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Linköping University Medical Dissertations
No. 1289
Canertinib-induced leukemia cell death signaling
– effects of a pan-ERBB inhibitor
Cecilia Trinks
Division of Cell Biology
Department of Clinical and Experimental Medicine
Faculty of Health Sciences, SE-581 85 Linköping, Sweden
Linköping 2012
© Cecilia Trinks, 2012
ISSN 0345-0082
ISBN 978-91-7519-983-2
Cover picture: Chamilly Evaldsson
Illustrations made by the author, unless otherwise specified.
Published articles have been reprinted with the permission of the copyright holder.
Printed by LiU-Tryck Linköping, Sweden, 2012
To myself!
I livets lopp finns det inget målsnöre.
De snören vi passerar är starten till ett nytt lopp.
SUPERVISOR
Thomas Walz, MD, Associate Professor
Division of Clinical Sciences
Faculty of Health Sciences
Linköping University, Linköping
CO-SUPERVISORS
Jan-Ingvar Jönsson, Professor
Experimental Hematology Unit
Faculty of Health Sciences
Linköping University, Linköping
Anna-Lotta Hallbeck, MD PhD
Division of Clinical Sciences
Faculty of Health Sciences
Linköping University, Linköping
OPPONENT
Dan Grandér, Professor
Department of Oncology and Pathology
Cancer Center Karolinska
Karolinska Institute, Stockholm
ABSTRACT
ABSTRACT
Acute myelogenous leukemia (AML) is the most common acute leukemia affecting
adults, the second most frequent leukemia in children, and remains one of the most
difficult to cure. Despite a substantial progress in understanding the pathogenesis of
AML, general and rather unspecific cytostatic drugs such as cytarabine and
anthracyclins still make up the cornerstones of therapy. Problems with these
protocols include toxicity and the occurrence of resistance to the drugs in many
patients. In order to extend the treatment options and ultimately improve survival
for patients with leukemia it is imperative to increase the therapeutic arsenal with
effective targeted therapies, preferentially with different mechanisms of action. AML
due to a substantial heterogeneity between patients and within the clones in the same
patient, as well as T-cell malignancies, are particularly difficult to treat since it is
almost impossible to eradicate all leukemic stem cells using chemotherapy, thus
there is a need to find more specific and effective treatments. Canertinib is a novel
tyrosine kinase inhibitor developed for the treatment of certain solid cancers and has
been designed to specifically inhibit all member of the ERBB-receptor family (ERBB1,
ERBB2, ERBB3 and ERBB4). However, there are indications that canertinib has a
broader specificity and it has not been tested on patients with leukemia.
The aim of this thesis was to investigate the anti-proliferative and pro-apoptotic
effects and mechanisms of canertinib in human leukemia cells, and more specifically
to clarify the cell death pathway and potential targets for the drug in these cells.
Canertinib treatment of leukemia cell lines resulted in an ERBB-independent
induction of the intrinsic apoptotic pathway and activation of caspase-10, -9, and -8
as a consequence of Akt and Erk inhibition. In the human T-cell leukemia cell line
Jurkat, the effects were associated to dephosphorylation of the lymphocyte-specific
proteins, Lck and Zap-70. However, as full-length ERBB receptors were absent in
leukemic cell lines other possible targets for canertinib were investigated. The FLT3
receptor, frequently mutated in AML, was discovered as a target since canertinib
inhibited FLT3 autophosphorylation and kinase activity as well as downstream
targets. The search for other possible proteins that might account for the effect
exerted by canertinib, lead to the discovery of a truncated form of ERBB2 in human
leukemic cells.
In conclusion, canertinib display promising anti-tumor effects on malignant
hematopoietic cells and might be used in future studies in combination with
conventional chemotherapy or other targeted therapies in the treatment of leukemia.
TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF PAPERS ............................................................................................ 9
PAPERS OUTSIDE THIS THESIS .................................................................. 10
ABBREVIATIONS ........................................................................................ 11
INTRODUCTION ......................................................................................... 13
CANCER ........................................................................................................ 13
Genetic alterations ....................................................................................... 13
Oncogenes and tumor suppressor genes ..................................................... 14
Cell cycle ...................................................................................................... 14
Apoptosis ..................................................................................................... 16
Leukemia ...................................................................................................... 19
RECEPTOR TYROSINE KINASES ..................................................................... 20
Receptor tyrosine kinases in cancer ............................................................. 20
The ERBB receptor family and its ligands ............................................. 20
The ERBB2 receptor ..................................................................................... 22
The ERBB2 receptor in hematopoietic cells .................................................. 24
The FLT3 receptor ..................................................................................... 24
Intracellular signaling proteins ............................................................... 25
The PI3-K / Akt / FoxO pathway ................................................................. 25
The Ras / MAPK pathway ........................................................................... 26
Non-receptor tyrosine kinases ...................................................................... 26
CANCER THERAPY ......................................................................................... 28
Chemotherapy .............................................................................................. 28
Targeted therapy .......................................................................................... 28
Small molecule inhibitors ........................................................................ 29
FLT3 targeted therapy ................................................................................. 30
ERBB targeted therapy ................................................................................ 31
TABLE OF CONTENTS
Strategies to apply kinase targeted therapy ................................................. 32
Resistance .................................................................................................... 33
Canertinib .................................................................................................... 35
AIMS OF THE THESIS ................................................................................. 37
MATERIALS AND METHODS .................................................................... 39
CELL LINES .................................................................................................... 39
PATIENT SAMPLES ......................................................................................... 39
WESTERN BLOT ANALYSIS ............................................................................. 40
FLOW CYTOMETRY ........................................................................................ 41
REVERSE-TRANSCRIBED POLYMERASE CHAIN REACTION, RT-PCR ............ 43
RESULTS AND DISCUSSION ...................................................................... 45
CONCLUSIONS ........................................................................................... 57
FUTURE ASPECTS ....................................................................................... 59
POPULÄRVETENSKAPLIG SAMMANFATTNING ....................................... 61
ACKNOWLEDGEMENTS ............................................................................. 64
REFERENCES ............................................................................................... 66
LIST OF PAPERS
9
LIST OF PAPERS
This thesis is based on the following papers, which will be referred to in the text by
their Roman numerals (I-IV):
I. Cecilia Trinks, Emelie A. Djerf, Anna-Lotta Hallbeck, Jan-Ingvar Jönsson,
Thomas M. Walz. The pan-ErbB receptor tyrosine kinase inhibitor
canertinib induces ErbB-independent apoptosis in human leukemia (HL-60
and U-937) cells. Biochemical and Biophysical Research Communications -BBRC
2010, 393(1):6-10.
II. Cecilia Trinks, Emelie A. Severinsson, Birgitta Holmlund, Anna Gréen,
Henrik Gréen, Jan-Ingvar Jönsson, Anna-Lotta Hallbeck ,Thomas M. Walz.
The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-
mediated cell-death in human T-cell leukemia (Jurkat) cells. Biochemical and
Biophysical Research Communications -BBRC 2011, 410(3):422-7.
III. Amanda Nordigården, Jenny Zetterblad, Cecilia Trinks, Henrik Gréen,
Pernilla Eliasson, Pia Druid, Kourosh Lotfi, Lars Rönnstrand, Thomas M.
Walz, Jan-Ingvar Jönsson. Irreversible pan-ERBB inhibitor canertinib elicits
anti-leukemic effects and induces the regression of FLT3-ITD transformed
cells in mice. British Journal of Haematology, 2011, 155(2):198-208.
IV. Cecilia Trinks, Birgitta Holmlund, Jan-Ingvar Jönsson, Thomas M. Walz.
Human leukemic cell lines express a truncated intracellular 160 kDa
ERBB2 receptor. Manuscript
PAPERS OUTSIDE THIS THESIS
10
PAPERS OUTSIDE THIS THESIS
1. Appelmelk B.J, Shiberu B, Trinks C, Tapsi N, Zheng P. Y, Verboom T,
Maaskant J, Hokke C.H, Schiphorst W.E, Blanchard D, Simoons-Smit I.M, van
den Eijnden D.H, Vandenbroucke-Grauls C. M, Phase variation in
Helicobacter pylori lipopolysaccharide. Infection & Immunity, 1998, 66(1):70-6
2. Hammarström S, Trinks C, Wigren J, Surapureddi S, Söderström M, Glass
C.K, Novel eicosanoid activators of PPAR gamma formed by RAW 264.7
macrophage cultures. Advances in Experimental Medicine and Biology, 2002,
507:343-9.
3. Djerf E.A, Trinks C, Abdiu A, Thunell L.K, Hallbeck A-L, Walz T.M, ErbB
receptor tyrosine kinases contribute to proliferation of malignant melanoma
cells: inhibition by gefitinib (ZD 1839). Melanoma Research, 2009, 19(3):156-66
4. Djerf Severinsson E.A, Trinks C, Gréen H, Abdiu A, Hallbeck A-L, Ståhl O,
Walz T.M, The pan-ErbB receptor tyrosine kinase inhibitor canertinib
promotes apoptosis of malignant melanoma in vitro and displays anti-tumor
activity in vivo. Biochemical and Biophysical Research Communications –BBRC,
2011, 414(3):563-8
ABBREVIATIONS
11
ABBREVIATIONS
Akt a serine/threonine kinase, protein kinase B (PKB)
Apaf-1 apoptotic protease-activating factor 1
Bad Bcl-2-antagonist of cell death
Bak Bcl-2 antagonist/killer-1
Bax Bcl-2-associated x protein
Bid BH3 interacting domain death agonist
Bim/BCL2L11 Bcl-2 interacting modulator, Bcl-2-like protein 11
Bcl-2 B-cell CLL/lymphoma 2
B-raf v-raf murine sarcoma viral oncogene homolog B1
CDK cyclin-dependent kinase
CDK4 cyclin-dependent kinase 4
DISC death-inducing signaling complex
DR death receptor
EGF epidermal growth factor
EGFR epidermal growth factor receptor
ERBB1 erythroblastic leukemia viral (v-erb-b) oncogene homolog
ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2
ERBB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3
ERBB4 v-erb-a erythroblastic leukemia viral oncogene homolog 4
Erk extracellular signal-regulated kinase
FADD Fas-associated death domain protein
FLT3 FMS-related tyrosine kinase -3
FoxO3a Forkhead box O3a
Grb2 growth-factor-receptor-bound protein 2
HER1-4 human epidermal growth factor receptor 1-4
ABBREVIATIONS
12
MAPK mitogen-activated protein kinase
MEK mitogen-activated protein kinase kinase /
extracellular signal-regulated kinase (Erk) kinase
NRG neuregulin
PARP poly-(ADP-ribose) polymerase
PI3-K phosphoinositide 3-kinase
PTB phosphotyrosine-binding
PTEN phosphatase and tensin homolog
Raf murine sarcoma viral oncogene homolog
Ras rat sarcoma viral oncogene homolog
RB retinoblastoma
SH2, SH3 Src homology 2 and 3 domains
Stat signal transducer and activator of transcription
TKI tyrosine kinase inhibitor
INTRODUCTION
13
INTRODUCTION
CANCER
Cancer is one of the most common diseases in the Western world. According to the
Swedish Cancer Registry, more than 55 000 patients are diagnosed with cancer
annually in Sweden. The incidence rate of cancer has increased by approximately 2%
each year during the last two decades. The increase is explained by the increasingly
elderly population and by screening and improved diagnostic methods [1].
Cancer is a large class of very different diseases where the cells are defected in
mechanisms controlling cell division and cell death. One goal for cancer research is to
understand the mechanisms that cause the transformation of a normal cell to a
malignant cell. Cancer cells are often able to produce growth factors and thereby
stimulate their own growth (autocrine stimulation). The cells could also be
insensitive to growth-inhibitory signals that might otherwise stop their growth, and
they are resistant to intrinsic programmed cell death (apoptosis). Cancer cells are also
characterized by limitless reproductive potential, sustained angiogenesis (blood
vessel growth), and the ability to invade local tissue and spread to distance sites
(metastasis) [2]. Additional hallmarks of cancer have been proposed such as
abnormal metabolic pathways, ability to evade the immune system, DNA instability
leading to accumulation of chromosome abnormalities, and modulation of
inflammatory response [3].
GENETIC ALTERATIONS
Genetic alterations leading to cancer may be inherited (germ-line mutation), or
induced by viral agents, ionizing irradiation, genotoxic compounds, or sporadically
induced by other means. A single genetic change is not sufficient for the
development of a malignant tumor; evidence indicates a multistep process of
alterations in several specific genes in cancer development [4]. Thus, tumors usually
contain a spectrum of cells with different genetic alterations and states of
differentiation, thereby exhibiting different malignant cell clones. This heterogeneity
results in differences in clinical behavior and response to treatment of cancer of the
same origin, making the treatment complicated [3].
INTRODUCTION
14
ONCOGENES AND TUMOR SUPPRESSOR GENES
Genetic alterations leading to the activation of oncogenes or loss of suppressor gene
function, may initiate the tumorigenic pathway that progressively drives the
transformation of normal into malignant cells through successive genetic alterations
in specific cellular genes. Oncogenes encode proteins such as transcription factors,
chromatin modulators, growth factors, growth factor receptors, signal transducers,
and apoptosis regulators. These proteins control cell proliferation, differentiation and
apoptosis. Oncogenes can be activated by structural alterations resulting from
mutations, translocations including gene fusion, or by amplification. Mutated
oncogenes normally results in constitutively active proteins, including tyrosine
kinases, which destabilize the normal regulatory mechanisms controlling signaling
pathways and thereby giving the cancer cells a survival or proliferative advantage
over normal cells [2-5].
There are a rapidly growing number of known oncogenes coding for activated
kinases such as Bcr-Abl, EGFR, c-Met, FLT3 and c-Kit. They contain an ATP- and
substrate-binding “pocket” in enzymatic kinase domains, which provide a well-
defined, conserved structure. These structures can be used for designing new specific
targeting drugs in order to inhibit these kinase activated pathways [6].
