Canertinib-Induced Leukemia Cell Death Signaling

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

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

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

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

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

9 PAPERS OUTSIDE THIS THESIS

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

10 ABBREVIATIONS

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 11 ABBREVIATIONS

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

12 INTRODUCTION

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].

13 INTRODUCTION

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

14 INTRODUCTION

(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].

G2 M

G0 S G1

Figure 1. The cell cycle with its phases: G1, S, G2, M, and the G0 phase. R= restriction-point

15 INTRODUCTION

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].

16 INTRODUCTION

Extrinsic pathway Fas L

plasma membrane Fas/CD95 FADD Caspase-8,-10 FADD Intrinsic pathway

Bid Bcl-2 tBid Bcl-2 Bak Bak Cellular stress Apaf-1 Caspase-9 Cyto C Caspase-3

PARP

APOPTOSIS

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].

17 INTRODUCTION

Table 1. Members of the Bcl-2 protein family are divided into anti-apoptotic or pro-apoptotic factors.

Anti-apoptotic factors Pro-apoptotic factors

• Bcl-2 • Bax, Bak

• Bcl-XL • Bim • Bcl-w • Bid, Bad, Noxa, Puma

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

18 INTRODUCTION 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].

19 INTRODUCTION

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].

20 INTRODUCTION

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].

21 INTRODUCTION

EGF AR BTC Growth TGF-α HB-EGF NRG1 NRG3 NRG2 NRG4 Factors Epigen EPR

Domain I X

Domain II

Domain III X

Domain IV

Transmembrane domain Tyrosine X Kinase

C-terminal phosphorylation sites

Gene ErbB1 ErbB2 ErbB3 ErbB4 Family HER1 HER2 HER3 HER4 EGFR Neu

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

22 INTRODUCTION 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].

ErbB2 ErbB3 ErbB2 – ErbB3 heterodimer

L L

ATP ATP ADP ADP P P P P P

PI3-K/Akt MAPK/Erk1/2PI3-K Stat

Survival Migration Adhesion Proliferation Differentiation

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]).

23 INTRODUCTION

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

24 INTRODUCTION 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].

Extracellular ligand binding domain

Juxtamembrane Internal tandem (JM) domain duplication (ITD)

Tyrosine kinase domain D835 mutation (TKD)

C-terminal domain

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

25 INTRODUCTION

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 26 INTRODUCTION 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].

27 INTRODUCTION

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;

28 INTRODUCTION

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

29 INTRODUCTION 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].

30 INTRODUCTION

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].

31 INTRODUCTION

Table 2. Tyrosine kinase inhibitors, their targets and the disease

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

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].

32 INTRODUCTION

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].

33 INTRODUCTION

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].

34 INTRODUCTION

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

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

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:

Cell line Origin (human) Cell line Origin FLT3 status HL-60 promyelocyte MV4:11 Human FLT3-ITD

U-937 promyelocyte monocytoid MonoMac-6 Human FLT3-V592A

Jurkat lymphoblasticT-cell leukemia FDC-P1 * Mouse wt, ITD, TKD

Daudi B-CLL Burkitt's lymphoma Ba/F3 * Mouse wt, ITD, TKD K-562 CML blast crisis THP1 Human wt FLT3 HEL erythrocyte leukemia NB4 Human wt FLT3 LCL4 EBV-transformed lymphoblastoid RS4;11 Human wt FLT3

232-B4 B-CLL * This cell line is FLT3 negative and was used as model for apoptotic and biochemical studies by MCF-7 Breast overexpressing different FLT3 versions.

PATIENT SAMPLES

FLT3 mutational data regarding ITD status in ten patient samples included in the study (Paper III).

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

39 MATERIALS AND METHODS

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

40 MATERIALS AND METHODS 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.

41 MATERIALS AND METHODS

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).

42 MATERIALS AND METHODS

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

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

45 RESULTS AND DISCUSSION 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

46 RESULTS AND DISCUSSION 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.

47 RESULTS AND DISCUSSION

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).

