1

Exploring sensitivity to

replication stress in

BRCA-deficient Triple

Negative Breast Cancer

Imene TABET

2020/2021

Genetic and phenotypic plasticity of cancer’s team

2

Triple Negative Breast Cancer (TNBC)

Introduction

The most aggressive from all breast cancer molecular subtypes :

ER-/PR-/HER2- :

15% of breast cancers

40% recur within 12 to 60 months

Treatment FCE100 (5FU/Epirubicin/Cyclophosphamide), little alternative in case of recurrence

1/20/2021 3

Defective Homologous Recombination repair (HRR)

Unrepaired DNA breaks

Sensitivity to Genotoxic drugs

30-35% of TNBC are

BRCA-deficient

TNBC and BRCAnessIntroduction

4

Introduction

ATR/ATM

ƔH2Ax

53BP1 NHEJ

BRCA1/CtIP

RAD51

S phase

Homologous Recombination Pathway

DSB

P

PCHK1

P

1) Damage Sensing

2) DNA Resection

3) Strand Invasion

4) DNA Repair

MRN

5

Genotoxic

chemicals

UV

IR

Exo

gen

ou

s Fa

cto

rs

End

oge

no

us

Fact

ors

DNA

replication

3D chromatin

structure

Free

Radicals

Replicativestress

Blocked DNA replication, and reduced origin firing

accumulation of single strand DNA stretches

HR involvment in replication stress response Introduction

6

We propose :

BRCA deficient tumors might be hypersensitive to replicative stress

BRCA deficient tumor

Replicative stress inducing drugs

Blocked replication

Accumulation of SSB and DSB

No BRCA

No RAD51 recruitment ?

No fork stabilization No repair ?

Cell cycle arrest and cell death ?

S/G2 ATR/CHK1checkpoint

Replicative Catastrophe ?

MitoticCatastrophe ?

7

SUM159 BRCA1 KO isogenic model is more sensitive to Gemcitabine

Gemcitabine in SUM159 BRCA1 WT vs. SUM159 BRCA1 KO

CRISPR/Cas9 isogenic models

BRCA1

RAD51

Tubuline0

20

40

60

80

100

120

0 4nm 13nm 40nm 130nm 400nm 1.3µM 4µM 13µM 40µM

A

Gemcitabine

SUM159 Cas9 BRCA1 WTSUM159 Cas9 BRCA1 KO

Results, In vitro

8

Comparative analysis of cell cycle distribution in SUM159 BRCA1 WT

Vs. SUM159 BRCA1 KO treated with Gemcitabine

Non Treated

0% 20% 40% 60% 80% 100%

WTKO

WTKO

WTKO

WT

KO

+2H

+8H

+24H

+48H

Gemcitabine

important accumulation of cells in G1, and much less G2 than NT

The percentage of SubG1 is twice as high in KO compared to the WT

EdU 30’

Wash EdU

Gemcitabine IC50 24h

Wash Gem

8H release

2H release

24H release

Fix cells Fix cells Fix cells Fix cells

48H release

1) By Flow Cytometry :

