When the MET receptor kicks in to resist targeted therapies

Abstract

Although targeted therapies have increased the life expectancy of patients with druggable molecular alterations directly involved in tumor development, the efficacy of these therapies is limited by acquired resistances leading to treatment failure. Most targeted therapies, including ones exploiting therapeutic antibodies and kinase inhibitors, are directed against receptor tyrosine kinases (RTKs) or major signaling hubs. Resistances to these therapies arise when inhibition of these targets is bypassed through activation of alternative signaling pathways. In recent years, activation of the receptor tyrosine kinase MET has been shown to promote resistance to various targeted therapies. This casts MET as important actor in resistance. In this review, we describe how the MET receptor triggers resistance to targeted therapies against RTKs such as EGFR, VEGFR, and HER2 and against signaling hubs such as BRAF. We also describe how MET can be its own resistance factor, as illustrated by on-target resistance of lung tumors harboring activating mutations causing MET exon 14 skipping. Interestingly, investigation of all these situations reveals functional physiological relationships between MET and the target of the therapy to which the cancer becomes resistant, suggesting that resistance stems from preexisting mechanisms. Identification of MET as a resistance factor opens the way to co-treatment strategies that are being tested in current clinical trials.

Full text

Oncogene (2021) 40:4061–4078
https://doi.org/10.1038/s41388-021-01835-0
REVIEW ARTICLE
When the MET receptor kicks in to resist targeted therapies
Marie Fernandes1●Philippe Jamme1●Alexis B. Cortot1,2 ●Zoulika Kherrouche1●David Tulasne 1
Received: 15 February 2021 / Revised: 26 April 2021 / Accepted: 7 May 2021 / Published online: 24 May 2021
© The Author(s), under exclusive licence to Springer Nature Limited 2021
Abstract
Although targeted therapies have increased the life expectancy of patients with druggable molecular alterations directly
involved in tumor development, the efficacy of these therapies is limited by acquired resistances leading to treatment failure.
Most targeted therapies, including ones exploiting therapeutic antibodies and kinase inhibitors, are directed against receptor
tyrosine kinases (RTKs) or major signaling hubs. Resistances to these therapies arise when inhibition of these targets is
bypassed through activation of alternative signaling pathways. In recent years, activation of the receptor tyrosine kinase
MET has been shown to promote resistance to various targeted therapies. This casts MET as important actor in resistance. In
this review, we describe how the MET receptor triggers resistance to targeted therapies against RTKs such as EGFR,
VEGFR, and HER2 and against signaling hubs such as BRAF. We also describe how MET can be its own resistance factor,
as illustrated by on-target resistance of lung tumors harboring activating mutations causing MET exon 14 skipping.
Interestingly, investigation of all these situations reveals functional physiological relationships between MET and the target
of the therapy to which the cancer becomes resistant, suggesting that resistance stems from preexisting mechanisms.
Identification of MET as a resistance factor opens the way to co-treatment strategies that are being tested in current clinical
trials.
Targeted therapies and the inevitable
associated resistances
Detection of genetic alterations in cancers, supported
mainly by improved genome sequencing, have led to the
discovery of activated oncogenes associated with tumor-
igenesis. These notably include genes encoding actors of
signaling pathways, such as receptor tyrosine kinases
(RTKs), and signaling hubs such as KRAS, BRAF, and
PI3K. Importantly, some tumors depend on a single
deregulated oncogene for their growth and/or survival. This
dependence, called oncogene addiction, explains the effi-
cacy of targeted therapies. The first example of this was
activation of the BCR–ABL gene in chronic myelogenous
leukemia, and therapies targeting the ABL kinase have led
to strong clinical responses [1]. Since then, many other
targeted therapies have proved effective as long as the
cancer cells depend on the targeted oncogene. Examples
include antagonist antibodies against HER2 or VEGFR,
tyrosine kinase inhibitors (TKIs) against EGFR or HER2
and inhibitors of the BRAF kinase. Such targeted therapies
have increased the life expectancy of patients suffering from
cancers such as lung cancer, breast cancer, and melanoma
[2], provided the tumor is clearly oncogene-addicted.
Despite these encouraging results, the therapeutic effi-
cacy of such inhibitors is limited by the systematic
appearance, after weeks or months of treatment, of acquired
resistances leading to treatment failure. Many studies have
aimed to identify resistance mechanisms in cell models or
by genome resequencing of the relapse tumors. Because
most targeted therapies are directed against RTKs and sig-
naling kinases, the first main mechanism of resistance
involves activation of alternative signaling pathways that
bypass driver inhibition. An example of this resistance
mechanism, called off-target resistance, is the case of lung
tumors harboring an activating EGFR mutation and treated
with an EGFR TKI: 10–20% of patients treated with a first-
generation EGFR TKI display EGFR-inhibition-bypassing
*David Tulasne
david.tulasne@ibl.cnrs.fr
1CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, UMR9020-
U1277—CANTHER—Cancer Heterogeneity Plasticity and
Resistance to Therapies, Univ. Lille, Lille, France
2Thoracic Oncology Department, CHU, Univ. Lille, Lille, France
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1038/s41388-
021-01835-0.
1234567890();,:
1234567890();,:

