The Potential Detrimental Impact of Galactic Cosmic Radiation on Central Nervous System and Hematopoietic Stem Cells

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THE POTENTIAL DETRIMENTAL IMPACT OF GALACTIC

COSMIC RADIATION ON CENTRAL NERVOUS SYSTEM AND

HEMATOPOIETIC STEM CELLS

RUTULKUMAR UPENDRABHAI PATEL

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Scott M. Welford, Ph.D

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

January, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of Rutulkumar Upendrabhai Patel

Candidate for the Doctor of Philosophy degree *.

(signed) Derek Taylor (Committee Chair)

Scott M. Welford (Dissertation Advisor)

Stanton L. Gerson (Committee Member)

Marvin Nieman (Committee Member)

Jennifer Yu (Committee Member)

(date) December 3rd, 2018

*We also certify that written approval has been obtained for any

proprietary material contained therein.

Dedication

I would like to dedicate this dissertation to my parents, Upendrabhai and

Ujvalakumari Patel, who supported my wishes and ambitions despite being lived most of their lives in a lower-middle class family income. They sacrificed a lot to make sure a better life for their children. I would also like to dedicate this to my two sisters, Ekta and Vanita, for their support and encouragement over the years.

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Table of Contents

Table of Contents ……………………………………………………………….. iv

List of Figures ………………………………………………………………….. viii

Acknowledgements ……………………………………………………………. xii

Abstract ……………………………………………………………….…………. 1

Chapter 1: Introduction and Background ………………………………….. 3

1.1 Radiation, DNA Damage, and Carcinogenesis …………………………... 3

1.1.1 Space Radiation Environment and Induction of DNA

Damage …………………………………………………………… 8

1.1.2 Radiation Induced Carcinogenesis ………………...... 10

1.2 Hematopoietic Stem Cell Niche and Functions …………………………… 12

1.2.1 Low-LET Irradiation and HSC Injuries …………………………. 16

1.2.2 High-LET Irradiation Impact on HSCs …………………………. 17

1.3 Importance of Mismatch Repair ……………………………………………. 20

1.3.1 Compromised MMR and Cancer …………………...... 25

1.3.2 MLH1, an Important MMR Component ………………………… 26

1.4 Radiation Exposure and Central Nervous System ……………………….. 27

1.4.1 Harmful Effects of Low-LET Irradiation on CNS ………………. 31

1.4.2 High-LET Irradiation Disrupts CNS functions …………………. 32

1.5 Statement of Purpose ……………………………………………………… 32

Chapter 2: Long-term deficits in behavior performances caused by low- and high-linear energy transfer radiation ……………………...... 37

2.1 Abstract ………………………………………………………………………. 37

2.2 Introduction ………………………………………………………………….. 39

2.3 Materials and Methods ……………………………………………………… 41

2.4 Results ………………………………………………………………………... 46

2.5 Discussion ……………………………………………………………………. 50

2.6 Acknowledgements ……………………………………………...... 55

Chapter 3: MMR deficiency does not sensitize or compromise the function of hematopoietic stem cells to low and high LET radiation …………….. 64

3.1 Abstract ………………………………………………………………………. 64

3.2 Introduction …………………………………………………………………... 66

3.3 Materials and Methods ……………………………………………………… 69

3.4 Results ………………………………………………………………………... 72

3.5 Discussion ……………………………………………………………………. 78

3.6 Acknowledgements …………………………………………………………. 81

Chapter 4: Mlh1 deficiency increases the risk of hematopoietic malignancy after simulated space radiation exposure ………………………………… 100

4.1 Abstract ……………………………………………………………………... 100

4.2 Introduction …………………………………………………………………. 102

4.3 Materials and Methods …………………………………………………….. 104

4.4 Results ………………………………………………………………………. 107

4.5 Discussion ………………………………………………………………….. 113

4.6 Acknowledgements ………………………………………………………… 117

Chapter 5: Age related loss of Mlh1 in hematopoietic stem cells accelerates tumorigenesis post simulated solar or galactic cosmic radiation exposure

………………………………………………………………………………………. 139

Chapter 6: Discussion and Future Directions ……………………………. 149

6.1 Tumorigenesis depends on LET of radiation source and Mlh1 status of HSCs

………………………………………………………………………………………. 152

6.2 Determine the impact of mixed beam GCR exposure on tumorigenesis of Mlh1 chimeric mouse model …………………………………………………………. 157

6.3 Define the mitigating potential of dietary polyamines as a countermeasure for

GCR induced tumorigenesis …………………………………………………... 161

6.4 Concluding Remarks ……………………………………………………... 163

References ……………………………………………………………………... 166

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List of Figures

Figure 1.1: Overview of DNA Damage, Repair Mechanisms, and Consequences

…………………………………………………………………………………………. 6

Figure 1.2: Overview of Hematopoietic Stem Cell Niche Components and

Hematopoiesis …………………………………………………………………… 14

Figure 1.3: Schematic Representation of Mismatch Repair Post Replication

………………………………………………………………………………………... 23

Figure 1.4: Symptoms and Timeline for the Development of IR-induced Brain

Injuries …………………………………………………………………………….. 29

Figure 2.1: Diminished activity is a late toxicity from low- and high-LET radiation

………………………………………………………………………………………... 56

Figure 2.2: Long-term motor coordination defects were revealed after low- and

high-LET radiation ……………………………………………………………….. 58

Figure 2.3: Low- and high-LET radiation cause long-term recognition memory loss

………………………………………………………………………………………... 60

Figure 2.4: Transient spatial memory loss is caused by γ-ray and 56Fe ion radiation

………………………………………………………………...... 62

Figure 3.1: High LET radiation induces similar long term damage to the bone marrow as γ radiation ……………………………………………………………. 82

Figure 3.2: High LET radiation is more damaging to clonogenic capacity of stem cells than low LET radiation, but independent of MMR status ………………. 84

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Figure 3.3: Blood counts demonstrate similar acute damage to the hematopoietic system across LET ………………………………………………………………. 86

Figure 3.4: Mlh1 knockout mice display enhanced sensitivity to IR

………………………………………………………………………………………... 88

Figure 3.5: Long term effects on hematopoiesis by IR is independent of MMR status ……………………………………………………………………………… 90

Figure 3.6: Defects in Mlh1 function do not enhance decreased competitive repopulation caused by IR ………………………………………………………. 92

Supplementary Figure 3.1: Myeloid CFU survival post radiation exposure

………………………………………………………………………………………... 94

Supplementary Figure 3.2: Lymphocyte counts in Mlh1+/+ and Mlh1-/- mice

………………………………………………………………………………………. 96

Supplementary Figure 3.3: Bone marrow cellularity in Mlh1+/+ and Mlh1-/- mice

………………………………………………………………………………………... 98

Figure 4.1: Long-term tumorigenesis assay post low- and high-LET radiation

exposure ………………………………………………………………………… 119

Figure 4.2: Histopathology of tumors from Mlh1+/+ and Mlh1+/- mice

………………………………………………………………………………………. 121

Figure 4.3: Immunohistochemistry of lymphomas from Mlh1+/+ and Mlh1+/- mice

………………………………………………………………………………………. 123

Figure 4.4: Microsatellite instability found in Mlh1+/+ and Mlh1+/- tumors

………………………………………………………………………………………. 125

Figure 4.5: Whole exome sequencing analysis of Mlh1+/+ and Mlh1+/- TRB

lymphomas ……………………………………………………………………… 127

Figure 4.6: Correlation between frequently mutated mouse TRB lymphoma genes

vs human leukemia genes ……………………………………………………... 129

Supplementary Figure 4.1: HSC acute functional assays post radiation exposure

………………………………………………………………………………………. 131

Supplementary Figure 4.2: HSC differentiation independent of Mlh1 status

………………………………………………………………………………………. 133

Figure 5.1: 1H ion and 28Si ion irradiation affects HSC acute function, but not long- term differentiation ……………………………………………………………… 143

Figure 5.2: Incidence of tumorigenesis in Mlh1+/+ and Mlh1+/- mice post 1H ion and

28Si ion exposure ……………………………………………………………….. 145

Figure 5.3: Gene expression profile of Mlh1+/+ vs Mlh1+/- TRB lymphomas

………………………………………………………………………………………. 147

Figure 6: Summary explains the detrimental impact of GCR on mouse brain and

hematopoietic stem cell ………………………………………………………... 150

Figure 6.1: The impact of LET and Mlh1 status on GCR induced tumorigenesis

………………………………………………………………………………………. 155

Figure 6.2: Generation of Mlh1-/- chimeric mouse model and study of tumorigenesis …………………………………………………………………… 159

Acknowledgements

I came to the United States in 2007 to pursue my career in the field of science and it was an exciting phase of my life to be in a vast country with plenty of opportunities around, but soon a honeymoon period was vanished and a real struggle began to find my place. Finding the first job was a really difficult task after graduating with Master’s degree and a minimal laboratory experience. At that time,

Dr. Rajendra Mehta gave me an opportunity to kick start my career at IIT Research

Institute, Chicago where I found my love for cancer research, and I knew that I had found my career path.

The second opportunity was given to me by Dr. Nancy Oleinick at the

CWRU, where she constantly supported and encouraged me to further pursue my education by getting into Ph.D. program. While working under excellent supervision of Dr. Oleinick, I met Dr. Scott Welford and came across a project funded by NASA, which immediately grabbed my attention. At that moment, I decided to pursue my Ph.D. under Dr. Welford’s mentorship. He taught me many lessons over the years but the importance of “asking the right question” to do a good science was the most insightful advice he ever gave me. Dr. Stanton Gerson, despite being extremely busy with multiple responsibilities, gave his precious time and advice throughout my journey. Dr. Derek Taylor, Dr. Marvin Nieman, and Dr.

Jennifer Yu were very instrumental in providing new perspective and kept me on the track throughout my dissertation. I received tremendous support from the department of pharmacology, my fellow lab members, and core facilities at CWRU,

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University Hospitals, Cleveland Clinic, University of Michigan, and University of

Miami.

Finally, the unconditional love and support I have received from my parents

(Upendrabhai and Ujvalakumari), my sisters (Ekta and Vanita), and friends. Most

importantly the unconditional support I have received from my wife Saloni Patel,

who is the most understanding person I have ever met by far. There isn’t enough

room to describe how lucky I feel to be in a place of ample opportunities and right

assistance during my career. I truly believe that no man or woman is self-made, we all are here because sacrifices and help we received from family, friends, and mentors. I would like to thank everyone from bottom of my heart who knowingly or unknowingly helped me to be where I am today.

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The Potential Detrimental Impact of Galactic Cosmic Radiation on Central

Nervous System and Hematopoietic Stem Cells

Abstract

RUTULKUMAR UPENDRABHAI PATEL

Space travel is associated with many primary hazards, but the risk associated with space radiation induced damage to the central nervous system

(CNS) and hematopoietic stem cells (HSCs) could be the limiting factor for the future exploration missions. The major component of space radiation is galactic cosmic radiation (GCR) and it is composed of high-linear energy transfer (LET) radiation. The lack of human data and poor understanding of high-LET radiobiology make current risk prediction models less reliable to assess the potential risk associated with long-term space travel. Thus, we irradiated C57bl/6 mice with a low-LET (γ-rays or 1H ions) or high-LET (28Si ions or 56Fe ions) ionizing radiation (IR) and performed a battery of behavioral tests at 5 and 9 month post exposure to study CNS defects. We found that each radiation source showed distinct effects on the behavioral tests performed, with most prominent impact on non-spatial memory, suggesting that the initial damage and subsequent molecular changes caused by each ion species were inherently unique.

MLH1 is a major component of MMR and a recent study has demonstrated that human CD34+ HSCs loses expression of MLH1 due to promoter hypermethylation as a function of age. Given that the upper end of astronauts are

~46 years old, HSCs with compromised MMR function will be exposed to high-LET space radiation leading to increase HSC malignancies. Therefore, we irradiated

Mlh1+/+ and Mlh1+/- mice with different doses of γ-rays, 1H, 28Si, or 56Fe particles.

We found that loss of Mlh1 significantly increases the incidence of lymphomagenesis in Mlh1+/- mice post IR compared to sham-irradiated Mlh1+/- or irradiated Mlh1+/+ mice. In addition, the early incidence and higher frequency of lymphomagenesis was dependent on radiation quality factor with a maximum impact observed at 28Si ion IR. Further, the molecular signature of the lymphomas revealed a striking correlation to human leukemias, implying the relevance of using a MMR compromised mouse model to recapitulate middle-aged human HSCs.

Collectively, our findings provide deeper insight into the long-term detrimental impact of space radiation induced behavioral deficits and HSC malignancy that may limit our future space exploration missions without tough interventions.

Chapter 1 – Introduction and Background

1.1 Radiation, DNA Damage, and Carcinogenesis

The field of radiology was born soon after the discovery of x-rays in 1896.

Shortly after the discovery of x-rays, radioactivity emitted by radium was detected

and used for the treatment of cancer and thus the emergence of the study of

radiobiology. Ionizing radiation (IR) contains enough energy to remove tightly

bound electrons from the orbits of atoms or molecules, causing ionization of

biological molecules such as DNA, protein, lipids, and carbohydrates. IR causes

damage through direct ionization of target atoms or molecules, called direct action,

or through indirect ionization of water molecules to produce free radicals that are

able to cause damage to target molecules, called indirect action. The majority of radiobiology experiments use x-rays or γ-rays that do not differ in nature or property, but x-rays are produced extranuclearly while γ-rays produced intranuclearly. In the case of x-rays or γ-rays, two thirds of the biological damage caused by indirect ionization due to sparse energy deposition occurs as photons passing through a cell. However, direct ionization dominates in the case of α- particles due to the dense ionization pattern of positively charged particles passing through a cell. The main difference between sparsely ionizing radiation and densely ionizing radiation is the amount of energy deposition occurring per unit length of the particle track, and is called linear energy transfer (LET) and measured in KeV/µm. X-rays or γ-rays are considered as a low-LET radiation due to less energy deposition per unit length of the track while α-particles are considered as a high-LET radiation due to large amount of energy deposited in a small distance.

Therefore, biological consequences of exposure to IR such as cell death, genetic mutations, and chromosomal aberrations are affected directly by radiation of different LET.

DNA is the major biomolecule of interest when it comes to IR induced cell damage or death. IR generates oxidative stress via formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in not only irradiated cells but also in their progeny for days after initial insult [1-4]. In fact, the concentration of

ROS and RNS along the tracks of irradiating particles have important consequences to the extent and nature of induced DNA damages such as DNA breaks, base modifications, and destruction of sugars in DNA [5-9]. Evidently, It has been shown that 1 Gy of low-LET γ-rays creates roughly 1300 base modifications, 1000 single strand breaks (SSB), and 40 double strand breaks

(DSB) in mammalian cell [10]. In addition, IR induces gross changes at the chromosomal level where aberrations such as dicentrics and ring formation in chromosomes and anaphase bridge formation in chromatids are lethal to cells.

Non-lethal chromosomal changes (translocations and small deletions) are also caused by IR leading to genomic instability rather than cell death. Collectively, many decades of research have shown that IR is a potent DNA damaging agent and causes biological changes that may takes hours, days, or months for cell killing, years for carcinogenesis, and even generations for hereditary damage.

Cells are equipped with potent DNA damage responses (DDR) and DNA repair proteins to execute the removal of DNA lesions. The DDR plays a crucial role in handling not only IR-induced damage but also chemical or metabolic stress

generated DNA damages. For instance, base excision repair (BER) is responsible

for correcting non-bulky damage to DNA bases, mismatch repair (MMR) is

responsible for correcting base:base mismatches or small insertion/deletion loops,

nucleotide excision repair (NER) is responsible for correcting multiple forms of

DNA damages including pyrimidine dimers and intrastrand crosslinks, and double

strand break repair is responsible for correcting DNA double stand breaks through

two highly conserved pathways: non homologous end-joining (NHEJ) and

homologous recombination (HR) (figure 1.1, adapted from [11]). Evidence suggests that DSBs are the most important lesion produced by IR and leads to cell death if unrepaired [12]. Therefore, the DDR is a central component of radiation induced DNA damage signaling and a damage tolerance mechanism that maintains a critical balance between cell survival and programmed cell death pathways.

Figure 1.1: Overview of DNA Damage, Repair Mechanisms, and

Consequences.

(A) Different types of DNA lesions instigated by common DNA damaging agents and DNA damage response pathways responsible for resolving these lesions.

(B) Acute effects of DNA damage on cell cycle progression and chronic effects of

DNA damage, including mutations and chromosome aberrations leading to cell death or disease such as cancer. Abbreviations: cis-Pt – cisplatin; MMC – mitomycin C; CPD – cyclobutane pyrimidine dimer; BER – base excision repair;

NER – nucleotide excision repair; HR – homologous recombination; EJ – end joining

Figure adapted from Hoeijmakers Nature 2001, 411:366-374

Figure 1.1: Overview of DNA Damage, Repair Mechanisms, and

Consequences.

Figure adapted from Hoeijmakers Nature 2001, 411:366-374

1.1.1 Space Radiation Environment and Induction of DNA Damage

One of the major underappreciated health concerns for the long-term deep

space missions is space radiation. While there are highly accurate predictions of

the types and doses of exposure in deep space from various Mars-bound vehicles,

we currently have a poor understanding of how space radiation will influence

human health directly, and thus we lack a predictable health risk model for deep

space endeavors. Space radiation consists of a broad spectrum of low-, medium-

, and high-LET radiation sources coming from galactic cosmic radiation (GCR),

solar energetic particles (SEP) emitted from the Sun, and trapped energetic

particles in the Van Allen radiation belts [13-15]. In particular, GCR are composed

of high-energy protons (85%), helium ions (14%), and nuclei of heavier elements

such as 12C, 16O, 20Ne, 28Si, 40Ca, 48Ti, and 56Fe ions, called HZE (high atomic number Z and energy E) particles (1%) [16]. HZE nuclei deposit large amount of energy throughout the track of each particle along with lateral extensions of low-

LET energetic electrons (δ-rays), which travel many microns from the original track

[17, 18]. SEPs and trapped energetic particles in the Van Allen radiation belts

include low to medium energy protons and electrons [16]. Therefore, space radiation is a harsh environment for astronauts and the potential risk associated with high-LET radiation exposure could limit our future endeavors.

Astronauts on the International Space Station (ISS) travel below and through the Van Allen belts, and are generally protected from SEPs and most of the GCR by the earth’s magnetic field and to a lesser extent by the shielding materials on the station itself, which can absorb lower energy protons and charged

8 particles. However, astronauts on deep space missions will be challenged by GCR and SEP radiation, occasional solar weather changes in the form of fairly high dose-rate solar particle events (SPEs), and the Van Allen radiation belts as they travel beyond low Earth orbit with only the spacecraft hull and contents to shield them [19]. Despite the low frequency of HZE ions, contributing to only 1% of GCR,

HZE ions deliver about 1–2 mSv whole body dose per day and have dense ionization patterns such that they are the main contributor to risk from GCR [16,

20]. Therefore, the deep space environment includes radiation exposure to HZE particles that we as a species have not endured, but will need to resolve before a

2–3 year exposure during a Mars or future deep space missions.

The spatial pattern of DNA lesions caused by HZE nuclei and low-LET irradiation are distinct in nature [18, 21, 22]. There are also qualitative differences in DNA damage induction and DDR between low- and high-LET irradiation [23-26].

For instance, high-LET IR creates complex cluster DNA damage that contains a combination of two or more types of damages (SSB, DSB, base modifications, and so on) within localized region of DNA [27, 28]. Studies have shown that HZE nuclei generate large γ-H2AX foci size and delayed removal of γ-H2AX foci post IR compared to low-LET IR [29]. Furthermore, HZE nuclei induce a much higher level of complex chromosomal rearrangements compared to sparsely ionizing IR [30-

32]. Emerging evidence suggests that there is a substantial difference in gene expression profile after high-LET compared to low-LET radiation exposure [33, 34].

To date, no experiments have shown that different DNA repair pathway machinery is evoked following high-LET vs low-LET radiation exposure. However, DDR

proteins, including ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and

Rad3-related (ATR), Artemis, and NBS1, could play a huge role in the repair of

complex cluster DNA damages generated by high-LET IR. Collectively, these data

suggest that high-LET IR is qualitatively and quantitatively different from terrestrial

radiation and hence could lead to a substantial genomic changes.

1.1.2 Radiation induced carcinogenesis

Irradiation has a potential to initiate and promote cancer formation and thus

is viewed as a complete carcinogen. Standard radiotherapy uses low-LET ionizing

radiation to eliminate tumor cells via damaging the DNA. At the same time,

exposure to DNA damaging agents raises the possibility to initiate normal cells

around the tumor and lead to formation of secondary tumors at a later stage of life.

The most extensive data on the devastating effects of low-LET radiation on human

health were derived from individuals exposed to the atomic bomb explosions in

Hiroshima and Nagasaki in 1945. The data revealed radiation-induced primary

solid cancers located to the breast, thyroid, colon, and lung [35]. In addition, two

of the most sensitive organ systems have proven to be the hematopoietic and the

gastrointestinal systems, with dose being the most discriminating factor between

onset and severity of the two. Analysis of survivors of the immediate bomb blasts

have found that the majority of IR-related deaths were associated with

hematopoietic failure caused by total body irradiation (TBI), including individuals

who received doses between 2.5–5 Gy [36-38]. For long-term survivors, increased

cancer risk has become a significant cause of death, with leukemia having the

10 highest relative risk of any malignancy [39]. Collectively, the data shows that low-

LET radiation exposure in human is associated with an increased risk to carcinogenesis.