48 RESULTS AND DISCUSSION

cell membrane

Lck Caspase-8,-10 Zap-70 ┴ LAT ERK1/2 Bid

AKT tBid Bcl-2 Bax Bax

Apaf 1 Caspase-9 Cyto C Caspase-3

PARP

APOPTOSIS

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

49 RESULTS AND DISCUSSION

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

50 RESULTS AND DISCUSSION

FoxO3a activating reagents and/or addition of BH3-mimetics (behaving like active Bim), this could provide interesting directions for future therapy.

FLT3-receptor FL FL R R cell membrane

canertinib canertinib

Caspase-8,-10

PI3-K Bid ERK1/2

tBid AKT Bcl-2 Bak Bak Bim ┴ FoxO3a Apaf-1 Caspase-9 Cyto C Caspase-3 Bim promoter PARP

APOPTOSIS

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

51 RESULTS AND DISCUSSION 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.

52 RESULTS AND DISCUSSION

exon intron DNA

1 2 3 4 5 6 7 8 ………………… 31

mRNA start codon Variant 1

1 2 3 4 5 6 7 8 ……………………… 31

Variant 2 start codon

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)

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.

53 RESULTS AND DISCUSSION

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.

54 RESULTS AND DISCUSSION

ErbB4 JM-a JM-a JM-b JM-b isoform: CYT-1 CYT-2 CYT-1 CYT-2

Extracellular domain (ECD) with ligand binding activity TACE

Transmembrane domain γ-secretase Intracellular domain (ICD) with tyrosine kinase PI3-K WW PI3-K WW

Signaling characteristics: • Release ICD • Release ICD • Release ECD • Release ECD • direct coupling • direct coupling to PI3K/Akt to PI3K/Akt • activation of • activation of • activation of • activation of MAPK MAPK MAPK MAPK

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

55 RESULTS AND DISCUSSION 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.

ErbB4 A M H U M H U M H U M H U C - CYT-1 - CYT-2

MCF-7 HL-60 U-937 Bp- B C ladder MCF-7 HL-60 U-937 C - 185 kDa - JM-a

- 80 kDa

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.

56 CONCLUSIONS

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

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.

59 FUTURE ASPECTS

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.

60 POPULÄRVETENSKAPLIG SAMMANFATTNING

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

61 POPULÄRVETENSKAPLIG SAMMANFATTNING 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.

62 POPULÄRVETENSKAPLIG SAMMANFATTNING

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!

63 ACKNOWLEDGEMENTS

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.

64 ACKNOWLEDGEMENTS

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.

65 REFERENCES

REFERENCES

[1] Cancer incidence in Sweden 2010. 2010, Stockholm, National Board of Health and Welfare. [2] Hanahan D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1): p. 57-70. [3] Hanahan D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74. [4] Rajagopalan H., et al., The significance of unstable chromosomes in colorectal cancer. Nat Rev Cancer, 2003. 3(9): p. 695-701. [5] Grander D., How do mutated oncogenes and tumor suppressor genes cause cancer? Med Oncol, 1998. 15(1): p. 20-6. [6] Griffin J., The biology of signal transduction inhibition: basic science to novel therapies. Semin Oncol, 2001. 28(5 Suppl 17): p. 3-8. [7] Kopnin B.P., Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biochemistry (Mosc), 2000. 65(1): p. 2-27. [8] Malumbres M. and M. Barbacid, To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer, 2001. 1(3): p. 222-31. [9] Vermeulen K., D.R. Van Bockstaele, and Z.N. Berneman, The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif, 2003. 36(3): p. 131-49. [10] Schwartz G.K. and M.A. Shah, Targeting the cell cycle: a new approach to cancer therapy. J Clin Oncol, 2005. 23(36): p. 9408-21. [11] Thompson C.B., Apoptosis in the pathogenesis and treatment of disease. Science, 1995. 267(5203): p. 1456-62. [12] Kerr J.F., A.H. Wyllie, and A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972. 26(4): p. 239-57. [13] Zhivotovsky B., et al., Caspases: their intracellular localization and translocation during apoptosis. Cell Death Differ, 1999. 6(7): p. 644-51. [14] Fulda S. and K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene, 2006. 25(34): p. 4798-811. [15] Lavrik I., A. Golks, and P.H. Krammer, Death receptor signaling. J Cell Sci, 2005. 118(Pt 2): p. 265-7.

