Nelly Babayan1, Bagrat Grigoryan2, Lusine Khondkaryan1, Gohar Tadevosyan1, Ruzanna Grigoryan1, Natalya Sarkisyan1

1Institute of Molecular Biology NAS of RA 2”CANDLE” Synchrotron Research Institute

Differences in DNA damage and repair in human cancer and normal cells

after ultrashort pulsed electron beam irradiation

Yerevan, 2019

Targeting the DNA Damage Response in CML cells

International Workshop ‘Ultrafast Beams and Applications’ 2-5 July 2019, Yerevan, Armenia

Every cell experiences up to 105 spontaneous or induced DNA lesions per day

Research overview: DNA damage response in cancer cells

Normal cell

No repair

Cell death

Tumor development

•Reduced DNA repair capacity

• Increased genetic instability

Targeting DNA damage response in cancer therapy

© Dr. Stephen P. Jackson. Biochemical Society Transactions Jun 01, 2009, 37 (3) 483-494; DOI: 10.1042/BST0370483

Synthetic lethal interactions in DNA repair genes implicated in cancer

© Helleday et al., Nature Reviews Cancer, 2008, vol 8, no. 3, pp. 193-204. DOI: 10.1038/nrc2342

Pathway Protein Syndrome Primary cancers HR BRCA1 breast, ovarian

BRCA2 Fanconi's anemia breast, ovarian

AD54B non-Hodgkin lymphoma, colon cancer

NHEJ MRE11 Ataxia-telangiectasia- like

disorder colorectal cancer

LIG4 LIG4 syndrome Leukemia

Artemis Omenn syndrome Lymphoma

NER XPA Xeroderma pigmentosum Skin cancers

ERCC1 cerebro-oculo-facio-skeletal syndrome

squamous cell carcinoma, head /neck

Crosslink repair FANCA, B, C, D2, E Fanconi's anemia Various

etc.

Olaparib treatment in BRCA-mut patients

© Ledermann, Lancet Oncol 2014

Targeting DDR in cancer radiotherapy

The Aim: DDR in cancer and normal cells after UPEB radiation

Radiation Source: AREAL

AREAL: Laser-driven radiofrequency gun-based linear pulsed electron accelerator Electron beam: - UHPDR - 1.6x1010 Gy/sec -pulse duration - 0.04x10-12 s - Electron energy – 3.6 MeV

Application - Potential alternative acceleration technology for ion radiotherapy - More precise investigation of damage mechanisms

Radiobiological Endpoints

DNA damage and repair

Comparison of DNA damage response (DDR) in

cancer and normal cells

Identification of target DNA repair genes/proteins for synthetic

lethality therapy

Endpoint: The level of DNA damage in cancer/normal cells

Method: Comet assay

K56

2

PBM

C

Control 1 Non-lethal 2 Sub-lethal 3 Lethal 4

Level of DNA damage in human normal (PBMCs, blood normal cells, female) and cancer (K562, blood cancer cells, female) cells after irradiation (0 h) at the non-lethal, sub-lethal and lethal doses

OTM - Olive Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail

OTM: 5.9±1.4

Viability 90-100%

Viability 90-100% OTM: 2,9±0.6

Viability 50%

Viability 50%

OTM: 170,5±6.7

OTM: 19,1±2.6

Viability 0-10%

Viability 0-10%

OTM: ----

OTM: 82.9±2.4

Endpoint: DNA repair in cancer/normal cells

Method: Comet assay

05

1015202530354045

0 2 4 6 8 10 12 14 16 18 20 22 24

OT

M

Dose, Gy

K562 (cancer cells) PBMC (normal cells)

The level of DNA-damage in PBMC (normal) and K562 cell line (cancer) cells after 3 hours of irradiation

OTM - Olive Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail

Endpoint: DNA repair kinetics in cancer/normal cells

Method: Comet assay

0

10

20

30

40

50

0 2 4 6 8 10 12 14 16 18 20 22 24

OT

M

Dose, Gy

K562 (cancer cells)PBMC (normal cells)

0 0.5 1 2 4 80

20

40

60

80

1000 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean

±S

EM

* *

*

* * *

0 0.5 1 2 4 80

50

100

150

200 0 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean

±S

EM

*

Comparison of the induced primary DNA-damage level and repair kinetics in PBMCs (a) and K562 (b) cells. *p<0.05 in comparison to corresponding control

PBMCs

K562 The level of DNA-damage in PBMC (blood normal cells, female) and K562 cell line (blood cancer cells, female) after 3 hours of irradiation

