Effects of G1T48, a novel orally bioavailable selective estrogen receptor degrader (SERD), and the CDK4/6 inhibitor, G1T38, on tumor growth in an animal
model of tamoxifen resistant breast cancer
Abstract
# 5641
Suzanne E. Wardell1, Jennifer G. Baker1, Robert M. Baldi1, Alexander P. Yllanes1, Taylor K. Krebs1, Jessica A. Sorrentino2, John E. Bisi2, Jay C. Strum2, John D. Norris1
50
G DC 810
ADZ9694
G 1T48
LASO
F u v e s tr a n t
100
50
50
40000
20000
0
-1 4
-1 2
-1 0
-8
-6
0
-1 4
-4
-1 2
L o g (M ) lig a n d
-1 0
-8
-6
0
-1 4
-4
-1 2
L o g (M ) lig a n d
-1 0
-8
-6
60000
60000
-4
0
-1 4
L o g (M ) lig a n d
Tam
4O H T
G W 5638
G W 7604
F u lv e s tr a n t
-1 2
-1 0
-6
L o g (M ) lig a n d
3
G1T48 inhibits ER-dependent MCF7 cell proliferation in
response to estrogen or growth factors.
MCF7 breast cancer cells were treated for 7 days with
M 17β-estradiol (A)
-8
-11
or 2 x 10 M insulin (B) in addition to ER ligands (10
– 10-5 M). Cell
proliferation was quantitated through detection of DNA content (Hoechst stain).
-1 2
-1 0
-8
B
B
M C F 7 p r o life r a tio n ( 0 .1 n M E 2 )
In s u lin ( 2 0 n M )
20000
15000
10000
60000
40000
-1 0
-8
-6
-4
-1 2
M C F 7 p r o life r a tio n ( 0 .1 n M E 2 )
-4
L o g (M ) lig a n d
15000
10000
60000
RFU
Ral
Baz
Laso
R U 5 8 ,6 6 8
F u lv e s tr a n t
-8
-6
40000
20000
5000
-1 0
G W 5638
G W 7604
R al
Baz
IFulvestrant
CI
0
-1 4
-4
-1 2
-1 0
-8
L o g ( M ) lig a n d
L o g (M ) lig a n d
M C F 7 p r o life r a tio n ( 0 .1 n M E 2 )
In s u lin ( 2 0 n M )
-6
-4
80000
25000
20000
15000
10000
G DC 0810
AZD9496
G 1T48
LASO
IC I
Fulvestrant
60000
RFU
G D C 0810
AZD 9496
G 1T48
F u lv e s tr a n t
40000
20000
5000
-1 0
-8
-6
L o g ( M ) lig a n d
-4
0
-1 4
-1 2
-1 0
-8
L o g (M ) lig a n d
-6
-4
Compound
wtER
Y537S
D538G
G1T48
2.1E-09
1.6E-08
1.5E-08
GDC0810
1.1E-08
1.3E-06
2.5E-08
Raloxifene
4.5E-10
4.5E-09
2.7E-10
2.6E-09
2.0E-08
4.8E-09
1.2E-09
1.0E-08
5.8E-09
Bazedoxifene
7.1E-10
2.9E-08
8.0E-09
Lasofoxifene
3.8E-10
7.1E-09
1.4E-09
RU58,668
2.9E-10
2.9E-09
1.6E-09
Tamoxifen
2.7E-07
>10mM
2.9E-09
4OH-tamoxifen
3.3E-09
3.3E-09
3.1E-09
GW5638
>10mM
>10mM
>10mM
GW7604
2.5E-08
>10mM
3
8
3
3
5
5
-D
-Y
250
F
C
M
M
G 1 T 4 8 ( 3 0 m g /k g )
*
500
+ G 1 T 3 8 ( 5 0 m g /k g )
G 1 T 4 8 ( 1 0 0 m g /k g q d )
*
*
p < 0 .0 5
7
7
F
C
tu m o r v o lu m e ( m m )
a v e ra g e
G
S
7
R
tE
-w
7
F
C
G 1T48
20
40
60
-1 2
-1 0
-8
-6
G DC810
15000
AZD9496
G 1T48
10000
0
-1 4
-4
7
-1 2
2.3E-08
-1 0
-8
-6
-4
L o g (M ) lig a n d
Compound
wtER
Y537S
D538G
G1T48
1.7E-11
6.1E-09
4.2E-10
Fulvestrant
7.7E-12
2.2E-09
6.2E-10
AZD9496
2.3E-12
4.3E-10
7.1E-11
GDC810
3.2E-11
2.2E-09
6.2E-10
G1T48, alone or in combination with CDK4/6 inhibitor
G1T38, inhibits the growth of estrogen-dependent MCF7
xenograft tumors.
