Dual lineage inhibition of ETV1 and KIT disrupts the ETV1 ... · PDF fileDual lineage...
1
Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates Imatinib antitumor effect in GIST Leili Ran 1 , Inna Sirota 1 , Zhen Cao 1 , Devan Murphy 1 , Shipra Shukla 1 , Ferdinando Rossi 4 , John Wongvipat 1 , William D. Tap 2 , Peter Besmer 4 , Cristina R. Antonescu 3 , Yu Chen 1,2,5 , and Ping Chi 1,2,5 1 Human Oncology and Pathogenesis Program, 2 Department of Medicine, 3 Department of Pathology, 4 Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. 5 Co–corresponding authors. References Summary Printed by Figure1 a) Representative H&E staining of the cecal masses and the cecums of Etv1 +/+ ;Kit ∆558V/+ and Etv1 -/- ;Kit ∆558/+ mice. M: mucosa; CM: circular muscle; LM: longitudinal muscle. Scale bars: 100 μm. b) Representative Ki67 IHC (proliferation) and tricrome staining(fibrosis)of the cecal tumors of 8-9 weeks old Etv1 flox/flox ; Kit Δ558V/+ ; Rosa26 CreERT2/+ mice treated with either corn oil or tamoxifen. Scale bars: 50μm. GEMM (Etv1 f/f ;Kit V558Δ/+ ;Rosa26 CreERT2/+ ) Cecal tumor (trichrome) Corn Oil Tamoxifen Cecal tumor (Ki67 IHC) Corn Oil Tamoxifen b ETV1 KIT Merge Corn Oil Tamoxifen GEMM (Etv1 f/f ;Kit V558Δ/+ ;Rosa26 CreERT2/+ ) Figure2 a) Representative immunofluorescence (IF) images of Etv1 (green) and Kit (red) protein in cecal tumors from Etv1 flox/flox ;Kit V558Δ/+ ; Rosa26 CreERT2/+ mice treated with tamoxifen or corn oil. Nuclei (DAPI, blue). Scale bars: 50 μm. b) Representative of ChIP-seq reads of ETV1, H3K4me1 and H3K4me3 at KIT transcription start site (H3K4me3) and enhancer regions (H3K4me1 and ETV1) in human GIST48 cells. c) ETV1 is knockdown with siRNA in GIST882 cells and ChIP for KIT enhancers were performed with anti-ETV1 antibody. Figure 2: ETV1 regulate KIT expression through binding to KIT enhancers KIT Normalized Peak Counts ETV1 H3K4me1 H3K4me3 0 0.005 0.010 0.015 0.020 0.025 % of Input Enhancer 1 Enhancer 2 Enhancer 3 siSCR siETV1 siSCR siETV1 siSCR siETV1 a b c Vehicle Imatinib MEK162 Imatinib+MEK162 Figure4 a) Tumor Size measurement of GIST T1 xenograft tumors after treatment. Mice were treated vehichle, Imatinib(80mg/kg, BID), MEK162(30mg/kg, BID), Imatinib(80mg/kg, BID)+MEK162(30mg/kg, BID). The result was presented as percentage tumor growth by normalizing to the first mean tumor volume measured for each group ± SEM (n = 10 mice/group). **P<0.005. b) Tumor Size measurement of GIST882 xenograft tumors after treatment. Mice were treated with vehichle, Imatinib(100mg/kg, BID), MEK162(30mg/kg, BID), dose 1(Imatinib, 100mg/kg, BID+MEK162, 10mg/kg, BID), dose 2 (Imatinib, 50mg/kg, BID+MEK162, 30mg/kg, BID) and dose 3 (Imatinib, 100mg/kg, BID+MEK162, 30mg/kg, BID) and presented as in a. *** p<0.001 c) Representative histology of GIST882 xenograft tumor after 1 month treatment. Scale Bars: 100uM. b Vehicle Imatinib MEK162 Imatinib+MEK162 Ki67 Trichrome Figure 5: Dual inhibition of KIT and ETV1 results in GIST GEMM tumor regression in vivo REFERENCE 1. Chi, P. et al. Nature 467, 849–853 (2010). 2. Verweij, J. et al.Lancet 364, 1127–34 (2004). 