Tumor suppressor genes encode proteins important for limiting proliferative and
metabolic processes, working as a break in normal cells. Mutations of tumor
suppressor genes frequently occur in cancer resulting in the inactivation of the
protein and thereby the loss of the inhibitory effect on proliferation. One such tumor
suppressor gene is PTEN (phosphatase and tensin homolog) which is responsible for
the regulation of the Akt pathway. Another example of a tumor suppressor gene is
the TP53 gene involved in the control of cell growth, cell division, DNA repair and
apoptosis [7].
CELL CYCLE
Dividing cells undergo the cell cycle, which includes a sequence of events by which a
cell duplicates its genetic material (as well as proteins and other macromolecules)
and divides into two identical daughter cells. The cell cycle is divided into four
phases: the “synthesis phase” (S phase), where the genetic material is duplicated, the
“mitosis phase” (M phase), where the duplicated chromosomes are distributed
equally into two daughter cells and the G1 (following M phase) and the G2 –phase
INTRODUCTION
15
(between S and M phase). In G1 cells prepare for DNA synthesis and in G2 cells are
getting ready for mitosis. Cells in G1 may, before commitment to DNA replication,
enter a resting state called G0 (Figure 1). Normal cells are usually found in G1 and G0.
Cells in G0 account for the major part of the non-growing, non-proliferating cells in
the human body [8].
Different classes of cellular proteins execute transition from one cell-cycle phase to
the next. Key regulatory proteins are the cyclin dependent kinases (CDKs), a family
of serine/threonine protein kinases that are activated at specific points of the cell
cycle and act as the main engines driving the cell cycle forward from one phase to the
next. The CDKs are positively regulated by cyclins and negatively regulated by
naturally occurring CDK inhibitors (CDKIs). Progression through the cell cycle is
controlled at different stages by so-called checkpoints. The R-point (Retinoblastoma
(Rb)-point) is a key checkpoint between the G1 and S phase. CDK4 phosphorylates
Rb and thereby induces the cell to pass the R-point into S-phase of the cell cycle. The
cell needs growth factors to be able to continue from G1 into S phase [8, 9].
In cancer cells the cell cycle may be dysregulated causing cells that overexpress
cyclins, or downregulate or lack the expression of CDKIs, to continue through the
cell cycle phases and undergo unregulated cell growth. These alterations may be
caused by deletions, mutations and/or promoter hypermethylation [10].
Figure 1. The cell cycle with its phases: G1, S, G2, M, and the G0 phase. R= restriction-point
G0
G1
MG2
S
R
INTRODUCTION
16
APOPTOSIS
Apoptosis or “programmed cell death” is a physiological cellular suicidal mechanism
that plays a central role in both development and homeostasis of tissues.
Dysregulation of the apoptotic process may result in a wide range of pathological
conditions. Cancer is one consequence of impaired apoptotic mechanisms that lead to
an insufficient removal of damaged cells [11]. The ability of cancer cells to avoid
apoptosis and continue to proliferate is one of the hallmarks of cancer. Thus, it is
important to understand the molecular mechanisms behind the regulation of
apoptosis and the proteins involved in this process since these regulatory proteins
may become major targets in cancer therapy development.
Apoptosis is characterized by distinct morphological changes including plasma
membrane blebbing, cell shrinkage, depolarization of the mitochondria, chromatin
condensation, and DNA fragmentation [12]. Several genes have been identified as
either inducers or repressors of apoptosis. In particular, the cysteine-related
proteases (caspases) are known to play key roles in the execution phase of cell death
through various apoptotic stimuli. The caspases are present in cells as inactive pro-
enzymes that are transformed into active form following proteolytic
cleavage/removal of the pro-domain [13].
There are two alternative pathways that initiate apoptosis: one is mediated by death
receptors on the cell surface, and is referred to as the “extrinsic pathway”; the other is
referred to as the “intrinsic pathway” and involves the Bcl-2 family of proteins that
regulate mitochondrial function (Figure 2). The two pathways converge on
downstream effector caspases and other key substrates in the apoptotic death process
[13, 14].
The extrinsic pathway is activated in response to multiple pro-apoptotic signals, such
as Fas-ligand (CD95L) and tumor necrosis factor alpha (TNFα). Upon ligand binding,
the death receptor (CD95) interacts via their intracellular domain, called death
domain (DD), with the adapter proteins such as Fas-associated death domain
(FADD). These adapter proteins also contain a second protein interaction domain,
the death effector domain (DED) that binds to a DED in initiator caspase-8 and
caspase-10 to form the death-inducing signaling complex (DISC). The DISC
formation activates caspase-8, which subsequently activates the effector caspase-3
(Figure 2). Effector caspases, like caspase-3, are responsible for the cleavage of
cellular proteins, such as cytoskeletal proteins resulting in the typical morphological
changes observed in cells undergoing apoptosis [15].
INTRODUCTION
17
Figure 2. The extrinsic and intrinsic pathways of apoptosis.
The intrinsic (mitochondrial) pathway is activated by induced cellular stress, such as
DNA damage, growth factor deprivation and oxidative stress. The Bcl-2 family of
proteins plays a central role in controlling the mitochondrial pathway. This family of
proteins is divided into anti-apoptotic proteins, such as Bcl-2 and Bcl-XL, and pro-
apoptotic proteins, such as Bak, Bax, Bid or Bim (Table 1). Bid (cleaved by caspase-8
to tBid), and Bim translocate from the cytosol to mitochondrial membranes to
stimulate oligomerization of Bak and Bax, which participates in formation of pores in
the outer mitochondrial membrane [16]. Cytochrome c then escapes from the
mitochondria through the pores into the cytosol of the cell and associates with
apoptotic protease-activating factor 1 (Apaf-1) and procaspase-9 to form a complex
called the apoptosome. Apoptosome formation triggers activation of caspase-9,
which further cleaves and activates the effector caspase-3, finally converging in the
same apoptotic end process as through activation of the extrinsic pathway, resulting
in selective destruction of subcellular structures and organelles, and DNA
fragmentation [17]. The anti-apoptotic members of the Bcl-2 family preserve
mitochondrial integrity by preventing activation of Bak and/or Bax until neutralized
by BH3-only proteins, such as Bim. Thus, Bcl-2 expression prevents the release of
cytochrome c from the mitochondria but also by binding Apaf-1 [14].
Fas L
Bid
tBid
Caspase-8,-10
Caspase-3
Caspase-9 Cyto C
Apaf-1
PARP
Bak
Bak
Bcl-2
Fas/CD95
Intrinsic pathway
FA
DD
FA
DD
APOPTOSIS
Extrinsic pathway
plasma membrane
Bcl-2
Cellular
stress
INTRODUCTION
18
Table 1. Members of the Bcl-2 protein family are divided into anti-apoptotic or pro-apoptotic factors.
The pro-apoptotic protein Bid connects the extrinsic and intrinsic pathways through
caspase-8 activation of Bid, which is transported to the mitochondria where it
promotes cytochrome c release [18]. The death receptor pathway and the
mitochondrial pathway are linked together by activating caspase-3. One target for
effector caspases is the enzyme poly-(ADP-ribose) polymerase (PARP) which is an
important DNA repair enzyme and was one of the first proteins identified as a
substrate for caspases. The ability of PARP to repair DNA damage is prevented
following cleavage of PARP by caspase-3 [19].
The sensitivity of cells to apoptotic stimuli may be dependent on the balance of pro-
and anti-apoptotic Bcl-2 proteins, which demonstrates the importance of these
intracellular proteins. Thus, when there is an excess of pro-apoptotic proteins the
cells are more sensitive to apoptosis, and when there is an excess of anti-apoptotic
proteins the cells will be more resistant, as the case as in many hematological
malignancies where Bcl-2 is upregulated by translocations, gene amplifications or
excessive receptor signaling [20].
Lysosomal involvement
Besides caspases, lysosomal proteases such as cathepsins are involved in apoptotic
cell death. The hallmark of cathepsin-mediated death-pathways is the lysosomal
membrane permeabilization (LMP) that results in the release of active cathepsins to
the cytosol. A wide range of apoptotic stimuli such as death receptor activation,
DNA damage and growth factor starvation can trigger LMP. In the cytosol
cathepsins are capable of triggering mitochondrial dysfunction with subsequent
caspase activation and cellular demise [21]. A link between the lysosomal and
mitochondrial pathway of apoptosis have been proposed. Cathepsins are able to
trigger Bax, which induce mitochondrial permeabilization in T-lymphocytes [22]. In
addition, Bid is generally processed and activated by caspases, but it has been
demonstrated that cathepsins are able to cleave Bid as well [23]. Caspase-9 is
Pro-apoptotic factorsAnti-apoptotic factors
• Bcl-2
• Bcl-XL
• Bcl-w
• Bax, Bak
• Bim
• Bid, Bad, Noxa, Puma
INTRODUCTION
19
generally known to be activated through the intrinsic apoptotic pathway, and has
been reported to play a dual role as an activator of effector caspases and LMP.
Caspase-9 may induce LMP whether it is cleaved and activated by caspase-8 or
activated in the apoptosome. Caspase-9 cleaved by caspase-8 differs from caspase-9
in the apoptosome because it fails to activate caspase-3. Thus, there is an ability of
caspase-8-cleaved caspase-9 to induce cell death without activating caspase-3.
Caspase-9 is a crucial link between caspase-8 and LMP [24].
LEUKEMIA
Leukemias are blood malignancies characterized by clonal proliferation of
hematopoietic precursor cells in the bone marrow, often associated with a
suppression of normal hematopoiesis. Leukemia cells migrate from the bone marrow
to the blood stream and may also expand throughout other tissues in the body. The
classification of human leukemias is based on the type of cell involved and the state
of maturity. Acute leukemias are characterized by the presence of rapidly
proliferating immature blasts whereas chronic leukemias consist of more mature
bone marrow precursors. Chronic leukemias may however transform into an acute
phase, clinically apparent as blast crises. The acute and chronic leukemias may be
further grouped as either myeloid or lymphoid depending on phenotypic
determinants and their respective progenitor origin [25].
The etiology of most leukemias is still mainly unknown although several possible
etiological factors such as certain chemotherapeutic agents, carcinogens in cigarette
smoke, occupational exposure to benzenes, viruses and other causes have been
suggested [25]. Several specific cytogenetic and molecular abnormalities have been
characterized in some acute, as well as in chronic leukemias. Acute leukemias
display a variety of genetic alterations such as translocations, inversions, deletions
and mutations in the genome. The first example of altered protein kinase signaling in
leukemia was the identification of the Bcr-Abl fusion protein, a constitutively
activated form of the ABL tyrosine kinase found in chronic myeloid leukemia (CML).
This tyrosine kinase protein results from the translocation of chromosome 9 and 22,
the Philadelphia chromosome [26].
INTRODUCTION
20
RECEPTOR TYROSINE KINASES
The human genome project revealed that 1.7% of the human genome encodes 518
kinases [27, 28]. Protein kinases are enzymes that play a key role in nearly all aspects
of cell biology. Dysregulation of protein kinases occurs in a variety of diseases
including cancer.
The functions of proteins are modified by these protein kinases by transferring
phosphate groups of adenosine triphosphate, ATP, or GTP to free hydroxyl groups
of amino acids on target proteins. The importance of phosphorylation as a
fundamental mechanism that controls cell physiology was established by the
pioneering work of Fisher and Krebs [29].
Most protein kinases phosphorylate serine and threonine residues, but a subset of
protein kinases selectively phosphorylates tyrosine residues. There are 90 protein
tyrosine kinases (PTKs), and they can be further divided into the two main
subgroups, 32 non-receptor, or cytosolic, tyrosine kinases and 58 receptor tyrosine
kinases (RTK). The cytoplasmic PTKs can be divided into nine subfamilies: the Src,
Csk, Ack, Fak, Tec, Fes, Syk, Abl and Jak classes. The RTK family consists of 20
subfamilies including, among others, the epidermal growth factor receptor (EGFR),
fibroblast growth factor receptor, insulin receptor and platelet-derived growth factor
receptor (PDGFR) [30]. The primary function is to mediate the flow of information
from the extracellular environment into the cell. These signals determine whether the
cell proliferates, differentiates, migrates or dies. RTKs are cell membrane proteins
consisting of a single transmembrane domain that separates an intracellular protein
kinase domain from an extracellular ligand-binding domain. Ligand-binding induces
receptor homo- or heterodimerization which is essential for activation of the tyrosine
kinase through cross-phosphorylation and subsequent recruitment of target proteins,
which initiate a complex signaling cascade that leads to distinct transcriptional
programs [31].
RECEPTOR TYROSINE KINASES IN CANCER
The ERBB receptor family and its ligands
The first RTK to be discovered was the epidermal growth factor receptor (EGFR),
reviewed in Carpenter et al. [32], which has yielded insight into the underlying
mechanism by which RTKs function [31] including the recruitment of second
messengers [33].
INTRODUCTION
21
There are four members of the EGFR/ERBB receptor family; the EGF receptor itself,
also known as ERBB1/HER1, ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4 (Figure
3). The four ERBB receptors share 40-45% sequence identity and are composed by
three distinct regions: an extracellular region consisting of four glycosylated
subdomains (I, II, III and IV), where subdomains II and IV are cysteine-rich regions
and subdomains I and III form the ligand-binding site; a transmembrane region
containing a single hydrophobic domain; and an intracellular region containing the
catalytic TK domain, which is responsible for the generation and regulation of
intracellular signaling (Figure 3) [34, 35].
Many neoplasms are associated with aberrant EGF receptor activation, which can
result from mutation of the receptor, its overexpression and/or from EGF receptor
stimulation through autocrine loops involving excess production of its ligand growth
factors [36].
