International Journal of

Molecular Sciences

Review
Tyrosine Kinase Receptors in Oncology
Jorge Esteban-Villarrubia 1,† , Juan José Soto-Castillo 1,† , Javier Pozas 1 , María San Román-Gil 1 ,
Inmaculada Orejana-Martín 1 , Javier Torres-Jiménez 1 , Alfredo Carrato 2 ,
Teresa Alonso-Gordoa 2, * and Javier Molina-Cerrillo 2
1 Medical Oncology Department, University Hospital Ramon y Cajal, 28034 Madrid, Spain;
elementjorge00@gmail.com (J.E.-V.); jj27sc@gmail.com (J.J.S.-C.); pozas.javier@gmail.com (J.P.);
mariasanro@gmail.com (M.S.R.-G.); inma.orejana@gmail.com (I.O.-M.); javier.torres.jim@gmail.com (J.T.-J.)
2 Medical Oncology Department, Ramón y Cajal Health Research Institute (IRYCIS), CIBERONC, Alcalá
University, University Hospital Ramon y Cajal, 28034 Madrid, Spain; acarrato@telefonica.net (A.C.);
javier.molinace@gmail.com (J.M.-C.)
* Correspondence: talonso@oncologiahrc.com
† These authors have contributed equally to this work.




Received: 19 October 2020; Accepted: 9 November 2020; Published: 12 November 2020


Abstract: Tyrosine kinase receptors (TKR) comprise more than 60 molecules that play an
essential role in the molecular pathways, leading to cell survival and differentiation. Consequently,
genetic alterations of TKRs may lead to tumorigenesis and, therefore, cancer development.
The discovery and improvement of tyrosine kinase inhibitors (TKI) against TKRs have entailed an
important step in the knowledge-expansion of tumor physiopathology as well as an improvement in
the cancer treatment based on molecular alterations over many tumor types. The purpose of this
review is to provide a comprehensive review of the different families of TKRs and their role in the
expansion of tumor cells and how TKIs can stop these pathways to tumorigenesis, in combination
or not with other therapies. The increasing growth of this landscape is driving us to strengthen
the development of precision oncology with clinical trials based on molecular-based therapy over a
histology-based one, with promising preliminary results.

Keywords: tyrosine kinase receptors; tyrosine kinase receptor inhibitors; tumorigenesis; research

1. Introduction
Since the discovery of tyrosine kinase receptors (TKR) in the 600 s, and their subsequent classification
by families, it has been revealed how the function of these receptors is key in different aspects of the
cell biology. In the meantime, it has been discovered how the alterations in the normal function of
these receptors can play a key role in the development of tumors, from early stages of the disease to
more advanced ones. The development of new drugs that are able to inhibit these receptors, and their
downstream pathways, has been one of the main milestones in cancer treatment. A great variety of
new TKR inhibitors (TKI) have been included in the therapeutic algorithm of different solid tumors
after confirming their efficacy and improvement in patients ‘survival.
The aim of this review is to provide a comprehensive insight of the different roles of the TKRs
within the cell biology, how their alteration can lead a tumorigenesis process and which drugs are
currently available or under development to stop tumor progression.

2. Roles of TKRs in Physiologic Cell Biology

There are nearly 60 different TKRs, which can be classified into 20 families according to their
structure and their ligand. In general, in the absence of a ligand, the receptors are docked in the plasmatic
membrane of the cell as monomers, which are the inactive form of the receptor. Ligand binding leads

Int. J. Mol. Sci. 2020, 21, 8529; doi:10.3390/ijms21228529 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 8529 2 of 48

to the homo or heterodimerization of these receptors and their activation by a variety of mechanisms.
Most of the receptors are activated by a process called transphosphorylation, while others are activated
by conformational changes caused by the interaction of the tyrosine kinase domains. Both processes
conclude in the phosphorylation of some tyrosine residues in the cytosolic side of the protein, which are
the docking sites for some intracellular signalling proteins. These proteins bind to a specific site of the
receptor by the reckoning of phosphotyrosine residues by a docking domain, which is usually the Src
homologue 2 (SH2) or phosphotyrosine binding (PTB) type.
The following table details some of the TKR with their demonstrated functions [1–17] (Table 1).

Table 1. Tyrosine kinase receptors, its ligands and most representative functions. Adapted from
Molecular biology of the cell, by Alberts B et al. [1].

Receptors and Most Representative

Ligand Cellular and Tissue Distribution
Representative Examples Functions

- Plasma membrane and cell junctions.

Stimulates survival, growth, - Wide expression: epithelial,
EGF (Epidermal EGFR (HER1), HER2, HER3, proliferation, endothelial, neuronal and glial, bone,
Growth Factor) HER4 and differentiation of adipose, liver,
various cell lines and cardiovascular cells.
Regulates carbohydrate - Vesicles and plasma membrane.
Insulin Insulin receptor metabolism and protein - All human cells, especially pancreas
synthesis
Stimulates cell growth and
IGF (Insulin-like survival. Regulation of - Vesicles and plasma membrane.
IGF-1R - Detected in all human cells.
Growth Factor) growth in young people and
has anabolic effects in adults.
- Plasma membrane, vesicles
NGF (Nerve Stimulates neural growth and cytosol.
NTRK1 (TrkA, TrkB, TrkC) - Adrenal gland, blood, central and
Growth Factor) and differentiation
peripheral nervous systems.

- Nucleoplasm, plasma membrane, cell

It stimulates survival, junctions (α), vesicles and
PDGF (Platelet-Derived growth, proliferation and additionally in Golgi apparatus (β)
PDGFRα, PDGFRβ
Growth Factor) migration of various cellular - Wide expression, especially
subtypes. ovarian (α)

It stimulates the - Golgi apparatus and

GM-CSF (Granulocyte plasma membrane.
proliferation of monocytes
Macrophage-colony GM-CSFR or GMRα/β - Wide expression, especially blood
and their differentiation to
stimulating factor) cells and placenta.
macrophages.
It stimulates the
FGF (Fibroblast FGFR1, FGFR2, FGFR3, proliferation of various cell - Plasma membrane.
Growth Factor) FGFR4 types and inhibits the - Detected in all human cells.
differentiation of other types.
- Plasma membrane, actin filaments (1),
nucleoplasm, cell junctions (3)
VEGF (Vascular Endotelial VEGFR-1 (FLT-1), VEGFR-2 - Wide expression, especially blood
Stimulates angiogenesis.
Growth Factor) (KDR), VEGFR-3 (FLT-4) compartment (mostly platelets),
skeletal muscle and placenta

Involved in the development - Plasma membrane

ALK (Anaplastic - Wide expression, especially the
ALK (CD246) and function of the central
Lymphoma Kinase) nervous system
nervous system.

Regulation of survival - Plasma membrane Golgi apparatus

GDNF (Glial (dopaminergic neurons), and nucleoplasm (1), vesicles (2),
GFRα1, GFRα2, GFRα3, cytosol (3)
cell-line derived growth of neurites, cell
GFRα4 - Wide expression, especially brain,
neurotrophic factor) differentiation and
migration. thyroid gland in GFRα2

- Plasma membrane
It intervenes in processes - Hematopoietic stem cells, germ cells,
SCF (Mast/Stem cell such as the survival of melanocytes, and Cajal cells of the
KIT (CD117) gastrointestinal tract, epithelial cells
growth factor) melanocytes, hematopoiesis
and gametogenesis. in skin adnexa, breast, and subsets of
cerebellar neurons
Int. J. Mol. Sci. 2020, 21, 8529 3 of 48

Table 1. Cont.

Receptors and Most Representative

Ligand Cellular and Tissue Distribution
Representative Examples Functions

- Plasma membrane, endoplasmic

Guides cell and axon reticulum, cytosol
Ephrin Eph receptors - Wide expression, especially in the
migration; angiogenesis.
nervous system and injured tissues.

Cell growth, survival, - Plasma membrane and additionally in

TAM family (Tyro3, MerTK, vesicles and actin filaments
Gas6, Protein S. differentiation. Regulation of
Axl) - Wide expression
systemic immunity

Ras and Rho are two monomeric guanosine 50 triphosphatases (GTPases) that are the most
responsible for relaying signals from the membrane receptors. There are various types in the human
cells, H, K and N being the most representative. These proteins are anchored to the cytosolic layer of
the plasmatic membrane, where they send signals mainly to the nucleus [18].
Ras is able to activate a complex of proteins called mitogen-activated protein (MAP) kinases.
This complex pathway is made of three proteins called Raf, mitogen-activated protein kinase kinase
(MEK) and extracellular signal-regulated kinase (ERK). The three-component module begins with
a MAP kinase called Raf. Ras recruits Raf to the plasmatic membrane and helps to activate it.
Raf then activates MEK, which, in turn, activates ERK. To date, there are six distinct groups of
MAPKs characterized in mammals; ERK1/2, ERK 3/4, ERK5, ERK7/8, Jun N-terminal (JNK)1/2/3 and
the p38 isoforms α/β/γ (ERK6)/δ [19]. ERKs phosphorylate many cytoplasmic and nuclear kinases,
phosphatases, transcription factors and cytoskeletal proteins. Sustained, but not transient activation
of ERK signalling, promotes phosphorylation and stabilization of For, Jun, Myc, Egr-1 and cyclin D1
genes, thereby promoting cell-cycle entry. This stimulation also represses the expression of genes which
inhibit proliferation; thus, controlling fundamental processes such as growth and proliferation [20–22].
The Rho family proteins are in charge of actin and microtubules of the cytoskeleton regulation.
The human Rho GTPase family consists of 20 proteins divided into eight subfamilies, Rac, Rho,
Cdc42 and RhoF/RhoD subfamilies being some of the most representative [23]. These proteins have
a central role in cell shape, polarity, motility, endocytosis and chemotaxis as they depend on the
actin cytoskeleton [24]. Rho proteins stimulate the activity of nucleation promoting factors (NPFs),
such as WASP, which in turn activates actin-related proteins 2/3 (Arp2/3) and formin. These proteins
are key in the regulation of stable new multimers of actin development [25]. Another important
Rho effector is Rho-associated protein kinase (ROCK) which is involved in actin-myosin II filament
assembly, cross-linking and generation of contractile forces by increasing the levels of phosphorylated
myosin light chain (pMLC). Contraction promotes membrane blebbing, movement of the cell body and
detachment of the cell rear [26]. Activation of Rho family proteins by TKR is key in the development of
the nervous system, as it is involved in the migration of the neural cells and the development of the
axons. The process of axon formation is guided by Eph and its TKRs, as the migration of neurons is
guided by Trk family proteins [27].
Another kinase involved in recruiting proteins and activating signaling pathways is the
phosphoinositide 3-kinase (PI 3-K). There are three classes of PI3K enzymes. Mammals express
four class I catalytic isoforms (p110α, β, γ and δ). p110α and p110β are expressed ubiquitously,
whereas expression of p110γ and p110δ is enriched in immune cells. Each one of these catalytic proteins
forms a dimer with a regulatory subunit that modulates the activity and sub-cellular localization
of the complex. PI-3K can be activated by Tyrosine kinase receptors (RTKs) by direct interaction
with the receptor, as three of the class I isoforms, (p110α, p110β, p110δ, known as the class Ia
subgroup) associate with regulatory subunits whose SH2 domains bind to phosphotyrosine residues
present in TKRs. Each isoform has a domain that can interact with members of the Ras or Rho
family. p110α, p110 γ and p110δ bind to Ras family members whereas p110β interacts with the
Rac/Cdc42 subfamily [28]. PI-3Kinases catalyze the formation of phosphoinositide (being PI(3,4,5)P3
Int. J. Mol. Sci. 2020, 21, 8529 4 of 48

the most important), which constitute a substrate of a wide range of proteins with PH domains,
which in turn act as second messengers of the signal. Those family proteins are, among others,
the Ser/Thr protein kinases, Btk/Itk/Tec subfamily, Insulin Receptor Substrate 1 (IRS-1), regulators
of small G-proteins, some cytoskeletal proteins or the mammalian phosphatidylinositol-specific
phospholipase C (PI-PLC) [28]
A relevant member of this family is Protein kinase B (PKB or Akt), which plays a key role
in cell survival and growth promotion in many cell types. Akt is regulated by PDK1 and mTOR
complex 2. mTOR phosphorylates Akt on serine 473, so it changes its conformation, and then
is phosphorylated by PDK1 on threonine 308. This process leads to the dissociation of Akt from
the plasma membrane and now is able to activate many other downstream effectors. There are
over 100 reported in the literature. These substrates are involved in cell proliferation, metabolism,
survival and motility. Glycogen synthase kinase 3 (GSK3) was the first substrate reported of Akt.
GSK3 is generally active in the absence of exogenous signals, and the substrates of GSK3 are usually
down-regulated or degraded under GSK3-mediated phosphorylation. Thus, growth factor signalling
trough Akt positively regulates these targets, such as the B cell Lymphoma (BCL-2) family member
Induced myeloid leukemia cell differentiation protein (MCL-1), c-myc and Bad. Bad is an anti-apoptotic
protein. This way, the activation of PI 3-K leads to the inhibition of the apoptosis, in turn, activating
cell survival [28–30]. GSK3 also regulates cellular metabolism, either by phosphorylation of metabolic
enzymes (GS) or indirectly by the regulatory inhibition of transcription factors such as Sterol regulatory
element-binding protein 1 (SREBP1c), Hypoxia-inducible factor 1-alpha (HIF1a), and Nuclear factor
erythroid 2-related factor 2 (NRF2) [31,32]. Another substrate of Akt is the forkhead box (FOXO)
family members, which are transcription factors. These transcription factors are usually localized in
the nucleus and Akt signalling leads to translocation of these proteins out of the nucleus and thus
inhibiting its transcriptional program. Akt, therefore, supresses transcription factors involved in the
induction of apoptosis (Bim, PUMA, Fasl, TRADD, TRAIL, NOXA, BCL6), cell-cycle arrest (p21, p27,
Cyclin G2, p15, p19), catabolism and growth inhibition (Sestrin3, MAP1LC), tissue-specific metabolic
changes (PEPCK, G6PC), autophagy (LC3, BNIP3), stem cell maintenance and terminal differentiation
(NeuroD), DNA repair (Gadd45), glucose and lipid metabolism (PPAR-γ), stress resistance (mnSOD),
pluripotency (OCT4, SOX2), immune response (CCR7, FOXP3) and other cellular processes [31,33,34].
Another PI3-K substrate is mammalian Target of Rapamycin (mTOR), which represents a family
of serine/threonine kinases that act through two distinct protein complexes, named mTORC1 and
mTORC2. These two proteins share some common subunits, such as the mTOR kinase, mLST8,
dishevelled, EGL-10, DEPTOR, Tel2 and Tti1. They are different in terms of rapamycin sensitivity
(mTORC1 is sensitive in contrast to mTORC2), binding components, sub-cellular localization, regulation
and downstream substrates [35]. mTOR1 contains the regulatory-associated protein of mTORC1
(RAPTOR), whereas mTORC2 contains the rapamycin-insensitive companion of mTOR (RICTOR) and
mSIN1. RICTOR is essential for the function, substrate recognition and cellular location of mTORC2,
while mSIN1 negatively regulates mTORC2 [36]. In terms of cellular localization, mTORC1 is localized
in endosomal and lysosomal membranes while mTORC2 is localized in plasmatic and ribosomal
membranes, where it interacts with other proteins as AGC, PKA, PKG and PKC and SGK family
kinases [37]. Both of them are regulated by several stimuli such as growth factors, hormones, hypoxia,
nutrients, but mTORC2 is more sensitive to extracellular growth factors whose molecular mechanism
is not elucidated yet [35,38]. The mTORC1 pathway is regulated by signalling pathways discussed
earlier, such as PI3K/Akt and Ras-MAPK. mTORC1 activation by Akt requires the activation of the
following steps referenced before (mTORC2 activation, phosphorylation of Akt in Ser473 by mTORC2
and at Thr308 by PDK1) and the phosphorylation of TSC2 by activated Akt, which inhibits TSC1 and
TSC2 combination. The activator of mTORC1, RHEB, is constitutively down-regulated by TSC1/2.
The inhibition of TSC1/2 leads to the release of RHEB and mTORC1 activation in lysosomes [35,39].
TSC2 can also be phosphorylated by ERKs from the Ras-MAPK pathway, leading to RHEB releasing and
mTORC1 activation [40]. Activated mTORC1 will activate another downstream effector, such as 4EBP1,
Int. J. Mol. Sci. 2020, 21, 8529 5 of 48

