Post on 02-Oct-2020
Tracking an AHR Regulatory Circuit in Cancer With AHR Inhibitors, CRISPR/Cas9 Knockdown, and Other Tricks
David H. Sherr, Ph.D.
AHR Activation With Environmental Ligands
CYP1B1
The AHR is Hyper-expressed and Hyper-active In Cancer
The AHR
AHR Activation With Endogenous Ligands
Endogenous
Ligands
Metabolized
Endogenous
Ligands
CYP1B1
Bad Stuff Happens
The AHR is Doing Bad Stuff in Cancer
The AHR
Invasion
Migration
Metastasis
“Stemness”
AHR-Specific CRISPR-Cas9 Strategy
lentiCRISPR v2 (Addgene)
puromycin
(Spacer Motif) (Spacer Motif)
WT sequence WT sequence
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
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
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
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
Design of Non-toxic AHR Inhibitors as Therapeutics
CB7993113 HP163
MDA-MB-231-BO(Very aggressive, bone tropic)
BP1
Vehicle
AHR Inhibitor Blocks Human TNBC Invasion In Matrigel
CB7993113
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
AHR Inhibitors Reduce TNBC and IBC Migration
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)
-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)
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
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
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”
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
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