BACKGROUND IN VITRO FGFR3 SIGNALING

Transcription

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F G F R 3 f u s io n
F G F R o v e r e x p r e s s io n
F G F R 3 m u ta t io n
FGFR3 wt
• Lucitanib GI50 was determined on 24 cell lines with FGFR
and/or PDGFR genomic alterations - including
amplifications, mutations, and fusions; and 25 nonFGFR/PDGFR aberrant cell lines (Fig. 2A). Mean GI50 for
cells with FGFR/PDGFR aberrations was significantly lower
than that for control cells (P<0.0001), suggesting that
FGFR/PDGFR genomic status is potentially correlated with
response to lucitanib in these models. However, several cell
MATERIALS & METHODS
lines without known FGFR/PDGFR aberrations were
• Cell proliferation assay: 3x103 cells per well were seeded in 96-well
sensitive to lucitanib through FGFR1 dependency due to
plates at day 0. Lucitanib was serially diluted, and incubated on cells
over-expression (e.g. JMSU1 [3] and NCI-H1048 [4]). The
for 72 hours. Cell viability was measured by Cell Titer Glo (Promega).
converse also appeared to be true; a subset of cell lines
• Phosphorylation analysis: 4x105 cells were seeded in 6-well plates at
day 0, and changed to 1% FBS media on day 1. On day 2, lucitanib
with FGFR/PDGFR aberrations were not sensitive to
was incubated for 1 hour and then 50 ng/mL FGF-1 was added for 15
lucitanib. This data was consistent with previous
minutes before cell lysate was collected. Phospho-FGFR3 ELISA
publications employing alternative FGFR inhibitors (e.g.
(R&D Systems) was performed according to the manufacturer’s
NCI-H2444 [5], J82 [6], and MFE319 [7]).
instructions and scaled proportionately for 384-well plates. PhosphoERK and phospho-AKT immunoblotting was resolved using NuPage • Lucitanib GI50 was determined in a panel of 12 bladder
(Life Technologies) gels, and stained with phospho-ERK, total-ERK,
carcinoma cell lines with (N=7) and without (N=5) genomic
phospho-AKT, and total AKT antibodies (Cell Signaling Technology).
FGFR3 fusion/mutations (Fig. 2B, 2C). Mean lucitanib GI50
Signals were detected using LiCor Imaging Systems.
for cells with FGFR3 fusion was significantly lower than that
• In vivo xenografts: RT4, RT112/84, and BN2289 studies were
for mutated and WT FGFR3 lines (0.076 vs. 4.1 vs. ~14
performed at Crown Bioscience, and SW780 at Charles River
µM, respectively; P<0.03). In addition, the JMSU1 cell line
Laboratories. Vehicle, sunitinib, and lucitanib were formulated in 0.5%
was also found to be sensitive to lucitanib (GI50 = 0.149
Methocel, and BGJ398 in PEG300/D5W 2:1, and given PO once a
day.
µM) due FGFR1 dependency [3].
AACR April 18-22, 2015; Philadelphia, Pennsylvania
Oncology Inc., San Francisco, CA 94158; 2Oncology Research and Development Unit,
Institut de Recherches Servier, 78290 Croissy-sur-Seine, France.
Fig. 4 Lucitanib demonstrates superior antitumor
efficacy to BGJ398 and sunitinib in FGFR3 gene
fusion xenograft models
B N 2 2 8 9 (F G F R 3 -T A C C 3 )
CONCLUSIONS
• Lucitanib is a FGFR1-3, VEGFR1-3 and
PDGFR
α/β
inhibitor
in
clinical
development for breast and lung cancer
R T 1 1 2 /8 4 (F G F R 3 -T A C C 3 )
V e h ic le
L u c ita n ib 2 0 m g /k g Q D
B G J 3 9 8 1 5 m g /k g Q D
S u n itin ib 4 0 m g /k g Q D
1000
B G J 3 9 8 1 5 m g /k g Q D
3
T u m o r v o lu m e
(M e a n m m  S E M )
1000
3
V e h ic le
100
1000
5
10
15
20
25
10
40
R T 4 (F G F R 3 -T A C C 3 )
S W 7 8 0 ( F G F R 3 - B A IA P 2 L 1 )
V e h ic le
V e h ic le
1000
B G J 3 9 8 1 0 /2 0 m g /k g Q D
B G J 3 9 8 2 0 m g /k g Q D
3
• Lucitanib potently inhibited tumor growth
in xenograft models carrying FGFR3 gene
fusions.