S6K1 and SREBP. This allows the eucaryotic initiation factors 3 and 4 (eIF-4E and eIF-3) release in order
to intensify ribosome genesis [35,41]. mTORC1 also regulates several proteins, such as HIF1α, PP2A,
glycogen synthase and STAT3. The promotion of protein, lipid and nucleotide synthesis is important
for the G1-S phase transition and cell-size [35,42]. In contrast, two effectors of mTORC2 are SGK and
protein kinase C (PKC). Activation of PKC will lead to changes in cytoskeleton, thus regulating cell
movements [35,38].
Another relevant pathway activated by TKR is the PKC/phospholipase C-γ. The phospholipase
acts on a phosphorylated inositol phospholipid (a phosphoinositide) called phosphatidylinositol
4,5-bisphosphate [PI(4,5)P2], which is present in the inner half of the lipid bilayer. The activated
phospholipase then cleaves the PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate (IP3)
and diacylglycerol. At this step, the signaling pathway splits into two branches. IP3 leaves the plasma
membrane and diffuses into the endoplasmic reticulum and binds to specific Ca2+ channels. This leads
to the release of the Ca2+ stored in the reticulum, and the increase of cytosolic Ca2+ concentrations+.
While this ion’s concentrations are raising, the other product, diacylglycerol, can act as a second
messenger, activating the PKC, so-called because it is Ca2+ dependent. The initial rise of cytosolic Ca2+
leads to its translocation to the plasma membrane to be activated by Ca2+ and diacylglycerol [1,43].
There are at least 10 different isoforms of PKC, some are ubiquitously expressed whereas others are
restricted to certain tissues. PKC is essential to smooth muscle contraction and proliferation [43].
Figure 1 summarizes which signaling pathways are activated by each receptor.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 51

Figure1.1.Above
Figure Aboveareare represented
represented the the interaction
interaction between
between various
various signaling
signaling pathwayspathways
activated activated
through
through kinase
tyrosine tyrosine kinase receptors
receptors (TKR) and(TKR) and involved
involved in tumor in proliferation.
tumor proliferation. (1) aOnce
(1) Once a ligand
ligand bindsbinds
to the
to the receptor, two STAT proteins are phosphorylated by JAK forming a dimer
receptor, two STAT proteins are phosphorylated by JAK forming a dimer which enters the nucleus, which enters the
nucleus, causing the transcription of target genes. (2) After the TKR is activated
causing the transcription of target genes. (2) After the TKR is activated by a ligand, Ras dimerizes by a ligand, Ras
dimerizes
and binds and
Raf, binds Raf, promoting
promoting Raf activation.
Raf activation. Active Raf Active Raf phosphorylates
phosphorylates and activates
and activates MEK1/2 MEK1/2
which
which induces ERK1/2 activation, leading to transcription activation. (3) PI3K
induces ERK1/2 activation, leading to transcription activation. (3) PI3K phosphorylates phosphatidyl phosphorylates
phosphatidyl inositol-bisphosphate
inositol-bisphosphate (PIP2) to PIP3, a(PIP2) to that
process PIP3,can
a process that by
be reversed canthebeaction
reversed by thePIP3
of PTEN. action of
causes
PTEN. PIP3 causes the activation of Akt in the plasma membrane, thereby activating
the activation of Akt in the plasma membrane, thereby activating the mTOR complex, one of the major the mTOR
complex, one
pathways of the in
involved major pathways involved
tumorigenesis. (4) PLCinhydrolyzes
tumorigenesis.
PIP2,(4)inPLC
thishydrolyzes
way forming PIP2, in this way
diacylglycerol
forming diacylglycerol (DAG) and PIP3, which activate PKC and intracellular
(DAG) and PIP3, which activate PKC and intracellular calcium mobilization, respectively. calcium mobilization,
respectively.

It is necessary for the cell to regulate the diversity of signals transduced by its receptors. Cells
are desensitized to an extracellular signal by receptor sequestration, receptor down-regulation,
receptor inactivation, inactivation of the signal protein and by the production of an inhibitory protein.
We will review how these processes regulate downstream signalling of the TKRs. Once activated in
the plasma membrane, TKRs are usually endocytosed. This endocytosis is regulated by extracellular
Int. J. Mol. Sci. 2020, 21, 8529 6 of 48

It is necessary for the cell to regulate the diversity of signals transduced by its receptors. Cells are
desensitized to an extracellular signal by receptor sequestration, receptor down-regulation, receptor
inactivation, inactivation of the signal protein and by the production of an inhibitory protein. We will
review how these processes regulate downstream signalling of the TKRs. Once activated in the
plasma membrane, TKRs are usually endocytosed. This endocytosis is regulated by extracellular
ligand concentration, as seen in EGFR. Higher ligand concentrations lead to receptor endocytosis by
non-clathrin mediated endocytosis (NCE) while lower concentrations lead to receptor endocytosis by
clathrin-mediated endocytosis (CME) [44,45]. The molecular mechanisms underlying CME are defined
with adaptor protein 2 (AP2) regulating EGFR clustering into clathrin-coated pits, initially required to
optimize receptor phosphorylation and especially constrain EGFR signalling [46]. Once the receptor
is internalized, EGFR is directed into early endosomes. At low ligand concentrations, the receptor
does not undergo ubiquitination and is returned to the plasma membrane, so the receptor can be
recycled. Through recycling, CME also serves to prolong and polarize EGRF signalling to specific
cell regions, which are key to achieving a productive proliferative response [44,47]. At high EGFR
concentrations, the receptor is ubiquitinated at the plasma membrane, thus leading to degradation at
lysosomes. The endosomal sorting complexes required for transport (ESCRT) recognizes ubiquitination
through a stepwise process leading to lysosomal endocytosis [48]. Higher EGFR concentrations also
trigger ubiquitination and the internalization of EGFR directly into the endoplasmic reticulum in a
non-clathrin-mediated process regulated by a protein called Reticulon-3, involved in the formation of
contact sites between the plasma membrane and endoplasmic reticulum [30]. In the early endosomes,
concentration of EGFR in the plasma membrane can also be unregulated, with the RNF11 transcription
factor being translocated into the nucleus and inducing transcription of genes required for EGFR
transport into the plasma membrane [44,49].
GTPases such as Ras and Rho are regulated by GTPase activating proteins (GAPs) and Guanine
nucleotide exchange factors (GEFs). GEFs activate GTPases by promoting the release of bound GTP,
which allows a new GTP to bind. Conversely increases the rate of GTP hydrolysis inactivating GTPases.
ERK plays a key role in its negative regulation of the MAPK pathway. Activated ERK can inhibit
Raf by direct phosphorylation and it is able to interfere with the coupling of Raf to a GEF called Sos,
thus avoiding its activation [21,50,51]. Activated ERK can also promote the transcription of MKPs,
which are able to dephosphorylate ERK activation sites, and the Sprouty family proteins, which interfere
with Ras and Raf activation [21,52,53]. Rho is also controlled by Guanine nucleotide dissociation
inhibitors (GDIs), which control Rho cycling between cytosol and membranes, holding them in an
inactive form in the cytosol. Rho GTPases can also undergo phosphorylation by PKA, ROCK1, Src and
Akt and, thus, be negatively regulated. Phosphorylation of RhoA by Erk2 facilitates ubiquitination of
RhoA, leading to its degradation in the proteasome [23,54,55].
PTEN (phosphatase and tensin homologue: MMAC/TEP1) is a negative regulator of the
PI-3K/Akt signalling. PTEN is a phosphoinositide phosphatase, acting as a direct antagonist of
PI-3K, dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. Cellular localization of PTEN is
crucial for its normal cellular function. In the cytoplasm, its main function is to regulate the cell
cycle, apoptosis, cell adhesion and migration by antagonizing PI-3K/Akt/mTOR pathway. In the
nucleus, PTEN plays a role in chromosomal stability and DNA double-strand break repair [56,57].
PTEN is regulated at a transcriptional level positively by EGR-1, PPAR-γ, ATF2 and p53, while is
negatively regulated by CBF-1, NF-κb, c-JUN and zinc-finger like proteins SNAIL and SLUG. At a
post-transcriptional level, phosphorylation of PTEN by casein kinase 2 and GSK3β results in a decrease
of phosphatase activity, but greater protein half-life [56,58]. mTORC1 is also an important regulator
of the PI-3K/Akt signalling pathway. mTORC1 activation promotes Insulin receptor substrate 1
and 2 (ISR1 and ISR2) degradation through multiple phosphorylation on serine residues. ISR1 and
ISR2 are scaffolding proteins that are able to link the insulin and IGF1 receptors to PI-3K activation.
This way, PI-3K activation is dampened [31,59]. S6K, a substrate of mTORC1, once activated is able
Int. J. Mol. Sci. 2020, 21, 8529 7 of 48

to phosphorylase RICTOR and Sin1, thus decreasing mTORC2-dependent phosphorylation of Akt

T450 [31,60].
It is important to note that these major pathways and their regulators already discussed are
cross-talking and redundant in cells. These cross-talking points are important to regulate downstream
components activated by other pathways. MAPK pathway and PI-3K/Akt pathway interact at many
points, as exemplified by Raf activation by Akt. Akt phosphorylases C-Raf and B-Raf, thus inhibiting
ERK activation by Raf [61]. In addition, Erk activation can suppress RTK induction of the PI-3K pathway
by phosphorylation of the GAB1 and GAB2 scaffolding proteins, in a similar way as we have discussed
before [31,62]. Both pathways converge to regulate several downstream effectors and regulators as
TSC2, FOXO, GSK3, RAPTOR and cellular processes [62]. However, both pathways can also act in
a cooperative manner. For example, MAPK interacting kinases (MNK1 and MNK2) phosphorylate
the cap-binding protein eIF4E to promote its ability to initiate transcription, and mTORC1-mediated
phosphorylation of the 4E-BP proteins stimulates their release from the inhibitory binding of eIF4E
and, thus, promoting transcription [62,63].
In summary, activation of TKRs gives rise to the activation of a series of complex signaling cascades,
which are connected to regulate basic cellular processes such as replication, growth, differentiation,
motility and communication with the cellular environment.

3. Roles of TKRs in Tumor Biology

As we have previously discussed, a main function of the TKR is to coordinate how the cells behave
in their environment, regulating mitosis, differentiation or apoptosis. This harmonization between
every member of an organism allows its survival and complexity. Alterations in RTKs can therefore
bring about an evolutionary advantage of a given cell that will ultimately lead to a cell line with higher
ability to survive in its environment. These evolutive advantages are summarized in the two basic
properties of a cancer cell: The ability to divide independently and to invade other tissues [64].
In vitro assays have shown that cancer cells exhibit an altered phenotype with aberrations in shape,
motility and in the way they integrate with their environment. It is characteristic of culture-grown
cancer cells that their growth and duplication are not inhibited by contact with other cells and the
absence of contact with the substrate, in contrast to normal cells. We can conclude that a characteristic
of cancer cells is the alterations in extracellular signal processing. The essential mechanism underlying
these aberrations are somatic and germline mutations of the DNA [1,64].
Every single gene is likely to have undergone mutation on about 1010 separate occasions in a
human. Tumor cells accumulate mutations at a higher rate than normal cells, and that is why they are
said to have genetic instability [65,66]. Thus, it is the progressive accumulation of mutations in key
genes of cellular biology what gives rise to the development of a tumor. These genes are divided into
proto-oncogenes and tumor-suppressor genes.
We will focus our attention on proto-oncogenes as some of these genes encode TKRs.
A proto-oncogene is a normal gene that encodes a protein implicated in cellular growth, differentiation,
or apoptosis, for example, the EGFR. There are plenty of mechanisms in which a proto-oncogene becomes
an oncogene, being the most relevant the deletion or point mutation in the coding region or in the
regulating region, the increase in the number of copies of the gene or chromosomic rearrangements [67].
One example is the amplification of ErbB2, which can form homo or heterodimers with other members of
the ErbB family. The increase in the density of these receptors in the plasma membrane can lead to their
dimerization in absence of a ligand and subsequent constitutive activation with sustained downstream
signaling [68,69]. Other examples are the mutations in the extra or intracellular domains of the protein.
Mutations in the extracellular domain of EGFR (i.e., EGFRvIII) can lead to a ligand-independent
activation, but these mutations also cause an altered phosphorylation of the receptor at Cbl-binding sites,
thus affecting receptor ubiquitination and turnover. This leads to increased signalling [70]. Mutations
in the intracellular kinase domain also cause ligand-independent activation, leading to a kinase domain
constitutively activated. For example, mutations in the activation loop of the EGFR receptor (L858R)
Int. J. Mol. Sci. 2020, 21, 8529 8 of 48

