24 July 2021
AperTO - Archivio Istituzionale Open Access dell'Università di Torino
Original Citation:
An 'in-cell trial' to assess the efficacy of a monovalent anti-MET antibody as monotherapy and in association with standard cytotoxics.
Published version:
DOI:10.1016/j.molonc.2013.12.006
Terms of use:
Open Access
(Article begins on next page)
Anyone can freely access the full text of works made available as "Open Access". Works made available under a Creative Commons license can be used according to the terms and conditions of said license. Use of all other works requires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law.
Availability:
This is the author's manuscript
This Accepted Author Manuscript (AAM) is copyrighted and published by Elsevier. It is posted
here by agreement between Elsevier and the University of Turin. Changes resulting from the
publishing process - such as editing, corrections, structural formatting, and other quality control
mechanisms - may not be reflected in this version of the text.
The definitive version of the text was subsequently published in
[Molecular Oncology, 8(2), 2014, 10.1016/j.molonc.2013.12.006]
You may download, copy and otherwise use the AAM for non-commercial purposes provided that
your license is limited by the following restrictions:
(1) You may use this AAM for non-commercial purposes only under the terms of the
CC-BY-NC-ND license.
(2) The integrity of the work and identification of the author, copyright owner, and publisher must
be preserved in any copy.
(3) You must attribute this AAM in the following format: Creative Commons BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/deed.en),
An ‘in-cell trial’ to assess the efficacy of a monovalent anti-MET antibody as monotherapy and in association
with standard cytotoxics.
Silvia Benvenuti
a, Alessandra Gentile
a, Luca Lazzaria, Addolorata Arnesano
a, Livio Trusolino
a,
b, Paolo M. Comoglioa,
b,
Corresponding author contact information, E-mail the corresponding author
aIRCC – Institute for Cancer Research at
Candiolo, Center for Experimental Clinical Molecular Oncology, 10060 Candiolo, Italy
bDepartment of Oncology,
University of Torino School of Medicine, 10060 Candiolo, Italy
Abstract
In clinical practice, targeted therapies are usually administered together with chemotherapeutics. However, little is known
whether conventional cytotoxic agents enhance the efficacy of targeted compounds, and whether a possible synergy
would be dictated by drug-sensitizing genetic alterations. To explore these issues, we leveraged the design of clinical
studies in humans to conduct a multi-arm trial in an ‘in-cell’ format. Using the MET oncogene as a model target and a
panel of genetically characterized cell lines as a reference population, we found that two different chemotherapeutic
regimens – cisplatin and 5-fluorouracil – exerted widespread cytotoxic activity that was not further enhanced by MET
inhibition with a monovalent anti-MET antibody. From a complementary perspective, targeted MET inhibition was
successful in a selected complement of cells harboring MET genomic lesions. In this latter setting, addition of
chemotherapy did not provide a therapeutic advantage. Mechanistically, chemotherapeutics did not influence the basal
activity of MET in cells with normal MET genomic status nor did they contribute to neutralize MET signals in cells with
MET amplification. These data suggest that tumors displaying MET aberrations achieve plateau responses by MET
monotherapy and do not receive further benefit by addition of cytotoxic treatments.
Keywords
MET tyrosine kinase; Monoclonal antibodies; Chemotherapy
1. Introduction
The advent of rationally targeted therapeutics has resulted in a new model of ‘cancer precision medicine’ whereby drug
treatment is matched to the presence or absence of defined molecular characteristics in the tumor of each patient
(Yauch and Settleman, 2012). This approach is fostered by accumulating genomic data – which steadily highlight new
potential druggable targets – and it is experimentally supported by preclinical platforms featuring extensive
multidimensional annotations, including vast collections of cancer cell lines and patient-derived xenografts (Barretina et
al., 2012, Bertotti et al., 2011, Garnett et al., 2012, Sharma et al., 2010 and Tentle et al., 2012). Notwithstanding the
impressive efficacy of some agents in genetically enriched subpopulations, and because of sensible cautionary issues
inherent in human experimentation, first-line targeted monotherapy in ‘target-positive’ patients remains daunting. In fact,
it is currently common practice to combine targeted therapies with a chemotherapy backbone, in order to guarantee a
‘one-fits-for-all’ basis likely to display some generic – but expected – activity. Inevitably, addition of chemotherapeutics
confounds the value of molecular lesions in predicting sensitivity or resistance to targeted drugs and precludes lucid
evaluation of whether response is attributable to selective blockade of the aberrant target, nonspecific cytotoxic activity
of the chemotherapeutic compound(s), or both. Accordingly, it remains to be established whether chemotherapy can
intensify the efficacy of targeted agents, or rational inactivation of the dominant oncoprotein in a genetically permissive
context suffices to reach a ‘point of no return’ beyond which chemotherapy becomes superfluous.
These open issues well apply to the case of the MET receptor tyrosine kinase. It is now established that, at least in
cellular and animal models, some cancer types rely on high-grade amplification of the MET oncogene – and the ensuing
hyper-activation of the encoded kinase – for their growth and survival, and this dependence results in drastic impairment
of cancer cell viability upon MET inactivation (‘oncogene addiction’: Bardelli et al., 2013, Bertotti et al., 2009, Comoglio et
al., 2008, McDermott et al., 2007 and Smolen et al., 2006). Such preclinical findings have been recently confirmed in
patients with MET-amplified esophago-gastric adenocarcinomas, who, albeit very rare (2%), have received clinical
benefit from therapy with MET small molecule inhibitors or antibodies ( Catenacci et al., 2011 and Lennerz et al., 2011).