M C F 7 x e n o g r a ft tu m o r g r o w th
V e h ic le
800
G 1 T 3 8 (5 0 m g /k g )
G 1 T 4 8 (3 0 m g /k g )
600
G 1 T 3 8 + G 1 T 4 8 (3 0 )
400
G 1 T 4 8 (1 0 0 m g /k g )
G 1 T 3 8 + G 1 T 4 8 (1 0 0 )
200
0
0
10
20
30
40
D atoy sday
o f tr
e a tm e n t
Treatment
28
M C F 7 tu m o r v o lu m e s @ d 4 1
800
600
400
*
200
0
* p < 0.05
T a m R tu m o r v o lu m e @ d 3 5
F u lv e s tr a n t
5000
C
SKBR3 breast cancer cells were transfected with an estrogen-responsive
reporter gene (3X-ERE-tata-Luc) together with wild type (wt) or mutant (Y537S
or D538G) ER expression plasmids prior to 18 hours of treatment with 17βestradiol (1.0 nM) and ER antagonists (10-11 – 10-5 M). Firefly and renilla
luciferase activity were then assessed using dual luciferase reagent. IC50
values were calculated in JMP 13.
AZD9496
G 1 T 4 8 ( 3 0 m g /k g q d )
*
4O HT
AZD9496
0
-1 4
G1T48 inhibits transcriptional activity of clinically
relevant ESR1 mutations.
Fulvestrant
80000
20000
0
-1 2
-6
In s u lin ( 2 0 n M )
25000
RFU
-8
L o g (M ) lig a n d
L o g ( M ) lig a n d
0
-1 2
-1 0
G 1 T 3 8 ( 5 0 m g /k g q d )
750
0
20000
L o g (M ) lig a n d
5
V e h ic le
0
G DC810
5000
20000
0
-1 4
0
10000
A v e r a g e tu m o r V o lu m e ( m m )
0
-1 2
R U 5 8 ,6 6 8
Tam
4O H T
IC I
Fulvestrant
10000
4O HT
15000
-6
80000
Tam
4O HT
G W 5638
G W 7604
F u lv e s tr a n t
20000
F u lv e s tr a n t
25000
10-10
A
30000
ER D 538G
20000
20000
-8
1000
E2
ER Y537S
40000
0
-1 4
V e h ic le
40000
80
d a y s o f tr e a tm e n t
R al
Baz
Laso
R U 5 8 ,6 6 8
F u lv e s tr a n t
80000
80000
150
T a m o x ife n re s is ta n t x e n o g ra ft tu m o rs
50000
Ovariectomized estrogen-treated female
nu/nu mice bearing MCF7 xenograft
tumors were randomized to treatment
with vehicle, G1T38 (50 mg/kg) or
G1T48 (30 or 100 mg/kg), alone or
together, p.o. daily for 28 days. 2-way
ANOVA comparison of average tumor
volumes throughout treatment, followed
by Bonferroni multiple comparison test,
indicated significant tumor growth
inhibition by all treatments, as well as
increased response to the combination
of G1T48 (30 mg/kg) and G1T38.
1500
3
100
L T E D P ro life r a tio n
G1T48 and G1T38 both inhibit the growth of tamoxifen
resistant (TamR) xenograft tumors
8
tu m o r v o lu m e (m m )
100
-6
L T E D P ro life r a tio n
In C e ll W e s te r n
G W 5638
G W 7604
R a lo x
BAZ
F u v e s tr a n t
150
R U 5 8 ,6 6 8
TAM
4O H T
F u lv e s tra n t
-8
L o g (M ) lig a n d
RFU
150
In C e ll W e s te r n
-1 0
A
MCF7 breast cancer cells with
stably integrated (doxycyclineinducible lentivirus) ESR1
mutants were treated for 7 days
(A) with vehicle or 10-10 M 17βestradiol or (B) with ER
antagonists (10-12 – 10-5 M). Cell
proliferation was quantitated
through detection of DNA content
(Hoechst stain). (C) IC50 values
were calculated in JMP 13.
M
-1 2
-05
RFU
LRP2
RFTN1
PMP22
SLFN5
YWHAZ
FHL1
SLC2A1
STC2
SLC6A14
RAPGFL
RGNEF
AGR2
SNX24
KRT13
SDK2
0
-1 4
-06
5000
MCF7 breast cancer
cells were plated in
media supplemented
with charcoal stripped
AZD9496
serum 48 hours prior to
GDC0810
18 hours of treatment
RU 58668
with ER ligands. mRNA
G1T48
expression of ER target
GW5638
genes previously found
Fulvestrant
to be predictive of ER
GW7604
Bazedoxifene S E R D / S E R M
Raloxifene
pharmacology was
17β-estradiol
analyzed by real time
4OH-tamoxifen qPCR analysis. Relative
Tamoxifen
mRNA levels were
Lasofoxifene
s u b j e c t e d t o
Vehicle
unsupervised clustering
in JMP 13.
40000
20000
-08
-07
RFU
1
RFU
-10
-09
25000
The ER target gene regulation profile of G1T48
resembles that of known SERDs.
