Ralf S Schmid1, Jeremy M Simon1, Mark Vitucci1, Robert S McNeill1, Ryan E Bash1, Andrea M Werneke1, Lauren Huey1, Kristen K White1, Matthew G Ewend1, Jing Wu1, C Ryan Miller1. 1. Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina (R.S.S., L.H., M.G.E., J.W., C.R.M.); Division of Neuropathology, Department of Pathology & Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina (M.V., R.S.M., R.E.B., A.M.W., K.K.W., C.R.M.); Carolina Institute for Developmental Disabilities and Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, North Carolina (J.M.S.); Department of Neurosurgery, University of North Carolina School of Medicine, Chapel Hill, North Carolina (M.G.E., J.W.); Department of Neurology and Neurosciences Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina (C.R.M.).
Abstract
BACKGROUND: Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.
BACKGROUND:Glioma stem cells (GSCs) from humanglioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.
Authors: Ed S Lein; Michael J Hawrylycz; Nancy Ao; Mikael Ayres; Amy Bensinger; Amy Bernard; Andrew F Boe; Mark S Boguski; Kevin S Brockway; Emi J Byrnes; Lin Chen; Li Chen; Tsuey-Ming Chen; Mei Chi Chin; Jimmy Chong; Brian E Crook; Aneta Czaplinska; Chinh N Dang; Suvro Datta; Nick R Dee; Aimee L Desaki; Tsega Desta; Ellen Diep; Tim A Dolbeare; Matthew J Donelan; Hong-Wei Dong; Jennifer G Dougherty; Ben J Duncan; Amanda J Ebbert; Gregor Eichele; Lili K Estin; Casey Faber; Benjamin A Facer; Rick Fields; Shanna R Fischer; Tim P Fliss; Cliff Frensley; Sabrina N Gates; Katie J Glattfelder; Kevin R Halverson; Matthew R Hart; John G Hohmann; Maureen P Howell; Darren P Jeung; Rebecca A Johnson; Patrick T Karr; Reena Kawal; Jolene M Kidney; Rachel H Knapik; Chihchau L Kuan; James H Lake; Annabel R Laramee; Kirk D Larsen; Christopher Lau; Tracy A Lemon; Agnes J Liang; Ying Liu; Lon T Luong; Jesse Michaels; Judith J Morgan; Rebecca J Morgan; Marty T Mortrud; Nerick F Mosqueda; Lydia L Ng; Randy Ng; Geralyn J Orta; Caroline C Overly; Tu H Pak; Sheana E Parry; Sayan D Pathak; Owen C Pearson; Ralph B Puchalski; Zackery L Riley; Hannah R Rockett; Stephen A Rowland; Joshua J Royall; Marcos J Ruiz; Nadia R Sarno; Katherine Schaffnit; Nadiya V Shapovalova; Taz Sivisay; Clifford R Slaughterbeck; Simon C Smith; Kimberly A Smith; Bryan I Smith; Andy J Sodt; Nick N Stewart; Kenda-Ruth Stumpf; Susan M Sunkin; Madhavi Sutram; Angelene Tam; Carey D Teemer; Christina Thaller; Carol L Thompson; Lee R Varnam; Axel Visel; Ray M Whitlock; Paul E Wohnoutka; Crissa K Wolkey; Victoria Y Wong; Matthew Wood; Murat B Yaylaoglu; Rob C Young; Brian L Youngstrom; Xu Feng Yuan; Bin Zhang; Theresa A Zwingman; Allan R Jones Journal: Nature Date: 2006-12-06 Impact factor: 49.962
Authors: Robert S McNeill; Ralf S Schmid; Ryan E Bash; Mark Vitucci; Kristen K White; Andrea M Werneke; Brian H Constance; Byron Huff; C Ryan Miller Journal: J Vis Exp Date: 2014-08-12 Impact factor: 1.355
Authors: Michael James Clark; Nils Homer; Brian D O'Connor; Zugen Chen; Ascia Eskin; Hane Lee; Barry Merriman; Stanley F Nelson Journal: PLoS Genet Date: 2010-01-29 Impact factor: 5.917
Authors: Mark Vitucci; Natalie O Karpinich; Ryan E Bash; Andrea M Werneke; Ralf S Schmid; Kristen K White; Robert S McNeill; Byron Huff; Sophie Wang; Terry Van Dyke; C Ryan Miller Journal: Neuro Oncol Date: 2013-06-27 Impact factor: 12.300
Authors: C J Herting; Z Chen; K L Pitter; F Szulzewsky; I Kaffes; M Kaluzova; J C Park; P J Cimino; C Brennan; B Wang; D Hambardzumyan Journal: Glia Date: 2017-08-24 Impact factor: 7.452
Authors: Jing Wu; Lauren N Frady; Ryan E Bash; Stephanie M Cohen; Allison N Schorzman; Yu-Ting Su; David M Irvin; William C Zamboni; Xiaodong Wang; Stephen V Frye; Matthew G Ewend; Erik P Sulman; Mark R Gilbert; H Shelton Earp; C Ryan Miller Journal: Neuro Oncol Date: 2018-01-10 Impact factor: 12.300
Authors: Onyinyechukwu Okolie; Juli R Bago; Ralf S Schmid; David M Irvin; Ryan E Bash; C Ryan Miller; Shawn D Hingtgen Journal: Neuro Oncol Date: 2016-06-13 Impact factor: 12.300
Authors: Mark Vitucci; David M Irvin; Robert S McNeill; Ralf S Schmid; Jeremy M Simon; Harshil D Dhruv; Marni B Siegel; Andrea M Werneke; Ryan E Bash; Seungchan Kim; Michael E Berens; C Ryan Miller Journal: Neuro Oncol Date: 2017-09-01 Impact factor: 12.300
Authors: Muayad F Almahariq; Thomas J Quinn; Pravin Kesarwani; Shiva Kant; C Ryan Miller; Prakash Chinnaiyan Journal: In Vivo Date: 2021 Jan-Feb Impact factor: 2.406
Authors: Robert S McNeill; Demitra A Canoutas; Timothy J Stuhlmiller; Harshil D Dhruv; David M Irvin; Ryan E Bash; Steven P Angus; Laura E Herring; Jeremy M Simon; Kasey R Skinner; Juanita C Limas; Xin Chen; Ralf S Schmid; Marni B Siegel; Amanda E D Van Swearingen; Michael J Hadler; Erik P Sulman; Jann N Sarkaria; Carey K Anders; Lee M Graves; Michael E Berens; Gary L Johnson; C Ryan Miller Journal: Neuro Oncol Date: 2017-10-19 Impact factor: 12.300