MET mainly activates anti-apoptotic pathways. This suggests that tumors exhibiting constitutive MET signaling may be
intrinsically poorly sensitive to chemotherapeutics, due to the incessant operativity of MET-dependent survival signals,
and that MET inhibition may potentiate the effects of chemotherapy in MET-amplified tumors. However, this assumption
has not been challenged experimentally.
Cancers exhibiting MET amplification account for 2–4% of the overall population (COSMIC
database:http://www.sanger.ac.uk). In most solid tumors, MET displays normal gene copy number but its expression
(and activity) can be transcriptionally induced by cues present in the tumor reactive stroma – such as inflammatory
cytokines and pro-angiogenic factors – and by exogenous stress stimuli such hypoxia or ionizing radiations ( De Bacco
et al., 2011, Pennacchietti et al., 2003 and Trusolino et al., 2010). In this setting, MET upregulation provides pro-survival
and pro-invasive advantages that exacerbate the tumor malignant phenotype (‘oncogene expedience’: Comoglio et al.,
2008). At present, it is not known whether chemotherapeutics, similar to treatments such as radiotherapy ( De Bacco et
al., 2011), can induce MET overexpression. If this were the case, MET inhibition could sensitize cancer cells to
chemotherapy in a wide spectrum of tumors, even in contexts in which MET is not genetically amplified.Prompted by
these quandaries, we decided to conduct an ‘in-cell trial’ in which we comparatively assessed the effects of MET
inhibition alone, chemotherapy alone, and a combination of chemotherapy and MET-targeted therapy in a panel of
cancer cell lines featuring normal or amplified levels of MET.
2. Material and methods
2.1. Cell lines and reagents
A549 (lung adenocarcinoma); NCI-H1975, NCI-H23, NCI-H522, NCI-H460 and HCC-78 (non-small cell lung cancer);
H2452, H226 and H28 (mesothelioma); H441 and H820 (lung papillary adenocarcinoma);
NCI-H322M (bronchio-alveolar carcinoma); SW620, SW48 and DLD1 (colorectal adenocarcinoma); HT29 (rectosigmoid
colon adenocarcinoma); MDA-MB-231 and MDA-MB-468 (breast adenocarcinoma); OVCAR8, OVCAR3, A2780 and
SKOV3, (ovarian adenocarcinoma); U-118 MG, SNB-75 and SF-268 (glioblastoma); CaKi (kidney carcinoma); HS746T
(gastric adenocarcinoma) cell lines were purchased from ATCC (Rockville, MD); EBC-1 (non-small cell lung cancer)
were acquired from HSRRB cell bank (Osaka, Japan); GTL16 (gastric adenocarcinoma) are a laboratory batch obtained
from limiting dilutions of MKN45 (Giordano et al., 1988). All cells were kept in culture for less than 8 weeks and used
between passage 2 and 20. Cells were grown in recommended media (Sigma Aldrich, St. Louis, MO) supplemented with
50 units/mL penicillin (Sigma Aldrich, St. Louis, MO), 50 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO), 2 mM
glutamine (Sigma Aldrich, St. Louis, MO) and 2% or 10% Foetal Bovine Serum (Lonza Sales Ltd, Basel, Switzerland) as
indicated. Cells were maintained at 37 °C in 5% CO2 atmosphere. Cisplatin (Sigma Aldrich, St. Louis, MO),
5-fluorouracil (Calbiochem, Merck Millipore, Darmstadt) and PHA-665752 (Tocris Cookson Ltd, Bristol UK) were
administered in 2% serum media and used at the indicated concentrations. MV-DN30 was kindly provided by Dr.
Petronzelli (Sigma Tau, Pomezia).
2.2. Immunoblots
Cells were lysed in EB buffer (10 mM Tris–HCl pH 7.4; 1% TRITON X-100; 5 mM EDTA; 150 mM NaCl; 10% Glycerol;
50 mM NaF; 2 mM Na3VO4; 1 mM Phenylmethanesulfonyl fluoride and a cocktail of protease inhibitors) protein
concentration was determined using a BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL). Equal amount of
proteins were loaded on to SDS-PAGE gels and transferred to nitrocellulose membrane supports (Hybond C+;
Amersham, Buckinghamshire, UK). The membranes were decorated with the following antibodies: mouse monoclonal
anti MET DL-21 (Prat et al., 1991); goat polyclonal anti actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit
polyclonal anti p44/42 MAPK (ERK1/2) (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti phospho
p44/42 MAPK (ERK1/2) (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti AKT (Cell Signaling
Technology, Danvers, MA, USA); rabbit polyclonal anti phospho AKT (Ser473) (Cell Signaling Technology, Danvers, MA,
USA); rabbit monoclonal anti phospho MET (Tyr1234/Y1235) (Cell Signaling Technology, Danvers, MA, USA); mouse
monoclonal anti vinculin (Sigma, St Louis, MO, USA). Anti mouse, anti rabbit, anti goat and protA peroxidase conjugated
secondary antibodies were from Amersham (Arlington Heights, VA). Signal detection was performed using ECL Western
Blotting Substrate (Promega Corporation, Madison, Wisconsin, USA).
2.3. Cell viability assay
Cell viability assays were performed in 96 well plates and low serum conditions (2%). Proliferation rates were assessed
using Cell Titre Glo Luminescence (Promega Corporation, Madison, Wisconsin, USA). All the experiments were
performed at least two times in triplicates or quadruplicates as indicated (Benvenuti et al., 2011).
