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Abstract
Hematopoiesis, the process by which the hematopoietic stem cells
and progenitors differentiate into blood cells of various lineages,
involves complex interactions of transcription factors that modulate
the expression of downstream genes and mediate proliferation and
differentiation signals. Despite the many controls that regulate
hematopoiesis, mutations in the regulatory genes capable of promot-
ing leukemogenesis may occur. The FLT3 gene encodes a tyrosine
kinase receptor that plays a key role in controlling survival, prolifera-
tion and differentiation of hematopoietic cells. Mutations in this gene
are critical in causing a deregulation of the delicate balance between
cell proliferation and differentiation. In this review, we provide an
update on the structure, synthesis and activation of the FLT3 receptor
and the subsequent activation of multiple downstream signaling path-
ways. We also review activating FLT3 mutations that are frequently
identified in acute myeloid leukemia, cause activation of more com-
plex downstream signaling pathways and promote leukemogenesis.
Finally, FLT3 has emerged as an important target for molecular thera-
py. We, therefore, report on some recent therapies directed against it.
Introduction
Recent advances in cell and molecular biology have revolutionized
our understanding of normal hematopoiesis. It is generally accepted
that survival, proliferation and differentiation are the three fundamen-
tal cellular processes that define normal hematopoietic cells. In acute
myeloid leukemia (AML), a heterogeneous disorder of the hematopoi-
etic progenitor cells, abnormalities have been identified that affect the
balance between cell proliferation, survival and differentiation. These
abnormalities result in the expansion of an abnormal stem cell clone.
Over the last few years, several studies have concluded that leukemo-
genesis is a process in which multiple events involving independent
genetic alterations in proto-oncogene or suppressor genes, together
with epigenetic or environmental factors, contribute to the develop-
ment of the full malignant phenotype.1The genes involved in leukemo-
genesis are related to various cellular functions, including ligand-
receptor interaction, signal transduction, intracellular localization, cell
cycle control and apoptosis. In detail, the oncogenic events that under-
lie the onset of leukemia are often divided into two classes of muta-
tions, following the two-hit model of leukemogenesis. Class I muta-
tions confer a proliferation and survival advantage to blast cells, typi-
cally as a result of aberrant activation of signaling pathways.
Otherwise, the class II mutations lead to an impaired differentiation
via interference with transcription factors or co-activators. The coop-
eration between these two main classes of mutations leads to the
emergence of leukemic cells capable of proliferation but not differen-
tiation.2In particular, epidemiological and genetic data have shown
that the majority of AML present more than one recurrent alteration,
including point mutations, gene rearrangements and chromosomal
translocations. Furthermore, it has been suggested that point muta-
tions in transcription factors are sufficient to confer a proliferative and
survival advantage to the leukemic clone.
Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene repre-
sent one of the most frequently identified genetic alterations that dis-
turb intracellular signaling networks with a role in leukemia patho-
genesis. FLT3 is a member of the class III receptor tyrosine kinase fam-
ily that also includes platelet-derived growth factor receptor (PDGFR),
macrophage colony-stimulating factor receptor (FMS) and stem cell
factor receptor (c-KIT), with which it shares the same structure.
Activating mutations in the FLT3 gene, including internal tandem
duplications (ITDs) and missense point mutations in the tyrosine
kinase domain (TKD), are the molecular abnormalities most frequent-
ly observed in the blood cells of AML patients. These mutations lead to
the overexpression or constitutive activation of the tyrosine kinase
receptor. Many studies indicate that patients with FLT3 mutations
have a worse prognosis than patients without FLT3 alterations. In par-
ticular, the presence of an FLT3-ITD correlates with an increased risk
of relapse and impaired overall survival. The effect on AML prognosis
Correspondence: Tiziana Grafone, Department of Onco-Hematology,
Fondazione di Ricerca e Cura Giovanni Paolo II, Campobasso, Italy.
Tel. +39.0874.312377 - Fax: +39.0874.312479.
E-mail: tiziana.grafone@rm.unicatt.it; tizianagrafone@yahoo.it
Key words: FLT3 receptor, internal tandem duplication, acute myeloid
leukemia, signal transduction networks, inhibitors.
Acknowledgements: the authors would like to thank the Italian Association
against Leukemia, Lymphomas and Myeloma (AIL) Campobasso for its con-
tinued support.
Conflict of interests: the authors declare no potential conflict of interests.
Received for publication: 20 December 2011.
Revision received: 6 March 2012.
Accepted for publication: 13 April 2012.
This work is licensed under a Creative Commons Attribution
NonCommercial 3.0 License (CC BY-NC 3.0).
©Copyright T. Grafone et al., 2012
Licensee PAGEPress, Italy
Oncology Reviews 2012; 6:e8
doi:10.4081/oncol.2012.e8
An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid
leukemia: biology and treatment
Tiziana Grafone,1Michela Palmisano,2Chiara Nicci,1Sergio Storti1
1Department of Onco-Hematology, Fondazione di Ricerca e Cura Giovanni Paolo II, Campobasso;
2San Raffaele Vita-Salute University, School of Molecular Medicine, Milano, Italy
[page 64] [Oncology Reviews 2012; 6:e8]
Oncology Reviews 2012; volume 6:e8
Review
of the FLT3-TKD mutation has not yet been clearly defined; in several
studies, the FLT3-TKD mutation did not seem to affect outcome while
other investigations showed opposite results.3In addition to cytogenet-
ic abnormalities detected at diagnosis, which are the most important
prognostic factor, FLT3 mutations are a significant independent prog-
nostic factor that can influence outcome in terms of survival and dura-
tion of complete remission, even in patients with a normal karyotype.4
Because of its prognostic relevance, the current World Health
Organization (WHO) classification recommends the assessment of
FLT3 mutation status for all patients with AML, particularly in cytoge-
netically normal AML. However, because FLT3 frequently accompanies
other genetic lesions, it is not included as a distinct entity in the 2008
revised WHO classification of myeloid neoplasms.5
Aberrantly activated FLT3 kinase is considered an attractive thera-
peutic target in AML. Therefore, specific small molecule FLT3 tyrosine
kinase inhibitors (TKI) have been developed for AML therapy and are
currently under investigation in the hope that they may revolutionize
AML treatment.
