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Hyperactivation of mTORC1 drives resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancersDhivya R. Sudhan1, Angel Guerrero-Zotano4, Helen Won8, Paula González Ericsson5, Alberto Servetto1,Kyung-min Lee1, Luigi Formisano4, Yan Guo7, Qi Liu6, Lisa N. Kinch3, Teresa Dugger4, James Koch4,
Richard E. Cutler, Jr.9, Alshad S. Lalani9, Richard Bryce9, Alan Auerbach9, Ariella B. Hanker1,2, Carlos L.Arteaga1,2
UTSW Simmons Comprehensive Cancer Center1, Department of Internal Medicine2, Howard Hughes Medical Institute3, University of Texas Southwestern Medical Center, Dallas TX; Department of Medicine4, Breast Cancer Program5, Vanderbilt-Ingram Cancer
Center; Center for Quantitative Sciences6, Vanderbilt University Medical Center, Nashville, TN; Comprehensive Cancer Center7, University of New Mexico, Albuquerque, NM; Memorial Sloan Kettering Cancer Center8, New York, NY; Puma Biotechnology
Inc.9,Los Angeles, CA.
Clinical background
Conclusions
B
• Tumor genomic profiling has identified patients with cancers
harboring activating ERBB2 (HER2) mutations that are sensitive to
HER2 targeted therapies.
• In the SUMMIT phase II ‘basket’ trial, a subset of patients with
ERBB2-mutant cancers exhibited significant clinical benefit from
treatment with the pan-HER irreversible tyrosine kinase inhibitor
(TKI) neratinib.
• However, durable responses to neratinib are few, suggesting
mechanisms of de novo and acquired drug resistance. Thus, we
sought to identify actionable mechanisms of resistance to neratinib.
Clinical response to neratinib in phase 2 SUMMIT ‘basket’ trial
• Neratinib-resistant HER2-mutant cells remained cross-resistant to other HER2 targeting agents. RNAseq
revealed significant enrichment of mTORC1 pathway in neratinib-resistant cells.
• Addition of the TORC1 inhibitor everolimus to neratinib as well as knockdown of RHEB or RPTOR
overcame resistance to neratinib.
• mTORC1 activation in neratinib resistance cells was achieved, at-least in part through RAS upregulation.
Knockdown of RAS suppressed S6 phosphorylation and restored sensitivity to neratinib.
• Patients with cancers harboring mTOR activating alterations did not exhibit clinical benefit from neratinib
compared to those without mTOR activating alterations.
• Addition of TORC1 inhibitors may improve the activity of irreversible HER2 TKIs against cancers with
HER2 activating mutations.
.
Combined neratinib and mTORC1 inhibition alone suppresses S6
phosphorylation in neratinib-resistant cells5637 5637-NR
Neratinib (1 uM)
Alpelisib (1 uM)
+ +
++
-
--
- + +
++
-
--
-
5637 5637-NR
+ +-- + +--
5637
+ +- + +--
5637-NR
-
- --- - --- - -- - ----
Everolimus (100 nM) ---- ---- - ++- - ++- - -- - ----
Selumetinib (1 uM) ---- ---- - --- - --- - ++ - ++--
p-ERBB2
(Y1248)
p-mTOR
(S2448)
p-S6
(S235/6).
mTOR
p-AKT
(S473)
p-S6
(S240/4)
AKT
p-ERK
S6
5637 5637-NR
Neratinib (1 uM)
Buparlisib (1 uM)
+ +
++
-
--
- + +
++
-
--
-
5637 5637-NR
+ +-- + +--
5637
+ +- + +--
5637-NR
-
- --- - --- - -- - ----
Everolimus (100 nM) ---- ---- - ++- - ++- - -- - ----
MK2206 (1 uM) ---- ---- - --- - --- - ++ - ++--
p-ERBB2
(Y1248)
p-mTOR
(S2448)
p-S6
(S235/6).
mTOR
p-AKT
(S473)
p-S6
(S240/4)
ERBB2
AKT
b-Actin
mTORC1 activation could be partly attributed to RAS pathway
upregulation
Neratinib-resistant HER2-mutant cells are cross-resistant to other
HER2 TKIs
(A) 12-point dose response curves of parental and neratinib-resistant 5637 and OVCAR8 cells treated with
neratinib or lapatinib. 6 days post-treatment, cells were counted on a Coulter counter. Neratinib-resistant cells
were generated over a period of 6-8 months through gradual dose escalation. (B) Crystal violet stained images of
parental versus neratinib-resistant 5637 and OVCAR8 cells treated with neratinib.