2.4. RNA extraction and quantitative real time PCR (Q PCR)
Total RNA was isolated using RNeasy Mini Kit (Quiagen, Gmbh, Hilden, Germany) according to manufacturer's
instructions. One microgram of RNA was retro-transcribed into cDNA using the High Capacity cDNA Reverse
Transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Q PCR was performed
using Taq Man Gene Expression Assays for HGF, MET, UBC and GUSB (Applied Biosystems, Foster City, CA) with
TaqMan PCR master mix (Applied Biosystems, Foster City, CA) and ABI PRISM 7900HT sequence detection system
(Applied Biosystems, Foster City, CA). All samples were analyzed in triplicates.
2.5. Apoptosis assay
Treated cells were detached and incubated with FITC-conjugated Annexin V (BD Bioscience). DAPI (Roche Applied
Science) was used to identify dead cells. Samples were analyzed on CYAN flow cytometer (CyAn ADP, Beckman
Coulter Life Sciences, Brea, CA, USA) equipped with 488 nm, 405 nm and 642 nm solid state lasers. Data were
collected and processed using Summit 4.3 software (Beckman Coulter Life Sciences).
2.6. In vivo cell transplantation and therapy
All animal procedures were performed according to protocols approved by the Italian Ministry of Health and by the
internal Ethical Committee for Animal Experimentation. EBC-1 were injected subcutaneously (1.5 × 106 cells/mouse)
into the posterior flanks of 6-week-old immunodeficient nu−/− female mice (Charles River Laboratories, Lecco, Italy).
When tumors reached the size of approximately 50 mm3 mice were randomized and assigned to four arms (n = 6) and
treated three times per week for 2 months as follow: placebo, MV-DN30 (20 μg/mice), CDDP (24 μg/mice) or MV-DN30
plus CDDP (20 μg/mice and 24 μg/mice respectively). Tumors were measured with a calliper and tumors' volume was
calculated at the indicated days using the formula: 4/3π × (d/2)2 × (D/2), where d and D are the minor and the major
tumor axis respectively. The general physical status of mice was monitored constantly. At the end of the experiments
mice were euthanized and tumors extracted.
2.7. Statistical analysis
Experiments were performed in triplicates or quadruplicates as indicated. Standard deviation (SD) or standard error
mean (SEM) of each result was reported. All statistical analyses were performed using Excel software (Microsoft Office
2010).
3. Results
3.1. ‘In-cell trial’ design
Typically, preclinical analysis of anticancer compounds involves testing them on a small number of cancer cell lines that
may be inadequate to capture the genetic heterogeneity of tumors on a population scale. Moreover, to the best of our
knowledge, systematic comparative assessment of the activity of targeted drugs with or without standard
chemotherapeutics has not been attempted so far. We therefore decided to gauge the sensitivity of a panel of 28 cancer
cell lines, representing diverse tumor types (Supplementary Table 1) and expressing different levels of MET protein
(Supplement
DN30 monoc
receptor she
DNA cross-l
Palumbo et
administered
artificial syste
(our unpublis
Figures 1 an
growth after 4
treatment arm
featuring at le
Figure 1.
Effect of anti
growth in a p
or placebo as
(a proxy for
tary Figure 1),
clonal antibod
dding and do
inking agent
al., 2013); a
d at a fixed 12
em of BaF3 c
shed observat
nd 2 depict a s
48 h of compo
m, responses
east 20% redu
MET MV-DN
panel of 28 hu
s adherent ce
cell numbers)
to four treatm
dy (MV-DN30
wn-regulation
( Loehrer an
and (iv) com
25 nM concen
cells exhibiting
ions). CDDP a
series of wate
ound administ
are independ
uction of viabi
N30 treatment
man cancer-d
lls. Cell growt
). Growth rate
ment modalitie
0), an enginee
(Pacchiana e
nd Einhorn, 1
mbination ther
ntration, a dos
g interleukin-3
and 5FU were
erfall plots sho
tration, as mea
ently ranked b
lity upon treat
on cancer ce
derived cell lin
h was measur
es are expres
es: (i) individua
ered antibody
et al., 2010);
1984); (iii) 5-f
rapy with MV
sage that was
3 independenc
e used at three
owing the effe
asured by AT
by cell numbe
tment were sc
ell growth. (A)
es. Each cell
red 48 h after
ssed as perce
al blockade of
y that binds t
(ii) cisplatin (c
fluorouracil (5
V-DN30 and
s found to ach
ce as a conse
e increasing c
ect of the vario
P cellular con
er compared w
ored as ‘respo
Waterfall plot
line was grow
treatment sta
entage over p
the MET onco
o MET with h
cis-diammined
5FU) a thymid
either CDDP
hieve 50% gro
equence of M
oncentrations
ous therapeut
tent (a proxy f
with values of
onders’.
t showing the
wn in the prese
art and measu
lacebo-treated
oprotein by th
high affinity a
dichloroplatinu
dylate syntha
P or 5FU. M
owth inhibition
MET ectopic ov
s (0.1, 1.0 and
tic regimens o
for cell numbe
placebo-treate
e effect of
MV-ence of MV-D
red by ATP ce
d cells. (B) D
e monovalent
and promotes
um, CDDP), a
ase inhibitor (
MV-DN30 was
n (IC50) in an
verexpression
10 μM).
on cancer cell
ers). For each
ed cells. Cells
-DN30 on cell
N30 (125 nM)
ellular content
ose-response
t
s
a
(
s
n
n
l
h
s
l
)
t
e
curves perfor
HS746T; and
receptor with
independent
Figure 2.