Structure of the FLT3 receptor
The FLT3 receptor (Fms-like tyrosine kinase 3), also known as FLK2
(fetal liver tyrosine kinase 2), STK-1 (stem cell tyrosine kinase 1) or
CD135, is encoded by the FLT3 gene located on chromosome 13q12.6-7
This gene consists of 24 exons and covers approximately 96 kb; the
exact size is unknown because of the presence of a large intron (>50
kb) located between exons 2 and 3.8-10 The length of the transcript is
3.7 kb and it contains a pseudogene in the open reading frame of 2979
bp.8The protein encoded is a transmembrane receptor of 933 amino
acids with a molecular weight of 155-160 kDa that belongs to the class
III family of receptor tyrosine kinase (RTK).8,11 The structure consists
of four regions: i) a N-terminal extracellular region (541aa) consisting
of five immunoglobulin-like domains, of which the three most distal
from the plasma membrane are involved in ligand binding, while the
proximal domains are involved in dimerization of the receptor; ii) a
transmembrane portion (21aa); iii) a juxtamembrane (JM) domain;
and iv) an intracellular C-terminal region (431aa) with a split kinase
domain. The two substructures of this domain are called N-lobe and C-
lobe and are connected by an interkinase domain. These lobes consist
of a TKD and are also indicated as first tyrosine kinase (TK1) and sec-
ond tyrosine kinase (TK2) domain, respectively9,12 (Figure 1). The
extracellular region is highly glycosylated and contains a binding
domain with high affinity for its ligand (FLT3 ligand or FL).13 The non-
glycosylated isoform has a molecular weight of 130-143 kDa and is not
associated with the plasma membrane.14
Synthesis and activation of FLT3
In normal bone marrow, FLT3 is selectively expressed on CD34+
hematopoietic stem cells and immature hematopoietic progenitors,
including the B-lymphoid progenitors, the myeloid precursors and
monocytes, but it is virtually absent from the erythroid progenitors,
playing an important role in the regulating processes of early
hematopoiesis.11,15-18 Furthermore, it has been experimentally shown
that CD34+ bone marrow cells can give rise to two different popula-
tions according to the level of FLT3 expression: cells expressing high
levels of the receptor mature in the colony forming unit granulocyte-
macrophage, while cells that express low levels preferentially mature in
the burst forming unit erythroid colonies.19 FLT3 is also expressed in
other hematopoietic organs such as spleen, liver, thymus, lymph nodes
and placenta, and blood-forming organs such as gonads and brain.20-22
The processes of activation, internalization and degradation of FLT3
occur in a similar manner to other members of the same class of RTKs.
The FLT3 gene transcription produces an mRNA that is translated into
the FLT3 protein. This passes through the compartments of endoplas-
mic reticulum and Golgi undergoing glycosylation, with the addition of
residues of N-acetylglucosamine, galactose, fucose and mannose to N-
terminal code, which promotes the localization of the receptor on the
plasma membrane.
Wild-type FLT3 (FLT3-WT) remains in the inactive monomeric form;
the binding of its ligand (FL or FLT3 ligand), probably in dimeric form,
induces receptor dimerization that promotes the phosphorylation of the
tyrosine kinase domain, activating the receptor and consequently the
downstream effectors. Once activated, the dimerized receptor bound to
the FL is rapidly internalized and degraded. The receptor remains in the
inactive conformation thanks to the steric inhibition mediated by the
JM domain. Upon FL binding, the receptor dimerization leads to the
exposure of phosphorylated acceptor site of the tyrosine kinase domain.
As previously indicated, the TKD of the FLT3 kinase is characterized by
two catalytic lobes: N lobe (TK1) and C lobe (TK2), connected by a flex-
ible peptide that can insert significant rotational movement of the
kinase domain. When the N lobe is rotated away from the carboxy-ter-
minal kinase domain, the receptor is in the catalytically inactive form. If
the N lobe is rotated towards the C lobe, the key catalytic residues of
[Oncology Reviews 2012; 6:e8] [page 65]
Figure 1. Schematic presentation of FLT3 receptor monomer.
ECD, extracellular domain; PM, plasma membrane; CP, cyto-
plasm; TM, transmembrane domain; JM, juxtamembrane
domain; TK1, first tyrosine kinase domain, N-lobe; KI, kinase
insert; TK2, second tyrosine kinase domain, C-lobe; AL, activa-
tion loop.
[page 66] [Oncology Reviews 2012; 6:e8]
both lobes are aligned and the kinase adopts an active conformation.12
The activating loop (A-loop), near to the C lobe, is a long flexible peptide
segment which folds between the two catalytic domains and contains 1-
3 tyrosine residues that can serve as a phosphorylation site (Figure 2).
When these tyrosines are not phosphorylated, the A-loop adopts a closed
conformation, folding back into the space between the two N and C lobes
and blocking access for peptide substrates and the binding of the adeno-
sine triphosphate (ATP). In the active state, when the tyrosines are
phosphorylated, the A-loop adopts an open conformation and does not
compromise the entry of the ATP and substrates.