High-LET irradiation, typical of the GCR, is more damaging than low-LET irradiation, such as x-rays or γ-rays, and thus risk prediction models for astronaut health effects need to be adjusted [40]. The major space radiation-associated health risks that have been identified in various experimental models here on Earth using ground-based particle accelerator facilities are degenerative damage to various organ systems (including the circulatory system, central nervous system, and bone, muscle and cartilage microarchitecture), cataract formation, and genetic mutations leading to cancer, which are all expected to occur in the relatively low dose environment of space [19]. Historically, NASA has adopted the goal of limiting increased risk of mortality by cancer for space travel to a 3%. Various studies have been carried out using mouse models of carcinogenesis to understand the types of cancers that might occur in humans in response to HZE ion exposure, such as mammary tumors, melanoma, hepatocellular carcinoma, intestinal colorectal cancer, and leukemia [41-43]. In particular, the radiosensitive nature of the hematopoietic system makes it a crucial target for investigating the impact of high-

LET irradiation in animal models in order to extrapolate the risk to humans in the space radiation environment. A better understanding of the harmful impact of space radiation will also allow us to design appropriately targeted radioprotectors, mitigators, and novel shielding designs and materials to protect astronauts for the future deep space missions.

1.2 Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) generate billions of cells every day throughout the life of vertebrates via the orchestrated processes of proliferation,

self-renewal, and differentiation into functional blood cells [44]. The health of HSCs depends on many factors and cellular players in the bone marrow (BM) niche, including perivascular cells, endothelial cells, osteoblasts, macrophages, and sympathetic nerves [45, 46] (figure 2.1). HSCs remain largely quiescent, effectively minimizing endogenous stress caused by cellular respiration and DNA replication processes [47]. HSC homeostasis is affected by niche signaling, niche location,

HSC stress and regeneration after injury, and even aging. However, internal and

external stresses continuously threaten HSC integrity and accumulation of genetic

instability in HSCs or in niche could lead to either stem cell failure or hematopoietic

malignancies. For instance, studies have shown that deletion of retinoic acid

receptor-gamma (RARγ) or mindbomb 1 (Mib1) within HSC microenvironment

induces myeloproliferation of wild-type cells in defective niche, suggesting a role of niche during disease formation [48, 49]. Similarly, constitutively active Wnt-

signaling pathway in osteolineage cells results in the development of acute myeloid

leukemia (AML) [50]. Therefore, HSC niche components play a critical role in

maintaining normal homeostasis and hold a key to promote proliferation and

expansion in response to disease.

Over the life of an organism on earth, HSCs are continuously challenged by

various stresses including terrestrial radiation, which is composed of up to 55% of

high-LET charged particles from radon [51]. Radiation exposure is associated with

12 causing not only genomic changes in HSCs but also influence extracellular matrix remodeling that has a direct impact on HSC functions. Thus, understanding normal hematopoietic homeostasis and responses to exogenous stressor, such as IR, can inform about how HSC will tolerate space radiation. In the context of cancer therapy, it is well appreciated that therapeutic radiation is used in the treatment of a broad spectrum of cancers including hematopoietic malignancies such as leukemia or lymphoma, and can lead to bone marrow injury [52]. From a radiobiology perspective, many laboratory studies have shown that the hematopoietic system is exquisitely radiosensitive, and damage caused by IR can lead to hematopoietic dysfunction or malignancies [37]. In addition, damage to the

HSCs themselves or damage to the supportive BM niche cells could lead to altered

HSC repopulation and bone marrow vascularization, exacerbating the harmful impact of IR [53, 54]. Collectively, many years of clinical experience and laboratory research have shown that IR is a potent stressor for the hematopoietic system, which can induce genomic instability leading to stem cell defects and cancer.

Figure 1.2: Overview of Hematopoietic Stem Cell Niche Components and

Hematopoiesis

Various cells involved in promoting stem cell maintenance, including stromal cells, adipocytes, endothelial cells, osteoclasts and osteoblasts, macrophages, and sympathetic neurons. Primitive stem cells have self-renewable and differentiation properties to produce multipotent progenitor cells that in turn give rise to myeloid and lymphoid progenitor cells. These progenitor cells further differentiate into lymphoid lineage cells (natural killer cell, T lymphocytes, and B lymphocytes) and myeloid lineage cells (neutrophil, basophil, eosinophil, monocyte/macrophage, platelets, and red blood cell) in order to maintain homeostasis.

Figure adapted from 2001 Terese Winslow (assisted by Lydia Kibiuk)

Figure 1.2: Overview of Hematopoietic Stem Cell Niche Components and

Hematopoiesis

Figure adapted from 2001 Terese Winslow (assisted by Lydia Kibiuk)

1.2.1 Low-LET irradiation and HSC injuries

Irradiation-induced damage leads to a hematopoietic malignancy in

humans, typically acute and chronic myeloid leukemias [55-57]. Apart from HSC

malignancies, IR exposure can also increase risk of local and systemic infections,

anemias, and potentially even susceptibility to other cancers through compromised immune function [58]. Several processes have been identified to have a role in IR- induced HSC injury, including (1) reduction in the number of HSCs due to apoptosis, senescence, or differentiation; and (2) damage to the bone marrow microenvironment affecting niche signaling [59-61]. IR is a potent activator of apoptosis and studies have shown that modulating the expression levels of a key anti-apoptotic protein, BCL-2, can protect the hematopoietic compartment against radiation-induced HSC death [62, 63]. Moreover, recent studies have investigated

a role of Puma, pro-apoptotic BH3-only protein, and shown that it plays a crucial role in IR-induced HSC apoptosis [64, 65]. Therefore, elucidating the mechanisms

of IR-induced HSC injury is important to understand the susceptible nature of

hematopoietic system.

Radiation also affects differentiation of HSCs, and a recent study has shown

that exposure to IR depletes HSCs by boosting differentiation into the lymphoid

lineage [66]. In the early 1960s, it was demonstrated that human diploid fibroblasts

have a limited growth capacity due to the finite length of telomeres and the

mechanical end-replication problem of DNA synthesis [67, 68]. The first evidence

of senescence in hematopoietic stem cells was observed in Bmi1-/- mice where

knockout mice developed progressive BM hypoplasia leading to early death [69,

70]. In fact, the study showed that IR treatment induces senescence in vitro and in vivo with increased expression of SA-β-gal (senescence associated β- galactosidase), p16, and Arf [60, 61]. Radiation induced senescence is well- described in relation with production of ROS in fibroblasts, and likely also highly relevant in the HSC setting [71-73]. In fact, it was found that ataxia-telangiectasia mutated ATM-/- mice exhibited progressive failure of hematopoietic function attributed to premature senescence due to increased production of ROS [73].

Finally, studies have also shown that IR not only affects HSCs directly, but also induces significant BM stroma (including fibroblasts) injury in a time and dose dependent manner [74-76]. Collectively, these data suggest that HSC compartment as a whole is quite sensitive to radiation exposure and would likely be even more sensitive to damage caused by HZE particle exposure.

1.2.2 High-LET Irradiation impact on HSCs

On the journey to Mars, every cell in an astronaut’s body will be hit by low and medium energy 1H ion approximately every 3–4 days, 4He nuclei every few weeks, and HZE particles every few months [19]. Radiation exposure from these sources during spaceflight could have profound short- and long-term negative impacts on human physiology including morbidity/mortality in astronauts [13, 27,

28]. These HZE particles create complex clustered DNA damage and the complexity of clustered DNA damage increases with increasing Z of HZE particles, rendering DNA repair even more difficult [13]. Unrepaired DNA damage in HSCs is capable of inducing either clonogenic death or substantial genomic instability

leading to cancer. Therefore, it is crucial to study the effect of HZE particles on

human health in order to understand the risk associated with human-based space

explorations.

The human hematopoietic system is highly sensitive to the development of

leukemia and lymphomas from exposure to low-LET, sparsely ionizing γ-rays. In

fact, IR-induced leukemias are a well-known outcome in the A-bomb survivors.

Protons, which are the most frequent ion species in the SEP and GCR fields in

space, have similar LET values to γ-rays, making them an important ion species to study the risk associated with space travel, with a general hypothesis that information gleaned from terrestrial radiation sources would be applicable. Thus, many studies have been carried out to compare the effects of protons with different energies on cell and animal models and the effects of TBI proton exposure on the hematopoietic system, with gamma radiation as a reference standard radiation.

Simulated SPE protons at a dose of 0.5 Gy or 1 Gy show significantly increased chromosomal aberration in lymphocytes up to 2 months post exposure compared to untreated mice [77]. C57BL/6J mice exposed to 1 Gy of 150 MeV/n 1H irradiation

show substantial BM injury resulting in significant lower frequency of HSCs long

term after the whole body exposure [78]. Sequential exposure to protons (1 GeV/n)

and heavy ion such as 56Fe ions (1 GeV/n) shows significant deleterious in vitro

impact on HSC colony formation [34]. These studies imply that low-LET SPE

protons may have short- and long-term harmful impact on human hematopoietic

system.

Though much lower in frequency than protons, the GCR field is also

composed of high-LET 4He nuclei and elements such as 12C, 16O, 20Ne, and 28Si

nuclei that are more poorly understood regarding impact on the hematopoietic

stem cell population. The growing interest and investment in heavy ion (hadron)

radiotherapy such as 12C ion therapy for cancer treatment also demands careful

determination of HZE ion effects on normal physiology. Heavy ion radiotherapy

takes advantage of the Bragg curve of the charged particle, which describes the

relationship of energy delivered versus distance, to deliver highly localized doses

precisely to a tumor at the particle’s so-called Bragg peak, thereby limiting

exposure to the normal tissue beyond the target area. However, the effect of heavy

ion exposure on normal circulating HSC or in the marrow within the treatment field

could in theory lead to DNA damage and even secondary malignancies. CD34+

stem cells from human placental/umbilical cord blood exposed in vitro to carbon

ion (290 MeV/n) at 0.5 or 1.5 Gy dose display severe damage to clonal growth of

myeloid hematopoietic stem/progenitor cells (HSPCs) compared to low-LET x-rays

[79]. Thus, there is a significant justification for the quest to understand the effects

of hadrons on the HSC on normal tissues as well.

The short- and long-term impact of HZE particles on the hematopoietic system was also studied recently in a rodent model. Acute effects of a 1 Gy TBI

16O ion irradiation (600 MeV/n) on C57BL/6J mice showed a significant decrease

in peripheral blood cells at two weeks that was correlated with substantial reduction

in HSPC clonogenic potential in an in vitro assay compared to sham-irradiated

controls [80]. Epigenetic changes, such as DNA methylation, are known to align

with HSC lineage commitment, and can demonstrate changes after significant

cellular stress and even correlate with carcinogenesis [42, 81, 82]. Exposure of

CBA/CaJ mice to doses less than 0.5 Gy of 28Si ions (300 MeV/n) results in an

increase chromosomal aberrations and a global inhibition of 5-

hydroxymethylcytosine in HSPCs 6 months post-irradiation compared to sham controls [83]. Furthermore, it was also shown that whole body 28Si ion exposure in

CBA/CaJ mice cause significantly higher levels of apoptotic cell death and

inflammatory response in BM cells 6 months post-exposure compared to sham-

irradiated mice [84]. These findings suggest that high-LET HZE particle irradiation

has short- and long-term effects on HSCs and are beginning to demonstrate some

unpredicted changes to HSC biology compared to standard x-rays and γ-ray

radiation. Beyond this exciting initial story, with an incompletely defined

mechanism, the effects of GCR on the hematopoietic system remain a significant

concern for space travel.

1.3 Importance of Mismatch Repair

The mismatch repair system plays a vital role in minimizing errors generated

during replication that escape proofreading. MMR is responsible for repair of base-

base mismatches and small insertion/deletion loops (INDELs) arising during

replication and failure of this mechanism leads to a threefold increase in mutational

frequencies [85-87]. The MMR contributes to fidelity of replication by the sequential

process of mismatch base identification, wrong base excision, and re-synthesis of

correct base. The MMR system achieves this feat with the help of different MMR

and accessory proteins. The MMR pathway starts with an identification of

mismatches occurring during replication by MutS heterodimers such as

MSH2/MSH6 (MutSα) or MSH2/MSH3 (MutSβ). The MutSα recognizes single

base mismatch or 1-2 nucleotide INDELs, while the MutSβ recognizes 3-6 bases

mismatches or small INDELs. The MutS heterodimers undergo an ADP-ATP

exchange driven conformational changes leading to recruitment of MutL

heterodimers. There are three different MutL heterodimers discovered so far;

MutLα (MLH1/PMS2 heterodimer), MutLβ (MLH1/PMS1 heterodimer), and MutLγ

(MLH1/MLH3 heterodimer) that belong to GHKL ATPase family [88]. Among all

MutL homologs, MutLα plays a prevalent role in MMR. The MutLα heterodimer

creates a nick into newly synthesized DNA, which allows EXO1, an endonuclease,

to load and degrade the DNA in a 5’ → 3’ direction. The single stranded DNA is

stabilized by replication protein A (RPA) post EXO1 exonuclease activity. The last

step includes filling the gap by polymerase δ followed by sealing the nick by DNA

ligase I (figure 3.1, adapted from [89]). The entire process is crucial to avoid

mutation accumulation in daughter cells post replication.

The core MMR proteins are not only involved in correction of errors during

replication but are also involved in homologous recombination and nonhomologous end joining of double strand breaks [90, 91]. Interestingly, MMR is

also found to be involved in mutagenic processes such as somatic hypermutation

(SHM), class switch recombination (CSR), and instability of trinucleotide repeats

(TNRs) [92]. For instance, our immune system is capable of generating a large

number of diverse antibody repertoires in order to deal with harmful antigens. To

achieve this goal, several mutagenic processes occurs at the immunoglobulin

locus that alters the genetic information in order to create sufficient diversity.

During the early developmental stage, B-cells produce a primary stock of low affinity IgM antibodies that go under a secondary diversification process that alters

the sequence and structure at the immunoglobulin genes. The second

diversification process requires SHM and CSR mechanisms to generate different

classes of antibodies [93-95]. In addition, MMR mediates DNA damage-induced

signaling, such as cell cycle arrest and apoptosis. Studies have shown that hMutSα

and hMutLα deficient cells are impaired in cell cycle arrest due to failure of p53

and p73 phosphorylation post DNA damage [96-100]. The involvement of MMR in

various processes underlies the importance of the pathway, and disruption of MMR

has been shown to associate with several diseases, including cancer.

Figure 1.3: Schematic Representation of Mismatch Repair Post Replication.

(1) Mismatch recognition: MMR process begins at G/T mismatch site by the

binding of MutSα (MSH2/MSH6 heterodimer). (2) Recruitment of machinery: Upon

binding, MutSα undergoes an ATP-driven conformational changes to recruit MutLα

(MLH1/PMS2 heterodimer). This complex can translocate in either direction on

DNA contour in order to encounter a strand break (gap between Okazaki fragments in lagging strand or nick introduced by PMS2 in leading strand). EXO1 and PCNA bind to MutSα/MutLα complex and initiate degradation that will terminate past the G/T mismatch site. (3) Re-synthesis and ligation: The resulting

single stranded DNA is stabilized by RPA. Finally, new DNA stretch synthesis

occurs by DNA polymerase δ and DNA ligase I seals the remaining nick.

Figure adapted from Sara Thornby Bak, Front. Genet., 21 August 2014.00287

Figure 1.3: Schematic Representation of Mismatch Repair Post Replication.

Figure adapted from Sara Thornby Bak, Front. Genet., 21 August 2014.00287

1.3.1 Compromised MMR and Cancer

A defective MMR pathway is associated with genomic instability due to mutation accumulation – a phenomenon known as hypermutator. Defective MMR

alters the length of microsatellites due to a lack of repair and thus generate

microsatellite instability (MSI). In the early 1990s, a subset of colorectal tumors

was discovered with a large number of mutations in microsatellite sequences,

which was subsequently explained by the documentation of loss of key MMR

genes in the tumors [101-104]. Ultimately, it was discovered that a compromised

MMR pathway predisposes to lynch syndrome (an autosomal dominant genetic

condition) due to a germline mutation in one of the four key MMR proteins – MLH1,

MSH2, MSH6, and PMS2 [101, 105-108]. HENRY T. LYNCH, a professor of

medicine at Creighton University Medical Center, characterized the syndrome in

1966 that accounts for ~5 percent of all colorectal cancer cases. However, MMR

malfunction due to germline mutations not only predisposes to colorectal cancer

alone but also increases the risk of endometrial, ovarian, gastric, small bowel,

urothelial, brain, hepatobiliary, pancreatic, bladder, prostate, and kidney cancers

[109-116]. In addition, somatic mutations in MMR genes are also found to be

associated with many human cancers, suggesting an important role of the MMR

pathway in carcinogenesis.

MMR deficient mice have displayed significantly higher levels of MSI and

increase susceptibility to cancer. There is a strong correlation between MMR

deficiency and MSI in human cancers, and thus MSI status of the tumor could have

a clinical importance. It is a well-known fact that radiation causes random

mutations in cells and in absence of functional MMR the effect could be more

pronounced. In fact, MMR deficient mice have shown increased sensitivity to

radiation induced malignancy due to a higher mutation rate in the genome [117].

The evidence suggests that patients undergoing radiotherapy for cancer treatment

could be at the risk of secondary tumor formation, especially in cases of MMR

compromised patients. In addition, preclinical and clinical data have shown chemo-

resistance due to MMR deficiency. Therefore, MMR status of the tumor has a

prognostic relevance in the clinic.

1.3.2 MLH1, an Important MMR Component

MLH1 is a common component of the MutL heterodimer complexes and

plays an essential role for downstream signaling events. Studies have shown that

MLH1-containing heterodimers are involved in signaling downstream MMR events,

such as EXO1, RPA, and proliferating cell nuclear antigen/POL30 [118-123].

Structural studies of MLH1 protein have shown the importance of an ATPase

domain on N-terminal for conformational changes and a binding domain on the C-

terminal for interaction with other MMR proteins [124-129]. MLH1 is an essential

component of MMR complex and lack of MLH1 expression is associated with

impaired MMR function. Complete loss of the Mlh1 gene is not lethal in mice,

however, knockout mice show elevated levels of MSI and significantly reduced tumor free survival.

The functional loss of MLH1 is one of the major contributor to hereditary

nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. In fact, germline

mutations in MLH1 are found in roughly 30% of Lynch syndrome patients and a majority of these mutations are deletions [106, 130]. Interestingly, sporadic cancer patients show epigenetic changes, in particular MLH1 promotor hypermethylation, which leads to silencing of MLH1. In addition, over expression of miR-155 in colorectal cancer has been shown to suppress MLH1 expression [131]. In recent years, the importance of MLH1 has also been found in other cancer types. Loss of

MLH1 was found in primary human cancers such as, gastric, esophageal, head and neck squamous cell carcinoma, and non-small cell lung cancer [132-136]. In fact, the lack of MLH1 expression due to epigenetic silencing was also revealed in recurrent glioblastoma and endometrial cancers [137, 138]. In mice, Mlh1 loss is associated with frequent mutations occurring in carcinogenic loci of neurofibromatosis 1 (Nf1) and ATR [139, 140]. Collectively, the data shows that epigenetic or mutational loss of MLH1 function is strongly associated with cancer formation and hence defining MLH1 role as a tumor suppressor.

1.4 Radiation Exposure and CNS

The central nervous system (CNS) comprises of the brain and spinal cord that consist of oligodendrocytes, astrocytes, microglia, neurons, and vasculature.

Neurons are the main anatomical unit of the brain, which forms a specialized network with the help of supporting cells in order to process and interpret the

information. In recent years, studies have shown that neurogenesis occurs in the

adult subventricular zone and hippocampus of mammals, which is associated with

cognitive activities [141, 142]. Many exogenous factors, such as stress, various

recreational drugs, pathological conditions, therapeutic agents, and irradiation

could affect different CNS functions leading to behavior changes. The complexity

of brain requires a readout in the form of behavior changes to determine whether

functions have been altered due to the interplay of contributing factors. Behavioral

effects are difficult to quantify, but there are various rodent behavior tests available

to measure different brain functions. For instance, few rodent tests are routinely

performed to study rodent behavior performances, such as (1) Open field test,

which measures locomotive activity and anxiety level [143], (2) Rotarod test, which measures endurance, balance, and motor coordination [144], (3) T-maze test and

Morris water task, which measure spatial memory and learning [145, 146], (4)

Novel object recognition, which measures non-spatial memory [147]. These

behavior tests examine a different region of the brain and play a vital role in

connecting behavior performance changes to brain damage.

Figure 1.4: Symptoms and Timeline for the Development of IR-induced Brain

Injuries

IR-induced brain injury is often detected post-fractionated partial or whole brain irradiation, which regularly manifested in the form of anatomical and functional deficits. Based on the time of clinical signs, radiation-induced brain injury is divided into three distinct phases (acute, early delayed, and late delayed injuries).

Figure adapted from Dana Greene-Schloesser, Front. Genet., 19 July

2012.00073

Figure 1.4: Symptoms and Timeline for the Development of IR-induced Brain

Injuries

Figure adapted from Dana Greene-Schloesser, Front. Genet., 19 July 2012.00073

1.4.1 Harmful Effects of Low-LET Irradiation on CNS

Several decades of research have shown that radiation exposure causes oxidative stress and neuroinflammation, leading to harmful impact on neurogenesis, brain microvascular, neuronal structure, and electrophysiology.

Evidence for deleterious effects of low-LET (x-rays or γ-rays) IR on CNS has been derived from patients undergoing radiotherapy with high doses of radiation [148].