3.Balachandran, V. P. & Dematteo, R. P. Surg. Oncol. Clin. N. Am. 22, 805–21 (2013). CONCLUSION •Etv1 is required for GIST tumor initation and maintanence in vivo. •Etv1 positively regulates KIT expression through binding to its enhancer. •Targeting ETV1-KIT feed forward circuits by combined therapy of MEK162 and imatinib leads to more enhanced inhibition of GIST growth than either single agent in vitro and in vivo. Tumor Weight (mg) 0 200 400 600 800 1000 ns P=0.03 P=0.01 P=0.04 Figure5 a) Cecal tumor weight meausrement of Etv1 f/f ;Kit V558Δ/+ mice treated with vehicle, MEK162(30mg/kg, BID), Imatinib(50mg/kg, BID), Imatinib(50mg/kg, BID)+MEK162(30mg/kg, BID) for 5 days. b) Representative trichrome staining and Ki67 immunohistochemistry staining of tumor masses 5days after treatment. Scale bars: 100uM. a b a Mutant KIT P P P ETV1 MAPK GIST + Imatinib MEK inhibitor: MEK162 0 500 1000 0 500 1000 0 500 1000 0 500 1000 0 62.5 125 1000 Imatinib (nM) MEK162(nM) KIT pKIT ERK1/2 ETV1 pERK1/2 ACTIN Cl-CASPASE3 b Figure 4: Dual inhibition of KIT and ETV1 results in GIST xenograft tumor regression in vivo *** 200 0 100 300 400 500 600 0 5 10 15 20 25 30 35 40 45 50 % original tumor size Days *** *** Vehicle Imatinib(100mg/kg) MEK162(30mg/kg) Imatinib + MEK162 (dose 1) Imatinib + MEK162 (dose 2) Imatinib + MEK162 (dose 3) b Figure3 a) Schematic model for GIST pathogenesis. b) Western blot of GIST882 cells treated with Imatinib and MEK162 for 60hrs. a GIST882 Vehicle MEK162(30mg/kg) Imatinib(80mg/kg) Imatinib(80mg/kg)+MEK162(30mg/kg) % original tumor size Days ** ** ** GIST T1 Figure 3: Dual inhibition of KIT and ETV1 synergistically induce apoptosis in GIST Gastrointestinal stromal tumor (GIST) is characterized by activating mutations of the KIT or PDGFRA receptor tyrosine kinases and originates from the interstitial cells of Cajal (ICCs), where KIT regulates its normal development and lineage specification. Despite the initial clinical success of imatinib, the majority of patients with advanced GIST develop imatinib resistance and die of their disease. The development of novel therapeutics that can improve the efficacy of first-line imatinib therapy and/or overcome imatinib resistance is imperative. We have previously identified ETV1, an ETS family transcription factor, as a lineage-specific survival master regulator for GIST and its precursor ICCs. Mutant KIT cooperates with ETV1 in GIST oncogenesis, in part by stabilizing the ETV1 protein through active MAP kinase signaling. Here, we demonstrate that ETV1 is required for GIST initiation and maintenance in vivo using compound genetically engineered mouse models (GEMMs). We have uncovered that ETV1 enhances KIT expression through direct binding to the KIT enhancers. Hence, ETV1 and mutant KIT form a positive feed-forward circuit and dual lineage dependence in GIST pathogenesis, where the ETV1 protein is stabilized by active KIT signaling and stabilized ETV1 augments KIT expression. Further, we demonstrate that inhibition of neither KIT (by imatinib) nor its downstream MAP kinase signaling (by MEK162, a MEK inhibitor) alone is sufficient to durably destabilize the ETV1 protein. Interestingly, the combined targeting of the dual lineage dependence of KIT by imatinib and ETV1 by MEK162 results in durable inhibition of the ETV1 protein and leads to significantly more growth suppression in vitro and complete tumor regression in vivo than either single agent. Our observations demonstrate that ETV1 is a novel therapeutic target in GIST. Importantly, the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more effective therapeutic strategy than imatinib alone in GIST clinical management. ABSTRACT RESULTS Cecum (Etv1 +/+ ;Kit V558Δ/+ ) Cecum (Etv1 -/- ;Kit V558Δ/+ ) H&E (4X) H&E (20X) M M M CM LM a Figure 1: Etv1 is required for the initiation of GIST development in vivo METHODS Genetic Engineered mouse modeling, tumor cell xenograft, Chromatin IP, Western blotting, Immunofluorescent, Immunohistochemistry.
Transcript of Dual lineage inhibition of ETV1 and KIT disrupts the ETV1 ... · PDF fileDual lineage...
Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates Imatinib antitumor effect in GIST
Leili Ran1, Inna Sirota1, Zhen Cao1, Devan Murphy1, Shipra Shukla1, Ferdinando Rossi4, John Wongvipat1, William D. Tap2, Peter Besmer4, Cristina R. Antonescu3, Yu Chen1,2,5, and Ping Chi1,2,5 1Human Oncology and Pathogenesis Program, 2Department of Medicine, 3Department of Pathology, 4Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. 5Co–corresponding authors.
References
Summary
Printed by
Figure1 a) Representative H&E staining of the cecal masses and the cecums of Etv1+/+;Kit∆558V/+ and Etv1-/-;Kit∆558/+
mice. M: mucosa; CM: circular muscle; LM: longitudinal muscle. Scale bars: 100 µm. b) Representative Ki67 IHC
(proliferation) and tricrome staining(fibrosis)of the cecal tumors of 8-9 weeks old Etv1flox/flox; KitΔ558V/+; Rosa26CreERT2/+
mice treated with either corn oil or tamoxifen. Scale bars: 50µm.
GEMM (Etv1f/f;KitV558Δ/+;Rosa26CreERT2/+)
Cecal tumor (trichrome)
Corn Oil
Tamoxifen
Cecal tumor (Ki67 IHC)
Corn Oil
Tamoxifen
b
ETV1 KIT Merge
Co
rn O
il
Ta
mo
xif
en
GEMM (Etv1f/f;KitV558Δ/+;Rosa26CreERT2/+)
Figure2 a) Representative immunofluorescence (IF) images of Etv1 (green) and Kit (red) protein in cecal tumors from
Etv1flox/flox;KitV558Δ/+; Rosa26CreERT2/+ mice treated with tamoxifen or corn oil. Nuclei (DAPI, blue). Scale bars: 50 µm. b)
Representative of ChIP-seq reads of ETV1, H3K4me1 and H3K4me3 at KIT transcription start site (H3K4me3) and
enhancer regions (H3K4me1 and ETV1) in human GIST48 cells. c) ETV1 is knockdown with siRNA in GIST882 cells
and ChIP for KIT enhancers were performed with anti-ETV1 antibody.
Figure 2: ETV1 regulate KIT expression through binding to
KIT enhancers
KIT
No
rma
lize
d P
ea
k C
ou
nts
ETV1
H3K4me1
H3K4me3
0
0.005
0.010
0.015
0.020
0.025
% o
f In
pu
t
Enhancer 1 Enhancer 2 Enhancer 3
siSCR siETV1 siSCR siETV1 siSCR siETV1
a
b
c
Vehicle Imatinib MEK162 Imatinib+MEK162
Figure4 a) Tumor Size measurement of GIST T1 xenograft tumors after treatment. Mice were treated vehichle,
Imatinib(80mg/kg, BID), MEK162(30mg/kg, BID), Imatinib(80mg/kg, BID)+MEK162(30mg/kg, BID). The result was
presented as percentage tumor growth by normalizing to the first mean tumor volume measured for each group ± SEM
(n = 10 mice/group). **P<0.005. b) Tumor Size measurement of GIST882 xenograft tumors after treatment. Mice were
treated with vehichle, Imatinib(100mg/kg, BID), MEK162(30mg/kg, BID), dose 1(Imatinib, 100mg/kg, BID+MEK162,
10mg/kg, BID), dose 2 (Imatinib, 50mg/kg, BID+MEK162, 30mg/kg, BID) and dose 3 (Imatinib, 100mg/kg,
BID+MEK162, 30mg/kg, BID) and presented as in a. *** p<0.001 c) Representative histology of GIST882 xenograft
tumor after 1 month treatment. Scale Bars: 100uM.
b
Vehicle Imatinib MEK162 Imatinib+MEK162
Ki6
7
Tri
ch
rom
e
Figure 5: Dual inhibition of KIT and ETV1 results in GIST
GEMM tumor regression in vivo
REFERENCE
1. Chi, P. et al. Nature 467, 849–853 (2010).
2. Verweij, J. et al.Lancet 364, 1127–34 (2004).
3. Balachandran, V. P. & Dematteo, R. P. Surg. Oncol. Clin. N. Am. 22, 805–21 (2013).
CONCLUSION •Etv1 is required for GIST tumor initation and maintanence in vivo.
•Etv1 positively regulates KIT expression through binding to its enhancer.