66 REFERENCES

[16] Brunelle J.K. and A. Letai, Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci, 2009. 122(Pt 4): p. 437-41. [17] Li P., et al., Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 1997. 91(4): p. 479-89. [18] Yin X.M., Bid, a BH3-only multi-functional molecule, is at the cross road of life and death. Gene, 2006. 369: p. 7-19. [19] Jeggo P.A., DNA repair: PARP - another guardian angel? Curr Biol, 1998. 8(2): p. R49-51. [20] Reed J.C., Bcl-2-family proteins and hematologic malignancies: history and future prospects. Blood, 2008. 111(7): p. 3322-30. [21] Boya P. and G. Kroemer, Lysosomal membrane permeabilization in cell death. Oncogene, 2008. 27(50): p. 6434-51. [22] Bidere N., et al., Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem, 2003. 278(33): p. 31401-11. [23] Chwieralski C.E., T. Welte, and F. Buhling, Cathepsin-regulated apoptosis. Apoptosis, 2006. 11(2): p. 143-9. [24] Gyrd-Hansen M., et al., Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9. Mol Cell Biol, 2006. 26(21): p. 7880-91. [25] Rodriguez-Abreu D., A. Bordoni, and E. Zucca, Epidemiology of hematological malignancies. Ann Oncol, 2007. 18 Suppl 1: p. i3-i8. [26] Reilly J.T., Receptor tyrosine kinases in normal and malignant haematopoiesis. Blood Rev, 2003. 17(4): p. 241-8. [27] Venter J.C., et al., The sequence of the human genome. Science, 2001. 291(5507): p. 1304-51. [28] Manning G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34. [29] Fischer E.H. and E.G. Krebs, Relationship of structure to function of muscle phosphorylase. Fed Proc, 1966. 25(5): p. 1511-20. [30] Robinson D.R., Y.M. Wu, and S.F. Lin, The protein tyrosine kinase family of the human genome. Oncogene, 2000. 19(49): p. 5548-57. [31] Schlessinger J., Cell signaling by receptor tyrosine kinases. Cell, 2000. 103(2): p. 211-25. [32] Carpenter G. and S. Cohen, Epidermal growth factor. J Biol Chem, 1990. 265(14): p. 7709-12. [33] Jorissen R.N., et al., Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res, 2003. 284(1): p. 31-53.

67 REFERENCES

[34] Roskoski R., Jr., The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun, 2004. 319(1): p. 1-11. [35] Stein R.A. and J.V. Staros, Evolutionary analysis of the ErbB receptor and ligand families. J Mol Evol, 2000. 50(5): p. 397-412. [36] Yarden Y. and M.X. Sliwkowski, Untangling the ErbB signalling network. Nat Rev Mol Cell Biol, 2001. 2(2): p. 127-37. [37] Yarden Y., The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer, 2001. 37 (Suppl 4): p. S3-8. [38] Fuller S.J., K. Sivarajah, and P.H. Sugden, ErbB receptors, their ligands, and the consequences of their activation and inhibition in the myocardium. J Mol Cell Cardiol, 2008. 44(5): p. 831-54. [39] Olayioye M.A., et al., The ErbB signaling network: receptor heterodimerization in development and cancer. Embo J, 2000. 19(13): p. 3159- 67. [40] Coussens L., et al., Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science, 1985. 230(4730): p. 1132-9. [41] King C.R., M.H. Kraus, and S.A. Aaronson, Amplification of a novel v-erbB- related gene in a human mammary carcinoma. Science, 1985. 229(4717): p. 974- 6. [42] Slamon D.J., et al., Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 1987. 235(4785): p. 177- 82. [43] Nielsen D.L., M. Andersson, and C. Kamby, HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors. Cancer Treat Rev, 2009. 35(2): p. 121-36. [44] Hynes N.E. and H.A. Lane, ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer, 2005. 5(5): p. 341-54. [45] Sliwkowski M.X., et al., Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin. J Biol Chem, 1994. 269(20): p. 14661-5. [46] Karunagaran D., et al., ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. Embo J, 1996. 15(2): p. 254-64. [47] Carraway K.L. and G.A. Kozloski, Conformational changes in receptor tyrosine kinase signaling: an ErbB garden of delights. F1000 Biol Rep, 2009. 1: p. 72.

