UNIVERSIDADE DE SAO~ PAULO FACULDADE DE MEDICINA DE RIBEIRAO~ PRETO PROGRAMA DE POS-GRADUAC¸´ AO~ EM GENETICA´ THA´ıSSARRAF SABEDOT Molecular classification of adult diffuse gliomas based on DNA methylation reveals subgroups of G-CIMP tumors associated with distinct clinical features Ribeir~aoPreto 2018 THA´ıSSARRAF SABEDOT Molecular classification of adult diffuse gliomas based on DNA methylation reveals subgroups of G-CIMP tumors associated with distinct clinical features Original version Doctoral thesis submitted to the Ribeir~ao Preto Medical School { FMRP-USP, in partial fulfillment of the requirements to obtain a doctoral degree (PhD) in Science. Area: Genetics Advisor: Prof. Dr. Houtan Noushmehr Ribeir~aoPreto 2018 Autorizo a reprodução e divulgação total ou parcial deste trabalho, por qualquer meio convencional ou eletrônico, para fins de estudo e pesquisa, desde que citada a fonte. I authorize the reproduction and total or partial dissemination of this study, by electronic or conventional forms, to research and teaching purposes, provided appropriately citation. Ficha catalográfica elaborada pela Biblioteca Central da USP Ribeirão Preto com os dados fornecidos pelo(a) autor(a) Sabedot, Thais Sarraf S115m Molecular classification of adult diffuse gliomas based on DNA methylation reveals subgroups of G-CIMP tumors associated with distinct clinical features. Ribeirão Preto, 2018. 121 p. : il. ; 30 cm Doctoral thesis (Doctorate Candidate - Program in Genetics) - Ribeirão Preto Medical School (FMRP/USP), USP, 2018. Area: Genetics Advisor: Noushmehr, Houtan 1. DNA methylation. 2. Glioma. 3. G-CIMP 4. Epigenetics 5. Bioinformatics Tese de autoria de Tha´ısSarraf Sabedot, sob o t´ıtulo \Molecular classification of adult diffuse gliomas based on DNA methylation reveals subgroups of G-CIMP tumors associated with distinct clinical features", apresentada `aFaculdade de Medicina de Ribeir~aoPreto da Universidade de S~aoPaulo, para obten¸c~aodo t´ıtulode Doutora em Ci^enciaspelo Programa de P´os-gradua¸c~aoem Gen´etica,aprovada em de de pela comiss~aojulgadora constitu´ıdapelos doutores: Prof. Dr. Institui¸c~ao: Presidente Prof. Dr. Institui¸c~ao: Prof. Dr. Institui¸c~ao: Prof. Dr. Institui¸c~ao: Prof. Dr. Institui¸c~ao: Dedicated to the memory of my biggest fan in the whole world, my beloved grandmother Irene Acknowledgements I would like to thank my parents and my family for loving me unconditionally and for making sure all my dreams come true. I would also like to thank my best friends Amanda, Isabel, Nat´alia,Tathi e Maritza. You have been by my side through all this journey and I can not thank you enough for your constant support. I am grateful for all my lab mates, more importantly my friends, from OMICs laboratory whom have taught me so much over the years that it is hard to condense in one paragraph. I would like to thank all employees and professors from the Department of Genetics at USP and from the Department of Neurosurgery at HFH, specially Nancy Takacs and Conni Kick. Also, I would like to thank Ana Valeria Castro, Laila Poisson, Ana de Carvalho, Jim Snyder, George Divine for all the stimulating discussions and insightful comments. In special, I am grateful for having Houtan Noushmehr as my mentor. There are no words to thank you enough for everything you did to me. Finally, I would like to thank S~aoPaulo Research Foundation (FAPESP) for the financial support and predoctoral fellowship (Processes # 2016/06488-3 and # 2016/12329- 5). Resumo SABEDOT, TS. Classifica¸c~aomolecular de gliomas difusos em adulto baseada em metila¸c~aodo DNA revela subgrupos de tumores G-CIMP associados com aspectos cl´ınicosdistintos. 2018. 121 f. Tese (Doutorado em Ci^encias){ Faculdade de Medicina de Ribeir~aoPreto, Universidade de S~aoPaulo, Ribeir~aoPreto, SP, 2018. Gliomas s~aotumores heterog^eneos,o que contribui para seu alto grau de mortalidade, apesar de avan¸cosna classifica¸c~aoe tratamento. Desde 2016, a incorpora¸c~aodo estado dos genes IDH e da integridade dos cromossomos 1p e 19q na classifica¸c~aode gliomas fornece aplica¸c~oescl´ınicasimportantes para o diagn´osticoe tratamento deste tumor; entretanto, a procura por assinaturas moleculares que possam refinar ainda mais os subtipos de glioma em subgrupos mais homog^eneos´eum esfor¸cocont´ınuo. Este estudo utilizou o maior n´umerode amostras de gliomas adultos (n=932) at´ea atualidade, variando dos graus II ao IV, a fim de definir subgrupos de glioma utilizando assinaturas de metila¸c~aodo DNA, indepentemente de grau e histologia. No total, 7 subtipos foram identificados: Classic- like, Mesenchymal-like, LGm6-GBM, PA-like, Codels, G-CIMP-low and G-CIMP-high. A maior parte dos subgrupos com IDH tipo selvagem, isto ´e,Classic-like, Mesenchymal-like, LGm6-GBM, possuem padr~aode baixa metila¸c~aodo DNA e um pior risco progn´ostico; caracter´ısticascl´ınicast´ıpicasde glioblastomas, o tipo mais agressivo de gliomas. Uma descoberta interessante foi a identifica¸c~aodo subgrupo PA-like dentre gliomas com IDH