MET gene amplification [3]. The second main mechanism
of resistance described is modification of the target itself by
additional molecular alterations which abolish the efficacy
of a targeted therapy. This resistance mechanism, called on-
target resistance, is also found in lung tumors harboring an
activating EGFR mutation: in about 50% of patients treated
with a first-generation EGFR TKI, there appears an addi-
tional EGFR mutation at residue T790, which leads to
resistance by interfering with TKI binding [4].
In recent years, the receptor tyrosine kinase MET has
emerged as an important actor in resistance to various tar-
geted therapies. In this review, we describe how the MET
receptor triggers resistance of various cancers to therapies
targeting RTKs such as EGFR, VEGFR, and HER2 or
signaling hubs such as BRAF. In physiological situations,
interestingly, functional relationships have been described
between MET and the bypassed target, suggesting that
resistance stems from preexisting mechanisms. We also
describe the recently observed on-target resistance of lung
tumors harboring activating MET mutations.
Presentation of the MET receptor
MET is an RTK produced predominantly in cells of epi-
thelial origin. It is activated by its stromal ligand, hepato-
cyte growth factor (HGF), also called scatter factor [5].
HGF and MET are essential to embryonic development,
since knockout of either one affects formation of the epi-
thelial organs, placenta, muscles, and neurons [6–9]. In
adults, the HGF-MET pair is involved in physiological
processes such as epidermal healing and liver regeneration
[10–13].
The extracellular part of MET contains an N-terminal
SEMA domain encompassing the αsubunit and the first amino
acids of the βsubunit, followed by a plexin–semaphorin–
integrin domain and four immunoglobulin-like domains. The
intracellular region contains a juxtamembrane domain, the
tyrosine kinase domain, and a C-terminal tail [14]. MET
interaction with its ligand favors its dimerization and autop-
hosphorylation of two tyrosine residues in the catalytic domain
(Y1234/Y1235). Other tyrosine residues are then phosphory-
lated, especially tyrosine 1003 in the juxtamembrane domain
and tyrosines 1349/1356 in the C-terminal tail [15,16]. The
phosphorylated C-terminal residues allow recruitment of
important effectors such as GAB1 and PI3K. This multi-
substrate docking site plays a key role in MET-induced bio-
logical responses, since its mutation in mice causes phenotypes
similar to those of MET-deficient mice [17]. Downregulation
of MET receptor activity is also an essential process, as it
prevents receptor oversignaling. All the negative regulatory
mechanisms evidenced so far target the juxtamembrane region
ofthecytoplasmicpart,encodedbyexon14[18].
Resistance to EGFR TKIs: the prototype of
resistance involving MET
In non-small cell lung cancer (NSCLC), mutations affecting
the kinase domain of EGFR (epidermal growth factor
receptor) and inducing constitutive activation of this
receptor are found in 10–40% of cases. MET and EGFR
have been shown to activate similar downstream pathways,
notably the MAPK and PI3K pathways [19]. Thus, inhibi-
tion of one of these two receptors can be compensated by
activation of the other. Direct crosstalk between MET and
EGFR has also been evidenced. It was first shown that
EGFR ligands such as TGFalpha and EGF activate MET
through EGFR–MET heterodimerization and that EGFR
and MET co-immunoprecipitate in tumor cells over-
expressing EGFR [20,21]. Ligand-independent activation
of MET by EGFR has also been demonstrated, in work
highlighting the role of G-protein coupled receptors in MET
transactivation in human carcinoma cells [22]. Inversely,
HGF has been found to induce transcription of Heparin-
Binding Epidermal Growth Factor (HB-EGF), a ligand of
EGFR [23]. MET can also activate EGFR indirectly,
through activation of SRC, which in turn activates EGFR
[24] (Fig. 1A). In addition, MET and HER-family members
often appear to be co-expressed in tumors [25,26]. EGFR
variants such as vIII EGFR have been found to induce MET
activation in glioblastomas [27].
The involvement of MET in resistance to EGFR-targeted
therapies was first observed in EGFR-mutated NSCLC. In
this type of cancer, MET activation through MET gene
amplification acts as a bypass pathway leading to resistance
to EGFR TKIs [3]: MET amplification causes HER3
transactivation, which in turn induces activation of the PI3K
pathway. In this setting, cell proliferation can be abolished
only when an EGFR TKI and a MET TKI are combined.
Importantly, although the resistance due to MET amplifi-
cation was first observed in preclinical models, it has now
been confirmed by molecular analysis of paired tumor
samples obtained before and after exposure to an EGFR
TKI. MET amplification is found in 5–22% of samples
showing acquired resistance to a first- or second-generation
EGFR TKI [3,28,29] (Fig. 1B). Variations in MET
amplification rates in the various series of post-progression
tumor samples may be due, in part, to the lack of a con-
sensual definition and technique for detection of MET
amplification [30]. Interestingly, clones displaying MET
amplification can be detected, at a very low rate, before any
treatment with an EGFR TKI, suggesting that these clones
are selected under therapeutic pressure [31]. Additionally,
MET amplification can occasionally but rarely occur toge-
ther with another mechanism of resistance to EGFR TKIs,
such as the EGFR T790M mutation [32]. Importantly, MET
amplification is also a mechanism of resistance to
4062 M. Fernandes et al.

Resistance mechanismsPhysiological interplay
P
Main treatment strategies
(Trial number or acronym)
MET
VEGFR
PTP1B
P
PTP1B
Hypoxia
P
bevacizumab
suninib
MET
HER2 HGF
trastuzumab
MET
expression
MET amplificaon
HGF expression
MET overexpression
HIF1a Signaling
Signaling
HCC, RCC
Breast cancer
CBA
MET EGFR Bispecific Anbodies MET/EGFR
-EMB-01 (NCT03797391)
-amivantanab (MARIPOSA/ PAPILLON)
-golvanib (NCT01332266)
Co-treatments MET TKI/EGFR TKI
-teponib/gefinib (INSIGHT)
-savolinib/osimernib (SAVANNAH/ ORCHARD)
-vannib/erlonib (MARQUEE)
Co-treatments MET TKI/ EGFR Ab
-capmanib/cetu ximab (NCT02205398)
-vannib/cetuximab (NCT01892527)
Co-treatments MET Ab/EGFR TKI
-emibetuzumab/erlonib (Chime)
Co-treatments HGF Ab/EGFR Ab
- rilotumumab/pan itumumab (NCT00788957)
Multarget TKI (MET/VEGFR/…)
-cabozannib (METEOR, MegaMOST, ACTION…)
-kaninib (NCT04093466)
-forenib (NCT00725764)
Co-treatments MET TKI/VEGFR TKI
-crizonib/axi nib (NCT01999972)
Co-treatments MET TKI/VEGFR Ab
-merisnib/ramucir umab (NCT02745769)
Co-treatments MET Ab/ VEGFR TKI
-onartuzumab/soraf enib (NCT01897038)
Co-treatments MET Ab/ VEGF Ab
- onartuzumab/bevacizumab (NCT01496742)
Co-treatment MET TKI/panHER TKI
-crizonib/dacominib (NCT01121575,
NCT01441128)
Co-treatments MET TKI/HER2 Ab
-cabozannib/trastu zumab (NCT02260531)
SRC P
HB-EGF
Dimerizaon
EGF
TGFα
or
HGF
HGF
cetuximab
panitumumab
MET amplificaon
Signaling
NSCLC HGF expression
1st generaon
erlonib
3rd generaon
osimernib
(T790M inhibion)
P
PP
PPP
BRAF PP2A P
MET amplificaon
PP2A
P
BRAF
BRAFi
HGF
HGF expression
PSer985
P
Signaling
MET
BRAF Melanoma
Co-treatement MET TKI/BRAF inhibitor
-crizonib/vemurafenib (NCT01531361)
-cabozannib/v emurafenib (NCT01835184)
-golvanib/sora fenib (NCT01271504)
Ser985
PPP
Fig. 1 Resistance mechanisms involving the MET receptor and
main therapeutic strategies. Illustrations summarizing examples of
physiological interplay (A), the corresponding resistance mechanisms
(B), and the main treatment strategies evaluated in clinical trials for
countering resistances due to EGFR/MET, VEGFR/MET, HER2/
MET, and BRAF/MET interplay (C). Hepatocellular carcinoma HCC,
Renal cell carcinoma RCC.
When the MET receptor kicks in to resist targeted therapies 4063