Most ligands of ERBB family receptors are synthesized as transmembrane precursors
that are proteolytically cleaved to release the soluble form of the peptide. The
peptide, comprising approximately 50 amino acids, contains an EGF-like or EGF-
homologous region which is required for ERBB binding and activation. The ERBB
ligands have been classified into three major groups based on their direct binding to
a particular ERBB family member (Figure 3). The first group consists of epidermal
growth factor (EGF), transforming growth factor alpha (TGFα) and amphiregulin
(AR) that bind exclusively to ERBB1. Membrane-bound forms of EGF and TGFα may
interact with receptors on the surface of adjacent cells (juxtacrine stimulation),
thereby contributing to cell-to-cell adhesion and cell-to-cell stimulatory interactions.
The second group of ERBB ligands is represented by heparin-binding EGF (HB-EGF),
betacellulin (BT) binding ERBB1 and ERBB4, and epiregulin (EPR) binding all
receptors except homodimers of ERBB2. The third group is composed of the
neuregulins (NRGs), and forms two subgroups based upon their capacity to bind
ERBB3 and ERBB4 (NRG1 and NRG2) or only ERBB4 (NRG3 and NRG4). ERBB2 is
an “orphan” for which there is no naturally occurring soluble ligand; however,
ERBB2 is a preferred hetero-dimerization partner and acts as a co-receptor [37, 38].
Ligand binding to ERBB receptors induces formation of homo- and heterodimers
leading to activation of the intrinsic kinase domain and subsequent phosphorylation
on specific tyrosine residues within the cytoplasmic tail. These tyrosine
phosphorylated residues serve as docking sites for downstream signaling molecules
and cytoplasmic messenger proteins involved in the action of the Akt, the mitogen-
activated protein kinase (MAPK) and Stat pathways [39].
INTRODUCTION
22
Figure 3. Epidermal growth factor family of ligands and the ERBB gene family. The names of the
receptor proteins are indicated. The inactive ligand-binding domain of ERBB2 and the inactive kinase
domain of ERBB3 are denoted with an X (modified from Roskoski [34])
The ERBB2 receptor
ERBB2 is a proto-oncogene located on chromosome 17q21, and encodes a 1255 amino
acid glycoprotein of 185 kDa [40]. ERBB2, also known as HER2 and neu, was initially
shown to be amplified in a human breast cancer cell line [41]. Since that time, ERBB2
amplification and ERBB2 protein overexpression have been linked to important
tumor cell proliferation and survival pathways [42]. Several drugs have been
developed to target the pathway and detection of ERBB2 has become a routine
prognostic and predictive factor in different types of cancer, such as breast cancer
[43].
As mentioned above, the ERBB2 receptor has no known ligand that might be
dependent on its fixed conformation where the ligand-binding site is buried and not
accessible for interaction. In spite of that, it is well established that ERBB2 is the
preferred docking partner among the other ligand-bound family members [44]. The
strength of ligand binding and signaling by heterodimers containing ERBB2 is
significantly greater than that of other homo- or heterodimers that do not recruit this
ErbB4
HER4
Gene
FamilyErbB1
HER1
EGFR
ErbB2
HER2
Neu
ErbB3
HER3
Transmembrane
domain
Domain IV
Domain III
Domain II
Domain I
Growth
Factors
EGF
AR
TGF-α Epigen
BTC
HB-EGF
EPR
NRG1
NRG2NRG3
NRG4
Tyrosine
Kinase
C-terminal
phosphorylation sites
X
X
XX
INTRODUCTION
23
receptor [45, 46]. The heterodimer with greatest oncogenic potential is that composed
of ERBB2 and ERBB3, where ERBB3 is a defective kinase capable of binding some of
the isoforms of the NRGs. By ligand binding and dimerization, ERBB3 becomes
transphosphorylated by ERBB2 causing several carboxyl-terminus phosphotyrosine
residues in each receptor to undergo phosphorylation and to interact with
intracellular signaling proteins (Figure 4) [47]. Second messengers containing SH2 or
PTB domains recognize site-specific phosphorylation (docking sites) in the C-
terminal of ERBB receptors. The type of growth factor that has bound to the receptor
may determine which tyrosine residues that become phosphorylated. This in turn
determines the identity of the signal transducers that are recruited. The tyrosine
residue and the surrounding amino acids of each ERBB family member are
specifically tailored to interact with a unique collection of proteins [39]. ERBB3
phosphotyrosines are able to recruit phosphatidylinositol 3-kinase (PI3K) and
thereby activate the Akt pathway, whereas ERBB2 phosphotyrosine residues have
the ability to activate the MAPK pathway [47, 48].
Figure 4. Ligand-induced receptor heterodimerization between ERBB2 with extended conformation
and the tyrosine kinase dead ERBB3 receptor. The intracellular tyrosine kinase domains are cross-
phosphorylated and form binding sites for intracellular molecules such as PI3-K/Akt, MAPK/Erk1/2
and Stat pathway resulting in different outcomes. Within the cytoplasmic tail, orange empty circles
represent nonphosphorylated tyrosine residues and orange circles with a P represent tyrosine
phosphorylation (modified from Carraway & Kozloski [47]).
PP
ATP
ADP
ATP
ADP
L
L
Survival
ProliferationMigration AdhesionDifferentiation
PP
P
ErbB2 ErbB2 – ErbB3 heterodimerErbB3
PI3-K
PI3-K/Akt
MAPK/Erk1/2
Stat
INTRODUCTION
24
The ERBB2 receptor in hematopoietic cells
Generally ERBB receptors are not expressed in hematopoietic cells, although low
levels of ERBB2 mRNA or protein have been observed in normal human
hematopoietic mononuclear cells from bone marrow, peripheral blood and umbilical
blood, and in some leukemic blasts derived from patients with myelodysplastic
syndrome (MDS) and AML [49]. Furthermore, full-length 185 kDa ERBB2 protein has
been demonstrated in blasts from patients with hematological malignancies
including B-cell acute lymphoblastic leukemia (B-ALL) [50]. The incidence of ERBB2
positivity in B-ALL appears to correlate with patient age, occurring in 3.4% of
children and 31% of adults [50, 51]. This ERBB2 expression is not related to gene
amplification, but may be related to transcriptional activation or post-translational
modifications. Moreover, patients with ERBB2 positivity are drug-resistant
suggesting that this may be a useful prognostic marker in B-ALL [51]. In contrast to
B-ALL, ERBB2 protein is not detected in Hodgkin’s disease, non-Hodgkin’s
lymphoma, AML and CML [52]. However, ERBB2 is expressed in CML where there
is evidence of B-lymphoblastic crisis. Interestingly, although ERBB2 protein is not
detectible in AML blasts, the EGFR kinase inhibitor gefitinib (Iressa) promotes the
differentiation of AML cell lines and primary AML blasts in vitro [53]. Furthermore,
the EGFR inhibitor erlotinib induces differentiation and apoptosis of EGFR-negative
myeloblasts in patients with MDS and AML [54]. There is a growing interest of the
ERBB family expression in leukemia and lymphoma. Matouk et al. found ERBB
family mRNA to be expressed in myeloma cell lines [55]. Similarly, Otsuki et al.
found plasma cell lines to have increased levels of mRNA for ERBB2/3/4, while only
the protein levels of ERBB2 were highly expressed [56].
The FLT3 receptor
In normal hematopoiesis, both tyrosine and serine/threonine kinases play essential
roles in proliferation, survival, and differentiation. A constitutive activation of
growth factor receptors or proteins participating in their downstream signaling
cascades may be a step in leukemogenesis providing a proliferative and/or survival
advantage. One example is the constitutive activation of the FMS-related tyrosine
kinase -3 (FLT3), a member of the class III receptor tyrosine kinase family, which also
includes c-Kit, PDGFRα and PDGFRβ. Mutations in the FLT3 gene, seen in
approximately 30 % of AML patients, are associated with a poor prognosis in terms
of patient survival. FLT3 receptor mutations include internal tandem duplication
(ITD) of the juxta-membrane domain and a point mutation of aspartate 835 to
INTRODUCTION
25
tyrosine in the activating loop in the tyrosine kinase domain (TKD) (Figure 5). Both
types of genetic alterations cause a constitutive activation of the TK activity of FLT3,
leading to dimerization and auto-phosphorylation of the receptor [57, 58]. This
results in a ligand-independent activation, promoting aberrant proliferation and
survival of hematopoietic progenitor cells leading to onset of malignancies including
AML, ALL and CML in lymphoblast crisis [26, 59]. FLT3 ligand, FL, binds to the
FLT3 receptor which stimulates several intracellular signaling molecules through
phosphorylation or association. These signaling proteins include the p85 subunit of
PI3-K, SHC, SHP2, GRB2, and Stat5. PI3-K activates the Akt pathway which results in
inactivation of the transcription factor FoxO3a through phosphorylation [60]. This
prevents transcription and translation to the pro-apoptotic protein Bim, and thereby
promotes cell survival [61].
Figure 5. Schematic presentation of the FLT3 receptor (modified from Takahashi [61]).
Intracellular signaling proteins
The PI3-K / Akt / FoxO pathway
Activation of receptors confers phosphorylation of intracellular tyrosine residues.
This, in turn, leads to recruitment of Src family members via their SH2 domains,
which in turn mediates activation of a second level of signaling molecules, including
Internal tandem
duplication (ITD)
D835 mutation
Tyrosine kinasedomain
(TKD)
Juxtamembrane
(JM) domain
Extracellular ligand
binding domain
C-terminal domain
INTRODUCTION
26
Ras, PI3-K, and Stats, either directly or via adaptor molecules. The PI3-K pathway
positively controlled by Ras and negatively regulated by the PTEN tumor suppressor
activates Akt, which specifically regulates cell survival and apoptosis [62].
Activated Akt phosphorylates a variety of proteins involved in survival pathways
leading to diminished cell apoptosis. For instance, Akt phosphorylates pro-caspase 9
and inhibits thereby the proteolytic processing of pro-caspase 9 [63]. Another
downstream target of Akt is the pro-apoptotic protein Bad. Akt phosphorylates Bad,
which results in blocked apoptosis.
In addition, the PI3-kinase/Akt pathway is the main regulator of the Forkhead box
(Fox) family of transcription factors involved in proliferation, cell survival, and
metabolic control. This family is highly conserved evolutionary and is expressed in
species ranging from yeast to human. Mammalian FoxO1, FoxO3 and FoxO4 contain
three highly conserved Akt phosphorylation sites and impaired phosphorylation of
these sites leads to increase transcriptional activity of FoxOs. Phosphorylation of Akt,
for instance as a consequence of receptor activation, leads to cytoplasmic localization
and decreased transcriptional activity of FoxO. This prevents transcription of pro-
apototic Bim and cell cycle inhibitor p27, promoting cell survival [64].
The Ras / MAPK pathway
The Ras/Raf/extracellular signal-regulated kinase (Erk) signaling pathway plays a
crucial role in almost all cell functions. Erk1/Erk2 (also known as p44/p42MAPK,
respectively) are two isoforms of Erk that belong to the family of mitogen-activated
protein kinases (MAPKs). These enzymes are activated through a phosphorylation
cascade that amplifies and transduces signals from the cell membrane to the nucleus.
Upon receptor activation, membrane bound GTP-loaded Ras recruits one of the Raf
kinases, A-Raf, B-Raf or C-Raf into a complex where it becomes activated. Raf then
phosphorylates the MEK1/2 (mitogen protein kinase kinse 1 and 2; MAP2K1 and
MAP2K2), which in turn activates Erk1/2. Finally, the activated Erks modulate many
cytoplasmic and nuclear targets that perform important biological functions such as
proliferation, differentiation, migration and death [65].
Non-receptor tyrosine kinases
In T-lymphocytes, protein tyrosine kinases play a role in the activation of cells
through various immunoreceptor molecules, such as the T-cell receptor (TCR)/CD3
complex. Engagement of the TCR leads to a rapid rise in intracellular protein
INTRODUCTION
27
tyrosine phosphorylation and the signal is mediated by the activation of the tyrosine
kinases Lck and Fyn. Phosphorylation of these immunoreceptor tyrosine-based
motifs creates docking sites for another tyrosine kinase, the ζ-chain associated
protein 70 kDa (Zap-70) kinase, leading to activation of a series of second messenger
cascades. These include phospholipase Cγ (PLCγ), the PI3-K pathway, and MAP
kinase cascades, and proinflammatory transcription factors [66-68].
A few studies indicate that tyrosine kinases might not only play a role in T-cell
activation but also in apoptosis [69-71]. For instance, Gruber et al. showed that Lck is
essential for apoptosis induction by chemotherapeutic drugs such as doxorubicin,
paclitaxel and 5-fluorouracil by regulating early steps of the mitochondrial apoptosis
signaling cascade, including caspase-activation [71]. Lck is essentially involved in
regulating Bcl-2 proteins such as Bak. In Lck-deficient cells, expression of Bak was
completely absent and by re-expression of Lck, Bak expression was transcritionally
triggered. Moreover, the truncated Bid (tBid) specifically activated Bak and further
cytochrome c release only from mitochondria of Lck-expressing cells [70]. Thus, Lck
controls steps of drug induced apoptosis signaling cascade and participate, as
mentioned above, in phosphoregulation of Erk1/2 and Akt [71].
The regulation of kinase activity in Lck is tightly controlled by conformational
changes arising from for instance; phosphorylation and dephosphorylation of two
critical tyrosine residues. Lck Tyr505 is phosphorylated by C-terminal src kinase (Csk)
when the protein is in an inactive conformation. Conversely, the CD45 tyrosine
phosphatase dephosphorylates Tyr505 allowing Lck to preserve an “open”
conformation which exposes the activation loop containing the activating tyrosine
residue [67]. Trans-phosphorylation of the Lck Tyr394 residue within the active loop
of the kinase domain promotes enzymatic activity. Active Lck phosphorylates CD3
and ζ-chain associated protein 70 kDa (Zap-70) kinase, which in turn, phosphorylates
the key adapter protein linker for activation of T-cells (LAT) and facilitates activation
of further downstream kinases and enzymes [67].