G DC 0810
AZD9496
G 1T48
F u lv e s tr a n t
60000
In C e ll W e s te r n
LTED MCF7 cells, a model of
aromatase resistance, were plated in
media supplemented with charcoal
stripped fetal bovine serum (for
complete removal of estrogens) for
48 hours and then treated for 7 days
with ER ligands (10-12 –10-7 M). Cell
proliferation was quantitated through
detection of DNA content (Hoechst
stain).
L T E D P ro life r a tio n
Results
G1T48 inhibits the growth of MCF7 breast cancer cells
expressing resistance-associated ESR1 mutation.
6
T u m o r V o lu m e ( m m )
Results
G1T48 inhibits the growth of long term estrogen
deprived (LTED) MCF7 breast cancer cells.
80000
-11
Results
3
Conclusions: G1T38, a novel CDK4/6 inhibitor, and G1T48, a novel SERD,
either alone or in combination, demonstrated highly potent inhibition of tumor
growth in an animal model of tamoxifen resistance. G1T48 also demonstrated
activity in models of endocrine resistance mediated by ER mutation
4
MCF7 breast cancer cells were treated with ER ligands (10-11 – 10-5 M) for 18
hours prior to fixation of cells and detection of ER levels by In-Cell Western.
RFU
Results: G1T48 treatment led to dramatic reductions in ER protein levels.
G1T48 significantly inhibited cellular proliferation of MCF7 breast cancer cells
bearing endocrine resistant ER mutations, ER-Y537S and ER-D538G.
Importantly, tumor growth inhibition was observed in mouse models of sensitive
and resistant human breast cancer when G1T48 was dosed as a single agent or
in combination with G1T38, a potent, selective CDK4/6 inhibitor.
G1T48 exhibits SERD activity comparable to other
clinically relevant SERDs.
2
% ER Remaining
Methods: Breast cancer cells expressing clinically relevant ESR1 mutations
(ER-Y537S, ER-D538G) were treated with G1T48, and mechanistically distinct
SERMs/SERDs and cellular proliferation was assessed by measuring DNA
content (Hoechst dye). Ovariectomized nu/nu mice bearing xenografts of
tamoxifen (TamR) resistant ER+ breast cancer were treated with G1T38 and
G1T48, alone or in combination, with clinically relevant comparators. Time to
progression and tumor volume were assessed over a 4 week dosing period.
Results
3
Background: The combination of targeting the CDK4/6 and ER signaling
pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the
treatment of ER positive breast cancer. However, the poor physicochemical
properties of fulvestrant require monthly intramuscular injections to patients,
which limit the pharmacokinetic and pharmacodynamic activity of the compound.
Therefore, an orally available compound that more rapidly reaches steady state
may lead to a better clinical response in patients. Here we report the preclinical
characterization of G1T48, a novel, orally bioavailable, non-steroidal small
molecule inhibitor of ERα, which is a potent, selective antagonist that down
regulates ERα in vitro and in vivo in ER-positive models of breast cancer.
Therapeutics, Inc., Research Triangle Park, North Carolina
RFU
Results
RFU
Abstract
2G1
RLU
University School of Medicine, Durham, North Carolina
RFU
1Duke
1000
500
0
*
*
*
*
Ovariectomized tamoxifen-treated
female nu/nu mice bearing TamR
xenograft tumors were randomized
to treatment with vehicle or G1T38
(50 mg/kg) or G1T48 (30 or 100 mg/
kg), alone or together, p.o. daily. 2way ANOVA comparison of average
tumor volumes throughout treatment,
followed by Bonferroni multiple
comparison test, indicated significant
tumor growth inhibition by all
treatments, as well as increased
response to the combination of
G1T48 (30 mg/kg) and G1T38.
Summary
• G1T48 is an orally bioavailable potent and efficacious antiestrogen with SERD
activity.
• G1T48
regulates ER target gene transcription in a manner similar to
fulvestrant, a pure antiestrogen with demonstrated efficacy in antiendocrine
refractory breast cancer.
• G1T48 inhibits growth factor mediated breast cancer cell proliferation.
• G1T48 inhibits the growth of long term estrogen deprived cells, a model of
aromatase resistance.
• G1T48
inhibits the transcriptional activity of ER mutants (Y537S, D538G)
associated with endocrine-refractory breast cancer.
• G1T48
inhibits the ligand-independent growth of breast cancer cells
expressing antiendocrine- resistant ER mutations (Y537S, D538G).
• G1T48, alone or in combination with the CDK4/6 inhibitor G1T38, inhibits the
growth of estrogen-dependent MCF7 xenograft tumors.
• G1T48
and G1T38 both inhibit the growth of tamoxifen resistant (TamR)
xenograft tumors
• G1T48 is currently completing IND enabling studies.
• G1T38 is currently being evaluated in combination with Faslodex in a Phase
1b/2a trial in ER+, HER2- breast cancer patients (NCT02983071).
Funding was provided by
G1 Therapeutics, Inc.,
via a sponsored research
agreement.