68 REFERENCES

[48] Hynes N.E. and G. MacDonald, ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol, 2009. 21(2): p. 177-84. [49] Leone F., et al., Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells. J Leukoc Biol, 2003. 74(4): p. 593-601. [50] Buhring H.J., et al., The receptor tyrosine kinase p185HER2 is expressed on a subset of B-lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia. Blood, 1995. 86(5): p. 1916-23. [51] Chevallier P., et al., Overexpression of Her2/neu is observed in one third of adult acute lymphoblastic leukemia patients and is associated with chemoresistance in these patients. Haematologica, 2004. 89(11): p. 1399-401. [52] Luftner D., et al., Lack of HER-2/neu overexpression in non-Hodgkin's lymphoma. Anticancer Res, 2004. 24(5B): p. 3233-7. [53] Stegmaier K., et al., Gefitinib induces myeloid differentiation of acute myeloid leukemia. Blood, 2005. 106(8): p. 2841-8. [54] Boehrer S., et al., Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study. Blood, 2008. 111(4): p. 2170-80. [55] Mahtouk K., et al., An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti- IL-6 antibody-induced apoptosis. Blood, 2004. 103(5): p. 1829-37. [56] Otsuki T., et al., Expression of HER family receptors and effects of anti-HER2- antibody on human myeloma cell lines. Int J Oncol, 2003. 23(4): p. 1135-41. [57] Abu-Duhier F.M., et al., Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol, 2001. 113(4): p. 983-8. [58] Nakao M., et al., Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia, 1996. 10(12): p. 1911-8. [59] Gilliland D.G. and J.D. Griffin, The roles of FLT3 in hematopoiesis and leukemia. Blood, 2002. 100(5): p. 1532-42. [60] Jonsson M., M. Engstrom, and J.I. Jonsson, FLT3 ligand regulates apoptosis through AKT-dependent inactivation of transcription factor FoxO3. Biochem Biophys Res Commun, 2004. 318(4): p. 899-903. [61] Takahashi S., Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications. J Hematol Oncol. 4: p. 13. [62] Nicholson K.M. and N.G. Anderson, The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal, 2002. 14(5): p. 381-95. [63] Cardone M.H., et al., Regulation of cell death protease caspase-9 by phosphorylation. Science, 1998. 282(5392): p. 1318-21.

69 REFERENCES

[64] Scheijen B., et al., FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins. Oncogene, 2004. 23(19): p. 3338-49. [65] Cagnol S. and J.C. Chambard, ERK and cell death: mechanisms of ERK- induced cell death--apoptosis, autophagy and senescence. Febs J, 2010. 277(1): p. 2-21. [66] Palacios E.H. and A. Weiss, Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene, 2004. 23(48): p. 7990-8000. [67] Salmond R.J., et al., T-cell receptor proximal signaling via the Src-family kinases, Lck and Fyn, influences T-cell activation, differentiation, and tolerance. Immunol Rev, 2009. 228(1): p. 9-22. [68] Wang H., et al., ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol, 2010. 2(5): p. a002279. [69] Belka C., et al., The tyrosine kinase Lck is involved in regulation of mitochondrial apoptosis pathways. Oncogene, 2003. 22(2): p. 176-85. [70] Samraj A.K., et al., The tyrosine kinase Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expression. Oncogene, 2006. 25(2): p. 186-97. [71] Gruber C., et al., Involvement of tyrosine kinase p56/Lck in apoptosis induction by anticancer drugs. Biochem Pharmacol, 2004. 67(10): p. 1859-72. [72] Au-Yeung B.B., et al., The structure, regulation, and function of ZAP-70. Immunol Rev, 2009. 228(1): p. 41-57. [73] Bagnyukova T.V., et al., Chemotherapy and signaling: How can targeted therapies supercharge cytotoxic agents? Cancer Biol Ther, 2010. 10(9): p. 839-53. [74] Gerber D.E., Targeted therapies: a new generation of cancer treatments. Am Fam Physician, 2008. 77(3): p. 311-9. [75] Nowell P.C. and D.A. Hungerford, Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst, 1960. 25: p. 85-109. [76] Rowley J.D., Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature, 1973. 243(5405): p. 290-3. [77] Blume-Jensen P. and T. Hunter, Oncogenic kinase signalling. Nature, 2001. 411(6835): p. 355-65. [78] Kantarjian H.M. and M. Talpaz, Imatinib mesylate: clinical results in Philadelphia chromosome-positive leukemias. Semin Oncol, 2001. 28(5 Suppl 17): p. 9-18.