The cytoplasmic tyrosine kinase Zap-70 is mainly expressed in T-cells [68]. The
importance of Zap-70 in TCR signaling and its predominantly T-cell-restricted
expression pattern make Zap-70 an attractive target for the inhibition of pathological
T-cell responses in disease. The tyrosine kinase domain of Zap-70 contains two
tyrosine residues, Tyr492 and Tyr493, which are phosphorylated after TCR
engagement. These tyrosines can be phosphorylated by Lck [72].
INTRODUCTION
28
CANCER THERAPY
CHEMOTHERAPY
Chemotherapy is the standard treatment for many types of leukemia. Even when a
cure is not possible, chemotherapy may prolong life of the patient. Chemotherapy for
leukemia is usually a combination of different drugs often with separate modes of
actions. In order to optimize treatment outcome and to prevents development of
drug resistance [73].
Chemotherapy works by destroying fast-dividing cells whether it is a cancer cell or a
normal cell. Therefore, chemotherapy eliminates not only the highly-proliferating
cancer cells but also normal proliferating cells, including hair follicles, cells in the
digestive tract and in the bone marrow. Drugs used for chemotherapy work by
different mechanisms: disturbing the proliferative machinery in the cells by toxic
effects on enzymes, causing structural changes in the DNA and RNA, disrupting
metabolic needs during proliferation, specifically disturbing the cell cycle and
selectively damage rapidly dividing cells during their DNA synthesis and the mitotic
spindle formation (S and M phases, respectively) [73]. In recent years, chemotherapy
has reached a plateau of efficiency as a primary treatment. The use of the “one
treatment fits all patients” principle entails that; many patients will get suboptimal
treatment. The new generation of anti-cancer drugs involves mechanisms that are
more targeted to cancer cells and their specific functions [74].
TARGETED THERAPY
Targeted therapy implies tailoring of cancer treatment to an individual patient’s
tumor. It has been a major goal of cancer research for many years to identify
intracellular oncogenic targets and to understand the signal transduction pathways
in which these targets participates, and to develop novel drugs able to specifically
target and block these, often aberrantly expressed, critical molecules. A large number
of the activated oncogenes are tyrosine kinases. Aberrant kinase signaling is a
recurrent mechanism implicated in cellular transformation. Discoveries connecting
the fields of viral oncogenes and human protein kinases stimulated discovery of
drugs targeting protein kinases, beginning with the identification of v-ABL from a
mouse leukemia virus. A specific chromosomal abnormality was identified in CML
that led to elucidation of the central role of the Bcr-Abl protein tyrosine kinase in
Philadelphia chromosome-positive CML [75, 76]. The creation of imatinib (Glivec;
INTRODUCTION
29
Novartis), a drug targeting the Bcr-Abl protein (Table 2), revolutionized cancer drug
design by showing that it is possible to kill the defective cells without any major
adverse effects in normal cells [77].
There are two major reasons for the development of targeted therapies: 1) by
targeting a unique characteristic of the tumor, cancer cells will be killed while normal
cells will be spared, thus providing effective cancer treatment with fewer side effects
and 2) if the target is essential for viability, the cancer cell will not easily become
resistant to the given therapy and thereby increasing the effectiveness of this type of
treatment.
Many RTK inhibitors have been developed, and more are under development.
Targeted therapies include monoclonal antibodies and small molecule inhibitors.
These drugs are currently a component of therapy for many common malignancies,
including breast, colorectal, lung and pancreatic cancers, as well as lymphoma,
leukemia and multiple myeloma [78]. Targeted therapies are generally better
tolerated than traditional chemotherapy, but they are associated with several adverse
effects, such as acneiform rash, cardiac dysfunction, thrombosis, hypertension and
proteinuria [74].
Small molecule inhibitors
Small molecule inhibitors interrupt cellular processes by interfering with the
intracellular signaling of tyrosine kinases. These inhibitors differ from monoclonal
antibodies in several ways: they are typically administered orally rather than
intravenously, they are metabolized by cytochrome P450 enzymes and are subject to
multiple drug interactions and in contrast to antibodies have half-lives of only hours
and require daily dosing [74]. In contrast to monoclonal antibodies, small molecules
have the ability to penetrate the plasma membrane and thereby inhibit receptors
expressed at the cell surface and target proteins located in the cytosol.
For cancers whose growth is driven by activated protein tyrosine kinases, so called
tyrosine kinase inhibitors (TKIs) have been developed to block abnormal signaling,
involved in cell proliferation and apoptosis. While some TKIs specifically inhibit one
or two tyrosine kinases, most of them are designed to inhibit several tyrosine kinases
in multiple signaling pathways, such as dasatinib, sorafenib and sunitinib (Table 2)
[79]. While imatinib was designed to selectively inhibit Bcr-Abl, this drug also shows
activity against several other kinases, including the RTKs PDGFR and c-Kit, as well
as colony stimulating factor 1 receptor (CSF1R) and Lck [80]. It is appreciated that the
INTRODUCTION
30
repertoire of potential targets of imatinib, contributes to the drug´s usefulness as a
therapeutic agent for cancer.
Small-molecule inhibitors are competitive antagonists that occupy the ATP-binding
site of the targeted receptor tyrosine kinase domain and compete for binding of ATP
and/or protein substrates. Potential targets include any tyrosine kinase with an
activating mutation; signaling molecules that are immediately downstream of
tyrosine kinases and are important for viability or proliferation signaling (for
example Ras and PI3-K); or other molecules that are not mutated but are
overexpressed in tumor tissues [81].
A variety of genetic abnormalities, including point mutations and in-frame tandem
duplications, have been identified in tyrosine kinase growth factor receptor genes
and linked to transformation. For instance, the c-Kit proto-oncogene encodes a cell
surface tyrosine kinase that functions as the receptor for stem cell factor. Active
mutations of c-Kit are an excellent candidate target in a few patients with AML.
Another attractive tyrosine kinase target is the FLT3 receptor, which is expressed
exclusively on hematopoietic cells. FLT3 is a proto-oncogene capable of transforming
cell lines in vitro to growth factor independence when mutated, as described
previously in this thesis [82].
FLT3 TARGETED THERAPY
AML is an aggressive hematological malignancy with short patient survival if not
treated. It is often associated to poor prognosis due to therapy induced mortality or
resistance to chemotherapy, reviewed in Weisberg et al. [83]. Compared to CML with
the characteristic Bcr/Abl fusion protein, AML is a multi-mutational leukemia and
therefore difficult to treat. Increased insight in specific pathogenic molecular
mechanisms has led to the development of novel therapeutic drugs. Due to the high
frequency of mutations in the FLT3 gene in patients with AML, FLT3 is an interesting
molecular target for treatment [84].
PKC412 is an example of an inhibitor, originally developed as an inhibitor of protein
kinase C, which acts on FLT3 inhibiting its kinase activity in AML [85, 86]. AG1295,
CEP701, MLN518, SU5416, and SU11248 are other examples of small-molecule
inhibitors that competitively inhibit ATP binding to FLT3 and show strong in vitro
efficacy on transformed FLT3-ITD expressing cell lines and primary AML blasts.
However, in clinical practice these inhibitors appear to have limited efficacy due to
development of resistant leukemic clones [84, 87].
INTRODUCTION
31
To overcome these problems of limited clinical efficacy, poor clinical response and
development of resistance associated with the currently used FLT3 targeting
inhibitors, novel inhibitors targeting FLT3 more effectively and/or inhibiting
alternative pathways are needed to enable sustained long-term therapeutic benefits.
ERBB TARGETED THERAPY
ERBB receptors have been implicated as key triggers of oncogenesis in various solid
tumors [88]. Therefore, these receptors represent potential molecular targets for
cancer therapy. A number of monoclonal antibodies directed towards the
extracellular ligand-binding domain, preventing ligand binding, have been
developed. One example is the ERBB2-targeting antibody trastuzumab (Herceptin ®)
that has improved treatment outcome in patients with breast cancer overexpressing
ERBB2 [43]. Another approach has been to target the intracellular tyrosine kinase
domain of the ERBB receptor using small-molecule ERBB tyrosine kinase inhibitors
such as gefitinib (ZD-1839; Iressa®) and erlotinib (OSI-774; Tarceva®) [89]. These two
inhibitors have previously been shown to attain clinical benefits in the treatment of
solid tumors such as lung cancer [90]. More recently, the pan-ERBB receptor tyrosine
kinase inhibitor canertinib (CI-1033) has been demonstrated to mediate growth arrest
in a number of solid cancer cell types and has shown promise in blocking cancer
growth when used in combination with DNA-damaging agents [91].
The EGFR-specific antibody, cetuximab (Erbitux®), is used for treatment of metastatic
colon cancer and head and neck cancer, where it is employed in combination with
chemotherapy or as a single drug. The antibody binds to the receptor with high
affinity and thereby blocks the ligand binding and induces receptor internalization
and degradation, resulting in downregulation of surface EGFR expression.
Cetuximab inhibits the growth and proliferation of cancer cells by blocking the G1
phase of the cell cycle and promoting programmed cell death [92].
The clear potential of ERBB-targeted therapies in the treatment of cancer, has led to
development of a variety of new agents targeting the extracellular ligand-binding
domain, the intracellular tyrosine kinase domain or the ligand.
There is evidence of feedback compensation upon the inhibition of a single target
within signaling networks. For example, inhibition of ERBB2 phosphorylation in
ERBB2 overexpressing human breast cancer cells induced by the EGFR tyrosine
kinase inhibitor gefitinib is followed by activation of ERBB3 and Akt, thus limiting
the inhibitory effect of gefitinib [93].
INTRODUCTION
32
Table 2. Tyrosine kinase inhibitors, their targets and the disease
STRATEGIES TO APPLY KINASE TARGETED THERAPY
To determine whether multiple single kinase inhibitors or a single multikinase
inhibitor is preferable in cancer therapy one must consider; 1) the extent and number
of expressed tyrosine kinases specific for the cancer type, 2) possibly pre-existing
resistance to tyrosine kinase inhibitors, 3) the bioavailability of the drug its
absorption, distribution, metabolism and excretion, 4) selectivity where tyrosine
kinase inhibitors are designed to specifically attack the ATP-binding site of tyrosine
kinases, 5) the tumor microenvironment involving the connective tissue and stroma
where for example growth factors and their cognate receptors may be upregulated,
and 6) the grade and type of toxicity in the choice of kinase inhibitors using them
alone or in combination [94].
The variability of tyrosine kinases between tumor types is high, but tumors of the
same histological type tend to have more similar receptor tyrosine kinase profiles,
with disease specific expression both in number and type of receptor. In cancer types
where few tyrosine kinases are overexpressed as in AML, the importance of each
kinase may be relatively higher than in other forms of cancer where many tyrosine
kinases are overexpressed. Specific targeting of these single kinases will present a
greater chance of an effective treatment compared with other tumors that have a
higher number of alterations in receptor tyrosine kinase expression [94].
Drug Known targets Disease Reference
Gefitinib ERBB1 NSCLC Muhsin et al. 2003
Erlotinib ERBB1 NSCLC Rocha-Lima et al. 2007
Lapatinib ERBB1, ERBB2 Breast cancer Nielsen et al. 2009
Canertinib ERBB1-4 Breast cancer Rocha-Lima et al. 2007
PKC412 FLT3 AML Weisberg et al. 2002
Imatinib BCR-ABL CML Fausel et al. 2007
Dasatinib BCR-ABL, SRC CML Gora-Tybor et al. 2008
Sorafenib RAF, FLT3, KIT AML Auclair et al. 2007
Nilotinib BCR-ABL CML Gora-Tybor et al. 2008
INTRODUCTION
33
In addition to the variability of expressed receptor tyrosine kinases between tumor
types and subtypes, the possibility exists that some receptor kinases are tumor
suppressor genes or that the role of a specific receptor tyrosine kinases may differ in
various types of cancer as well as between cancer cells and normal cells.
It is important to focus on selectivity for tumor cells compared to normal cells. The
sensitivity to for example gefitinib treatment is dependent on particular genetic
alterations observed only in tumor cells, thereby minimizing the effect on normal
cells. Non-small-cell lung cancer patients for instance are selected for gefitinib and
erlotinib based on their mutational status; the treatment-predictive deletion in EGFR
exon 19 and EGFR L858R point mutation are highly associated with a non-smoking
female patient of Asian origin [95, 96]. It has been suggested that the most important
condition for an inhibitor to achieve specificity for a particular kinase, is the ability to
adapt to multiple conformational states of the enzyme. This ability seems to be more
important than differences in sequence of the kinase domain or differences in
interactions with binding site residues [97]. One example is erlotinib, which is shown
to be dependent on the recognition and high affinity binding of many conformations
of EGFR. Another example of a specificity mechanism is that of dasatinib, which is
suggested to inhibit Abl, c-Kit, Src and Lck because of its ability to recognize multiple
conformational states of many different targets [97].
RESISTANCE
Eventually most cancer cells will develop resistance to the given therapy
independent of the nature of the anti-cancer drug, including TKIs. Malignant tumors
can develop resistance in several ways such as; secondary mutations of the tyrosine
kinase (Bcr-Abl in CML, FLT3 in AML and EGFR in NSCLC), gene amplification and
thereby overexpression of the protein kinase (Bcr-Abl in CML and c-Kit in GIST),
activation of other signaling pathways and overexpression of kinases downstream
the targeted kinase (PDGFR mutation in c-Kit mutated GIST), differential expression
of the drug transporters (i.e. P-glycoprotein), mediating active cellular influx and
efflux of the drug [98].
There are different approaches to avoid development of resistance. To develop
inhibitors that bind the protein with higher affinity and/or to develop a combination
of inhibitors, who have different mutation profiles, might be effective to prevent
development of resistant clones [99].
INTRODUCTION
34
Moreover, resistance may depend on overexpression of the target kinase. In these
cases inhibiting a kinase downstream of the tyrosine kinase receptor, in addition to
the target receptor itself, will be much more effective because these downstream
kinases are probably not amplified. Thus, a single multi kinase inhibitor would be
preferred, but these downstream kinases may not be specific for cancer cells and
inhibition will result in toxicity to normal cells [83].
