THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

BRAIN EFFECTS

INTRODUCTION

?Do bystander effects exist in the brain?

Cranial RT is a well-established treatment modality that is of paramount importance, and approximately 200,000 patients receive cranial RT yearly

RT leads to a variety of side effects including debilitating cognitive declines, learning and memory deficits, changes in visual motor processing, quantitative skills and attention

IR damages normal brain tissues through a variety of poorly understood mechanisms and results in profound cognitive impairment and significant life-long disability

IR can elicit molecular and cellular responses in cells and tissues that were not hit directly by IR but rather received a distress signal from irradiated cells – the bystander effects

Currently, there is a lack of comprehensive evidence on the existence and nature of bystander effects in the brain

INTRODUCTION

IR was shown to affect hippocampus and the prefrontal cortex (PFC).

The hippocampus is one of the two active sites of neurogenesis in the mammalian brain. The hippocampus plays important role in memory.

The prefrontal cortex (PFC) the most anterior region of the frontal lobe, is involved in “executive” functions such as planning, decision-making, behavioural inhibition, and working memory.

These functions are affected post RT.

METHODS

sham treated

head exposed

liver exposed

males females males femalesmales females

4 days 14 dayspost-exposure

Molecular analysis•Gene expression•Small RNA expression•DNA methylation•Protein analysis

Behavioral analysis Neuroanatomy

EXPERIMENTAL SET-UP

DOSE: 1 Gy

To the surface of the tray!

METHODS

What is the precise dose to the brain?

The yellow oval indicates the approximate location of the oval hole in the lead shielding, i.e., the region exposed to the primary radiation. All other regions were

shielded.

METHODS

DIRECT

LIVER IRVERY

SMALL DOSES!!

METHODSNEUROANATOMYGolgi-Cox stained coronal sections were used to measure PFC thickness and to study spine density and dendritic morphology. Dendritic morphology (i.e. branching and length) reflects the organization of brain connectivity.

To measure spine density, the distal dendrites of individual neurons were traced from Golgi-Cox stained brain sections, using a camera lucida mounted on a microscope.

BEHAVIOURAL TESTINGWe used tasks sensitive to hippocampal and/or prefrontal injury - the object location memory task and the Morris water task

Object Location Memory Testing – to measure how well rats encode the location of particular objects among an array of objects

Morris Water Task – a well-established behavioural procedure widely used to study spatial learning and memory

RESULTS

Effect of radiationHead Liver

Spines Branch length Spines Branch length

Par1AMale - - - - -

Female - - - -

Par1BMale - - - - - -

Female - - - - -

Cg3AMale * - - -

Female -

Cg3BMale - - -

Female - - - -

AIDMale - - - -

Female - -

HPCMale - - - -

Female - - * - -

Both head and liver irradiation affect morphology

RESULTS

Effect of sex (being male)Head Liver

Spines Branch length Spines Branch length

Par1ANo radiation - - - - - -

Radiation - -

Par1BNo radiation - - - - - -

Radiation - - - -

Cg3ANo radiation * *

Radiation - -

Cg3BNo radiation - - - - - -

Radiation - - - *

AIDNo radiation - -

Radiation -

HPCNo radiation * - -

Radiation - - - -

Females are more affected than males

RESULTS

RESULTS

We also identified differentially expressed genes between treatments for 2 brain regions: prefrontal cortex and hippocampus

Treatments analysed: • sham-irradiated• liver-irradiated/brain bystander

Brain regions Females Males

Hippocampus - 1

PFC 22 1

No overlap between males and females

KEY RESULTS

A predicted gene (ENSRNOG00000043197) was the only DE gene found in PFC, hippocampus and cerebellum. It was strongly up-regulated in these brain regions.

EFFECTS OF SCATTER IR ON THE BRAIN

0.115 cGy

EFFECTS OF SCATTER IR ON THE BRAIN

0.115 cGy

EFFECTS OF SCATTER IR ON THE BRAIN

FEMALES MALES

EFFECTS OF SCATTER IR ON THE BRAIN

EFFECTS OF SCATTER IR ON THE BRAIN

EFFECTS OF SCATTER IR ON THE BRAIN

Epigenetically mediated

They affect behavior

They are sex-specific

Studies are needed to analyze clinical repercussions of RT-induced effects

EFFECTS OF SCATTER IR ON THE BRAIN

ACKNOWLEDGEMENTSCollaborators:

Bryan Kolb, CCBNGerlinde Metz, CCBNRobbin Gibb, CCBN

Richelle Mychasiuk, CCBN Arif Muhammad, CCBN

Shakhawat Hossain, CCBNGreg Silasi, CCBN

Esmaeel Ghassrodhashti, JACCCharles Kirkby, JACC

AJ Ghose, JACCIgor Kovalchuk/Bio

Youli Yao/BioOlena Babenko, CCBN

William Bonner, NCI/NIHOlga Martin, Peter Maccallum Cancer Center, AustraliaJacob Adserballe, the Family Pet Hospital of Lethbridge

Rochellys Diaz-Heijtz, Karolinska Uni, SwedenIgro Pogribny, NCTR

CIHR Institute of Gender and Health – Chair Program

Kovalchuk group

Slava Ilnytsky

Bo Wang

Dongping Li

Anna Kovalchuk

Rocio Rodriguez-Juarez

Rommy Rodriguez-Juarez

Andrey Golubov

Lidia Luzhna

Corinne Sidler

Rafal Woicicki

Dee Goyal

Alumni

Darryl Hudson

Jason Novak

Jan Tamminga

Kristy Kutanzi

Igor Koturbash

Mike Lowings

Jonathan Loree

James Meservy

Natasha Singh

Joel Stimson

Munima Alam

Paul Walz

Jody Filkowski

Matt Merrifield

“Epigenetic profiling for the identification of novel targets for therapeutics in refractory leukemia in children”