osimertinib, a third-generation EGFR TKI designed to tar-
get even T790M-bearing EGFR. This is true both when
osimertinib is used after failure of a first EGFR TKI [33,34]
and when it is used as first-line treatment [35] (Fig. 1B). In
colorectal cancer, MET amplification is responsible for
primary and acquired resistance to monoclonal antibodies
against EGFR. In patients whose tumors initially respond to
cetuximab or panitumumab and later progress without any
evidence of a KRAS mutation, MET amplification may be a
frequent mechanism of resistance [36] (Fig. 1B).
In addition to conferring resistance to EGFR TKIs, MET
amplification promotes an aggressive phenotype in EGFR-
mutated cells: increased cell proliferation, anchorage-
independent growth, and migration, leading to an
increased capacity to metastasize [37]. Patients displaying
MET amplification as a mechanism of resistance to EGFR
TKIs are more prone to develop new metastases and to have
a shorter time on treatment [37,38].
Beside to MET amplification, in both EGFR-mutated and
EGFR-wild-type cell lines, the MET ligand HGF has been
also shown to induce resistance to EGFR inhibitors,
including anti-EGFR antibodies and EGFR TKIs [39,40]
(Fig. 1B). On a larger scale, HGF can promote resistance to
several targeted therapies in models of oncogene addiction,
including EGFR- and BRAF-mutated cells [41]. In vitro,
HGF causes activation of the PI3K-AKT pathway via
GAB1, without recruitment of HER3 [31,39]. Transient
exposure to HGF is sufficient to promote clonal expansion
of MET-amplified clones among EGFR-mutated cells [31].
In a study where tumor samples were obtained from patients
with EGFR-mutated NSCLC after tumor progression on an
EGFR TKI, 61% of the samples displayed elevated levels of
HGF [42]. Patients showing tumor progression on an EGFR
TKI showed higher serum levels of HGF than patients with
no progression, and elevated serum HGF levels were sig-
nificantly associated with shorter progression-free and
overall survival [43]. HGF-induced resistance to EGFR
inhibitors could be overcome with mTOR inhibitors [44]or
by combining an EGFR TKI with either an antiangiogenic
agent [45] or a PI3K inhibitor [46].
Lastly, MET mutations have also been implicated in
acquired resistance to EGFR TKIs in EGFR-mutated
NSCLC. Suzawa et al. reported the case of an EGFR-
mutated NSCLC patient who, at the time of progression on
erlotinib, had developed both a T790M EGFR mutation and
a MET mutation leading to exon 14 skipping, and was later
stabilized with a combination of osimertinib and the MET
TKI crizotinib [47]. Furthermore, MET kinase mutations
have been proposed as a potential mechanism of acquired
resistance to EGFR TKIs in EGFR-mutated NSCLC [48].
Clinical studies have evaluated the combined use of an
EGFR TKI and a MET TKI in patients with MET-induced
resistance to EGFR inhibitors, mostly in EGFR-mutated
NSCLC (Table 1and Fig. 1C). MET-dependent resistance
was variously defined in these different studies, according
to different criteria (gene copy number (GCN) gain or the
ratio of MET GCN to centromeres) and thresholds. Com-
bining osimertinib with the specific MET TKI savolitinib, in
patients with MET amplification defined as GCN ≥5or
ratio ≥2, yielded an objective response rate of 30% in those
who had been treated previously with osimertinib and
64–67% in those who had been treated previously with a
first- or second-generation EGFR TKI [49]. In a randomized
phase II study, a combination of gefitinib and tepotinib, a
MET-specific TKI, was evaluated in MET-altered, EGFR-
mutated NSCLC patients having acquired resistance to an
EGFR TKI. In patients with MET amplification (GCN ≥5or
ratio ≥2), the median OS was 37.3 months for patients
treated with gefitinib and tepotinib, as compared to
13.1 months in those treated with chemotherapy [50]. In
another study, evaluating the combination of gefitinib and
capmatinib in patients with tumors progressing on EGFR
TKIs, the authors found antitumor activity to depend on the
level of MET amplification [51]. Other agents such as
amivantamab, a bispecific monoclonal antibody targeting
both EGFR and MET, have shown encouraging results in
MET-deregulated NSCLC, in both in vivo and clinical
studies [52,53].
Resistance to antiangiogenic VEGF and
VEGFR inhibitors
Angiogenesis, which ensures oxygenation and nutrient
uptake, is required for tumor growth. Therefore, investiga-
tors have developed therapies targeting angiogenic factors
such as VEGFR 1 and its ligand the VEGF. Since 2005, the
FDA has approved these therapies for gastrointestinal
stromal tumors, advanced renal cell carcinoma (RCC),
thyroid carcinoma, hepatocellular carcinoma (HCC), and
lung cancer [54]. The agents used include TKIs against
VEGFR (e.g., sorafenib, sunitinib, pazopanib, and vande-
tanib) and monoclonal antibodies such as bevacizumab and
ramucirumab (directed against VEGF and VEGFR2,
respectively).
Unfortunately, resistances to these inhibitors also arise.
They seem due, in part, to adaptive responses induced by
intratumoral hypoxia resulting from treatment with anti-
angiogenic compounds. MET receptor involvement in the
adaptive response was highlighted in the early 2000’s:
hypoxia-induced factor 1 (HIF1a) promotes MET expres-
sion leading to increased survival, migration, and angio-
genesis [55]. Resistance to VEGF/VEGFR inhibitors is thus
proposed to involve the MET receptor as a consequence of
targeted-therapy-induced hypoxia. Furthermore, MET and
VEGFR2 have been found to interact directly. This
4064 M. Fernandes et al.

Table 1 Clinical trials evaluating multitarget agents or treatment combinations.
HGF/MET inhibitor combined with Target of TKI (inib) or Antibody (mab) Identifier Acronym Phase Indication Ref.
VEGFR/MET
Cabozantinib MET/VEGFR2/Ret/Kit/Flt-1,3,4/Tie2/AXL TKI NCT03541902 CABOSUN II Metas. RCC [129]
NCT01755195 II Soft tissue sarcomas
NCT04134390 CABOMAYOR II Metas. RCC
NCT03425201 NICARAGUA I/II With Niraparib advanced UC
NCT04116541 MegaMOST II Malignant solid tumor
NCT04316182 ACTION II HCC
NCT01865747 METEOR III Metastatic RCC [68,130]
Kanitinib MET/VEGFR TKI NCT04093466 I Advanced solid tumor
Foretinib MET/VEGFR2/RON/AXL TKI NCT00725764 II Neoplasms, head and neck
Crizotinib Axitinib MET/ALK/ROS TKI +VEGFR TKI NCT01999972 Ib Advanced solid tumors [64]
Golvatinib Lenvatinib MET/VEGFR TKI +pan-VEGFR TKI NCT01433991 I/II Glioblastoma/melanoma
Merestinib Ramucirumab Multitarget MET TKI +anti-VEGFR2 NCT02745769 I Advanced cancers
Emibetuzumab Ramucirumab anti-MET +anti-VEGFR2 NCT02082210 I/II Advanced cancer
Onartuzumab Sorafenib anti-MET +VEGFR TKI NCT01897038 I Advanced HCC
Bevacizumab anti-MET +anti-VEGF NCT01496742 II NSCLC [131]
pan-HER
Crizotinib Dacomitinib MET/ALK/ROS TKI +pan-HER TKI NCT01121575 I NSCLC [80]
NCT01441128 I Advanced NSCLC
Foretinib Lapatinib MET/VEGFR2 TKI +pan-HER TKI NCT01138384 II Metas. Breast cancer [132]
HER2
Cabozantinib Trastuzumab MET/AXL/VEGFR2 TKI +anti-HER2 NCT02260531 II Breast cancer (brain Metas.) [133]
EGFR/MET
LY3164530 anti-MET/EGFR bispe.Ab NCT02221882 I Neoplasms/neoplasm Metas. [128]
EMB-01 anti-MET/EGFR bispe.Ab NCT03797391 I/II Advanced/Metas. Solid tumors
Emibetuzumab Erlotinib anti-MET +EGFR TKI NCT01900652 Chime II NSCLC
Savolitinib Osimertinib MET TKI +EGFR TKI NCT03778229 SAVANNAH II EGFRm+/MET+NSCLC after
Osimertinib
[134]
NCT03944772 ORCHARD II Advanced NSCLC after Osimertinib
Tivantinib Erlotinib MET TKI +EGFR TKI NCT01244191 MARQUEE III NSCLC [135]
Cetuximab MET TKI +anti-EGFR NCT01892527 II EGFR Resist/MET High; Metas. CC
Glumetinib Osimertinib MET TKI +EGFR TKI NCT04338243 I/II Metas. T790M negative and MET
amplif. NSCLC
Capmatinib Gefitinib MET TKI +EGFR TKI NCT01610336 II EGFR mut., MET amplif. NSCLC
after EGFRi
When the MET receptor kicks in to resist targeted therapies 4065

