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6 8 10 12 14 16 18 20 22 24 26
Tum
or V
olum
e (m
m3 )
Days After Tumor Inoculation
Vehicle
TVB-3166 30 mg/kg
TVB-3166 60mg/kg
Tumor Growth Tumor Volume
TGI (%): 14 10
• PIK3CA Q546R mutant • Adenocarcinoma
Vehicl
e
TVB-316
6 (30
)
TVB-316
6 (60
)0
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Tum
or G
row
th (%
)
Tumor Volume
TGI (%): 57
Tumor Growth
19 *
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Tum
or V
olum
e (m
m3 )
Days after Tumor Inoculation
TVB-3166 60 mg/kg p.o.
TVB-3166 100 mg/kg p.o.
Vehicle
*
Vehic
le
TVB-316
6 30 m
pk
TVB-316
6 100
mpk
-100
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Tum
or G
row
th (%
)
• KRAS G12D mutant • Adenocarcinoma
* p<0.05
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TVB-3166
(60)
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Cub
ic m
illim
eter
s
Day after Treatment Start
Vehicle
TVB-3166 (60 mg/kg); po/qd
TGI (%): 87
• KRAS, EGFR wild type • Stage IV, brain metastasis
Tumor Volume Tumor Growth
Vehicl
e
TVB-3166
(60)
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illim
eter
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Day
Vehicle
TVB-3166 (60 mg/kg); po/qd
Tumor Volume For CTG-0160
TGI (%): 50
Tumor Volume Tumor Growth
• KRAS, EGFR wild type • MET mutant • mTOR over-expressing • PTEN null • Squamous cell carcinoma
Conclusions and Status
Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models Timothy S. Heuer1, Richard Ventura1, Joanna Waszczuk1, Kasia Mordec1, Julie Lai1, Russell Johnson1, Lily Hu1, Haiying Cai1, Allan Wagman1, Steve Smith1, Douglas Buckley1,
Stanley T. Parish2, Elizabeth Bruckheimer2, and George Kemble1 3-V Biosciences1, Menlo Park, CA, and Champions Oncology2, Baltimore, MD
Introduction Results
Metabolic and Signaling Pathway Interaction
• 3-V Biosciences lead, oral FASN inhibitor has initiated Phase I clinical trails for the treatment of solid tumors
• Fatty acid synthase (FASN) catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH
• Palmitate and palmitate-derived lipids function in vital cellular processes such as energy metabolism, and cellular membrane biosynthesis
• Palmitate is conjugated directly to specific proteins as a mechanism to affect protein localization and activation
• FASN tumor expression has been found to be increased in a stage-dependent manner with high expression associated with diminished patient survival
• FASN activity promotes the tumorigenic capacity of cells by multiple mechanisms including enhanced macromolecular biosynthesis and glucose metabolism, cell growth and survival signal transduction, cellular stress response, and resistance to chemotherapeutic agents
• FASN inhibition can restore sensitivity to chemotherapeutics • In vitro and in vivo studies in preclinical tumor models demonstrate that FASN
inhibition reduces tumor cell proliferation and induces apoptosis • Preclinical studies have discovered biomarker candidates and provide insight into
FASN inhibition anti-tumor mechanisms of action
Lipid Regulation of Protein Function • Membrane-associated lipid rafts localize proteins for cell signaling
– Protein palmitoylation required for architecture and signal transduction • Lipid-associated protein activation fuels cancer cell growth and survival
– Ras, Raf, RTKs (e.g. EGFR, ErbB2), Akt, etc. • FASN inhibition is a mechanism to inhibit vital tumor-cell-activated pathways regulated
by lipid modification or lipid association
• Metabolic and signal transduction pathway cross-talk reprograms tumor cell metabolism
• Tumor cells require increased energy and macromolecular biosynthesis
• FASN inhibition blocks cellular lipid synthesis and signal transduction
Assay% TVB)3166%IC50%(µM)%
Hu%FASN%Biochem% 0.042%
%CALU)6%Cell%Palmitate% 0.081%
CALU)6%Cell%Viability% 0.061%
FASN Inhibition Disrupts Lipid Rafts and Palmitoylated Protein Localization
• Discontinuous, lower intensity lipid raft staining following FASN inhibition • N-Ras localization altered following FASN inhibition
Lipid rafts N-Ras Merge
DMSO 96 H
TVB-3166, 1 µM 96 H
Figure 1. CALU-6 in vitro immunoflouresent staining of lipid rafts (cholera toxin) and N-Ras. CALU-6 cells were treated with TVB-3166 for 96 hours in Advanced MEM media with 1% charcoal-filtered FBS.