promotes de-stabilization of the steady-state, thus converting to a more active state. These alterations
also impair receptor ubiquitination affecting receptor trafficking [71,72]. These mutations that impair
receptor intracellular trafficking and degradation/turnover can help to prolong the propagation of the
signal and to locate receptors at specific membrane sites, stimulating cell motility and the formation of
invadopodia [73,74]. Another example is the EML4-ALK fusion gene, resulting from the inversion of
the short arm of the chromosome 2, gives rise to the expression of a chimeric protein with the tyrosine
kinase domain constitutively activated [75]. Definitely, overactivation of the TKR represents a key
pathogenic factor in the development of cancer.
The pathogenic role of TKR is not related only to activating or disrupting mutations but also to
“non-canonical” activation of the receptors. Stressing stimuli, such as UV radiation, hypoxia or ionizing
radiation. UV radiation leads to EGFR phosphorylation by p-38 MAPK at serine/threonine residues,
which trigger receptor endocytosis and storage at endosomes. These receptors are not degraded and
can be recycled again to the plasma membrane as p-38 MAPK is inactivated [44,76]. Hypoxia may
lead to an upregulation of the EGFR gene transcription in the absence of genetic alterations [77].
Ionizing radiations increases EGFR expression and surprisingly, receptor endocytosis. However, as the
receptor is endocytosed, is phosphorylated by PKC, impairing receptor ubiquitination and promoting
its translocation to the nucleus.
Nuclear localization of the EGFR can be induced by EGF binding, Akt pathway, ionizing radiation
and alkylating drugs as cisplatin [78]. Nuclear EGFR can bind to transcription factors as STAT3 to
increase transcription several genes as iNOS, c-Myc and Cyclooxygenase-2 [78–80]. RNA helicase
seems to be another nuclear-EGFR partner involved in EGFR-regulation of cyclin D1 [81]. EGFR can
also play an important role in DNA repair, interacting with DNA-dependent protein kinase (DNA-PK)
that leads to the repair of double-strand breaks of the DNA. Other described partners are p53 and
MDC1 [82,83].
Furthermore, oncogenic signaling pathways can induce metabolic reprogramming in cancer cells
supporting tumor growth. For example, EGFR has been involved in the regulation of several metabolic
processes like biosynthesis of fatty acids, pyrimidines and glucose metabolism [84,85]. This is achieved
indirectly by phosphorylating transcription factors like Myc, PI3K-Akt dependent nuclear translocation
of sterol regulatory binding protein 1 (SREBP-1), phosphorylation of stearoyl-CoA desaturase-1,
amongst others [86–88]. TKRs also collaborate in the metabolic drift to glycolysis as the main source of
energy in cancer cells, known as the Warburg effect. GLUT-1, one of the main glucose transporters
of the membrane, can be stabilized in the cell surface by the action of the PI3K/AKT/mTOR pathway,
activated by EGFR mutant receptors [89]. EGFR also regulates the expression of Hexokinase 1 (HK1)
and the activity of pyruvate kinase M2 (PKM2), two relevant enzymes in the glycolytic pathway [90].
The resulting increase in lactic acid, inhibits the activity of T-lymphocytes, supporting tumor immune
escape [90].
The role of the TKRs is not only important in the tumor cell. Growing attention is given to the
tumor microenvironment and its role in tumor progression. The tumor microenvironment is composed
of stromal cells as fibroblasts, endothelial cells and adipocytes; immune cells such as lymphocytes,
macrophages, monocytes and neutrophils; the extracellular matrix composed by macromolecules such
as proteoglycans, structural proteins and glycoproteins; and other several components as growth
factors, cytokines, chemokines and antibodies [91].
Angiogenesis is a hallmark of cancer, in response to a need of oxygen and nutrients from the
bloodstream. Tumor vascularization requires cooperation between cancer cells, vascular endothelial
cells, pericytes, BM-derived precursor cells, tumor-associated macrophages, mesenchymal stem cells
(MSCs) and cancer-associated fibroblasts (CAFs). The main molecule responsible for angiogenesis is
VEGF, although other important regulators are PDGF, FGF, placental growth factor and angiopoietin-1.
Cancer cells are the main VEGF producers, although the other cell types described can also produce
it [91–93]. VEGF transcription is stimulated under hypoxic conditions via HIF-transcription factors.
VEGF binding to its receptor activates PI-3K/Akt/mTOR pathway at endothelial cells regulating
Int. J. Mol. Sci. 2020, 21, 8529 9 of 48

its replication, differentiation and motility [37]. PDGF stimulated angiogenic process activates
PI-3K/Akt/mTOR pathway, regulating the maturation of the newly formed vessels by attracting smooth
muscle cells and pericytes [94].
CAFs are another important cellular population in tumors. These fibroblasts are different
from normal fibroblasts and are present in aberrantly high numbers [92]. TGFβ, MCP1, PDGF,
FGF have been implicated in CAF activation. Their origins are unclear, as there are studies
suggesting an endothelial-to-mesenchymal transition origin [95] while others suggest its origin
in epithelial-to-mesenchymal transition [96]. In tumors, their functions range from promoting tumor
proliferation, angiogenesis, secretion of pro-inflammatory factors to tumor immunosuppression.
CAFs are able to secrete VEGF and growth factors mentioned before [92].
Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSC) are
important regulators of the tumorigenesis. Macrophages are attracted to the edge of the tumor
by hypoxia-induced secretion of endothelin-2 and VEGF, as discussed before. Then, macrophage
secretion of growth factors as CSF-1 and EGF can trigger the acquisition of an invasive phenotype
by tumor cells [97]. TAMs also secrete VEFG, VEGF-C and VEGF-D. These last two correlate
peritumoral inflammation and lymphangiogenesis [98]. TAMs usually show an M2 phenotype,
playing an additional tumorigenic role by secretion of type II cytokines and, thus, promoting immune
evasion [76,77]. MDSC can also promote tumor vascularization [99], but their key role is disrupting
normal immunosurveillance by impairing antigen presentation by dendritic cells, T-cell activation,
M1-macrophage polarization and inhibition of NK cytotoxicity [92].
In summary, the genetic mutations of the TKR give rise to aberrant proteins, constitutively active,
or constitutively deregulated; which alters the transmission of signals in the cell, altering important
cellular functions such as replication, migration and differentiation. This gives an evolutionary
advantage with respect to the normal cells beyond the control of the usual regulatory mechanisms of
cell proliferation. TKRs are also important in the regulation of the tumor microenvironment, where
they play key pathogenic roles.

4. Tyrosine Kinase Receptor Inhibitors Developed for Cancer Treatment

As of 22 July 2020, there are 61 tyrosine kinase inhibitors approved by the Food and Drug
Administration (FDA) for the treatment of haematological and oncological malignancies with TKR
disorders (http://www.brimr.org/PKI/PKIs.htm) (Table A1).
In the last two decades, a large number of drugs have been developed with the specific role of
blocking intracellular signal pathways, that we could classify into two groups: monoclonal antibodies
targeted against the self-receptor (extracellular portion) and small molecules with inhibitory activity
against peptides harboring the tyrosine kinase function (intracellular signaling). In this section,
we focus on drugs which directly inhibit the tyrosine kinase domain of the transmembrane receptor
and have approval for the treatment of advanced solid tumors. A summarizing table of the different
mutations of TKRs described below is supplied in Appendix A.

4.1. Epidermal Growth Factor Receptor (EGFR)

Targeting of EGFR family has been widely used as an effective therapeutic resource against
cancer, since the discovery of an intimate involvement of this molecular pathway in cell proliferation,
angiogenesis and motility. Specifically, the EGF receptor-1 (HER1 or ErbB1) and -2 (HER2 or ErbB2)
blockade aims to inhibit the subsequent transduction signal and sequential steps: RAS/RAF/MEK,
PI3K/Akt/mTOR, PKC, Src, and Jak/Stat. The majority of these transmembrane receptors behave as
oncogenes when an activating mutation or overexpression occur, and become highly responsible of
cell growth and survival “without limits” [100].
EGFR is involved in the oncogenic process of multiple malignancies. However, currently, we only
have TKIs against this receptor for the treatment of a few cancers (especially, lung cancer). Only some
EGFR activating mutations (exon 19 deletions, L858R point mutation in exon 21), and some resistance
Int. J. Mol. Sci. 2020, 21, 8529 10 of 48

mutations are sensitive to therapeutic blockade with TKIs, so the identification of a driver mutation in
EGFR before starting any systemic treatment is mandatory [100,101]. The two previous cited mutations
account for 85% of all EGFR mutations, which lead to the kinase domain activation without the need
for ligand binding [102]. Both mutations reduce the affinity of the kinase for adenosine triphosphate
(ATP), allowing TKI to bind it.
The estimated incidence of EGFR mutation in advanced non-small cell lung cancer (NSCLC) is
about 15–20% in the white race, and higher among the Asian female, and nonsmoker population [103].
The first EGFR TKIs were gefitinib and erlotinib (first generation, reversible binding). Subsequently,
came afatinib and dacomitinib (second generation, irreversible binding). Gefitinib is a small
molecule that selectively inhibits EGFR tyrosine kinase by binding to the ATP site, inhibiting receptor
autophosphorylation and signal transduction. Three phase III trials (IPASS, West Japan Oncology
Group 172 and North–East Japan Study Group 002) compared gefitinib with chemotherapy in advanced,
EGFR-positive (activating mutations), Non Cell Samall Lung Cancer (NSCLC) patients, who were
treatment-naïve. Gefitinib significantly improved the progression-free survival (PFS) and the objective
response rates (ORR) in the three trials, although the difference in overall survival (OS) was not
statistically significant [104–107]. Further analysis reported that EGFR overexpression did not seem
to benefit from gefitinib. Combinations of gefitinib plus chemotherapy are not a standard in this
setting. Gefitinib was approved by the FDA (2003) for the first-line treatment of patients with
EGFR-positive NSCLC.
Erlotinib, with a similar mechanism of action to gefitinib, also demonstrated an increased
PFS (versus platinum-based chemotherapy) in three trials: OPTIMAL (plus better ORR), EURTAC
and ENSURE, but no statistically significant difference in OS was detected [108–110]. Erlotinib was
approved by the FDA (2004) for the first-line treatment of EGFR exon 19 deletions or exon 21 substitution
mutations in NSCLC. In 2010, the FDA extended the erlotinib indication as maintenance therapy for
patients with advanced NSCLC whose disease had not progressed after four cycles of platinum-based
chemotherapy in the first-line setting (EGFR mutation not previously identified). This decision was
based on data from the phase III trial SATURN, which showed significantly better PFS with erlotinib
maintenance versus placebo (45 weeks vs. 13 weeks, Hazard ratio [HR] 0.71) [111]. In 2016, the FDA
modified the initial indication and limited the susceptible mutations to exon 19 deletions or exon 21
L858R substitution, because of the reported lack of survival benefit with erlotinib in the non-mutant
population (results of the IUNO trial) [112]. In May 2020, erlotinib was also approved (in combination
with ramucirumab) for first-line treatment of advanced NSCLC with EGFR exon 19 deletions or exon
21 (L585R) mutation. The RELAY trial compared the combination against erlotinib monotherapy,
and reported a better PFS (19.4 months vs. 12.4 months, HR 0.59), although ORR were similar between
the two arms [113].
In addition, erlotinib (combined with gemcitabine) is approved for the first-line and maintenance
treatment of advanced pancreatic cancer, without targeting EGFR alteration [114].
Afatinib is a potent, selective, and irreversible inhibitor of the EGFR, HER2 and HER4 kinases.
In advanced EGFR-positive NSCLC patients, afatinib showed a significant increase both in PFS and
ORR, compared with platinum-based chemotherapy, in two large phase III trials: Lux-Lung 3 and
Lux-Lung 6 [115,116]. Initially, OS rates were similar between the therapeutic options, but later,
the combined analysis of these trials reported a statistically significant benefit in OS, but only in NSCLC
with exon 19 deletion [117]. Regarding afatinib, the alterations G719X (exon 18 point mutation, 3–4%
of all EGFR mutations), L861Q (exon 21 point mutation, 2% of all EGFR mutations) and S768I (exon
20 point mutation) are sensitive [118–121], while exon 20 insertions are resistant [122]. Afatinib was
approved by the FDA (2013) for first-line treatment of EGFR-positive NSCLC (including the S768I,
L861Q, G719X mutations).
Dacomitinib is an irreversible pan-HER inhibitor (HER1, HER2 and HER4). Compared with
gefitinib, dacomitinib increased the PFS and OS rates in the ARCHER 1050 trial [123]. FDA approved
Int. J. Mol. Sci. 2020, 21, 8529 11 of 48

dacomitinib for the first-line treatment of patients with metastatic NSCLC and EGFR exon 19 deletion
or exon 21 L858R substitution.
The vast majority of NSCLC with EGFR sensitizing mutations eventually develop resistance to
TKIs therapy, with a median time to progression (TTP) of approximately 10–12 months. In about
60% of cases, resistance is associated with a secondary T790M mutation in exon 20 of the EGFR gene.
Other reported mechanisms of acquired resistance are: amplification of MET, HER2 and CRKL genes
(20%), mutations in PI3K, KRAS or BRAF, activation of the AXL TKR, or histological transformation to
small cell lung cancer (SCLC) [124–127]. Some of these molecular events are considered activating
bypass tracks, because downstream signalling pathways are eventually stimulated, regardless of EGFR
tyrosine kinase activity inhibition.
The previous survival results were surpassed with the arrival of osimertinib, a third generation,
orally available, and irreversible TKI EGFR mutant-selective variants. Osimertinib inhibits a broad
range of EGFR mutant forms, including the T790M. In a phase I/II study, osimertinib demonstrated a
significant response rate of 61% and median PFS of 10 months in patients with NSCLC harboring a
T790M mutation whose disease progressed on other EGFR-inhibiting therapy [128]. These favourable
results allowed the FDA approval (2017) for the treatment of NSCLC after progression on after
EGFR-TKI therapy. In the AURA3 trial, a randomized and phase III trial, osimertinib was compared
with platinum-therapy plus pemetrexed, in the same setting of the disease. Osimertinib increased
median PFS (10.1 months vs. 4.4 months, HR 0.3) and ORR (71% vs. 31%), and also demonstrated
higher efficacy in central nervous system (CNS) disease [129,130].
The FLAURA trial (2019) tested osimertinib in the front-line treatment of EGFR-mutated advanced
NSCLC (versus gefitinib or erlotinib). Newly, both median PFS and median OS were higher with
osimertinib (18.9 months vs. 10.2 months, HR 0.46; 38.6 months vs. 31.8 months, HR 0.8, respectively).
Disease control at CNS was ratified [131]. Due to FLAURA results, the FDA approved osimertinib as the
standard therapy in the front-line setting, including EGFR-mutant NSCLC with uncommon mutations.
Some resistant mechanisms to osimertinib have been described, such as the KRAS amplification,
the EGFR C797S mutation (which has not been seen in resistance to first-generation drugs) and the
MET amplification [132,133].
TAS6417, tarloxotinib (TH-4000) and luminespib (Hsp90 inhibitor; AUY922) are novel inhibitors
of EGFR, especially directed to exon 20 insertions. [134–136].