Table 1 (continued)
HGF/MET inhibitor combined with Target of TKI (inib) or Antibody (mab) Identifier Acronym Phase Indication Ref.
Erlotinib MET TKI +EGFR TKI NCT02468661 I EGFR mutated, MET positive NSCLC
Nazartinib MET TKI +EGFR TKI NCT02335944 I/II EGFR-mutant NSCLC
NCT03333343 I EGFR-mutant NSCLC
Cetuximab MET TKI +anti-EGFR NCT02205398 I MET pos.; metas. RCC/HNSCC after
Cetuximab
EGFR/MET
Cabozantinib Erlotinib MET TKI +EGFR TKI NCT03213626 II EGFR +MET Expressiong metas. PA
Tepotinib Gefitinib MET TKI +EGFR TKI NCT01982955 INSIGHT II NSCLC [50,136]
Osimertinib MET TKI +EGFR TKI NCT03940703 INSIGHT 2 II Osimertinib relapsed MET amplif. NSCLC
Cetuximab MET TKI +anti-EGFR NCT04515394 II Colorectal neoplasms
Foretinib Erlotinib Multitarget MET TKI +EGFR TKI NCT01068587 I/II Advanced or metastatic NSCLC [137]
Onartuzumab Erlotinib anti-MET +EGFR TKI NCT01887886 III EGFR Mutated, MET pos. NSCLC [138]
Amivantamab Lazertinib anti-MET/EGFR bispe.Ab +EGFR TKI NCT04487080 MARIPOSA III EGFR-Mutated NSCLC
NCT02609776 CHRYSALIS I Advanced NSCLC
NCT04077463 I Advanced NSCLC
Carboplatin-
Pemetrexed
anti-MET/EGFR bispec.Ab +chemo NCT04538664 PAPILLON III Advanced or Metas. NSCLC with EGFR
Exon 20 Ins.
Golvatinib Cetuximab anti-MET +anti-EGFR NCT01332266 I/II SCC of the head and neck
Rilotumumab Panitumumab anti-HGF +anti-EGFR NCT00788957 I/II Metastatic CC [139]
Ficlatuzumab Gefitinib anti-HGF +EGFR TKI NCT01039948 I/II NSCLC [140]
Erlotinib anti-HGF +EGFR TKI NCT02318368 FOCAL II NSCLC
BRAF
Crizotinib Vemurafenib MET/ALK/ROS TKI +B-Raf inhibitor NCT01531361 I Advanced cancers with BRAF mut
Cabozantinib Vemurafenib Multitarget MET TKI +B-Raf inhib. NCT01835184 I Solid tumors or melanoma
Golvatinib Sorafenib MET/VEGFR TKI +Multitarget TKI Raf-1/B-
Raf/VEGFR-2, 3…
NCT01271504 I/II HCC
Overview of the clinical trials registered at ClinicalTrial.gov (November 2020) evaluating the safety and/or efficacy of EGFR, HER2, VEGFR, or BRAF inhibitors (TKIs or antagonist monoclonal
antibodies) in combination with MET or HGF inhibitors (TKIs or antagonist monoclonal antibodies). For each clinical trial, the identifier number, acronym, phase, main indications, and published
references are indicated.
RCC Renal cell carcinoma, HCC hepatocellular carcinoma, NSCLC non-small cell lung cancer, CC colorectal cancer, HNSCC head and neck squamous cell carcinoma, PA pancreatic
adenocarcinoma, SCC squamous cell carcinoma, UC urothelial cancer, Chemo. chemotherapy, Meta. metastatic or metastasis, amplif. amplification, Mut. mutated, progres. progression, Resist.
resistant, pos. positive.
4066 M. Fernandes et al.