Resistance to gefitinib and erlotinib is caused by a secondary mutation in the EGFR
gene, such as T790M, but also by K-ras mutation and overexpression of
phosphorylated Akt [100]. Another mechanism for resistance of NSCLC to gefitinib
and erlotinib was the amplification of MET. This tyrosine kinase receptor has the
ability to induce ERBB3 phosphorylation without the involvement of EGFR and
ERBB2, resulting in the activation of PI3-K/Akt signaling [101]. A combination of
MET inhibitors and EGFR inhibitors could therefore offer an effective treatment for
patients that are resistant to gefitinib and erlotinib [102].
It has been shown that PI3-K/mTOR blockers may override resistance against
irreversible ERBB inhibitors such as canertinib (ERBB1-4) and pelitinib (ERBB1 and
ERBB2) in breast cancer cells [103]. Upregulated phosphorylation of Akt may serve
as a biomarker for resistance against irreversible ERBB inhibitors. Therefore, co-
application of ERBB- and mTOR inhibitors may be an option for treatment of breast
and ovarian cancer.
Dasatinib, which is a multi-kinase TKI approved for the treatment of CML patients
who are resistant or intolerant to imatinib, targets the Abl-kinase, c-Kit, PDGFR and
various members of the Src-kinase family such as Lck in T-cell leukemia [67, 104].
Another multi-kinase inhibitor is sorafenib. Like dasatinib, sorafenib targets multiple
molecules and related pathways (including c-Kit, PDGFR, as well as Raf-MEK-Erk
signaling), and is of interest in AML for its capacity to antagonize deregulated
signaling induced by the FLT3-ITD receptor [104].
Costa and co-workers showed that upregulation of the pro-apoptotic protein Bim
correlated with gefitinib-induced apoptosis in lung cancer cells. The T790M mutation
in EGFR blocked the gefitinib-induced apoptosis but was overcome by the
irreversible TKI CL-387,785. This suggests that induction of Bim may have a role in
the treatment of TKI-resistant tumors [105].
INTRODUCTION
35
CANERTINIB
Canertinib, also called CI-1033, PD-183805 (Pfizer), was designed as a pan-ERBB
tyrosine kinase inhibitor. It inhibits all four ERBB receptor family members.
Canertinib is an irreversible inhibitor that binds covalently to specific cysteine
residues in the ATP-binding pocket such as cysteine 773 of EGFR, cysteine 784 of
ERBB2 and cysteine 778 of ERBB4 thereby blocking the ATP binding site in the
kinase domain of ERBB proteins, preventing their kinase activity and downstream
signaling, it also prevents transmodulation of ERBB3 [106]. The covalent binding of
canertinib results in prolonged suppression of ERBB activity [107].
Since canertinib blocks signaling through all members of the ErbB receptor family it
is more efficient and has a broader antitumor effect than inhibitors that only prevent
signaling from one of the ErbB receptors. Studies of human cancer cell lines indicate
that canertinib results in potent and sustained inhibition of ERBB tyrosine kinase
activity, thereby inhibition of Akt and MAPK pathways [108, 109].
Canertinib has been shown to inhibit growth and induce apoptosis in several cancer
cell lines and xenografts [110-112]. In clinical studies canertinib has been shown to
have acceptable side-effects. However, in phase II studies canertinib was only able to
show modest effects on breast cancer and NSCLC patients [113, 114]. Therefore, it is
important to identify the patients that are the most likely to respond to treatment.
Canertinib is evaluated in clinical trials in the treatment of different solid cancers but,
to our knowledge, not in hematopoietic malignancies.
Canertinib seems to be a promiscuous drug, a multi-kinase inhibitor, which is able to
bind not only to the ERBB receptor family, but also to intracellular proteins. For
instance, the Src kinase family consists of eight members, five of which are mainly
expressed in hematopoeitic cells, Blk, Hck, Lck, Fyn, and Lyn, where the Lck protein
seems to have a stronger binding to canertinib as shown in a protein binding assay
[115].
36
AIMS OF THE THESIS
37
THE PRESENT INVESTIGATION
AIMS OF THE THESIS
The overall goal of the present study was to gain an increased molecular
understanding of targeted therapy and drug-induced cell death-mechanisms in
leukemia. This resulted in formulation of the following objectives:
to investigate the anti-proliferative and/or pro-apototic effects of the pan-
ERBB receptor tyrosine kinase inhibitor canertinib on leukemic cells.
to examine if human leukemic cell lines express ERBB receptor family
members.
to investigate the mechanism of action of canertinib and whether it acts on
a specific target or if it displays off-target effects, or alternatively if there
are any ERBB receptor targets available.
to determine anti-proliferative and cell death-inducing efficacy of
canertinib in vitro and in vivo in AML cells expressing mutated FLT3
receptors.
38
MATERIALS AND METHODS
39
MATERIALS AND METHODS
The principles of some methods used in this thesis are described here.
CELL LINES
This thesis was based on cell lines as follows:
PATIENT SAMPLES
FLT3 mutational data regarding ITD status in ten patient samples included in the
study (Paper III).
* This cell line is FLT3 negative and was used as model for apoptotic and biochemical studies by overexpressing different FLT3 versions.
Cell line Origin (human)
HL-60 promyelocyte
U-937 promyelocyte monocytoid
Jurkat lymphoblasticT-cell leukemia
Daudi B-CLL Burkitt's lymphoma
K-562 CML blast crisis
HEL erythrocyte leukemia
LCL4 EBV-transformed lymphoblastoid
232-B4 B-CLL
MCF-7 Breast
Cell line Origin FLT3 status
MV4:11 Human FLT3-ITD
MonoMac-6 Human FLT3-V592A
FDC-P1 * Mouse wt, ITD, TKD
Ba/F3 * Mouse wt, ITD, TKD
THP1 Human wt FLT3
NB4 Human wt FLT3
RS4;11 Human wt FLT3
Patient # ITD length (bp)* % ITD of blast **
I 162 25
II 40 11
III 36 57
IV 27 19
V 51 32
VI 66 31
VII 21 38
VIII 93 48
IX 0 n.a.
X 0 n.a.
* FLT3-ITD (length in number of base pairs).
** Mutant FLT3-ITD DNA as percent of total DNA content.
n.a., not applicable
MATERIALS AND METHODS
40
WESTERN BLOT ANALYSIS
In paper I-IV, Western blot was used and is a method for separation and
characterization of proteins. Western blot analysis was introduced by Towbin et al. in
1979, and is also called immunoblotting because antibodies are used to specifically
detect different protein antigens [116]. The initial separation procedure followed by
the specificity of the antibody-antigen interaction enables a target protein to be
identified from a complex protein mixture (protein lysate).
First the gels are prepared with different pore size depending on the size of the
protein. The size of the pore in the gel is inversely related to the amount of
acrylamide used. Thus, a 7% polyacrylamide gel has larger pores than a 12%
acrylamide gel. Acrylamide mixed with bisacrylamide forms a crosslinked polymer
network when the polymerizing agent, ammonium persulfate (APS), is added.
TEMED (tetramethylenediamine) catalyzes the polymerization reaction by
promoting the production of free radicals by APS. Laemmli buffer containing β-
mercaptoethanol is added to the protein lysate and then the lysate is boiled for 4 min.
The proteins are denatured during the boiling, whereby cleavage of disulfide bonds
enables a protein to completely unfold. Using SDS-PAGE, sodium dodecyl sulfate
(SDS) is added to the gel, to the protein samples and to the buffer which is in the tank
where the gel will be running, thus SDS is in the complete system making all proteins
negatively charged. Protein lysates are added to the gel and an electric current is
applied, making the negatively charged proteins move in the gel from the negative
cathode towards the positive anode. The proteins are size-separated in the
approximate range of the molecular mass, thus the smaller proteins migrate faster
than the larger proteins. The separated proteins are transferred from the gel and
blotted onto a membrane such as polyvinylidene difluoride (PVDF) which is applied
on the gel surface. When an electric field is applied, the proteins move from the gel
and onto the surface of the membrane, where the proteins become tightly attached.
The result is a membrane with the protein pattern that was originally in the gel. After
transfer, membranes are incubated with a protein solution such as milk or bovine
serum albumin, to block all free binding sites on the membrane. This is done to
prevent the antibody from binding non-specifically. The protein solution also
contains Tween-20, which is a detergent that improves the antigen-antibody binding.
The membrane is incubated with a primary antibody directed towards the specific
protein of interest. Thereafter membranes are washed to remove all non-bound
antibodies to minimize the non-specific background labeling. Subsequently, the
membrane is incubated with the secondary antibody which binds to the primary
MATERIALS AND METHODS
41
antibody. The secondary antibodies used in this thesis are conjugated with
horseradish peroxide (HRP). The membrane is washed to remove excess antibodies.
Then enhanced chemoluminescence (ECL) is used which produce a signal with HRP
that may be detected using an ECL film.
FLOW CYTOMETRY
Flow cytometry is a well-known method that is used to measure multiple parameters
in a single cell. The fluidic system transport particles or cells from a suspension
through the cytometer. Intracellular molecules and membrane markers can be
stained with different fluorochromes and analyzed simultaneously in the flow
cytometer. The cells pass through a light source one at the time. Light is scattered
upon passing through the cells and the fluorochromes are excited to a higher energy
state. This energy released in form of emitted light, is detected together with the
scattered light by several detectors. Forward-scattered light (FSC) is proportional to
the surface area or the size of the cell, whereas Side-scattered light (SSC) is
proportional to the granularity or internal complexity of a cell (i.e. shape of the
nucleus, or the amount and type of cytoplasmic granules). By plotting FSC against
SSC, different cell populations can be visualized and a region of interest is defined,
called gating. The population of interest is then shown in a histogram and the mean
fluorescence intensity (MFI) can be analyzed by statistics. Mean fluorescence values,
from 15 000 cells/ sample, were determined. Gating was performed using FSC and
SSC, excluding cell debris. Autofluorescence of unstained cells was routinely
analyzed and subtracted from the fluorescence values of the stained cells. Unspecific
binding was analyzed by isotype antibodies.
Cell cycle distribution
In paper I and II the cell cycle distribution of the cell population before and after
treatment with canertinib was measured. The staining of DNA with propidium
iodide (PI) was chosen and is a method developed by Vindelov et al. [117]. PI is not
able to penetrate intact cell membrane therefore a detergent such as trypsin is used to
make the membrane more permeable. Next, a trypsin inhibitor is added that prevent
trypsin from breaking down the cells, thereafter staining of the DNA in the cell
nuclei with PI. Flow cytometric measurement results in a DNA histogram where data
from each cell is analyzed to determine their location in the cell cycle.
MATERIALS AND METHODS
42
Detection of cell death
In paper I-III induction of apoptosis was investigated in leukemia cell lines after
canertinib treatment, but also after caspase-8, -9 and -10 inhibtion or
lysosomal/cathepsin inhibition together with canertinib treatment (Paper I-III).
During the apoptotic process the membrane phospholipid phosphatidylserine is
transferred from the cytoplasmic surface of the cell membrane to an outer side
location, where it serves as a signal for phagocytosis [118]. Annexin-V is a
phospholipid-binding protein with a high affinity for phosphatidylserine, thus cells
in early and middle stages of apoptosis can be detected. In order to follow the
binding of Annexin-V to phosphatidylserine, a fluorochrome called phycoerythrin
(PE) was labelled to Annexin-V. A concomitant staining with the DNA binding dye
7-amino-actinomyocin D (7-AAD) which only enters cells with permeabilized
membrane.
By using both Annexin V and 7-AAD cells that are viable (Annexin V-PE negative
and 7-AAD negative), early apoptotic (Annexin V-PE positive and 7-AAD negative),
late apoptotic or dead (Annexin V-PE positive and 7-AAD positive), mechanically
destroyed cells (7-AAD positive and Annexin V-PE negative) can be distinguished
from each other [119].
Presence of ERBB2 protein
In paper IV we used flow cytometry to demonstrate the presence of ERBB2 protein in
U-937, HL-60 and Jurkat cells. Indirect immunofluorescent labeling was performed,
first an antibody directed towards the receptor of interest was used then a secondary
fluorochrome-conjugated antibody that recognizes the Fc-part of the primary
antibody was utilized. The antibody for ERBB2 recognized intracellular part of the
receptor. Therefore, an IntraStain and Permeabilisation kit (K2311, DakoCytomation)
was used. First reagent A was used to fix the cells and then reagent B was used to
permeabilize the membrane, allowing the antibody to enter the cells.
The ERBB2 antibody recognizes the extracellular part of the receptor. Then a
fluorescein-isothiocyanate (FITC)-conjugated Goat-anti-Mouse F(ab’)2 (F0479,
DakoCytomation) was added, the cells were fixed with paraformaldehyde and the
samples were analyzed on a FACSCalibur (BD Biosciences).
MATERIALS AND METHODS
43
REVERSE-TRANSCRIBED POLYMERASE CHAIN REACTION, RT-PCR
In paper I-IV, RT-PCR was used and is a sensitive method for the detection of mRNA
expression levels. Traditionally RT-PCR involves two major steps. First the reverse
transcription (RT) is performed in which single stranded RNA is reverse transcribed
into complementary DNA (cDNA) by using total cellular RNA, a reverse
transcriptase enzyme, a random primer, deoxynucleotides triphosphates (dNTP) and
an RNase inhibitor. The RT reaction is also called the first strand cDNA synthesis.
For a RT-reaction in this thesis, 2 micrograms (µg) of RNA was used. The RNA was
first incubated with the primer at 70 C° to denature RNA secondary structure and
then quickly chill on ice to let the primer anneal to the RNA. Other components of
RT, as mentioned above, were added to the reaction. RT reaction is extended at 42 C°
for 1 h thereafter the reaction is heated at 70 C° to inactivate the enzyme. The
resulting cDNA was used as templates for PCR amplification using primers specific
for one or more genes.
