Mechanism of Acquired Temozolomide Resistance in Glioblastoma AAcHU$ETS MAssACHu N by O F TEScHN OLO NGY E MAY 2 8 2014 Jose L. McFaline-Figueroa LIBRARIES B.S. Chemistry University of Puerto Rico at Mayaguez, 2006 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY JUNE 2014 © 2014 Massachusetts Ir4tute of Technology Signature redacted Signature of Author artment of Biology izt%014 Signature redacted Certified by Leona u. Samson Professor of Biological Engineering and Biology Signature redactedThesis Supervisor Certified by Forest. M. White Professor of Biological Engineering and Biology Thesis,Supervisor Signatu re redacted K_ Accepted by \XAmy E. Keating Associate Professor of Biology Co-Chair, Biology Graduate Committee Mechanism of Acquired Temozolomide Resistance in Glioblastoma by Jose L. McFaline-Figueroa Abstract Glioblastoma (GBM) is the most common and malignant form of brain cancer. After aggressive treatment, therapy resistant tumors inevitably recur. However, the molecular mechanisms underlying such resistance remain unclear. We isolated GBM cells resistant to temozolomide (TMZ), the frontline chemotherapy agent for GBM, and observed modest decreases in the mismatch repair (MMR) components MSH2 and MSH6. The modest decrease in MSH2, and relatively modest decrease in MSH6, did not seem sufficient to account for the very large increase in TMZ resistance. However, shRNA-mediated modulation of MSH2 and MSH6 levels in vitro confirmed that such decreases in MSH2 and MSH6 provide a potent mechanism for TMZ resistance. We demonstrate in an in vivo GBM mouse model that minor changes in MSH2 suppress TMZ-induced tumor regression, and moreover, show that even minor decreases in MSH2 transcript levels correlate with decreased survival in TMZ treated GBM patients. These modest changes in MMR are unlikely to alter classical markers of MMR deficiency, namely microsatellite instability and a mutator phenotype. Our results suggest that the involvement of MMR deregulation in mediating TMZ resistance is likely to be much more prevalent than previously appreciated. Additionally, we have employed phosphoproteomic network analysis to identify changes at the signaling network level that accompany the acquisition of TMZ resistance. Through mathematical and computational approaches, we identified changes that suggest increased PDGFR and integrin/FAK1 signaling in response to repeated TMZ exposure. Additionally, kinase motif analysis identified widespread alterations in phosphorylation of peptides containing motifs associated with the CDK/MAPK kinase family. Currently, we are applying molecular biology techniques to investigate the effects of these altered cellular signals on MMR activity and the sensitivity of GBM cells to TMZ. Thesis Supervisor: Leona D. Samson Title: Professor of Biological Engineering and Biology Thesis Supervisor: Forest M. White Title: Professor of Biological Engineering 2 Acknowledgements I begin by expressing my deepest gratitude to my advisors Leona D. Samson and Forest M. White for their support, motivation and guidance throughout my graduate career, which has allowed me to grow as a scientist and a teacher. I would like to thank the members of my committee Jackie Lees and Mike Hemann for their counsel and contributions to my project. I am grateful for all of my friends and colleagues from the Samson and White laboratories for their endless advice, encouragement and fun times. Additionally, I would like to thank all of my friends from the Biology, Biological Engineering and Koch Institute communities for making my time as a graduate student both educational and extremely entertaining. I would like to thank my family. To my beautiful wife Nelly, thank you for all of your support through out the years. Thanks to Ana, Jos6 Enrique, Cristina and Jose Guilllermo for taking me into their family. To my uncles Agnelis, Dahri, Manolo, Lizette and cousins Raul, Mariela and Daniel thank you for your encouragement and friendship. Thanks to my brother Ricky and my sister Jennifer for an absolute fantastic time growing up and continued support. To my little brother Luis Manuel for letting me be a kid again. To my parents, Magaly and Berto for innumerable things, especially for always pushing me to go forward and achieve my dreams. Thanks to my gradparents Casilda, Octavio, Natividad and Luis, for their inspiration and for being the best cheerleaders for my siblings and myself. All of you have allowed me to be where I am today and I find myself truly blessed and grateful. 3 Table of Contents A b s tra c t .............................................................................................................................. 2 Acknowledgements ...................................................................................................... 3 Table of Contents ....................................................................................................... 4 List of Figures ...................................................................................................... 7 L is t o f T a b le s .............................................................................................................. 1 0 List of Abbreviations ........................................................................................... 11 Chapters...... ....................................................................................................... 14 Chapter 1: Introduction.......................................................................................... 16 W HO grade IV gliom a or Glioblastom a M ultiforme................................................ 16 Classification of GBM subtypes............................................................................. 17 Current standard of care for patients with initial GBM presentation ..................... 20 Temozolomide, the main chemotherapeutic agent in the treatment of GBM ........... 21 DNA repair pathways that alter sensitivity to TMZ................................................ 24 Genetic changes and signaling network nodes proposed to alter the sensitivity of G BM cells to TMZ................................................................................................ 26 Glioblastoma cancer stem cells and response of GBM to therapy........................ 28 Temozolom ide resistance in recurrent GBM ........................................................ 31 Overview of the current study ............................................................................... 32 F ig u re s ...................................................................................................................... 3 4 R e fe re n c e s ............................................................................................................... 