Effect of CDD
effect of CDD
increasing co
after treatme
treated cells.
addicted can
respectively)
of 5FU in co
rmed using inc
d on control c
hout it being a
experiments p
DP and 5FU t
DP (A) and 5F
oncentrations
ent start and m
(C) Waterfall
ncer cells. Cel
. Growth rates
mbination wit
creasing conc
cells: A549 (e
amplificated –
performed in t
treatment on g
FU (B) on canc
(0.1–1.0–10
measured by
plot showing
ls were incuba
s are expresse
h MV-DN30 o
centrations of
expressing ph
not addicted)
triplicates (A)
growth of a pa
cer cell growth
μM) of either
ATP cellular c
the effect of C
ated with: plac
ed as percent
on growth of n
MV-DN30 (1.9
hysiological le
) and A2780 (
and quadrupli
anel of human
h. 28 human c
CDDP or 5F
content. Grow
CDDP in com
cebo; CDDP
tage over plac
not addicted c
9–500 nM) on
evels of MET)
(not expressin
icated (B). Err
n cancer-deriv
cancer-derived
U as adheren
wth rates are
bination with a
(10 μM) or MV
cebo-treated c
cancer cells. C
n MET addicte
), CaKi (expre
ng MET). Data
ror bars repres
ed cell lines. W
d cell lines wer
nt cells. Cell g
expressed as
anti MET antib
V-DN30 plus C
ells. (D) Wate
Cells were inc
ed cells:
EBC-essing constit
a are means ±
sent standard
Waterfall plots
re grown in th
growth was a
s percentage o
body on grow
CDDP (125 n
erfall plot show
cubated with:
1, GTL16 and
tutively active
± SD of three
deviations.
s showing the
e presence of
ssessed 48 h
over
placebo-th of not MET
nM and 10 μM
wing the effect
placebo; 5FU
d
e
e
e
f
h
-T
M
t
U
(10 μM) or M
placebo-treat
3.2. Effects o
Consistent w
of the cell li
GTL16, HS74
gene amplific
inhibition ( Be
Responsiven
concentration
MET gene co
and A2780 (
manner and
without MET
3.3. Effects o
Different from
or 5FU was
cellular toxic
cells proved t
1 and Figure
small molecu
constitutive s
have therape
For combinat
(10 μM). In a
sensitize cell
substantially
slight additive
PHA-665752
MV-DN30 plu
ted cells. Data
of anti-MET mo
with the notion
nes tested w
46T and, to a
cation, further
ertotti et al., 2
ness to MV-D
ns (1.9–500 n
opy number: A
(not expressi
to various deg
amplification
of standard ch
m targeted ME
more evenly
ity increasing
to be particula
e 2A, B). EBC
ule inhibitor (M
survival pathw
eutic relevance
torial treatmen
all lines, com
s without MET
improve resp
e activity in G
2 as an alterna
s 5FU (125 n
a are means ±
onotherapy
that targeted
were largely re
higher extent
r attesting to in
009, McDerm
DN30 was m
nM) using MET
A549 (express
ng MET). In
grees, with EB
were complet
emotherapy a
ET blockade, a
distributed an
as a function
arly sensitive t
-1 cells also s
McDermott et a
ways that may
e in patients w
nts, we electe
bination thera
T amplification
ponse to MV-D
GTL16 ( Figur
ative way to ha
nM and 10 μ
± SD of three i
therapies are
efractory to M
t, EBC-1 (Figu
ncreased MET
ott et al., 2007
more precisely
T-amplified ce
sing normal lev
all MET-amp
BC-1 being the
tely resistant t
alone and in co
and in line wit
nd on average
n of dosage (
to MET inhibit
scored as ‘bes
al., 2007). It is
y limit respon
with tumors ex
d to use MV-D
apy was not s
n to chemothe
DN30 in
MET-e 3A and B).
alt MET activit
M respectivel
independent e
e efficacious o
MV-DN30 mon
ure 1A). Notab
T gene copy n
7 and Smolen
y evaluated
ell lines
(EBC-vel of MET), C
plified cell line
e most sensit
to MV-DN30, e
ombination wi
th induction of
e more prono
Figure 2A an
tion, but were
st responders
s tempting to s
nsiveness to s
xhibiting intens
DN30 togethe
superior to be
erapy ( Figure
-amplified cel
This latter re
ty ( Figure 3C
ly). Growth ra
experiments p
only in genetic
notherapy, wi
bly, these thre
number as a
et al., 2006).
in dose-respo
-1, GTL16 and
CaKi (overexp
es, MV-DN30
ive to MET ina
even at high a
th MET inhibit
f pervasive cy
ounced than th
d B). We note
substantially
s’ in a large-sc
speculate that
standard cyto
se MET amplif
r with the high
est agent mon
2C and D); si
l lines, with no
sult was conf
and D).
ates are expre
performed in tr
cally defined t
th relative se
ee responders
molecular pre
onse assays
d HS746T) as
ressing MET,
reduced viab
activation ( Fig
antibody conce
tion
totoxic effects
hat observed
e, anecdotally
refractory to e
cale monother
t ‘MET addicti
otoxic agents,
fication.
her concentrat
notherapy: ad
milarly, additio
o effect in HS
firmed using th
ressed as per
riplicates.
tumor subsets
ensitivity displ
s all harbor hig
edictor of sens
with increas
s well cell line
without gene
bility in a do
gure 1B). At v
entrations ( Fi
s, response to
for MV-DN30
y, that EBC-1
either CDDP o
rapy screenin
ion’ in EBC-1
an assumpti
tion of either C
dition of
MV-on of chemoth
S746T and EB
he small mole
rcentage over
s, the majority
ayed only by
gh-grade MET
sitivity to MET
sing antibody
es with normal
amplification)
se-dependent
variance, cells
gure 1B).
o either CDDP
0, with overall
and HS746T
or 5FU (Figure
g with a MET
cells sustains
on that could
CDDP or 5FU
DN30 did not
herapy did not
BC-1 and only
ecule inhibitor
r
y
y
T
T
y
l
)
t
s
P
l
T
e
T
s
d
U
t
t
y
r
Figure 3.
