discovery of tumor types highly susceptible to fasn ......pakt (s473) 100 60 42 66 75 78 70 70 70...
TRANSCRIPT
www.postersession.com
0
100
200
300
400
500
600
700
800
900
1000
0 2 4 6 8 10 12 14 16 18 20 22
Tum
or V
olum
e (m
m3 )
Days After Dosing Start
Vehicle
TVB-3166 (60 mg/kg); po/qd
Paclitaxel (10 mg/kg); iv/q4d
TVB-3166 (60 mg/kg); po/qd + Paclitaxel (10 mg/kg); iv/q4d
Tumor Volume For CTG-0165
0
200
400
600
10 12 14 16 18 20 22 24 26 28 30 32
Tum
or S
ize(
mm
3)
Days after Tumor Inoculation
Vehicle QD ! 20 + Q4d x 5 p.o. + i.v.
TVB-3166 30 mg/kg QD ! 20 p.o.
TVB-3166 60 mg/kg QD ! 20 p.o.
TVB-3166 100 mg/kg QD ! 20 p.o.
TVB-3166 + Paclitaxel 60 mg/kg + 10 mg/kg QD ! 20 + Q4d x 5 p.o. + i.v.
Paclitaxel 10 mg/kg Q4d x 5 i.v.
0
200
400
600
800
1000
1200
1400
1600
1800
2000
7 9 11 13 15 17 19 21 23 25 27
Tum
or V
olum
e (m
m3)
Days post Tumor Inoculation
Vehicle
TVB-3166 60mg/kg
TVB-3166 + Paclitaxel 60 mg/kg + 10mg/kg
Paclitaxel 10 mg/kg
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
6 8 10 12 14 16 18 20 22 24 26 28 Tu
mor
Vol
ume
(mm
3 )
Days After Tumor Inoculation
Vehicle
TVB-3166 30 mg/kg
TVB-3166 60mg/kg
Paclitaxel 10mg/kg
TVB-3166 + Paclitaxel 60mg/kg + 10mg/kg
0
200
400
600
800
1000
1200
1400
1600
1800
2000
12 14 16 18 20 22 24 26 28 30 32
Tum
or S
ize
(mm
3)
Days post Tumor Inoculation
!"#$%&"'((')*'+&,-'./01)'2'.3/04'5676'2'$686'
9!:(;)44'4*'<-,=-')*'+&,-'./01)'5676'
>?%&$@?A"&')*'<-,=-')*'+&,-'.3/04'$686'
/7%"@?A"&'BCD+E@'F'<-,=-'@7'4'<-,=-'GH7<'>I(/)3')*'+&,-'.J/0;2E$K-&"'C7E$K-'7K'>I(/1*'$686'
9!:(;)44'2'>?%&$@?A"&'4*'<-,=-'2')*'<-,=-')*'+&,-'./01)'2'.3/04'5676'2'$686'
9!:(;)44'2'/7%"@?A"&'4*'<-,=-'2'?CD+E@'F'<-,=-'@7'4'<-,=-'GH7<'>I(/)3')*'+&,-'./01)'2'L.J/0;2E$K-&"'C7E$K-'7K'>I(/1*M'5676'2'$686'
c-Myc
p!-cateninS675
!-catenin
pAKTS473
"-tubulin
LRP6
0 30 60 100 TVB 2640 (mg/kg) FASN
FASNLRP6
B-Cate
nin
pB-Cate
ninc-M
yc
pAKT (S47
3)0
25
50
75
100
125
!"#$%&'%$()*&+,-
./%01%&2
34%1556$7
86$#/%01%&234%1556$7&95&!"#$%&'%$()*&(6)*&!:8;<=>?&!%1/)#17)
60 mg/kg 100 mg/kg
100% = Vehicle
Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Marie O’Farrell, Douglas Buckley, and George Kemble
3-V Biosciences, Menlo Park, CA
Introduction
FASN Inhibition of Tumor Cell Viability is an On-Target Effect
• 3-V Biosciences’ lead, oral FASN inhibitor is in Phase I clinical trials for the treatment of solid tumors
• Fatty acid synthase (FASN) catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH
• Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells
• FASN expression increases with tumor progression in human tumors and associates with chemoresistance, metastasis, and diminished patient survival in many tumor types.
• Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival
• Studies to understand the mechanisms of action and biological consequences of FASN inhibition are guiding the discovery of tumors highly dependent on FASN and biomarkers for assessment of pharmacodynamic activity and patient selection
• Inhibition of the AKT and Wnt/β-catenin pathways, including TCF-promoter-regulated genes such as c-Myc, provide examples of mechanistic responses to FASN inhibition that identify biomarker candidates
Additive and Synergistic Inhibition of Xenograft Tumor Growth by FASN Inhibition in Combination with Taxanes
TVB-2640 Inhibits COLO-205 Rat Xenograft Tumor Growth in Association with β-Catenin, c-Myc and pAKT Inhibition
Figure 4. Combination treatment of patient-derived and cell-line-derived xenograft tumors with TVB-3166 and paclitaxel (or docetaxel) shows synergistic tumor growth inhibition and tumor regression. TGI was calculated as the percentage of tumor growth, relative to tumor size at the start of treatment in drug-treated groups compared to vehicle-treated groups. The efficacy studies were performed by Champions Oncology and Crown Biosciences.
Conclusions and Status
Mechanism of Taxane Synergy Insights: FASN Inhibition Affects β-tubulin Expression and Microtubule Organization
• TVB-2640 a first-in-class oral FASN inhibitor, is in Phase I clinical development for the treatment of solid tumors.
• TVB-2640 demonstrates dose-dependent single agent tumor growth inhibition in the COLO-205 rat xenograft tumor model.
• Tumor growth inhibition by TVB-2640 is associated with inhibition of β-catenin, c-Myc and AKT and modulation of tumor gene expression.
• FASN inhibition combined with paclitaxel or docetaxel shows synergistic tumor growth inhibition, including tumor regression in many different xenograft tumor models that include NSCLC, ovarian, and prostate tumors types.
• The mechanism of FASN-taxane synergy likely includes: (1) decreased expression of β-tubulin and disrupted microtubule organization as a result of FASN inhibition; (2) taxane-mediated sensitization of tumor cells to modulation of gene expression by FASN inhibition.
• In-vitro and in-vivo evaluation of additional, potent drug combinations is ongoing
Figure 6. CTG-0165 NSCLC PDX xenograft tumors (Champions Oncology) were harvested after 20 days of once-daily, oral treatment as indicated (6 hours post final dose). RNA isolation and data analysis was performed at 3-V Biosciences. RNA sequencing (RNASeq-25, Illumina, Inc.) was performed by Expression Analysis (Durham, NC). Differential gene expression data analysis was performed at 3-V Biosciences using Partek Genomics Suite software (St. Louis, MO).
Figure 1. Cell-based assays for palmitate synthesis and cell viability show alignment of IC50 values in CALU-6 tumor cells. MRC5 lung fibroblasts show inhibition of palmitate synthesis but minimal effect on viability. Palmitate assay measures incorporation of 13C into palmitate from 13C sodium acetate. Cell viability is measured using the Cell Titer Glo assay following treatment with TVB-3166 for 7 days in Advanced MEM media with 1% charcoal-stripped FBS. Absolute fatty acid levels were measure using Metabolon’s FAME analysis.
Results NSCLC Cell Line - Inhibition of palmitate synthesis and cell viability
Lung Fibroblast Cell Line - Inhibition of palmitate synthesis, not cell viability
-3 -2 -1 0 1 20
20
40
60
80
100
Log TVB-3166 Concentration (!M)
Palm
itate
Syn
thei
s or
Via
bilit
y (%
)
TVB-3166 CALU-6 Palmitate Synthesis
TVB-3166 CAU-6 Cell Viability
TVB-3166 CALU-6 Viability with 25 !M Exogenous Palmitate
CALU-6
TVB-3166 Concentration (µM)
% V
iab
ilit
y or
Palm
itat
e Sy
nth
esis
-3 -2 -1 0 1 20
20
40
60
80
100
Log TVB-3166 Concentration (!M)
Palm
itate
Syn
thei
s or
Via
bilit
y (%
)
TVB-3166 Cell ViabilityTVB-3166 Palmitate Syntheis
MRC5Cell Viability
Palmitate Synthesis
Cell Viability + Exogenous Palmitate
TVB-3166 Concentration (µM)
Fatty Acid Levels Align with Sensitivity to FASN Inhibition
!""
#!!""
$!!""
%!!""
&!!""
'!!!""
'#!!""
'$!!""
'#" '$" '%" '&" #!" ##" #$" #%" #&" (!"
