tracking an ahr regulatory circuit in cancer with ahr...
TRANSCRIPT
![Page 1: Tracking an AHR Regulatory Circuit in Cancer With AHR ...nas-sites.org/emergingscience/files/2018/02/02_02-Sherr.pdfFeb 02, 2018 · lentiCRISPR v2 (Addgene) puromycin (Spacer Motif)](https://reader036.vdocument.in/reader036/viewer/2022081617/6023351ea042402377698214/html5/thumbnails/1.jpg)
Tracking an AHR Regulatory Circuit in Cancer With AHR Inhibitors, CRISPR/Cas9 Knockdown, and Other Tricks
David H. Sherr, Ph.D.
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AHR Activation With Environmental Ligands
CYP1B1
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The AHR is Hyper-expressed and Hyper-active In Cancer
The AHR
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AHR Activation With Endogenous Ligands
Endogenous
Ligands
Metabolized
Endogenous
Ligands
CYP1B1
Bad Stuff Happens
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The AHR is Doing Bad Stuff in Cancer
The AHR
Invasion
Migration
Metastasis
“Stemness”
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AHR-Specific CRISPR-Cas9 Strategy
lentiCRISPR v2 (Addgene)
puromycin
(Spacer Motif) (Spacer Motif)
WT sequence WT sequence
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AHR
b-actin
CYP1B1
Naive DMSO FICZ
AHR
b-actin
CYP1B1
Naive DMSO FICZ
Sum149 MDA-MB231
AHR
b-actin
Naive DMSO FICZ
Hs578T
Western blot:
Confirmation of Human AHR Knockdown/Knockout
with CRISPR/Cas9
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1.0 1.4
52.1
1.0 0.8
63.2
0.1 0.2 0.20
10
20
30
40
50
60
70
Naïve DMSO FICZ
CYP1A1
WT
Crispr Ctr
AhR KO
1.0 0.8
7.9
1.0 1.1
10.2
0.1 0.1 0.2
0
2
4
6
8
10
12
Naïve DMSO FICZ
CYP1B1WT
Crispr Ctr
AhR KO
1.0 1.0
29.7
1.0 1.1
13.5
0.0 0.0 0.00
5
10
15
20
25
30
35
Naïve DMSO FICZ
CYP1A1WT
Crispr Ctr
AhR KO2#
1.01.3
3.0
1.01.2
2.6
0.1 0.20.3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Naïve DMSO FICZ
CYP1B1WT
Crispr Ctr
AhR KO2#
1.0 1.3
82.5
1.0 3.0
116.8
0.2 0.1 0.10
20
40
60
80
100
120
140
Naïve DMSO FICZ
CYP1A1WT
CrisprCtr
AhR KO
1.0 1.6
13.3
1.03.0
36.8
0.0 0.0 0.2
0
5
10
15
20
25
30
35
40
45
Naïve DMSO FICZ
CYP1B1WT
CrisprCtr
AhR KO
Sum149 MDA-MB231Hs578T
No
rma
lize
d R
ela
tive
RN
A e
xp
res
sio
n
AHR Knockout with Crispr-Cas9 Ablates AHR Activity
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20x magnification
AHR or CYP1B1 Knockout Reduces an Invasive Phenotype
in SUM149 Inflammatory Breast Cancer Cells
Matrigel SUM149 Day 6
WT Crispr Ctr
AHR KO CYP1B1 KO
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WT Crispr Ctr
Crispr AHR KO Crispr CYP1B1 KO
Matrigel Hs578T at Day 3
20x magnification
AHR or CYP1B1 Knockout Reduces an Invasive Phenotype in
Hs578T Triple Negative Breast Cancer Cells
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Design of Non-toxic AHR Inhibitors as Therapeutics
CB7993113 HP163
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MDA-MB-231-BO(Very aggressive, bone tropic)
BP1
Vehicle
AHR Inhibitor Blocks Human TNBC Invasion In Matrigel
CB7993113