3 8 Chapter II: Response of glioblastoma cells to acute and periodic temozolomide exposure.....................................................................................................................50 Int ro d u c tio n ............................................................................................................... 5 0 Materials and m ethods ......................................................................................... 51 4 R e s u lts ...................................................................................................................... 5 8 p53 status alters the magnitude and the resolution of TMZ-induced G2/M arrest but not the sensitivity of GBM cells to acute TMZ exposure.............................. 59 Generation of TMZ resistant p53 proficient and p53 deficient GBM cells by periodic exposure to escalating doses of TMZ .................................................. 60 The TMZ resistant phenotype is specific for 06-meG formation and does not confer resistance to ionizing radiation or 1,3-bis-(2-chloroethyl)-1-nitroso-urea (B C N U ) ................................................................................................................. 6 2 TMZR3 cells do not express increased M GMT protein ....................................... 63 MMR protein levels and activity are deregulated in TMZR3 cells ............ 64 D is c u s s io n ................................................................................................................ 6 5 F ig u re s ...................................................................................................................... 6 7 R e fe re n c e s' .............................................................................................................. 8 9 Chapter III: Minor decreases in MSH2 leads to major changes in the response of glioblastom a to chem otherapy............................................................................ 94 In tro d u c tio n ............................................................................................................... 9 4 M aterials and methods ......................................................................................... 96 R e s u lts .................................................................................................................... 1 0 1 Very limited knockdown of MSH2 protein levels leads to extensive TMZ chemoresistance in GBM cells in vitro................................................................101 Small reductions in Msh2 decrease the in vivo response of GBM tumors to TMZ tre a tm e n t................................................................................................................. 1 0 3 MSH2 transcript levels are predictive for the overall survival of TMZ treated primary GBM patients ......................................................................................... 104 D is c u s s io n .............................................................................................................. 1 0 6 F ig u re s .................................................................................................................... 1 1 1 5 R e fe re n c es ............................................................................................................. 1 3 1 Chapter 4: Phosphoproteomic profiling of parental and TMZR3 GBM cells.......136 In tro d u c tio n ............................................................................................................. 1 36 Materials and Methods ........................................................................................... 138 R es u lts .................................................................................................................... 1 4 1 Phosphoproteomic profiling of parental and TMZ resistant GBM cells............... 141 ANOVA analysis identifies phosphotyrosine sites that vary significantly by p53 status and/or TMZ sensitivity..............................................................................142 Identification of phosphoserine/threonine sites that vary significantly by p53 status and/or TMZ sensitivity .............................................................................. 145 Kinase enrichment and substrate motif analysis of TMZR3 GBM cells................ 146 D is c u s s io n .............................................................................................................. 1 49 F u tu re g o a ls ........................................................................................................ 1 5 1 F ig u re s .................................................................................................................... 1 5 3 R ef e re n c e s ............................................................................................................. 1 7 1 Chapter V: Discussion ............................................................................................ 178 Key concepts and conclusions ............................................................................... 178 Increased MGMT activity is not selected for in TMZR3 GBM cells .......................... 179 Minor decreases in MMR components alter the sensitivity of GBM to TMZ........... 180 Exploring the role of additional DNA repair and damage tolerance pathways on the resistant phenotype of TMZR3 GBM cells ............................................................... 182 Systems level profiling of TMZ sensitive and resistant GBM cells ......................... 184 R ef e re n c e s ............................................................................................................. 1 8 9 6 List of Figures Figure 1.1 Survival differences amongst GBM subtypes as classified by mRNA e x p re s s io n . ............................................................................................................... 3 4 Figure 1.2 Survival differences amongst GBM subtypes as classified by miRNA e x p re s s io n . ............................................................................................................... 35 Figure 1.3 TMZ is non-enzymatically metabolized to the reactive methyldiazonium ion.. 36 Figure 1.4 Fate of TMZ induced 06-meG lesions. ....................................................... 37 Figure 2.1 p53 status does not alter the sensitivity of GBM cells to TMZ..................... 67 Figure 2.2 TMZ treatment induces an accumulation of cells at 4N two cell cycles post- treatment in Control and p53kd cells. .................................................................. 68 Figure 2.3 BrdU incorporation and DNA content staining reveals robust cell cycle changes in Control and p53kd cells after acute TMZ exposure............................ 69 Figure 2.4 Gating used for the quantitation of cell cycle phase accumulation and cell cycle distribution of untreated Control and p53kd cells. ....................................... 