Effect of CDD
5FU (10 μM)
(10 μM) as s
Cell prolifera
means ± SD
0.05 by Stud
The evidence
contrast with
MET is inhib
induces tran
signals that
radiotherapy
chemotherap
produce sign
reduced MET
usually acco
likely due to
demonstrated
Figure 4.
DP and 5FU t
as single age
single agents o
ation rates we
D of three inde
ent's t test; n.s
e that MV-DN
h the observat
bited, indepen
scriptional up
prevent cell d
is enhanced
py, time-cours
nificant chang
T protein leve
mpanied by M
o the more p
d, MET expres
treatment on g
ents or in com
or in combinat
ere determine
ependent expe
s.: statistically
N30 and chem
tion that other
ndent of MET
pregulation an
death induced
d. In cohere
se experiment
ges in MET e
els, as observ
MET protein d
pronounced cy
ssion was upr
growth of MET
bination (pane
tion (panels C
ed 48 h after
eriments perfo
y not significan
motherapy did
r cytotoxic ins
T genomic sta
nd catalytic a
d by irradiatio
ence with the
ts on A549 a
xpression, bo
ed for NCI-H4
downregulatio
ytostatic activ
regulated by io
T addicted ce
els A and B) a
C and D) on g
treatment sta
ormed in triplic
nt.
not cooperate
sults, such as
atus ( De Ba
activation of M
n, and MET i
e finding tha
and NCI-H441
oth at the mR
441 treated w
on ( Boccaccio
vity of CDDP
onizing radiati
lls. Effects of
and of PHA-66
rowth of HS74
art and meas
cates. Error ba
e in cells expr
ionizing radia
acco et al., 20
MET; increase
inhibition curt
at MET inhib
revealed tha
RNA and prot
with CDDP ( F
o et al., 1994
compared w
on ( Figure 4B
MV-DN30 (12
65752 (100 nM
46T, GTL16 a
ured by ATP
ars represent
ressing physio
ation, are in fa
011). Mechan
ed MET activ
ails such sign
ition did not
at treatment w
ein levels ( F
Figure 4A and
4), this decrea
with 5FU (see
B).
25 nM), CDDP
M), CDDP (10
and EBC-1 (M
cellular conte
standard dev
ological levels
fact more detr
nistically, ioniz
vity conveys
nals so that th
t exacerbate
with CDDP or
Figure 4A and
d B). Since gro
ased expressi
e Figure 2). A
P (10 μM) and
μM) and 5FU
MET addicted).
ent. Data are
viations. *: P <
s of MET is in
rimental when
zing radiation
anti-apoptotic
he efficacy of
response to
r 5FU did not
d B), or even
owth arrest is
on of MET is
As previously
d
U
.
e
<
n
n
n
c
f
o
t
n
s
s
y
Effect of CD
A549 and NC
1, 6 or 24 h
keeping gene
independent
A549 and NC
8, 24 or 48 h
protein level
loading. – re
transcriptiona
Lack of syne
signaling lev
baseline pho
not further in
tumors elicits
cytotoxics. B
was only mo
apoptotic re
administratio
readouts).
DP and 5FU
CI-H441 cells
after treatme
es. Graphs sh
experiments
CI-H441 cells
h after treatm
was checked
epresent place
al upregulation
ergy between
vel: treatment
osphorylation o
nfluenced by
s maximal dow
Biologically, th
oderately incre
esponse of ‘M
n of chemoth
treatment on
treated with C
ent beginning
how the fold c
performed in
treated with C
ment beginning
d probing the m
ebo treated ce
n.
MET inhibitio
with MV-DN3
of distal trans
CDDP or 5F
wn-modulation
e rate of apo
eased by co-tr
MET addicted
herapeutics; t
MET transcri
CDDP (10 μM)
and examined
change of trea
triplicates. (B
CDDP (10 μM
g and proteins
membranes w
ells. Irradiated
on and standa
30 effectively
sducers such
U ( Figure 5A
n of downstrea
optosis induce
reatment with
d’ EBC-1 wa
this is similar
iption. (A) Tim
) or 5FU (10 μ
d by Q PCR.
ated cells ver
B) Time cour
) or 5FU (10 μ
s were separa
with anti MET
d cells (expos
ard chemothe
suppressed c
as Akt and M
A). This sugg
am pathways
ed by anti-ME
CDDP or 5FU
as stronger
r to that obse
me course exp
μM) in low ser
Results were
rsus not treate
rse experimen
μM) in low ser
ated on SDS
antibody. Act
ed to 10 Gy f
erapy in MET
constitutive M
MAPK. Howev
gests that ind
that cannot b
ET monothera
U ( Figure 5B
following tre
erved in the
periment asse
rum conditions
e normalized
ed cells. Data
nt assessing M
rum conditions
PAGE gels (8
tin was used a
for 12 h) were
-amplified cel
MET autophos
er, this overa
ividual MET b
be additively a
py, as assess
). (We note a
atment with
initial screen
essing MET m
s (2%). RNA w
on UBC and
a are means ±
MET protein
s (2%). Cells
8%) as total e
as control for
e used as co
lls was also e
sphorylation a
all signal neut
blockade in M
aggravated by
sed by annex
again, ‘en pass
MV-DN30 th
n using viabili
mRNA level in
was extracted
GUSB house
± SD of three
expression in
were lysed 1,
extracts. MET
equal protein
ntrols of MET
evident at the
nd abrogated
ralization was
MET-amplified
y conventional
xin-V staining,
sant’, that the
han following
ty assays as
n
d
e
e
n
,
T
n
T
e
d
s
d
l
,
e
g
s
Figure 5.