70 REFERENCES

[79] Arora A. and E.M. Scholar, Role of tyrosine kinase inhibitors in cancer therapy. J Pharmacol Exp Ther, 2005. 315(3): p. 971-9. [80] Karaman M.W., et al., A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol, 2008. 26(1): p. 127-32. [81] Pytel D., et al., Tyrosine kinase blockers: new hope for successful cancer therapy. Anticancer Agents Med Chem, 2009. 9(1): p. 66-76. [82] Downing J.R., Targeted therapy in leukemia. Mod Pathol, 2008. 21 Suppl 2: p. S2-7. [83] Weisberg E., et al., FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat, 2009. 12(3): p. 81-9. [84] Kindler T., D.B. Lipka, and T. Fischer, FLT3 as a therapeutic target in AML: still challenging after all these years. Blood. 116(24): p. 5089-102. [85] Weisberg E., et al., Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell, 2002. 1(5): p. 433-43. [86] Nordigarden A., et al., BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3. Blood, 2009. 113(10): p. 2302-11. [87] Shipley J.L. and J.N. Butera, Acute myelogenous leukemia. Exp Hematol, 2009. 37(6): p. 649-58. [88] Yarden Y., J. Baselga, and D. Miles, Molecular approach to breast cancer treatment. Semin Oncol, 2004. 31(5 Suppl 10): p. 6-13. [89] Baselga J. and L.A. Hammond, HER-targeted tyrosine-kinase inhibitors. Oncology, 2002. 63 Suppl 1: p. 6-16. [90] Harari P.M., Epidermal growth factor receptor inhibition strategies in oncology. Endocr Relat Cancer, 2004. 11(4): p. 689-708. [91] Rocha-Lima C.M., et al., EGFR targeting of solid tumors. Cancer Control, 2007. 14(3): p. 295-304. [92] Baselga J., The EGFR as a target for anticancer therapy--focus on cetuximab. Eur J Cancer, 2001. 37 Suppl 4: p. S16-22. [93] Sergina N.V., et al., Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature, 2007. 445(7126): p. 437-41. [94] Broekman F., E. Giovannetti, and G.J. Peters, Tyrosine kinase inhibitors: Multi- targeted or single-targeted? World J Clin Oncol, 2011. 2(2): p. 80-93. [95] Riely G.J., et al., Update on epidermal growth factor receptor mutations in non-small cell lung cancer. Clin Cancer Res, 2006. 12(24): p. 7232-41.