The radiation exposure damage includes acute brain injuries (<1 month), early delayed (1-6 months), and late delayed injuries (>6 months) post irradiation (figure

4.1, adapted from [148]). Patient undergoing high doses of radiotherapy leads to

CNS injuries, including early delayed effects such as demyelination, attention deficits, and short-term memory loss and late delayed effects such as vascular damage, white matter necrosis, and cognitive impairment. The pediatric patients undergoing radiotherapy have showed behavior changes and the neurocognitive effects even at lower doses of radiation [149, 150]. In particular, children with brain tumor have shown marked decline in intelligence and various academic achievements such as reading, spelling, mathematics, and attention functioning post radiation treatment [151, 152]. Similarly, pediatric leukemia patients undergoing low dose whole body radiation exposure have exhibited low intelligence scores [153, 154]. The majority of cognitive defects in these patients were observed at high doses during long-term follow-up post radiotherapy.

However, cognitive impairments are also found with individuals receiving lower doses of IR. For instance, Atomic bomb and Chernobyl accident survivors

receiving ≤ 2 Gy show evidence of memory and cognitive deficits. Overall, the

31 evidence clearly shows that accidental or therapeutic exposure to low-LET radiation affects CNS similar to those seen during aging or in some neurological disorders.

1.4.2 High-LET Irradiation Disrupts CNS Functions

Space radiation is composed of HZE nuclei that cause distinct cellular damage to living cells and possess even a greater risk to the CNS. Due to lack of human data for high-LET radiation exposure, we strictly have to rely on animal and cell models to understand the deleterious effects of space radiation. The experimental studies of high-LET radiation suggested that during missions, early and late risks to the CSN will be imminent and could limit our future long-term exploration missions. However, these experiments were performed with few of the ion species present in space at high dose and dose-rate, which makes these results difficult to extrapolate to humans in order to better design the risk models.

Nonetheless, these animal models can provide a valuable insight into how cosmic rays negatively influences the CNS functions in order to design better radioprotective strategies

Many animal studies were performed to illustrate HZE particles impact on the CNS, and their dependencies on factors such as dose, dose-rate, ion species

(LET), sex, age, and time after the exposure. However, a majority of studies were performed with acute exposure of relatively high dose with few ion species (1H,

28Si, and 56Fe), which is not close to what astronauts will experience on their way

32 to the deep space missions. For instance, studies have shown that 56Fe ion IR in rodents affects their cognitive functions even up to 9 months post exposure [155-

157]. Similarly, exposure to 28Si particles has shown significantly higher anxiety levels and lower cognitive learning and memory functions in mice and rats post IR

[157, 158]. In addition, animals exposed to low doses of 4He or 28Si or 48Ti ions has shown cognitive impairments up to few months post IR [159-161]. An extensive number of mechanistic studies using various types of neurons, other brain cells, and rodent models have shed light on possible molecular changes leading to the

CNS functional deficits. Altered neurogenesis, cell lose due to apoptosis or autophagy, neuroinflammation, oxidative and radical damages due to ROS and

RNS, morphological changes in neurons, and fluctuation in neurotransmitter levels have all been associated with negative impacts of high-LET radiation on the CNS

[142, 162-169]. Collectively, these data suggest that HZE ions possess a greater risk to the CNS and thus could be major limiting factor for future deep space missions.

1.5 Statement of Purpose

The studies were aimed to determine harmful impacts of space radiation on

CNS and HSCs, in particular the MMR deficient HSCs. The goal was to answer three major questions; (1) What are the long-term behavioral changes, such as anxiety, locomotive activity, spatial and non-spatial memory, caused by high-LET radiation exposure? (2) To what extent high-LET radiation exposure and compromised MMR are responsible for altered short- and long-term functions of

HSCs? (3) Does MMR deficient HSCs lead to hematopoietic malignancies post

high-LET radiation exposure? If so, is there a difference in frequency and incidence

of HSC malignancies compared to low-LET γ-rays? Together, understanding these

questions will shed a light on the detrimental impact of high-LET radiation exposure on CNS and HSCs, help us design better risk models for the space travel, and give insight into how to protect astronauts from these radiation sources during the future deep space missions.

To study the impact of high-LET IR on the CNS, we used C57BL/6 mice and irradiated them with low-LET γ-rays or 1H ions (1000 MeV/n) and high-LET

28Si ions (300 MeV/n) or 56Fe ions (600 MeV/n). The experiment was carried out to identify different behavior changes occurring long-term (up to 9 months) after the exposure, mainly due to the fact that many previous studies were focused on

CNS damage and behavioral changes immediate or short-term after the radiation exposure. A significant impact was found on activity levels and non-spatial memory, regardless of radiation source. However, each ion species caused a distinct detrimental impact on a battery of behavior tests, suggesting that molecular damage and subsequent response instigated by each ion species was different in nature and hence their effects on the CNS. These long-term behavioral studies in animal model will help us identify potential brain damage that could happen in astronauts post missions and may allow us to put protective measures in place before serious damage could occur.

Previously, our group has shown that MLH1 expression in HSCs diminish due to promoter hypermethylation as a function of age [170, 171]. The finding is

very important to the NASA community due to the fact that a majority of astronauts

belong to the middle-aged group. To comprehend the impact of age-related loss

of MLH1 in HSCs, we used Mlh1+/+, Mlh1+/-, and Mlh1-/- mice to understand the

HSC functional deficit due to Mlh1 loss in presence and absence of irradiation. We

irradiated Mlh1+/+, Mlh1+/-, and Mlh1-/- bone marrow cells with different doses of

low-LET γ-rays or 1H ions (1000 MeV/n) and high-LET 56Fe ions (600 MeV/n). The

acute HSC functions, measured by colony forming unit (CFU) assay, complete

blood counts, and competitive repopulation assays, were impaired by increasing

doses of IR with LET dependency. Similarly, the long-term HSC functions were

significantly affected by 56Fe ion IR, compared to low-LET IR, causing the most

harmful impact. However, Mlh1 status of the HSCs did not play any role in the

short- or long-term assays, suggesting that loss of Mlh1 does not affect HSCs

functional viability.

Finally, we employed a MMR deficient mouse model (Mlh1+/-) that correlates with the middle-aged human population to study HZE particles induced HSC malignancies. We demonstrated that Mlh1+/- mice are more sensitive to low-LET,

such as γ-rays and 1H ions, radiation-induced lymphomagenesis compared to sham-irradiated Mlh1+/- or irradiated Mlh1+/+ mice. In addition, we found that high-

LET IR, such as 28Si or 56Fe, caused a significantly higher and early incidence of

lymphomagenesis compared to Mlh1+/- mice irradiated with low-LET IR. MSI analysis revealed that lymphomas were MMR deficient, suggesting that loss of

Mlh1 leads to lymphomagenesis. Interestingly, the majority of lymphomas were B- cell in origin and upon whole exome sequencing, we discovered that Mlh1+/-

lymphomas showed a significantly higher levels of SNVs and INDELs compared to wildtype lymphomas. Upon further analysis, we discovered that a distinct pattern of mutations in carcinogenic loci was dependent on Mlh1 status and irradiating ion species, implying a specific role of each factor leading to lymphomagenesis. These

findings suggest that middle-aged astronauts will be venerable to space radiation

induced HSC malignancies post long-term deep space mission.

Collectively, we discovered that HZE particles have a greater risk to cause

a long-term CNS defects along with a long-term HSC related lymphomagenesis.

The study also demonstrated that high-LET radiation exposure possess even a

greater risk to loss of Mlh1 induced HSC malignancy, which was previously

unknown. However, we used a single ion species at a time to irradiate our mice,

which will not be the case for astronauts during their long-term deep space mission. Therefore, we proposed future experiments with multiple ions at a low dose-rate to closely simulate space conditions. The implications of current experiment results and future studies will be immense in making the future missions safer and more predictable.

Chapter 21 Long-term deficits in behavior performances caused by low- and high- linear energy transfer radiation

2.1 Abstract

Efforts to protect astronauts from harmful galactic cosmic radiation (GCR) require a better understanding of their effects on human health. In particular, little is known about the lasting effects of GCR on the central nervous system (CNS) leading to behavior performance deficits. Previous studies have shown that high linear energy transfer (LET) radiation in rodents leads to short-term declines in a variety of behavior tests. However, the lasting impacts of low-, medium-, and high-

LET radiation on behavior are not fully defined. Therefore, we exposed C57BL/6 male mice to 100 cGy or 250 cGy of γ-rays (LET of ~ 0.3 KeV/µm), 10 cGy or 100 cGy of 1H at 1000 MeV/n (LET of ~ 0.2 KeV/µm), 28Si at 300 MeV/n (LET of ~ 69

KeV/ µm), or 56Fe at 600 MeV/n (LET of ~ 180 KeV/ µm), and collected behavior

metrics at 5 months and 9 months post exposure for analysis of differences

between radiation quality and dose. A significant effect of radiation dose was

observed on recognition memory and activity levels measured 9 months after

exposure regardless of radiation source. In contrast, we found that each ion

species has a distinct impact on anxiety, motor coordination, and spatial memory

at extended time points. Even though 28Si and 56Fe are both regarded as high-LET particles, they have different detrimental impacts on behavior. In summary, our findings suggest that GCR radiation not only affects the CNS in the short term but

37 also has lasting damaging effects on the CNS that can cause sustained declines in behavioral performances.

1 A version of this chapter was accepted for publication in the Radiation

Research journal on September 29, 2017.

2.2 Introduction

The National Aeronautics and Space Administration (NASA) is working

towards the ambitious goal of sending astronauts to Mars and return them safely

back to Earth by 2030. The journey will take roughly three years including time on

the surface of Mars for research purposes. Risk assessment for long-term space travel requires a greater understanding of how human physiology is affected by outer space factors such as microgravity, emotional stress, and radiation. During a long-term deep space mission outside the earth’s magnetosphere, astronauts will be exposed to harmful space radiation that is composed of galactic cosmic radiation (GCR), solar particle events (SPEs) and trapped energetic particles in the Van Allen radiation belt [13-15]. Although only ~1 % of GCR is due to nuclei of heavier particles such as 12C, 16O, 20Ne, 24Mg, 26Al, 28Si, and 56Fe (called HZE,

high atomic number (Z) and energy (E) charged ions), it is extremely difficult to

protect astronauts from GCR by current spacecraft shielding materials [16, 19].

During a mission to Mars, it has been calculated that every cell in astronaut’s body

will be hit by one proton ion every few days, and by one heavy ion once per month,

ultimately delivering a whole-body radiation dose of 1 Sievert (Sv) [172, 173]. Thus,

space radiation remains one of the major emphases of the Human Research

Program of NASA in order to better protect our astronauts before mission launch

approaches.

Exposure to high-LET space radiation increases an astronaut’s risk to a

variety of health problems, including cancer, damage to the central nervous system

(CNS), degenerative damage to the circulatory system, loss of bone mass and

cartilage micro-architectural changes, increase in inflammatory response, and

cataract formation [174-179]. Initial observations of CNS effects by heavy ion irradiation come from past NASA missions, but the data are insufficient for extrapolation to a Mars mission for two major reasons: 1) previous missions have been short in length with a small astronaut population; and 2) the majority of missions were carried out in low earth orbits where astronauts were protected by earth’s magnetic field from potent GCR exposure. Non-human primates (NHPs) have been used to study acute and late effects of X-rays and proton ion radiation on earth for long-term cancer risk, but significant costs and an extensive follow up periods have made NHPs difficult to use in behavior performance tasks [180].

Therefore, rodent models have become popular for studying detrimental effects of

GCR on physiology.

Rodents sent to space have likewise shown deficits in hematopoietic,

immune, and CNS functions that indicate harmful effects of space travel [181-183].

Rodent behavior studies in simulated space radiation environments on earth have

demonstrated that heavy ion radiation damages the CNS as well. For example,

56Fe ion exposure in rats has been reported to lead to short and long-term

hippocampal dependent spatial memory loss [155, 156], and low doses of 48Ti ions

at 1000 MeV/n (>20 cGy) and 16O ions at 600 MeV/n (>30 cGy) results in cognitive

impairment [159, 160]. Animals exposed to low LET 4He ion radiation at 1000

MeV/n (>10 cGy) revealed disruption in a variety of cognitive tasks [161]. Together,

the data clearly show that radiation exposure poses a significant threat to the CNS,

and that considerations for astronaut cognitive functions on deep space missions

40 need to be made. What is not clear, however, is the lasting effect of radiation exposure on the CNS, and how radiation of different qualities compared across a standard set of behavior tests including motor-coordination and cognitive memory tasks.

The purpose of the present study was to examine the effects of low-, medium-, and high-LET radiation on behavior performances at long times post exposure to determine if generalizations can be made between different radiation qualities, and if the observed effects are sustained over multiple long-term observation points. Therefore, animals were exposed to 100 cGy or 250 cGy of terrestrial γ-ray radiation 137Cs with an LET of ~ 0.3 KeV/µm, and compared to 10 cGy or 100 cGy of 1H ion radiation at 1000 MeV/n (LET of ~ 0.2 KeV/µm), 28Si ion radiation at 300 MeV/n (LET of ~ 69 KeV/ µm), or 56Fe ion radiation at 600 MeV/n

(LET of ~ 180 KeV/ µm). Behavior tests were then performed at 5 and 9 month post exposure.

2.3 Materials and Methods

Animals

All animals were handled in accordance with the National Institutes of

Health and institutional animal care guidelines along with approval of Institutional

Animal Care and Use Committee at Case Western Reserve University and

Brookhaven National Laboratory (BNL). Animals were bred and maintained at the

Case Western Reserve University Animal Research Core (ARC) facility where they

were weaned at ~1 month of age. The animals were kept in ventilated micro-

isolator cages and fed regular mouse chow diet and sterilized water. Male C57BL/6

mice were used throughout the study to reduce variations from estrous cycles on

behavior tests.

Particle Irradiation

Adult C57BL/6 male mice (~3 months old) were shipped from Case Western

Reserve University to BNL in two traveling groups (sham plus 56Fe; and sham plus

the other ions) for logistical reasons, and allowed to habituate 4-7 days prior to

radiation. Animals were divided into 10 groups of 10 mice per group, including the

two sham groups. One group of 10 sham irradiated mice (1st control group) was

used to compare to 56Fe ion irradiated mice, and a separate group of 10 sham

irradiated mice (2nd control group) was used to compare γ-ray, 1H ion, and 28Si ion

irradiated mice because they were exposed on different trips to BNL. On the day

of the experiment, the animals were transferred from the BNL animal facility to the

NASA Space Radiation Laboratory (NSRL), and placed into a plastic pie-shaped

holding container that minimized animal movement while keeping them well

ventilated. The animal holding container was carried to the NSRL beam line where

it was placed in a vertical orientation of relative to the 20x20 cm beam line. The

animals received a whole body radiation dose of 10 cGy or 100 cGy with different

charged particles (n = 10 mice/dose), including 1H (1000 MeV/n), 28Si (300 MeV/n),

and 56Fe (600 MeV/n) at a dose rate of 5-50 cGy/minute. Twenty animals were

irradiated with 100 cGy or 250 cGy γ-rays (n = 10 mice/dose) in a Shepherd Mark

I irradiator at BNL. After exposure, all mice were transferred back to the BNL

animal facility where they remained for at least 7 days before returning to Case

Western Reserve University for the remainder of the study.

Behavior Tests

The Open Field test was performed by placing the animals in a square

plastic 50x50 cm apparatus closed with 50 cm high plastic walls, and activity was

recorded by a camera attached using ANY-maze video tracking software (Stoelting

Co. IL). The field was digitally divided into two areas by the software: inner area

(30 x30 cm) and outer periphery (10 cm gallery). The mice were kept in the field

for 10 minutes and the data were analyzed with two metrics: total distance travelled

(meters) as an activity measure, and percentage of time spent in the inner area as

an anxiety measure. All tests were conducted under 4 x 4-W lamp light conditions

during light phase of the day at similar times.

The Rota-rod (Columbus Instruments, OH) test was used to measure motor

coordination and balance. Each mouse was trained prior to the experiment by

placing the mouse on the cylinder-shaped rod for 60 seconds twice; the first time

at 0 rpm (revolutions per minute) and the second time at a constant 4 rpm speed.

After the initial trial period, each mouse was placed on the rod set to an

accelerating speed (speed increase of 0.1 rpm per second starting from 4 rpm for

three separate trials. The average maximum latency to fall (seconds) of the three

individual trials carried out within 2 hours was calculated for each mouse and used

as a parameter to assess motor coordination.

The Novel Object Recognition test as a memory task is based on the innate

preference of mice to spend more time exploring a new object than a previously

seen familiar object. The test apparatus was a regular mouse cage containing

bedding material. For training, two identical objects were placed at different

corners of the cage and each mouse was allowed to investigate the objects for 5

minutes, before returning to their home cages. Twenty-four hours later (long-term

memory), the mice were again placed in a cage with two objects, one from the

training period and one new object, and observed for 5 minutes. The total amount

of time spent sniffing or contacting each object was recorded and scored using

ANY-maze video tracking software. A memory score was calculated by dividing

sniffing times of unfamiliar and familiar objects and multiplying by 100 (*50% = a

chance level).

The T-maze, a Plexiglas T-shaped maze (with 60 cm length arms) was

utilized for a spatial memory task, in which mice investigate an unfamiliar arm

versus a familiar arm in order to assess their environment. Each mouse was placed

in the apparatus for 8 minutes and allowed to explore the maze while keeping one

arm of the T closed. Following 8 minutes of exploration, the mouse was placed

back in the original cage until the next day. Twenty-four hours later, the mouse was put back into the T-maze for 5 minutes during which both arms were kept open. Each mouse movement was video recorded from above and analyzed. For the memory performance score, the amount of time spent in the previously blocked arm was used to calculate the percentage unfamiliar arm choice (*50% = a chance level).

Statistical Analysis

Data were reported as mean ± SEM (standard error of mean) for all groups

in each experiment. All behavior test data of three groups (control, low, and high

dose) were analyzed by one-way analysis of variance (ANOVA) followed by a subsequent Newman-Keuls multiple comparison adjustment using Prism software

(Prism 5). Results were considered significant only when irradiated versus sham irradiated mice showed p values less than 0.05.

Table 1: Animal body weights 5 months after radiation exposure.

Individual group’s body weights (gm)

Control γ-ray IR 1H ion IR 28Si ion IR 56Fe ion IR

1st 2nd 100 250 10 100 10 100 10 100

cGy cGy cGy cGy cGy cGy cGy cGy

n 9 10 10 10 10 10 10 10 10 10

AVG 40.40 38.35 40.06 39.53 38.59 33.22 41.83 37.71 40.45 42.64

SEM 1.59 0.81 1.2 0.86 0.62 1.37 1.2 1.4 1.95 1.26

Table 2: Animal body weights 9 months after radiation exposure.

Individual group’s body weights (gm)

Control γ-ray IR 1H ion IR 28Si ion IR 56Fe ion IR

1st 2nd 100 250 10 100 10 100 10 100

cGy cGy cGy cGy cGy cGy cGy cGy

n 9 10 10 10 10 10 10 10 10 10

AVG 44.65 42.47 46.02 44.80 45.19 38.54 46.74 41.65 42.56 43.98

SEM 1.58 1.04 1.4 1.05 0.85 1.94 1.4 1.09 2.08 0.95

2.4 Results

Diminished activity is a late toxicity from low- and high-LET radiation.

An Open Field test was performed to measure both activity and anxiety

levels in mice treated with sham radiation, low-LET, and high-LET radiation.

C57BL/6 male mice were irradiated at ~3 months of age with γ-ray, 1H, 28Si, and

56Fe, and a battery of behavior tests were carried out at 5 months and 9 months

after exposures (Figure: 1A). Body weights of all mice were measured at both time

points post exposure and no significant differences were noted between the groups

(Table 1 & 2). The animals exposed to 100 cGy and 250 cGy γ-ray radiation displayed significantly lower activity levels compared to sham irradiated mice at both time points [5 months: F2,27 = 16.90, p<0.0001; 9 months: F2,27 = 15.73,

p<0.0001; figure 1B]. Similarly, animals exposed to 10 cGy or 100 cGy of 1H ion

radiation or 28Si ion radiation revealed significantly lower activity levels compared

1 to sham irradiated mice measured at both time points [ H ion – 5 months: F2,27 =

28 15.08, p<0.0001; 9 months: F2,27 = 7.67, p=0.0023 and Si ion – 5 months: F2,27 =

6.54, p=0.0048; 9 months: F2,27 = 4.39, p=0.0224; figure 1D, 1F]. However, mice

exposed to 10 cGy or 100 cGy 56Fe ion radiation did not show significant

differences in activity levels compared to control mice at either time point [5

months: F2,27 = 2.67, p=0.0878; 9 months: F2,27 = 0.26, p=0.7761; figure 1H].

Anxiety-like behavior was also measured. We found that animals exposed

to γ-ray, 1H ion, and 56Fe ion radiation did not have significant differences in anxiety

levels in comparison to sham irradiated animals at either time point [figure 1C, 1E,

and 1I]. In contrast, animals exposed to 10 cGy or 100 cGy 28Si ion radiation did

show significantly higher anxiety levels as shown by a lower time spent in the inner

area at 9 month post exposure but not at 5 months [5 months: F2,27 = 0.51,

p=0.6041; 9 months: F2,27 = 4.71, p=0.0176; figure 1G]. Together, we found that

animals exposed to γ-ray, 1H ion, and 28Si ion radiation showed significant dose

dependent decreases in activity levels, but the same effect was not observed in

animals exposed to 56Fe ion radiation. In addition, 28Si radiation altered anxiety

levels in a dissimilar manner to γ-ray, 1H ion, and 56Fe ion radiation.

Long-term motor coordination defects were revealed after low- and high-LET radiation.