")*+
,-"./01"2+
+(3"
4567"581-")*+,-"9:,;*<5=,:"
Fig.2. Antitumor Activity of TVB-0001 and TVB3166 as a Single Agent or in Combination with Irinotecan in the Treatment of Colo205 Colon cancer xenograft model in nude rats
!"#$%&"'(')*'+',-'./0/'
1!234-5('6('789:8')*'+',-'./0/'
1!234-5('-('789:8')*'+',-'./0/'
1!234-5(',(('789:8')*'+',-'./0/'
Mean Tumor Volume Individual Tumor Growth
TVB-2640 Induces Gene Expression Changes in COLO-205 Rat Xenograft Tumors In Association with Treatment Efficacy
Vehicl
e
30 m
g/kg
60 m
g/kg
100 m
g/kg0
2000
4000
6000
8000
RASA1
TVB-2640 Dose
Mea
n Tr
ansc
ript
Num
ber Vehicle
30 mg/kg60 mg/kg100 mg/kg
Vehicl
e
30 m
g/kg
60 m
g/kg
100 m
g/kg0
500
1000
1500
2000
ACSL1
TVB-2640 Dose
Mea
n Tr
ansc
ript
Num
ber Vehicle
30 mg/kg60 mg/kg100 mg/kg
Vehicl
e
30 m
g/kg
60 m
g/kg
100 m
g/kg0
500
1000
1500
2000
FABP5
TVB-2640 Dose
Mea
n Tr
ansc
ript
Num
ber Vehicle
30 mg/kg60 mg/kg100 mg/kg
Vehicl
e
30 m
g/kg
60 m
g/kg
100 m
g/kg0
50
100
150
BCL2L14
TVB-2640 Dose
Mea
n Tr
ansc
ript
Num
ber Vehicle
30 mg/kg60 mg/kg100 mg/kg
!-catenin
LRP6
pAKT
Vp 20 25 29 38 47 74 79 g4p Vp Animal #
% Vehicle
p!-cateninS675
"-tubulin
c-Myc
FASN
FASN 100 157 117 108 122 106 209 139 125 93LRP6 100 85 68 54 78 56 61 67 57 95
B-catenin 100 82 68 62 86 81 72 76 78 99pB-catenin 100 63 50 49 76 94 87 51 59 115
cMyc 100 21 20 19 48 56 38 32 37 126pAKT (S473) 100 60 42 66 75 78 70 70 70 113
!-tubulin 100 96 85 80 100 96 105 97 97 105% Tumor Growth 467 190 103 183 188 269 600 179 467
Tumor Size (mm3) 356 288 538 749 436 972 681
Figure 2. Tumor growth and pharmacodynamic analysis of of COLO-205 tumors. Female Rowett nude rats (NIH-Foxn1rnu ), 10-12 weeks of age, were inoculated subcutaneously at the right flank with COLO-205 tumor cells (2 x 107) in 0.2 mL of PBS with matrigel (1:1). Tumor growth inhibition (TGI) was calculated as the percentage of tumor growth, relative to tumor size at the start of treatment in drug-treated compared to vehicle-treated groups. The Mann-Whitney U test was used to assess statistical significance. In-life phase of the efficacy studies were performed by Crown Biosciences (Santa Clara, CA; Beijing, China). Western blot analysis of tumor lysates was performed at 3V Biosciences.
MRC-5
HUVECH19
75PC-3
HCT-116
CALU-6
Colo 205
22Rv1
10% FBS
22Rv1
1% FBS
0
20000
40000
60000
80000
100000
120000
Cell Line
Fatt
y A
cids
(nM
ole/
1B C
ells
)
FAME Data 12192014 Reorg - All Cell Lines
SFA DMSOSFA 3166
PUFA DMSOPUFA 3166
Palmitate DMSOPalmitate 3166
Low HighFASNi Sensitivity
DMSO TVB-3166 0.1 µM
Paclitaxel 1 nM TVB-3166 +Paclitaxel
!-tubulin mRNA Expression
DMSO
Paclita
xel 1
nM
TVB-3166
1 !M
TVB-3166
+ Pac
litaxe
l0
10000
20000
30000
40000
TUBB_LS Mean
Treatment
mR
NA
Qua
ntity
DMSOTVB-3166 1 !MPaclitaxel 1 nMTVB-3166 + Paclitaxel
** p<0.001
**
-4
1
+4
Apoptosis & Tubulin-Associated Lipid and Glucose Metabolism
VEGFC
CASP1
TUBB2ATUBB3
EIF4E3CASP7
CASP4TUBA3D
POTEF
POTEE
POTEM
PARP10
LIPASG
K1
TUBB8
APOL3
APOL2
EIF5A2
TUBA8SQ
LELPIN
1CERK
LPIN3
GAPD
H
VEGFA
NO
TCH3
VEGFB
EIF4BACACARPS6KA2M
AGL1
LPIN2
PDK3
PDK1
PGK1
HK2
Vehicle
TVB-3166
Paclitaxel
Combination
Combined FASN-Paclitaxel Inhibition Induces PDX mRNA Changes in Apoptosis, Metabolism, and Tubulin-Associated Genes
Vehicle TVB-3166
Paclitaxel TVB-3166+ Paclitaxel
7 days Treatment TVB-3166 + Paclitaxel vs Vehicle • p<0.01 • 1.5 fold change • 1143 genes • Counter selection for
paclitaxel-induced genes (n=151)
Figure 5. Immunofluorescence of β-tubulin in 22Rv1 cells shows decreased expression and disrupted cellular organization following treatment with TVB-3166 for 72 hours. Imaging was performed using a Zeiss LSM510 confocal microscope (100X objective). β-tubulin mRNA expression was measured by RNA sequencing (RNASeq-25, Illumina, Inc.) following 48 hours of TVB-3166 treatment. RNA sequencing was performed by Expression Analysis (Durham, NC).