B
56
37
56
37
NR
Neratinib
OV
CA
R8
OV
CA
R8
NR
-
5637 HER2S310F extracellular domain mutation; OVCAR8 HER2G776V kinase domain mutation
1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2
0
5 0
1 0 0
1 5 0
L a p a t in ib d o s e re s p o n s e - 5 6 3 7N R
c e lls
lo g [L a p a tin ib ] , u M
Via
bil
ity
(P
erc
en
t)
p a re n ta l
N R 5 6 3 7
IC 5 0 : p a re n ta l ~ 1 5 0 n M
N R c e lls > 5 0 0 0 n M
1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2
0
5 0
1 0 0
1 5 0
N e ra t in ib d o s e re s p o n s e - 5 6 3 7N R
c e lls
lo g [N e ra t in ib ] , u M
Via
bil
ity
(P
erc
en
t)
5 6 3 7
5 6 3 7 N R
IC 5 0 p a re n ta l 1 4 n M
N R c e lls ~ 8 0 0 n M
Neratinib dose response – OVCAR8NR cells
1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2
0
5 0
1 0 0
1 5 0
lo g [N e ra t in ib ] , u M
Via
bil
ity
(P
erc
en
t)
O V C A R 8
O V C A R 8 N R
IC 5 0 p a re n ta l ~ 1 2 0 n M
N R c e lls > 1 M
1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2
0
5 0
1 0 0
1 5 0
L a p a t in ib d o s e re s p o n s e - O V C A R 8N R
c e lls
lo g [L a p a tin ib ], u M
Via
bil
ity
(P
erc
en
t)
O V C A R 8
O V C A R 8 N R
A
B
Neratinib resistant HER2-mutant cells sustain S6 phosphorylation in the
presence of neratinib
(A) Immunoblots of parental and neratinib-resistant 5637
and OVCAR8 cells treated with increasing
concentrations of neratinib. 24 h later, whole cell lysates
were probed for key members of ERBB pathway.
(B) Immunoblot analysis of parental and neratinib-
resistant 5637 and OVCAR8 cells treated with increasing
concentrations of neratinib for 24 hours.
Identification of mTORC1 as a potential driver of neratinib resistance
(A) RNA-seq based gene set enrichment analysis of pathways significantly upregulated in neratinib treated
parental vs. neratinib-resistant cells. (B) Connectivity map analysis to identify drugs that could potentially reverse
expression of resistance associated genes. (C) Enrichment plots for mTOR pathway related genes in neratinib
treated parental vs. neratinib-resistant cells.
((A) Immunoblot analysis of parental and neratinib-resistant 5637 cells treated with indicated drugs
combinations for 24 hours; alpelsib (PI3K-alpha specific inhibitor), everolimus (mTORC1 inhibitor), selumetinib
(MEK1/2 inhibitor), Buparlisib (pan-PI3K inhibitor), MK2206 (AKT inhibitor). (B) Heatmaps representing 12-
point dose response assays of 5637NR and OVCAR8NR cells treated with indicated single agent or drug
combination. (C) Representative images of cells seeded in a 12 well plate, treated with indicated drug
combination every 72 hours. (D) Immunoblot analysis of neratinib treated 5637NR and OVCAR8NR cells
transfected with indicated siRNA. (E) Growth assay of neratinib treated cells transfected with indicated
siRNAs. (F) Growth assay of TSC2 knockdown 5637, OVCAR8, MCF7 HER2L755S and HER2V777L cells treated
every 3 days with indicated concentrations of neratinib.