Also, the state of phosphorylation of two key tyrosine residues of the
JM domain is implicated in the activation and regulation of enzymatic
activity of the receptor. The JM domain adopts a wedge shape that sta-
bilizes the inactive state by establishing strong interactions with the
rest of the molecule and preventing the rotation of the N lobe to C lobe.
In the inactive form, the JM domain is close to the A loop which, there-
fore, cannot adopt an open conformation (Figure 2).
The binding of FL promotes the dimerization and the concomitant
juxtaposition of the cytoplasmic domain of the FLT3 receptor. Once the
dimer is formed, the trans-phosphorylation of specific tyrosine
residues of the JM domain takes place, allowing activation of the
kinase. When at least one of the JM tyrosines is phosphorylated, this
domain cannot fold properly to maintain the inactive state, and the
activating loop adopts an open conformation. This exposes the phos-
phate acceptor site residues of the catalytic domain. This conformation
favors the entry of the ATP and the binding of the substrates, followed
by the phosphorylation and the activation of downstream proteins. The
protein dimerization stabilizes these conformational changes and pro-
motes further activation of the receptor.
On the other hand, the kinase activity is negatively modulated by
the tyrosine phosphatase that dephosphorylated the JM domain:
when the phosphatase removes the phosphates, the JM domain is in
the proper position around the kinase domain and correctly fits on its
autoinhibitory site.12,23 The phosphorylation of FLT3 occurs after 5-15
min by the binding of FL, and the receptor-ligand complex is internal-
ized and degraded after 20 min of stimulation. The frequency of FLT3
production, its rapid degradation and the downstream effects partic-
ipates in a complex feedback loop that regulates normal receptor
activity.20
FLT3 ligand
The exposure of FLT3 at its ligand is a crucial step in regulating the
activity of the receptor. FL is a type 1 transmembrane protein, a mem-
ber of a small family of growth factors that stimulate proliferation and
differentiation of hematopoietic cells.24-25 There are three known iso-
forms: i) a 30-kDa glycoprotein with four transmembrane alpha-helix,
an aminoterminal domain and a small cytoplasmic region, which is the
most commonly expressed form, that binds to and activates the recep-
tor; ii) a soluble, biologically active form, generated by the cut of the
transmembrane isoform; and 3) a soluble form generated by alternative
splicing that creates a premature stop codon.12,21-23,26 FL is expressed by
most tissues, including blood-forming organs (spleen, thymus, periph-
eral blood and bone marrow), prostate, ovary, kidney, lung, colon, small
intestine, testes, heart and placenta, with a higher level of expression
in peripheral blood mononuclear cells. The wide expression of FL is in
contrast to the limited pattern of expression of FLT3, suggesting that
FLT3 expression is the limiting step in determining the tissue-speci-
ficity of receptor activation.27-29
FL activity is minimal when it acts alone, but it strongly synergizes
Review
Figure 2. Activation of FLT3. A) Inactive conformation; B) Active conformation. Juxtamembrane domain (yellow), activation-loop
(green), catalitic loop (red). P, phosphorylation site; N, N-lobe, TK1 domain; C, C-lobe, TK2 domain; ITD, internal tandem duplica-
tions; JM-PMs, point mutation in the juxtamembrane domain; TKD, point mutation in the tyrosine kinase domain; FL, FLT3 ligand.
with a number of other cytokines. In acute myeloid leukemias, FLT3
stimulation by its ligand promotes the proliferation and survival of
leukemic blasts that express the receptor.21,30-31 The hematopoietic pro-
genitors can be stimulated by the local secretion of FL or from direct
contact with FL expressed on the surface of mononuclear cells, indicat-
ing the possibility of controlling the FLT3 activation through a
paracrine loop or an autocrine feedback control.13,20
Signal transduction networks activated by
FLT3-WT
FLT3 has a crucial role in many regulatory processes of hematopoi-
etic cells, including phospholipid metabolism, transcription, prolifera-
tion, apoptosis, and establishing a connection with the RAS pathway; it
is also involved in leukemogenesis14,20,30-33 (Figure 3). FLT3-WT causes
the activation of signal transduction networks mainly through
phospatidylinositol-3-kinase (PI3K) and the cascade of RAS, supporting
the activation of AKT (protein kinase B, PKB), signal transducer and
activator transcription factor (STAT) and extracellular-signal regulated
kinase 1 and 2 (ERK1/2).34 Activated FLT3 is associated with growth
factor receptor bound protein-2 (GRB2), a linker protein that binds to a
diverse repertoire of signaling proteins, through SHC (Src homology 2
containing protein) via the SH2 domain. The adaptor protein GRB2 also
contains an SH3 domain capable of binding proline-rich residues of
other proteins, such as SOS (guanine nucleotide exchange factor),
stimulating the dissociation of GDP and the subsequent binding of GTP
to RAS. This leads to activation of RAS, which stimulates downstream
effectors RAF, MAPK/ERK kinase and RSK (90-kDa ribosomal protein
S6 kinase). These effectors activate CREB (cyclic adenosine
monophosphate response element-binding protein), ELK and STAT
leading to the transcription of genes involved in proliferation. Both the
PI3K/AKT and RAS/ERK pathways are often activated in parallel and
probably interact with many other anti-apoptotic and cell cycle proteins,
such as WAF1, KIP1 and BRCA1. Activated FLT3 transduces the signal
through the association and phosphorylation of various other cytoplas-
mic proteins, including PLCγ1 (phospholipase Cγ1), regulatory protein
of the metabolism of phosphatidylinositol, VAV and FYN.