Analysis of M
chemotherap
or 5FU (10 μ
phospho ME
membranes
reprobing the
DN30 (125 n
cytometry an
experiments.
Finally, we t
EBC-1 cells
and (iv) MV-D
DN30 potent
alone was as
MET-amplifie
were obtaine
Figure 6.
Effect of MV
injected subc
When tumors
and treated a
24 μg/mice r
indicated day
report standa
4. Discussio
The first hint
tumors bearin
randomized
MET signaling
py. (A) ECB-1
μM) for 48 h.
ET and then
with anti phos
e same memb
nM) alone or i
nalysis of Ann
. Error bars re
ransferred the
and, after 15
DN30 + CDDP
tly inhibited tu
s effective as
ed cells was m
ed using 5FU a
V-DN30 and C
cutaneously on
s volume was
as follow: (i) pl
respectively).
ys using a ca
ard errors.
on
that targeted
ng drug-sensi
phase 3 trial c
g and apopto
cells were tre
Cellular extra
reprobed wi
spho MAPK a
branes. Vincul
in combinatio
exin V versus
present stand
e ‘in-cell trial’
days, random
P. As expecte
umor growth. A
the combinati
maximal and n
as chemothera
CDDP as sing
n the right flan
s approximate
lacebo; (ii) MV
Mice were tre
alliper. Values
therapy in so
tizing mutatio
comparing the
sis rates in r
eated with MV
acts were run
th anti MET
and anti phosp
lin was used a
n with either
s DAPI followi
dard deviations
to an in vivo
mized into four
d, and consist
Again in acco
on with CDDP
not further incr
apeutic drug (
le agents and
nks of immuno
ly 50 mm3 (15
V-DN30 (20 μg
eated three tim
represent the
olid cancers ca
ns – emerged
e EGFR smal
response to M
V-DN30 (125 n
n on SDS PA
antibodies.
pho AKT antib
as control for
CDDP (10 μM
ing 48 h of tre
s. *: P < 0.05
o xenograft se
r treatment arm
tent with the i
ordance with t
P, reinforcing
reased by con
( Supplementa
d in combinati
ocompromised
5 days post-in
g/mice); (iii) C
mes per week
e mean tumo
an be more e
d from studies
ll molecule inh
MET inhibition
nM) alone or in
GE gels and
MET signalin
bodies. Total
equal loading
M) or 5FU (10
eatment. Data
by Student's t
etting. Immun
ms: (i) placeb
n vitro data, C
the in vitro ex
the notion tha
ncomitant che
ary Figure 2).
ion on growth
d mice to indu
njection) mice
CDDP (24 μg/m
k for two mont
or volume of e
ffective than c
in advanced n
hibitor gefitinib
alone or in
n combination
analyzed by
ng cascade w
MAPK and AK
g. (B) ECB-1 c
0 μM) and apo
a are means ±
test.
nocompromise
o; (ii) MV-DN3
CDDP was poo
xperiments, m
at the inhibitory
motherapy ( F
h of tumor xen
ce the formati
were random
mice) and (iv)
ths and tumor
each experime
chemotherapy
non-small cell
b versus a ca
combination w
n with either C
western blott
was analysed
KT levels wer
cells were trea
optosis determ
± SD of three
ed mice were
30 alone; (iii)
orly effective,
monotherapy w
y effect of ME
Figure 6). The
nografts. EBC
ion of experim
mized into four
MV-DN30 + C
rs volume me
ental group a
y – but only in
lung cancer (
arboplatin-pacl
with standard
CDDP (10 μM)
ing using anti
d probing the
re determined
ated with
MV-mined by flow
e independent
injected with
CDDP alone;
whereas
MV-with MV-DN30
ET targeting in
e same results
C-1 cells were
mental tumors.
r arms (n = 6)
CDDP (20 and
easured at the
and error bars
n patients with
(NSCLC). In a
litaxel doublet
d
)
i
e
d
-w
t
h
;
-0
n
s
e
.
)
d
e
s
h
a
t
as first line treatment in advanced NSCLC, pre-planned sub-group analysis indicated that progression-free survival was
significantly longer for gefitinib than chemotherapy in patients with EGFR mutant tumors, and significantly longer for
chemotherapy than gefitinib in patients with EGFR wild-type tumors ( Mok et al., 2009). This and other findings
contributed to FDA approval of EGFR inhibitor monotherapy for the initial treatment of patients with EGFR
mutation-positive metastatic NSCLC. In this vein, again in advanced NSCLC, single-agent inhibition of ALK by crizotinib has been
recently shown to be superior to standard chemotherapy in ALK-translocated cases ( Shaw et al., 2013).
The notion that targeted therapy can outperform standard cytotoxics in genetically defined patient subpopulations does
not imply that addition of chemotherapy is unproductive. At least in principle, one could argue that the effects of selective
target inhibition can be potentiated by less specific but potentially more aggressive pro-apoptotic insults, such as those
triggered by chemotherapeutics. Although empirical, this line of thinking has informed clinical practice, and combination
therapies with targeted drugs and conventional agents are now commonplace despite the risk of cumulative toxicity.