In the second step of RT-PCR a single piece of DNA is amplified and to millions of
copies of a particular sequence [120]. DNA replication in vitro is performed in cycles,
each consisting of: denaturation, annealing and extension. Usually the numbers of
cycles used are between 30 and 40. During denaturation, heating (95 C°) separates
double-stranded DNA into two single strands. This is possible because the hydrogen
bonds linking the bases to one another are weak and break at high temperatures. In
this thesis the goal was to replicate a target sequence of approximately 100-1000 base
pairs unique to the ERBB receptor proteins being studied. The two primers used
were short, 20-30 bases, and targeted the DNA sequence of interest by annealing to
the complementary sequence in the amplified DNA. The annealing temperature in
any RT-PCR procedure is usually between 40 and 65 C°, depending on the length
and base sequence of the primers. This allows the primers to anneal to the target
sequence with high specificity. After the annealing process the temperature is raised
to 72 C° and the enzyme Taq DNA polymerase, which is active at high temperatures
(thermostable), starts to extend the primer with soluble dNTPs copying the sequence
of the attached single strand DNA. The extension begins at the 3’ end of the primer
and the enzyme synthesizes in the 5’ to the 3’ direction and creates a complementary
strand. Two new double stranded DNA molecules, identical to the original DNA
region are produced. The cycle is repeated 30-35 times and the amount of DNA is
doubled further with every cycle. The PCR samples are then loaded on a 1.3%
agarose gel (Invitrogen, Paisley, Scotland UK), size-separated and stained with
ethidium bromide for detection of the PCR products on a UV-table.
44
RESULTS AND DISCUSSION
45
RESULTS AND DISCUSSION
PAPER I AND II
Canertinib inhibits growth and induces apoptosis in human leukemia cell lines
This study was initiated to determine whether the pan-ERBB receptor tyrosine kinase
inhibitor, canertinib, displays any anti-neoplastic effect on human leukemia cells. It
had previously been shown that epidermal growth factor (EGF) receptor tyrosine
kinase inhibitors such as gefitinib and erlotinib induce differentiation and apoptosis
in human malignant myeloid cells [53, 54, 121]. Our results show that treatment of
leukemic cell lines (HL-60, U-937 and Jurkat) with canertinib significantly inhibits
cell growth in a dose dependent manner as well as induces an accumulation of cells
in the G1 phase of the cell cycle. In addition, canertinib clearly induced apoptotosis in
the cell lines as determined by the Annexin V method and cleavage of poly-(ADP-
ribose) polymerase (PARP).
Mechanisms of apoptotic cell death in leukemia cells
Since canertinib induced apoptosis in leukemic cell lines, the effect and underlying
mechanisms was investigated with primary focus on the involvement of caspases,
intracellular signaling proteins and lysosomal contribution (Paper II). As an
experimental model, Jurkat T-cell leukemia cells were chosen, mainly due to the
multiple previous studies employing Jurkat cells to study the apoptotic process.
Canertinib induced caspase-mediated apoptosis at low doses. The pattern of
apoptotic signaling employed both initiator caspases, including caspase-8, -9 and -10,
as well as effector caspases, including caspase-3. Time-response experiments showed
an activation of Bid and caspase-3, -8, and -9 within 3 h as well as an activation of
caspase-10 within 6 h by canertinib treatment, indicating primarily activation of the
intrinsic apoptotic pathway (Paper II, Figure 3a). A decreased level of tBid was
identified at 6 h, presumably due to the short half-life of tBid, in accordance with the
canertinib-induced caspase-8 activation. Canertinib-induced activation of caspase-8
was enzymatically active enough to cleave substrate like Bid [122]. As previously
described, caspase-9 is an initiator caspase of the intrinsic pathway and activates
downstream effector caspases such as caspase-3 and caspase-8. Activated caspase-8,
in turn, induces cleavage of Bid and forms a positive feedback loop that amplifies the
apoptotic signaling [123]. In addition, Guerrero and co-workers have demonstrated
RESULTS AND DISCUSSION
46
that caspase-3, -8 and -10 are capable of acting downstream of caspase-9 [124].
Caspase-10 is generally a candidate for binding to the death receptor (CD95) in
Jurkat cells but it is also involved in the intrinsic pathway since it is able to cleave Bid
to tBid [122, 125]. Since caspase-10 was activated later than the other caspases we
suggest the latter role for caspase-10 in our study.
Reduction of apoptosis was demonstrated in canertinib-treated Jurkat cells using
specific caspase-8, -9 and -10 inhibitors as shown by Annexin V analysis. This result
was confirmed by western blot experiments using increasing concentrations of the
specific inhibitors resulting in decreasing levels of active caspase-8 and -10. In
contrast, caspase-9 was still active at a concentration of 10 µM caspase-9 inhibitor
and the activity could not be further reduced using up to 40 µM of the inhibitor.
Camptothecin, which was used as a positive control for the intrinsic pathway of
apoptosis [126], showed a similar pattern for the caspase-9 activity. Caspase-9 has
previously been found both in its inactive as well as in its active form in the
mitochondria of camptothecin-treated Jurkat cells. However, the authors could not
conclude if the activation occurred in the cytosol or in the mitochondria [126]. Others
have suggested that caspase-9 is directly activated in the mitochondria [13].
Lysosomal involvement
It has been shown that lysosomal cathepsins can either mediate caspase-independent
apoptosis or be involved in caspase-dependent apoptosis. Earlier data showed that
caspase-9 is able to promote lysosome-mediated cell-death through an apoptosome-
independent mechanism [24]. We show no evidence of cathepsin cysteine proteases
in canertinib-induced apoptosis, since the cathepsin cysteine protease inhibitor E64d
did not affect the canertinib-induced apoptosis. However, FasL-induced apoptosis
was readily decreased by the inhibitor indicating that canertinib and FasL initiates
different apoptotic pathways. Hence, we conclude that lysosomal proteases are not
involved in canertinib-induced apoptosis in Jurkat cells.
ERBB receptor expression in leukemia cells
The myeloid cell lines used in this thesis were found to express mRNA for ERBB2/3/4
receptors, but did not express any ERBB1 receptor mRNA as determined by RT-PCR
(Paper I, Figure 3 A). The lymphoid cell line Jurkat demonstrated the presence of
mRNA for ERBB2 and ERBB3, but not ERBB1 or ERBB4 transcripts. However, we
could not demonstrate any protein expression of either ERBB receptor, even
RESULTS AND DISCUSSION
47
following neuregulin, NRG1-β1, treatment (Paper I, Figure 4 B), by using Western blot
analysis with several different commercially available ERBB-antibodies.
Canertinib affects specific signaling proteins in T-cell leukemia cells
If leukemia cells are devoid of ERBB receptors, how come they undergo apoptosis
after treatment with canertinib? Most likely canertinib is able to act on important
signaling kinases present in the cell. Canertinib has been shown to interact with
several intracellular proteins as identified from a binding assay [115]. One of these
targets to which canertinib bound with high affinity, was Lck involved in T–cell
activation. Besides participating in T-cell activation, Lck might also be involved in
the induction of apoptosis, since Lck has been shown to be important in drug-
induced apoptosis [71, 127]. When we treated Jurkat cells with canertinib, total Lck
protein expression gradual decreased, dependent of increasing concentrations of the
drug, possibly by Lck degradation (Paper II, Figure 4 C and D). Concomitantly
dephosphorylation or degradation of the active and inactive form of Lck Y394 and
Y505 respectively was also shown. Others have shown that Lck is essential for
apoptosis induction regulating steps of the mitochondrial apoptotic signaling
cascade, including caspase and Bak activation in Jurkat cells [71]. However, the
kinase activity of Lck is not necessary for its pro-apoptotic effect [70]. According to
another study, where the multikinase sorafenib induced apoptosis by targeting Lck
in primary human T-cells [128], it is possible that canertinib confers chemotherapy
sensitivity through activation of pro-apoptotic pathways involving Lck in Jurkat
cells.
Downstream of Lck, Zap-70 has been shown to be phosphorylated in an Lck-
dependent manner on its tyrosine residue Tyr493 [68, 72]. The importance of Zap-70
in T-cell signaling and its predominantly T-cell-restricted expression pattern makes
Zap-70 an attractive target in canertinib-mediated effects.
In fact, we could demonstrate that Zap-70Tyr493 is inhibited by canertinib treatment in
both a dose- and time-dependent manner in Jurkat cells. The inhibition of
phosphorylated Zap-70 occurs already at the first dose- and time-point of canertinib
treatment (Paper II, Figure 4 C and D). This is possibly a result of: i) the decreased Lck
protein expression, ii) Zap-70 is a potential target for canertinib or iii) canertinib
affects other proteins upstream of Lck and Zap-70.
RESULTS AND DISCUSSION
48
Canertinib inhibits survival-signaling pathways
In our study caspase-9 seems to play an essential role in canertinib-induced
apoptosis since it is unphosphorylated and cleaved to its active form. This is possibly
a result of Akt and Erk1/2 inhibition, because these intracellular signaling proteins
are inhibited earlier than the caspase activation occurs. Others have shown that
inactivated Akt allows cleavage of Bid and procaspase-9 which results in caspase-3
activation and increased induction of apoptosis [129]. Canertinib has also been
shown to significantly suppress constitutive activation of Akt and Erk1/2 in ERBB-
positive solid tumors [130]. We could show that canertinib induced a gradual
inhibition of phosphorylated Akt in a dose- and time-dependent manner, whereas
inhibition of constitutive Erk1/2 activity was already observed within the first time-
point (15 min) and Erk1/2 activation reappeared within 3-6 h (Paper II, Figure 4B).
This pattern of Akt and Erk1/2 expression has previously been shown in AML, where
inhibition of Erk1/2 activity either induces apoptosis in leukemic cells or sensitizes
the cells to chemotherapy induced cytotoxicity [131]. In the majority of primary acute
leukemic cells, the Erk1/2 pathway is constitutively active which might explain the
active Erk1/2 expression in untreated Jurkat cells [132, 133]. Gruber and colleagues
also showed that phosphorylation of Erk1/2 was significantly reduced in Lck-
deficient Jurkat cells [71].
This supports the hypothesis that canertinib inhibits Lck and thereby suppress activation of
Akt and Erk1/2. We believe canertinib induces a receptor block and/or a promiscuous
inhibition of several intracellular targets resulting in a decreased pLck, Lck, pZap-70, pAkt
and pErk1/2 activity in Jurkat cells (Figure 6).
RESULTS AND DISCUSSION
49
Figure 6. Proposed model of the mechanisms by which canertinib induces apoptosis in leukemia cells
based on the observations in paper I and II. The intrinsic pathway is activated including caspase-8 and
-10. The signaling pathways Akt and Erk1/2 are inhibited and the upstream lymphocytic specific
proteins Lck and Zap-70 are affected by canertinib treatment.
PAPER III
Anti-leukemic effects of canertinib are associated with Bim-mediated apoptosis through
inhibition of FLT3 signaling and regression of FLT3-transformed cells in mice
The results presented above together with studies from other groups indicate that
tyrosine kinase inhibitors (TKIs) targeting ERBB receptor family display anti-
leukemic effects despite the lack of the receptor expression [54, 121, 134, 135]. When
we initiated our study, we identified canertinib as an effective inhibitor of FLT3
signaling in hematopoietic and leukemic cell lines. These results are described in
paper III, in which we studied the efficiency of canertinib to induce antiproliferative
effects and cell death in cells with activated FLT3 receptor.
We used human leukemic cell lines MV4;11 and MonoMac-6, expressing FLT3-ITD
and FLT3-JM-PM respectively. The cells became apoptotic upon treatment with
canertinib. Cell lines expressing non-mutated FLT3 (THP1, NB4, RS4) required a
significantly higher concentration to reach an equal level of cell death. In addition,
we tested the effects of canertinib on AML patient samples harbouring FLT3
mutations (ITDs of different length varying in size from 21-162 base pairs and with
ERK1/2
AKT
Bid
tBid
Caspase-8,-10
Caspase-3
Caspase-9 Cyto C
Apaf 1
PARP
Bax
Bax
Bcl-2
APOPTOSIS
cell membrane
Lck
Zap-70
LAT
┴
RESULTS AND DISCUSSION
50
FLT3-ITD expression levels between 11-57% of the blast cell DNA). Treatment with
canertinib decreased the viability of the AML cells in correlation to the expression
level of FLT3-ITD. Cell death was measured by Annexin V staining, PARP cleavage
and increasing amount of the pro-apoptotic protein Bim and caspase-3.
Interestingly, blast cells from patients with AML ablated by canertinib expressed
stem cell markers, such as CD33 and CD34, and had apparent blast cell morphology.
These cells also revealed a higher FLT3-ITD expression levels. Thus, the therapeutic
efficacy was demonstrated in leukemic cell lines expressing mutated FLT3 and in transformed
progenitor cells expressing FLT3-ITD as well as in primary leukemic blasts from patients
diagnosed with de novo AML.
To prove that canertinib acts directly on the FLT3 receptor, we also studied
downstream targets associated to FLT3. We could demonstrate downregulation of
phosphorylated Akt and Erk1/2, as well as a block in FLT3 receptor
autophosphorylation and kinase activity. Since transcription factor FoxO3a is a
downstream target of Akt, we analyzed and found that FoxO3a was
dephosporylated and thereby activated by canertinib in FDC-P1/FLT3-ITD cells.
Consequently, pro-apoptotic Bim, a transcriptional target of FoxO3a, was
upregulated by canertinib in FLT3-ITD cells. Using RNA interference in the FLT3-
ITD-transformed progenitor cells, we could demonstrate that Bim was required for
canertinib-induced apoptosis.
In vivo anti-leukemic activity of canertinib was determined in a transplantation
model, where syngenic mice were intravenously injected with FLT3-ITD expressing
FDC-P1 cells and thereafter treated either with canertinib or control. In canertinib-
treated mice, expansion of FLT3-ITD cells was significantly prevented.