PI3K activity is probably regulated through various interactions
between FLT3, adaptor proteins SHC, one or more protein SHIP (SH2-
containing inositol 5-phosphatase).35-36 Although PI3K activity is
involved in the metabolism of phospholipids, it can also act as a regu-
lator of proliferation in a competitive binding of phosphorylated pro-
tein SHC, SHP2 (SH2-containing phosphotyrosine phosphatase 2) and
GRB2. The p85 subunit of PI3K kinase is associated with a protein of
100kDa and 120kDa represented by GAB2 (GRB2-associated binder 2)
and CBL (casitas B-lineage lymphoma) proto-oncogene and, finally,
with CBL-b, in a complex with SHP2 and SHIP. Receptor stimulation
results in the phosphorylation of SHC proteins and CBL, with the for-
mation of a complex with p85 subunit, and induces tyrosine phospho-
rylation of GAB1 and GAB2 and their association with SHP2 and
GRB2.37 The PI3K stimulates downstream proteins such as PDK1 (pro-
[Oncology Reviews 2012; 6:e8] [page 67]
Review
Figure 3. Signaling pathways activated by FLT3-WT.
[page 68] [Oncology Reviews 2012; 6:e8]
tein kinase 1-dependent phosphoinositol-3) and AKT that phosphory-
lates mTOR (mammalian target of rapamycin), which activates the
transcription of key genes through the activation of p70S6Kinase
(p70S6K) and the inhibition of 4E-BP1 (eucaryotic transcription initi-
ation factor 4E-binding protein).38-39 As p70S6K and 4E-BP1 regulate
protein translation, activation of p70S6K results in an increase in
overall protein synthesis and induction of cell survival. In addition, the
activation of AKT blocks apoptosis through the phosphorylation of pro-
apoptotic protein BAD (BCL2 antagonist of cell death, Bcl-XL/Bcl-2-
association death promoter). Therefore, the pro-survival function of
FLT3 is mediated by the phosphorylation of the pro-apoptotic BAD pro-
tein, the induction of anti-apoptotic BCL-2, and the prevention of the
induction and activation of pro-apoptotic Bax.40 PI3K may still be
deregulated by low-level expression of PTEN (phosphatase and tensin
homolog deleted on chromosome 10).41 Among the important down-
stream targets of AKT, there are also members of the forkhead tran-
scription factor family, including FoxO3. FoxO3 is involved in the tran-
scription of apoptosis and cell cycle regulatory genes; the phosphoryla-
tion of these factors by AKT inactivates its function. The forkhead
transcription factors control the survival of hematopoietic cells
through their transcriptional activity on gene promoters of pro-apop-
totic protein BIM and FAS-L, and it may influence the cell cycle pro-
gression through the transcription of cyclin dependent kinase
inhibitors, such as p27kip1 and cycline D. Via the PI3K, FL activates
anti-apoptotic signals in hematopoietic progenitors in a cytokine-
dependent manner. The activation of PI3K and AKT is a major down-
stream pathway of intracellular signal transduction by which cytokines
can support the survival of several cell-types.41-43
FLT3 and acute myeloid leukemia
FLT3-WT is expressed at high levels in a spectrum of several hema-
tologic malignancies: 93% of AML cases, almost 100% of B-cell acute
lymphoblastic leukemia (B-ALL), 87% of T-cell acute lymphoblastic
leukemia (T-ALL) and in a small percentage of cases of chronic
myeloid leukemia in blast crisis and chronic lymphoid leukemia. This
suggests that the overexpression of this receptor may play a role in the
survival and proliferation of leukemic cells.44-45 This abnormal expres-
sion is also found in human leukemia cell lines46-47 and, when com-
bined with FLT3 ligand expression, leads to constitutive phosphoryla-
tion/activation of the receptor. These data indicate that the signaling
of FLT3-WT may be important in certain subtypes of leukemia and may
be involved in signaling pathways in an autocrine, paracrine or
intracrine manner.37
In recent years, it has been shown that somatic activating mutations
of the FLT3 gene are the most common genetic abnormalities in AML
and have a significant impact on prognosis.44
About 30% of cases of AML at diagnosis have mutations of the FLT3
gene; in particular, 70% of patients with normal karyotype and 35% of
patients with t(15;17), and the frequency is higher in de novo AML
(26%) than in secondary AML (9%). Female patients are affected more
frequently, and these mutations are associated with hypercellularity
and a higher incidence of recurrence.48
Genetic alterations of FLT3 cause the expression of a constitutively
activated tyrosine kinase receptor in AML blasts, and this activation is
implicated in leukemogenesis, regulating the functional characteris-
tics of leukemic blasts.49 In AML patients, two main types of activating
mutations have been identified: ITD in the region coding for the JM
domain, determined by the insertion of repeated amino acid
sequences, and point mutations that cause amino acid substitution in
the activation loop of the TKD.50-52
Internal tandem duplications
The ITDs are variable duplications of a number of bases that are
multiples of three, preserving the reading frame, in exons 14 and 15,
coding for the juxtamembrane domain.53 These duplications, whose
length can vary from 3 to more than 400 base pairs, result in the inser-
tion of repeated amino acid sequences in variable positions of the JM
domain, and are not identifiable through traditional cytogenetic meth-
ods (Figure 2). Often there is also the insertion of three or six addition-
al base pairs of unknown origin that leads to the addition of one or two
new amino acids before the repeat region.22 The ITDs are formed when
a fragment of the coding region for the JM domain is duplicated and
inserted in direct head-to-tail orientation.20 Most ITDs occur at the 5’
of the JM domain (carboxy-terminal region of the domain) in exon 14;
however, recent research has established that approximately 33% of
ITDs occur within the tyrosine kinase domain.54 In addition, a new
class of activating point mutations in the JM domain (JM-PMs) was
reported in AML patients; although these mutations lead to constitutive
autophosphorylation, their biological and clinical role still remains to
be clarified55-56 (Figure 2).