Examples of FDA-approved combinations include the anti-HER2 monoclonal antibody trastuzumab and the mitotic
inhibitor paclitaxel in the first-line treatment of HER2-amplified metastatic breast cancer ( Slamon et al., 2001);
trastuzumab and a cisplatin-5FU doublet in HER2-positive advanced gastric carcinoma ( Bang et al., 2010), and the
anti-EGFR antibody cetuximab and cisplatin-5FU in head and neck cancer ( Vermorken et al., 2008). Although most of such
combinations have received preclinical experimental validation in xenograft mouse models, their real impact in terms of
therapeutic synergy awaits confirmation.
MET inhibitors are currently being tested in several investigational trials, but none of them has been certified for clinical
use and conclusions about their efficacy remain immature (Gherardi et al., 2012). Schematically, studies with MET
inhibitors are structured according to two major designs. On one side, recruitment is limited to ‘target-positive’ cases –
for instance, patients with MET-amplified esophago-gastric adenocarcinoma or MET-mutant papillary renal carcinoma –
and treatment involves targeted monotherapy, usually in patients who have failed prior cycles of conventional
chemotherapy. On the other side, recruitment does not occur on a molecular basis and usually MET inhibitors are
administered together with standard cytotoxics; trials of this kind are ongoing in NSCLC, colorectal tumors, and
triple-negative breast cancer.
Based on these premises we sought to dissect the relative contribution of anti-MET targeted therapy versus standard
chemotherapy to tumor growth inhibition and systematically coupled response annotation with the assessment of MET
genomic status. To do this we embraced a reductionist approach in cancer cell lines, while trying to accommodate the
informative merits of clinical trials. Hence, we selected a reference population – a panel of cancer cell lines of different
tumor origin and with different genomic makeups, including some with MET gene amplification – and designed a
multi-arm study in which each cancer cell line was comparatively challenged with anti-MET monotherapy, single-agent
chemotherapy, and combination therapy.
A first series of experiments demonstrated that chemotherapy was overall superior to specific MET blockade in cell lines
with normal activity (and genetically wild-type forms) of MET. This is not surprising, as lack of genetic lesions responsible
for target hyperactivity usually predicts lack of efficacy of targeted intervention. The unexpected finding is that
neutralization of MET, which commonly exerts cytoprotective activities in response to exogenous stress stimuli, did not
drastically potentiate the effects of chemotherapy. The reason for this inadequacy is intrinsic to the mode of action of
chemotherapeutic agents (at least those tested in this study). Both CDDP and 5FU did not induce transcriptional
upregulation of MET, which is one stereotyped mechanism whereby cancer cells adapt to adverse events, including
other cytotoxic therapeutic modalities such as ionizing radiation (De Bacco et al., 2011). The clinical relevance of this
observation is straightforward: patients with tumors that are routinely treated with radiotherapy are likely to benefit from
MET inhibition, irrespective of MET genetic status. At variance, MET inhibition is probably futile in patients with MET
wild-type tumors that receive standard chemotherapy. It cannot be excluded that, in the in vivo situation,
chemotherapeutics could activate MET signaling in spite of their inability to boost MET expression. Indeed, the
inflammatory phenotype that is commonly produced by standard chemotherapeutics might lead to increased local
bioavailability of the MET ligand HGF in the tumor reactive stroma, with consequent paracrine activation of MET and
emission of MET-driven pro-survival signals in cancer cells ( Bhowmick et al., 2004). Our ‘in-cell’ approach indirectly
tackled this issue by performing viability assays in the presence of serum, which contains HGF, recapitulates in part the
trophic composition of the tumor microenvironment, and sustains tonic activation of MET signaling. Our repeated
observation that MET inhibition does not dramatically influence the outcome of chemotherapy, even in a context of basal
MET activity, suggests that interception of MET activity is unlike to reinforce the effects of conventional cytotoxics also in
the clinical setting.
Another point of interest of this investigation is that impairment of tumor growth in MET-amplified cells appeared to be
saturated by MET inhibition, with no additional activity of chemotherapy. Again, this is somehow unexpected because
nullification of MET-regulated survival signals is supposed to exacerbate cell death in the presence of pro-apoptotic
stimuli. It is likely that blockade of the dominant oncoprotein in a context of oncogene addiction is so catastrophic that
any other insult becomes superfluous. Accordingly, MET obstruction resulted in massive abrogation of MET-dependent
downstream signals, with no further impact by addition of cytotoxic agents. When translated into the clinic, this finding
supports the use of anti-MET monotherapy in all cases in which a genetic basis for MET hyperactivity is evident, with
salient consequences as far as toxicity issues and economic sustainability are concerned.
Cancer cell line-based drug sensitivity screens are emerging as crucial tools to inform therapeutic decisions (Sharma et
al., 2010 and Trusolino and Bertotti, 2012). Their implementation is likely to become increasingly important as the pace
of discovery of new investigational compounds is increasing and the rationale for drug combinations receives continuous
support from mechanistic studies. Our work provides formal demonstration that MET targeted monotherapy is maximally
effective per se whenever tumors exhibit the relevant genetic lesion and is dispensable – even when combined with
chemotherapy – in genetically inappropriate contexts. These results add weight to the emerging paradigm that clinical
trials need to rely on tumor molecular characterization as a prerequisite for patient stratification and should progressively
dismiss post-hoc, evidence-based conclusions.
Authors' contributions
SB and PMC conceived and designed the experiments; SB, AA and LL performed the experiments; SB, LL and AG
analyzed the data; LT and PMC wrote the paper.
Conflict of interest
The authors disclose no potential conflicts of interest.
Financial support
Paolo M. Comoglio: AIRC ‘IG’ n. 11852 and AIRC ‘Special Program Molecular Clinical Oncology 5xMille’ n. 9970; Livio
Trusolino: AIRC ‘IG’ n. 10116.