This study provides mechanistic insight to how canertinib causes apoptosis of FLT3
expressing leukemic cells. It adds canertinib as a potential drug for future therapy of
AML patients diagnosed with FLT3 mutations. In recent years, multiple FLT3
inhibitors have been described in the literature. Some of these are clearly more active
than canertinib, and at significantly lower concentrations. However, initial studies
describe limiting clinical effectiveness, poor clinical response and development of
resistance. To overcome these problem, novel inhibitors targeting FLT3 more
effectively and/or inhibiting alternative pathways is needed to enable sustained long-
term therapeutic benefits. Although additional studies are necessary, canertinib
could turn out to be an efficient drug due to its multi-kinase activity and its mode of
action via activation of FoxO3a and activation of Bim (Figure 7). Combined with
RESULTS AND DISCUSSION
51
FoxO3a activating reagents and/or addition of BH3-mimetics (behaving like active
Bim), this could provide interesting directions for future therapy.
Figure 7. Proposed model for the anti-proliferative action of canertinib in AML cells expressing
activated FLT3 receptor. Canertinib induces an inhibition of the signaling cascade and an activation of
the pro-apoptotic proteins resulting in increased activation of FoxO3a and thereby upregulation of
Bim, subsequently stimulating the mitochondrial pathway of apoptosis.
PAPER IV
Expression of a non-glycosylated truncated ERBB2 receptor in human leukemia cell lines.
During the course of our work, we came across an interesting finding: evidence for
the presence of an N-terminally non-glycosylated ERBB2 receptor protein in human
leukemia cells, where the mRNA consists of an alternative in-frame start codon.
An ERBB2 receptor protein of approximately 160 kDa was demonstrated in several
human leukemia cell lines (Paper IV, Figure 1). The extracellular (ec) domain of the
protein was not found on the cell surface, and surprisingly neither in the cytoplasm.
However, the intracellular (ic) domain was clearly identified, suggesting that a part
ERK1/2
PI3-K
AKT
canertinib canertinib
R RFLFL
Bid
tBid
Caspase-8,-10
Caspase-3Caspase-9 Cyto C
Apaf-1
PARP
FLT3-receptor
Bak
Bak
Bcl-2
┴FoxO3a
Bim promoter
Bim
APOPTOSIS
cell membrane
RESULTS AND DISCUSSION
52
or a complete protein sequence in the ec domain of the ERBB2 receptor was deleted
(Paper IV, Figure 2).
Previous studies have identified truncated forms of ERBB receptors in several solid
cancers from different tissues such as lung and breast. For example, the wild type
ERBB2 185 kDa protein has been shown to exist as a truncated form detected in
patients with advanced breast cancer. This N-terminally truncated ERBB fragment,
p95ERBB2, possesses in vitro kinase activity and its presence in breast cancer
specimens has been shown to correlate with a poor prognosis and dismal treatment
outcome in patients treated with the ERBB2 specific antibody trastuzumab
(Herceptin ®) [136]. The EGFR/ERBB2 dual drug lapatinib was shown as an effective
treatment to p95ERBB2-positive tumors [137].
The ERBB2 receptor DNA consists of 31 exons where exon 5-31 are transcribed and
further translated into the full-length receptor protein of 1255 amino acids (Figure 8).
In contrast, the truncated ERBB2 receptor in Jurkat cells consists of exon 1-4 and exon
6-31 (Paper IV, Figure 4 A-C). Others have shown that lack of exon 5 results in a
putative in-frame start codon in exon 6, which generates in an ERBB2 protein with
1225 amino acids [138, 139]. The lack of exon 5 in ERBB2 mRNA may be explained by
alternative splicing with a mutation in the exon-intron junction splice sites and/or an
alternative promoter in the ERBB2 receptor sequence. In previous studies, a novel
transcript of ERBB2 was identified containing additional 5’ sequences demonstrating
“new” exons in the ERBB2 gene with a novel promoter further upstream of the
general transcriptional start site for full-length ERBB2 receptor [138, 139].
Other splice variants of ERBB2 have previously been described. For example, the
truncated HER2-ECD is a truncated 100 kDa protein, which encodes the extracellular
domain (ECD) of ERBB2 (subdomains I-IV) and is expressed in gastric cancer cells
[140, 141]. Another variant is the soluble truncated 68 kDa protein called Herstatin,
consisting of the first 340 amino acid of ERBB2 (subdomain I and II) and is expressed
in normal human liver and kidney tissues [142, 143]. Both the 100 kDa and 68 kDa
variant are capable of binding to the full-length ERBB2 receptor and thereby
inhibiting tumor cell growth.
RESULTS AND DISCUSSION
53
Figure 8. Genomic organization of the human ERBB2 gene and transcript variants.
Deletion of exon 5 encoding 31 amino acids corresponding to 3.6 kDa, is not
sufficient to relate the difference in molecular weight between the 160 kDa and 185
kDa ERBB2 proteins. One plausible explanation is structural changes in the truncated
ERBB2 protein. Therefore, the glycosylation status was investigated. Exposing cells to
tunicamycin disrupts glycosylation of newly synthesized proteins. To examine the N-
glycosylation of the truncated ERBB2 protein, Jurkat and U-937 cells were exposed to
tunicamycin. This resulted in an equal molecular mass of the protein (~160 kDa) as
before the treatment. The control cells treated with tunicamycin showed an ERBB2
protein with a molecular mass identical to the truncated ERBB2-receptor protein
(Paper IV, Figure 4).
Glycosylation is important for protein tertiary structure and protein folding, which
occurs in the endoplasmic reticulum, ER, and further in the Golgi apparatus. Exon 5
(31 amino acids) may possibly code for the signal peptide, which is important for
entering the ER. Therefore, the ERBB2 mRNA lacking exon 5 is translated to a protein
probably in the free ribosomes in the cytoplasm, and further as a non-glycosylated
protein unable to localize to the membrane.
1 2 3 4 5 6 7 8
DNA
………………… 31
mRNA
5 6 7 8 ……………………… 31
1 2 3 4 6 7 8 ……………………… 31
Protein
Variant 1 = Isoform a 1255 aa (wt ERBB2)
Variant 2 = Isoform b 1225 aa (truncated ERBB2)
Variant 1
Variant 2
1 2 3 4
exon intron
start codon
start codon
RESULTS AND DISCUSSION
54
ADDITIONAL RESULTS NOT INCLUDED IN PAPER I-IV
Expression of ERBB4 receptor isoforms in human leukemia cells
The fourth member of the ERBB receptor family, ERBB4, has been shown to be
overexpressed in several solid cancers such as breast and colon [144, 145]. ERBB4 is
expressed as at least four different isoforms as a consequence of tissue-specific
alternative splicing [146]. ERBB4 has two different splice regions, extracellular
juxtamembrane (JM) region and intracellular cytoplasmic (CYT) region (Figure 9).
Alternative exon splicing within the JM region produces two different isoforms, JM-a
and JM-b. The JM-a isoforms, but not the JM-b isoforms, have a cleavage site for
tumor necrosis factor-α converting enzyme (TACE) and can therefore release a 120
kDa ERBB4 ectodomain protein. An 80 kDa intracellular cytoplasmic domain (ICD)
fragment is released from the cell membrane after cleavage induced by γ-secretase.
Further, this intracellular ERBB4 protein accumulates in the nucleus where it may
regulate gene expression and/or does it accumulate in the mitochondria where it acts
as a pro-apoptotic protein [147-149]. The intracellular region, an exon coding for 16
amino acids is either included (isoform CYT-1) or excluded (isoform CYT-2) from
full-length ERBB4 mRNA. This exon codes for a PI3-K binding motif and a WW
domain-binding motif. Thus alternative splicing of ERBB4 generates diversity in
receptor signaling resulting in different cellular responses. MAPK pathway is an
example of a signaling cascade that can be activated by all four ERBB4 isoforms,
whereas PI3-K pathway activation occurs only in CYT-1 isoforms [146].
The involvement of ERBB4 in cancer cell biology differs, some of the isoforms seem
to be important in cancer cell signaling and therefore represent cancer drug targets
while targeting of other ERBB4 isoforms may be disadvantageous due to their
possible anti-proliferative effects [145, 149].
In the first paper in this thesis we demonstrated ERBB4 transcripts in HL-60 and U-
937 cells. The two identified products are depicted in Figure 10 A, and demonstrate
the two intracellular isoforms of ERBB4, CYT1 and CYT2. The respective product
was sequenced confirming the presence of CYT-1 and CYT-2 mRNA (data not
shown). The human breast cancer cell line, MCF-7 cells expressed mRNA for the
CYT-1 and CYT-2 isoform of ERBB4 and was used as a positive control.
RESULTS AND DISCUSSION
55
Figure 9. Schematic illustration of structure and differential signaling properties of alternatively
spliced ERBB4. JM, juxtamembrane; CYT, cytoplasmic; white rectangles in the extracellular JM
domains, sequence specific for the JM-a isoforms; grey rectangles in the CYT domains, sequence
specific for the CYT-1 isoforms (modified from Paatero & Elenius [150])
Western blot experiment using a specific antibody directed towards the intracellular
domain of ERBB4 revealed that no full-length ERBB4-receptor (185 kDa) was
expressed in either HL-60 or U-937 cells (Figure 10 B). However, an 80 kDa ERBB4
protein band was identified suggesting that the ERBB4-receptor in HL-60 and U-937
might represent the JM-a isoform. PCR analysis showed a band for extracellular
juxtamembrane JM-a (Figure 10C). JM-a includes 23 amino acids that confer a
proteinase cleavage site in the receptor that is missing from the alternative 13 amino
acids in the JM-b isoform. The positive control MCF-7 contains all four isoforms of
the ERBB4 receptor.
The ERBB4 receptor protein was detected by flow cytometry analysis using a specific
ERBB4 antibody against the intracellular domain (data not shown). In addition, the
ERBB4 receptor was detected by immunhistochemistry (IHC), demonstrating a
significant intracellular expression of the receptor (data not shown).
The different isoforms of ERBB4 promote different cellular responses in vitro.
Localization of a cleavable ERBB4 isoform in the nucleus of breast cancer cells seems
•Release ICD
• Release ECD
• direct coupling
to PI3K/Akt
• activation of
MAPK
• Release ICD
• Release ECD
•direct coupling
to PI3K/Akt
• activation of
MAPK
• activation of
MAPK
Signaling
characteristics:
ErbB4
isoform:
JM-a
CYT-1
JM-a
CYT-2
JM-b
CYT-1
JM-b
CYT-2
Extracellular domain (ECD)
with ligand binding activity
Intracellular domain (ICD)
with tyrosine kinase
Transmembrane domain
PI3-KWW
PI3-KWW
• activation of
MAPK
TACE
γ-secretase
RESULTS AND DISCUSSION
56
to associate with worse prognosis when compared to patients with breast cancer cells
expressing the full-length ERBB4 at the cell surface [150]. If ERBB4 has any
prognostic or treatment predictive role in human leukemia remains to be elucidated.
Figure 10. Expression of ERBB4 JM-a CYT-1 and JM-a CYT-2 isoforms in U-937 cells and HL-60 cells.
A) PCR analysis with primers designed for the two intracellular isoforms, CYT-1 and CYT-2. B)
Western blot experiments using an ERBB4 antibody directed towards the intracellular domain of the
receptor demonstrate a full-length receptor (185 kDa) and shorter band at 80 kDa. C) PCR analysis of
the extracellular JM-a isoform in HL-60 and U-937 cells. MCF-7 cells were used as a control for the
ERBB4 receptor in all experiments.
Bp-
ladder MCF-7 HL-60 U-937 C
- JM-a
MCF-7 HL-60 U-937
- 185 kDa
- 80 kDa
- CYT-1
- CYT-2
ErbB4
M H U CM H U M H U M H UA
B C
CONCLUSIONS
57
CONCLUSIONS
Based on the findings in this thesis, we conclude as follows:
Canertinib inhibits growth and induces caspase-mediated apoptosis in human
leukemia cells, mainly by the intrinsic apoptotic pathway.
Canertinib inhibits activated FLT3 receptor tyrosine kinase, which leads to
apoptosis due to upregulation of the pro-apoptotic protein Bim through
activation of transcription factor FoxO3a.
In cells lacking FLT3 expression, canertinib induces ERBB receptor
independent apoptosis at higher doses.
The mechanisms of canertinib-induced apoptosis in Jurkat cells as a model for
T-cell malignancies involve dephospholylation of important lymphocyte-
specific molecules (Lck and Zap-70) as well as inhibition of survival kinases
(Akt and Erk), suggesting that canertinib may act as a multi-kinase inhibitor.
Human leukemia cell lines express a non-glycosylated truncated ERBB2
receptor protein – the function of these remains to be investigated.
Concluding remarks
Several lines of evidence indicate that tyrosine kinase inhibitors with blocking effects
on the ERBB-receptor family and/or the FLT3 receptor may serve as possible
treatment strategies when developing new drugs for patients with AML. This thesis
may contribute with novel ways to understand the mechanisms of drug-induced
apoptosis of AML and T-cell leukemia cells. These pro-apoptotic effects of canertinib
on human leukemia cells, offers an alternative therapeutic approach to interfere with
these malignancies. There is still no pharmacologic inhibitor available for Zap-70.
Such an inhibitor could have a therapeutic potential in transplantation,
autoimmunity and possibly in B-CLL. We speculate that apoptosis by canertinib
treatment could be enhanced by addition of conventional cytostatic drugs, indicating
that this pan-ERBB inhibitor may give therapeutical benefits in leukemic patients.
The potential role for truncated ERBB2 receptors in leukemia as well as in normal
hematopoiesis needs further investigation.