The ITD mutation results in the ligand-independent dimerization and
phosphorylation of the receptor causing phosphorylation of the WT
receptor expressed by the same cell. While an unmutated receptor in the
JM domain has an -helix conformation that blocks the activation of the
kinase and can inhibit the self-dimerization, the presence of an ITD can
prevent the association between the JM domain and the kinase domain,
exposing it to constitutive activation.44 The presence of the duplication
leads, therefore, to weak auto-inhibitory activity of the juxtamembrana
domain, resulting in a conformational change from an inactive to a cat-
alytically active state, even in the absence of the ligand.12
The conformational change in the JM domain promotes the ligand-
independent dimerization and the constitutive autophosphorylation
and activation of the receptor, resulting in cytokine-independent prolif-
eration and in blocking myeloid differentiation of hematopoietic pro-
genitors due to the inappropriate activation of signal pathways.57 It has
been shown that the ITD and WT kinase receptors regulate downstream
proteins in different ways: the mutated receptor activates the RAS and
PI3K pathways in a manner similar to FLT3-WT. But, in this case, the
activation of STAT5 (signal transducer and activator of transcription 5)
plays a more critical role: STAT5 phosphorylated by FLT3-WT does not
bind to the DNA, cells harboring FLT3-ITD have a high level of STAT5
phosphorylation and they show the binding of this transcription factor
to DNA.20,23,58 Activation of STAT5 is critical for cell growth in associa-
tion with the activation of MAPK, and its anti-apoptotic function is
mediated by transcriptional regulation of cycline D1, BCL-XL,59 PIM
serine-threonine kinases, p21WAF1/CIP1 (inhibitor of cyclin-dependent
kinases) and c-MYC.60-64
Through a mechanism that is still not fully understood, FLT3-ITD is
also able to inhibit the function of silencing mediator of retinoic acid
tyroide hormone receptors (SMRT), a co-repressor that interacts with
promyelocitic leukemia zinc finger (PLZF) and eight twenty one (ETO),
and is involved in blocking proliferation through silencing of target
genes involved in the regulation of cell growth.65
It has been reported that FLT3-ITD expression in Ba/f3 cells resulted
in the activation of AKT and in the concomitant phosphorylation of the
Forkhead family member FoxO3a. FoxO3a phosphorylation on threo-
nine 32 through FLT3-ITD signaling promotes its translocation from
the nucleus to the cytoplasm. Specifically, FLT3-ITD expression pre-
vents FoxO3a-mediated apoptosis and the upregulation of p27Kip1 and
Bim gene expression, suggesting that the oncogenic tyrosine kinase
can negatively regulate FoxO transcription factors through FoxO3a
phosphorylation, leading to the suppression of its function. This then
Review
promotes the survival and proliferation of AML cells.14,64-66
There are marked differences in expression and function of several
myeloid transcription factors among the wild-type and mutated recep-
tors. In FLT3-ITD expressing cells, the transcription factors PU.1, a
member of the ETS family of transcription factors (EZB transforma-
tion-specific sequence), and C/EBP (CCAAT/enhancer-binding pro-
tein), both implicated in normal myeloid development, are repressed.67
Furthermore, the effects of FLT3-ITD on the activation of downstream
signaling may be increased as a result of inhibition of cellular phos-
phatases, such as SHP-1, which can lead to amplification of the prolif-
erative and anti-apoptotic effects.37,68
In addition, Tickenbrock et al. reported that FLT3-ITD induces
expression of the receptor Frz-4 (Frizzled-4), making it active even in
the absence of its natural ligand Wnt.69 The Wnt signaling pathway has
important functions in cell fate decisions during embryonal develop-
ment and in the adult organism, and it is implicated in hematopoietic
stem cell self-renewal and proliferation. Deregulation of its activity is
implicated in carcinogenic processes, and several molecules down-
stream of Wnt act as either tumor suppressors or proto-oncogenes.69
The activation of Frz-4 regulates the stability of the transcriptional
coactivator b-catenin that, in the absence of Wnt ligand in the cyto-
plasm, is associated with protein APC (adenomatous polyposis coli),
Axin and the serine-threonine kinase GSK 3b. After Wnt binding, the
non-phosphorylated b-catenin accumulates in the cytoplasm, translo-
cates into the nucleus and acts as a transcriptional coactivator of tran-
scription factors TCF/LEF, target genes for transcription, including c-
myc and cyclic D1. Activation of TCF transcriptional activity by FLT3-
ITD is functionally relevant for the transforming activity of the mutant
receptor. FLT3-ITD induces higher levels of b-catenin expression, asso-
ciated with increased stability and, therefore, promotes the TCF/LEF-
dependent transcriptional activity in the absence of Wnt ligand. The
activation of this signal cascade is involved in leukemic progression
since it causes a significant increase in cell proliferation.69 The FLT3-
ITD receptor can still maintain its ability to respond to FL. Indeed, in
the presence of the ligand, there is additional AKT phosphorylation and
increased MAPK activation (Figure 4).
Tyrosine kinase domain mutations
The second type of mutations found in the FLT3 receptor is the
replacement of an amino acid residue in the TKD (Figure 2). These
substitutions are caused by missense mutations in the exon 20, involv-
ing the codons aspartic acid 835 (D835) and isoleucine 836 (I836). At
least five different substitutions have been identified in the D835
codon: i) mainly tyrosine (D835Y), given by the mutation GAT →TAT;
ii) less frequently, histidine (D835H), given by GAT →CAT mutation;
iii) valine (D835V), given by the mutation GAT →GTT; vi) glutamate
(D835E); and v) asparagine (D835N). The I836 codon is mutated to
methionine (I836M) and asparagine (I836N).70-71
In addition, approximately 0-5% of de novo AML20,37,44,70 harbor a
mutation in exon 20 that results in the insertion of a glycine and ser-
ine between amino acids 840 and 841. Recently, the replacement of
[Oncology Reviews 2012; 6:e8] [page 69]
Review
Figure 4. Signaling pathways activated by FLT3-ITD.