3
30
D a y s p o s t - t u m o r im p l a n t
Fig.3 Lucitanib inhibits FGFR3 pathway activation in
FGFR3 fusion cell lines
20
100
• The antitumor efficacy of lucitanib was
superior to the angiogenesis inhibitor
sunitinib (VEGFR/PDGFR inhibitor) and
the selective FGFR inhibitor BGJ398 in all
xenograft models examined.
100
10
A
20
30
40
10
20
R T 1 1 2 F G F R 3 -T A C C 3
30
40
R T 1 1 2 /8 4 F G F R 3 - T A C C 3
1 .5
+ F G F 1 b a s e lin e
1 .5
1 .0
- F G F 1 b a s e li n e
0 .5
0 .0
1 .0
BN2289
0 .5
10 0
10 1
10 2
10 3
10 4
10
-1
10
0
10
c o n c (n M )
1
10
2
10
3
10
RT112/84
RT4
SW780
% TGI
P vs
BGJ398
% TGI
P vs
BGJ398
% TGI
P vs
BGJ398
% TGI
P vs
BGJ398
vehicle
-
-
-
-
-
-
-
-
lucitanib
931
0.385
921
0.0001
821
0.046
961
0.990
BGJ398
811
-
29
-
641
-
921
-
sunitinib
ND
ND
761
0.010
781
0.502
851
0.990
0 .0
1 0 -1
4
c o n c (n M )
w /o F G F 1
w/ FGF1
w /o F G F 1
R T 4 F G F R 3 -T A C C 3
w/ FGF1
S W 7 8 0 F G F R 3 - B A IA P 2 L 1
4
1 .5
• Lucitanib preferentially inhibited the
growth of cancer cell lines with FGFR and
PDGFR gene amplification, mutation
and/or fusion.
• In bladder carcinoma cell lines with
FGFR3
fusions,
lucitanib
inhibited
pFGFR3 with a mean IC50 of 0.070 µM
and cell proliferation with a GI50 of 0.076
µM.
100
0
lucitanib GI50 (M)
0.055
0.06
0.075
0.115
0.149
0.168
>5
>5
>5
>5
>5
>5
p F G F R 3 ( n g /m L )
1
cell line
FGFR status
RT112/84
FGFR3-TACC3
RT112
FGFR3-TACC3
SW780
FGFR3-BAIAP2L1
RT4
FGFR3-TACC3
JMSU1
FGFR1 dependent
639-V (3D) FGFR3 R248C
5637
WT
HT1197
WT
HT1376
WT
J82
FGFR3 K650E
T24
WT
UMUC3
WT
• These results support the
development of lucitanib in
harboring FGFR translocations.
clinical
tumors
3
p F G F R 3 ( n g /m L )
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• A Phase 1/2 clinical study [1] showed compelling activity of
lucitanib in solid tumors and, in particular, a 50% (6 of 12
patients) RECIST partial response rate in breast cancer
patients with FGF-aberrant (FGFR1 and/or FGF3/4/19
gene amplified) tumors. On-going clinical studies are
further defining the activity of lucitanib in breast
(NCT02053636; NCT02202746; ISRCTN23201971) and
lung cancer (NCT02109016). FGFR gene fusions /
translocations
(e.g.
FGFR3-TACC3)
resulting
in
constitutively active FGFR signaling and tumor proliferation
are observed in a wide variety of cancers [2] and inhibition
of FGFR signaling in FGFR translocated tumors with
lucitanib may be a potential new therapeutic strategy.
C
G I 5 0 s f o r b l a d d e r c a r c i n o m a c e l l l in e s
Figure 1: Lucitanib kinase inhibition
profile (DiscoveRx KINOMEscan)
2
Table 1: Lucitanib kinase binding profile
B
1
0.26
W
PDGFRβ
1
0.43
0 .0
G
PDGFRα
none
0 .1
C
7.1
PDGFR 
0 .2
4
VEGFR3
FGFR3
1
0 .3
/8
1.1
2
VEGFR2
1
1
2
p F G F R 3 ( n g /m L )
51
FGFR2
1Clovis
TUMOR XENOGRAFT EFFICACY STUDIES
• FGFR3 phosphorylation (pFGFR3) analysis was performed
using four bladder carcinoma cells harboring FGFR3
fusions with and without the addition of exogenous of FGF1 (Fig. 3A). Lucitanib was seen to inhibit pFGFR3 with
mean IC50 of 70 nM with FGF-1. Interestingly, RT4 and
SW780 showed constitutively active pFGFR3 which was
unchanged by FGF-1 addition; while RT112 and its
derivative RT112/84 increased phosphorylation levels with
FGF-1 ligand addition.