58
FUTURE ASPECTS
59
FUTURE ASPECTS
Investigate resistance mechanisms in drug-resistant leukemia cells
We would like to isolate leukemia cell lines that are resistant to gefitinib and
canertinib to investigate the mechanisms of resistance. To study if any cross-
resistance between different tyrosine kinase inhibitors exists would be important. In
addition, development of leukemia cells resistant to canertinib and investigate
whether the resistant mechanisms differ in comparison with gefitinib resistant cells
could identify alternative routes of resistance.
It would also be interesting to develop cell lines resistant to PKC412 (FLT3-TKI) and
study the resistance mechanisms. In addition, it would be interesting to study the
effect of canertinib treatment on PKC412-resistant cell lines. We are currently testing
this in cell lines expressing PKC412-resistant FLT3-ITD harboring N676 mutation
[151].
Examine the function and location of a truncated ERBB2 in leukemia cells
In the last paper of this thesis (Paper IV) a truncated ERBB2 receptor protein was
identified in leukemia cells. We have not yet demonstrated the cellular localization,
but the protein is probably located in the cytoplasm or in the nucleus of the cell. It
would be interesting to investigate the localization but also the function of the
protein in leukemia cells. The truncated ERBB2 receptor is not overexpressed and we
have not been able to show any phosphorylation of this receptor. In spite of that, the
truncated ERBB2 might be able to act as an adaptor protein for other signaling
pathways. We have investigated if the truncated ERBB2 mRNA is present in human
AML cells and blood cells from healthy donors, but we could not identify any
alternative spliced mRNA of ERBB2. However, this study was performed with a
limited numbers of AML patient cells. It is also possible that only a fraction of the
AML blasts express truncated ERBB2. Additional samples from patients with AML
and other leukemias, as well as from healthy donors, are needed to elucidate the
presence of any truncated ERBB2 receptor protein and also its role in malignant or
normal hematopoietic cells.
FUTURE ASPECTS
60
Investigate the function of an intracellular ERBB4 receptor in leukemia cells
The intracellular ERBB4 protein accumulates in the nucleus where it may regulate
gene expression. In addition, ERBB4 accumulates in the mitochondria where it could
act as a pro-apoptotic protein in breast cancer cells [147-149]. Hence, it would be of
interest to investigate the role and location of the ERBB4 intracellular protein in
human leukemia cells, and also the involvement of this receptor protein in
canertinib-induced apoptosis.
POPULÄRVETENSKAPLIG SAMMANFATTNING
61
POPULÄRVETENSKAPLIG SAMMANFATTNING
Leukemi (blodcancer) är en elakartad sjukdom som angriper de blodbildande
cellerna i benmärgen och kännetecknas av att omogna vita blodkroppar frisätts och
ansamlas i blodet där de ökar okontrollerat i antal. Leukemier indelas vanligtvis i två
huvudgrupper, akut och kronisk leukemi. Årligen insjuknar i Sverige ca 1000
personer, varav ca 100 barn, i leukemi (2011). För vuxna ökar risken att insjukna i
leukemi med stigande ålder. Akut leukemi förekommer i alla åldrar medan kronisk
leukemi drabbar framförallt äldre individer. Barn drabbas oftast av akut leukemi och
insjuknar vanligtvis före 5 års ålder.
Orsakerna till att leukemi uppstår är okända, men bland yttre faktorer misstänks
joniserad strålning vara viktig för utveckling av akut leukemi och kronisk myeloisk
leukemi. Miljögifter och andra kemikalier tros kunna bidra till kronisk lymfatisk
leukemi och lösningsmedel såsom benzen kan orsaka akut leukemi. I cirka 6% av alla
akuta leukemi fall uppstår sjukdomen till följd av tidigare behandling med
cytostatika och/eller strålning.
De senaste decennierna har det gjorts stora framsteg i behandlingen av leukemi. Den
vanligaste behandlingen mot leukemi är cytostatika, cellhämmande eller celldödande
medel. Ibland ges dessa i kombination med strålbehandling och/eller
stamcellstransplantation. Vid akut leukemi syftar behandlingen till att bota
patienten. Vid kronisk leukemi är syftet inte primärt att bota men istället att
kontrollera sjukdomen och hålla patienten symptomfri, enstaka fall av bot finns
dock.
Inte sällan utvecklar dock leukemiceller motståndskraft (resistens) mot given terapi.
Konsekvensen blir att patienten tvingas byta terapi eller helt avsluta behandling om
alternativ saknas. Mekanismerna för resistensutveckling är ofullständigt kartlagda.
En mutation eller annan typ av aktiverande genförändring i de proteiner som
förmedlar tillväxtstimulerande signaler kan främja cellernas överlevnad och öka
deras motståndskraft mot programmerad celldöd (apoptos). Mutationer kan också
drabba tyrosinkinasreceptorer eller signalproteiner som förmedlar signaler till cellens
inre.
Dessa receptorer kan användas för utveckling av nya läkemedel för att på ett
effektivare sätt behandla leukemier. Verkningsmekanismen hos sådana läkemedel är
att konkurrera bort ATP, dvs. cellens energimolekyl, från tyrosinkinasets
POPULÄRVETENSKAPLIG SAMMANFATTNING
62
aktiveringsdel varmed aktiviteten hos receptorn stängs av, därmed förhindras
signalerna att överföras till andra signalproteiner inne i cellen. Detta resulterar i
blockerad celldelning och aktivering av programmerad celldöd.
Under senare år har flera nya typer av målsökande läkemedel utvecklats mot
leukemi, exempelvis så kallade tyrosinkinashämmare (TKI). TKI utövar sin anti-
leukemiska effekt genom att blockera ett specifikt enzym (tyrosinkinas) vilket
förhindrar den okontrollerade celldelningen eller har en direkt avdödande effekt på
leukemiceller.
I det föreliggande avhandlingsarbetet (Paper I-III) har effekten av
tyrosinkinashämmaren, canertinib (CI-1033), studerats med avseende på tillväxt,
celldöd och intracellulär signalering hos humana leukemiceller in vitro. Därutöver
(Paper IV) har en stympad tillväxtfaktorreceptor (ERBB2) identifierats i humana
leukemicellinjer.
Multi-tyrosinkinashämmaren canertinib hämmar tillväxten och inducerar så kallad
programmerad celldöd (apoptos) i odlade leukemiceller (HL-60 och U-937) i ett dos-
och tidsberoende mönster. Canertinibs avdödande effekt uppfattades som en ”off-
target” verkan eftersom något målprotein (ERBB 1-4) för canertinib inte kunde
identifieras i de studerade leukemicellerna.
För att närmare kartlägga de specifika mekanismerna som är ansvariga för
programmerad celldöd i canertinib-behandlade leukemiceller användes den
etablerade leukemicellinjen Jurkat (T-cells leukemi). Apoptosmaskineriet innehåller
många kaspaser (enzymer) som aktiveras av signaler utifrån men även från signaler
inuti cellen. Vi har kunnat identifiera vilka kaspaser som är aktiverade under
canertinib behandling och även kunnat påvisa hämning av intracellulära
signaleringsvägar som har betydelse för T-cellens tillväxt och överlevnad.
Vidare har vi studerat effekten av canertinib och dess verkningsmekanismer vid akut
myeloisk leukemi (AML) i cell linjer, celler från AML patienter och i möss. Ett viktigt
protein i leukemi celler är en tyrosinkinas receptor vid namn FLT3, den är förändrad
(muterad) i upp till 30 % av alla patienter med AML och förekomst av denna
mutation ger patienten en sämre prognos. Canertinib visar sig ha bättre effekt på
leukemiceller som besitter muterade FLT3 receptorer än på de som inte är muterade.
Vi har även visat att canertinib påverkar andra viktiga intracellulära proteiner som
reglerar cellernas tillväxt och överlevnad.
POPULÄRVETENSKAPLIG SAMMANFATTNING
63
I detta avhandlingsarbete har vi försökt klargöra på vilket sätt leukemiska celler dör
under behandling med målsökande behandlingar. Våra studier kan visa sig vara av
betydelse för fortsatt läkemedelsutveckling för att öka chansen till bot för patienter
med leukemi.
Vi har i detta arbete också identifierat en stympad form av ERBB2 receptorn i
humana leukemiceller. Vid karaktärisering av receptorn visades att den dels saknar
den extracellulära ligandbindande delen, men den saknar även en del av sin struktur
som är viktig för att proteinet skall hitta till sin plats i cellmembranet. ERBB2
proteinet förefaller i stället vara lokaliserad inne i cellen. Proteinet visade sig
dessutom sakna viktiga sockermolekyler (icke glykosylerat) vilket indikerar att
receptorn saknar förmågan att signalera på egen hand. Det stympade proteinets
funktion är fortfarande okänd och behöver närmare utredas. Möjligen kan den
stympade ERBB2-receptorn utgöra ett icke tidigare identifierat målprotein för
canertinib i leukemiceller. Fortsatta studier behövs för att identifiera ytterligare
förändrade proteiner och deras respektive funktioner i leukemiceller då de kan visa
sig vara viktiga som terapeutiska mål, och därmed vidare förbättra behandlingen av
leukemi.
Sammanfattningsvis har studierna i denna avhandling bidragit till att öka kunskapen
om tyrosinkinashämmaren canertinibs potential som anti-leukemisk behandling
samt dess verkningsmekanismer. Dessutom har en stympad ERBB2-receptor variant
identifierats som möjligen i framtiden kan tjäna som ett terapeutiskt mål vid
behandling av leukemi. Förhoppningsvis!
ACKNOWLEDGEMENTS
64
ACKNOWLEDGEMENTS
Jag skulle vilja rikta ett varmt tack till alla som har hjälpt och stöttat mig under min
tid som forskarstuderande. Speciellt vill jag tacka:
Thomas Walz, huvudhandledare, för din generositet och för att Du gav mig
möjlighet att skriva denna avhandling och gav mig frihet att utvecklas till en
självständig forskare. Tack för den här tiden!
Jan-Ingvar Jönsson, bi-handledare, för att du förde mig in i leukemins värld. Tack för
ditt stöd och dina goda råd, för din uppmuntran, entusiasm och inspiration. Du är
full med energi och en stor idéspruta. Tack för all hjälp!
Anna-Lotta Hallbeck, bi-handledare, för din kunskap vid granskning av manus.
Åke Wasteson, för att du kritiskt har granskat manus och varit ett stöd i gruppen.
Emelie Severinsson, kollega och medförfattare. Alltid glad och positiv, noga och
hjälpsam. Utan dig skulle livet framför datorn varit en katastrof.
Birgitta Holmlund, kollega, rumskamrat och medförfattare. Du är en stjärna på
labbet! Kunnig, glad, förstående och stöttande i alla tänkbara stunder.
Kerstin Willander, kollega och rumskamrat, tack för att du alltid finns att prata med,
jag uppskattar din kunskap och ditt sällskap.
Annelie Lindström och Maria Wester, kollegor och vänner på och utanför jobbet.
Tack för allt roligt vi haft tillsammans såsom Blodomloppet, cross-training, F&S och
andra utflykter. Fortsättning följer…..
Elisavet Koutzamani och Anna Gréen, fd rumskamrater. Vad vore livet utan skratt
och gråt?...
Anneli Karlsson, Pia Wegman och Liselotte Ydrenius, för fikastunder, jympa, en
trygghet under forskartiden och för ett bra samarbete med undervisningen.
Alla kollegor, tack för trevligt sällskap och intressanta diskussioner, Kristina, Anita
S., Anita L., Lotta B., Chamilly, Eva, Anna L, Vivian, Ingemar, Anders, Björn,
Richard, Hanna, Susana, Christine, Sara, Viviana, Jacob, Narges, Gizeh, Veronica.
Pia Druid, du är en klippa på att labba, tack för all hjälp och roliga historier under
alla år.
ACKNOWLEDGEMENTS
65
Annette Molbäck, för all hjälp med rening och sekvensering av mina PCR-produkter.
Karin Öllinger, för att du introducerade mig in i apoptosens värld.
Birgit Olsson, för att du hjälpt till med beställningar.
Lena Wigren, Chatarina Malm, Anette Wiklund och Monica Hardmark för att ni har
ställt upp och hjälpt till med administrativa frågor.
Håkan Wiktander för att du snabbt ställer upp och fixar sådant som bl.a. gått sönder.
Lena och Anna för att ni alltid är så trevliga och ser till att allt är diskat.
Och till sist, några av de som ger mig ett liv utanför universitetet…………….
Mina barndoms- och är fortfarande kompisar, Martina, Nadine, Jessica, Jenny,
Maria och Susann med familjer, för att ni finns och har funnits hela min uppväxt
vilket har präglat mycket av vad och vem jag är idag. Martina för att du ”pushat”
mig till att träna och genomföra Tjejklassikern, Göteborgsvarvet, Tjejmilen mm, utan
dig skulle det inte blivit av. Vänner är ett skafferi förmögenhet.
Mina kära kusiner Karin, Mats, Anna och Anders med familjer, för allt stöd och allt
kul vi hittar på utanför jobb och alla måsten. Faster och farbror Gunilla och Leif för
er generositet och gästvänlighet.
Marianne och Tommy, min mamma och pappa, som har stöttat mig i allt jag gjort och
alltid finns till hands när man behöver hjälp och goda råd under alla år. Utan ert stöd
hade jag aldrig varit där jag är idag.
Min bror Robert med familj; Marika, Gustav och Alva, för ert stöd och att ni finns i
mitt liv.
Janne, för alla kloka ord och stöttning och Julia, för din livsglädje och min syster
Jenny som också var min bästa vän, en god lyssnare och stöttade mig in i det sista.
Min allra käraste syster, Du finns alltid hos mig i mitt hjärta.
Kenneth, för att du underlättar vardagsbestyren när det behövs.
Min familj, Ulf och Adam min lilla solstråle, som hjälpt mig i med och motgångar. Ett
speciellt tack för allt stöd och uppmuntran de senaste månaderna.
Denna avhandling har finansierats av ALF-medel, FORSS-medel, Cancerfonden och
Barncancerfonden.
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