interaction is involved in activated-VEGFR-mediated
downregulation of MET phosphorylation through
increased association of PTP1B phosphatase with MET.
Hence, VEGFR2 inhibition by the anti-VEGF bevacizumab
restores MET phosphorylation [56] (Fig. 1A). This finding
makes MET one of the main suspects in resistance to
VEGF/VEGFR inhibitors.
Meanwhile, many TKIs targeting two or more RTK-
family members have been developed. Cabozantinib,
among others, belongs to this multikinase inhibitor family.
Its targets notably include VEGFR and MET [57]. Such
inhibitors have been used in both preclinical models and
patients to evidence the involvement of MET in resistance
to VEGFR inhibition. They constitute promising tools for
treating resistant patients.
Especially for metastatic RCC, antiangiogenic therapy is
a major treatment modality. Unfortunately, about 20% of
patients are refractory to this approach, and a large majority
of remaining patients develop resistance [58]. Interestingly,
patient treatment with the VEGFR TKI sunitinib appears
associated with increased MET phosphorylation without
any concomitant increase in MET expression. In RCC cell
lines, chronic sunitinib treatment also leads to MET acti-
vation, associated with increased motility and invasion. In a
mouse model, both co-treatment of tumor xenografts with a
VEGFR-targeting and a MET-targeting TKI and treatment
with cabozantinib, which targets both, prevented growth of
sunitinib-resistant tumors, confirming the involvement of
MET activation in resistance [59,60] (Fig. 1B).
In HCC, the VEGFR inhibitor sorafenib, which also
inhibits several targets, has been approved for first-line
treatment [61] (Table 1). For patients, however, the survival
benefit of sorafenib treatment is limited. Preclinical testing
in animal models has revealed that the initial decrease in
angiogenesis under VEGFR inhibition is followed by rapid
revascularization, associated with a more invasive and
metastatic behavior [62]. Similarly, although mice xeno-
grafted with HCC cells show inhibition of tumor growth
when treated with a VEGF antagonist antibody, this effect is
concomitant with increased metastasis [63]. It is associated
with an increase in both HIF1a and MET expression, sug-
gesting that the hypoxia associated with VEGF-targeting
therapy promotes MET expression. The involvement of the
MET receptor in this resistance was evidenced by the effi-
cacy of co-treatment with an anti-VEGF Ab and a MET
TKI, which prevents both tumor growth and metastasis.
Interestingly, the multikinase inhibitor NZ001 under
development, which inhibits both VEGFR2 and MET,
induces a similar response [63]. Unfortunately, the results of
the first study of combination treatment with axitinib (a
selective inhibitor of VEGFR 1–3) and crizotinib failed to
demonstrate a clinical benefit for patients with advanced
RCC [64].
In NSCLC, bevacizumab is currently the only anti-
angiogenic agent approved for first-line treatment. Yet the
clinical benefits of these therapies for NSCLC are modest,
giving rise to both intrinsic and eventually acquired resis-
tances [65]. In preclinical models, xenograft tumors derived
from NSCLC cell lines showed, upon treatment with var-
ious VEGFR TKIs, acquired resistance associated with
increased HGF and MET expression leading to its phos-
phorylation. Ectopic expression of HGF in NSCLC cell
lines reproduced this resistance, and co-treatment with
VEGFR and MET TKIs overcame it, confirming the
potential role of HGF in causing resistance. Interestingly,
plasma analysis of NSCLC patients enrolled in clinical trials
evaluating the efficacy of the VEGFR TKI vandetanib
revealed an association between elevated circulating levels
of HGF and poor prognosis [66].
In neuroblastoma, likewise, MET activation by its ligand
may be a resistance mechanism, as the IGR-N91 neuro-
blastoma cell line, resistant to the VEGFR TKI axitinib,
displays elevated levels of HGF and of the phosphorylated
form of MET. In addition, treating the parental cell line with
HGF triggers resistance to axitinib. Consistently with these
observations, the growth of tumors derived from these
neuroblastoma cell lines was inhibited by co-treatment with
the VEGFR- and MET-targeting TKI cabozantinib, this
effect being associated with decreased angiogenesis and
increased apoptosis [67].
Following the positive results from the phase 3
METEOR trial [68] (Table 1and Fig. 1C), the multikinase
TKI cabozantinib was approved by the FDA in 2016, first
for treatment of patients with advanced RCC having
received prior antiangiogenic therapy and for first-line
treatment. It is unclear, however, whether the efficacy of
cabozantinib is due to its ability to prevent the appearance
of resistance mechanisms involving interplay between
VEGFR and MET or to other causes.
Resistance to anti-HER2 in breast cancer
The HER2 receptor is overexpressed in ~15–30% of inva-
sive breast cancers and also in gastric and gastroesophageal
junction adenocarcinomas. This overexpression is associated
with reduced disease-free and overall survival. HER2+
breast tumors generally display constitutive tyrosine phos-
phorylation, signalling pathway activation, and a high rate of
cell growth [69].
The development of anti-HER2 agents such as mono-
clonal antibodies against HER2 (trastuzumab), TKIs (lapa-
tinib, afatinib, dacomitinib able to inhibit HER-family
RTKs), and an antibody-cytotoxic agent conjugate (trastu-
zumab emtasine) has dramatically improved breast cancer
patient care. Yet in the majority of patients, tumor
When the MET receptor kicks in to resist targeted therapies 4067

progression is observed in the year after initiation of tras-
tuzumab treatment [70]. Mechanisms of resistance to anti-
HER2 agents are wide-ranging. Resistance can be asso-
ciated with loss of HER2 through either decreased receptor
expression, masking of its extracellular domain or shedding
of the trastuzumab target domain through the action of
proteases [69]. Furthermore, one frequently observes
alterations in signalling pathways downstream of HER2,
such as activating mutations in PI3K/AKT/mTOR pathway
members [71] or SRC [72].
The involvement of the MET receptor in resistance to
anti-HER2 treatments was evidenced by transcriptomic
analysis of a HER2-positive breast cancer cohort (Fig. 1B).
Interestingly, tumors resistant to anti-HER2 treatments
showed higher MET expression [25]. MET amplification is
observed in approximately one-fourth of HER2+breast
cancer cases and is associated with a higher risk of trastu-
zumab therapy failure [73]. Similarly, HER2+gastric
cancer studies also revealed MET amplification as a
mechanism of resistance to the pan-HER inhibitor afatinib
[74,75]. Consistently with these clinical observations,
trastuzumab treatment of HER2-positive breast cancer cell
lines led to increased MET expression, involved in the
observed acquired resistance [25].
Interestingly, the interplay between HER2 and MET is
key to explaining how MET—particularly its over-
expression—promotes resistance to HER2 inhibition.
Indeed, trastuzumab efficiently inhibits HER2 homo-
dimerization but not HER2 heterodimerization with other
RTKs. Yet, the MET receptor is able to dimerize with
HER2, and this interaction is not inhibited by trastuzumab.
Consequently, the HER2/MET interaction leads to cross-
activation of the two receptors and to induction of both
RTK downstream signaling pathways [76] (Fig. 1A). This
co-activation triggers synergistic action promoting cell
invasion in epithelial cell models [77].
HGF overexpression has also been detected in resistant
tumors [25] and can be associated with trastuzumab failure
[73]. In HER2+metastatic gastric cancers, Takashi et al.
evidenced a significant association between a low serum
level of HGF at trastuzumab pretreatment and a positive
response, a high level being a factor of poor prognosis [78].
Furthermore, an increased serum level of HGF, as compared
to pretreatment, was observed in cases of trastuzumab
failure, suggesting that serum HGF could be used as a
tracker of the response to trastuzumab.
In the light of MET involvement in resistance to HER2
inhibition, strategies combining MET TKI treatment with
HER2 inhibition by an antibody or a TKI were first eval-
uated in cell models. Shattuck et al. found MET inhibition
to restore sensitivity to trastuzumab treatment in HER2+
breast cancer cell lines [25]. Similarly, in HER2-amplified
gastric cancer cell lines rendered resistant to afatinib by
MET amplification, co-therapy against HER2 and MET was
found to inhibit tumor growth [75]. A combination of
lapatinib and foretinib (a multikinase inhibitor of MET and
VEGFR) was likewise found to reverse resistance to lapa-
tinib [79]. These data support the relevance of developing
anti-MET strategies to fight resistance to therapies targeting
HER2 (Table 1and Fig. 1C). Unfortunately, the phase I
study (NCT01121575) that investigated the effect of com-
bining dacomitinib, a pan-HER inhibitor, with crizotinib in
patients with advanced-NSCLC showed limited antitumor
activity and substantial toxicity [80].
Resistance to BRAF inhibitors in melanoma
and anaplastic thyroid carcinoma
More than half of metastatic melanomas display BRAF-
activating mutations, mainly BRAF-V600E, leading to
constitutive activation of the MEK–ERK signaling pathway
[81]. These patients can be treated with a combination
including a BRAF inhibitor (BRAFi) such as vemurafenib.
Such treatments have proved effective in about 50% of
patients, but various innate and acquired resistances can
lead to treatment failure [82,83]. Many resistance
mechanisms have been described, including reactivation of
the MAPK signaling pathway or upregulation of PI3K-
AKT-mTOR [84].
The involvement of HGF/MET signaling in resistance to
BRAFis was first described in two back-to-back papers
published in Nature in 2012. The objective of both studies
was to investigate, in a large panel of cell lines with various
oncogene addictions, whether sensitivity to targeted thera-
pies could be overcome either by co-culturing with stromal
cells [85] or by adding various RTK ligands [41]. Both
studies revealed HGF as an effective inducer of resistance to
BRAFi in melanoma cell lines expressing the BRAF-
V600E oncogene (Fig. 1B), by preventing cell death and by
restoring activation of the MEK–ERK and PI3K–AKT
signaling pathways. The MET TKI crizotinib was able to
prevent this response, demonstrating that HGF-induced
resistance was dependent on MET.
HGF expression analysis in a cohort of patients with
BRAF-V600E melanoma revealed its expression in the
tumor-associated stroma, with an increase of this expression
after treatment with BRAFi [85]. Also, an increase in circu-
lating HGF in BRAF-mutant melanoma patients was asso-
ciated with shorter progression-free and overall survival [41].
Besides resistances involving HGF expression, an acti-
vating BRAF mutation (G469A) was recently found to
coexist with MET gene amplification in a patient-derived
xenograft from a liver metastasis. Although MET amplifi-
cation usually leads to constitutive MET activation, the
receptor was found, unexpectedly, to be inactivated. Its
4068 M. Fernandes et al.