•Targeting ETV1-KIT feed forward circuits by combined therapy of MEK162 and
imatinib leads to more enhanced inhibition of GIST growth than either single agent in
vitro and in vivo.
Tu
mo
r W
eig
ht
(mg
)
0
200
400
600
800
1000
ns
P=0.03
P=0.01
P=0.04
Figure5 a) Cecal tumor weight meausrement of Etv1f/f;KitV558Δ/+ mice treated with vehicle, MEK162(30mg/kg, BID),
Imatinib(50mg/kg, BID), Imatinib(50mg/kg, BID)+MEK162(30mg/kg, BID) for 5 days. b) Representative trichrome
staining and Ki67 immunohistochemistry staining of tumor masses 5days after treatment. Scale bars: 100uM.
a
b
a Mutant KIT
P P
P
ET
V1
MAPK
GIST
+
Imatinib
MEK inhibitor: MEK162
0 500 1000 0 500 1000 0 500 1000 0 500 1000
0 62.5 125 1000 Imatinib (nM)
MEK162(nM)
KIT
pKIT
ERK1/2
ETV1
pERK1/2
ACTIN
Cl-CASPASE3
b
Figure 4: Dual inhibition of KIT and ETV1 results in GIST
xenograft tumor regression in vivo
***
200
0
100
300
400
500
600
0 5 10 15 20 25 30 35 40 45 50
% o
rig
ina
l tu
mo
r s
ize
Days
***
***
Vehicle
Imatinib(100mg/kg)
MEK162(30mg/kg)
Imatinib + MEK162 (dose 1)
Imatinib + MEK162 (dose 2)
Imatinib + MEK162 (dose 3)
b
Figure3 a) Schematic model for GIST pathogenesis. b) Western blot of GIST882 cells treated with Imatinib and
MEK162 for 60hrs.
a
GIST882
Vehicle
MEK162(30mg/kg)
Imatinib(80mg/kg)
Imatinib(80mg/kg)+MEK162(30mg/kg)
% o
rig
ina
l tu
mo
r s
ize
Days
** **
**
GIST T1
Figure 3: Dual inhibition of KIT and ETV1 synergistically
induce apoptosis in GIST Gastrointestinal stromal tumor (GIST) is characterized by activating mutations of the KIT or
PDGFRA receptor tyrosine kinases and originates from the interstitial cells of Cajal (ICCs),
where KIT regulates its normal development and lineage specification. Despite the initial
clinical success of imatinib, the majority of patients with advanced GIST develop imatinib
resistance and die of their disease. The development of novel therapeutics that can improve
the efficacy of first-line imatinib therapy and/or overcome imatinib resistance is imperative.
We have previously identified ETV1, an ETS family transcription factor, as a lineage-specific
survival master regulator for GIST and its precursor ICCs. Mutant KIT cooperates with ETV1
in GIST oncogenesis, in part by stabilizing the ETV1 protein through active MAP kinase
signaling. Here, we demonstrate that ETV1 is required for GIST initiation and maintenance
in vivo using compound genetically engineered mouse models (GEMMs). We have
uncovered that ETV1 enhances KIT expression through direct binding to the KIT enhancers.
Hence, ETV1 and mutant KIT form a positive feed-forward circuit and dual lineage
dependence in GIST pathogenesis, where the ETV1 protein is stabilized by active KIT
signaling and stabilized ETV1 augments KIT expression. Further, we demonstrate that
inhibition of neither KIT (by imatinib) nor its downstream MAP kinase signaling (by MEK162,
a MEK inhibitor) alone is sufficient to durably destabilize the ETV1 protein. Interestingly, the
combined targeting of the dual lineage dependence of KIT by imatinib and ETV1 by
MEK162 results in durable inhibition of the ETV1 protein and leads to significantly more
growth suppression in vitro and complete tumor regression in vivo than either single agent.
Our observations demonstrate that ETV1 is a novel therapeutic target in GIST. Importantly,
the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more
effective therapeutic strategy than imatinib alone in GIST clinical management.
ABSTRACT
RESULTS
Cecum (Etv1+/+;KitV558Δ/+) Cecum (Etv1-/-;KitV558Δ/+)
H&
E (
4X
) H
&E
(2
0X
)
M M
M
CM
LM
a
Figure 1: Etv1 is required for the initiation of GIST
development in vivo
METHODS Genetic Engineered mouse modeling, tumor cell xenograft, Chromatin IP, Western blotting,
Immunofluorescent, Immunohistochemistry.