70 Figure 2.5 Quantitation of cell cycle phase accumulation in TMZ treated Control and p53kd cells as measured by BrdU incorporation and DNA content staining. ........... 71 Figure 2.6 TMZ treatment leads to Chk2 and H2AX activation in Control and p53kd GBM c e lls ........................................................................................................................... 7 2 Figure 2.7 Generation of an in vitro model of acquired TMZ resistance in GBM.......... 73 Figure 2.8 TMZR3 cells obtained from a p53 deficient background display increased p lo id y . ....................................................................................................................... 7 5 Figure 2.9 TMZR3 GBM cells exhibit decreased G2/M accumulation two cell cycles post- T M Z ex p os u re ........................................................................................................... 7 7 Figure 2.10 TMZR3 GBM cells exhibit decreased H2AX activation two cell cycles post- T M Z ex p os u re ........................................................................................................... 7 8 7 Figure 2.11 TMZR3 GBM cells display cross-resistance to MNNG but not to IR or BCNU.80 Figure 2.12 The TMZ resistant phenotype in TMZR3 GBM cells is not due to increased repair of 06-m ethylguanine lesions...................................................................... 82 Figure 2.13 TMZR3 GBM cells exhibit decreased MMR component levels................... 84 Figure 2.14 Decreased MMR component levels in TMZR3 GBM cells correlates with decreased M M R activity ....................................................................................... 85 Figure 3.1 Panel of MSH6 and MSH2 knockdown GBM cells........................................ 111 Figure 3.2 Modulation of MSH6 and MSH2 levels and TMZ sensitivity. ........................ 112 Figure 3.3 Extent of G2/M accumulation post-TMZ exposure correlates with sensitivity in M S H 6 kno ckdow n ce lls. ......................................................................................... 114 Figure 3.4 Extent of G2/M accumulation post-TMZ exposure correlates with sensitivity in M S H 2 kno ckdow n ce lls. ......................................................................................... 115 Figure 3.5 Relationship between MSH levels and response to TMZ. ............................ 116 Figure 3.6 Small decreases in MSH2 protein alter MSH6 protein levels and lead to decreased m ism atch repair activity........................................................................ 117 Figure 3.7 Decreased MMR activity correlates with TMZ resistance in MSH6 and MSH2 knockd ow n G B M ce lls. ........................................................................................... 1 18 Figure 3.8 Model for MSH induced decreased MMR activity. ........................................ 119 Figure 3.9 Investigating the effects of minor Msh2 decreases on the response of GBM tu m o rs to T M Z ........................................................................................................ 12 0 Figure 3.10 Small decreases in Msh2 confer a growth advantage to GBM tumors after T M Z c h a lle n g e ........................................................................................................ 12 2 Figure 3.11 MSH2 levels are predictive for the survival of TMZ treated GBM patients.. 124 Figure 3.12 Distribution of patient survival in TMZ treated TCGA GBM patients........... 125 Figure 3.13 MSH2 and MSH6 are predictive for survival in TMZ treated 95th percentile G B M p at ie n ts .......................................................................................................... 12 6 8 Figure 3.14 MGMT levels are predictive of survival in TMZ treated 95th percentile GBM p at ie n ts ................................................................................................................... 1 2 8 Figure 3.15 Overall survival of GBM patients stratified by MSH3, MLH1 and PMS2 tumor tra n s c rip t le v e ls ....................................................................................................... 12 9 Figure 4.1 Phosphoproteomic profiling of parental and TMZR3 GBM cells. .................... 153 Figure 4.2 Hierarchical clustering of phosphopeptides identified in this study. ............. 155 Figure 4.3 ANOVA approach for identifying phosphopeptides that vary significantly by p53 status or T M Z treatm ent. ................................................................................. 156 Figure 4.4 pY containing phosphopeptides that vary due to p53 status......................... 158 Figure 4.5 pY containing phosphopeptides that vary due to TMZ sensitivity. ................ 159 Figure 4.6 Grouping of pY containing phosphopeptides that vary due to TMZ sensitivity.160 Figure 4.7 Hierarchical clustering of pS/T containing peptides identified as varying by p53 status o r T M Z sensitivity. ................................................................................. 162 Figure 4.8 Select pS/T containing sites that vary due to p53 status or TMZ sensitivity. 163 Figure 4.9 K-means clustering of pS/T sites that vary due to TMZ sensitivity................ 164 Figure 4.10 K-means clustering of pS/T sites that vary due to p53 status. .................... 165 9 List of Tables Table 2.1 Parental and TMZR3 GBM cells accumulate equal 06-meG levels upon TMZ e x p o s u re ................................................................................................................... 8 7 Table 2.2 shRNA constructs used in this study............................................................. 88 Table 3.1 shRNA constructs used in this study.............................................................. 130 Table 4.1 Results of Kinase Enrichment analysis of proteins containing phosphorylation sites that vary due to TMZ sensitivity. .................................................................... 166 Table 4.2 Sequence motifs enriched in phosphorylation sites that vary significantly due to T M Z se n s itiv ity .................................................................................................... 16 7 Table 4.3 Sequence motifs enriched in all pS/T containing phosphopeptides sites identified in this study prior to ANOVA filtering....................................................... 168 Table 4.4 Sequence motifs enriched in k-means clusters of phosphorylation sites that vary significantly due to TMZ sensitivity. ................................................................ 169 Table 4.5 Sequence motifs enriched in k-means clusters of phosphorylation sites that vary significantly due to p53 status......................................................................... 170 10