The Rota-rod test as a motor coordination and muscle strength test, illustrates a selective effect of radiation on mouse behavior. We found a significant decline in motor coordination by measuring latency to fall (seconds) in animals exposed to 100 cGy, but not 250 cGy, of γ-ray radiation compared to untreated control animals at 5 months [5 months: F2,27 = 4.67, p=0.0181] and animals

exposed to 250 cGy, but not 100 cGy, of γ-ray radiation compared to untreated control animals at 9 month post exposure [9 months: F2,27 = 4.37, p=0.0228; figure

2A]. We also evaluated learning of the test by measuring the slope of the learning

curves of the three individual trials (latency to fall in seconds) of irradiated vs sham-

irradiated mice. We found learning defects in motor coordination at 5 months [F2,27

= 5.543, p=0.0096], but not at 9 months [F2,27 = 2.2787, p=0.1218], for  irradiated

mice post exposure similar to motor coordination. In contrast, animals exposed to

10 cGy, but not 100 cGy, of 1H ion radiation showed significant decreases in motor coordination compared to control animals at 5 month post exposure [5 months:

1 F2,27 = 6.76, p=0.0042]. Animals exposed to 10 cGy, but not 100 cGy, of H ion radiation also displayed modestly decrease in motor coordination at 9 month post

exposure [9 months: F2,27 = 3.61, p=0.0407; figure 2B]. Mice exposed to 100 cGy,

but not 10 cGy, of 28Si ion radiation revealed significantly impaired motor

coordination only at 9 months but not at 5 months after exposure [5 months: F2,27

28 = 1.99, p=0.1559; 9 months: F2,27 = 3.81, p=0.0348; figure 2C]. Si ion exposed mice also showed learning defects measured at 9 months [F2,27 = 3.374,

56 p=0.0492], but not at 5 months [F2,27 = 2.73, p=0.1071], post exposure. Fe ion

irradiated animals did not show a defect in motor coordination long-term after

exposure to radiation compared to sham irradiated animals at either time points

(figure 2D). Collectively, Rota-rod testing revealed a generally significant decline in motor coordination and/or muscle strength in the low LET radiation species at the lower dose but not higher dose, and a difference in response between the two high LET species.

Low- and high-LET radiation cause long-term recognition memory loss.

The Novel Object Recognition test is a non-spatial recognition memory task.

Animals exposed to 100 cGy or 250 cGy γ-ray radiation revealed a significant

decline in recognition memory at 9 month post exposure but not at 5 months [5

months: F2,27 = 2.49, p=0.1021; 9 months: F2,27 = 3.80, p=0.0350; figure 3A].

However, animals exposed to 10 cGy or 100 cGy 1H ion radiation showed

significant declines in recognition memory at both time points after exposure

compared to unirradiated animals [5 months: F2,27 = 4.68, p=0.0180; 9 months:

28 56 F2,27 = 4.24, p=0.0251; figure 3B]. Similarly, Si ion and Fe ion irradiated animals at 10 cGy or 100 cGy revealed significant losses of recognition memory long-term

28 after exposure compared to sham irradiated animals [ Si ion – 5 months: F2,27 =

56 14.42, p<0.0001; 9 months: F2,27 = 6.90, p=0.0038 and Fe ion – 5 months: F2,27

= 8.22, p=0.0016; 9 months: F2,27 = 8.45, p=0.0014; figure 3C & 3D]. Therefore,

animals exposed to different doses of 1H ion, 28Si ion, or 56Fe ion radiation exhibited similar effects of recognition memory loss at both time points after radiation exposure, but a similar reduction in recognition memory after γ-ray

radiation was only observed at 9 months after exposure.

Transient spatial memory loss is caused by γ-ray and 56Fe ion radiation.

To investigate long-term spatial memory, a T-maze test was performed. We

found a significant decline in spatial memory of animals exposed to γ-ray radiation

at 100 cGy, but not 250 cGy at 5 month post exposure [F2,27 = 5.72, p=0.0085;

figure 4A], while this decline was not seen at 9 months post exposure [F2,27 = 0.22,

p=0.8004]. 1H ion (10 and 100 cGy groups) and 28Si ion (10 and 100 cGy groups)

irradiated mice showed no differences in spatial memory performances compared

1 to sham irradiated mice at either time point [ H ion – 5 months: F2,27 = 1.696,

28 p=0.2024; 9 months: F2,27 = 2.604, p=0.0925 and Si ion – 5 months: F2,27 =

0.8193, p=0.4514; 9 months: F2,27 = 0.4707, p=0.6296; Figure: 4B & 4C], though

the profiles of the 1H graphs looked similar to the -ray five month point with low

dose effects but not high dose effects. In contrast, animals that were exposed to

10 cGy, but not 100 cGy of 56Fe ion radiation revealed a significant decline in

spatial memory at 5 month post exposure [F2,27 = 3.66, p=0.0397] that were no

longer significant at 9 months [F2,27 = 0.88, p=0.4278; figure 4D]. In conclusion,

spatial memory performance in the T-maze showed effects that were dose, ion, and time of assessment-dependent.

2.5 Discussion

The majority of behavior studies of the impact of GCR on the CNS have been performed with 56Fe ion irradiated mice at short time intervals post radiation

exposure [184]. The short-term impact of GCR on cognitive function is not the only

risk factor during orbital space missions, but rather late CNS risks such as

dementia, Alzheimer’s disease, and premature aging could also be a potential risk

factor for humans on deep space missions. Therefore, our study was designed to

look at long-lasting motor coordinative and cognitive changes post radiation to

understand the impact of low, medium, and high-LET radiation on the CNS. We

observed that different ion species can differentially impact behavior metrics

assessed at 5 months and 9 months after exposures.

In the current study, we found a prominent impact of low, medium, and high-

LET radiation on recognition memory defects up to 9 months after the exposure.

A novel object recognition test is known to be activity-independent task represents

a performance of non-spatial memory or recognition memory [147]. A recently

published study showed that C57BL/6J male and female mice exposed to less

than 50 cGy of 56Fe (600 MeV/n) radiation caused hippocampal dependent recognition memory defects two weeks after exposure [185]. Similarly, in our study

we found that mice exposed to 1H, 28Si, and 56Fe irradiation showed significant

declines in recognition memory at 5 and 9 months after radiation exposure, but

only at 9 months in mice exposed to γ-rays. Our data demonstrate that mice are more susceptible to radiation induced declines in recognition memory after extended periods of time regardless of radiation source. One potential hypothesis, to explain this mechanism, may be the loss of memory due to structural defects in the CNS, which has been shown in a rodent model exposed to HZE particles. Mice exposed to protons at 10 or 100 cGy showed a significant changes in the number of dendritic branches and dendritic length in the hippocampal region after one month compared to control mice [186]. In addition, another study has shown that the hippocampus is important for non-spatial and spatial memories, and lesion sizes in the hippocampus are directly correlated to loss of spatial versus non-

spatial memory [187]. In order to affect spatial memory, larger lesions are required,

and this indicates that spatial memory relies on larger hippocampal regions than

recognition memory. Studying structural HZE-induced damage, at extended time

intervals post exposure is thus an important area of future mechanistic study.

Interestingly, our data showed that spatial memory declines caused by low-

, medium-, and high-LET radiation exposure is not as profound as the decline in

recognition memory. In published studies, rats irradiated with whole-body 56Fe

(1000 MeV/n) ion radiation at 150 cGy demonstrated spatial memory impairment

one month after exposure [155]. Similarly, we found that mice exposed to γ-rays

and 56Fe ion radiation showed significant declines in spatial memory after 5

months, but not after 9 months. This reversal of memory impact seen at 9 months

could be explained by active neurogenesis occurring in hippocampal region of

adult mouse brain. In fact, recent studies discovered active neurogenesis occurring

in dentate gyrus, and changes in neurogenesis was linked to hippocampal

dependent cognitive impairments [142, 165, 188]. Moreover, we observed a

significant decline in spatial memory at 100 cGy of γ-rays and 10 cGy of 56Fe ion irradiated mice. A similar impact of low dose 56Fe versus high dose of γ-rays could be explained by a higher relative biological effectiveness (RBE) of HZE particles compared to sparsely ionizing γ-ray radiation. In contrast, mice exposed to either

1H or 28Si ion radiation showed no impact on spatial memory at either time point.

It has been shown that mice exposed to 100 cGy 56Fe ion radiation demonstrated

loss of neuronal progenitor cells in the hippocampal region 3 months post exposure

[189]. Therefore, further analysis of neurogenesis alteration at long time post

radiation exposure could shed a light on mechanism by which different radiation

sources induce different spatial memory deficits. Notably, changes in T-maze

behavior can also be indicative defects of cognition in addition to memory, which

would also point to hippocampal damage, and may explain why the animals show

not just a lack of preference for the novel arm, but indeed a preference for the

familiar arm in some cases [190].

In short term studies, mice exposed to 56Fe (1000 MeV/n) ion irradiation at

10 cGy, 50 cGy, or 200 cGy have been reported to display no impact on motor

coordination up to 2 months after exposure [191]. Similarly, we found no

impairment in motor coordination at either time point when mice were exposed to

10 cGy or 100 cGy of 56Fe ion radiation. Intriguingly, as a side note, even though

the same mice were examined at 5 and 9 months, the controls in the second test

did not reveal a memory of the test four months prior for any study except for the

activity measurement of the open field test, allowing conclusions to be drawn with

equal weight from each test time point. The sham-irradiated animals from the 56Fe

irradiations do however show differences in overall activity when compared to the

second sham group that may mask some iron effects. Notably, mice exposed to

100 cGy of γ-rays or 10 cGy of protons showed significant impairment in motor

coordination at 5 months, while 28Si ion irradiated mice showed motor coordination

defects at 9 months. Early biochemical studies have shown that radiation alters

nerve signaling pathways, specially K+-evoked release of dopamine in irradiated groups [192-194]. Insufficient dopamine in humans has been associated with

Parkinson’s disease leading to motor defects. Motor coordination defects observed

in our study could be associated with reduced dopamine levels in brain. The

findings suggest that impairment caused by each ion species is different, and the

mechanism by which low- vs high-LET radiation manifest their impacts is distinct.

We observed that mice exposed to γ-rays, 1H, or 28Si ion radiation, but not 56Fe ion

radiation, showed significantly lower activity levels compared to sham irradiated

mice up to 9 months after exposure. Lower activity levels in irradiated mice suggest

that radiation induced premature aging could be a driving factor in these mice. In

addition, reduction of locomotor activity observed in sham-irradiated mice at 9

months compared to those at 5 months is primarily due to aging effect [195].

Moreover, the open field test also revealed significantly higher anxiety levels in

mice exposed to 28Si ion radiation at 9 months, but no impact of other ion species

at either time points post exposure. Thus, even though 28Si ion and 56Fe ion

radiation are both considered high-LET, we found that each ion has a distinct

impact on activity, anxiety, and motor coordination, suggesting they do not impact

biological functions in the same manner.

In summary, we found that radiation exposure causes a long-lasting

damage on the CNS region regulating locomotor activity as well as on the

hippocampal memory function. 56Fe ion radiation caused the majority of damage

in cognitive functions, but had no impact on anxiety, activity, or motor coordination;

28Si ion radiation caused a significant impact on activity, anxiety, motor

coordination, and non-spatial memory, but no impact in spatial memory; 1H ion

radiation caused significant impairment in activity, motor coordination, non-spatial

memory but no impact in anxiety or spatial memory; and γ-rays showed damaging

impact on all behavior performances except anxiety. These differences in behavior

patterns caused by each ion species could be explained by the way low- and high-

LET radiation deposit energy into the cells. In the case of low-LET γ-rays and proton exposure, dispersed ionization damage is generated by stochastic energy distribution of randomly encountered photons or protons. In the case of high-LET radiation such as 28Si ion or 56Fe ion exposure, nearly all of the energy deposited

in localized regions of their main tracks and delta-ray penumbra [18, 196].

Mechanism based studies looking at changes in neuro-inflammation, oxidative

damage, neurogenesis, and neuronal plasticity, are required to understand the

pattern of behavior performances associated with each ion species long-term after radiation exposure. The combined effects of all radiation species that exist in the

GCR spectrum to which astronauts will be exposed on the journey to Mars will be necessary to accurately quantify risks to cognitive behaviors at extended time points.

2.6 Acknowledgements

This research was funded by National Aeronautics and Space Administration

(NASA) grant NNJ13ZSA001N. The authors are grateful to all members of NASA

Space Radiation Laboratory and support staff at Brookhaven National Laboratory, and to Tom Petereson, Jr., for his generosity.

Figure 2.1: Diminished activity is a late toxicity from low- and high-LET radiation.

(A) Time-line indicating irradiation and a battery of behavior tests performed over a yearlong study. (B) Activity, and (C) Anxiety level in animals after exposure to

low-LET γ-ray radiation at 100 cGy and 250 cGy.(D) Activity, and (E) Anxiety level

1 in animals after exposure to 10 cGy and 100 cGy H ion radiation at 1000 MeV/n.

(F) Activity, and (G) Anxiety level in animals after exposure to 10 cGy and 100 cGy

28 Si ion radiation at 300 MeV/n. (H) Activity, and (I) Anxiety level in animals after

56 exposure to 10 cGy and 100 cGy Fe ion radiation at 600 MeV/n. γ-rays, 1H ion,

and 28Si ion irradiated mice were compared to one sham-irradiated group while separate sham-irradiated group was used to compared 56Fe ion irradiated mice.

Data in open field test were analyzed by one-way ANOVA followed by Newman-

Keuls to control for multiple comparisons in generating p-values in individual

graphs. All data are presented as mean ± SEM; * indicates significant differences

compared to the control groups; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-

significant.

Figure 2.1: Diminished activity is a late toxicity from low- and high-LET radiation.

Figure 2.2: Long-term motor coordination defects were revealed after low-

and high-LET radiation.

Latency to fall in seconds (average of three individual trials per mouse) was

measured in animals after exposure to (A) 100 cGy and 250 cGy γ-ray radiation,

1 (B) 10 cGy and 100 cGy H ion radiation at 1000 MeV/n, (C) 10 cGy and 100 cGy

28 56 Si ion radiation at 300 MeV/n, and (D) 10 cGy and 100 cGy Fe ion radiation at

600 MeV/n. γ-rays, 1H ion, and 28Si ion irradiated mice were compared to one sham-irradiated group while separate sham-irradiated group was used to compared 56Fe ion irradiated mice. Data in rota-rod test were analyzed by one-way

ANOVA followed by Newman-Keuls to control for multiple comparisons in

generating p-values in individual graphs. All data are presented as mean ± SEM;

* indicates significant differences compared to the control groups; *p < 0.05; **p <

0.01; ***p < 0.001; ns = non-significant.

Figure 2.2: Long-term motor coordination defects were revealed after low- and high-LET radiation.

Figure 2.3: Low- and high-LET radiation cause long-term recognition

memory loss.

Percentage memory score was calculated by preference ratio for an unfamiliar

object to familiar object after exposure to (A) 100 cGy and 250 cGy γ-ray radiation,

1 (B) 10 cGy and 100 cGy H ion radiation at 1000 MeV/n, (C) 10 cGy and 100 cGy

28 56 Si ion radiation at 300 MeV/n, and (D) 10 cGy and 100 cGy Fe ion radiation at

by a one-way ANOVA followed by Newman- Keuls to control for multiple

comparisons in generating p-values in individual graphs. All data are presented

as mean ± SEM; * indicates significant differences compared to the control

groups; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant.

Figure 2.3: Low- and high-LET radiation cause long-term recognition memory loss.

Figure 2.4: Transient spatial memory loss is caused by γ-ray and 56Fe ion

radiation.

Spatial memory was measured by calculating ratio of total time spent in unfamiliar

arm over familiar arm after exposure to (A) 100 cGy and 250 cGy γ-ray radiation,

1 (B) 10 cGy and 100 cGy H ion radiation at 1000 MeV/n, (C) 10 cGy and 100 cGy

28 56 Si ion radiation at 300 MeV/n, and (D) 10 cGy and 100 cGy Fe ion radiation at

ANOVA followed by Newman-Keuls to control for multiple comparisons in

generating p-values in individual graphs. All data are presented as mean ± SEM;

* indicates significant differences compared to the control groups; *p < 0.05; **p <

0.01; ***p < 0.001; ns = non-significant.

Figure 2.4: Transient spatial memory loss is caused by γ-ray and 56Fe ion radiation.

Chapter 32 MMR deficiency does not sensitize or compromise the function of hematopoietic stem cells to low and high LET radiation.

3.1 Abstract

One of the major health concerns on long-duration space missions will be radiation exposure to the astronauts. Outside the earth’s magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events

(SPE) that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel.

The hematopoietic system is one of the most radiation-sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and

56Fe radiation on HSC function in Mlh1+/+ and Mlh1-/- marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity.

2 A version of this chapter was accepted for publication in the Stem Cell

Translational Medicine journal on March 20, 2018.

3.2 Introduction

Exposure to ionizing radiation (IR) is considered to be one of the major risk

factors during space activities, especially long-duration space missions [28, 197].

Astronauts on missions to the International Space Station, the moon, or Mars will

be exposed to IR with effective total doses in the range of 5 to 200 cGy (centigray)

based on projected mission scenarios [198, 199].The primary components of

space radiation are galactic cosmic rays (GCR) and radiation from solar particles

[200]. This space radiation consists of 85% protons, 14% helium nuclei and 1%

high-energy, high-charge (HZE) particles, including oxygen (16O), carbon (12C), silicon (28Si), and iron (56Fe) ions [201]. HZE radiation is of particular concern

because it causes high linear-energy transfer (high-LET) damage in biological targets and induces repair-refractory clustered DNA damage in cells [28, 202-205].

These types of damage directly affect cell survival and genomic integrity in

surviving cells [206]. In addition, it is estimated that for every cell traversed by a

potentially lethal HZE nucleus (e.g. 56Fe), another 32 cells are hit by δ rays of

decreased energy that could induce non-lethal mutations [207]. 56Fe has been

thought to be the most biologically important HZE particles, since it may be the

single largest naturally occurring particle [201].

The hematopoietic system is one of the most radiosensitive tissues of the

body [37]. Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and are

responsible for generation and maintenance of multiple cell lineages in the blood

supply [208]. It is well known that total body irradiation (TBI) affects both mature

blood cells and hematopoietic stem/progenitor cells and causes both acute

radiation hematopoietic syndrome and long-term BM injury [38, 209]. HZE

particles like 56Fe ions have been shown to be more toxic than γ rays with lethal

doses (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-days) of 5.8

Gy compared with 7.25 Gy for γ rays. Mice irradiated with a lethal dose of 56Fe

showed significantly lower white blood cell (WBC) recovery at 4 weeks post-IR,

compared to γ-IR mice [210]. 56Fe-IR caused loss of hematopoietic

stem/progenitor cells immediately after IR, which was maintained for up to 8 weeks

[211]. In addition, protons, 28Si ions and 56Fe ions are also known to induce

hematopoietic malignancies such as AML, though not necessarily with greater

efficiency than γ irradiation [42, 212].

Exposure of mice to 1 Gy 56Fe results in highly complex chromosome

aberrations, including dicentrics, as well as translocations, insertions and acentric

fragments, which is unlike the damage from γ radiation. Cells exhibiting these

aberrations disappear rapidly after exposure, probably as a result of death of

heavily damaged cells. Cells with apparently simple exchanges as their only

aberrations, appear to survive longer than heavily damaged cells. Eight weeks

after exposure, the frequency of cells showing cytogenetic damage was reduced to

less than 20% of the levels evident at 1 week. These results indicate that exposure

to 1 Gy 56Fe produces heavily damaged cells, a small fraction of which appear to

be capable of surviving for relatively long periods [213, 214]. In addition, exposure

to low doses of 56Fe resulted in significant epigenetic alterations involving

methylation of DNA, and expression of repetitive elements, which is also unique to

high LET radiation [215]. Therefore, while damage from terrestrial γ radiation and

X-rays forms the basis our understanding of cellular responses to DNA damage,

GCR provides a unique cellular stress and highlights gaps in current models describing the response of biological systems to space radiation.

IR causes not only DNA strand breaks, but also nucleotide base and sugar damage [216]. Analyses of gamma rays and 56Fe induced murine AML samples have identified similar molecular changes, including biallelic loss and/or mutation of PU.1. Microsatellite instability (MSI), a commonly observed marker of DNA mismatch repair (MMR) deficiency, has been observed in 42% of AML samples induced by gamma rays or 56Fe [217]. MMR is an essential DNA repair pathway responsible for maintaining genomic integrity primarily by removing base mismatches and small insertion/deletion loops (IDLs) introduced during replication or under genomic stress [218]. In humans, MMR gene defects (most notably in the

MLH1, MSH2, MSH6, PMS2 genes) and MSI have been most closely associated with Lynch Syndrome [101, 105, 219], but are also found in an increasing number of tumor types [220]. MMR deficiency has also been identified in primary and secondary hematopoietic malignancies and in leukemia and lymphoma cell lines

[221-223]. Exposure of MMR-deficient mice to gamma radiation results in hypermutability compared to wild type mice, and much of this hypermutability can be attributed to induced instability of simple sequence repeats [224]. Interestingly, recent results from our laboratory have demonstrated that acquired MMR deficiency and increased MSI in human hematopoietic stem and progenitor cells is age-related, attributable to progressive loss of Mlh1 through promoter hypermethylation [170, 171]. The data show that as many as 30% of HSCs in

healthy individuals have lost MLH1 by 45 years of age, which is well within the age

range of current and former astronauts. Therefore, TBI may pose an

unappreciated risk to astronauts on deep space missions if they have significant

numbers of MMR defective HSCs.