K
ErbB3 ErbB2
Cytoplasm p85
Extracellular
NRas PI3K AKT
FASN Inhibition Blocks Signal Transduction and Induces Apoptosis
FASN Inhibition Blocks Anchorage-Independent Tumor Cell Growth
Figure 3. Soft agar colony formation assays show inhibition of anchorage-independent growth by FASN inhibition in lung, prostate, and colon tumor cell lines. Cells were treated with TVB-3166 for 21-28 days in IDEM plus 10% FBS.
Figure 2.FASN inhibition in MDA-MB-468 cells blocks β-catenin and AKT signaling. Annexin V flow cytometry shows induction of apoptosis. Cells were treated with TVB-3166 for 96 hours (Western) or 120 hours (Annexin V) in Advanced MEM media with 1% CF FBS.
MDA-MB-468
- 0 52 77
DM
SO TVB-3166 (µM)
2 0.2
0.02
β-Catenin pS675
β-Catenin
PARP- Cleaved
% Inhibition
% inhibition
% Induction
% Inhibition Akt pS473
- 0 52 77
- 0 49 60
- 39 124 187
FASN Inhibition Induces mRNA Expression Changes in Tumor Cell Growth, Survival, and Metabolism Pathways
• Lipid, sterol, glucose, metabolism signatures • Apoptosis, cell cycle, DNA replication signatures • Concerted modulation of pathway-associated gene expression
DNA Replication, Cell Cycle, Mitosis
Vehi
cle
1.0 µM
0.1 µM
IDH2
DHF
R
CDK2
MCM
6
CCNB
2
PCNA
CDC7
FGFR
3
AURK
A
SKP2
RRM
2 M
CM2
MCM
4 CC
NE2
PLK4
CDK1
CCNB
1
CCNA
1
BUB1
TGFB
2
RCF4
MCM
3
DHF
R
BUB1
ETS2
NFAT
C2
KIF2
C M
KI67
Apoptosis Lipid, Sterol, Glucose Metabolism,
ACLY
AACS
INSI
G1
LIPI
N1
ELO
VL6
ACLY
SCD
ACAC
A AC
SS2
ACSL
1
EIF4
EBP1
EIF5
CASP
4
ACSL
3 EP
B41
EIF1
IDH1
EIF5
ACSL
3
LIPI
N1
FASN
LDLR
CASP
1 AN
XA7
DNA
JA3
CASP
4 CA
SP1
BCL2
A1
BCL2
L13
TP53
BP2
PRKA
CB
PPP1
R15A
EGFR
PRKC
A
DUS
P6
DUS
P5
DUS
P1
SQLE
Figure 4 Affymetrix HU133 Plus 2.0 microarray analysis of gene expression changes induced by FASN inhibition. PANC-1 tumor cells were treated with TVB-3166 for 72 hours in Advanced MEM plus 1% CF FBS and L-glutamine. Unsupervised hierarchical clustering and pathway enrichment analysis of 911 genes with significant TVB-3166 treatment-dependent variance (FDR <0.0001).
TVB-3166 Oral PK and Inhibition of De Novo Lipogenesis
TVB-3166 Inhibits In Vivo Tumor Growth
Figure 5 TVB-3166 plasma exposure in Sprague Dawley rats following oral dosing at 30 mg/kg. Inhibition of de novo lipogenesis in Sprague Dawley rats, measured 8 hours following oral dosing at 10 or 30 mg/kg. Lipogenesis is measured as incorporation of deuterium from D2O into newly synthesized palmitate (Kinemed Inc., Emeryville, CA).
Figure 6 . TVB-3166 inhibits growth of patient-derived and cell-line-derived xenograft tumors. Tumor growth inhibition (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.