4.2. HER2
In breast cancer, HER2 overexpression is a poor prognostic factor, given the reported higher
disease recurrence and death rates. It is considered a predictive factor for targeted therapy response,
because the identification of high HER2 overexpression has allowed to detect patients who most
benefit from these targetable drugs. At present, detection of HER2 overexpression is a standard in the
diagnostic approach of breast cancer.
HER2 receptor activates through heterodimerization with other receptors, but external ligand
binding is not necessary. This leads to activation of the following pathways, promoting cell survival
and proliferation [137]. In this “HER2-blocking setting”, the approved TKIs by the FDA are lapatinib,
neratinib and tucatinib.
Lapatinib is a dual and reversible TKI of EGFR and HER2, also with activity against Akt, Erk-1 and
-2. Lapatinib has been tested in HER2-positive, both hormone receptor (HHRR)-positive and -negative
metastatic breast cancer (MBC), and became the first TKI approved in this setting. In postmenopausal
women with HER2-positive and HHRR-positive MBC (who had received no previous treatment
with either trastuzumab nor aromatase inhibitors), the EGF30008 trial tested the combination of
lapatinib plus letrozole (versus placebo plus letrozole) [138]. The combined treatment improved
median PFS (progression risk was reduced by 29%), but not OS. These results supported the FDA
approval. In addition, lapatinib has been combined with trastuzumab plus an aromatase-inhibitor
(AI) in the ALTERNATIVE trial, which focused on postmenopausal women with HER2-positive and
Int. J. Mol. Sci. 2020, 21, 8529 12 of 48

HHRR-positive MBC, but after receiving prior trastuzumab and endocrine therapy [139]. PFS and ORR
were improved with the triplet (compared with trastuzumab plus an AI), but it remains unclear when is
the most appropriate moment to add lapatinib in the therapeutic algorithm of those patients. This study
has been recently retracted due to numerical corrections in some secondary efficacy analyses [140].
In the population with HER2-positive and HHRR-negative MBC, the EGF 104990 trial compared
lapatinib plus trastuzumab against lapatinib alone, and the combination significantly improved the
PFS (11 weeks vs. 8 weeks, HR 0.74) and the OS (14 months vs. 10 months, HR 0.74) in the population
who had received one or more prior trastuzumab-containing regimens [141,142]. Nevertheless,
its approval by the FDA (2007) includes the combination with capecitabine after progression on
standard chemotherapy (anthracyclines and taxanes) and trastuzumab in the advanced disease setting.
The benefit of this doublet was showed in a phase III trial of 399 patients with HER2-positive MBC,
who were randomly assigned to receive lapatinib plus capecitabine versus capecitabine alone. PFS
significantly improved with the combination (6 months vs. 4 months), but not OS (75 weeks vs.
65 weeks) [143,144].
Neratinib is an irreversible TKI of EGFR, HER-2 and HER-4. It locks on the cysteine residue
in the ATP-binding pocket, to irreversibly inhibit the downstream intracellular pathways. First of
all, in the adjuvant setting, one year of neratinib treatment demonstrated significant improvement of
the 5-year invasive DFS rate (ExteNET trial) in women with HER2-positive breast cancer who had
completed neoadjuvant and adjuvant chemotherapy plus trastuzumab, compared with placebo [145].
In 2017, this was the first FDA approval for neratinib, extending the adjuvant treatment after receiving
trastuzumab-based therapy. Later, in the advanced setting, neratinib was approved, in combination
with capecitabine, for the treatment of women with HER2-positive MBC who have received two or
more previous regimens, based on the results of the NALA study [146]. This phase III trial randomized
621 patients to receive neratinib plus capecitabine versus lapatinib plus capecitabine. Twelve-months
PFS rate was higher in the experimental arm (29% vs. 15%), although OS results were not statistically
different (21 months vs. 18.7 months). Interestingly, neratinib has shown efficacy as a single agent in
the metastatic setting (breast cancers carrying HER-2 LSS5 mutation) and combined with paclitaxel in
a phase II trial [137,147]. Both lapatinib and neratinib are valid options (combined with capecitabine),
even though efficacy data are higher with the second doublet [148,149].
Recently, tucatinib has emerged as a new TKI called to change the current therapeutic landscape
of HER2-positive MBC. Tucatinib is an orally bioavailable inhibitor of HER-2, which selectively
blocks the intracellular TK domain, exerting a minimal inhibition of EGFR. This distinct mechanism
of action substantially modifies the side effects. In the initial phase I trial (2018), tucatinib plus
capecitabine and trastuzumab demonstrate a promising response rate, even in a remarkable population
with CNS metastases [150]. Afterwards, the HER2CLIMB trial (phase II, double-blinded and
placebo-controlled) compared tucatinib plus capecitabine and trastuzumab against placebo plus
capecitabine and trastuzumab) in heavily-pretreated women with HER2-positive MBC (median of four
previous regimens) [136]. The triplet improved the 12-months PFS rate (33% vs. 12%, HR 0.54) and
the 2-years OS rate (45% vs. 27%, HR 0.66) in the overall population, as well as in patients with CNS
disease, whose 12-months PFS rate was clearly higher with the tucatinib-containing regimen (24.9% vs.
0%). In 2020, tucatinib has been approved, in combination with trastuzumab and capecitabine, for the
treatment of advanced HER2-positive MBC after one or more prior HER2-based regimens.
Interestingly, HER2 mutations have been detected in lung cancer too (approximately 1 to 3% of
NSCLC tumors). Insertions in exon 20 are the most common alteration [151,152]. In 2016, Mazières et
al. published the results of a European cohort of 101 patients with NSCLC harboring HER2 mutations
and treated with neratinib, afatinib and lapatinib (among other HER2-targeted therapies), setting a
precedent for the treatment with TKI in HER2-positive lung cancer. However, data are not mature
enough for its clinical use [153].
Poziotinib and pyrotinib are other HER2-targeted drugs currently under investigation in early
phase trials [137,154,155].
Int. J. Mol. Sci. 2020, 21, 8529 13 of 48

The following table briefly summarizes the main EGFR and HER2 inhibitors developed and under
development to date (Table 2).

Table 2. EGFR and HER2 inhibitors, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
Gefitinib (ZD1389) Iressa EGFR NSCLC
Erlotinib (OSI-774) Tarceva EGFR NSCLC
Afatinib (BIBW2992) Tovok Erb1/2/4 NSCLC
Osimertinib (AZD-9291) Tagrisso EGFR NSCLC
Dacomitinib (PF299804) Visimpro Pan-HER NSCLC
Phase II trial (NCT00752206, NCT00607594,
Saracatinib (AZD0530) EGFR, Src NCT01267266) for osteosarcoma, gastric, prostate and
other solid tumors
Lapatinib (GW572016) Tykerb EGFR/HER2 Breast cancer
Neratinib (HKI-272) Nerlynx HER2 Breast cancer
Icotinib EGFR NSCLC (China)
Poziotinib (HM781-36B) EGFR/HER2/HER4 Phase II trial (NCT02979821) for NSCLC
Phase II trial (NCT03805841) for NSCLC and solid
Tarloxotinib (TH4000) EGFR/HER2
tumors harboring ERBB/NRG1 gene fusions
Lazertinib (YH25448) EGFR Phase III (NCT04248829) trial for NSCLC
AZD3759 EGFR Phase II/III (NCT03653546) trial for NSCLC.
Phase I trial (NCT02500199) for HER2 positive
Pyrotinib (SHR-1258) EGFR/HER2
solid tumors.
Avitinib (AC0010MA) EGFR Phase I/II (NCT02330367) clinical trial for NSCLC
Sapitinib (AZD8931) EGFR/HER2/HER3 Phase I/II trial (NCT01862003) for colorectal cancer.
Rociletinib (CO-1686) EGFR Phase III (NCT02322281) for NSCLC
TAS6417 EGFR/HER2/HER3 Phase I/IIa trial (NCT04036682) for NSCLC
Phase II/III (NCT03093870, NCT03130790) for billiard
Varlitinib (ASLAN001) EGFR/HER2/HER4 tract cancer, gastric cancer and hepatocarcinoma (Phase
Ib; NCT03499626)
Phase Ib (NCT04510415) and phase II (NCT03228277)
Olmutinib (HM61713) EGFR
clinical trials for NSCLC
Nazartinib (EGF816) EGFR Phase I/II (NCT02108964) clinical trial for NSCLC
Mavelertinib (PF-06747775) EGFR Phase II trial (NCT02349633) for NSCLC
Naquonitinib (ASP8273) EGFR Phase I (NCT02113813) trial for NSCLC
Ibrutinib (PCI-32765) Imbruvica EGFR, BTK Phase I/II (NCT02321540) trial for NSCLC, MCL, CLL.
EAI001 EGFR Preclinical
EAI045 EGFR Preclinical

4.3. Anaplastic Lymphoma Kinase (ALK)

The discovery of ALK involvement in cancer has changed the therapeutic approach of multiple
neoplasms. In physiological circumstances, the ALK gen encodes for a transmembrane receptor with
tyrosine kinase activity. This TKR belongs to the great insulin-receptor family. Its activation promotes
receptor dimerization, which, in turn, will send the activating signal towards the following intracellular
pathways: PI3K/Akt/mTOR, ERK, Stat3, RAS/MEK/Erb.
In solid tumors, alterations of ALK are especially involved in NSCLC oncogenesis, in such a
dependent way that it has been called “addictive pathway”. Characteristically, ALK is altered through
rearrangements that create fusion oncogenes (although also amplification and point mutations have
been described) [156]. Such rearrangements involve the ALK gene loci on chromosome 2 and are
found in 5–6% of NSCLC. The best-known rearrangement juxtaposes the 50 end of the echinoderm
microtubule-associated protein-like 4 (EML4) gene with the 30 end of the ALK gene, resulting in
the EML4-ALK fusion oncogene. Other gene partners can fuse with ALK: KIF5B, KLC1, TPR, HIP1,
DCTN1, SQSTM1, NPM1, BCL11A, y BIRC6.
In the same way as EGFR, detection of ALK rearrangements is mandatory before starting any
systemic therapy for advanced NSCLC, since it constitutes a predictive (dramatic) factor of response to
TKI drugs. From a clinical point of view, ALK-positive NSCLC patients include never or light smokers,
younger aged, and adenocarcinomas (with signet ring or acinar histology) [157,158].
Int. J. Mol. Sci. 2020, 21, 8529 14 of 48

There are several FDA-approved ALK-targeted TKIs: Crizotinib, ceritinib, alectinib, brigatinib
and lorlatinib.
Crizotinib was the first ALK inhibitor that got the approval by the FDA (2011). It is a first-generation
TKI, which selectively inhibits the tyrosine kinase domain and some oncogenic forms (including ALK
fusions, such as with the NPM1 and BCL11A genes, and selected mutations). Likewise, crizotinib
also inhibits the tyrosine kinase activity of c-MET (in fact, crizotinib was originally developed as a
MET inhibitor), ROS1, Tropomyosine Receptor Kinase (TRK) and Recepteur d’Origine Nantais (RON).
Initially, crizotinib was tested in patients with advanced ALK-positive NSCLC who has progressed
on platinum-based chemotherapy, showing better 1-year PFS rate than chemotherapy [159]. Later,
in the PROFILE 1014 phase II trial, crizotinib was compared against pemetrexed plus either cisplatin
or carboplatin in the first-line setting [160]. Both median PFS and ORR were higher with crizotinib
(10.9 months vs. 7 months, HR 0.45; 74% vs. 45%, respectively). Following analysis proved OS
improvement after crossover adjustment with crizotinib over chemotherapy (HR 0.35) [161].
Despite these findings, novel ALK TKIs were developed and demonstrated better survival
outcomes in the frontline treatment and after progression on crizotinib.
Ceritinib is a highly selective, second-generation TKI of ALK. It inhibits autophosphorylation
of wild-type ALK, ALK fusion proteins and ALK point mutations. Ceritinib showed superiority
over chemotherapy (platin plus pemetrexed) in the ASCEND-4 trial [162]. Both median PFS and
ORR were better with ceritinib (16.6 months vs. 8.1 months, HR 0.55; 72.5% vs. 26.7%, respectively),
including patients with CNS metastases. In patients without CNS disease, the PFS rates were even
better (26.3 months vs. 8.3 months). Ceritinib was approved by the FDA in 2014 for the first-line
treatment of advanced ALK-positive NSCLC.
Alectinib is another second-generation TKI of ALK, potent and highly selective against the tyrosine
kinases of the receptor. It also blocks some ALK fusion proteins and the gatekeeper mutation L1196M
(a known resistance mechanism) and RET (Rearranged during transfection). At first, the J-ALEX trial
compared alectinib against crizotinib in a Japanese population with advanced ALK-positive NSCLC,
who were chemotherapy-naïve or had received one previous chemotherapy regimen [163]. The median
PFS was higher with alectinib. After that, the global study ALEX faced alectinib versus crizotinib in
untreated ALK-positive NSCLC patients [164]. After a follow-up period of approximately 28 months,
the PFS rate was significantly higher with alectinib (35 months vs. 11 months, HR 0.43). The HR for
CNS progression was surprisingly better with alectinib (0.16) [165]. OS results are not yet mature.
These data supported the FDA approval in 2015.
Secondary mutations arise as a resistance mechanism after exposure of tumor cells to ALK
inhibitors, and most of them are within the ALK tyrosine kinase domain (“ALK-dependent mechanisms
of resistance”), inducing the inability to inhibit the encoded tyrosine kinase. L1196M (the most common),
G1269A, C1156Y, F1174L, 1151Tins, L1152R, S1206Y and V1180L are some of these acquired mutations
that confer resistance to crizotinib [166]. This is particularly important, since in approximately 50% of
cases, the CNS is the first site of disease progression. “ALK-independent resistance mechanisms” are
EGFR, IGF-R1 or c-KIT mutations.
Both ceritinib and alectinib can be administered after progression on crizotinib. The ASCEND-5
study randomized 231 patients to receive ceritinib or chemotherapy [167]. All had advanced
ALK-rearranged NSCLC and all had received prior chemotherapy and crizotinib, with disease
progression after that. Ceritinib increased median PFS (5.4 months vs. 1.6 months, HR 0.49) and ORR
(39.1% vs. 6.9%). In addition, in the ALUR trial, 107 patients with advanced ALK-positive NSCLC
and previously treated with platinum-based chemotherapy and crizotinib were randomly assigned to
alectinib or chemotherapy [168]. The median PFS was higher with alectinib (7.1 months vs. 1.6 months,
HR 0.32). Two additional phase II trials support these results, and, also, reported rates of CNS disease
control higher than 80% [169,170]
Brigatinib is a next-generation ALK inhibitor, whose anti-TK spectrum encompasses EGFR (mutant
forms), ROS1, and Insulin Growth-Factor Receptor-1 (IGF-R1). Brigatinib prevents autophosphorylation
Int. J. Mol. Sci. 2020, 21, 8529 15 of 48

of ALK and Stat3 downstream signalling pathway proteins. In preclinical studies, brigatinib
demonstrated increased inhibitory capacity over some ALK resistance mutations, compared with
crizotinib, ceritinib and alectinib (including G1202R, the most recalcitrant one, and L1196M), and also
against non-ALK kinases [171]. In the first-line treatment of advanced ALK-positive NSCLC, brigatinib
was approved (2017) based on the results of the phase III trial ALTA 1L. Two hundred seventy-five
patients were randomly assigned to brigatinib or crizotinib, and both the 12-months PFS rate and the
ORR among patients with brain metastases were higher with brigatinib (67% vs. 43%, HR 0.49; 78%
vs. 26%, respectively) [172]. Subsequently, the ALTA trial provided evidence of brigatinib benefit
in patient’s tumors refractory to crizotinib. In this phase II trial, 222 patients whose disease had
progressed after crizotinib were assigned to receive different doses of brigatinib (90 mg vs. 180 mg),
demonstrating better median PFS and median OS with the double dose (9.2 months vs. 16.7 months;
29.5 months vs. 34.1 months, respectively) [173]. Furthermore, the median response duration at CNS
was higher with 180 mg (9.4 months vs. 16.6. months).
Finally, lorlatinib is a third-generation TKI of ALK, which competitively and selectively inhibits
ALK kinases and the c-ROS oncogene. In preclinical studies, lorlatinib showed sustained inhibition
of mutated and non-mutated variants of ALK, including the G1202R and I1171T resistant variants,
which confer refractoriness to crizotinib, ceritinib, alectinib and brigatinib [174–176]. Lorlatinib clinical
efficacy was demonstrated in a phase II study (B7461001), in which 198 patients with ALK-positive
metastatic NSCLC previously treated with, at least, one ALK inhibitor, received lorlatinib [177].
After crizotinib, the ORR with lorlatinib reached 40%, with a median PFS of 11.1 months; after one
or two second-generation ALK TKIs, ORR was higher among patients with ALK mutations [178].
Taking these results into account, the FDA approved lorlatinib in 2018 for the treatment of advanced
ALK-positive NSCLC which has progressed on crizotinib and, at least, another ALK TKI; or, after a
second-generation ALK TKI in the frontline setting.

