Beyond the beam: the multi-faceted applications of radiation in cancer biology Restricted; Files Only

Abstract

The discovery of radiation significantly furthered the field of cancer biology. Radiation has proven itself to be a valuable tool as it can both cause and treat cancer. This dissertation aims to emphasize the employment of radiation to i) uncover novel molecular interactions and ii) understanding therapeutic options.

Glioblastoma multiforme (GBM) is the most fatal adult brain tumor and is commonly radioresistant via an amplification of the PI3K/AKT pathway. AKT phosphorylates histone H3 at threonine 45 (pH3T45) when DNA is damaged, but the functionality is unknown. Here, we utilize radiation to elucidate the role of pH3T45 in the GBM DNA damage response. We show PI3K pathway-activated GBM cell lines harboring exogenous phospho-null mutants exhibit hindered damage signaling via decreased H3K36me3 in nucleosome-incorporated histones and non-homologous end-joining protein foci formation. We also observe defective damage resolution and reduced cell clonogenicity and proliferation with no additional effect by radiation. Finally, differential expression analysis suggests genetic loss of pH3T45 dysregulates hallmark pathways, potentially resulting in significant phenotype changes. These data suggest pH3T45 loss may contribute to GBM radioresistance.

           Furthermore, we demonstrate the important application of radiation in understanding potential cancer therapies. Vγ9Vδ2 T cells represent a promising cancer therapy platform; however, intravenous administration of human Vγ9Vδ2 T cells manufactured under GMP-compliant, serum-free conditions do not migrate from circulation to NSG mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to characterize human γδ T cell migration and direct them to therapeutic sites in mice. We found conditioning mice with radiation increases the percentage of γδ T cells accumulating in the BM. Additionally, γδ T cells enter the BM through passive migration from circulation, not homing. We then leveraged the chemoattraction between the highly expressed chemokine receptor, CCR2, on γδ T cells and its secreted ligand, CCL2, by IFNγ-primed MSCs (γMSCs), to induce homing of γδ T cells in vivo. γδ T cells significantly traffic to BM- and neuroblastoma tumor-injected γMSCs. Here, we uncovered fundamental migration properties of these cells. Overall, this dissertation highlights radiation in cancer treatment, in elucidating mechanisms, and understanding principles governing innate cell product development.

Table of Contents

Abstract

Acknowledgements

Table of Contents

List of Figures and Tables

 

Chapter 1: Introduction…………………………………………………………………….….1

1.1 Radiation………………………………………………………………………….….2

1.2 Radiation as a Tool……………………………………………………………….….4

1.3 Challenges that Remain………………………………………………………….…14

Chapter 2: Key players in the radiation-induced DNA damage response….……………..19

2.1 Abstract……………………………………………………………………………..20

           2.2 Introduction…………………………………………………………………………20

           2.3 The Key Players of the DDR……………………………………………………….24

           2.4 Discussion…………………………………………………………………………..38

Chapter 3: Loss of histone H3 threonine 45 phosphorylation decreases H3K36me3 to abrogate the radiation-induced DNA damage in glioblastoma multiforme…………….…39

           3.1 Abstract………………………………………………………………………….….40

           3.2 Introduction……………………………………………………………………...….41

           3.3 Materials and Methods……………………………………………………………...45

           3.4 Results…………………………………………………………………………........51

           3.5 Discussion………………………………………………………………………..…70

Chapter 4: Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells..…..75

4.1 Abstract………………………………………………………………………..……76

           4.2 Introduction…………………………………………………………………………77

           4.3 Materials and Methods………………………………………………………..…….80

           4.4 Results………………………………………………………………………...…….91

           4.5 Discussion…………………………………………………………………..……..108

           4.6 Supplemental Figures……………………………………………………..……….113

Chapter 5: General discussion……………………………………………………..………..118

           5.1 Summary of Results……………………………………………………….………119

           5.2 Implications of Findings…………………………………………………………...121

           5.3 Limitations and Future Directions………………………………………...…...…..123

           5.4 Conclusions…………………………………………………………………….….127

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell
Keyword	glioblastoma multiforme gamma delta T cells immunotherapy DNA damage radiation epigenetics
Committee Chair / Thesis Advisor	H. Trent Spencer, Emory University
Committee Members	Kelly Goldsmith, Emory University Sunil Raikar, Emory University Curtis Henry, Emory University Christopher Doering, Emory University

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