71 REFERENCES

[96] Pao W., et al., EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A, 2004. 101(36): p. 13306-11. [97] Verkhivker G.M., Exploring sequence-structure relationships in the tyrosine kinome space: functional classification of the binding specificity mechanisms for cancer therapeutics. Bioinformatics, 2007. 23(15): p. 1919-26. [98] Rubin B.P. and A. Duensing, Mechanisms of resistance to small molecule kinase inhibition in the treatment of solid tumors. Lab Invest, 2006. 86(10): p. 981-6. [99] Shah N.P., Loss of response to imatinib: mechanisms and management. Hematology Am Soc Hematol Educ Program, 2005: p. 183-7. [100] Kosaka T., et al., Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res, 2006. 12(19): p. 5764-9. [101] Engelman J.A., et al., PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib. Cancer Res, 2007. 67(24): p. 11924-32. [102] Broekman F., E. Giovannetti, and G.J. Peters, Tyrosine kinase inhibitors: Multi- targeted or single-targeted? World J Clin Oncol. 2(2): p. 80-93. [103] Brunner-Kubath C., et al., The PI3 kinase/mTOR blocker NVP-BEZ235 overrides resistance against irreversible ErbB inhibitors in breast cancer cells. Breast Cancer Res Treat, 2011. 129(2): p. 387-400. [104] Lainey E., et al., Tyrosine kinase inhibitors for the treatment of acute myeloid leukemia: delineation of anti-leukemic mechanisms of action. Biochem Pharmacol, 2011. 82(10): p. 1457-66. [105] Costa D.B., et al., BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med, 2007. 4(10): p. 1669-79; discussion 1680. [106] Fry D.W., et al., Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci U S A, 1998. 95(20): p. 12022-7. [107] Smaill J.B., et al., Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor. J Med Chem, 1999. 42(10): p. 1803-15.

72 REFERENCES

[108] Djerf E.A., et al., ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839). Melanoma Res, 2009. 19(3): p. 156-66. [109] Djerf Severinsson E.A., et al., The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo. Biochem Biophys Res Commun, 2011. 414(3): p. 563-8. [110] Ako E., et al., The pan-erbB tyrosine kinase inhibitor CI-1033 inhibits human esophageal cancer cells in vitro and in vivo. Oncol Rep, 2007. 17(4): p. 887-93. [111] Nyati M.K., et al., Radiosensitization by pan ErbB inhibitor CI-1033 in vitro and in vivo. Clin Cancer Res, 2004. 10(2): p. 691-700. [112] Slichenmyer W.J., W.L. Elliott, and D.W. Fry, CI-1033, a pan-erbB tyrosine kinase inhibitor. Semin Oncol, 2001. 28(5 Suppl 16): p. 80-5. [113] Rixe O., et al., A randomized, phase II, dose-finding study of the pan-ErbB receptor tyrosine-kinase inhibitor CI-1033 in patients with pretreated metastatic breast cancer. Cancer Chemother Pharmacol, 2009. 64(6): p. 1139-48. [114] Janne P.A., et al., Multicenter, randomized, phase II trial of CI-1033, an irreversible pan-ERBB inhibitor, for previously treated advanced non small- cell lung cancer. J Clin Oncol, 2007. 25(25): p. 3936-44. [115] Fabian M.A., et al., A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol, 2005. 23(3): p. 329-36. [116] Towbin H., T. Staehelin, and J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A, 1979. 76(9): p. 4350-4. [117] Vindelov L.L., I.J. Christensen, and N.I. Nissen, A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 1983. 3(5): p. 323-7. [118] Fadok V.A., et al., Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol, 1992. 148(7): p. 2207-16. [119] Pfaffel-Schubart G., C. Scalfi-Happ, and A. Ruck, Early and late apoptotic events induced in human glioblastoma cells by Hypericin PDT. Medical Laser Application, 2008. 23: p. 25-30. [120] Mullis K., et al., Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol, 1986. 51 Pt 1: p. 263-73.