Though the effects of protons and HZE ions on normal mouse

hematopoietic systems have been characterized at the effector cell level [225,

226], the impacts on HSC function are not fully known. Importantly, in humans

demonstrating diminished MMR capacity, the potential for loss of HSC function

and/or malignant transformation may be greater [170]. Therefore, the role of MMR

in response to γ and GCR radiation damage needs to be carefully examined. In

this study, we used Mlh1-deficient mice to investigate the importance of the MMR

system on the response of HSCs to protons and 56Fe, in comparison to γ radiation,

with an aim to investigate the risk of hematopoietic failure in astronauts on deep

space missions. In short, we find that while heavy ions are more damaging than

gamma radiation to HSC functionality, no additional deficits in hematopoietic

function were identified. The data suggest that the greater risk of MMR deficient

HSCs likely lies in malignant transformation rather than hematopoietic failure.

3.3 Materials and Methods

Mice

B6.129-Mlh1tm1Rak/NCI heterozygous mice were obtained from NCI and

then mated to produce Mlh1+/+ and Mlh1-/- mice for the study. C57BL/6J mice were

purchased from Jackson Laboratory, congenic strain B6.SJL-

PtprcaPepcb/BoyCrCrl (BoyJ, CD45.1) mice were obtained from Charles River

Laboratory. All the mice were group housed in ventilated microisolator cages in a

specific pathogen-free facility. Mice had ad libitum access to food (Laboratory

Rodent Diet 5LOD, Lab Diet, St. Louis, MO) and water. The animal housing room

was maintained on a 12:12h light:dark cycle and constant temperature (72 ± 2˚ F).

Male and female mice between 8 and 16 weeks of age were used for the study.

All mouse studies were approved by the Institutional Animal Care and Use

Committee at Case Western Reserve University (Cleveland, OH), and Brookhaven

National Laboratory (BNL) (Upton, NY).

Radiation

Two to three month old, male and female, and Mlh1+/+ and Mlh1-/- mice were

used for the study. Proton and 56Fe irradiation were performed at the NASA Space

Radiation Laboratory (NSRL) at Brookhaven National Laboratories. After 7 days

of acclimation, the mice were exposed to TBI with 1 Gy of protons (1000 Me/V) or

56Fe (600 MeV/n). Gamma irradiations were performed at BNL using a 137Cs

source for Mlh1+/+ and Mlh1-/- animals, and at Case Western Reserve University

for recipient mice irradiated with 11 Gy. Sham irradiated animals that traveled to

BNL were used as controls. One set of sham irradiated Mlh1+/+ and Mlh1-/- mice

was used to compare 1H ion and 56Fe ion irradiated mice while second set of sham irradiated Mlh1+/+ and Mlh1-/- mice were used to compare γ-rays irradiated mice

because they were exposed on different trips to Brookhaven National Laboratory.

Peripheral Blood and BM Collection

Peripheral blood (PB) was collected from the submandibular vein using a

heparinized hematocrit capillary tube. Complete blood count was measured by a

Hemavet 950 FS that gives 5-part differentiation with 20 parameters. For BM

collection, mice were sacrificed roughly three months after radiation exposure, the

femur and tibia were harvested and immediately flushed with Phosphate-buffered

saline (PBS) containing 2% fetal bovine serum (FBS) using 21 and 27-gauge

needles and syringes to collect the BM. The number of BM cells was counted using

a hemocytometer.

Colony-forming unit (CFU) assays

BM mononuclear cells (BM-MNCs) were cultured in methylcellulose

medium containing cytokines, including mouse interleukin 3, human interleukin 6,

mouse stem cell factor, and human erythropoietin, MethoCult GF M3434 (Stem

Cell Technologies, Vancouver, BC). Total numbers of CFU colonies were scored

on day 7, according to the manufacturer’s protocol. Two separate experiments of

three plates each per radiation exposure were performed, and combined results

were shown in the results.

Flow cytometry

Flow cytometry was performed on a BD LSRII (BD Biosciences, San Jose,

CA), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).

Antibodies include CD45.2 (clone 104), CD45.1 (clone A20), Ly-6G (Gr-1, clone

RB6-8C5), CD11b (Mac-1, clone M1/70), CD45R/B220 (clone RA3-6B2), CD3

(clone 500A2), and Ter119/Ly76 (clone Ter-119), Sca1 (Ly-6A/E, clone D7), c-Kit

(CD117, clone 2B8), CD48 (clone HM48-1, BD Bioscience) and CD150 (clone

TC15-12F12.2, Biolegend, San Diego, CA).

Competitive repopulation assay

All mice were sacrificed roughly three months post radiation exposure and

BM cells were collected for competitive repopulation assay. 2x106 BM cells from mice (CD45.2) of each genotype were mixed with the same number of wild type

(CD45.1) competitor BM and transplanted into lethally irradiated (11Gy) BoyJ recipients (CD45.1) through lateral tail veins. The recipients were monitored and analyzed for hematopoietic reconstitution and lymphoma development. Peripheral blood was collected 8 weeks and 16 weeks post BM transplant to measure percentage contribution of CD45.2 cells in circulation.

Statistical Analysis

Statistical analysis was performed using GraphPad 5.0. Student’s t tests were used to determine the significance of pairwise comparisons; ANOVA were used for dose response analyses, and log-rank tests were used to analyze survival curves. p<0.05 was the measure of statistical significance.

3.4 Results

High LET radiation induces similar long term damage to the bone marrow as

γ radiation.

In order to begin to assess the effects of high LET radiation on MMR defective HSCs, we first sought to establish the effects of 56Fe compared to γ radiation on stem cell function. Various studies in the literature have demonstrated

72 that 56Fe and other high LET sources can damage hematopoiesis with different relative biological effectiveness (RBE) [226, 227]. We thus assessed bone marrow cellularity; enumerated Sca1+, c-Kit+, Lin- (SKL) cells; measured proliferation; and determined repopulation capacity all at a latent time point. As seen in figure 1A, at three months after exposure to 1 Gy of γ or 56Fe radiation, bone marrow cellularity was unchanged in the animals compared to control animals. In contrast, the SKL populations in the animals dropped by greater than 50% in both the γ and

56Fe groups. Notably, there was no significant difference between the two radiation sources. HSCs are known to rapidly reenter the cell cycle from quiescence in response to hematopoietic stresses in order to regain homeostasis. By three months, however, both the irradiated cohorts displayed HSC proliferation that was identical to the non-irradiated animals, as evidenced by BrdU incorporation (Figure

1C). The behavior was similar with the broader SKL population as well as the more primitive CD48- SKL cells. Finally, we measured the functionality of the irradiated HSCs by performing a competitive transplantation assay using unirradiated marrow injected into lethally irradiated hosts. We observed that the irradiated marrow was functionally challenged and had grafting potential that was reduced nearly 50% in both γ and 56Fe cohorts, indicating similar RBEs at the 1

Gy dose (Figure 1D). With these data in hand, we could now address the role of

MMR function.

High LET radiation is more damaging to clonogenic capacity of stem cells than low LET radiation, but independent of MMR status.

Exposure of HSCs to multiple forms of ionizing radiation, including space

radiation, is known to reduce clonogenic capacity [211]. To determine whether loss

of MMR plays a role in BM vitality after exposure to γ, protons (1000 MeV/n) or

56Fe radiation (600 MeV/n), clonogenic assays were performed on isolated bone

marrow in a standard colony forming assay. As expected, high LET radiation

effected a greater decrease in colony formation than γ or proton radiation across

a range of doses from 0.1 to 2.5 Gy (Figure 2A-C). In addition, 56Fe ion exposure

clearly caused a greater decrease in the number of BFU-E and CFU-GM than low

LET γ or proton radiation (supplementary figure 1A-1I). We did not, however observe significant differences between the wild type and Mlh1 knockout marrow,

suggesting that MMR function does not contribute to acute effects of IR on

clonogenicity in vitro.

Blood counts demonstrate similar acute damage to the hematopoietic

system across LET.

To assess the acute effects of IR on MMR competent and deficient marrow

in vivo, we performed TBI with γ, protons or 56Fe at 1 Gy, and performed regular

blood counts for up to 30 days. The response of specific effector cells to IR has been well documented, with lymphocytes being the most sensitive cells. As seen in Figure 3A-C, total WBCs displayed significantly more sensitivity to any form of

IR compared with red blood cells and platelets (Figure 3D-I), which here did not

decrease substantially during the 30-day follow up. There were no significant

differences between the different radiation sources, however, all three effected

similar transient decreases in lymphocytes (supplementary figure 2A-2C). Again,

no differences were observed between Mlh1 competent and deficient marrow.

Long term effects on hematopoiesis by IR is independent of MMR status.

In order to assess latent or long term effects of high and low LET radiation on hematopoiesis, TBI was performed on cohorts of both wild type and Mlh1-/- mice

with 1 or 2.5 Gy of γ radiation, 1 Gy of proton radiation, or 1 Gy of 56Fe. Three

months after exposure, we planned to analyze the frequencies and numbers of

different hematopoietic cell populations in BM by flow cytometry, and measure

HSC functionality by transplantation assays. We first, however, noted survival

statistics of the cohorts. As previously published, Mlh1-/- animals are tumor prone,

with lymphomas and gastrointestinal tumors being the most prevalent

malignancies [228] and γ irradiation of Mlh1 deficient mice is known to accelerate

tumorigenesis [117]. With a three-month follow-up, we found that 43% of the null

animals developed malignancy while none of the control, wild type mice developed

any tumors. To break the numbers down by treatment group, 19% (4 of 21)

unirradiated null mice, 64% (7 of 11) 1 Gy γ irradiated mice, 45% (5 of 11) 2.5 Gy

γ irradiated mice, 33% (3 of 10) of 1 Gy proton irradiate mice, and 30% (3 of 9) 1

Gy 56Fe irradiated mice were euthanized for morbidity due to tumor formation within three months after exposure (Figure 4A-C). The only statistically significant increase in malignancy was in the 1 Gy γ group (p=0.0155, Log-rank). Presumably, had the animals been given longer times to develop tumors, as has been done in other studies, our data would have shown increased tumorigenesis in the other

radiation groups as well. Additionally, we found only lymphomas in our cohorts,

also likely due to the short follow up, as found previously [117].

Our primary interest was in determining the function of the MMR-defective

HSCs at extended times after radiation. Therefore, at three months after exposure,

all surviving mice were euthanized, and marrow was harvested and assessed. We

first quantified the SKL cells. As seen in Figure 5A-C, γ, proton, and 56Fe irradiated animals demonstrated significant decreases in SKL cells compared to unirradiated control mice. The γ irradiated animals displayed dose dependent decreases in

SKL cells of up to 56% of non-irradiated animals at 2.5 Gy (p<0.0001 for Mlh1+/+,

and p=0.0002 for Mlh1-/-; one-way ANOVA). In pairwise comparisons, we noted

that unlike the wild type mice, the Mlh1-/- null mice did not show significantly reduced SKL cells in the marrow at the 1 Gy dose, potentially suggesting protection against IR (p<0.0001 for wild type, p=0.25 for Mlh1-/-; student’s t tests); but any protection was lost at the higher dose where both genotypes were similar.

Likewise, proton radiation led to a significant decrease in SKL cells in the wild type

(p<0.0001, student’s t test), and Mlh1-/- animals (p<0.0001, student’s t test). 56Fe

irradiation resulted in indistinguishable decreases of SKL cells in both genotypes

(p<0.05 for both, student’s t tests). In addition, BM cellularity was unchanged

between Mlh1+/+ and Mlh1-/- mice (supplementary figure 3). In summary, three months after IR, significant decreases can be observed in SKL cell numbers in the

bone marrow of exposed mice, but Mlh1 function does not appear to play a role in

this response.

We next looked at hematopoietic progenitor cells (HPCs) by gating for

Sca1-, c-Kit+, Lin− cells. Here we observed that there were no detectable differences in any of the radiation source groups, at any dose, and for either genotype (Figure 5D-F). These data suggest that the SKL cells that remain functional three months after IR are sufficient to produce the normal levels of progenitor cells that are required to maintain the hematopoietic system.

Finally, we tested the colony forming potential of the marrow in standard

CFU assays, by embedding bone marrow derived cells in methylcellulose with a variety of stem cell cytokines and enumerating colonies after 7 days (Figure 5G-

H). In agreement with our observations of SKL cells, we found that γ radiation led to statistically significant drops in CFU in a dose dependent manner for both genotypes (p<0.0001 for wild type and Mlh1-/-, ANOVA). For proton radiation at 1

Gy, both genotypes displayed significant drops in CFU, 26.8% for wild type

(p<0.0001, student’s t test) and 31.6% for Mlh1-/-, (p<0.0001 student’s t test).

Surprisingly, 56Fe radiation did not cause any measureable defects in clonogenic capacity, which is in contrast to the observed effects on SKL cells, and could be due to an iron specific hyperproliferation in the SKLs. Importantly, all of the observed effects were independent of Mlh1 status.

Defects in Mlh1 function do not enhance decreased competitive repopulation caused by IR

As a long term, functional measure of hematopoiesis, we conducted competitive repopulation studies comparing irradiated marrow of each genotype to wild type, unirradiated marrow. The concept is that if long term functional defects

exist, the irradiated and/or Mlh1 defective marrow will contribute less efficiently to

repopulation of the marrow after lethal IR, and can be demonstrated by flow

cytometry of peripheral blood [229, 230]. We therefore mixed competitor bone

marrow cells from CD45.1 mice at a 1:1 ratio with irradiated wild type, or Mlh1-/-

marrow from each irradiation exposure, and transplanted the cells into lethally

irradiated hosts. At 8 and 16 weeks after transplant, peripheral blood was analyzed and revealed marked decreases in competition of the irradiated marrow from all sources. Much like we observed with the SKL data, γ irradiation led to a dose dependent decrease in competition from 1 and 2.5 Gy (p<0.0001, ANOVA) treated mice of both genotypes, at both 8 and 16 weeks (Figure 6A, B). For proton irradiation, similar decreases were seen with 1 Gy in both genotypes, at both time points (Figure 6C, D), and no differences were observed between the genotypes.

Finally, we tested the 56Fe irradiated marrow and found reduced but consistent

effects of IR on the marrow functionality (Figure 6E). Together the data confirm

that long term damage occurs after low and high LET radiation, but that Mlh1 status

is inconsequential to the function of the marrow.

3.5 Discussion

In the current study, we investigated the effects of low and high LET IR on

HSCs in vitro and in vivo with the goal of evaluating the significance of MMR defects that are characteristic of aging individuals who could be exposed during space travel. We found primarily that MMR, as a function of Mlh1 gene presence or absence, is not a relevant factor for hematopoietic functionality after radiation.

We assessed clonogenic capacity of bone marrow harvested immediately after

irradiation, as well as clonogenic capacity in marrow harvested three months after

irradiation with γ, proton or 56Fe sources and found almost identical responses,

regardless of Mlh1 status. We assessed blood counts of the animals for up to 30 days post exposure and similarly found that Mlh1 wild type and knock out animals responded indistinguishably. Finally, we assessed SKL cells and HPCs in the bone marrow at three months after exposure, and also measured transplantability of the bone marrow at three months and found notable decreases in stem cell number and function due to radiation, but not due to Mlh1 status. Together the data support the conclusion that MMR status is not a relevant variable for function of the hematopoietic system after exposure to γ or space radiation, and thus does not contribute added risk to astronauts.

Effective DNA repair is an essential function for the fidelity of organisms.

HSCs are no exception to this rule, and indeed it has been elegantly shown that

HSCs that lack efficient repair as they age, through loss of Ligase IV or Ku70, and

thus non-homologous end joining, display reduced hematopoietic stem cell

functions [231, 232]. Studies of animals engineered to harbor defective nucleotide

excision repair or telomere maintenance show similar age-associated defects

[231]. However, defects in these major repair pathways, unlike MMR defects, are

relatively low in frequency [170, 233]. Ionizing radiation poses a greater threat to

HSC biology due to the relative facility of exposure in modern society, and the

exquisite sensitivity of the HSCs to DNA damaging stress [234]. Clearly, the

79 combination of DNA repair defects, in the presence of IR stress would be predicted to compromise HSC functions even further.

Tissue kinetics contribute significantly to the time of manifestation of radiation induced damage. The hematopoietic system is extremely sensitive to ionizing radiation, demonstrating depletion of functional cells within hours to days

[36], and an associated proliferative response of progenitor cells in the same time frame [235]. Latent tissue damage, however, implies depletion of stem cells, or a continued source of cell stress that prolongs damage manifestation. In the current studies, both explanations are likely to contribute. We observed depletion of HSCs three months after exposure, as well as decreased repopulation capacity of the remaining stem cells in a competitive transplant assay. Previously, elevated levels of reactive oxygen species correlating with decreased HSCs have been observed at 22 weeks after exposure to proton radiation, in line with our observations here

[78]. Added mutational load, which is known to occur with MMR defects and radiation exposure [224], however, did not exacerbate the latent phenotype, at least regarding HSC function. While we measured functional cells by complete blood counts and found no effect of Mlh1 status, it is possible that some lineage differences exist that were not detected. Most notably, though, decreased stem cell function at latent times could be due to stem cell exhaustion caused by excessive induced proliferation in the acute phase immediately after exposure, and remains a risk to hematopoiesis that is shown here to be independent of MMR function.

Therefore, the predominant additional risk to the hematopoietic system of

MMR defective individuals is likely to remain in the development of malignancies.

The identification of MMR defective cancers outside of the gastrointestinal family in which they were first appreciated continues to rise, highlighting the importance of MMR to tumor suppression [220, 236]. Further studies will be required to assess the role of Mlh1 loss in the carcinogenic process in the hematopoietic lineages to most accurately assess risk for the purposes of deep space missions.

3.6 Acknowledgements

This study was supported by NASA NNX14AC95G, and the Cytometry &

Microscopy and Radiation Resources Shared Resources of the Case

Comprehensive Cancer Center (P30CA043703). We would like to thank members of NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory for help and support of our studies. We further thank Thomas F. Peterson, Jr., for his generosity.

Figure 3.1: High LET radiation induces similar long term damage to the bone

marrow as γ radiation

Low and high LET radiation have similar effects on hematopoiesis at 3 months post IR. Bone marrow cellularity (A), SKL numbers (B), and HSC proliferation (C) as measured by BrdU incorporation at three months post exposure to 1 Gy of γ or

56Fe. (D) Competitive transplantation assay of marrow harvested from irradiated

cohorts at three months against unirradiated healthy marrow.

Figure 3.1: High LET radiation induces similar long term damage to the bone marrow as γ radiation

Figure 3.2: High LET radiation is more damaging to clonogenic capacity of stem cells than low LET radiation, but independent of MMR status

MMR status does not contribute to clonogenic capacity of HSCs after radiation. γ

(A), proton (B), and 56Fe (C) irradiated wild type and Mlh1 knockout BM cells in

CFU assays after exposure to 0.1, 0.5, 1, or 2.5 Gy of radiation (n=6, number of plates used for each radiation exposure).

Figure 3.2: High LET radiation is more damaging to clonogenic capacity of stem cells than low LET radiation, but independent of MMR status

Figure 3.3: Blood counts demonstrate similar acute damage to the

hematopoietic system across LET

Blood counts of wild type and Mlh1 knockout animals demonstrate radiation

induced changes, but no Mlh1 associated defects. Complete blood counts were

performed at 10 days prior to, and 2, 10, 20, and 30 days after exposure to

indicated doses of γ (A, D, G), proton (B, E, H) or 56Fe (C, F, I) sources. White

blood cells (WBC), Red blood cells (RBC), and Platelets, are shown (n=6, number

of Mlh1+/+ or Mlh1-/- mice used for each radiation exposure).

Figure 3.3: Blood counts demonstrate similar acute damage to the hematopoietic system across LET

Figure 3.4: Mlh1 knockout mice display enhanced sensitivity to IR Mlh1 knockout mice display enhanced sensitivity to ionizing radiation. Survival plots of cohorts of mice of wild type or Mlh1 knockout genotypes exposed to 1 Gy of γ (A), proton (B), or 56Fe (C) radiation (n=9-12, number of Mlh1+/+ or Mlh1-/- mice used for each radiation exposure).

Figure 3.4: Mlh1 knockout mice display enhanced sensitivity to IR.

Figure 3.5: Long term effects on hematopoiesis by IR is independent of MMR

status

Latent effects of radiation are independent of MMR status. Quantification of SKL

cells in wild type and Mlh1 deficient animals three months after exposure to γ (A),

proton (B), and 56Fe (C) radiation at indicated doses. Quantification of HPC cells

in wild type and Mlh1 deficient animals three months after exposure to γ (D), proton

(E), and 56Fe (F) radiation at indicated doses. Quantification of colony formation

of bone marrow preparations in wild type and Mlh1 deficient animals three months

after exposure to γ (G), proton (H), and 56Fe (I) radiation at indicated doses.

Marrow was collected from between 4 and 12 animals.

Figure 3.5: Long term effects on hematopoiesis by IR is independent of

MMR status.

Figure 3.6: Defects in Mlh1 function do not enhance decreased competitive

repopulation caused by IR

Radiation, but not Mlh1 deficiency, reduces competitive capacity of the bone

marrow after exposure. Measurement of percentage contribution of CD45.2

positive cells to peripheral blood at 8 (A, C, and E) and 16 (B and D) weeks after

competitive transplant of 1 Gy of γ (A and B), proton (C and D), and 56Fe (E) irradiated marrow of indicated genotypes.

Figure 3.6: Defects in Mlh1 function do not enhance decreased competitive repopulation caused by IR

Supplementary Figure 3.1: Myeloid CFU survival post radiation exposure.

Myeloid CFU survival post radiation exposure. (A-C) Number of total myeloid CFU,

BFU-E, and CFU-GM post different doses of gamma-rays IR. (D-F) Number of total myeloid CFU, BFU-E, and CFU-GM post different doses of proton ion IR. (G-I)

Number of total myeloid CFU, BFU-E, and CFU-GM post different doses of 56Fe ion IR.

Supplementary Figure 3.1: Myeloid CFU survival post radiation exposure.

Supplementary Figure 3.2: Lymphocyte counts in Mlh1+/+ and Mlh1-/- mice.