activation was rescued upon BRAF inhibition. This sug-
gests an interplay between these two oncogenes [86], a view
supported mechanistically by the fact that BRAF signaling
inhibits the PP2A phosphatase known to dephosphorylate
Ser985 of MET. Since Ser985 phosphorylation inhibits
MET kinase activity, BRAF activation leads to inhibition of
MET activity, and the MET amplification remains func-
tionally dormant. Treatment with a BRAFi reactivates this
alteration, causing resistance to this targeted therapy (Fig.
1A). However, this elegant mechanism have to be con-
firmed in other patients. On the basis of all these data, phase
I clinical trials are ongoing to evaluate the use of MET TKIs
and BRAFis in advanced BRAF-mutated cancers, including
melanomas (Table 1and Fig. 1C).
In anaplastic thyroid carcinoma (ATC), BRAF-V600E is
again the most common oncogenic driver, often associated
with a TP53 alteration [87]. A recent clinical trial of
vemurafenib treatment for ATC showed significant respon-
ses, but these were followed by resistance [88]. To investi-
gate the underlying resistance mechanisms, an in vivo mouse
model of ATC was developed, based on both thyroid-
specific TP53 inactivation and doxycycline-dependent
BRAF-V600E expression [89]. Dox withdrawal induced
complete tumor regression, followed by high-rate recurrence,
constituting an elegant model of resistance. Analysis of
tumors resistant to BRAF blockade revealed focal amplifi-
cation of chromosome 6, causing both MET and HGF
overexpression. The involvement of MET activation in the
resistance process was then demonstrated in the model by
effective inhibition of tumor growth by MET inhibitors [89].
In thyroid cancer cell lines with BRAF-V600E mutation,
acquired resistance was likewise associated with MET
amplification leading to reactivation of the PI3K/AKT sig-
naling pathway [90]. Further study is required, however, to
confirm the existence of this resistance mechanism in patient.
In metastatic colorectal cancer, BRAF mutations are found
in about 10% of patients. Yet in a phase II clinical trial,
selective BRAFis such as vemurafenib showed only a
minimal effect against BRAF-mutant CRC [91]. This ineffi-
cacy as compared to the results obtained in melanoma
patients appeared associated with reactivation of EGFR sig-
naling [92]. This led to evaluating, in clinical trials, co-
treatment with BRAFi and EGFR-antagonist antibodies [93].
In the context of these co-treatments, novel mechanisms of
resistance were revealed, including amplification or mutation
of KRAS and also MET amplification. Pietrantonio et al. [94]
report that a patient co-treated with the anti-EGFR antibody
panitumumab and the BRAFi vemurafenib displayed disease
progression after an initial response. This resistance to tar-
geted co-treatment was associated with MET amplification
[36]. In the same study, interestingly, MET overexpression in
colorectal cancer cells was found to cause resistance to
BRAF and EGFR inhibition. Subsequent co-treatment of the
patient with vemurafenib and the MET TKI crizotinib proved
effective. This confirmed MET gene amplification as a
resistance mechanism in this context [94]. As previously
mentioned, this observation has to be confirmed in other
patients in order to assess how widespread this mechanism is.
On-target resistance in METexon14 NSCLC
patients
Although MET has been identified as a driver oncogene in
several well-characterized situations [95], it was not until
this year that the clinical use of MET-targeting therapies
was approved for treatment of NSCLC [96,97] (Supple-
mentary Table S1).
Novel MET-gene alterations, called METex14 mutations,
have recently been found in about 3% of lung tumors
[98,99]. All these alterations affect splice sites of MET exon
14 and lead to in-frame exon 14 skipping and subsequent
deletion of its regulatory juxtamembrane domain [18]. This
domain contains several negative regulatory sites, including
the ligand-dependent phosphorylated tyrosine 1003 and
serine 985. The Y1003 is involved in the recruitment of the
E3 ubiquitin ligase CBL involved in MET ubiquitination, a
molecular mechanism underlying intracellular RTK traf-
ficking [100] and Ser985 phosphorylated by protein kinase
C is involved in feedback downregulation of the MET
kinase activity [101]. Juxtamembrane domain contains also a
caspase cleavage site (ESVD1002), leading upon apoptotic
stress to generation of a cytoplasmic fragment able to
amplify cell death through mitochondrial permeabilization
[102–104].
Importantly, objective responses to several MET-
targeting TKIs have been observed in NSCLC patients
harboring METex14 mutations. This was the case, for
instance, in two phase II trials: VISION (evaluated tepoti-
nib) and GEOMETRY (evaluated capmatinib). In these
trials, about half of the METex14 patients showed an
objective response, and the median duration of the response
exceeded 11 months [97,105]. These results have led the
Japanese Ministry of Health to approve tepotinib for the
treatment advanced METex14 NSCLC. In the US, tepotinib
treatment has received breakthrough therapy designation
and capmatinib has been approved for patients with meta-
static METex14 NSCLC.
Thus, in the last few years, various MET TKIs (crizoti-
nib, glesatinib, capmatinib, tepotinib) have been used and
have shown efficacy in clinical trials or as off-label treat-
ments for METex14 NSCLC patients [96,97]. Yet after
initial responses and as expected for targeted therapies,
resistances have systematically been observed. Many
resistance mechanisms have been described, mostly invol-
ving activation of the usual suspects such as other RTKs or
When the MET receptor kicks in to resist targeted therapies 4069

