morgan, J. E., Bushby, K. & Muntoni, F. (2011). Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 378, 595605. 200. Goemans, N. M., Tulinius, M., van den Akker, J. T., Burm, B. E., Ekhart, P. F., Heuvelmans, N., Holling, T., Janson, A. A., Platenburg, G. J., Sipkens, J. A., Sitsen, J. M., Aartsma-Rus, A., van Ommen, G. J., Buyse, G., Darin, N., Verschuuren, J. J., Campion, G. V., de Kimpe, S. J. & van Deutekom, J. C. (2011). Systemic administration of PRO051 in Duchenne's muscular dystrophy. The New England journal of medicine 364, 1513-22. 201. Marnett, L. J. & Plastaras, J. P. (2001). Endogenous DNA damage and mutation. Trends in genetics : TIG 17, 214-21. 202. Lopez-Bigas, N., Audit, B., Ouzounis, C., Parra, G. & Guigo, R. (2005). Are splicing mutations the most frequent cause of hereditary disease? FEBS letters 579, 1900-3. 203. Cartegni, L., Chew, S. L. & Krainer, A. R. (2002). Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature reviews. Genetics 3, 285-98. 204. D'Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V. M., Bird, T. D. & Schellenberg, G. D. (1999). Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. Proceedings of the National Academy of Sciences of the United States of America 96, 5598-603. 205. Lalonde, E., Ha, K. C., Wang, Z., Bemmo, A., Kleinman, C. L., Kwan, T., Pastinen, T. & Majewski, J. (2011). RNA sequencing reveals the role of splicing polymorphisms in regulating human gene expression. Genome research 21, 545-54. 206. Nembaware, V., Wolfe, K. H., Bettoni, F., Kelso, J. & Seoighe, C. (2004). Allelespecific transcript isoforms in human. FEBS letters 577, 233-8. 207. Gregory, S. G., Schmidt, S., Seth, P., Oksenberg, J. R., Hart, J., Prokop, A., Caillier, S. J., Ban, M., Goris, A., Barcellos, L. F., Lincoln, R., McCauley, J. L., Sawcer, S. J., Compston, D. A., Dubois, B., Hauser, S. L., Garcia-Blanco, M. A., Pericak-Vance, M. A. & Haines, J. L. (2007). Interleukin receptor alpha chain (IL7R) shows allelic and functional association with multiple sclerosis. Nature genetics 39, 1083-91. 208. Mazzucchelli, R. & Durum, S. K. (2007). Interleukin-7 receptor expression: intelligent design. Nature reviews. Immunology 7, 144-54. 149 209. Benzeno, S., Narla, G., Allina, J., Cheng, G. Z., Reeves, H. L., Banck, M. S., Odin, J. A., Diehl, J. A., Germain, D. & Friedman, S. L. (2004). Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1. Cancer research 64, 3885-91. 210. Narla, G., DiFeo, A., Yao, S., Banno, A., Hod, E., Reeves, H. L., Qiao, R. F., Camacho-Vanegas, O., Levine, A., Kirschenbaum, A., Chan, A. M., Friedman, S. L. & Martignetti, J. A. (2005). Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread. Cancer research 65, 5761-8. 211. Narla, G., Difeo, A., Reeves, H. L., Schaid, D. J., Hirshfeld, J., Hod, E., Katz, A., Isaacs, W. B., Hebbring, S., Komiya, A., McDonnell, S. K., Wiley, K. E., Jacobsen, S. J., Isaacs, S. D., Walsh, P. C., Zheng, S. L., Chang, B. L., Friedrichsen, D. M., Stanford, J. L., Ostrander, E. A., Chinnaiyan, A. M., Rubin, M. A., Xu, J., Thibodeau, S. N., Friedman, S. L. & Martignetti, J. A. (2005). A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer research 65, 1213-22. 212. Wohlke, A., Philipp, U., Bock, P., Beineke, A., Lichtner, P., Meitinger, T. & Distl, O. (2011). A one base pair deletion in the canine ATP13A2 gene causes exon skipping and late-onset neuronal ceroid lipofuscinosis in the Tibetan terrier. PLoS genetics 7, e1002304. 213. Goguel, V., Wang, Y. & Rosbash, M. (1993). Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing. Molecular and cellular biology 13, 6841-8. 214. Cornelis, S., Tinton, S. A., Schepens, B., Bruynooghe, Y. & Beyaert, R. (2005). UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein. Nucleic acids research 33, 3095-108. 