4.4. ROS1
c-ROS is another proto-oncogene which encodes a transmembrane type-1 receptor with tyrosine
kinase activity, very structurally similar to the ALK receptor. It is a member of the insulin-receptor
family (without clearly identified ligands) and its activation generates signalling cascades whose
objective remains unclear. Naturally, it is highly expressed in kidney, cerebellum, small bowel and colon.
Some molecular alterations have gained attention in the last years, specially ROS1 fusions,
present in 2–3% of the lung adenocarcinomas, as well as in cholangio, gastric and colonic
carcinomas [179]. They are present in spitzoid neoplasms, glioblastoma multiforme and inflammatory
myofibroblastic tumors, too. Approximately 30 genes have been identified as ROS1 fusion partners,
some of them were also known for being ALK and RET fusion partners as well. The acquisition of
these fusion genes induces the constitutive activation of the tyrosine kinase activity of the receptor,
which in term deregulates cellular processes as differentiation, proliferation and survival. Genes most
frequently described as ROS1 fusion partners in NSCLC are CD74 (most frequent), SLC34A2, TPM3,
SDC4 and EZR [180]. Interestingly, every ROS1 fusion partner activates different signalling pathways
depending on the heterodimeric partner. However, the mutations in ROS1 can constitute a resistance
mechanism in patients treated with crizotinib as first-line treatment of ALK-rearranged NSCLC,
as shown by G2032R mutation [181]. Typically, ROS1 fusion NSCLCs are present in women without
known tobacco consumption.
For diagnostic purposes, ROS1 fusions are mutually-exclusive with other driver mutations.
Crizotinib is one of the two drugs approved for the treatment of tumors with ROS1 rearrangements.
Shaw et al., published the results of crizotinib in a cohort of 50 patients with ROS1-rearranged NSCLC,
showing an ORR of 72% and a median PFS of 19.2 months [182]. No correlation between the type of
ROS1 rearrangement and response to crizotinib was found. Later, update on survival rates from the
PROFILE 1001 trial confirmed the previous response rate and showed a median duration of response of
24.7 months and a median OS of 51.4 months [183]. In 2016, crizotinib was approved for the treatment
Int. J. Mol. Sci. 2020, 21, 8529 16 of 48

of advanced ROS1-positive NSCLC patients, who were treatment-naïve and those who had received
previous chemotherapy.
Entrectinib is a pan-TRK inhibitor with affinity against ROS1 and ALK too. This drug is approved
for the treatment of ROS-1 rearranged NSCLC because of the results of the pooled analysis of the
ALKA-372-001, STARTRK-1 y STARTRK-2 trials [184]. In these trials, treatment with Entrectinib in
patients with locally advanced or metastatic ROS1-fusion positive NSCLC offered a benefit in terms
of a 77% of ORR, a median duration of response of 26.6 months and a median PFS of 19 months.
Due to its high intracranial penetration, entrectinib is preferred for treatment of advanced disease with
CNS metastases.
Given the structural homology between ALK and ROS1, ceritinib, brigatinib and lorlatinib show
promising activity against ROS1 fusions. Ceritinib showed near to 62% of response rates and a median
PFS of 9.3 months in a phase II clinical trial [185]. In patients who have progressed to crizotinib, there is
data from a phase I/II trial favoring the use of lorlatinib, with a 35% response rates [186].
As a future view, repotrectinib is a new TKI designed to potently inhibit some secondary and
resistant mutations of ALK, ROS1 and TRKA-C (for example, D2033N in ROS1, G595R in TRKA and
G623R in TRKC) [187]. Its antitumoral activity has been proven in preclinical and cells models.

4.5. Neurotropic Tropomyosine Receptor Kinase (NTRK)

NTRK 1, 2 and 3 genes encode three proteins called TRKA, TRKB and TRKC, respectively.
These proteins are TKR involved in neural development. Each one of them contains a ligand-binding
extracellular domain, a transmembrane region and a unique intracellular kinase domain. Each receptor
has its preferential ligand: TRKA with neurotrophin nerve growth factor (NNGF), TRKB with
brain-derived neurotrophic factor (BDNF) and neurotrophin-4, and TRKC with neurotrophin-3.
TRKA, TRKB and TRKC have been identified as oncogenic drivers in a variety of malignancies.
The most frequent pathogenic mechanism described are rearrangements [188]. Mutations or gene
amplifications have been described, but seem to have no role in carcinogenesis. Rearrangements
usually occur between the C-terminal end of the tyrosine kinase domain with the N-terminal end of the
fusion partner, and this is usually secondary to chromosomic inversions, deletions or translocations.
The fused protein has no ligand-binding domain, and dimerisation and phosporylation ocurr in an
independent manner.
NTRK fusions characteristically appear in rare tumors, like secretory carcinoma of the breast,
fibrosarcoma infantile and salivary gland carcinoma. In more frequent tumors like NSCLC, colorectal
and thyroid cancer, melanoma or gliomas, the prevalence of these alterations is very low [189].
For example, in NSCLC, it is approximately 1%.
Larotrectinib and entrectinib are the only drugs approved by the FDA for the treatment of
advanced solid tumors with NTRK fusions, and are approved for previously treated patients. Efficacy of
larotrectinib was demonstrated in various phase I and II clinical trials. Hong et al. reported the first
data of anti-tumoral activity in 70 patients with harboring NTRK gene fusions [190]; Later, Drilon et al.
published the results of the phase I/II trial [191]. Larotrectinib showed an ORR of 75%; with a median
follow-up of 9.4 months, 86% of the patients continued treatment with the drug. The 12-months
PFS rate was 55%. The largest and most recent publication of larotrectinib focused on its long-term
efficacy and safety. Included 159 patients with multiple types of neoplasms, reporting ORRs of 79%
(including 16% of complete responses), concluding that NTRK rearranged tumors are highly sensitive
to larotrectinib [192].
Entrectinib was also approved in base of the combined results of various early-phase trials
(ALKA-372-001, STARTRK-1 y STARTRK-2). As we have discussed before, entrectinib is able to inhibit
TRKA, TRKB y TRKC. Its activity in solid tumors harboring NTRK fusions was evaluated in 54 patients,
reaching an ORR of 57% (including 7.4% of complete responses) and a median duration of response of
10 months [193].
Int. J. Mol. Sci. 2020, 21, 8529 17 of 48

Cabozantinib minimally inhibits TRK, specifically TRKB, but it has not been approved in this
setting. Crizotinib, ponatinib and nintedanib show different affinity against TRK.
Resistance mechanisms to TRK inhibitors published so far essentially lie in secondary mutations
of the kinase domain (G595R, A608D and G667S in TRKA, G623R and G696A in TRKC). LOXO-195,
TPX-0005 and ONO-539055 are next-generation TRK inhibitors developed to potentially overcome
these acquired resistances, showing promising activity in in vitro assays [188,194].
The following table briefly summarizes the main ALK, ROS1 and NTRK inhibitors developed and
under development to date (Table 3).

Table 3. ALK, ROS1 and NTRK inhibitors, target and main clinical indications or trials.

Approved Clinical Indications or

Name (Code) Trade Name Targets
Clinical Trial Study
Crizotinib (PF 2341066) Xalkori ALK, MET, ROS1 NSCLC
Ceritinib (LDK378) Zykadia ALK, IGF-1R, ROS1 NSCLC
Alectinib (CH5424802)Alecensa ALK, RET NSCLC
Brigatinib (AP 26113) Alunbrig ALK, ROS1, EGFR NSCLC
Lorlatinib (PF-06463922) Lorbrena ALK, ROS1 NSCLC
ALK, MET, Axl, ABL, EPHA2 LTK, Phase II (NCT01625234) and phase III
Ensartinib (X-396)
ROS1, SLK (NCT02767804) trials for NSCLC
Phase II trial (NCT02920996) for
Merestinib (LY2801653) NTRK, MET, ROS1, FLT3, Axl NSCLC and solid tumors with NTRK
fusion proteins.
Phase I/II trial for solid tumors and
Belizartinib (TSR-011) ALK, TRKA/B/C lymphomas with NTRK
fusion proteins
Solid tumors with NTRK fusion
Entrectinib (RXDX-101) Rozlytrek TRKA/B/C, ROS1
proteins, ROS1-positive NSCLC
Larotrectinib (LOXO-101) Solid tumors with NTRK
TRKA/B/C
Vitrakvi fusion proteins
Phase I/II trial (NCT03093116) for
Repotrectinib (TPX-0005) ROS1, TRKA/B/C, ALK solid tumors with NTRK fusion
proteins and ROS1-positive NSCLC
Phase I trial (NCT02675491) for solid
Taletrectinib (DS-6051b) ROS1, TRKA/B/C tumors with NTRK and ROS1
fusion proteins.
Phase I/II trial (NCT03215511) for solid
Selitrectinib (BAY2731954) TRKA/B/C
tumors with NTRK fusion proteins.
Phase II trial (NCT01225172) for
BMS-754807 TRKA/B, Insulin receptor, MET
breast cancer.

4.6. Fibroblast Growth Factor Receptor (FGFR)

Four highly conserved transmembrane TKR form the FGFR family, corresponding to the FGFR 1, 2,
3, and 4. FGFR dimerization precedes the phosphorylation signal of the tyrosine kinase domain, which,
in turn, leads to signal transduction (RAS/RAF/MAPK, PI3K/Akt/mTOR, etc.). In non-cancer cells,
FGFR is involved in intracellular signaling cascades that regulate functions of homeostasis, embryonic
development, endocrine and repair functions.
FGFR dysregulation has been related to oncogenic processes, especially with tumor progression
and resistance mechanisms to classical chemotherapy. FGFR aberrations have been described in
many tumors (including receptor amplification, mutations, and chromosomal translocations) [195].
FGFR1 amplifications have been found in squamous NSCLC, in SCLC, and in MBC (15% of the
HHRR-positive, 5% of the triple-negative subtype). FGFR2 amplifications have been discovered in
5-10% of gastric cancers and in 4% of triple-negative breast cancer. FGFR mutations are more common
in FGFR2 and FGFR3, predominantly occurring in the ligand-binding and transmembrane domains
of the receptor. The former is found in 10-12% of endometrial carcinomas, in 4% of NSCLC and
gastric cancers, and in 2% of urothelial cancers. FGFR3 mutations are found in approximately 75% of
non-muscle invasive urothelial carcinomas and in 15% of high-grade urothelial carcinomas. Activating
Int. J. Mol. Sci. 2020, 21, 8529 18 of 48

gene fusions have also been described: involving FGFR3, especially in glioblastoma and bladder cancer,
and FGFR2, in approximately 15% of cholangiocarcinomas (and, less commonly, in lung, thyroid,
and prostate cancers).
Currently, many drugs have multikinase inhibitory activity and partially block FGFR, but only
two specifically targeted TKIs against FGFR have been approved by the FDA for clinical use: erdafitinib
and pemigatinib.
Erdafitinib is a pan-FGFR inhibitor. It binds to the receptor, inhibits its phosphorylation
and avoids the signal transduction. Erdafitinib demonstrated clinical efficacy in the BCL2001
study [196]. This open-label and nonrandomized phase II trial tested erdafitinib in 99 patients
with advanced urothelial carcinoma and known FGFR3 mutations and FGFR2/FGFR3 fusions.
Previous immunotherapy was allowed. The ORR was 40% (59% in the immunotherapy-treated
subgroup) and the median PFS was 5.5 months (13.8 months in the immunotherapy-treated subgroup).
Given these positive results, in 2019 erdafitinib became the first-ever FGFR kinase inhibitor approved
for the treatment of patients with advanced or metastatic urothelial carcinoma with susceptible FGFR2
or FGFR3 genetic alterations, after progression on platinum-based chemotherapy.
Pemigatinib is a selective inhibitor of the FGFR types 1, 2 and 3 so, subsequently, inactivates the
related signal transduction pathways. Its safety and antitumor activity were tested in the single-arm
FIGHT-202 trial, which included patients with advanced cholangiocarcinoma who had progressed
to, at least, one previous line of systemic treatment. FGFR2 fusions or rearrangements, other FGFR
abnormalities, and FGFR normal status were included [197]. Surprisingly, among the population
with FGFR2 fusions or rearrangements, 36% of patients responded to treatment and 80% achieved,
at least, stable disease. In the population with other FGF/FGFR alterations, 40% of patients reached
stable disease. Because of these results, in April 2020, the FDA approved pemigatinib for the treatment
of patients with unresectable locally advanced or metastatic cholangiocarcinoma previously treated,
and with FGFR2 gene fusion or rearrangements.
Others multi-kinase inhibitors, such as lenvatinib, nintedanib, pazopanib, ponatinib,
and regorafenib target FGFR with variable affinity, but they have no indication for
FGFR-driven treatment.
The following table briefly summarizes the main FGFR inhibitors developed and under
development to date (Table 4).