A B 5637NR
Enrichment score:0.42; q =0.001
OVCAR8NR
Enrichment score:0.35; q = 0.003
B CA
TN
F
sig
nalin
g v
ia N
FkB
Allo
gra
ft r
eje
ct i
on
Inf l
am
mato
ry r
esp
on
se
mT
OR
C1 s
ign
alin
g
E2F
targ
ets
Kra
s s
ign
alin
g
IL2 S
tat5
sig
nalin
g
Co
mp
lem
en
t
Ch
ole
ste
rol h
om
eo
sta
sis E
MT
Hyp
oxia
Ap
ical ju
nct i
on
INF
resp
on
se
IL6 J
ak S
tat
sig
nalin
g
1 .5
2 .0
2 .5
3 .0
1 0 -4
1 0 -3
1 0 -2
1 0 -1
5 6 3 7 p a re n ta l v s N R - tre a te d
No
rm
ali
ze
d e
nric
hm
en
t s
co
re
(N
ES
)
FD
R q
va
lue
N E S
F D R q v a lu e
E2F
targ
ets
G2 M
ch
eckp
oin
t
MY
C t
arg
ets
V1
MY
C t
arg
ets
V2
Sp
erm
ato
gen
esis
Ch
ole
ste
rol h
om
eo
sta
sis
Kra
s s
ign
alin
g
Oxid
at i
ve p
ho
sp
ho
ryla
t io
n
Mit
ot i
c s
pin
dle
An
dro
gen
resp
on
se
Po
r tein
secre
t io
n
Inf l
am
mato
ry r
esp
on
se
mT
OR
C1 s
ign
alin
g
IL2 S
tat5
sig
nalin
g
TN
F
sig
nalin
g v
ia N
FkB
EM
T
UV
resp
on
se
0
1
2
3
4
1 0 -3
1 0 -2
1 0 -1
O V C A R 8 p a re n ta l v s N R - tre a te d
No
rm
ali
ze
d e
nric
hm
en
t s
co
re
(N
ES
)
FD
R q
va
lue
N E S
F D R q v a lu e
Combined neratinib and mTORC1 suppression overcomes
neratinib resistance
C D
B
C R P R S D P D
0
5 0
1 0 0
Pe
rc
en
t
m T O R a c tiv a tin g a lte ra tio n s
n o n -m T O R a c tiv a tin g a lte ra tio n s
(A-C) Outcomes of patients enrolled in SUMMIT ‘basket’ trial based on mTOR pathway alteration status. Source:
cBioPortal SUMMIT (Nature 2018). (D) Immunoblot analysis of DV90, SNUC2A, MCF7 HER2L755S and
HER2V777L cells treated with indicated concentrations of neratinib, everolimus or combination for 24 hours. (E)
Viability assay to test synergy between neratinib and everolimus. Cells were treated with increasing
concentrations of single agents or drug combinations. Staining intensities were quantified colorimetrically and
combination indices were determined using Chou-Talalay test. (F-H) mTOR pathway mutations acquired in
cancers progressing on neratinib.
A
B
A
C
D E F
A B
(A) Growth of 5637NR tumors
treated with vehicle, neratinib (40
mg/kg), everolimus (5 mg/kg) or
drug combination. (B) Percent
change in volume of individual
tumors in each treatment arm
shown in (A). (C) H-scores for
pS6 levels based on IHC analysis
of tumors shown in (A). (D)
Representative images from (C),
DC
0
5 0
1 0 0
Re
lati
ve
ce
ll n
um
be
r
(pe
rc
en
t)
- - - -+ + + +N era tin ib
(200 nM )
Co
ntr
ol
siR
NA
HR
AS
siR
NA
KR
AS
siR
NA
NR
AS
siR
NA
5 6 3 7N R
0
5 0
1 0 0
Re
lati
ve
ce
ll n
um
be
r
(pe
rc
en
t)
- - - -+ + + +N e ra t in ib
Co
ntr
ol
siR
NA
HR
AS
siR
NA
KR
AS
siR
NA
NR
AS
siR
NA
O V C A R 8N R
(A) Immunoblot analysis of 5637NR and OVCAR8NR cells treated with neratinib, buparlisib (pan-PI3kinase
inhibitor), trametinib (MEK inhibitor) or everolimus (mTOR inihibitor) for 24 h. (B) Enrichment plots for RAS
pathway related genes in neratinib treated parental vs. neratinib-resistant cells (C) Active-RAS pulldown assay in
parental and neratinib resistant 5637 and OVCAR8 cells treated with neratinib for 6 hours. (D) Growth assay of
neratinib treated cells transfected with indicated siRNA.
A
CD
E
F G H