Lymphocyte counts Mlh1+/+ and Mlh1-/- mice pre and post (A) γ-rays, (B) 1H ion IR, and (C) 56Fe ion IR.

Supplementary Figure 3.2: Lymphocyte counts in Mlh1+/+ and Mlh1-/- mice.

Supplementary Figure 3.3: Bone marrow cellularity in Mlh1+/+ and Mlh1-/- mice.

Bone marrow cellularity in Mlh1+/+ and Mlh1-/- mice three months after exposure to

IR is unchanged with no impact of Mlh1 status.

Supplementary Figure 3.3: Bone marrow cellularity in Mlh1+/+ and Mlh1-/- mice.

Chapter 43

Mlh1 deficiency increases the risk of hematopoietic malignancy after

simulated space radiation exposure

4.1 Abstract

Cancer-causing genome instability is a major concern during space travel

due to exposure of astronauts to potent sources of high-linear energy transfer

(LET) ionizing radiation. Hematopoietic stem cells (HSCs) are particularly

susceptible to genotoxic stress, and accumulation of damage can lead to HSC

dysfunction and oncogenesis. Our group recently demonstrated that aging human

HSCs accumulate microsatellite instability coincident with loss of MLH1, a DNA

Mismatch Repair (MMR) protein, which could reasonably predispose to radiation-

induced HSC malignancies. Therefore, in an effort to reduce risk uncertainty for

cancer development during deep space travel, we employed an Mlh1+/- mouse

model to study the effects high-LET 56Fe ion space-like radiation. Irradiated Mlh1+/-

mice showed a significantly higher incidence of lymphomagenesis with 56Fe ions

compared to γ-rays and unirradiated mice, and malignancy correlated with

increased MSI in the tumors. In addition, whole exome sequencing analysis

revealed high SNVs and INDELs in lymphomas being driven by loss of Mlh1 and

frequently mutated genes had a strong correlation with human leukemias.

Therefore, the data suggest that age-related MMR deficiencies could lead to HSC malignancies after space radiation, and that countermeasure strategies will be required to adequately protect the astronaut population on the journey to Mars.

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3 A version of this chapter was accepted for publication in the Leukemia journal on

October 1, 2018.

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4.2 Introduction

The success of manned missions to outer space depends on many factors,

including overcoming health risks such as space radiation. Space radiation is

composed of protons and high (H) atomic number (Z) and energy (E) (HZE)

charged ions that arise from Solar Particle Events (SPEs), Galactic Cosmic

Radiation (GCR), and the Van Allen radiation belts [13-15]. In particular, GCR is

composed of 90% of protons, 9% of alpha particles (4He nuclei), and ~1% nuclei

of HZE particles such as 12C, 16O, 20Ne, 24Mg, 26Al, 28Si, and 56Fe ions [16, 19].

These particles have a broad range of LET characteristics (densities of induced

ionization events along particle tracks). The extent to which differences in LET

relate to different types of health risks remains largely unknown, and current

mitigation strategies and shielding materials are ineffective to protect astronauts

from HZE radiation due to the penetrance of the particles. In addition, there is

incomplete understanding of the radiobiology of HZE particles and a lack of

accurate risk assessment models, which puts future human-based space missions

in question.

A major space radiation-induced health risk to astronauts is tumorigenesis.

Cancer fatality risk prediction is an important consideration for deep space

missions for government agencies including the National Aeronautics and Space

Administration (NASA). Data for low-LET radiation-induced cancer risk in humans

come from epidemiological studies of Japanese A-bomb survivors, radiotherapy patients, and occupational radiation workers [150]; while data for high-LET

radiation rely mostly animal modeling. Various studies performed using mice have

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identified cancers such as mammary tumors, hepatocellular carcinoma, colorectal

cancer, and leukemia as being HZE-induced [41-43]. The data show clear differences between high- and low-LET radiation, both in tumor type and incidence.

Radiation-induced lymphomas and leukemia represent a significant concern for astronauts during space travel due to the efficiency of radiation induced hematopoietic malignancies.

Ionizing radiation (IR) produces a variety of DNA damage products that are repaired by multiple DNA damage response (DDR) processes. The DNA mismatch repair (MMR) pathway is part of the DDR that fixes mismatches generated by DNA polymerase during replication, but also repairs base damage from a variety of stresses including radiation [237, 238]. In particular, MMR eliminates IR-induced buildup of 8-oxoguanine lesions to prevent adenine misincorporation during DNA replication [239-241]. MMR defects in tumors are associated with microsatellite instability (MSI) – gain or loss of nucleotides from microsatellite tracks in DNA. MSI

is classically associated with colorectal cancers where loss of functional MMR

components is frequently found, and tumor cells are said to display a mutator

phenotype indicating the lack of a key caretaker pathway [242, 243]. MMR could

thus play a radioprotective tumor suppressor role, a concept supported by studies

that have shown enhanced induction of intestinal carcinogenesis in MMR defective

mice exposed to oxidative stress [244], and others that have found induction of a

preleukemic state in HSCs [245]. MMR consists of seven different proteins, including MLH1, which is crucial for bringing repair machinery to mismatch repair sites. Studies have found epigenetic silencing of MLH1 in cancers such as

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glioblastoma multiforme, endometrial, lung, and head and neck squamous

carcinomas [138, 246-248]. Therefore, loss of MLH1 may predispose cells to

become cancerous, particularly if exposed to high-LET ionizing radiation.

A recent study by our group demonstrated that MSI accumulates in human

HSCs as a function of age, with loss of MLH1 by promoter hypermethylation [170,

171]. Given that upper end of astronauts are ~46 years old, HSCs with deficient

MMR function will likely be exposed to space radiation. We thus sought to

characterize the interaction between loss of MLH1 and exposure to high-LET

radiation in the induction of hematopoietic malignancies. Using Mlh1+/- mice, that

exhibit MSI [249], exposed to 100 or 250 cGy of low-LET γ-rays and 10 or 100 cGy of high-LET 56Fe ion particles, we find that Mlh1 status does not have an impact

on long-term HSC function. However, Mlh1 allelic deficiency significantly increases the risk of hematopoietic malignancy after γ-ray or 56Fe ion radiation with

associated loss of Mlh1 function determined by high levels of single nucleotide

variants (SNVs)/insertions and deletions (INDELs) in resulting tumors.

4.3 Materials and Methods

Animals

Institutional Animal Care and Use Committee approved protocols were

followed at Case Western Reserve University (CWRU) and Brookhaven National

Laboratory (BNL). The Mlh1+/- strain B6.129-Mlh1tm1Rak/NCI was acquired from the

National Cancer Institute at Frederick [249]. All animals were bred and maintained

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at the CWRU Animal Research Core. All mice had ad libitum access to food

(Laboratory Rodent Diet 5LOD, Lab Diet, St. Louis, MO) and water. The animal housing room was maintained on a 12:12h light:dark cycle and constant temperature (72 ± 2° F).

Particle irradiation

Adult B6.129-Mlh1tm1Rak male and female mice (~12 weeks) were shipped to BNL roughly one week prior to irradiation. The animals were divided into 10 groups of ~ 40 animals, including sham-irradiated Mlh1+/+ and Mlh1+/-. On the day

of exposure, animals were arranged into an animal pie-shaped holder and placed perpendicular to a 20x20 cm beam line to expose with 10 or 100 cGy of 600 MeV/n

56Fe ions at a dose rate of 5-50 cGy/minute. Additional animals were exposed to

100 or 250 cGy of γ-rays in a Shepherd Mark I irradiator-containing 137Cs at BNL.

Bone marrow (BM) cells were irradiated at NSRL for clonogenic survival assays

and competitive repopulation assays. Mlh1+/+ and Mlh1+/- mice (5 animals per genotype) were sacrificed on site, and bone marrow cells were harvested and irradiated with 0, 10, 50, 100, or 250 cGy of 600 MeV/n 56Fe ions. Additional BM

cells were irradiated with 0, 10, 50, 100, or 250 cGy of γ-rays.

Clonogenic survival assay

Irradiated Mlh1+/+ and Mlh1+/- BM cells were plated with complete

methylcellulose media (MethoCultTM GF M3434 or MethoCultTM M3630,

STEMCELL Technologies) to measure survival by colony forming unit (CFU)

assay. M3434 media was used for myeloid colony formation, and M3630 media

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for pre-B lymphoid assays. All assays were performed twice with three replicates

(50,000 cells/plate for myeloid CFU and 250,000 cells/plate for lymphoid CFU),

and counted between 7-14 days post-plating.

Histology and Immunohistochemistry

Animals were euthanized at first signs of morbidity and tumors were

collected. All tumors were fixed in 10% formaldehyde for 24 hours followed by

immersion into 70% ethanol until processed and sectioned. Hematoxylin and eosin

(H&E) stains were performed and then analyzed at the In Vivo Animal Core facility

at the University of Michigan. Selected lymphomas were further analyzed by

immunohistochemistry (IHC) with B220 (B-cell marker), CD3 (T-cell marker), or

F4/80 (histiocyte marker) antibodies.

Microsatellite instability

Tumors were assessed for four mononucleotide repeats (mBat-26, mBat-

37, mBat-59, and mBat-64) [250]. Amplification of each mononucleotide repeat

was performed separately by PCR. Detection of amplified PCR fragments was

performed on an Agilent TapeStation and analyzed by TapeStation Analysis

Software A.02.01 SR1. Each marker length (deletion or addition of nucleotides)

measured by the software was compared to marker length of a normal tissue to

identify each marker as being stable or unstable. The classification of microsatellite

instability was accomplished by calculating the number of unstable markers for

each tumor sample. We classified tumors as MSI stable, low, or high based on

numbers of these markers with instabilities being 0/4, 1/4, or >1/4, respectively.

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Whole-exome sequencing

Whole-exome sequencing (WES) was carried out by using a Truseq Exome

library prep kit according to manufacturer’s protocol, and a 2x75bp HS run was

performed using an Illumina HiSeq2500. Sequencing quality was assessed using

FastQC (ver.11.5). Trimmomatic (ver.0.32) was used to remove sequence

adapters and low quality leading and trailing bases from reads [251]. Filtered and trimmed reads were aligned to reference genome mm10 using the Burrows-

Wheeler Aligner (ver.0.7.12) algorithm [252]. Refinement of reads alignment was performed using GATK (ver.3.4.0) analysis toolkit, including PCR duplicated removal, local INDEL realignment, and base recalibration [253]. For variant calling, we performed individual tumor sample calling using Mutect2, against the sample from normal mouse tissue as normal reference [254]. Final SNVs and INDELs were selected with stringent criteria and final variants were annotated using

VariantAnnotation (ver. 1.20.3) R package [255].

4.4 Results

Mlh1 heterozygosity significantly increases high-LET radiation induced

malignancy.

Knockout animals are known to be tumor prone, and thus do not phenocopy

aged people; in contrast, Mlh1+/- animals exhibit relatively low spontaneous

tumorigenesis in spite of partial loss of MMR function [117]. During follow-up, mice

were euthanized at the onset of signs of morbidity or the appearance of visible

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tumors (figure 1A). We found a significant reduction in tumor-free survival of Mlh1+/-

mice irradiated with 100 or 250 cGy of γ-ray vs. sham-irradiated Mlh1+/- mice or

irradiated Mlh1+/+ mice (p<0.0001, figure 1B) Interestingly, we observed

significantly increased mortalities in Mlh1+/- mice exposed to 10 or 100 cGy 56Fe

ions vs. sham-irradiated Mlh1+/- or irradiated Mlh1+/+ mice (p<0.0001, figure 1C).

Indeed, the biological impact of 100 cGy of 56Fe ions exceeded 100 cGy of γ-rays

(p=0.0470). In addition, Mlh1+/- mice exposed to 100 cGy 56Fe ion IR showed a

significant lower tumor free survival compared to mice exposed to 10 cGy 56Fe ion

IR (p=0.0456). In contrast, we observed no significant increases in tumorigenesis

of Mlh1+/+ mice regardless of the type of radiation used. To gain insight into the

time of onset of disease, the same data with exposed animals grouped by

genotype were used to estimate 70% survival times: sham-irradiated Mlh1+/- mice,

513 days; Mlh1+/- mice irradiated with 100 or 250 cGy of γ-rays, 446 and 444 days;

Mlh1+/- mice irradiated with 10 or 100 cGy of 56Fe ion irradiation, 424 and 385 days;

and all Mlh1+/+ mice, regardless of being irradiated, time not reached (figures 1D-

1F, supplementary table 1). Thus, loss of Mlh1 enhances radiation-induced tumorigenesis that heavily depends on radiation quality.

Mlh1 deficiency increases the incidence of lymphomagenesis after low and

high LET radiation.

It has been shown that loss of Mlh1 is associated with a higher incidence of

lymphomas and gastrointestinal tumors in animal models [228, 256]. Therefore,

we next examined tumors collected from Mlh1+/+ and Mlh1+/- mice by hematoxylin

and eosin staining to determine tumor types and if radiation exposure altered the

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distribution of tumor types formed. Histopathology analysis revealed different

tumor types, but lymphoma was found to be the most common tumor (figure 2A).

Sporadic age-related tumors such as hepatocellular adenomas (HCA),

hepatocellular carcinomas (HCC), histiocytic sarcoma (HS), and other rare tumors

(figure 2B-2F; supplementary table 2) were also observed. The analysis

determined that ~40% of total tumors found in Mlh1+/+ cohorts were lymphomas

(figure 2G). Interestingly, we observed a significant difference in tumor type

distribution between Mlh1+/+ mice treated or not with low- or high-LET radiation

(p=0.0447). In contrast, ~80% of tumors of Mlh1+/- cohorts were lymphomas (figure

2H). Further, Mlh1+/- cohorts revealed significantly higher incidence of multiple

tumors per mouse compared to Mlh1+/+ cohorts (p=0.0288, figure 2I). These data

argue that Mlh1 deficiency increases incidence mostly of hematopoietic

malignancies after IR, independent of radiation quality (i.e. LET).

Mlh1+/- cohorts have higher incidence of T-cell rich B-cell lymphomas.

Lymphomas can be classified based on immunophenotype. The majority of lymphomas show immune cell infiltrates in the tumor microenvironment, which is associated with profound influence on disease pathology [257]. Therefore, we decided to further explore the lymphomas based on IHC analysis. We used CD3,

B220, and F4/80 to discern T cell, B cell, and macrophage/histiocytes in the tumors, respectively. Staining patterns revealed six different types of lymphoma that include T-cell rich B-cell (TRB) lymphoma, B-cell lymphoma, T-cell lymphoma, histiocytic sarcoma, B/T mixed lymphoma, and T-cell/histiocyte rich B-cell lymphoma (figure 3A-3F; supplementary table 3). We found 40-60% of lymphomas

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were TRB lymphomas in the Mlh1+/+ mice (figure 3G). Interesting, we observed

roughly 30% of lymphomas were histiocytic sarcoma in sham-irradiated and γ-

irradiated Mlh1+/+ mice, whereas no histiocytic sarcomas were found in 56Fe

particle irradiated Mlh1+/+ mice. Similarly, we observed that the majority of

lymphomas were TRB lymphomas in all treatment groups of Mlh1+/- mice (figure

3H). Collectively, the data show that TRB lymphomas were common in Mlh1+/+ and

Mlh1+/- mice regardless of radiation type and that infiltrating T-cells might play a role in the process of lymphomagenesis.

Mlh1+/- tumors exhibit elevated levels of microsatellite instability.

Loss of MMR strongly correlates with MSI in many human cancers [220].

Therefore, we anticipated that mononucleotide repeats would be highly

susceptible to MSI in Mlh1+/- tumors compared to Mlh1+/+ tumors, particularly

because heterozygosity of Mlh1 has been shown to associate with decreased DNA

repair [170, 249]. The average sizes of four mononucleotide markers were

measured from MMR-proficient control samples and shown as peak values in

figures 4A-4D. MSI analysis showed that a majority of markers had a deletion of

one or more nucleotides in the stretch of mononucleotide repeats in Mlh1+/- tumors

(figure 4E-4H). We found that roughly 80% of Mlh1+/- tumors showed high MSI,

while only 2% of these tumors showed stable MSI. In contrast, we found that

roughly 45% of Mlh1+/+ tumors were high MSI, and that 55% were either stable or low MSI. Thus, MSI differed in Mlh1+/+ vs. Mlh1+/- tumors (p=0.0048, figure 4I).

Interestingly, we observed no change in high MSI of Mlh1+/- tumors by different

radiation types, including sham-irradiated (figure 4J). Collectively, the data show

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that MSI associates with tumorigenesis in both the Mlh1 wild type and

heterozygous mice, and hence MMR status could be a potential risk stratification

marker for individuals exposed to high LET ionizing radiation.

Significantly elevated levels of SNVs and INDELs appear in Mlh1+/-

lymphomas.

MMR deficiency is associated with a mutator phenotype. In particular, loss

of Msh2 and Mlh1, key components of MMR, have been shown to increase

mutational frequency in newborn mice and during different stages of

embryogenesis [258, 259]. After verifying high MSI in Mlh1 heterozygous tumors,

we decided to further analyze TRB lymphomas by WES to study SNV/INDEL

patterns in wildtype vs heterozygous lymphomas. The WES analysis revealed a

significant increase in mutation rate of Mlh1+/- compared to Mlh1+/+ TRB

lymphomas arising from sham-, γ-rays, or 56Fe ion IR (p<0.0001, figure 5A, 5B).

Surprisingly, radiation exposure showed no further increase in number of SNVs of

irradiated cohorts compared to sham-irradiated cohorts, regardless of Mlh1 status

(p=0.9225, figure 5A,5B). In addition, WES analysis showed significantly higher

INDELs in the Mlh1+/- irradiated cohorts compared to Mlh1+/+ irradiated cohorts

(p=0.0314, figure 5A, 5C). The data suggest that Mlh1 heterozygosity was

associated with higher SNVs, while INDELs were correlated with irradiation plus

loss of Mlh1.

For further analysis, we identified frequently mutated genes in Mlh1+/+ and Mlh1+/-

cohorts based on type of radiation exposure. To examine the role of recurring

mutations occurred at specific loci, we defined a gene as frequently mutated if it

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was found to be mutated in ≥ 40% of at least one cohort. The analysis revealed

that a significantly higher number of frequently mutated genes were found in

Mlh1+/- cohorts compared to Mlh1+/+ cohorts of TRB lymphomas (p<0.0001, figure

5F, 5G). Mlh1 heterozygosity not only increased the mutation rate, but the

repeated nature of mutations occurring at the same loci suggests importance of

these genes in tumorigenesis. In fact, we compared frequently mutated genes to

well-defined cancer causing genes and discovered that ~13% of the genes in each

cohort of Mlh1+/- TRB lymphomas were associated with cancer (supplementary

table 4). Collectively, WES analysis not only revealed higher SNVs and INDELs in

Mlh1+/- TRB lymphomas, but also that mutations occurred frequently in genes responsible for tumorigenesis.

High LET radiation induces a unique spectrum of genetic alterations in genes associated with human leukemia.

The C57BL/6 mouse model is a useful resource for studying radiation- induced cancers if parallels can be drawn between the mechanisms of radiation- induced tumorigenesis of mouse lymphomas and human leukemias. Many studies have shown that expression changes in genes such as Ikaros, Bcl11b, and Epha7 occur in both mouse lymphomas and various types of human leukemias [260-263].

Therefore, we asked whether genes frequently mutated in TRB lymphomas are relevant to human leukemia, and whether 56Fe ions produced unique mutations

compared to γ-rays. We identified 8 and 39 recurrently altered human leukemia

genes in Mlh1+/+ and Mlh1+/- TRBs, respectively (figure 6A, 6B). Interestingly, irradiated cohorts showed different gene mutational patterns compared to sham-

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IR tumors, suggesting a distinct pathway leading to lymphomagenesis. For

instance, high rates of somatic mutations in Cbl, Huwe1, Runx1, and Ttn genes

were found in all Mlh1+/- cohorts. In contrast, some genes were found mutated in

specific treatment groups: Jag1, Kit, Nup214, and Pik3cd were prominently found

mutated in the sham-IR cohort; Dnmt3a and Myb were prominently found in γ-ray

cohort; and Myc was only found in 56Fe ion IR cohort. In addition, the majority of

the mutations were nonsynonymous in nature (figure 6C). Thus sequence

analyses of TRB lymphomas suggests that common mechanisms underlie these

mouse lymphomas and radiation induced human leukemias, and strengthens the

position that MLH1 defects will predispose space radiation-exposed astronauts to

disease development.

4.5 Discussion

The impact of age-associated MMR defects to the risk of space radiation-

induced malignancies has not been previously assessed. The current study

provides evidence that loss of Mlh1 in HSCs, which occurs as a function of age in

normal healthy individuals [170] leads to a significantly higher incidence of

tumorigenesis after exposure to high LET radiation, and that the incidence is

dependent on the type of radiation exposure. At the same time, we observed no

significant changes in acute hematopoietic functions of Mlh1+/- vs Mlh1+/+ BM cells

measured by CFU and competitive repopulation assays (supplementary figure 1).

Further, long-term differentiation potential of HSCs was also unaffected by Mlh1

status (supplementary figure 2). Thus, the critical observation described here is

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that MMR defective animals are cancer prone when exposed to cosmic radiation.

Mlh1+/- mice show increased incidence of lymphomagenesis compared to Mlh1+/+

mice, and MSI is coincident with tumorigenesis in all cohorts. WES analysis of the

tumors revealed a significantly higher rate of SNVs/INDELs in Mlh1

haploinsufficient TRB lymphomas along with strong evidence of recurrent gene

mutations occurring in carcinogenic and leukemogenic genes. The data are in

agreement with the observation that MMR deficiency due to Msh2 loss has been

shown to promote a preleukemic state without affecting HSC repopulation function

[245]. Together, our studies demonstrate that low- and high-LET radiation induce

elevated tumorigenesis in Mlh1 deficient contexts that could alter the risk paradigm

for astronauts on deep space missions.