Acknowledgments
We thank Melisa Milan for passionate bench work and Dr. Elena Casanova for supervising the apoptosis experiments
(FACS analysis); Dr. Francesca De Bacco for reagents and suggestions and Dr. Raffaella Albano for inestimable
technical assistance. We thank Antonella Cignetto and Michela Bruno for secretarial help and Daniela Gramaglia for
assistance in grant management.
Appendix A.
The following
Supplement
MET express
from all cell l
levels were c
loading. The
HS746T and
should be no
increased M
specific inhib
NOT MET-ad
protein level
Supplement
Listed the hu
Cell line NCI-H820 NCI-H441 NCI-H1975 NCI-H23 NCI-H522 HCC-78 EBC-1 NCI-H460 NCI-H322M NCI-H226 NCI-H2452 NCI-H28 A549 MDA-MB-231 MDA-MB-468 A2780 OVCAR8 OVCAR3 SKOV3 HT29 SW620 SW48 DLD1 SF-268 U-118 MG SNB-75 CaKi HS746T GTL16. Supplement
g are the supp
tary Figure 1
sion levels in t
lines listed in
checked by pr
cell lines test
EBC1) -which
oted that also
ET copy num
bitors (McDerm
ddicted cells.
was checked
tary table 1
uman cancer-d
Hystotype Lung Papillary A Lung Papillary A Non-Small Cell Non-Small Cell Non-Small Cell Non-Small Cell Non-Small Cell Non-Small Cell Bronchio-alveo Mesothelioma Mesothelioma Mesothelioma Lung Adenocar Breast Adenoca Breast Adenoca Ovarian Adeno Ovarian Adeno Ovarian Adeno Ovarian Adeno Rectosigmoid C Colorectal Aden Colorectal Aden Colorectal Aden Glioblastoma Glioblastoma Glioblastoma Kidney Carcino Gastric Adenoc Gastric Adenoctary data
plementary dat
the panel of h
Supplementa
robing the me
ted proved to
h harbor high
o NCI-H441 d
mber and, acc
mott et al. Pro
Cells were ly
probing the m
derived cell lin
Adenocarcinoma Adenocarcinoma Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer lar Carcinoma rcinoma arcinoma arcinoma carcinoma carcinoma carcinoma carcinoma Colon Adenocarcin nocarcinoma nocarcinoma nocarcinoma oma carcinoma carcinoma
ta related to th
uman cancer
ary Table 1. T
embranes with
express differ
grade MET g
displayed high
cordingly, do n
oc Natl Acad S
ysed and prot
membranes wit
es representin
noma
his article:
cell lines use
Total extracts w
h anti MET an
rent amount o
ene amplifica
h levels of M
not experienc
Sci U S A 10
teins separate
th anti
ng diverse tum
ed to conduct a
were separate
ntibody. Actin
f receptors. A
tion ¬express
MET expressio
ce reduction o
4, 19936-199
ed on SDS PA
mor types use
a multi-arm tri
ed on SDS PA
was used as
s expected, M
ed the highes
on. However,
of their viabilit
41, 2007). NC
AGE gels (7.5
d in the ‘in cel
ial. Proteins w
AGE gels and
a control for
MET-addicted
st amount of M
these cells d
ty when treate
CI-H441 cells
5%) as total e
ll trial’.
were extracted
d MET protein
equal protein
cells (GTL16,
MET protein. It
do not harbor
ed with
therefore are
extracts. MET
d
n
n
,
t
r
-e
T
Supplement
Effect of MV
injected subc
When tumors
placebo; (ii)
respectively)
days using a
standard erro
References
Bang, Y.J., V
Satoh, T., Ap
chemotherap
junction canc
Bardelli, A., C
Zecchin, D., A
A., Gambaco
Giordano, S.
cancer. Canc
Barretina, J.,
G.V., Sonkin
Valbuena, J.,
Aspesi Jr., P
Sougnez, C.,
G., Ardlie, K.
T.R., Morriss
predictive mo
tary Figure 2
V-DN30 and 5
cutaneously on
s volume was
MV-DN30 (2
. Mice were t
a calliper. Va
ors.
Van Cutsem, E
prile, G., Kuliko
py versus chem
cer (ToGA): a
Corso, S., Ber
Apicella, M., M
orta, M., Diaz J
, Siena, S., 20
cer Discov. 3,
Caponigro, G
, D., Reddy, A
, Mapa, F.A., T
., de Silva, M.
, Onofrio, R.C
, Chan, V., My
sey, M.P., Sell
odelling of ant
5FU as single
n the right flan
s approximate
20 μg/mice);
treated three
alues represen
E., Feyereislov
ov, E., Hill, J.,
motherapy alo
phase 3, open
rtotti, A., Hobo
Migliardi, G., G
Jr., L.A., Saus
013. Amplifica
658e673.
G., Stransky, N
A., Liu, M., Mu
Thibault, J., B
, Jagtap, K., J
., Liefeld, T., M
yer, V.E., Web
ers, W.R., Sc
ticancer drug s
De agents and
nks of immuno
ely 50 mm3 m
(iii) 5FU (15
times per we
nt the mean
va, A., Chung,
Lehle, M., Ru
one for treatme
n-label, rando
or, S., Valtorta
Galimi, F., Lau
sen, M., Velcu
ation of the ME
N., Venkatesan
rray, L., Berge
Bric-Furlong, E
Jones, M.D., W
MacConaill, L
ber, B.L., Port
hlegel, R., Ga
sensitivity. Na
Days after injectio