Post on 16-Feb-2022
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 ?
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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
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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
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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
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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
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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
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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
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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