Table 4. FGFR inhibitors, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
Erdafitinib (JNJ-42756493)
FGFR1/2/3/4 Urothelial bladder cancer
Balversa
Nintedanib (BIBF-1120) Vargatef FGFR1/2/3 Idiopathic pulmonary fibrosis, NSCLC
Pemigatinib (INCB054828)
FGFR1/2/3 Cholangiocarcinoma
Pemazyre
Under development in SQCLC (NCT03762122), breast cancer
Rogaratinib (BAY 1163877) FGFR1/2/3 (NCT04483505), urothelial carcinoma (NCT03473756), sarcoma
GIST (NCT04595747), and gastric cancer (NCT04077255)
Under development in urothelial carcinoma (NCT03123055,
Vofatamab (B-701) FGFR 3
NCT02401542)
Under development in urothelial carcinoma (NCT04197986),
Infigratinib (BGJ398) FGFR1/2/3 breast cancer (NCT04504331), cholangiocarcinoma
(NCT03773302), and glioblastoma (NCT04424966)
Under development in urothelial carcinoma (NCT04045613),
Derazantinib (ARQ 087) FGFR1/2/3 gastric cancer (NCT04604132) and cholangiocarcinoma
(NCT03230318)
Under development in NSCLC (NCT01824901), breast cancer
AZD4547 FGFR1/2/3 (NCT01202591), gliomas (NCT02824133), urothelial carcinomas
(NCT02546661) and gastro-esophageal cancer (NCT01457846)
Under development in breast cancer (NCT03344536) and other
Debio 1347 FGFR1/2/3
solid tumors (NCT03834220)
Int. J. Mol. Sci. 2020, 21, 8529 19 of 48

4.7. c-KIT
c-Kit is a proto-oncogene present on chromosome 4, which encodes a receptor with tyrosine
kinase activity (KIT receptor). Its ligand is the Stem Cell Factor (SCF). KIT has three domains: one
extracellular, for the ligand binding, one transmembrane, and one cytoplasmic with the tyrosine kinase
function. Physiologically, binding of the SCF ligand allows the dimerization of two KIT receptors and
their autophosphorylation, to initiate downstream signalling.
KIT is physiologically involved in fertility processes, intracellular homeostasis, differentiation
of hematopoietic cells, melanogenesis and development of other cell lines (erythrocytes, mast cells,
interstitial cells of Cajal, and sweat glands cells). Its oncogenic role in gastrointestinal stromal
tumors (GIST) was studied in 1998 by Hirota and colleagues, demonstrating that KIT activating
mutations occurred in the absence of natural ligand [198]. In approximately 80–85%, the KIT
receptor is overexpressed, and this aberration is associated with an activating mutation of the c-Kit
protooncogen [199]. This finding marked a milestone in the history of molecular medicine and set the
basis for targeted therapies.
It is estimated that 80–85% of GISTs show some c-Kit activating mutations. Additional c-Kit
alterations have also been described in various neoplasms: leukemias, melanoma, germ cell carcinomas,
adenoid cystic carcinomas, and other human cancers.
As we mentioned, the gain activity of c-Kit has been associated with activating mutations (mostly)
and overexpression. The main mutations of c-Kit (in GIST) are present in exons 11 (65%), 9 (15%),
17 (2%) and 13 (2%), and they encode distinct molecular regions of the receptor (juxtamembrane
domain, extracellular dimerization domain, activation loop domain, TKI and ATP-binding pocket,
respectively) [199]. However, not all c-Kit mutations are activating. For instance, c-Kit activating
mutations in melanoma induce apoptosis. Characteristically, each neoplasm presents its own c-Kit
mutations, which may vary in the location of the exon and in the type of mutation. It is well known
that those tumors harboring a c-Kit mutation and, as a consequence, boost the TKR function, are those
which can benefit from the targeted therapy.
Imatinib is a potent antineoplastic agent which inhibits the tyrosine-kinase activity of the
Bcr-Abl fusion protein, KIT, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), PDGFR Beta
(PDGFRB), DDR1, DDR2 and CSF-1R. Its structure mimics ATP, so it competitively joins to the
c-Kit binding site, avoiding phosphorylation of the substrate and the consequent signalling cascade.
Imatinib administration in patients with GIST led to a dramatic, rapid and sustained clinical response in
tumors that, historically, were considered “chemo-resistant”. The first trial to test imatinib in advanced
GIST was published by Demetri et al. It was a phase II trial which randomly assigned 147 patients
to receive either 400 mg or 600 mg of imatinib mesylate [200]. In 81.6% of the overall population,
imatinib achieved disease control (53.7% of objective response plus 27.9% of stable disease). Imatinib
was approved in 2001 for the treatment of advanced or unresectable GIST. A long-term analysis
revealed that 18% of patients were still receiving imatinib after 9.4 years of follow-up [201]. At the
2014 annual meeting of the American Society of Clinical Oncology (ASCO), a preliminary analysis of
the SWOG S0033 trial showed that 26% of patients survived 8 years or more, and the estimated 10-year
survival rate was approximately 22% [202].
Proliferation and survival GIST cells are TKR-dependent processes, that is the reason why
these tumors are commonly denominated “KIT addicted”. This phenomenon explains the need for
continuous treatment with imatinib. In this regard, dose increasing has not obtained a clear benefit in
OS [203,204]. A meta-analysis of some studies showed that the presence of an exon 9 mutation was the
only significant predictive factor for benefiting from a higher dose, as the better PFS and OR rates were
reported with 800 mg daily [205].
In the locally advanced setting of GIST, imatinib is approved for the adjuvant treatment of resected
GIST with a high risk of relapse [206].
The vast majority of patients improves with imatinib. However, 5% of patients have primary
resistance to imatinib 400 mg, and 10% will develop early resistance to the treatment, defined as
Int. J. Mol. Sci. 2020, 21, 8529 20 of 48

progression in the first 6 months of therapy [206]. One the one hand, these patients probably do not have
mutations in c-Kit (in that case, within exon 9) or in PDGFRA genes. On the other hand, the acquisition
of resistance to imatinib is basically related to secondary mutations in the c-Kit gen, an event that
usually occurs two years after the treatment starts [203,207]. These resistance mechanisms can be
divided into: KIT primary mutations, KIT ATP-binding pocket secondary mutations, KIT activation
loop secondary mutations, and acquired loss of KIT oncoprotein expression.
Sunitinib and regorafenib are multikinase inhibitory drugs (detailed in the “Vascular Endothelial
Growth Factor Receptor” section), and both are approved for the treatment of advanced GIST after
progression to imatinib. Sunitinib (compared with placebo) showed clinical activity in patients with
refractory GIST in a phase III trial: median PFS was higher with sunitinib (27 weeks vs. 6 weeks),
but not the median OS [207]. There appears to be a greater clinical benefit in patients with the KIT
exon 9 mutation and in patients without the KIT or PDGFRA mutation. The above results allowed its
approval by the FDA in 2009. Later, the FDA approved regorafenib for the treatment of advanced
GIST refractory to imatinib and sunitinib. This was based on the results of the phase III trial GRID,
of 199 patients who progressed on sunitinib: compared to placebo, regorafenib increased median PFS
(4.8 months vs. 0.9 months, HR 0.27), with no apparent improvement in OS [208].
Serrano et al. published (in cell cultures) the variety of acquired resistances in the KIT gen as a
mechanism of resistance to imatinib [209]. In their work, they observed that V654A (exon 13) and
T670I (exon 14) mutations were more sensible to sunitinib, while activating loop mutations D816E and
D820A (exon 17), especially when the primary mutation occurred in exon 11, were more sensible to
regorafenib. Head to head comparisons between sunitinib and regorafenib have demonstrated that
sunitinib was more active to block KIT exon 13 V654A ATP-binding pocket mutant.
Several KIT TKIs are currently under research and some of them have already provided significant
results of safety and efficacy (for example, nilotinib, dasatinib, sorafenib, and pazopanib) [210].

4.8. Platelet-Derived Growth Factor Receptor (PDGFR)

PDGFR is another receptor with tyrosine kinase activity, whose initial researches were born
from a better understanding of the VEGFR functioning. The PDGF constitute a set of polypeptide
growth factors (called A, B, C and D), and which are fundamentally involved in the natural process of
angiogenesis, although they also enhance the growth of fibroblasts, smooth muscle cells and glial cells
derived from platelets. Its main receptor is PDGFR. There are two different types of class III receptors:
Alpha (PDGFRA) and Beta (PDGFRB). Numerous studies have revealed the close correlation between
PDGF and VEGF: most PDGF proteins play an important role in angiogenesis, directly stimulating
endothelial cell proliferation and VEGF secretion. Apparently, the most important function of PDGFB
is to recruit perivascular cells during angiogenesis.
At the tumor level, PDGF is known to take part in autocrine stimulation of cancer cells, angiogenesis
stimulation, and control of interstitial cells growth.
In vitro, constitutive phosphorylation of PDGFRA has been shown to exhibit ligand-independent
kinase activity [211]. Furthermore, the following signalling pattern is very similar to that activated
by KIT.
Six to eight percent of GIST show activating mutations of PDGFRA [212]. The most frequently
mutated exons are 18 (82.5%), 12 (13.7%), and 14 (3.7%) [213]. Exon 18 encodes the activation loop in
the second tyrosine kinase domain. As we mentioned before, imatinib preserves inhibitory activity
against PDGFR, but sensitivity to imatinib in PDGFR-mutated GIST is variable. The intrinsic resistance
of the D842V mutation (substitution) in exon 18 is relevant, which is the most common (62%) and
shows negative response rates: 68% disease progression in the largest study. Other exon 18 mutations
(D846Y, N848K, Y849K) are sensitive to imatinib [214,215]. In conclusion, imatinib is still the initial
therapy for patients with PDGFRA mutant GIST.
In addition to GIST, imatinib is approved for the treatment of recurrent, unresectable, or metastatic
dermatofibrosarcoma protuberans (DFSP). Almost all cases of DFSP have the peculiar chromosomal
Int. J. Mol. Sci. 2020, 21, 8529 21 of 48

translocation t(17:22), which subjects control of PDGFB to the collagen type I Alpha 1 (COL1A1)
promoter [216,217]. This change finally results in constitutive activation of the PDGFB. Several reported
clinical cases, two prospective trials and a systematic review provided high efficacy of imatinib in
unresectable locally advanced or metastatic DFSP (and in the neoadjuvant setting, where some clinical
trials have been published) [218–222].
Avapritinib (BLU-285) is a potent and selective TKI of KIT and PDGFRA, specifically targeting to
some mutations at exons 11 and 17 of KIT, as well as D842 mutations of PDGFR. It is considered the
gold-standard treatment for advanced GIST with D842V mutation. Different doses of daily avapritinib
(300 mg and 400 mg) were tested in the phase I trial NAVIGATOR, which included 56 patients with
advanced D842V-mutated GIST [223]. The reported response rate was 88% and the 2-years OS rate
was 81%. These data justified the FDA approval in January 2020, indicating avapritinib for adults with
metastatic GIST harboring a PDGFRA exon 18 mutation (including D842V mutations).
Ripretinib is a switch dual TKI that inhibits both wild-type and mutant forms of KIT and PDGFRA,
avoiding the switch from inactive to active conformations. The activity range of ripretinib encompasses
exons 9, 11, 13, 14, 17 and 18 mutations (in c-Kit), as well as exons 17 and 18 mutations (in PDGFRA).
Ripretinib also targets the PDGFRA D842V and D816V resistance mutations, and inhibits other
molecules, such as PDGFRB, VEGFR2, TIE2, and BRAF. In the INVICTUS trial, 129 patients with
advanced GIST who had progressed to imatinib, sunitinib and regorafenib, were randomly assigned to
receive ripretinib or placebo [224]. Ripretinib improved median PFS (6 months vs. 1 month, HR 0.15),
ORR (9% vs. 0%) and median OS (15 months vs. 6 months, HR 0.36). These impressive results allowed
its approval by the FDA in May 2020 for the treatment of advanced GIST after progression to 3 or more
previous TKIs (including imatinib).
In addition to the drugs previously mentioned, many other TKIs target PDGFR. Most share
VEGFR inhibition, so they are discussed in the “Vascular Endothelial Growth Factor Receptor” section.
The following table briefly summarizes the main c-KIT and PDGFR inhibitors developed and
under development to date (Table 5).

Table 5. c-KIT and PDGFR inhibitors, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
GIST, CML (Chronic Myeloid Leukemia), ALL (Acute
PDGFR, c-Kit,
Imatinib (STI-571) Glivec lymphocytic leukemia), dermatofibrosarcoma protuberans,
v-Abl
myelodisplasic síndrome, leukemias.
Avapritinib (BLU-285) Ayvakit PDGFR, c-Kit GIST
Ripretinib (DCC-2618) Qinlock c-Kit, PDGFR GIST
c-Kit, PDGFR, Under development in NSCLC (NCT01357395) and other solid
Amuvatinib (MP-470)
Flt3 tumors (NCT00894894)
Dasatinib (BMS-354825) Sprycel c-Kit, Abl, Src CML

4.9. c-MET
MET or hepatocyte growth factor receptor (HGFR) is a TKR distributed along multiple epithelial
subtypes of the organism, and it is essential for embryonic development and tissue repair. Its main
ligand is the Hepatocyte Growth Factor (HGF). In physiologic conditions, MET forms a heterodimer
consisting of an external alpha subunit, a transmembrane beta subunit, and four immunoglobulin-like
domains. The intracellular region of MET contains tyrosine kinase activity. MET has its own
downregulation activity, whose protein sequence (juxtamembrane region) is encoded in exon 14 [225].
Skipping mutations in these loci are characteristic of NSCLC (3% of adenocarcinomas and 20% of
sarcomatous carcinomas) and, molecularly, lead to excessive receptor activation due to reduction of
MET protein degradation (the aim of the juxtamembrane region), promoting a sustained activation
of paracrine and autocrine signals. This event has been associated with a worse prognosis [226].
Amplifications of c-MET and its receptor overexpression have also been described. Such alterations
Int. J. Mol. Sci. 2020, 21, 8529 22 of 48

generate overactivation of various intracellular signalling pathways, such as Stat3, PI3K/Akt, mTOR,
and RAS/MAPK [227].
Several TKI block MET, but only capmatinib specifically targets this substrate. Capmatinib is an
oral TKI which selectively blocks the c-MET protooncogene, preventing both receptor phosphorylation
and signal transduction. The GEOMETRY-mono-1 clinical trial tested capmatinib in 97 patients with
advanced MET-exon-14-skipping mutated NSCLC, reaching an impressive ORR of 68% and a median
PFS of 9.7 months [228]. In the overall capmatinib-treated population, ORR and median PFS were 41%
and 5.4 months, respectively.
Other studies have reported favorable results with capmatinib in this setting [229]. With these
data, capmatinib obtained the FDA approval in May 2020 for the treatment of patients with advanced
NSCLC and a MET exon-14-skipping mutation.
Crizotinib, as explained above, is able to inhibit MET. Some studies have tested crizotinib in
patients with MET exon-14-altered NSCLC, including the skipping-mutated population, with good
results [230–232]. In 2018, the FDA granted Breakthrough Therapy designation for crizotinib for the
treatment of patients with metastatic NSCLC with exon 14 alterations and disease progression on or
after platinum-based chemotherapy.
Cabozantinib (multi-TKI), tepotinib (MET-selective TKI), glesatinib (multi-TKI) and savolitinib
(MET-selective TKI) are other MET inhibitors that are under research in clinical trials [233,234].
The following table briefly summarizes the main MET inhibitors developed and under
development to date (Table 6).