• Lucitanib inhibition of FGFR3 downstream signaling was
evaluated in FGFR3 fusion bladder carcinoma cells (Fig.
3B). Complete inhibition of pERK and a partial decrease of
pAKT were observed with increasing concentrations of
lucitanib, correlating with decrease in pFGFR3. Addition of
FGF-1 ligand also increased both pERK and pAKT levels.
The lucitanib mean IC50 for inhibiting pERK were 33 and 46
nM without and with FGF-1, respectively. Results for
RT112/84 were comparable to RT112 (data not shown).
p F G F R 3 ( n g /m L )
41
VEGFR1
FGFR1
3
1
FGFR3
Lucitanib : 395 non mutant kinases
11 interactions mapped at 100nM
S(35) = 0,028
T
FGFR2
4
S
21
5
L u c ita n ib G I 5 0 (  M )
FGFR1
L u c it a n ib G I 5 0 f o r c e ll lin e s
T
Kd (nM)
A
R
Kinase
Fig. 2 Lucitanib preferentially inhibits growth of cell
lines with FGFR- or PDGFR- genomic aberrations
2
1
0
1 .0
0 .5
0 .0
1 0 -1
10 0
10 1
10 2
10 3
10 4
1 0 -1
10 0
10 1
c o n c (n M )
w /o F G F 1
B
0
0.3
1
3
10
w/ FGF1
30
10 2
10 3
1
P< 0.0001 (treatment vs vehicle)
10 4
c o n c (n M )
w /o F G F 1
Without FGF-1
w/ FGF1
With FGF-1 50ng/mL
100
300 1000
0
0.3
1
3
10
30
100
300 1000 nM lucitanib
Table 2: Efficacy of lucitanib compared to BGJ398 and sunitinib in FGFR3 gene fusion
xenograft models
p-AKT
SW780
• Lucitanib is a tyrosine kinase inhibitor that selectively
inhibits fibroblast growth factor receptors 1-3 (FGFR1-3),
vascular endothelial growth factor receptors 1-3 (VEGFR13) and platelet derived growth factor receptors (PDGFR)
α/β. (Table 1; Fig.1).
IN VITRO FGFR3 SIGNALING ANALYSIS
t-AKT
p-ERK
t-ERK
p-AKT
RT112/84
CELLULAR VIABILITY ASSAYS
t-AKT
p-ERK
t-ERK
p-AKT
RT4
BACKGROUND
G I5 0 s (  M )
784
Nonclinical activity of the FGFR, VEGFR and PDGFR
inhibitor lucitanib in FGFR3 translocated tumor models
R
Abstract
Minh Nguyen1, Kevin K. Lin1, Mike F. Burbridge2,
Andrew D. Simmons1, Thomas C. Harding1
t-AKT
p-ERK
t-ERK
• Lucitanib anti-tumor activity was evaluated in four human
xenograft models with FGFR3 fusions—three bladder
carcinoma cell line models (RT112/84, RT4, and SW780)
and a glioblastoma PDX (BN2289; CrownBio) (Fig. 4; Table
2). Lucitanib dosed at 20 mg/kg QD demonstrated potent
growth inhibition in all four models tested (P<0.0001). The
selective FGFR inhibitor BGJ398 (15 or 20 mg/kg) was also
active in SW780, RT4 and BN2289 demonstrating FGFR
dependence of these tumors, but only slightly impacted
tumor growth in RT112/84 xenograft model. Lucitanib had a
greater antitumor activity in 4/4 models as compared to
BGJ398 and in 3/3 models as compared to sunitinib.
REFERENCES
1. Soria et al., (2014), Ann. Oncol. 25(11):2244-51.
2. Stransky et al., (2014), Nat. Comm. (5):5006.
3. Tomlinson et al., (2009), Cancer Res.
69(11):4613–20.
4. Wynes et al., (2014), Clin Cancer Res.
20(12):3299-309.
5. Dutt et al., (2011), PLoS One 6(6): e20351.
6. Tomlinson et al., (2007), Oncogene 26:58895899.
7. Konecny et al., (2013), Mol Cancer Ther.
12(5):632-42.
Electronic copies of posters for other
lucitanib studies can be accessed via
the link below
http://www.clovisoncology.com/product
s-companion-diagnostics/lucitanib/

BACKGROUND IN VITRO FGFR3 SIGNALING

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