downstream signaling hubs [106,107]. Some resistances,
however, are due to on-target mutations, essentially in the
MET kinase domain. In the context of this review we can
thus state that MET resists its own targeting.
To date, seven mutations have been clinically described,
after treatment with a MET TKI, in patients harboring
METex14 mutations: H1067, H1112, G1181, L1213,
F1218, D1246, and Y1248 [108–118]. These mutations
usually emerge after only a few months of treatment with
crizotinib, glesatinib, or capmatinib (Table 2and Fig. 2A).
The presence of multiple mutations in the kinase domain
has also been observed [108,109,114,116,117]. For
example, D1246N/H has been found with Y1248H [109]
and G1181R can be present with L1213V, D1246H/N, and
Y1248H/S [116] in the patient’s circulating DNA.
In the recent study of Recondo et al. among 20
METex14-mutation-harboring patients who experienced
resistance to a MET TKI, 7 (35%) displayed on-target
resistances due to MET mutations, the other resistances
being off-target ones [116].
Most of the MET mutations recently found to be asso-
ciated with resistance to TKIs were already known as MET-
activating mutations, notably in renal cancer [119–122].
Detection of these mutations in tumors suggests that they
may play a direct role in resistance through ligand-
independent MET activation.
Resistance to MET TKIs has also been investigated in
cell models expressing METex14 and/or displaying
METex14 oncogene dependence. In Ba/F3 cells engi-
neered to depend, for their growth, on ectopic expression
of an oncogene rather than on a cytokine, 12 MET resis-
tance mutations were revealed, including five at residues
previously spotted in patients (G1181, L1213, F1218,
D1246, and T1248) [123]. Interestingly, mutations at
positions 1246 and 1248 were involved in resistance to
type I TKIs, which bind the active form of MET, while
mutations at 1213 and 1218 were involved in resistance to
type II TKIs, which bind the inactive form. This study thus
suggests that the contribution of specific mutations to
resistance may depend on the type of TKI [123]. These
Table 2 Acquired MET
mutations after MET TKI in
METex14 NSCLC, review of
clinical data.
Ref. Sample type before
resistance
TKI Sample site at
resistance
MET mutation post TKI
[109] cfDNA Crizotinib cfDNA D1246N; D1246H; Y1248H
[112] Tumor Crizotinib Metastasis Liver D1246N
[118] Tumor Crizotinib cfDNA G1181R; D1246H;
D1246A; Y1248H
[110] Tumor Crizotinib Tumor +cfDNA D1246N, Y1248H/S, G1181R,
(+glenatinib →L1213V)
[113] Tumor +cfDNA Crizotinib cfDNA D1246N
[108]–Crizotinib –D1246N
[108]–Crizotinib Tumor +cfDNA Y1248H; D1246N; D1246S
–Glesatinib cfDNA D1246N; G1181R; L1213V
–Glesatinib Tumor +cfDNA H1067Y; L1213V; L1213F
–Crizotinib cfDNA D1246N
[117] cfDNA Crizotinib cfDNA Y1248S; F1218I
cfDNA Crizotinib cfDNA Y1248H; D1248N
cfDNA Crizotinib cfDNA L1213V
cfDNA Crizotinib cfDNA D1246H
[116] Tumor biopsy Glesatinib Tumor +cfDNA H1112Y; L1213V
Tumor biopsy Crizotinib Tumor +cfDNA G1181R; L1213V; D1246H/N;
Y1248H/S
Tumor biopsy Crizotinib Tumor D1246H
Tumor biopsy Crizotinib cfDNA D1246N
Tumor biopsy Capmatinib Tumor +cfDNA D1246N
Tumor biopsy Crizotinib Tumor +cfDNA Y1248C
Tumor biopsy Glesatinib-
Crizotinib
Tumor MET Amplification
This table compiles the data available from recent publications describing MET kinase mutations identified
in METex14 patients treated with the indicated MET TKI. Site and type of the collected samples before and
after resistance are indicated.
cfDNA Circulating free DNA.
4070 M. Fernandes et al.

data are also in agreement with the view that a con-
formational change induced by a particular mutation in the
kinase domain may interfere with binding of particular
MET TKIs. For instance, the resistance mutations at
positions 1181 and 1248 are consistent with steric hin-
drance of crizotinib binding (crizotinib being a type I
TKI), while the resistance mutations at positions 1213 and
1218 are consistent with steric hindrance of binding of
merestinib, a type II TKI (Fig. 2B). Interestingly, MET
variants with resistance mutations against type I TKIs were
found to be sensitive to type II inhibitors, and vice versa
[123]. This suggests that resistance might be overcome
through the complementary activity of the MET TKIs.
Similar results were obtained in NIH3T3 cells, where
expression of a MET variant displaying both an exon 14
deletion and a D1246N or Y1248C mutation was found to
drive resistance to type I but not type II inhibitors [110]. In
the same publication, the authors report a patient with a
METex14mutationwhodisplayedresistancetocrizotinib,
associated with the presence of the following additional
MET mutations: D1246N, Y1248H/S, and G1181R. On
the basis of results obtained in cell models, treatment was
switched from the type I inhibitor crizotinib to the type II
inhibitor glesatinib. This led to regression of some meta-
static localizations, with disappearance of the Y1248H/S
mutation. This disappearance was concomitant, however,
with the appearance of an L1213V mutation [110], iden-
tified in a study by Fujino et al. [123] as a mutation leading
to resistance to type II TKIs.
On-target resistance has also been described in the con-
text of MET gene amplification. A case report describes a
patient with an initial EGFR-activating mutation who
METex14
MET TKI
crizonib
glesanib
capmanib
H1067Y
H1112Y
G1181R
L1213V/F
F1218I
D1246H/N/A/S
Y1248H/N/S/C
Acve MET kinase
with crizonib type I inhibitor
Inacve MET kinase
with meresnib type II inhibitor
A
CB
MET WT
Y1248
DFG
in conformaon
crizonib
G1181
DFG
out conformaon F1218
meresnib
L1213
Fig. 2 On-target resistance to
MET TKIs in NSCLC
displaying MET exon
14 skipping. A Illustration of
MET WT and MET exon 14,
with the positions of the on-
target resistance mutations that
appear within the kinase domain
upon treatment with MET TKIs.
B3D structure images of the
active form of MET kinase in a
complex with the type I inhibitor
crizotinib (DOI: 10.2210/
pdb2WGJ/pdb). C3D structure
images of the inactive form in a
complex with the type II
inhibitor merestinib (DOI:
10.2210/pdb4EEV/pdb). The
images were created with Mol*,
an online software of the
Research Collaboratory for
Structural Bioinformatics
Protein Data Bank (www.rcsb.
org)[141]. The position of the
DFG motif of the ATP pocket in
the in or out conformation is
colored in yellow, residue
G1181 in blue, Y1248 in red,
L1213 in pink, and F1218
in green.
When the MET receptor kicks in to resist targeted therapies 4071