[page 70] [Oncology Reviews 2012; 6:e8]
tyrosine with a cysteine codon 842 (Y842C) and asparagine 841 with
istidine (N841I) was discovered in patients with cytogenetic abnormal-
ities who relapsed.72 Rarely, insertions of nucleotides and complex
changes have been identified in the TKD domain deletions, but the
sequence remains in frame. The amino acids replaced belong to the
activation loop, which blocks the access of the ATP and the substrate to
the kinase domain when the receptor is inactive. TKD mutations inter-
fere with the inhibitory effect of the loop and lead to constitutive kinase
activation, conferring growth factor independent proliferation through
the activation of downstream target RAS/MAPK, PI3K/AKT and
STAT5.41,73 In contrast to ITD, TKD mutations are not able to inhibit the
transcription factors C/EBP and PU.1.74
Prognostic significance of FLT3 mutations in
acute myeloid leukemia
Approximately 25-35% of adult AML patients and 10-17% of pediatric
patients have ITD mutations in the FLT3 receptor, with a higher asso-
ciation with M3 subtype. In particular, these involve the variant BCR3,
and M5 subtype with a lower frequency in M2, M6, and M7 sub-
types.30,75-78 FLT3-ITD mutations are also closely correlated with specif-
ic cytogenetic abnormalities such as t(15;17) and MLL gene alter-
ations. They are highly associated with increased white blood cell
counts, high percentage of peripheral blood and bone marrow blasts,
indicating that the mutation has a biological effect on proliferation.79
In addition, a significant proportion of patients (9-23%) have more
than one ITD mutation, reflecting an underlying genetic instability.
FLT3-ITD has been found mainly in the heterozygous state, but there
is also evidence of a partial or complete loss of wild-type allele; a high
ratio of ITD/WT is a further adverse clinical prognostic factor. Indeed,
the presence of a hemizygous genotype ITD/- that is found in 1% of
pediatric patients and in 5% of adult patients is associated with a dis-
tinct phenotype with a significantly worse clinical outcome.80 The pres-
ence of an ITD in adults has no impact on achieving complete remis-
sion (CR) but it is significantly correlated with an increased risk of
relapse (RR), and reduced disease free and overall survival (OS).20,44,81
Therefore, after the karyotype, FLT3-ITD was found to be the most sig-
nificant independent prognostic factor in predicting survival in
patients under the age of 60 years.
In 2005, Falini et al. described a novel mutation within the NPM1
gene detected in 35% of AML patients. Like FLT3-ITDs, NPM1 muta-
tions are significantly associated with cytogenetically normal AML, and
a significant proportion of patients carry both FLT3-ITD and NPM1mut.
NPM1 mutations are associated with a high rate of CR, an increase in
event-free survival, and favorable OS. However, these positive effects
are lost in the presence of a coexisting FLT3-ITD. Whether the genotype
NPM1mut/FLT3-ITD is associated with intermediate or poor outcome is
still under question.82
FLT3-TKD mutations occur more frequently in AML patients with
MLL gene duplication, like the ITDs. The incidence of mutations in
TKD is significantly lower than that of ITDs, with a frequency of 6-10%
in AML, 2-5% in myelodysplastic syndromes (MDS) and 2-8% in
ALL.70,83 Probably because of their low frequency, the prognostic impact
of these mutations, both in terms of RR and OS, has not been defini-
tively determined, but it was thought that it is still associated with
reduced OS in intermediate risk patients under the age of 60 years;
instead, there is no significant association with age, WBC count, per-
centage of blasts or cytogenetic profile.14,22,37,84-85
The prognostic significance of the differences between the ITDs and
TKD mutations, which are supposed to have the same functional con-
sequences, such as loss of auto-inhibitory control and constitutive
kinase activity, might reflect different levels of constitutive activation.
This might also be a consequence of the different signaling pathways
triggered or might indicate that the ITD mutation is simply a marker of
cellular genetic instability that causes other unknown mutations.44,70
The differences in the signaling pathway of FLT3 mutations could,
therefore, have important implications for their transforming ability
and for the design of mutation-specific therapeutic approaches.86
All changes in the kinase domain, however, induce same alterations
in downstream signaling and have similar biological consequences
other than tandem duplication. This suggests that ITDs and TKD muta-
tions can be considered functionally different classes with different
transforming potential and different sensitivity to the action of
inhibitors.86 Both mutations (FLT3-ITD-TKD) on the same allele are
detected in a rare percentage of AML patients at diagnosis (1-2%), with
a worse clinical outcome than patients with single mutations. The dual
expression of mutants in hematopoietic cells causes resistance to tyro-
sine kinase inhibitors and cytotoxic drugs. This is because the altered
receptor shows increased kinase activity and causes cell cycle arrest at
the G2/M checkpoint, and hyperactivity of STAT5 and its anti-apoptotic
target genes BCL-XL and RAD51, already identified to form the basis of
the drug resistance mechanism.87
A second alteration may be acquired in the course of the disease or
during prolonged exposure to TK inhibitors, leading to the emergence
of a resistant phenotype to the low affinity receptor inhibitor or
increased catalytic activity.87-88
Targeted-therapies for FLT3 mutated cells
The main objective in the treatment of AML is inducing remission
and preventing relapse. Conventional therapy is based on the adminis-
tration of cytotoxic agents with the aim of reducing and eventually
eradicating the leukemic population, and allowing the remaining stem
cells to repopulate the bone marrow. Targeted therapy is a new type of
cancer treatment using drugs directed against specific genetic or other
abnormalities related to the leukemic cell clone. It is thought to dimin-
ish toxicity in healthy tissues and to increase the specificity of the tar-
get malignant cells.89 This therapy interferes with specific molecular
pathways that are important for the genesis and/or maintenance of the
malignant phenotype. This is in contrast to conventional chemothera-
py agents which interfere with some aspects of the global cellular
machinery, shared by non-malignant and malignant cells.
