Divergence from the human astrocyte developmental trajectory in glioblastoma Restricted; Files Only

Abstract

Properties of early embryonic development are frequently recycled in cancer, including the acquisition of a highly plastic stem-like cell state, uncontrolled cell growth and proliferation, and adaptation to a harsh microenvironment. This mirroring of development is evident in glioma, where tumors reflect the differentiation hierarchies that underpin normal glial cell formation during neurodevelopment. Glioblastomas (GBM), the most severe glioma class, harbor cells that resemble immature progenitor populations, including oligodendrocyte, neural, and astrocyte precursor cells, and demonstrate the capacity to transition between these cell states. Given the close parallels between neurodevelopment and GBM cell programs, utilizing maps of normal glial lineages could inform us about how tumor cells hijack and progress along developmental trajectories to promote tumor survival. However, glial differentiation and maturation are challenging to delineate as it peaks between late gestational and early postnatal ages, a window of development that is hard to capture in humans due to limited primary tissue samples and suboptimal two-dimensional in vitro model systems. To overcome this hurdle, we leveraged human cortical organoids (hCOs), a three-dimensional in vitro model of the developing human cortex, to generate a comprehensive molecular timeline of human astrocyte maturation. We then projected this developmental trajectory onto GBM astrocyte-like tumor cells to identify how astrocyte development is recapitulated in GBM. We maintained hCOs in culture for nearly two years, profiling the chromatin- and transcriptome-level changes in hCO astrocytes at 10 discrete time points. In doing so, we found three molecularly distinct stages of maturation, including a novel intermediate stage that may serve as a key lineage determination cell state. This particular intermediate stage of maturation was consistently and highly expressed in astrocyte-like cells from GBM tumors, potentially serving as an “attractor” maturation state, where malignant tumor cells thrive. When looking at maturation signature across the diverse tumor cohort, we discovered that astrocyte-like cells from tumors harboring an IDH1 mutation were substantially more mature compared to IDH1-wildtype tumors, suggesting that the IDH1 mutation may directly or indirectly preserve astrocyte maturation state. We hypothesized that conserved mature molecular programs may be related to IDH1-mutant-associated DNA hydroxymethylation (5hmC) patterns and found an enrichment of 5hmC in differentially expressed maturation gene sets. Together, these experiments describe a novel astrocyte developmental program that is preferentially activated in human GBM revealing new facets of tumor biology and therapeutic targeting to explore.

Table of Contents

CHAPTER 1: INTRODUCTION 1

1.1 Overview and organization 2

1.2 Glial development and maturation 3

1.2.1 The rise of glial cells: how, when, and where 3

1.2.1.1 Molecular regulators of the gliogenic switch 4

1.2.1.2 Extrinsic regulators of gliogenesis 6

1.2.1.3 Populating the CNS: where and when 8

1.2.2. Glial cell maturation 9

1.2.2.1 Astrocyte maturation 9

1.2.2.2 Oligodendrocyte maturation 11

1.2.3 Tipping the scales: making astrocytes vs oligodendrocytes 12

1.2.3.1 Evidence of a shared glial precursor 12

1.2.3.2 Diverging molecular programs 14

1.3 Cancer as an echo of glial development 17

1.3.1 Glioblastoma 18

1.3.2 Other diffuse and lower-grade gliomas 22

1.3.2.1 Diffuse glioma 23

1.3.2.2 Pilocytic astrocytoma 24

1.3.3 Diffuse midline glioma 25

1.4 How do glioma cells move across developmental time? 29

1.4.1 Where along the normal developmental trajectory do gliomas start? 29

1.4.2 When in developmental time do glioma cells reside and thrive during tumor progression? 31

1.4.3 When in developmental time are glioma cells capable of moving towards with intervention? 33

1.5 Summary and thesis objectives 36

CHAPTER 2: DIVERGENCE FROM THE HUMAN ASTROCYTE DEVELOPMENTAL TRAJECTORY IN GLIOBLASTOMA 39

2.1 Abstract 40

2.2 Introduction 41

2.3 Results 44

2.3.1 A molecular map of human astrocyte maturation in cortical organoids 44

2.3.2 Molecular differences between IDHwt tumor and margin astrocyte lineage cells 46

2.3.3 Projecting GBM astrocytes onto a normal human astrocyte maturation trajectory 49

2.3.4 Subtype-specific molecular signatures in GBM tumor astrocyte lineage cells 50

2.3.5 The contribution of DNA methylation to IDHwt and IDH1mt maturation differences 52

2.4 Discussion 85

2.5 Materials and methods 90

CHAPTER 3: DISCUSSION 108

3.1 Summary 109

3.2 Key findings and future directions 110

3.2.1 Human astrocyte maturation: filling in the gaps 110

3.2.2 What can the principles of astrocyte maturation tell us about GBM biology? 111

3.2.3 Can maturation TFs be harnessed for therapeutic intervention? 114

3.2.4 Revisiting the GBM cell of origin 117

3.2.5 Developmental differences across glioma subtypes 120

3.3 Conclusion 124

APPENDIX: ONGOING AND FUTURE FUNCTIONAL EXPERIMENTS 125

Abstract 126

Introduction 127

Results 128

Overexpressing TF candidates in hCSs 128

Mapping molecular maturation programs onto published fetal datasets 129

The effects of D2HG on astrocyte maturation 130

Discussion 139

Methods 142

REFERENCES 148

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, Neuroscience
Parola chiave	Glioma Glia Cancer Neurodevelopment
Committee Chair / Thesis Advisor	Sloan, Steven, Emory University
Committee Members	Katz, David, Emory University Kukar, Thomas, Emory University Spangle, Jennifer, Emory University Read, Renee, Emory University

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