tipo selvagem, o qual compartilha aspectos gen^omicos similares a astrocitoma piloc´ıtico, um glioma pedi´atricobenigno com bom quadro cl´ınicoentre gliomas com IDH tipo selvagem. Codels, os quais abragem pacientes com muta¸c~aoem IDH e codele¸c~aodos cromossomos 1p e 19, possuem o melhor progn´osticodentre os gliomas difusos em adultos. Uma descoberta importante em rela¸c~aoa gliomas com muta¸c~aoem IDH, por´emsem codele¸c~aodos cromossomos 1p e 19q, foi a estratifica¸c~aode gliomas com fen´otipo metilador de ilhas CpG (G-CIMP) em G-CIMP-low, com n´ıveis mais baixos de metila¸c~aodo DNA e pior quadro cl´ınico,e G-CIMP-high, com n´ıveis mais altos de metila¸c~aodo DNA e melhor risco progn´ostico.Curiosamente, o grau de metila¸c~aodo DNA (-low e -high) estava associado com altera¸c~oesdistintas em elementos regulat´oriose modifica¸c~oesde histona aberrantes na regi~aopromotora de genes do ciclo celular. Estes achados consolidaram a import^ancia cl´ınicada epigen´etica,particularmente da metila¸c~aodo DNA, em gliomas, como tamb´emlevantou a possibilidade de que a sobrevida m´ediaruim de G-CIMP-low pode ser associada a elementos regulat´orios.Al´emdisso, a hip´otese de que enhancers ativos podem agir na regula¸c~aog^enica de G-CIMP-low fornece mais evid^enciasde que elementos regulat´oriospodem levar `amaior agressividade e prolifera¸c~aode G-CIMP-low. Este estudo visa 1) identificar e caracterizar subtipos de gliomas difusos em adultos baseados na metila¸c~aodo DNA, e 2) avaliar a associa¸c~aoentre modifica¸c~oesde histona com um subtipo mais agressivo de G-CIMP. Palavras-chaves: Metila¸c~aodo DNA. Glioma. G-CIMP. Epigen´etica.Bioinform´atica. Abstract SABEDOT, TS. Molecular classification of adult diffuse gliomas based on DNA methylation reveals subgroups of G-CIMP tumors associated with distinct clinical features. 2018. 121 p. Thesis (PhD of Science) { Ribeir~aoPreto Medical School, University of S~aoPaulo, Ribeir~aoPreto, SP, 2018. Gliomas are heterogeneous tumors which contribute to their high mortality despite ad- vancements in classification and treatment. As of 2016, the incorporation of IDH status and the integrity of chromosomes 1p and 19q to glioma classification have provided impor- tant clinical application for diagnostics and treatment; however, the search for molecular signatures that further refine glioma subtypes into more homogeneous subgroups is an ongoing effort. This study used the largest sample cohort (n=932) of adult gliomas to date, ranging from grades II to IV, in order to define gliomas subgroups using DNA methylation signatures, independent of histopathological grading. In total, 7 subtypes were identified: Classic-like, Mesenchymal-like, LGm6-GBM, PA-like, Codels, G-CIMP-low and G-CIMP-high. Most IDH -wildtype subgroups, e.g. Classic-like, Mesenchymal-like and LGm6-GBM, had low DNA methylation pattern and a poor outcome, typical of glioblastomas, the most aggressive phenotype of gliomas. An interesting finding was the identification of the PA-like subgroup within IDH -wildtype samples, which shared similar genomic features with pilocytic astrocytoma, a rare pediatric benign glioma, with a good overall survival (OS) among IDH -wildtype gliomas. Codels, which comprise IDH mutant gliomas with codeletion of chromosomes 1p/19q have the best OS across all adult gliomas. An important finding regarding IDH mutant gliomas with no codeletion of chromosomes 1p/19q, was the further segregation of the Glioma-CpG Island Methylator Phenotype (G-CIMP) into G-CIMP-low, with lower levels of DNA methylation and worse OS, and G-CIMP-high, characterized by higher DNA methylation profile and better OS. Interest- ingly, the degree of G-CIMP methylation (-low and -high) was associated with distinct alterations in regulatory elements and aberrant histone modifications at promoter regions of cell cycle genes. These findings consolidated the clinical importance of epigenetics, particularly DNA methylation, in gliomas, as well as the possibility that aggressive OS in G-CIMP-low may be driven by regulatory elements. Moreover, our results suggest that active enhancers that might be acting in gene regulation in G-CIMP-low provide more evidence of the regulatory elements that might be driving aggressiveness and proliferation in G-CIMP-low. This study aims 1) to identify and characterize adult diffuse glioma DNA methylation subtypes, and 2) evaluate the association of histone modifications with a more aggressive G-CIMP subtype. Keywords: DNA methylation. Glioma. G-CIMP. Epigenetics. Bioinformatics. List of Figures Figure 1 { Schematic flow chart of the study design from chapter 2. 30 Figure 2 { Venn diagram with overlapping CpGs between both Illumina platforms. 32 Figure 3 { Schematic description of the methodology to identify epigenetically regulated genes................................ 36 Figure 4 { Glioma DNA methylation subtypes..................... 41 Figure 5 { IDH -specific DNA methylation subtypes.................. 42 Figure 6 { G-CIMP characterization.