Some of the main inhibitors for targeted therapy of AML include:
hypomethylating agents, multidrug resistance modulators, biological
agents, and agents that modulate intracellular signaling pathways
(Table 1).
During the past decade, the identification of a constitutively active
form of FLT3 as a possible mechanism to promote the progression of
leukemogenesis has suggested exploiting this receptor as a therapeu-
tic target, fueling the study and research for selective FLT3 tyrosine
kinase inhibitors. Many of these, however, have inhibitory activity
against other RTKs due to the structural homology of this receptor and
causing possible side-effects because they bind to the active site of pro-
tein competing for the binding site for ATP. The mechanism of inhibi-
tion of tyrosine kinase inhibitors is similar, although they have a dif-
ferent structure, because it can bind the protein in a transition state,
from the active to the inactive form.90
In this context, several promising compounds are undergoing clini-
cal and experimental assessment. Lestaurtinib and midostaurin,
known as CEP-701 and PKC412, respectively, are the most extensively
studied FLT3 inhibitors and are of all clinical trials in AML the most
advanced. Lestaurtinib is a multitargeted tyrosine kinase inhibitor that
Review
pre-clinical studies have shown to strongly inhibit FLT3 at nanomolar
concentrations. In a phase I/II trial that examined patients with refrac-
tory or relapsed AML and FLT3 activating mutation, this agent showed
a good tolerability profile and clinical activity. Fourteen patients
received lestaurtinib 60 mg twice daily. Clinical activity was observed in
5 patients, including significant reductions in bone marrow and
peripheral blood blasts from 25% to less than 5% at Day 28 of therapy.
Lestaurtinib-related toxicities were minimal: grade ½ nausea and eme-
sis (41% and 29%, respectively) and grade ¾ generalized weakness
(18%).91 In a second separate phase II trial, lestaurtinib 60 mg twice
daily was tested as first-line treatment in elderly patients with AML not
considered fit for standard chemotherapy. Unlike the previous study,
similar adverse events were observed. Clinical response has shown
transient hematologic responses in 3 of 5 patients (60%) with FLT3
mutation and in 5 of 23 patients with FLT3 wild type.92-93 Other phase
III studies are ongoing to determine the future of lestaurtinib as a
treatment option for patients with FLT3-ITD AML, both in monotherapy
and in combination with cytotoxic drugs. However, the results from a
randomized trial of salvage chemotherapy followed by lestaurtinib for
patients with FLT3 mutation in AML in first relapse are not encourag-
ing. This trial reported that lestaurtinib treatment after chemotherapy
did not increase response rate or prolong survival.94
Midostaurin, initially developed as a protein kinase C inhibitor,
showed inhibitory activity against class III RTKs, such as c-KIT, FMS,
PDGFR, VEGFR 2, as well as FLT3. As a derivate of staurosporine,
midostaurin is biologically active by binding to the ATP-binding pocket of
FLT3 so that it inhibits activation and tyrosine autophosphorylation.
Midostaurin safety and tolerability were examined in a phase I study.95 A
recent phase IIb trial reported on administration of midostaurin at two
different dosages (50 or 100 mg twice daily) in 95 patients with AML or
MDS with either FLT3-WT or FLT3 mutant. The investigators reported
that the response rate of peripheral blood or bone marrow was 71% in
FLT3 mutant patients and 42% in FLT3-WT patients. One partial response
(PR) occurred in a patient with FLT3-mutant receiving the 100 mL dose
regime.96 After these encouraging results, a multi-center phase III study
has been started to test midostaurin in combination with daunorubicin
and cytarabin in newly diagnosed FLT3 mutant AML patients.97
The MEK/MAPK pathway is a signaling cascade involved in the control
of hematopoietic cell proliferation and differentiation. Downregulation
of MEK phosphorylation inhibits proliferation and induces apoptosis of
primary AML blasts.65 Sorafenib (BAY 43-9006) is a small oral molecule
originally designed as an inhibitor of Raf-1 kinase targeting the
RAF/MEK/ERK pathway. It has inhibitory properties against a number of
other kinases, including FLT3 and vascular endothelial growth factor
receptor.98-99 In pre-clinical studies, sorafenib induced dephosphoryla-
tion of MEK1/2 and ERK and induced apoptosis in AML cells.100 In a
mouse leukemia model with mutant FLT3, sorafenib reduced leukemic
burden and prolonged survival.101 A phase I study showed that sorafenib
was well tolerated when investigated as single agent against FLT3-
mutant AML. Although patients treated with one cycle of sorafenib
achieved a marked decrease in peripheral blood and bone marrow blasts,
this response was transient.102 In newly diagnosed cases of AML patients
under the age of 65 years, the combination of sorafenib 400 mg twice
daily with cytarabine and idarubicin has produced a high rate of CR in
FLT3-ITD patients (93%) and inhibited FLT3 signaling.103
AC220 is the most recent kinase inhibitor of FLT3 under clinical
investigation.104 AC220 inhibits FLT3 at low nanomolar concentrations
in cellular assays and is highly selective when screened against the
majority of the human kinome. Zarrinkar et al. showed that AC220 is
much more selective than CEP-701, PKC-412, MLN-518, sunitinib or
sorafenib.105 AC220 inhibits FLT3 activity in vivo, significantly extend-
ed survival in a mouse model of FLT3-ITD AML at doses as low as 1
mg/kg when given orally once a day, eradicated tumors in an FLT3-
