Enhancing Oncolytic Reovirus with Doxorubicin for Triple-Negative Breast Cancer Therapy Open Access

Abstract

Breast cancer is the leading cause of cancer-related deaths in women in the United States.

The triple-negative (TNBC) subtype associates with higher rates of relapse, decreased survival,

aggressive metastatic disease, and limited therapeutic options. Mammalian orthoreovirus

(reovirus) selectively infects and kills transformed cells, and a serotype 3 reovirus (T3C$) is in

clinical trials to assess oncolytic efficacy against several cancers. To engineer a reovirus with

increased oncolysis against TNBC, we coinfected MDA-MB-231 TNBC cells with Type 1 Lang

(T1L), Type 2 Jones (T2J), and Type 3 Dearing (T3D) reoviruses. After serial passage of cells, we

isolated the reassortant r2Reovirus that is composed of T1L and T3D genes. r2Reovirus infects

TNBC cells more efficiently and reduces cell viability with faster kinetics than parental T1L or

T3D and T3C$. Pretreating cells with topoisomerase inhibitors, including doxorubicin, enhances

r2Reovirus infectivity and cytotoxicity in TNBC cells and leads to stimulation of DNA damage

response pathway activation and Type III interferon production. To develop a mechanism of

codelivery of drug and virus, we conjugated doxorubicin to r2Reovirus (reo-dox). Reo-dox induces

cytotoxicity in TNBC cells more efficiently than r2Reovirus alone. Conjugation has minimal

effects on virus biology, and host response to reo-dox is altered compared virus alone or with

exogeneous doxorubicin. Crosslinked doxorubicin retains the ability to damage DNA and promote

damage response pathway activation. Importantly, r2Reovirus and reo-dox significantly reduce

primary TNBC tumor burden in vivo, with greater reduction in metastatic burden after reo-dox

inoculation. Crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug

delivery to cells targeted by the virus, making it a viable approach for combination therapy against

TNBC. Together, these studies identify a new reassortant reovirus with improved oncolysis against

TNBC that can be enhanced by classical combination with topoisomerase inhibitors or by novel

chemical conjugation. Adapting viruses to target tumor cells is an important step in generating

improved individualized therapies to address tumor heterogeneity, and novel drug delivery

mechanisms that enhance combination therapy and anti-tumor immune responses will benefit the

long-term systemic response to treatment, improving quality of life and care of patients.

Table of Contents

Abstract

Acknowledgements

Tables of Contents

List of Figures and Tables

List of Abbreviations

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

CANCER .................................................................................................................................................. 2

Breast cancer ......................................................................................................................................... 2

Triple-negative breast cancer ................................................................................................................ 3

DOXORUBICIN ....................................................................................................................................... 4

Topoisomerase ...................................................................................................................................... 6

DNA double strand break response ...................................................................................................... 7

ONCOLYTIC VIRUSES .......................................................................................................................... 8

REOVIRUS............................................................................................................................................... 9

Reovirus discovery and isolation ........................................................................................................ 10

Reovirus cell entry .............................................................................................................................. 11

Virus-host response ............................................................................................................................. 13

Reovirus as an oncolytic ..................................................................................................................... 13

SUMMARY AND SCOPE ..................................................................................................................... 19

Chapter 2: Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and

Topoisomerase Inhibitors ............................................................................................................ 20

ABSTRACT ............................................................................................................................................ 22

IMPORTANCE ....................................................................................................................................... 23

INTRODUCTION .................................................................................................................................. 24

RESULTS ............................................................................................................................................... 27

DISCUSSION ......................................................................................................................................... 38

MATERIALS AND METHODS ............................................................................................................ 44

ACKNOWLEDGEMENTS .................................................................................................................... 52

Chapter 3: Doxorubicin Conjugation to Reovirus Improves Oncolytic Efficacy in Triple-Negative

Breast Cancer ............................................................................................................................... 79

GRAPHICAL ABSTRACT .................................................................................................................... 81

ABSTRACT ............................................................................................................................................ 82

INTRODUCTION .................................................................................................................................. 83

RESULTS ............................................................................................................................................... 86

DISCUSSION ......................................................................................................................................... 96

MATERIALS AND METHODS .......................................................................................................... 103

ACKNOWLEDGEMENTS .................................................................................................................. 112

AUTHOR CONTRIBUTIONS ............................................................................................................. 113

Chapter 4: Discussion and Conclusions ................................................................................................ 130

MODEL OF FINDINGS ....................................................................................................................... 134

FUTURE DIRECTIONS ...................................................................................................................... 141

Appendix: Unpublished Data ................................................................................................................. 144

References ................................................................................................................................................ 148

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Virology
Keyword	reovirus conjugation doxorubicin oncolytic virus breast cancer
Committee Chair / Thesis Advisor	Bernardo Mainou, Emory University
Committee Members	Gregory Lesinski, Emory University Mehul Suthar, Emory University Curtis Henry, Emory University Periasamy Selvaraj, Emory University

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