developed primary resistance to an EGFR TKI through
MET amplification [124]. Co-treatment with an EGFR TKI
and the MET TKI savolitinib led to a dramatic clinical
response. After several months of co-treatment, however,
the patient experienced a relapse associated with detection
of an additional MET D1246V mutation, previously
described as an activating mutation [125]. In the same
study, interestingly, the authors demonstrated with recon-
stituted MET mutations in cell lines that the D1246V
mutant is resistant to type I TKIs, including savolitinib, but
not to type II MET inhibitors. This finding led to adapting
the treatment to an EGFR TKI and cabozantinib, a type II
MET TKI, to which the progressive lung disease responded
dramatically [124].
Taken together, case reports and cell-based resistance
assays allow proposing a flowchart for sequential treatment
of METex14 lung cancer patients. Mutations causing
resistance to type I TKIs, including at least the D1246H/N
and Y1248H/N/S/C mutations, can be overcome with type
II MET TKIs. Conversely, mutations causing to type II
TKIs, including at least the L1213F and F1218I mutations,
can be overcome with type I TKIs (Fig. 3).
Conclusion and prospects
A review of the literature identifies MET activation as an
effective mechanism of resistance to targeted therapies against
several RTKs including EGFR, HER2, and VEGFR (Fig. 4).
Inthesethreecases,theresistancecanbequalified as a sub-
stitution resistance, since MET can replace another RTK. One
would think that the substitution “works”because of major
similarities between the downstream signaling pathways of
MET and of the replaced RTK. Yet when MET replaces
EGFR in the context of resistance to an EGFR TKI, only a
subset of signaling pathways seems involved in the resistance.
First, in cell models of oncogene addiction, specific targeting
of either MET or EGFR with a TKI led to similarly altered
phosphoprotein profiles and to largely common transcriptomic
modifications. Reductions in the phosphoproteome were
mainly dependent on RAS and PI3K, suggesting that the MET
and EGFR oncogene addictions involve similar signaling hubs
restricted to a few pathways [126]. The central role of RAS
and PI3K signaling in resistance was latter demonstrated by
the fact that mutations in and amplification of KRAS and PI3K
are widespread mechanisms of resistance to both EGFR and
MET TKIs [127]. Therefore, at least for EGFR/MET sub-
stitution, the resistance seems supported by restricted common
downstream signaling pathways including the RAS and PI3K
pathways.
Resistance through substitution also seems supported by
preexisting functional relationships between RTKs, includ-
ing their heterodimerization, cross-activation, and negative
feedback loops. This is exemplified by the interplay
between VEGFR and MET. The MET receptor participates
in the adaptive response to hypoxia, since MET, like
VEGFR, is an angiogenic receptor. Thus, hypoxia due to
VEGFR inhibition leads to MET re-expression, which in
turn supports resistance [55]. Furthermore, as activated-
VEGFR negatively regulates MET phosphorylation through
the phosphatase PTP1B, MET is directly activated upon
VEGFR inhibition [56]. Thus, resistance mechanisms
appear to rely both on similarities in RTK signaling and on
preexisting functional relationships between RTKs. These
observations suggest that acquired resistance may arise not
only through random phenomena but also through oriented
processes. First, resistance can emerge through selection of
the molecular alteration that is most appropriate for effec-
tive substitution. Second, resistance can be initiated by
breaking of a negative feedback loop by the targeted ther-
apy, leading to activation of the resistance factor.
The second main mechanism of resistance involving
MET is exemplified by resistance to BRAFis in melanoma
and thyroid carcinoma (Fig. 4). In these cancers, MET
activation through gene amplification can bypass inhibition
of BRAF. This mechanism is thus called bypass resistance.
Interestingly, as in the case of substitution resistance, the
existence of a functional relationship between BRAF and
MET may favor establishment of the resistance: BRAF-
inhibitor-triggered disruption of the negative feedback
exerted by BRAF on MET through PP2A promotes MET
activation.
METex14
MET amplif.
Type I TKI
Type II TKI
Type II TKI
METex14 +
D1246H/N
First line TKI On-target
Resistance
Second line TKIInial MET
alteraon
METex14 +
Y1248H/N/S/C
Type I TKI
METex14 +
L1213F
METex14 +
F1218I
crizonib
capmanib
teponib
savolinib
cabozannib
meresnib
glesanib
Fig. 3 Flowchart for potential sequential treatments of METex14
lung cancer patients. Results of case reports and cell-based resistance
assays have led to proposing a flowchart for sequential treatment of
METex14 lung cancer patients with either type I or type II MET TKIs.
4072 M. Fernandes et al.

For all substitution and bypass resistances, the existence
of powerful MET TKIs opens the way to potential co-
treatments. In resistant cell lines and experimental animal
tumorigenesis, co-targeting of MET and of a second
oncogenic RTK (VEGFR, EGFR, or HER2) has proven
effective. Multiple trials are in progress to investigate co-
targeting with two inhibitors or with multitarget agents, and
results are pending (Table 1). Interestingly, some clinical
trials are specifically designed to evaluate co-treatment
efficacy in the expected resistance situation. For example,
clinical trials are evaluating EGFR/MET TKI co-treatment
as a way to counter resistance associated with MET gene
amplification. One should note, however, that co-treatment
studies considering molecular MET alterations as stratifiers
remain the exception. It is also important to mention the
possibility of targeting two receptors with a single com-
pound such as cabozantinib, a TKI that inhibits both
VEGFR and MET, and the bispecific antibodies which
target both EGFR and MET [52,128].
The third mechanism of resistance involving MET is its
own molecular alteration as a consequence of targeted
therapy against MET (Fig. 4). Such on-target resistance has
been observed in the context of METex14 mutations in lung
cancer: resistance arises through additional MET mutations
within the kinase domain. In this situation, the mutation
interferes directly with TKI-MET binding. Because type I
and type II MET inhibitors bind, respectively, to either the
active or the inactive kinase domain, different MET muta-
tions occur according to the type of treatment. In several
patient cases, these observations have led to proposing a
sequence of treatments to overcome resistance [110,124].
These case reports open the way to possible adaptation of
MET-targeting therapies according to the type of on-target
resistance (Fig. 3).
Basic knowledge of RTKs, accumulated from their dis-
covery in the early 80’s, has led to the development of
targeted therapies which have profoundly changed the
treatment of eligible patients. Much has notably been
learned about the complex mechanisms which underlie
RTK activation and downstream signaling. In parallel with
improvements in targeted therapy, the focus of scientific
research on RTKs in general and on the MET receptor in
particular has shifted gradually from basic studies to applied
research, on the false premise that RTK signaling is suffi-
ciently well known, since RTK targeting is effective. Yet
the emergence of resistances has revealed how shaky that
premise is. It now appears that most resistances are based on
preexisting functional links between target and resistance
factor. Hence, extensive functional data are needed to fully
understand resistance mechanisms. This includes—but is
not limited to—the study of RTK co-receptors, of signaling
hubs shared by RTKs, and of negative feedback loops
between RTKs and actors of the signaling pathways. Con-
stant cross feeding between basic and clinical research will
thus be required to meet future challenges imposed by
resistance to targeted therapies.
Acknowledgements This work was supported by the CNRS, the
Institut Pasteur de Lille, and INSERM, and by grants from the
“Cancéropôle Nord-Ouest”, the “Ligue Contre le Cancer, Comités
Nord et Aisne”, the “Agence nationale de la recherche”and the
“Institut National du Cancer”.
Compliance with ethical standards
Conflict of interest The author declares no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Off Target Resistance
EGFR
(NSCLC)
HGF
MET
amplificaon /overexpression
Signaling
HGF expression
VEGFR
(HCC,RCC)
HER2
(breast cancer)
Ab
BRAF
BRAFi
METex14
(NSCLC)
On Target Resistance
L1213V/F
F1218I
D1246H/N/A/S
Y1248H/N/S/C
Type II
TKI
Type I
TKI
Signaling
«Bypass»
« Substuon »
TKI
(Melanoma)
Fig. 4 Off- and on-target
resistances involving the MET
receptor. Illustrations
summarizing the main interplay
and resistance mechanisms
involving the MET receptor.
HCC hepatocellular carcinoma,
RCC renal cell carcinoma,
NSCLC non-small cell lung
cancer.
When the MET receptor kicks in to resist targeted therapies 4073

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