After high-LET iron particle exposure, nearly all energy deposition occurs in confined regions of the cell near the particle track and associated δ-ray penumbras, causing dense local ionization and clustered DNA lesions [18, 196].

Thus, the likelihood of repair of DNA damage and survival of cells is significantly reduced for most cell types following the same doses of high-LET compared to low-LET irradiation. In our studies, we observed a significantly higher impact of high-LET 56Fe particles on HSC acute functions compared to low-LET γ-rays, regardless of Mlh1 status. Similarly, we found that radiation exposure significantly accelerated tumorigenesis in Mlh1+/- mice compared to wild type mice, and that

high LET radiation was markedly more effective. The findings are in agreement

with work from the Weil group and others which showed higher incidence of

tumorigenesis in animals exposed to high-LET IR compared to low-LET γ-rays

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exposure [42, 212, 264, 265]. Mlh1+/- mice exposed to 100 cGy 56Fe ion IR were

reduced to 70% survival ~130 and ~60 days earlier compared to sham-IR and 100 cGy γ-rays exposed Mlh1+/- mice, respectively. Collectively, these findings suggest

that loss of Mlh1 and high-LET radiation exposure together are responsible for not

only higher frequency but early incidence of tumorigenesis.

Radiation induced damage by high-LET sources may have an indirect role

in leading to tumorigenesis. Late-occurring chromosomal aberrations and global

DNA methylation in hematopoietic stem/progenitor cells have been shown after

28Si ion irradiation [83]. Kennedy, et al, have also observed altered methylation in

bronchial epithelial cells after 56Fe and 28Si exposure, contributing to lung cancer,

which in theory could also contribute to the mechanism of loss of MLH1 expression

in HSCs [266]. Mice exposed to high-LET 16O (600 MeV/n) ions showed

significantly higher level of ROS in HSCs three months after irradiation, suggesting

that cells experience continuous damage stress [80, 267]. Continuous ROS levels

in HSCs post irradiation could lead to mutation accumulation in absence of

functional MMR and may explain our observation of significantly higher SNVs in

all cohorts of Mlh1+/- TRB lymphomas. However, we did not detect differences in

SNVs between sham and irradiated cohorts, which may be due to the longer time

taken by the sham-IR cohort to reach to 70% survival hence allowing extra time to

accumulate SNVs. In addition, we discovered significantly higher mean INDEL size

(≥5 and ≥10 base pairs) in all Mlh1+/- cohorts compared to Mlh1+/+ cohorts, implying

Mlh1 plays a role not only in MMR but also in double strand break repair (figure

5D, 5E), which has been suggested in other models [268, 269]. Collectively, the

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WES analysis suggests that Mlh1 loss is strongly associated with high mutational

burden in lymphomas, and high mean INDELs size could be due to Mlh1

involvement in repair mechanism other than MMR.

We observed not only a high mutation rate in Mlh1 haploinsufficient

lymphomas, but also frequent mutations occurring in carcinogenic loci. Studies

have shown that Mlh1 loss is associated with frequent mutations occurring in the loci of NF1 and ATR [139, 140]. Similarly, we found frequent mutations in Nf1 and

Atr along with 12 other carcinogenic genes (Met, Cacna1d, Ptprd, Nbea, Gnaq,

Cntnap2, Csmd3, Pabpc1, Lrp1b, Zfhx3, Dcc, and Ctnna2) across all cohorts of

Mlh1+/- TRB lymphomas. In addition, each cohort of Mlh1+/- TRB lymphoma

showed radiation-specific gene mutational profiles. For instance, well-defined

carcinogenic genes such as Cdh1, Eps15, Was, Atp2b3, Cdh11, and Myc were

predominantly mutated in 56Fe ion IR Mlh1+/- cohort while the γ-ray Mlh1+/- cohort

revealed frequent mutations in genes such as Ddx6, Tsc2, Raf1, Nt5c2, Crebbp,

Tfe3, Stat3, Map2k1, Dnmt3a, Bcor, Map3k1, and Arid2. Critically, we observed

an enrichment of mutations in the Mlh1+/- lymphomas that also occur in human

leukemias. The data also revealed radiation quality specific effects, such as the

observation of Myc mutation exclusively in 56Fe, Myb mutation exclusively in γ-

rays, and Nup214 mutation predominately in sham irradiated Mlh1+/- lymphomas.

It is unclear at this point what mechanism would lead to gene-specific mutations,

but the observation is similar to one recently published by Porada and colleagues

when human HSCs were exposed to HZE radiation that showed enrichment in

mutations in leukemia-associated genes within 24 hours of exposure [34].

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Therefore, our study suggests that age-related MLH1 loss in astronaut HSCs results in a preleukemic state that can be exacerbated by high-LET radiation exposures received during space travel.

Increased use of high-LET radiotherapy also raises concern for therapy- related malignancies in patients with MMR defects, both in the hematopoietic system and beyond. Although further studies will be required to better characterize the molecular nature of tumors formed in our studies, and what types of doses and

LET are sufficient for enhancing tumor development, the results should be interpreted carefully, as astronauts in outer space may be exposed to several types of HZE particles with different fluences and energies. Future studies will be required to assess the effects of medium LET species and subsequently mixed ion beam fields and lower dose-rates to better mimic space radiation. In summary, the data suggest that loss of Mlh1 in HSCs, either genetically or as a function of age, could play a critical role in sensitizing humans to space-radiation induced HSC malignancies. Further studies will be required to more accurately calculate risks, both for missions into outer space and for patients undergoing current proton or future carbon-ion radiotherapy.

4.6 Acknowledgements

This research was funded by NASA grant NNX14AC95G. The authors are grateful to all members of NASA Space Radiation Laboratory and support staff at

Brookhaven National Laboratory, in particular to Adam Rusek, Chiara La Tessa,

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and Peter Guida, for their assistance. The authors are also thankful to shared

resources of the Case Comprehensive Cancer Center including Radiation

Resources, Integrated Genomics, Cytometry & Microscopy, and Hematopoietic

Biorepository & Cellular Therapy. We also thank the generosity of Thomas F.

Peterson, Jr.

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Figure 4.1: Long-term tumorigenesis assay post low- and high-LET radiation

exposure.

(A) Schematic representation of long-term tumorigenesis assay design. Tumor free survival of Mlh1+/+ and Mlh1+/- mice post (B) 100 or 250 cGy γ-rays, or (C) 10

or 100 cGy 56Fe ions (n=36-44, number of Mlh1+/+ or Mlh1-/- mice used for each

radiation exposure). (D) Tumor free survival of Mlh1+/+ mice post 0, 100 or 250 cGy

γ-rays, or 10 or 100 cGy 56Fe ions. (E) Tumor free survival of Mlh1+/- mice post 0,

100 or 250 cGy γ-rays, or 10 or 100 cGy 56Fe ions. (F) Days post-irradiation to reach 70% survival.

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Figure 4.1: Long-term tumorigenesis assay post low- and high-LET radiation exposure.

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Figure 4.2: Histopathology of tumors from Mlh1+/+ and Mlh1+/- mice.

(A) Lymphoma in sections of liver, characterized by sheets of neoplastic

lymphocytes infiltrating and effacing normal hepatic parenchyma (arrowheads)

(40X, bar = 20um). (B) Histiocytic sarcoma composed of round to spindyloid

neoplastic cells with occasional multinucleate giant cells (arrowhead) (20X, bar =

50um). (C) Hepatocellular carcinoma composed of lobules, cords, and trabeculae

of atypical hepatocytes replacing normal parenchyma (bar = 50um). (D)

Hemangiosarcoma composed of sheets and bundles of spindle-shaped cells forming haphazard vascular channels (arrowhead) lined by neoplastic endothelial cells (40X, bar = 20um). (E) Harderian gland adenoma characterized by an expansile proliferation (arrowhead) of tubules and acini of fairly well differentiated glandular epithelial cells (bar = 100um). (F) Ovarian granulosa cell tumor composed of solid lobules and nests of neoplastic cells often forming rudimentary follicular structures (arrowhead) (40X, bar = 20um). (G) Percentage tumor distribution based on histology of tumors collected from Mlh1+/+ mice treated with

sham-, γ-, or 56Fe ion irradiation. (H) Percentage tumor distribution based on

histology of tumors collected from Mlh1+/- mice treated with sham-, γ-, or 56Fe ion

irradiation. (I) Aggressive cancer measured by percentage of mice with multiple

tumor types or same tumor type in multiple organs. Histopathology was performed

on 13-27 tumors of Mlh1+/+ origin and 18-44 tumors of Mlh1+/- origin. Tumor

distribution was analyzed by Chi-square and multiple tumor incidence was

analyzed by two-way ANOVA; ns = non-significant.

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Figure 4.2: Histopathology of tumors from Mlh1+/+ and Mlh1+/- mice.

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Figure 4.3: Immunohistochemistry of lymphomas from Mlh1+/+ and Mlh1+/-

mice.

(A) B-cell lymphoma in a mesenteric lymph node shows diffuse and strong positive

membrane immunoreactivity for B220 antibody. (B) T-cell lymphoma in mesenteric lymph node shows diffuse membrane and cytoplasmic immunoreactivity to CD3 antibody. (C) Histiocytic sarcoma in the liver shows strong and diffuse membrane immunoreactivity to F4/80 antibody. (D-F) The majority of neoplasms had an immunophenotype of T-cell rich, B-cell lymphomas, characterized by a dominant population of neoplastic B cells immunoreactive to B220 antibody (D), with a minority population of well-differentiated T-cells immunoreactive to CD3 antibody

(E), and only a few resident macrophages illustrated by F4/80 immunoreactivity

(F). (A-F) 40X, bar = 20um. (G) Distribution, based on immunohistochemistry, of lymphomas collected from Mlh1+/+ mice treated with sham-, γ-, or 56Fe ion

irradiation. (H) Distribution, based on immunohistochemistry, of lymphomas

collected from Mlh1+/- mice treated with sham-, γ-rays, or 56Fe ion irradiation. IHC

was performed on 8-12 lymphomas of Mlh1+/+ origin and 15-31 lymphomas of

Mlh1+/- origin.

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Figure 4.3: Immunohistochemistry of lymphomas from Mlh1+/+ and Mlh1+/- mice.

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Figure 4.4: Microsatellite instability found in Mlh1+/+ and Mlh1+/- tumors.

Stable MSI (MSI-S) was found in control tissue (Mlh1+/+) via the markers mBat-26

(A), mBat-37 (B), mBat-59 (C), and mBat-64 (D). Similarly, high MSI (MSI-H) was

observed in Mlh1+/- tumor sample also via mBat-26 (E), mBat-37 (F), mBat-59 (G),

and mBat-64 (H). MSI distribution in Mlh1+/+ vs Mlh1+/- tumors (I). MSI-H

distribution found in tumors of irradiated Mlh1+/- mice (J). Number of Mlh1+/+ and

Mlh1+/- tumors used for the analysis were 15 and 43, respectively. Distributions were tested using Chi-square tests.

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Figure 4.4: Microsatellite instability found in Mlh1+/+ and Mlh1+/- tumors.

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Figure 4.5: Whole exome sequencing analysis of Mlh1+/+ and Mlh1+/- TRB

lymphomas.

(A) Number of SNVs and INDELs found in each TRB lymphoma arising from sham-

, γ-rays, or 56Fe ion irradiation. (B) Average number of SNVs per Mlh1+/+ and

Mlh1+/- cohorts. (C) Average number of INDELs per Mlh1+/+ and Mlh1+/- cohorts.

(D) Size of INDELs ≥ 5 bp in each cohort of Mlh1+/+ and Mlh1+/- TRB lymphomas.

(E) Size of INDELs ≥ 10 bp in each cohort of Mlh1+/+ and Mlh1+/- TRB lymphomas.

Venn Diagram shows number of frequently mutated genes found in (F) Mlh1+/+,

and (G) Mlh1+/- cohorts. P values were determined by a two-way ANOVA model.

Data plotted are means ± SEM.

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Figure 4.5: Whole exome sequencing analysis of Mlh1+/+ and Mlh1+/- TRB lymphomas.

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Figure 4.6: Correlation between frequently mutated mouse TRB lymphoma genes vs human leukemia genes.

Heatmap represents human leukemia genes also found to be frequently mutated in (A) Mlh1+/+, and (B) Mlh1+/- mouse TRB lymphoma cohorts. Solid aqua lines in each Heatmap represent actual mutational frequency of a gene in that particular cohort. (C) Different types of mutations (mis-sense, non-sense, frameshift, intron, and silent) found in each gene of Mlh1+/- TRB lymphomas.

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Figure 4.6: Correlation between frequently mutated mouse TRB lymphoma genes vs human leukemia genes.

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Supplementary Figure 4.1: HSC acute functional assays post radiation exposure.

HSC acute functional assays. (A) CFU assay showing percentage survival of total myeloid (M) and lymphoid (L) CFU of Mlh1+/+ and Mlh1+/- bone marrow cells

irradiated with 0, 10, 50, 100, or 250 cGy of γ-rays. (B) CFU assay showing

percentage survival of total myeloid (M) and lymphoid (L) CFU of Mlh1+/+ and

Mlh1+/- bone marrow cells irradiated with 0, 10, 50, 100, or 250 cGy of 56Fe ions.

Two separate experiments of three plates each per radiation exposure were

performed, and combined results were shown. Competitive repopulation assay

showing percentages of CD45.2 cells in peripheral blood 4 (C) and 10 (E) weeks

post transplantation into lethally irradiated host mice of Mlh1+/+ or Mlh1+/- donor BM

cells irradiated with 0 or 100 cGy of γ-rays, or for (D) and (F), 0 cGy or 100 cGy of

56Fe ions. Six mice per group used for competitive repopulation assay. P values (*

<0.05, *** < 0.001) were determined by a two-way ANOVA model with Bonferroni adjustments for multiple comparisons. Data plotted are means ± SEM.

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Supplementary Figure 4.1: HSC acute functional assays post radiation exposure.

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Supplementary Figure 4.2: HSC differentiation independent of Mlh1 status.

HSC differentiate independent of Mlh1 status. (A) Total WBC (K/µl), (C) RBC (M/

µl), and (E) platelets (K/µl) at 5 months and 9 months post 0, 100, or 250 cGy γ- rays irradiated Mlh1+/+ or Mlh1+/- mice. (B) Total WBC (K/µl), (D) RBC (M/ µl), and

(F) platelets (K/µl) at 5 months and 9 months post 0, 10, or 100 cGy 56Fe ion irradiated Mlh1+/+ or Mlh1+/- mice. 36-44 mice per each treatment group were used for the experiment. P value insignificance was determined by two-way ANOVA modeling with Bonferroni adjustments. Data are means ± SEM.

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Supplementary Figure 4.2: HSC differentiation independent of Mlh1 status.

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Supplementary Table 4.1: Pairwise log-ranked tests of survival differences

Treatment Pairwise log-rank tumor-free survival comparison p values groups Mlh1+/+ Mlh1+/- 0 Mlh1+/+ Mlh1+/+ Mlh1+/+ Mlh1+/+ 0 cGy cGy 100 cGy 250 cGy 10 cGy 100 cGy γ-rays γ-rays 56Fe ion 56Fe ion Mlh1+/- 0 0.0049 - X X X X cGy Mlh1+/- 100 < 0.0040 < 0.0001 X X X cGy γ-rays 0.0001 Mlh1+/- 250 < 0.0051 X < 0.0001 X X cGy γ-rays 0.0001 Mlh1+/- 10 < 0.0034 X X < 0.0001 X cGy 56Fe 0.0001 ion Mlh1+/- 100 < < 0.0001 X X X < 0.0001 cGy 56Fe 0.0001 ion

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Supplementary Table 4.2: H & E analysis revealed types of tumors in

Mlh1+/+ and Mlh1+/- mice

Treatment groups Tumor types in mice (n = number of mice with tumor) (n = total number of Lymphoma Histiocytic HCA/HCC Others mice/group) (n) Sarcoma (n) (n) (n) Sham-IR Mlh1+/+ (44) 6 3 4 0 Sham-IR Mlh1+/- (36) 13 3 2 0 γ-rays IR Mlh1+/+ (84) 9 6 3 6 γ-rays IR Mlh1+/- (77) 31 4 0 2 56Fe ion IR Mlh1+/+ (88) 12 0 10 5 56Fe ion IR Mlh1+/- (79) 36 6 1 1

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Supplementary Table 4.3: IHC analysis revealed types of lymphomas in

Mlh1+/+ and Mlh1+/- mice

Treatmen Type of lymphomas in mice (n = number of mice with lymphoma) t groups T-cell rich B-cell T-cell T/Histiocyt B/T mixed Histiocyti (n = total B-cell Lymphom Lymphom e rich B- Lymphom c number Lymphom a (n) a (n) cell a (n) Sarcoma of a(n) Lymphoma (n) mice/gro (n) up) Sham-IR 3 0 1 0 1 3 Mlh1+/+ (44) Sham-IR 7 3 0 1 1 3 Mlh1+/- (36) γ-rays IR 4 0 2 0 0 3 Mlh1+/+ (84) γ-rays IR 13 7 5 0 2 2 Mlh1+/- (77) 56Fe ion 7 1 1 3 0 0 IR Mlh1+/+ (88) 56Fe ion 22 1 1 2 0 5 IR Mlh1+/- (79)

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Supplementary Table 4.4: Frequently mutated well-defined cancer genes found mutated in TRB lymphomas

Mlh1+/- Well-defined cancer genes found mutated in TRB cohorts lymphomas Sham-IR TLX3, RBM15, CTNND2, SETD2, ERG, MET, NUP214, SFPQ, SMO, GPC5, ARHGEF12, CDK12, WHSC1, GNAS, RBM10, BCLAF1, DICER1, ALK, FBXO11, TPM3, PRDM16, LSM14A, MYH9, BCL9, SND1, ATR, KDM5A, KDM6A, BCL9L, RUNX1, PIM1, ACVR2A, MTOR, HIST1H4I, UBR5, LEF1, MAP2K2, SIX2, CSMD3, FGFR2, KIT, EPHA7, LRP1B, MYH11, NF1, RUNX1T1, ELL, BCL11B, GOPC, CLTC, PRDM1, COL3A1, CACNA1D, SS18, EXT1, TNC, KMT2D, PTPRD, HNRNPA2B1, BIRC6, NBEA, CBL, AXIN2, BCR, FOXL2, GNAQ, STAT5B, ACVR1, SF3B1, ANK1, TEC, CNTNAP2, CHD4, TRRAP, LHFP, KMT2A, MECOM, PDE4DIP, ROBO2, PABPC1, FOXP1, ZFHX3, PAX3, CUX1, BCL11A, DCC, CTNNA2, APC, PRPF40B, TPR, RANBP2, AR, ARID1A, AFF3, ERBB4

γ-rays IR PTPRK, CACNA1D, SETD2, DDX5, MET, PTPRD, GPC5, NBEA, CBL, GNAQ, CAMTA1, DDX6, ATR, TSC2, CNTNAP2, CHD4, LHFP, RUNX1, MED12, KMT2A, SRC, RAF1, NT5C2, CREBBP, TFE3, CSMD3, SET, STAT3, PABPC1, LRP1B, ZFHX3, MAP2K1, DNMT3A, RUNX1T1, DCC, CTNNA2, APC, BCOR, AR, ARID1B, MAP3K1, ARID2, ARID1A, PRDM1, MYB

56Fe ion IR ERBB4, PTPRK, CACNA1D, DDX5, MET, PTPRD, NBEA, TPM3, CDH1, GNAQ, ATR, KDM6A, CNTNAP2, MED12, RB1, SRC, CSMD3, SET, PABPC1, LRP1B, EPS15, ZFHX3, NF1, WAS, ATP2B3, DCC, CTNNA2, CDH11, MYC, ARID1B

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Chapter 5

Age related loss of Mlh1 in hematopoietic stem cells accelerates tumorigenesis post simulated solar or galactic cosmic radiation exposure

We demonstrated in chapter 3 and 4 that Mlh1 deficiency does not affect short- and long-term HSC functions but lack of functional MMR does sensitize mice to lymphomagenesis in presence and absence of IR. The finding is very important to NASA due to the fact that future long-term missions will involve astronauts spending more time in the deep space. However, the tumorigenesis study was performed with only 56Fe ion IR and thus we decided to further strengthen our findings with two more HZE particles from GCR spectrum. Proton ion is the most prominent species of charged particle in GCR while 28Si ion is as frequent as 56Fe ion among HZE particles. Therefore, we performed HSC functional and tumorigenesis assays by irradiating Mlh1+/+ and Mlh1+/- mice with 10 or 100 cGy

1H ion (1000 MeV/n) or 28Si ion (300 MeV/n).

To determine if Mlh1 deficiency affects HSC functions, we irradiated bone marrow cells collected from Mlh1+/+ and Mlh1+/- mice with 0, 10, 50, 100, or 250 cGy of 1H ion or 28Si ion. BM cells were plated to form myeloid CFU and counted in order to determine impact of IR and Mlh1 status of HSCs. We discovered that high-LET 28Si ion caused more damage to BM cells and hence a significantly less myeloid CFU compared to 1H ion irradiated BM cells (figure 5.1A and 5.1B). As anticipated, Mlh1 status did not affect acute function of HSC measured by CFU assay. In addition, HSC differentiation was measured by complete blood count at

5 and 9 month post Mlh1+/+ and Mlh1+/- mice irradiated with 1H ion or 28Si ion. At

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these time points, irradiation impact on HSC differentiation was undetermined

along with no effects of Mlh1 status on long-term differentiation (figure 5.1C –

5.1H). As shown in chapter 3 and 4, loss of Mlh1 did not affect HSC functions and

survival.