Vehicle
TVB-2640 30 mg/kg
TVB-2640 60 mg/kg
TVB-2640 100 mg/kg
PCA
Mean Tumor Volume Individual Tumor Growth
Figure 3. COLO-205 rat xenograft tumors were harvested after 17 days of once-daily, oral treatment with TVB-2640 or vehicle (2 hours post final dose). RNA isolation and data analysis was performed at 3-V Biosciences. RNA sequencing (RNASeq-25, Illumina, Inc.) was performed by Expression Analysis (Durham, NC). Differential gene expression data analysis was performed at 3-V Biosciences using Partek Genomics Suite software (St. Louis, MO).
0 100 200 300 400 500 600 7000
25
50
75
100
125
!"#$%&'%$()*&+,-
./01.&$%&2
34!&562%7889$
:
./01.&;:<&234!&562%7889$:&=8&!"#$%&'%$()*&(9)*&!>?/@ABC&!%7;)#7:)
!"#$!%&'()*+,-./
100% = no growth with drug treatment
Vehicle Mean (n=10)
Group 4: 100 mg/kg TVB-2640, QDx17
0 100 200 300 400 500 600 7000
25
50
75
100
125
!"#$%&'%$()*&+,-
./0.&$%&1
23!&451%6778$
9
./0.&:9;&123!&451%6778$9&<7&!"#$%&'%$()*&(8)*&!=>?@ABC&!%6:)#69)
!"#$!%&'()*+,-./
Vehicl
e
TVB-2640
(30)
TVB-2640
(60)
TVB-2640
(100
)0
100
200
300
400
500
600
700
800
Treatment Group
Tum
or G
row
th (%
)
TGI (%): 46 7458!!"#$%$$&'(!!")#$%$$&*(!")$%$''+(
Vehicl
e
TVB-3166
(60)
Paclit
axel
10 m
g/kg Q
4D
Docetax
el 8m
g/kg Q
7D
TVB-3166
+ D
TVB-3166
+ P
0
500
1000
1500
Treatment Group
Tum
or G
row
th (%
)
TGI (%): 17 5248 81 76
Tumor Growth (%) Day 20
Vehic
le
TVB-316
6 (60
)
TVB-316
6+ P
ac (6
0+10
)
Paclit
axel
(10)
0
100
200
300
400
500500
1000
1500
2000
2500
Tum
or G
row
th (%
)
TGI (%): 6314 183
Vehicl
e
TVB-3166
(60)
TVB-316
6 + P
ac 60
+10
Paclit
axel
(10)
0
200
400
600
800
1000
Tum
or G
row
th (%
)
TGI (%): 0 60115
Vehicl
e
TVB-3166
(30)
TVB-3166
(60)
TVB-3166
(100
)
TVB-316
6 + P
ac 60
+10
Paclit
axel
(10)
0
100
200
300
400
500
Tum
or G
row
th (%
)
TGI (%): 83130745946
Vehicl
e
TVB-316
6 (30
)
TVB-316
6 (60
)
TVB-3166
+ Pac
(60+
10)
Paclita
xel (1
0)0
1000
2000
3000
4000
Tum
or G
row
th (%
)
TGI (%): 21 9715 57
CALU-6 NSCLC Cell Line
22Rv1 CRPC Tumor Cell Line
OVCAR-8 Ovarian Tumor Cell Line
A549 NSCLC Cell Line
CTG-0165 NSCLC PDX