Cancer is the second most common cause of death, after accidents, in children

Pediatric cancers include

• Acute leukemia• Brain tumors• Lymphoma

• Neuroblastoma• Wilm’s tumor

• Rhabdomyosarcoma• Retinoblastoma• Osteosarcoma

• Ewing’s sarcoma

The cellular components of the blood are derived from hematopoietic stem cells, which give rise to lymphoid stem cells

and myeloid stem cells

Myeloid stem cells differentiate into erythrocytes, platelets, monocytes, macrophages and eosinophils

Lymphoid stem cells differentiate into B-cells and T-cells

Infant leukemia is:

Rare

Under-Researched

Acute (AML ~15%, ALL >80%)

Deadly

Devastating

Costly

Sorting pediatric leukemia stem cells for genomic and epigenomic analyses

Blast

CD38 A750

CD34

FIT

C

100

101

102

103

104

100

101

102

103

104

34- 38+78.62%

34- 38-3.08%

34+ 38+16.70%

34+ 38-0.36%

Blast

CD38 A750

CD34

FIT

C

100

101

102

103

104

100

101

102

103

104

34- 38+91.23%

34- 38-7.44%

34+ 38+1.10%

34+ 38-0.10%

Blast

CD38 A750

CD34

FIT

C

100

101

102

103

104

100

101

102

103

104

34+ 38-12.22%

34+ 38+72.28%

34- 38-0.32%

34- 38+1.36%

Conducting genomic, epigenomic and signalome analyses

OncoFinder TM is a unique platform that utilizes a variety of original algorithms to score intracellular signaling pathways, to analyze in a comprehensive way thousands of molecular interactions in cancer cells, and to model activity of anticancer drugs in cancer cells of an individual patient.

VT vs normal = numerous pathways affected

2945 genes up-regulated, involved in:• cell cycle control• proliferation• DNA repair

2772 down-regulated, involved in:• deregulated apoptosis • deregulated differentiation • down-regulated immune response

RESULTS

INCREASED PROLIFERATIVE CAPACITY

ALTERED APOPTOSIS

ERK1/2

pERK1/2

p38

PCNA

CYCLIN A

CYCLIN B

NOTCH

nor

mal

VT

VT

Cell cycle control, proliferation → pro-survival

Leukocyte differentiation

PROTEIN ANALYSIS CONFIRMS GENE EXPRESSION DATA

MRE11

CASPASE 3

norm

al

VT

VT

DNA repair

Apoptosis Note: while caspase 3 is induced, it is not cleaved

KU70

γH2AX

PROTEIN ANALYSIS CONFIRMS GENE EXPRESSION DATA

norm

al

VT

VT norm

al

VT

VT

DNMT1

DNMT3a

MeCP2

pH3

H3K9me3

H4K20me3

DNA methylation

Repressive histone modifications

EPIGENETIC CONTROL – RELATED PROTEINS

Dr. James ThomasAnna Kovalchuk

Dr. Yaroslav IlnytskyyDr. Andrey Golubov

Ms. Rocio Rodriguez-Juarez

Dr. Aru NarendranDr. Karl Riabowol

Dr. Aarthi JayanthanDr. Yibing Ruan

Iwona Auer-GrzesiaDerrick Rancourt

Gregor ReidTony Truong

ACKNOWLEDGEMENTS

ATM-Directed Precision Therapyin Non-Small Cell Lung Cancer

Lead: Gwyn Bebb

Team: Olga KovalchukSusan Lees-

MillerDon MorrisMing TsaoTyler Williamson

832 Stage IV

56.1 % Referred to Medical Oncologist

50.7 % Saw a Medical Oncologist

23.1% Received Systemic Chemotherapy

362 Stage IIIA/B

37% Received Radical Chemo/Radiation

Systemic Treatment in Metastatic NSCLCTom Baker Cancer Centre (2003-2006)

Albertans with advanced NSCLC are under-treated

0 50 100 150Survival (Months)

Palliative RT

Radical RT

Kaplan-Meier survival estimates, by chemort

63% MOS = 11.9 mo

37% MOS = 23.7 mo

Stage IV survival according to treatmentStage III survival according to treatment

Most often this is due to poor performance status

Precludes guideline-recommended treatment

Untreated Albertans with advanced NSCLC have poor outcomes

23% MOS 16 mo

3.7 mo

0 25 50 75Survival (Months)

ATM: A critical gene for DNA repair

ATM ATM

DNA repairApoptosisCell cycle arrest

Cells with ATM Cells without ATM

Mutation accumulationGenetic instability

Cancer transformation

p53

DNA damaging agent

ATM loss in cancers

Breast Cancer (40%)Poor overall survival

Head and Neck CancersPoor overall survival

Mantle Cell LymphomaIncreased radiation

sensitivitySynthetic lethality

Lung Cancer (22%)Poor overall survivalBetter chemo responseGastric Cancer

Increased radiation sensitivity

Synthetic lethality

Lung CancerIncreased radiation

sensitivitySynthetic lethalityChemo sensitivity

In the lab In the clinic

ATM in resected NSCLC

22% of patients have low relative ATM associated with poorer survival outcomes

CLUSTER 1 CLUSTER 2

Hierarchical clustering of global gene expression in NSCLC cell lines

Pathways that are differentially expressed in cluster 2 as compared to cluster 1

KEGG analysis of differentially regulated pathway - example of pathways and genes that are differentially expressed.