Next, we hypothesized that, due to Mlh1+/- mice showed a significantly

higher and early incidence of lymphomagenesis in 56Fe ion irradiated mice, the

impact of 1H ion or 28Si ion would be similar. Post IR, we followed mice up to 18 months and euthanized any mouse with a visible sign of distress or palpable tumor.

We found that Mlh1+/+ mice irradiated with any dose or IR source showed no

significant increase in the tumor formation due to radiation exposure (figure 5.2A).

In contrast, Mlh1+/- mice showed sensitization to radiation-induced tumorigenesis

as displayed in chapter 4 (figure 5.2B). In fact, we revealed a significant higher

incidence of deaths due to tumor formation in Mlh1+/- mice irradiated with 100 cGy

28Si ion IR compared to 100 cGy 1H ion IR (p=0.0001). The finding shows that high-

LET IR is more effective in prompting tumorigenesis compared to low-LET 1H ion

IR in MMR compromised mice. In addition, histology and immunohistochemistry

analysis revealed a majority of tumors in Mlh1+/- mice were lymphomas, in

particular T-cell rich B-cell lymphomas (figure 5.2C and 5.2D). Collectively, the

data revealed that incidence of lymphomagenesis in MMR deficient mice was LET

dependent.

To determine gene expression profile of each T-cell rich B-cell lymphoma

cohorts, we decided to run RNA-sequencing analysis. The RNA-sequencing

experiment was carried out to detect differentially expressed genes and to

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determine which signaling pathways are involved in the process of tumorigenesis.

To study this, we collected RNAs from snap frozen TRB lymphomas arising from

Mlh1+/+ and Mlh1+/- mice irradiated with sham-, 1H ions, or 28Si ions. To identify

differentially expressed genes, each TRB lymphoma gene expression profile was

compared to Mlh1+/+ and Mlh1+/- lymph nodes collected from healthy age-matched sham-irradiated mice. The hierarchical clustering analysis revealed that all control

age-matched lymph node samples clustered together with a similar gene

expression profile (figure 5.3A). In contrast, TRB lymphomas gene expression profiles were discrete from control lymph nodes and no further distinction was

observed between irradiation sources or Mlh1 status of TRB lymphomas (figure

5.3A). To visualized the similarity and differences between TRB lymphoma cohorts, we plotted the same gene expression profile data by grouping samples to their respective groups. The data showed that irradiated TRB lymphoma cohorts are distinct from sham-irradiated Mlh1+/- cohort, suggesting that tumorigenesis processes associated with irradiation are unique in nature (figure 5.2B).

Further, we looked at number of differentially expressed genes in each cohort to identify how many genes are differentially expressed between cohorts.

We determined that over 85% of differentially expressed genes are found to be commonly associated with all cohorts, regardless of Mlh1 status or irradiation source (figure 5.3C). Interestingly, we observed that roughly 8% of differentially expressed genes are only associated with irradiated cohorts with no correlation found between low- and high-LET IR. Similarly, we also observed that roughly 11% of differentially expressed genes are only found to be associated with sham

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irradiated cohort. The data suggest that there are common genes involved in the

process of tumorigenesis, but enough differences exist between irradiated vs

sham-irradiated cohorts that could be the reason for early and higher frequency of

tumorigenesis found in 28Si ion irradiated Mlh1+/- mice. In fact, gene ontology

analysis revealed that processes, for instance DNA repair, mRNA and rRNA

processing, ribosome biogenesis, and RNA splicing, were commonly involved in all TRB lymphoma cohorts. In contrast, differentially regulated genes belong to

adherens junction and anchoring junction were predominately found in irradiated

TRB lymphoma cohorts, but not in sham-irradiated TRB lymphoma cohort.

In conclusion, we showed that high-LET 28Si ion irradiation causes

significantly early and higher incidence of lymphomagenesis compared to low-LET

1H ion irradiated Mlh1+/- mice. The RNA-sequencing analysis revealed that high

number of differentially expressed genes are commonly found in each lymphoma.

In addition, the analysis also detected that signaling pathways among the different

cohorts were predominantly similar with a few signaling pathways distinct between

the cohorts.

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Figure 5.1: 1H ion and 28Si ion irradiation affects HSC acute function, but not

long-term differentiation

CFU assay showing percentage survival of total myeloid CFU of Mlh1+/+ and

Mlh1+/- bone marrow cells irradiated with 0, 10, 50, 100, or 250 cGy of (A) 1H ion

with 1000 MeV/n, and (B) 28Si ion with 300 MeV/n. Two separate experiments of

three plates each per radiation exposure were performed, and combined results

were shown. HSC differentiate measured by (C) Total WBC (K/µl), (E) RBC (M/

µl), and (G) platelets (K/µl) at 5 months and 9 months post 0, 10, or 100 cGy 1H

ion irradiated Mlh1+/+ or Mlh1+/- mice. HSC differentiate measured by (D) Total

WBC (K/µl), (F) RBC (M/ µl), and (H) platelets (K/µl) at 5 months and 9 months

post 0, 10, or 100 cGy 28Si ion irradiated Mlh1+/+ or Mlh1+/- mice. 35-43 mice per each treatment group were used for the experiment.

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Figure 5.1: 1H ion and 28Si ion irradiation affects HSC acute function, but not long-term differentiation

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Figure 5.2: Incidence of tumorigenesis in Mlh1+/+ and Mlh1+/- mice post 1H ion and 28Si ion exposure

Animals were sacrificed at the onset of signs of morbidity or the appearance of visible tumors. (A) Tumor free survival of Mlh1+/+ mice post 0,10, or 100 cGy of 1H ion or 28Si ions IR. (B) Tumor free survival of Mlh1+/+ mice post 0,10, or 100 cGy of

1H ion or 28Si ions IR. (C) Percentage tumor distribution based on histology of tumors collected from Mlh1+/- mice treated with sham, 1H ion, or 28Si ion irradiation.

(D) Distribution based on immunohistochemistry of lymphomas collected from

Mlh1+/- mice treated with sham, 1H ion, or 28Si ion irradiation.

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Figure 5.2: Incidence of tumorigenesis in Mlh1+/+ and Mlh1+/- mice post 1H ion and 28Si ion exposure

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Figure 5.3: Gene expression profile of Mlh1+/+ vs Mlh1+/- TRB lymphomas

The RNA-sequencing analysis of T-cell rich B-cell lymphomas. (A) Cluster analysis of differentially expressed genes found in each control and TRB lymphoma samples. (B) Cluster analysis of differentially expressed genes found in each cohort of TRB lymphomas. (C) Venn diagram represents common and unique number of differentially expressed genes found between different cohorts.

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Figure 5.3: Gene expression profile of Mlh1+/+ vs Mlh1+/- TRB lymphomas

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Chapter 6 – Discussion and Future Directions

Our findings identify a potential long-term detrimental impact of galactic

cosmic radiation on astronauts’ brains and hematopoietic stem cells through three

distinct mice studies. We have demonstrated: (1) high-LET space radiation have a

lasting impact on mouse brain, which was translated into behavioral changes such

as decreased in activity and memory levels, and increased anxiety levels up to 9

months post radiation exposure, (2) Mlh1 deficient mouse model, that represents

middle-aged human HSCs, showed no impact on short- and long-term HSC

functions, (3) however, loss of Mlh1 significantly increased incidence of

lymphomagenesis post high-LET 28Si or 56Fe ion IR compared to low-LET γ-rays

or 1H ion IR (figure 6). We are the first one to show the lasting harmful impact of

HZE particles on mouse behavior performances up to 9 months post IR. In addition, age related genomic changes in HSC, such as loss of MLH1 expression in human HSC, was found to be more detrimental in long-term radiation-induced lymphomagenesis. Furthermore, WES and RNA-sequencing analysis have revealed striking similarity between mouse lymphomas and human leukemias at molecular level, suggesting a potential risk to space radiation induced HSC malignancy in future astronauts. Collectively, these findings highlight the importance of lasting impacts of HZE particles on brain and the hematopoietic stem cells and call for further investigation to understand the molecular mechanisms underlining these harmful effects.

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Figure 6: Summary explains the detrimental impact of GCR on mouse brain

and hematopoietic stem cell

Animals were irradiated with different doses of 1H ion (1000 MeV/n), 28Si ion (300

MeV/n), or 56Fe ion (600 MeV/n) particles to study the lasting impact of space radiation exposure on the CNS and HSCs. The summary explains what were the long-term harmful effects of GCR exposure on behavior performances, short- and

long-term impact on HSC functions, and long-term impact on HSC malignancies.

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Figure 6: Summary explains the detrimental impact of GCR on mouse brain and hematopoietic stem cell

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6.1 Tumorigenesis depends on LET and Mlh1 status

Previous studies have shown increased incidence of carcinogenesis post

high-LET radiation exposure compared to low-LET x-rays or γ-rays in animal

models. The most extensive study was carried out by Fry, Alpen, and coworkers

to determine the effects of LET on radiation-induced Harderian gland tumors, as a

typical epithelial tumor model. The study showed increased incidence of

tumorigenesis with increasing LET up to 56Fe ions (600 MeV/n) or 40Ar ions (570

MeV/n) irradiation with LET ≤ 190 KeV/µm [270]. Michael Weil group has shown that CBA/CaJ mice are extremely sensitivity to 56Fe ion (1000 MeV/n) induced

hepatocellular carcinoma (HCC), but not acute myeloid leukemia, compared to

mice exposed to γ-rays [42]. The further analysis has revealed that 28Si ion caused

slightly higher incidence of HCC and HCC metastasis to lungs compared to 56Fe

ion IR. Similarly, we observed a significantly higher incidence of tumorigenesis in

Mlh1 wildtype mice exposed to 100 cGy dose of different LET ions with a maximum

effect observed post 28Si ion IR (p=0.038, figure 6.1A and 6.1B). The findings

suggest that carcinogenesis increases with increasing LET up to a certain value

and reaches saturation level beyond which the cell killing becomes more prominent

outweighing the cell transformation.

The epigenetic and genetic changes play a crucial role in HSC self-renewal

and differentiation to the lymphoid or myeloid lineages. DNA methylation and

histone marks, such as H3K4 and H3K27 methylation, have certain essential roles

in gene regulations and chromatin organization [271, 272]. For instance, DNA

methyltransferase (Dnmt1) regulates distinct patterns of methylation in long-term

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HSC, multipotent progenitor, and lineage-restricted progenitors in order to express

discrete gene families to control these populations [273]. Accumulating evidences

have suggested that aging reduces HSC functions, specifically biased towards

myeloid lineage due to a loss of DNA methylation [274, 275]. In fact, recent studies

have identified specific sites of age-dependent hypermethylation in human blood

samples, which also correlated with genes found hypermethylated in various

cancers [276, 277]. Similarly, our group recently identified that MLH1 expression

decreases in CD34+ human stem cell population due to promoter

hypermethylation, as a function of age [170, 171]. In many human cancers,

specifically in lynch syndrome, loss of MLH1 expression was found in a strong

agreement with the tumor formation. Mlh1 deficient mouse model has shown

increased incidence of lymphomagenesis. Similarly, our experiments have shown

a significantly lower tumor-free survival due to lymphomagenesis and the

incidence was dependent on radiation quality factor, i.e. LET (figure 6.1C). The

radiation-induced lymphomagenesis was found to be LET dependent in Mlh1+/-

mice with highest number of deaths caused by 28Si ion IR, similarly as seen in

wildtype mice (figure 6.1D). In addition, 28Si ion irradiated Mlh1+/- cohort reached to 70% survival mark quicker than Mlh1+/- cohorts irradiated with low-LET γ-rays

or 1H ions (figure 6.1E). Furthermore, we observed that Mlh1 heterozygous cohorts

have significantly higher incidence of multiple lymphomas per mouse compared to

Mlh1 wildtype cohorts, independent of IR sources (p=0.0078, figure 6.1F).

Collectively, the data from Mlh1 heterozygous cohorts showed that incidence of

153 tumorigenesis was dependent on LET with maximum effects reaching at 28Si ion

IR.

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Figure 6.1: The impact of LET and Mlh1 status on GCR induced tumorigenesis The different impact of low- and high-LET radiation exposure on Mlh1+/+ and

Mlh1+/- mice were studied by looking at tumor-free survivals, incidence of

lymphomagenesis, and occurrence of multiple tumors per mouse. To show the

impact of LET, only one dose of IR (100 cGy) was chosen to make comparison

less complicated. Tumor-free survival of (A) Mlh1+/+ and (C) Mlh1+/- mice post 100

cGy IR. Percentage deaths due to tumorigenesis in (B) Mlh1+/+ and (D) Mlh1+/-

cohorts. (E) Time to reach 70% survival in different Mlh1+/- cohorts. (F) Multiple

tumor formation per mouse in Mlh1+/+ and Mlh1+/- mice exposed to different IR

source.

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Figure 6.1: The impact of LET and Mlh1 status on GCR induced tumorigenesis

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6.2 Determine the impact of mixed beam GCR exposure on Mlh1 chimeric

mouse model

The previous carcinogenesis experiments were performed with Mlh1+/- mice in order to recapitulate HSCs of middle-aged astronauts. However, the entire mouse was heterozygous for Mlh1 gene that does not entirely correlate with middle-aged human population. Therefore, chimeric mouse model will be generated in order to get Mlh1 wildtype to Mlh1 knockout ratio 50:50 only in HSC

compartment while keeping the rest of the mouse wildtype for Mlh1. The Mlh1

chimeric model will allow us to closely recapitulate human aged HSC in mouse model for carcinogenesis study. Many studies have established different doses

(sub-lethal to lethal) of x-rays or γ-rays irradiation to achieve various percentage of bone marrow chimerism in mouse model. However, the whole body irradiation for myeloablation could cause potential damage to other vital organs and tissues with the increase susceptibility to secondary infections [278]. Busulfan conditioning

is well tolerated in mice to achieve a high-level of BM chimerism with minimal toxicity compared to myeloablation doses of irradiation. Therefore, Busulfan conditioning will be used to generate chimeric mice contains ~50% Mlh1 knockout

BM in Mlh1 wildtype mice (figure 6.2). Mlh1 chimeric mice will be used for

tumorigenesis assays to study the long-term impact of space radiation exposure.

In previously performed studies, we used 10 or 100 cGy IR dose of different

ion species as a single exposure. However, astronauts will be exposed to a mixed

field of HZE particles at a lower dose and dose-rate throughout their journey to the

deep space. To closely simulate the space radiation conditions, NASA space

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radiation laboratory (NSRL) at Brookhaven national laboratories has recently

created a mixed filed GCR simulator (60% 1H; 20% 4He; 10% 16O; 10% 56Fe) [279].

Mlh1 chimeric mice will be exposed to 10 cGy single dose or 10 cGy protracted

dose (2 cGy/day for five consecutive days) of mixed beam. Again, 40 Mlh1

chimeric mice per group will be used, and the results will be compared directly to

sham- and 10 cGy 56Fe ion IR groups from previously performed studies. Here, we

aim to study the effects of single and protracted exposures of simulated GCR beam

on Mlh1 chimeric mouse model to extend our knowledge and improve on the risk estimates to astronauts on the deep space missions.

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Figure 6.2: Generation of Mlh1-/- chimeric mouse model and study of

tumorigenesis

Three steps process is proposed to generate Mlh1 chimeric mouse model for

future tumorigenesis study: (1) Collection of BM cells from donor CD45.1 Mlh1-/-

mice, (2) Busulfan conditioning to prepare recipient CD45.2 Mlh1+/+ mice for BM

transplant, (3) Intravenous tail vein injection of CD45.1 Mlh1-/- BM cells into

busulfan conditioned recipient CD45.2 Mlh1+/+ mice followed by simulated mixed- beam GCR exposure.

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Figure 6.2: Generation of Mlh1-/- chimeric mouse model and study of tumorigenesis

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6.3 Define the mitigating potential of dietary polyamines as a countermeasure for GCR induced tumorigenesis

There are preventive measures that can be used in order to reduce GCR exposure and its harmful impact, including administration of drugs or dietary supplements, choice of an appropriate time for space flight, and crew selection.

Irradiation protection in cells can be achieved by squelching ROS/RNS and augmenting DNA repair to reduce cell deaths by IR. Traditional antioxidants have shown to be moderately useful in minimizing the harmful effects of low-LET IR on humans, and could also be useful as an adjunct therapy to minimize cell damages created by δ-rays coming off of HZE particles core. For instance, resveratrol has shown ameliorating effects against IR by acting as a strong antioxidant and a potent activator of Sirtuin1 (Sirt1) in mice [280]. In fact, antioxidants have shown to play a key role in prevention of cancer if used for a prolonged period [281, 282].

Hence, the development of radioprotective compounds has historically been of high interest to the Department of Defense, and the attention was recently caught by the NASA to protect their astronauts from harmful space radiation exposure.

The only FDA approved drug for radiation protection is amifostine that thought to function primarily as an antioxidant [283]. In contrast, amifostine has a side effects, such as hypotension and vomiting that limit regular use for astronauts during their long-term space missions [284]. Many radioprotective compounds have been studies for decades with little to no success in getting pass the clinical trials by

FDA. However, the search remains to find pharmacological modulators that hold the promise to improve the efficacy of radioprotection with minimal side effects.

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Polyamines (putrescine, spermidine, and spermine) are positively charged

small molecules in the cell that bind to negatively charged macromolecules, such

as lipids, proteins, and nucleic acids [285]. It has properties like chromatin

condensation to decrease the size of target DNA through compaction and oxygen

radical scavenging to reduce ROS/RNS in irradiated cells [286]. In fact, recent

findings have demonstrated that dietary polyamines increase lifespan and

cardiovascular health in many species [287, 288]. Therefore, we propose that a

polyamine could increase organism lifespan by acting as radioprotector and thus increase tumor-free survival. To evaluate the effects of polyamines on space radiation-induced tumorigenesis, groups of roughly 40 Mlh1-/- chimeric mice will be

irradiated with 10 cGy of single or protracted (2 cGy/day for five consecutive days)

mixed-beam GCR at NSRL. Polyamines will be dosed to the appropriate groups of mice for the length of the study in the drinking water (3 mM spermidine). All

animals will be checked for chimerism by collecting blood at every 8 weeks till the

end of study in order to measure successful chimerism. Any mice displaying

morbidity will be euthanized and assessed histologically for malignancy. Together

with additional sham-IR group with and without polyamine treatment will allow us

to distinguish beneficial impact of polyamines post GCR exposure.

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6.4 Concluding Remarks

Since the dawn of space exploration, NASA and other space agencies around the world have wondered how far in deep space humans can go, survive, and still return to earth safely. NASA’s manned missions to the moon landed the first humans on the surface in 1969. However, more than 45 years have passed since that initial triumph, and we have not yet returned to the moon or sent humans beyond our celestial partner. NASA’s next ambitious manned missions includes sending astronauts to the Moon and eventually Mars and back, a journey that will take roughly two to three years including time on the surface for research purposes, significantly longer than the 10-day journeys of the Apollo missions and beyond the protection afforded by the Earth’s magnetosphere. Long-term space travel requires a deeper understanding of the effects of the space environment on human physiology, particularly the impact of space radiation. Only through pushing our primarily earth-based experimental models to the extreme can we begin to gain insight into how we will fare so far from home.

A major obstacle to space travel is long-term GCR exposure and even if we reduce the uncertainties of space radiation induced risks, the effective countermeasures to ionizing radiation will remain the ultimate goal of a long-term space travel. There are three different ways to minimize terrestrial radiation exposure: proper shielding, increase the distance from the radiation source, and reduce the time spent in the radiation field. However, future space travel time will likely to be increased and distance from the GCR will be an unavoidable task.

Despite continuous improvements in spacecraft shielding materials and designs,

163 exposure to SEPs and GCR cannot be completely eliminated due to the simple issue of weight limitation. Current shielding materials are useful only during low earth orbit missions but become ineffective when it comes to GCR exposure. The thickness of the spacecraft required to fully shield against all incident radiation would not be practically possible, and becomes complicated by the fact that GCR ions result in fragmentation of the hull materials and can deliver similar cumulative doses of multiple secondary fragments typically with high-LET values inside the spacecraft as well [289, 290]. In this case, liquid hydrogen would be an ideal candidate for the shielding material, but it is a low temperature liquid with a highly explosive nature making it an impractical material. NASA is working towards designing a better shielding materials but it will not be sufficient enough to protect astronauts completely from harmful HZE nuclei.

Currently, we are also facing an another challenge of translating experimental results into reliable risk models due to two fundamental issues. First, the fact that majority of experimental studies were carried out using a single ion species at higher dose and dose-rate, which will be not the case for astronauts traveling in space for the long-term mission. Second, the lack of human data on high-LET radiation exposure and hence strict reliance on experimental animal models for risk assessment prediction. Previous animal models have not considered astronauts age related genomic changes as one of the major driving factor for carcinogenesis, and hence gross underestimation of risk associated with high-LET radiation induced carcinogenesis. It has been known that stem cells

(skin, gut, or hematopoietic origin) in our body loses their fitness as we age due to

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genomic changes and mutation accumulation in the genome. In fact, our recent findings have demonstrated that age related loss of a single gene in HSCs, such as MLH1, is strongly associated with a significant increase in tumorigenesis in

animal model. Therefore, age related loss of other genes in HSCs should be taken

into consideration for future studies, especially cell-cycle, DNA damage response, and chromatin modifier genes known to play a critical role in radio sensitization.

Our constant efforts to improve existing animal models with help of genetic engineering will benefit us to design better risk assessment models, sophisticated

shielding materials, and radioprotectors/mitigators that will alleviate the potential

detrimental impact of space radiation during future long-term missions.

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