Table 6. MET inhibitors, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
NSCLC, under development in solid tumors harboring
Capmatinib (INC280) Tabrecta MET
MET mutations.
Under development in NSCLC (NCT02864992) and colorectal
Tepotinib (EMD 1214063) MET
cancer (NCT04515394)
Under development in NSCLC (NCT04270591) and other solid
Glumetinib (SCC244) MET
tumors (NCT03457532)
Savolitinib (AZD6094) MET Under development in solid tumors harboring MET mutations.
Under development in clear cell sarcoma (NCT03132155) and
AMG 337 MET
other solid tumors (NCT01253707)
Under development in gliomas (NCT02978261), NSCLC
(NCT04258033), renal cell carcinoma and hepatocellular
Bozitinib (PLB-1001) MET
carcinoma (NCT03655613), and other solid tumors
(NCT03175224)
MET, AXL, Under development in NSCLC (NCT02920996), biliary tract
Merestinib (LY2801653)
DDR1, DDR2 cancer (NCT02711553) and other solid tumors (NCT03027284)
Under development in multiple solid tumors harboring
Tivantinib (ARQ197) MET
MET mutations.
MET, Tie-2,
Foretinib (GSK1363089) Under development in several solid tumors.
VEGFR3
Under development in GIST (NCT02847429), glioma
Crenolanib (CP-868596) PDGFR, FLT3
(NCT01393912), and esophagogastric carcinoma (NCT03193918)

4.10. RET
RET oncogene was identified from studies of T-cell lymphomas DNA. This chimeric gene (due to
recombination of unlinked DNA sequences) encodes a transmembrane fusion protein with tyrosine
kinase intracellular domain characteristically ligated to a cadherine domain in the extracellular region.
Its natural ligands are members of the glial cell-line derived neurotrophic factor (GDNF) family, such as
GDNF, neurturin, artemin, and persephin.
Oncogenic activity of RET was first described on papillary thyroid carcinomas (PTC).
RET rearrangements and the proto-oncogen PTC are present in 5–40% of the cases [235]. This oncogenic
mechanism seems to be dependent on large somatic rearrangements with a variety of activating genes,
which in turn allows the constitutive dimerization of the chimeric proteins RET/PTC. In addition,
Int. J. Mol. Sci. 2020, 21, 8529 23 of 48

point mutations in RET are responsible for the constitutive activation of its tyrosine kinase activity in
inherited MEN-2 syndrome, and in 30–50% of patients with sporadic medullary thyroid carcinoma
(MTC) [236–238].
Selpercatinib is a selective RET inhibitor. It also targets VEGFR1, VEGFR3 and FGFR1-3 (although
it is more specific for RET). Multicohort, phase I/II LIBRETO-001 trial allowed the approval of
selpercatinib in RET-rearranged NSCLC and in advanced RET-mutant MTC and RET fusion-positive
thyroid cancer which require systemic therapy [239–242]. One hundred and forty-three patients with
MTC, treatment-naïve or previously treated with cabozantinib and/or vandetanib, received selpercatinib,
reaching an ORR of approximately 70% and a 12-months PFS rate superior to 80%. In RET-fused
previously treated differentiated thyroid carcinomas (DTC) (N = 19), the ORR was of 79%. In regard to
NSCLC, 39 treatment-naïve patients with RET-rearranged NSCLC achieved an ORR of 85%, including
those with CNS metastatic disease. RET rearrangements have been detected in nearly 1–2% of lung
adenocarcinomas. The confirmatory phase III trials currently undergoing are the LIBRETTO-431
and LIBRETTO-531.
Other RET-specific TKIs, like pralsetinib (BLU-667, which selectively inhibits RET), are being
tested in the phase I dose-escalation and phase II expansion ARROW trial, with promising results,
showing an ORR of 73% in RET-mutant MTC patients and an ORR of 91% in 11 patients with RET-fused
DTC [243]. Interestingly, the TPX-0046, a potent and selective next-generation TKI with activity against
SRC kinase, has emerged as a targeted agent to potentially overcome resistance to first-generation
selective RET inhibitors [244,245].
Alectinib, cabozantinib, lenvatinib, ponatinib, regorafenib, sorafenib, sunitinib and vandetanib
are other potent TKI with nonselective RET inhibitory activity, but have been used to block this
target. At present, sorafenib and lenvatinib are approved in radioiodine refractory DTC [246,247].
Cabozantinib and vandetanib are approved for the treatment of metastatic MTC [248,249].
The following table briefly summarizes the main RET inhibitors developed and under development
to date (Table 7).

Table 7. RET inhibitors, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
Selpercatinib (LOXO-292) Retevmo RET Thyroid cancer and NSCLC
Pralsetinib (BLU-667) Gavreto RET NSCLC
Under development in solid RET-mutated tumors
BOS172738 RET
(NCT03780517)
TAS0953/HM06 RET Under development in solid RET-mutated tumors
RET, Under development in solid RET-mutated tumors
TPX-0046
SRC (NCT04161391)

4.11. Vascular Endothelial Growth Factor Receptor (VEGFR)

VEGFR represent a family of three receptors, named VEGRF1, VEGFR2 and VEGFR3, being the
most important from a biological point of view VEGFR2, as it is mainly expressed in the vascular
endothelium. As we have discussed in previous paragraphs, VEGFR regulates the development
of new blood vessels, in a process called angiogenesis, and in the regulation of its permeability,
as we have discussed before [91–93]. This group of tyrosine kinase inhibitors are commonly called
as antiangiogenics.
The structure of the receptor consists of seven immunoglobulin-like domains, which form the
extracellular part of the receptor; the transmembrane domain and the intracellular tyrosine kinase
domain. The receptor can be activated by different ligands, such as VEGF A/B/C/D, placenta growth
factor (PlGF), parapoxvirus VEGFE, snake venom VEGFF and neuropilins NRP1 and NRP2 [250] or by
mechanical stimulus coming from the environment, in a process called mechanotransduction [251].
Int. J. Mol. Sci. 2020, 21, 8529 24 of 48

This leads to the activation of PI3K/AKT/mTOR pathway, GTPases like RHO and RAC1 and eNOS1,
regulating cell proliferation, survival, motility and vascular permeability [60,93,252].
It is particularly important to point out that most of the drugs listed below are multi-TKI with
affinity to one or more TKRs. The drugs that are currently approved are listed below:
Sunitinib is an inhibitor of PDGFR (α and β), VEGFR (1, 2 and 3), KIT, Fms-like tyrosine kinase
3 (FLT3), CSF-1R and RET. Approved by FDA and EMA for the treatment of advanced GIST (as we
cited before), advanced renal cell carcinoma (mRCC) and progressive, well-differentiated pancreatic
neuroendocrine tumors (pNET). In these indications, sunitinib significantly prolonged TTP and OS in
imatinib-refractory GIST [208], PFS, ORR and OS (patients who crossed over from IFN-α to sunitinib
were censored) in mRCC, compared with IFN-α [253]; and PFS in pNETs in comparison to placebo [254].
Pazopanib shows activity against VEGFR (1-3), PDGFR (α and β), FGFR and cKIT. Approved
by FDA and EMA for the treatment of mRCC and soft-tissue sarcoma who have received prior
chemotherapy. This decision was based on phase III trials that demonstrated superiority against
placebo and non-inferiority against sunitinib [255,256]. In soft tissue sarcoma, approval was based on
the PALLETTE study, which included many different types of soft tissue sarcoma except adypocitic
sarcoma, chondro and osteosarcoma, and DFSP [257].
Tivozanib is a TKI which selectively inhibits VEGFR1-3 at picomolar concentrations. Approved by
EMA for the treatment of mRCC which are VEGFR and mTOR inhibitors-naïve and after progression to
cytokine therapy. This approval was based on the TIVO-1 trial, with benefit in PFS, but not in OS [258].
Axitinib is another selective inhibitor of VEGFR 1-3. Approved by FDA and EMA for the treatment
of mRCC, as monotherapy after progression to a prior treatment based on the AXIS trial in which a
benefit in PFS was observed against sorafenib but did not improve OS [259]. In the first-line setting,
axitinib is approved in combination with pembrolizumab based on the results of the MK426 trial [260]
or with avelumab based on the results of the JAVELIN Renal 100 trial [261]
Cabozantinib is able to inhibit c-MET, VEGFR2, RET, TRKA, TRKB and Axl. Cabozantinib is
approved by the FDA and EMA for the treatment of intermediate and poor risk mRCC in the first-line
setting based on the CABOSUN phase II trial [262], and in all risk groups after progression to another
TKI based on the METEOR phase III trial [263]; in hepatocellular carcinoma after treatment with
sorafenib, under the Cabometyx® brand [264]; and progressive, unresectable locally advanced or
metastatic MTC under the Cometriq® brand [248]. They are not interchangeable as the formulation is
not equal. Cabometyx® comes as a tablet and Cometriq® as a capsule.
Lenvatinib is an inhibitor of VEGFR (1–3), FGFR (1–4), PDGFRα, c-KIT and RET. Approved by
FDA and EMA for the treatment of patients with locally recurrent or metastatic, progressive, radioactive
iodine-refractory DTC [265]; in combination with everolimus, for the treatment of patients with mRCC
following one prior anti-angiogenic therapy [266] and for the first-line treatment of patients with
unresectable hepatocellular carcinoma [267].
Sorafenib shows activity against VEGFR (1–3), PDGFRβ, Flt-3, c-KIT, RET and Raf. Inhibition
of Raf has apoptotic effect in tumoral cells [268,269]. Sorafenib was the first systemic treatment to be
approved for the treatment of advanced hepatocellular carcinoma, based on the SHARP study [270].
It is also approved for locally recurrent or metastatic, progressive, DTC refractory to radioactive iodine
treatment; with benefit on PFS but not in OS and for the treatment of good and intermediate-risk
mRCC after progression to cytokine treatment and [246,271].
Regorafenib: The structure of this compound is similar to sorafenib, but it differs in the addition
of a fluorine atom in the proximal phenyl ring. The activity is similar to Sorafenib being able to
inhibit Raf, TIE-2, PDGFR, VEGFR, RET and CSF-1R [272,273] A Approved by the FDA and EMA
for the treatment of patients with metastatic colorectal cancer who have been previously treated
with, or are not considered candidates for available therapies, which include fluoropyrimidine-based
chemotherapy, an anti-VEGF therapy and an anti-EGFR therapy [274]; locally advanced, unresectable
or metastatic gastrointestinal stromal tumor who have been previously treated with imatinib mesylate
Int. J. Mol. Sci. 2020, 21, 8529 25 of 48

and sunitinib malate, offering benefit in PFS but not in OS [208]; and hepatocellular carcinoma who
have been previously treated with sorafenib [275].
Nintedanib: This drug shows activity against VEGFR (1-3), PDGFR (α and β), FGFR (1-3), TRK,
Flt-3 and RET. Approved by the EMA for the treatment of NSCLC with adenocarcinoma histology,
locally advanced, metastatic of locally recurrent after first line-chemotherapy, based on the result of the
LUME-Lung-1 trial, with modest benefit in PFS and OS in combination with Docetaxel [276].
Vandetanib: Inhibitor of VEGFR, EGFR and RET. Vandetanib is approved by the FDA and EMA
for the treatment of symptomatic or progressive MTC patients with unresectable locally advanced or
metastatic disease [249].
Other antiangiogenics that deserve our attention are apatinib (also known as rivoceranib),
fruquintinib and anlotinib. Apatinib is approved for the treatment of advanced gastric carcinoma in
China based on the results of a phase II trial [277]. The results of a phase III with rivoceranib failed
to show benefit in OS but the result of secondary endpoints as PFS were positive, and the drug is
under FDA evaluation for approval [278]. In a similar way, fruquintinib is approved in China for the
treatment of advanced colorectal cancer and anlotinib for the treatment of non-squamous cell lung
cancer [279,280].
The following table briefly summarizes the main antiangiogenics developed (Table 8) and under
development to date (Table 9). Another table on the main antiangiogenics and their affinity for each
receptor is presented in Appendix B (Table A2).

Table 8. Antiangiogenics, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
Sunitinib (SU011248) Sutent VEGFR1/2/3, FLT3, Kit, PDFGRβ GIST, RCC, pNET
Pazopanib (GW786034) Votrient VEGFR1/2/3, PDGFRα/β, FGFR cKIT. RCC, soft-tissue sarcoma.
Tivozanib (AV-951) Fotivda VEGFR1/2/3 RCC
Axitinib (AG013736) Inlyta VEGFR1/2/3, c-KIT RCC
Cabozantinib (BMS-907351) c-MET, VEGFR2, RET, TRKA, TRKB and
RCC, HCC, MTC
Cabometyx, cometriq Axl.
VEGFR1/2/3, FGFR1/2/3/4, PDGFRα,
Lenvatinib (E7080) Lenvima RCC, DTC, HCC
c-KIT, RET
VEGFR1/2/3, PDGFRβ, Flt-3, c-KIT, RET,
Sorafenib (BAY43-9006), Nexavar HCC, DTC, RCC
Raf
Regorafenib (BAY73-4506) VEGFR1/2/3, Raf, TIE-2, PDGFR, RET,
CRC, GIST, HCC
Stivarga CSF-1
VEGFR1/2/3, PDGFRα/β), FGFR1/2/3,
Nintedanib (BIBF1120) Vargatef NSCLC
TRK, Flt-3 RET
Vandetanib (ZD6474) Caprelsa VEGFR, EGFR and RET. MTC
Apatinib (YN968D1) VEGFR2 Gastric cancer (China)
Fruquintinib (HMPL-013) VEGFR1/2/3 CRC (China)
Anlotinib (AL3818) VEGFR2/3, c-Kit NSCLC (China)
Motesanib (AMG-706) VEGFR1/2/3, c-Kit, RET, PDGFR Under development in several solid tumors
Linifanib (ABT-869) VEGFR1/2, CSF-1R, FLT3, c-Kit Under development in several solid tumors
Under development in NSCLC (NCT02544633) and other
Glesatinib (MGCD-265) VEGFR1/2/3, MET, RON, Tie-2
solid tumors
Cediranib (AZD2171) VEGFR1/2/3, c-Kit, PDGFRβ, FGFR1 Under development in several solid tumors
Multiple clinical trials mainly in renal cell carcinoma
Dovitinib (CHIR-258) FGFR1/2/3, VEGF, c-Kit, FLT3 (phase III), breast cancer, hepatocellular cancer,
endometrial cancer and GIST
Tivozanib (AV-951) VEGFR1/2/3, EphB2, PDGFR Under development in several solid tumors
Vatalanib (PTK787) VEGFR1/2/3, PDGFR, c-Kit Under development in several solid tumors
Brivanib (BMS-540215) VEGFR1/2, FGFR1 Under development in several solid tumors
Apatinib (YN968D1) VEGFR2, RET Under development in several solid tumors
Ponatinib (AP24534) VEGFR2, PDGFR, FGFR1 Under development in several solid tumors
LY2874455 VEGFR2, FGFR1/2/4 Under development in solid tumors (NCT01212107)
Golvatinib (E7050) VEGFR2, MET Under development in several solid tumors
Under development in gastric cancer (NCT00952497,
Telatinib (BAY 57-9352) VEGFR2/3, c-Kit, PDGFR
NCT03817411) and other solid tumors (NCT03175497)
Lucitanib (E-3810) VEGFR1/2/3, FGFR1/2 Under development in several solid tumors
Under development in neuroendocrine tumors
(NCT04579679), thyroid cancer (NCT04524884), biliary
Sulfatinib VEGFR1/2/3, FGFR1, CSF1R
tract carcinoma (NCT03873532) and other solid tumors
(NCT04579757, NCT02549937)
Int. J. Mol. Sci. 2020, 21, 8529 26 of 48

Table 9. Other TKIs, target and main clinical indications or trials.