Gene Expression Pathway ANOVA Analysis Pathway Name Database Enrichment
p-valueSteroid biosynthesis kegg 5.55E-09
Cell cycle kegg 5.47E-07
Metabolic pathways kegg 0.0001
DNA replication kegg 0.0002
p53 signaling pathway kegg 0.0003
One carbon pool by folate kegg 0.0004
Terpenoid backbone biosynthesis kegg 0.0005
Insulin signaling pathway kegg 0.0009
Viral carcinogenesis kegg 0.0016
Aminoacyl-tRNA biosynthesis kegg 0.0023
HIF-1 signaling pathway kegg 0.0037
NF-kappa B signaling pathway kegg 0.0053
Pyruvate metabolism kegg 0.0085
Glycine, serine and threonine metabolism kegg 0.0138
Biosynthesis of unsaturated fatty acids kegg 0.0155
Folate biosynthesis kegg 0.0156
Glycolysis / Gluconeogenesis kegg 0.0166
Glutathione metabolism kegg 0.0169
2-Oxocarboxylic acid metabolism kegg 0.0200
Mismatch repair kegg 0.0228
Propanoate metabolism kegg 0.0261
Dorso-ventral axis formation kegg 0.0271
Selenocompound metabolism kegg 0.0311
Pyrimidine metabolism kegg 0.0336
Gap junction kegg 0.0506
Mineral absorption kegg 0.0632
Vitamin B6 metabolism kegg 0.0655
Fatty acid biosynthesis kegg 0.0655
Sterol Biosynthesis Up-Regulation Cell Cycle Down-Regulation
AKT
mTOR Raptor
PI3K
FASN
P
RTK
Palmitate
SREBP1C
Glucose
Pyruvate Citrate
Acetyl CoA
FASN Malonyl CoA NADPH
Glutamine
NADPH
KRAS
DMSO TVB-3166 (2µM)
6.9% Apoptosis 26.7% Apoptosis
CTG-0165 NSCLC PDX CTG-0160 NSCLC PDX
87% Tumor Growth Inhibition 2 of 3 tumors: 50% regression
50% Tumor Growth Inhibition
Vehicl
e
TVB-3166
(60)
0
200
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Cub
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eter
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Day
Vehicle
TVB-3166 (60 mg/kg); po/qd
TGI (%):
• KRAS G12S mutant • Stage IV • Adenocarcinoma
Tumor Volume Tumor Growth
47
-20
-10
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20
0 2 4 6 8 10 12 14 16 18
Bod
y W
eigh
t Cha
nge
(%)
Day
Vehicle
TVB-3166 (60 mg/kg); po/qd
TVB-3166 is well tolerated
CTG-0165 Body Weight Change CTG-0743 NSCLC PDX
47% Tumor Growth Inhibition
PANC-1 Tumor Cell Line
57% Tumor Growth Inhibitionp=.0431
22Rv1 Tumor Cell Line
14% Tumor Growth Inhibition
• Clinical development of TVB-2640, a first-in-class oral FASN inhibitor, is initiated • TVB-3166 demonstrates single agent activity in tumor cell line and patient-
derived xenograft tumor models • FASN inhibition effects on tumor cell biology include: (1) membrane and protein
localization disruption, (2) signal transduction inhibition, (3) concerted gene expression pathway modulation, (4) apoptosis induction, and (5) anchorage-independent tumor cell growth inhibition
• FASN inhibition-mediated pathway modulation informs selection of drug combinations and discovery of mechanisms of action and biomarker candidates
• Biomarker discovery for patient and expansion cohort selection is proceeding • In vitro and in vivo evaluation of multiple, potent drug combinations is ongoing
DMSO
TVB-3166 (µM)
0.1
1.0
CALU-6 LnCAP 22Rv1 HT-29
Inhibition of Deuterium-Labeled De Novo Lipogenesis
Vehicl
e
TVB-316
6 (10
)
TVB-316
6 (30
)0
2
4
6
Deu
teriu
m-la
bele
d Pa
lmita
te (%
)
3.31
1.49 1.01
10#
100#
1000#
10000#
0# 4# 8# 12# 16# 20# 24#
Plasma&Co
ncen
tra.
on&(n
g/mL)&
Time&(H)&
Mean&Rat&Plasma&Concentra.on&Following&Oral&Administra.on&of&TVBA3166&
TVB+3166#PO#(10#mg/kg)#
TVB+3166#PO#(30#mg/kg)#