Results, In vitro

9

Gemcitabine induced cell death in SUM159 BRCA1 KO

confirmed

Cell death continues to increase In the BRCA1 KO isogenic model at +48, and +72h

Plate cells 24h

TRT Gem IC5024h24h

Release

24h 48h 72h

FACS: Annexin V/PI

Gemcitabine IC50 24H

Results, In vitro

10

SUM159 BRCA1 KO display disrupted HR upon

Gemcitabine treatment

Results, In vitro

RAD51

RAD51

DAPI RAD51

GEM

+2G

EM+8

GEM

+24

GEM

+48

NT

SUM159 BRCA1 KO

DAPI RAD51

GEM

+2G

EM+8

GEM

+24

GEM

+48

NT

SUM159 BRCA1 WT

SUM159 BRCA1 WT

SUM159 BRCA1 KO

Weak RAD51 foci formation in the SUM159 BRCA1 KO cells

11

NTNTNT +2 +2 +2 +8 +8 +8

+24+24+24 +48+48

0

20

40

60

80

100

SUM B1KO GEM 500Nm N1+N2

% o

f cells

wit

h >

10 B

RA

C1/R

AD

51/5

3B

P1 f

oci

NT +2H +8H +24H +48HNTNTNT +2 +2 +2 +8 +8 +8

+24+24+24 +48+48

0

20

40

60

80

100

SUM B1KO GEM 500Nm N1+N2

% o

f cells

wit

h >

10 B

RA

C1/R

AD

51/5

3B

P1 f

oci

NT +2H +8H +24H +48H

SUM159 BRCA1 KO seem to engage in a non homologous

recombination pathway upon Gemcitabine treatment

BRCA1

RAD51

53BP1

Gemcitabine IC50 24H Gemcitabine IC50 24H

SUM159 BRCA1 WTSUM159 BRCA1 KO

Results, In vitro

Progressive increase of cells with 53BP1 foci in SUM159 BRCA1 KO

12

SUM159 BRCA1 KO display persistent DNA damage

upon Gemcitabine treatment

Results, In vitro

SUM159 BRCA1 WT SUM159 BRCA1 KO

SUM159 BRCA1 WT SUM159 BRCA1 KO

% of gH2AX positive cells doesn't decrease even at +48h post release in SUM159 BRCA1 KO

13

DAPI RPA32ƔH2aX MERGE

ƔH2aX+/RPA32-

SUM159 B1 WT

SUM159 B1KO

SUM159 BRCA1 KO display more gH2AX+/RPA- cells upon Gemcitabine

treatment

Results, In vitro

Potential Replicative Catastrophe in

the SUM159 BRCA1 KO especially at

+48h

Gemcitabine IC50 24H

14

DAPI RPA32ƔH2aX MERGE

ƔH2aX+/RPA32-

SUM159 B1 WT

SUM159 B1KO

SUM159 BRCA1 KO display more gH2AX+/RPA- cells upon Gemcitabine

treatment

Results, In vitro

Potential Replicative Catastrophe in

the SUM159 BRCA1 KO especially at

+48h

Gemcitabine IC50 24H

DAPIR

PA

32

ƔH2aX

MER

GE

SUM159 BRCA1 WT SUM159 BRCA1 KO

DAPI

RP

A3

2

ƔH2aX

MER

GE

15

SUM159 BRCA1 KO present more mitotic aberrations upon

Gemcitabine treatment

% of micronuclei increases between +24h and +48h in the SUM159 BRCA1 KO

Gemcitabine IC50 24H

WT Gem+72h KO Gem+72h

Results, In vitro

16

BRCA1 deficient (Hypermethylated) PDX’s tumor volume decreases

under gemcitabine treatment

In vitro results confirmed in vivo

BRCA1

HSP70

Results, In vivo

B4122 BRCA1 METH

No

rm.T

V(%

)

16,21%

No

rm.T

V(%

)

B3804 BRCA1 WT

Ctl NT

Gem 50mg/kg

1/20/2021 Designed by PoweredTemplate.com 17

BRCA1P

ATR/ATM

ƔH2AxCHK2

CHK1 P

DSB

Replication arrest/Stalled forks

Less efficient fork protection, less efficient repair

Accumulation of unrepaired DNA breaks and single stranded DNA

gH2ax accumulation =

Pan-nuclear

RPA pool exhaustion= Replicative catastrophe

CyclineB/CDK1G2/M Cell cycle checkpoint

Mitotic entryMitotic catastrophe

GemcitabineConclusion

Thank you for your attentionCharles Theillet

Claude Sardet

Carolina Velazquez

Esin Orhan

Lise Fenou

William Jacot

Beatrice Orsetti

Genevieve Rodier

Stanislas Dumanoir

Stephanie Arnould

Chloe Fallet

Sophie Baize

Laua Boudarel

Mehdi Zaroual