dependent mouse xenograft model at 10 mg/kg, and strongly inhibited
FLT3 activity in primary human cells.105 A phase I study of single agent
AC220 in 76 patients with FLT3-WT and FLT3-ITD in relapsed/refracto-
ry AML confirmed its significant clinical activity; 13% of patients
achieved CR and 17% achieved PR. AC220 induced a rapid and durable
response of up to 67 weeks, and higher overall response (56%) and CR
(28%) rates were observed in FLT3-ITD mutations compared to FLT3-
WT.106 Interim data from a phase II study of AC220 as single agent in
relapsed/refractory patients with FLT3 mutations were presented at the
2011 Congress of the European Hematology Association. The clinical
response data reported that a group of 53 patients achieved a CR rate
of 45% and an additional 25% achieved PR.107
Another approach in target therapy is the development of anti-FLT3
monoclonal antibody, such as IMC-EB10 that blocks signaling by bind-
ing to the receptor and induces antibody-dependent cell-mediated cyto-
toxicity. Pre-clinical studies have shown the antiproliferative effects of
IMC-EB10 against both wild-type and mutant FLT3 AML models.102
So far, several clinical trials have shown that the use of FLT3
inhibitors as monotherapy, although well tolerated, is limited by tran-
[Oncology Reviews 2012; 6:e8] [page 71]
Review
Table 1. Summary of the main inhibitors for acute myeloid leukemia targeted therapy.
Agents Target and mechanisms of actions
Hypomethilating agents Hypermethylation at CpG islands within promoter region can lead to silencing of tumor-suppressor genes
and gene inactivation, acting as an alternative mechanism to deletions and mutations; the methylation of
tumor-suppressor genes and silencing of DNA are chromatin remodeling factors that can contribute to
carcinogenesis.
MDR modulators Over-expression of multidrug resistance 1(MDR1) gene, encoding P-glycoprotein, reduces the intracellular
drugs accumulation, causing resistance to chemotherapy treatment.
Biological agents Anti-CD33 monoclonal antibodies (trigger downstream signaling cascade and lead to antiproliferative
effect); histone deacetylases inhibitors (HDAC removing the acetyl group causes a tighter binding of
histones to DNA, preventing gene transcription).
Agents that modulate intracellular RTKs, docking and adapter proteins and transcription factors are the classes of proteins involved in
signaling pathways the signaling, and an inappropriate function of the members of each group was associated with
hematological malignancies. Several aberrations have been described in RTKs genes: c-abl, c-fms, flt3, c-kit,
PDGFR
α
/
b
, and consequently inappropriate activation of downstream signaling cascades, such as Jak-Stat,
Ras/MAPK e PI3K/AKT. Each of these proteins may be a therapeutic target, as well as factors directly involved
in the regulation of apoptosis (BCL-2, NFkB, caspases, cyclins, inhibitors of cyclins, transcription factors).
MDR, multidrug resistance; RTK, receptor tyrosine kinase; PI3K, phospatidylinositol-3-kinase; AKT, protein kinase B (PKB).
[page 72] [Oncology Reviews 2012; 6:e8]
sient or partial clinical response. Furthermore, there is growing evi-
dence to suggest that this partial clinical response is associated with
multiple factors of resistance to FLT3 inhibitors.108 Consequently, in
attempt to overcome resistance, several agents in combination with
standard chemotherapy are under clinical investigation.
Discussion
The identification of FLT3 expression levels and its molecular muta-
tions represent new opportunities in the treatment of AML. Over the
last decade, the biology and the function of the wild-type and mutated
FLT3 receptor have been well characterized. Equally, the relationship
between FLT3 and new molecular alterations, such as NPM1, is well
known. Various mechanisms of FLT3 activations may be present in dif-
ferent AML patients: FLT3 mutations are associated with a constitutive
tyrosine kinase activity, consequently the inhibition of phosphorylated
targets is growing in importance.109 Up to now, different compounds,
including lestaurtinib, sunitinib, midostaurin and AC220 have been
investigated in vitro and in vivo as FLT3 inhibitors.59 However, clinical
trials have produced only partial and transitory results. Therefore, addi-
tional data are required to optimize treatment and, in particular, to
investigate the underlying causes of resistance to FLT3 inhibitors, such
as the role of the deregulation signaling pathways, the aberrant expres-
sion of antiapoptotic proteins or the acquisition of genetic mutations.
Certainly, the role of the FLT3 ligand should be taken into consideration
since recent evidence suggests that the ITD-mutated receptor is heav-
ily influenced by FL.110 As reported by Sato et al., the FL ligand inter-
feres with the ability of FLT3 inhibitors to block FLT3 signaling, at least
in vitro.111 Current clinical trials are combining FLT3 inhibitors with
conventional chemotherapy in an attempt to increase the cytotoxic
effect against leukemia cells and reverse the poor prognosis for AML
patients with FLT3 mutations. But treatment of patients with
chemotherapy leads to high levels of FL that may be responsible for the
generally poor level of in vivo FLT3 inhibition. Despite the considerable
progress made in understanding the molecular mechanisms underly-
ing the onset of AML, there is still no definitive cure. Therefore, based
on our acquired knowledge, the challenge for the future will be to
define appropriate therapeutic strategies that take into account the
complex biological system of AML with an FLT3-ITD mutation.
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Review