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Sum149
AHR or CYP1B1 Knockout Reduces SUM149 IBC Migration
No
rmalized
op
en
are
a
*p<0.05, **p<0.01 vs. WT at 24h
0.0
0.2
0.4
0.6
0.8
1.0
1.2
WT CrisprCtr
CYP1B1KO
AhR KO
0 h 24 h
*
**
Crispr Ctr Crispr AHR KOCrispr CYP1B1 KO
24 h
WT
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AHR Inhibitors Reduce TNBC and IBC Migration
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EMT PCR Array
Sum149 (AHR KO vs. Control)
-14.9
-9.3-6.8-6.3-5.2-4.5-4.0-3.6-3.2-3.0-2.6-2.5-2.1-2.1-2.0-2.0
20.4
4.8 4.1 4.0 3.7 2.7 2.6 2.6 2.6 2.5 2.3
-20
-15
-10
-5
0
5
10
15
20
25
WN
T5B
MM
P9
MM
P2
OC
LN
ES
R1
CA
MK
2N
1
ZE
B1
GS
C
MA
P1
B
WN
T5A
CD
H2
IG
FB
P4
CD
H1
KR
T1
9
TG
FB
1
NU
DT
13
KR
T1
4
SP
AR
C
CO
L1A
2
FG
FB
P1
MM
P3
BM
P2
SE
RP
INE
1
WN
T11
NO
DA
L
RG
S2
CO
L3A
1
Fo
ld c
ha
ng
es
(re
lati
ve
to
Co
ntr
ol)
AHR Knockout Generates Robust Transcriptional Data
(Epithelial to Mesenchymal Transition Array)
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-43.2
-27.2
-7.3 -6.1 -5.9-4.3 -4.2 -3.7 -3.2 -2.6 -2.5 -2.4 -2.3 -2.3 -2.3 -2.2 -2.1 -2.1
3.7 2.9 2.5 2.4 2.4 2.1
-50
-40
-30
-20
-10
0
10
Fo
ld c
han
ges (
rela
tive t
o C
on
tro
l)
breast cancer resistance protein" (BCRP): multidrug resistance efflux transporter
No
rmali
zed
Rela
tive R
NA
exp
ressio
n
AHR Knockout Generates Robust Transcriptional Data
(Cancer Stem Cell Array)
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An AHR Transcriptional Profile from AHR KO’d IBC is Strongly
Associated with Tumor Infiltrating Leukocytes (TILS)
AHR activity (FDR<0.05 only, n=644 genes) vs infiltrating neutrophils (breast cancer) or macrophages (oral cancer)
Infi
ltra
tin
g N
eu
tro
ph
ils
AHR Activity Score
Infi
ltat
ing
Ne
utr
op
hils
r=0.63p<10-16
AHR Activity Score
r=0.60P<4 x 10-36
Infi
ltat
ing
Mac
rop
hag
es
0.0 0.2 0.4 0.6 0.8 1.0
Human Breast Cancers Human Oral Cancers
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AHR AHR Knockout Prevents Oral Tumor Growth
Concommitant with an Increase in Tumor-Associated
Macrophages (TAMs)
% C
D4
+T
Ce
lls
% C
D8
+T
Ce
lls
% C
D1
1b
+ M
DS L
C
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Summary
1. CRISPR/Cas9 Knockdown/Knockout is wicked good*.2. Robust knockout results in robust biologic and molecular changes.3. Robust biologic and molecular changes increase confidence in
additional (therapeutic) applications.4. CRISPR/Cas9 Knockdown/Knockout is wicked good*.
*New England-ish for “extremely effective”
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Acknowledgments
Sherr Lab (BU & BU-WHOI SRP*)Yan Wang, Ph.D.
Elizabeth Stanford, Ph.D.Olga Novikov, (M.D., Ph.D.)
Jessica Kenison-White
Monti Lab (BU & BU-WHOI SRP)Stefano Monti
Amy LiLiye Zhang
Hahn Lab (WHOI/BU-WHOI SRP)Mark Hahn
Sibel Karchner
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SUM149 Crispr AHR KO and CYP1B1 KO
AhR
b-actin
CYP1B1
Naive DMSO FICZ
1 1
52
1 1
63
0.1 0.2 0.2
12 13
211
0
50
100
150
200
250
Naïve DMSO FICZ
SUM149 CYP1A1
WT
Crispr Ctr
AhR KO
CYP1B1 KO