215. Mitchell, S. A., Brown, E. C., Coldwell, M. J., Jackson, R. J. & Willis, A. E. (2001). Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras. Molecular and cellular biology 21, 3364-74. 216. He, X., Pool, M., Darcy, K. M., Lim, S. B., Auersperg, N., Coon, J. S. & Beck, W. T. (2007). Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro. Oncogene 26, 4961-8. 217. Ke, S. & Chasin, L. A. (2011). Context-dependent splicing regulation: exon definition, co-occurring motif pairs and tissue specificity. RNA biology 8, 384-8. 218. Min, H., Turck, C. W., Nikolic, J. M. & Black, D. L. (1997). A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes & development 11, 1023-36. 219. Foong, A. W., Saw, S. M., Loo, J. L., Shen, S., Loon, S. C., Rosman, M., Aung, T., Tan, D. T., Tai, E. S. & Wong, T. Y. (2007). Rationale and methodology for a population-based study of eye diseases in Malay people: The Singapore Malay eye study (SiMES). Ophthalmic epidemiology 14, 25-35. 150 220. Lavanya, R., Jeganathan, V. S., Zheng, Y., Raju, P., Cheung, N., Tai, E. S., Wang, J. J., Lamoureux, E., Mitchell, P., Young, T. L., Cajucom-Uy, H., Foster, P. J., Aung, T., Saw, S. M. & Wong, T. Y. (2009). Methodology of the Singapore Indian Chinese Cohort (SICC) eye study: quantifying ethnic variations in the epidemiology of eye diseases in Asians. Ophthalmic epidemiology 16, 325-36. 221. Frazer, K. A., Ballinger, D. G., Cox, D. R., Hinds, D. A., Stuve, L. L., Gibbs, R. A., Belmont, J. W., Boudreau, A., Hardenbol, P., Leal, S. M., Pasternak, S., Wheeler, D. A., Willis, T. D., Yu, F., Yang, H., Zeng, C., Gao, Y., Hu, H., Hu, W., Li, C., Lin, W., Liu, S., Pan, H., Tang, X., Wang, J., Wang, W., Yu, J., Zhang, B., Zhang, Q., Zhao, H., Zhou, J., Gabriel, S. B., Barry, R., Blumenstiel, B., Camargo, A., Defelice, M., Faggart, M., Goyette, M., Gupta, S., Moore, J., Nguyen, H., Onofrio, R. C., Parkin, M., Roy, J., Stahl, E., Winchester, E., Ziaugra, L., Altshuler, D., Shen, Y., Yao, Z., Huang, W., Chu, X., He, Y., Jin, L., Liu, Y., Sun, W., Wang, H., Wang, Y., Xiong, X., Xu, L., Waye, M. M., Tsui, S. K., Xue, H., Wong, J. T., Galver, L. M., Fan, J. B., Gunderson, K., Murray, S. S., Oliphant, A. R., Chee, M. S., Montpetit, A., Chagnon, F., Ferretti, V., Leboeuf, M., Olivier, J. F., Phillips, M. S., Roumy, S., Sallee, C., Verner, A., Hudson, T. J., Kwok, P. Y., Cai, D., Koboldt, D. C., Miller, R. D., Pawlikowska, L., Taillon-Miller, P., Xiao, M., Tsui, L. C., Mak, W., Song, Y. Q., Tam, P. K., Nakamura, Y., Kawaguchi, T., Kitamoto, T., Morizono, T., Nagashima, A., Ohnishi, Y., Sekine, A., Tanaka, T., Tsunoda, T., et al. (2007). A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-61. 222. Wagner, E. J. & Garcia-Blanco, M. A. (2002). RNAi-mediated PTB depletion leads to enhanced exon definition. Molecular cell 10, 943-9. 151 [...]... Extracellular domain Cytoplasmic domain Figure 1: Domain organization of receptor tyrosine kinases Receptor tyrosine kinases consist of an extracellular domain that contains a ligand-binding site, a transmembrane helix 2 and a cytoplasmic domain The cytoplasmic domain contains a kinase active site that catalyzes tyrosine phosphorylation Because tyrosine kinases play important roles in signal transduction... non-receptor tyrosine kinases In an inactive state, the kinase activity of ABL1 and SRC are repressed by intramolecular interactions8 Mutating the Src homology-3 (SH3) domain of ABL1 has been shown to activate its kinase activity These results suggest that the SH3 domain of ABL1 is able to participate in intramolecular interactions to inhibit ABL1’s kinase activity9 Apart from intramolecular interactions,... have demonstrated that oncogenic kinases, such as BCRABL1 and EGFR, play