SSBs and DSBs

Phosphorylation of Histone H2AX at DNA Double-Strand Breaks

γH2AX is a key regulator of the DNA damage response

DNA double strand breaks (DSB) are considered the most lethal form of DNA damage

© Fernandez-Capetillo et al., 2004

Endpoint: DNA DSBs and repair kinetics in cancer and normal cells

Method: Flow cytometric analysis of γ-H2AX

The representative dot-plot (a1, b1) and histogram (a2, b2) of the level of γ-H2AX in PBMCs (a) and K562 cells (b) after sub-lethal (4 Gy) dose of irradiation

a1 a2 b1 b2

The kinetics of γ-H2AX foci formation in cancer (a) and normal (b) cells after lethal (8 Gy) and sub-lethal (4 Gy) doses of irradiation. *- p<0.05 in comparison with non-irradiated cells

a b

*

* *

* * *

* Foci_KCL

30 min 1 h 4 h 24 h0

10

20

30

40

4 Gy8 Gy

% o

f cell

s

Foci_PBMC

30 min 1 h 4 h 24 h0

20

40

60

80

4 Gy8 Gy

Time

% o

f cell

s

K562 PBMCs

Ionizing radiation induced-DNA SSBs/DSBs repair pathways

Non-Homologous End Joining NHEJ

Homologous Recombination Repair HRR

Base Excision Repair BER

FAST Error-prone

Core protein: DNA-PK

Slow Free of errors

Core protein: MRE11

FAST Free of errors

Core protein: APEX1

DNA SSBs/DSBs repair pathway activation accompanied with cell cycle arrest

©https://openi.nlm.nih.gov/detailedresult?img=PMC3413124_gks315f5&req=4

0 0.5 1 2 4 80

20

40

60

80

1000 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean±

SEM

* *

*

* * *

Endpoint: The activation of repair pathways in cancer/normal cells at the

non-lethal level of DNA damages Method: ELISA

Normal cells Cancer cells

DNA-PKMRE11

APEX1

0

0.5

1

1.5

0 h30 min 1 h

4 h

HRR >> BER >> NHEJ

0 0.5 1 2 4 80

50

100

150

200 0 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean

±S

EM

*

NHEJ >> HRR >> BER

DNA-PKMRE11

APEX1

0

0.5

1

1.5

0 h30 min 1 h

4 h

0 0.5 1 2 4 80

20

40

60

80

1000 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean±

SEM

* *

*

* * *

Normal cells Cancer cells

BER >> NHEJ >> HRR

0 0.5 1 2 4 80

50

100

150

200 0 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean

±S

EM

*

NHEJ = HRR >> BER DNA-PK

MRE11APEX1

0

0.5

1

1.5

0 h30 min 1 h

4 h DNA-PKMRE11

APEX1

0

0.5

1

1.5

0 h30 min 1 h

4 h

PI

Cco

unts

PI

Cco

unts

Endpoint: The activation of repair pathways in cancer/normal cells at the

sub-lethal level of DNA damages Method: ELISA

0 0.5 1 2 4 80

20

40

60

80

1000 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean±

SEM

* *

*

* * *

Normal cells Cancer cells

NHEJ >> HRR >> BER

0 0.5 1 2 4 80

50

100

150

200 0 h 30 min 1 h 4 h 24 h

Dose, Gy

OT

M, M

ean

±S

EM

*

BER >> NHEJ = HRR DNA-PK

MRE11APEX1

0

0.5

1

1.5

0 h30 min 1 h

4 h DNA-PKMRE11

APEX1

0

0.5

1

1.5

0 h30 min

1 h4 h

PI

Cco

unts

PI

Cco

unts

Endpoint:

The activation of repair pathways in cancer/normal cells at the lethal level of DNA damages

Method: ELISA

Conclusions/Recommendations To increase the sensitivity of

cancer cells (CML) vs. normal cells

Target for synthetic lethality

Inhibition of DNA-PK (NHEJ pathway)

Normal cells HRR >> BER >> NHEJ

Cancer cells NHEJ >> HRR >> BER

Effective DNA repair

Non-effective DNA repair

Non-lethal dose of irradiation

1

1.1

1.2

1.3

1.4

1.5

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Lev

el o

f rep

air

path

way

ac

tivat

ion

Post-irraidation time, h

DNA-PKMRE11APEX1

0.5

0.7

0.9

1.1

1.3

1.5

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Lev

el o

f rep

air

path

way

ac

tivat

ion

Post-irradiation time, h

DNA-PKMRE11APEX1

Cancer cells

Decrease the time of dose delivery or fractionation of sub-

lethal dose Normal cells

Thanks for Your Attention !!!

Acknowledgements

Dr., Prof. V. Tsakanov and Dr. B. Grigoryan – “CANDLE” Synchrotron Research Institute Dr., Prof. A. Osipov - A.I. Burnazyan Federal Medical and Biophysical Center, Moscow, RF Dr. Prof. R. Aroutiounian – Yerevan State University