Name (Code) Trade Name Targets Approved Clinical Indications or Clinical Trial Study
Under development in breast cancer (NCT03854903) and
Bosutinib (SKI-606) SRC, STAT3
other solid tumors (NCT03023319)
MAPK,
Pexmetinib (ARRY-614) Under development in solid tumors (NCT04074967)
Tie-2
Dubermatinib (TP-0903) Axl Under development in solid tumors (NCT02729298)
Under development in breast cancer (NCT03184558),
Bemcentinib (BGB324) Axl pancreatic cancer (NCT03649321), and SNCLC
(NCT03184571)
Axl,
Sitravatinib (MGCD516) Under development in several solid tumors
VEGFR3
Axl, c-MET,
Ningetinib (C31H29FN4O5) Under development in NSCLC (NCT03758287)
VEGFR2

5. Discussion
The development of TKI against different TKR has become an important part of progress in the
landscape of cancer treatment and has opened a stimulating field of research in precision oncology.
The increasing availability of genomic, transcriptomic, proteomic and metabolomic data has offered us
a broad vision of the heterogeneity and complexity of an individual tumor, driving us to an era of
molecular-based therapy over a histology-based one [281].
There is a growing tendency to perform a tumor-agnostic strategy to treat cancer. This consists
on initial screening with next-generation sequencing (NGS) to detect multiple potential targets for
specific molecules. We can distinguish between basket and umbrella trials: In the former, patients are
selected based on the same molecular alteration in spite of the tumor origin, whilst in the latter, they are
selected based on the same tumor type or location and then subdivided depending on the molecular
alteration identified. The tumor-agnostic strategy allows us to offer a gene alteration-targeted therapy
in different kind of tumors or diverse therapies within the same histology. One of the main limitations
is that the same driver mutation may require different therapeutic approaches among diverse cancer
types because of the specificities in resistance mechanisms. It is important to underline the complexity
of designing this type of studies, the high costs involved, the difficulty in recruiting patients and the
accomplishment of regulatory agencies requirements for the drug approval [281–283].
One example of a basket trial is the NCI-MATCH (the NCI’s Molecular Analysis for Therapy
Choice): a non-randomized phase II study for advanced refractory solid tumors, lymphomas, or
multiple myeloma who have progressed to previous treatment. They are subdivided into 37 substudies
depending on the identified druggable mutation, showing interesting preliminary results in terms
of ORR. Another interesting basket trial is the NCI-MPACT (the NCI’s Molecular Profiling-Based
Assignment of Cancer Therapy), a phase II study for advanced refractory solid tumors which are tested
for 20 genes belonging to three molecular pathways broadly known to be involved in tumorigenesis
-MAPK, PI3K and DNA repair- and then randomized 2:1 to different therapies depending on the
mutation found. No results have been posted to date (NCT01827384) [283,284].
The idea of giving the right treatment for the right patient at the right time has still a long way
ahead to be validated by positive results in the basket or umbrella-designed trials due to the limitations
presented above. To date, there are 3 FDA-approved tumor-agnostic therapies: pembrolizumab in the
case of microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) and, very recently,
also for mutational burden-high (TMB-H) (≥10 mutations/megabase (mut/Mb)) solid tumors and
larotrectinib or entrectinib for patients with unresectable or metastatic solid tumors with NTRK fusion.
Other candidates are selpercatinib or pralsetinib based on the results in lung and thyroid RET-altered
cancers. [285].
It is essential to promote the research of targeted gene panels and the analysis of tumor tissue and/or
circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) through liquid biopsies.
Int. J. Mol. Sci. 2020, 21, 8529 27 of 48

Furthermore, reliable predictive biomarkers to foresee the response or resistance to certain therapies are
becoming critical in order to choose the correct treatment for each patient. All these studies are available
through NGS techniques and the difference lies in the quantity of genes analysed: approximately 20 to
500 genes in panels, 22,000 human protein-coding genes in whole-exome sequencing (WES) and 3.3
billion bases of the human genome in whole-genome sequencing (WGS) [281,286].
The improvement of technology has led to consider comprehensive genomic profiling (CGP)
of tissue through NGS as the standard of care for sequencing multiple tumor samples. The clinical
potential of NGS, with its short- and long-read applications, covers from diagnostic to prognosis by
using small pre-surgical biopsies or even fine-needle-aspirations. It is not only important its utility in
treatment, by identifying drug targets, but also the resistance alterations. These applications have been
incorporated to different clinical studies at different stages: initial screening, disease progression or as
longitudinal monitoring of molecular variations. This is possible since NGS may be used in tumor
biopsies but also in ctDNA or CTCs, which helps to avoid monitoring the mutations with repeated
biopsies and the subsequent risks and discomfort that this causes to patients [281,287].
Initially, TKIs were very unselective, addressing several different TKRs. This is known as
multi-TKIs (MKI), which can range from targeting a small number of kinases to being highly
promiscuous. It is thought that inhibiting different pathways at the same time would provide
superior efficacy and safety profiles. Although this approach remains to be true, novel selective TKIs
have been developed in the past years in order to avoid the off-target effects of non-selective MKIs,
and, also, hoping that specifically targeting driver mutations can result in better outcomes and less
toxicity when compared to non-selective MKIs. For instance, preliminary results from selective RET
inhibitors have shown impressive results in lung cancer and MTC [288] However, the use of highly
selective TKIs does not always lead to tumor response, and even when it does, a considerable number
of patients relapse due to the appearance of acquired resistance mutations. This is why there is still a
constant need of research in order to overcome this resistance so that patients can be offered subsequent
treatment lines.
There is a physiologic rationale to combine immunotherapy with TKIs in cancer treatment.
Angiogenesis inhibition seems to potentiate the antitumor activity of checkpoint inhibitors by increasing
immune cells infiltration into tumors. In murine models, simultaneous inhibition of VEGF and
PD-1 synergistically increased T-cell infiltration into tumor microenvironment. The initial efforts to
combine TKIs and immunotherapy, which took place in metastatic RCC, yielded poor results due
to unacceptable toxicity. The arrival of more selective VEGF inhibitors, such as axitinib, allowed
the design of randomized clinical trials combining this molecule with checkpoint inhibitors, such as
pembrolizumab and avelumab, showing a benefit in overall survival when compared to standard
treatment, therefore becoming the treatment of choice in patients with metastatic RCC [260,261,289–293].
The combination of TKIs with chemotherapy has also been explored. Despite showing promising
results in preclinical models, the combination of TKIs with chemotherapy (platinum-based) in
EGFR-mutated non-small cell lung cancer, does not seem to be beneficial in terms of PFS and OS [294].
The combination of TKIs has also being evaluated in the past few years. Cell line models suggested
that using specific TKIs could enhance intracellular concentrations of a targeted TKI, hence overcoming
resistance and increasing efficacy. This rationale has been put to test in several clinical trials, especially
in EGFR-mutated NSCLC, with the aim to overcoming or delaying drug resistance in these patients.
In a recent study, third-generation EGFR inhibitor osimertinib—current first-line treatment in this
setting—has been combined with first-generation gefitinib, showing a similar toxicity profile, with
survival outcomes yet to be published. In the same subset of patients, the phase Ib TATTON trial
tested the combination of osimertinib with MEK1/2 inhibitor selumetinib or MET inhibitor savolitinib,
showing acceptable tolerability [295–298]. Cancer is a dynamic reality and tumor drivers to be targeted
may change. Biologic heterogeneity found within the primary tumor and metastases is another
challenge to face for tailor-made treatment.
Int. J. Mol. Sci. 2020, 21, 8529 28 of 48

6. Conclusions
TKRs play a key role in the molecular pathways that lead to cell mitosis and differentiation.
Therefore, genetic alterations in TKRs provide the cell with a survival advantage, which leads to
tumorigenesis. The inhibition of these targets using specific agents has proven to be successful in
cancer treatment. In recent years, increasingly selective molecules have been developed showing very
satisfactory survival outcomes in clinical trials. Moreover, combination regimes (with other TKIs or
immunotherapy) seem to have a synergistic effect and to further ameliorate the survival of cancer
patients. It is imperative to standardize tumor genotyping in order to offer the most selective and
effective treatment against specific molecular targets.

Author Contributions: J.E.-V.; J.J.S.-C.; J.P.; M.S.R.-G.; I.O.-M.; J.T.-J.; T.A.-G. and J.M.-C. contributed substantially
for conceptualization, methodology, validation, investigation and writing. Supervision: A.C.; T.A.-G.; J.M.-C.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors wish to thank the patients who participated and are currently participating in
the studies mentioned and their families.
Conflicts of Interest: T.A.-G. declares consultant, advisory or speaker role for IPSEN, Adacap, Roche, Pfizer,
Sanofi, Bayer, Janssen, Astellas, BMS, MSD, Astrazeneca. J.M.-C. declares consultant, advisory or speaker role for
IPSEN, Roche, Pfizer, Sanofi, Janssen, BMS. The rest of authors declare no conflict of interest.

Abbreviations
MDPI Multidisciplinary Digital Publishing Institute
DOAJ Directory of open access journals
TLA Three letter acronym
LD linear dichroism
Int. J. Mol. Sci. 2020, 21, 8529 29 of 48

Appendix A
Table A1. Main TKR mutations distributed by various types of solid and hematological tumors. [124,126,151,152,156,171,179,195,198,199,237,238,299–302].

TKR Mutations Most Representative Tumors Other Described Alterations in Tumors

EGFR (primary mut.)
Colorectal carcinoma (EGFR S464L, G465R, I491M, EGFR S492R)
Exon 19 (del), exon 21 (L858R, L861Q), exon 18 (G719X), exon 20
NSCLC (15–20%) Pancreatic carcinoma
(ins)
NSCLC Head and neck cancer (EGFR amplifications)
EGFR (secondary mut.)
Glioma (EGFR A289V, R108K, G598V, T263P)
Exon 20 (T790M), D761Y, T854A, L747S
Breast cancer (HER2 amplification negative; HER2 D769Y, D769H, R896C, V777L,
HER2 mut.
NSCLC (1–3%) G309E, V842I, S310F, S310Y, C311R; HER2 inframe deletion (755-759), inframe
Exon 20 (ins)
insertion (780GSP), inframe insertion (781GSP)
Anaplastic large cell lymphoma (other not ALK L1196M; ALK F856S, A348D)
ALK rearrangement
Glioblastoma (ALK fusions)
EML4-ALK, KIF5B-ALK, KLC1-ALK, HIP1-ALK
NSCLC (5–6%) Inflammatory myofibroblastic tumors (ALK C1156Y)
ALK (secondary mut.)
NSCLC (5–6%) Diffuse large B-cell lymphoma (ALK G1269A)
Exon 23 (L1196M, S1206Y), exon 25 (G1269A), exon 22 (I1171T),
Esophageal squamous cell carcinoma (ALK L1152R)
V1180L, F1174L
Colorectal carcinoma (ALK F1245C)
Cholangiocarcinoma (other not FGFR2 fusion; FGFR2 N549H, V564F, K659M,
NSCLC (Squamous) (FGFR1 amplif., 17%) L617V, K641R, R565A)
SCLC (FGFR1 amplif., 6%) Bladder cancer (oncogenic mutations others not FGFR3 fusions)
Breast cancer (FGFR1 amplif., 15%) Endometrial carcinoma (FGFR2 M536I, M538I, I548V, N550, E566G, L618M, K660E,
FGFR1-4
Cholangiocarcinoma (FGFR2 fusions) S252W, N550K, V565I)
Urothelial carcinoma (FGFR2-3 fusions) NSCLC (Squamous) (other not FGFR1 amplification; FGFR2 W290C, S320C, K660;
Gastric cancer (FGFR2 amplif., 5–10%) FGFR3 S249C, G691R)
Myeloma multiple (FGFR3 K650, Y373C, V555M)
AML (KIT D816V, N822K)
c-KIT mut. Systemic mastocytosis (KIT D816V)
GIST (80–85%)
Exon 11, exon 9, exon 17, exon 13 Thymic carcinoma (KIT H697Y)
Cutaneous melanoma (KIT amplif.)
Cutaneous melanoma (PDGFRA V658A, P577S, R841K, H845Y, G853D)
PDGFR mut. Renal carcinoma (PDGFRA overexpression)
GIST (6–8%)
Exon 18, exon 12, exon 14 Myelodisplasic syndrome (PDGFRA fusion)
Dermatofibrosarcoma protuberans (translocation t(17:22))
Renal carcinoma (MET H1112R)
MET skipping mut.
NSCLC (3%) NSCLC (MET amplif.)
Exon 14
Colorectal, gastric carcinoma (MET amplif.)
NSCLC, cholangiocarcinoma, gastric carcinoma (ROS1 NSCLC (ROS1 G2032R)
ROS1
fusions, 1–2%) Inflammatory myofibroblastic tumor
RET Papillary thyroid carcinoma (RET rearrangement, 5–40%) MEN-2 syndrome, sporadic medullary thyroid carcinoma (RET mutations)
Int. J. Mol. Sci. 2020, 21, 8529 30 of 48

Appendix B

Table A2. Inhibitory concentrations (IC50) in nmol for targets with multi-targeted tyrosine kinase inhibitors [303–313].

VEGFR1 VEGFR2 VEGFR3 PDFGRa PDGFRb c-KIT RET Flt-3 MET CSF-1R FGFR1 FGFR2 FGFR2 FGFR4 Axl
Sunitinib 2 9 17 7 8 7 10 250 - 890 830 - - - 9
Pazopanib 10 30 47 71 81 74 - - - - 140 - 130 80 -
Tivozanib 30 6 15 40 49 78 - 2550 550 - 525 - 1250 1400 -
Axitinib 0.1 0.2 0.1–0.3 5 1.6 1.7 >1000 >1000 - 73 100 - - - -
Cabozantinib - 0.035 - - 234 4.6 5.2 11.3 1.3 - 5294 - - - 7
Lenvatinib 22 4 5.2 25 39 0.7 12 - 1900 - 46 - - 43 -
Sorafenib 26 90 20 - 57 68 - 33 - - 580 - - - -
Regorafenib 13 4.2 46 - 22 7 1.5 - - - 202 - - - -
Nintedanib 34 21 13 59 65 - - 26 - - 69 37 100 610 -
Vandetanib - 40 110 - - - 100 - - - - - - - -
Int. J. Mol. Sci. 2020, 21, 8529 31 of 48

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