an important role in malignant transformation, a rational approach to treat kinase- driven cancers is to develop targeted therapies against the kinases driving these diseases Small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies are two classes of drugs that have been developed to target oncogenic kinases In. .. JAK-STAT pathways are downstream pathways activated by tyrosine kinases When these pathways are constitutively activated, they can mediate malignant transformation by deregulating cell proliferation, inhibiting the induction of apoptosis and enhancing telomerase activity MAP kinase pathway The MAP kinase pathway consists of a phosphorylation cascade that regulates gene transcription The phosphorylation... Mechanism of receptor tyrosine kinase activation The epidermal growth factor receptor (EGFR) is an example of a receptor tyrosine kinase EGFR is activated upon binding of the epidermal growth factor to the receptor5 This leads to the recruitment of downstream signaling proteins such as rat sarcoma (RAS) protein and phosphatidylinositol 3 -kinase (PI3K) Activation of these signaling proteins is essential for... tyrosine phosphorylation generates binding sites for proteins containing Src homology-2 (SH2) and protein tyrosine- binding (PTB) domains4 Two families of tyrosine kinases exist within cells They are the receptor tyrosine kinases as well as the non-receptor tyrosine kinases Receptor tyrosine kinases are transmembrane glycoproteins that are able to phosphorylate on tyrosine residues within the receptors and... monitoring70 1.5 MOLECULAR BASIS OF RESISTANCE TO TYROSINE KINASE INHIBITORS Generally, the mechanisms of resistance to TKIs can be grouped into two main categories First, resistance mechanisms can be oncogenic kinase- dependent such as mutations in the kinase domain that reduce the binding efficiency of TKIs, or overexpression of the 13 oncogenic kinases Second, factors mediating resistance to TKIs can... signaling proteins that associate with the receptors These kinases consist of a glycosylated extracellular domain that is responsible for binding to ligands, a transmembrane helix and a cytoplasmic domain that harbor tyrosine kinase activity as well as additional regulatory residues that are subjected to phosphorylation (Figure 1)1 Exterior Cell membrane Cytoplasm Transmembrane helix Kinase catalytic... promoting cell proliferation and survival6 Non-receptor tyrosine kinases are another class of tyrosine kinases Unlike receptor tyrosine kinases, non-receptor tyrosine kinases do not possess a transmembrane domain and 3 they are usually found in the cytoplasm7 Abelson murine leukemia viral oncogene homolog 1 (ABL1), v-src sarcoma viral oncogene homolog (SRC) and Janus kinases (JAKs) are examples of non-receptor... factors11 1.2 THE ROLE OF TYROSINE KINASES IN CANCER Numerous studies have demonstrated that tyrosine kinases play an important role in the development as well as the progression of cancer2 ; 12; 13 Although the activity of tyrosine kinases are tightly regulated in normal cells, these kinases are usually targets of oncogenic mutations, which generate a constitutively activated tyrokine kinase that can . membrane Cytoplasm Extracellular domain Cytoplasmic domain Transmembrane helix Kinase catalytic region Figure 1: Domain organization of receptor tyrosine kinases. Receptor tyrosine kinases. role of tyrosine kinases in cancer …………………………………… 4 1.3 Tyrosine kinase inhibitors as therapeutic agents in cancer ……………… 9 1.4 Clinical resistance to tyrosine kinase inhibitors …………………………. FUNCTIONAL EFFECTS OF A NOVEL BIM DELETION POLYMORPHISM IN MEDIATING RESISTANCE TO TYROSINE KINASE INHIBITORS IN CANCER JUAN WEN CHUN B.Sc. (Hons), NATIONAL UNIVERSITY OF SINGAPORE