73 REFERENCES

[121] Moon D.O., et al., Molecular mechanisms of ZD1839 (Iressa)-induced apoptosis in human leukemic U937 cells. Acta Pharmacol Sin, 2007. 28(8): p. 1205-14. [122] Kim S.M., et al., Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL. Toxicol Appl Pharmacol, 2009. 241(2): p. 210-20. [123] Xu G. and Y. Shi, Apoptosis signaling pathways and lymphocyte homeostasis. Cell Res, 2007. 17(9): p. 759-71. [124] Guerrero A.D., M. Chen, and J. Wang, Delineation of the caspase-9 signaling cascade. Apoptosis, 2008. 13(1): p. 177-86. [125] Park S.J., et al., Taxol induces caspase-10-dependent apoptosis. J Biol Chem, 2004. 279(49): p. 51057-67. [126] Sanchez-Alcazar J.A., et al., Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells. Cell Death Differ, 2000. 7(11): p. 1090-100. [127] Belka C., et al., The tyrosine kinase lck is required for CD95-independent caspase-8 activation and apoptosis in response to ionizing radiation. Oncogene, 1999. 18(35): p. 4983-92. [128] Zhao W., et al., Sorafenib inhibits activation of human peripheral blood T cells by targeting LCK phosphorylation. Leukemia, 2008. 22(6): p. 1226-33. [129] Stiles B.L., PI-3-K and AKT: Onto the mitochondria. Adv Drug Deliv Rev, 2009. 61(14): p. 1276-82. [130] Nelson J.M. and D.W. Fry, Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition. J Biol Chem, 2001. 276(18): p. 14842-7. [131] Milella M., et al., Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J Clin Invest, 2001. 108(6): p. 851-9. [132] Sinha D., et al., Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors up-regulates Akt and prevents apoptosis. J Biol Chem, 2004. 279(12): p. 10962-72. [133] Pereira N.A. and Z. Song, Some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity. Biochem Biophys Res Commun, 2008. 377(3): p. 873-7. [134] Trinks C., et al., The pan-ErbB receptor tyrosine kinase inhibitor canertinib induces ErbB-independent apoptosis in human leukemia (HL-60 and U-937) cells. Biochem Biophys Res Commun, 2010. 393(1): p. 6-10.

74 REFERENCES

[135] Trinks C., et al., The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells. Biochem Biophys Res Commun, 2011. 410(3): p. 422-7. [136] Scaltriti M., et al., Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst, 2007. 99(8): p. 628-38. [137] Scaltriti M., et al., Clinical benefit of lapatinib-based therapy in patients with human epidermal growth factor receptor 2-positive breast tumors coexpressing the truncated p95HER2 receptor. Clin Cancer Res, 2010. 16(9): p. 2688-95. [138] Nezu M., et al., Identification of a novel promoter and exons of the c-ERBB-2 gene. Biochem Biophys Res Commun, 1999. 258(3): p. 499-505. [139] Tal M., et al., Human HER2 (neu) promoter: evidence for multiple mechanisms for transcriptional initiation. Mol Cell Biol, 1987. 7(7): p. 2597-601. [140] Scott G.K., et al., A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol, 1993. 13(4): p. 2247-57. [141] Aigner A., et al., Expression of a truncated 100 kDa HER2 splice variant acts as an endogenous inhibitor of tumour cell proliferation. Oncogene, 2001. 20(17): p. 2101-11. [142] Doherty J.K., et al., The HER-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor. Proc Natl Acad Sci U S A, 1999. 96(19): p. 10869-74. [143] Hu P., et al., Sequestering ErbB2 in endoplasmic reticulum by its autoinhibitor from translocation to cell surface: an autoinhibition mechanism of ErbB2 expression. Biochem Biophys Res Commun, 2006. 342(1): p. 19-27. [144] Kountourakis P., et al., Prognostic significance of HER3 and HER4 protein expression in colorectal adenocarcinomas. BMC Cancer, 2006. 6: p. 46. [145] Junttila T.T., et al., Cleavable ErbB4 isoform in estrogen receptor-regulated growth of breast cancer cells. Cancer Res, 2005. 65(4): p. 1384-93. [146] Junttila T.T., et al., Erbb4 and its isoforms: selective regulation of growth factor responses by naturally occurring receptor variants. Trends Cardiovasc Med, 2000. 10(7): p. 304-10. [147] Ni C.Y., et al., gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science, 2001. 294(5549): p. 2179-81. [148] Carpenter G., ErbB-4: mechanism of action and biology. Exp Cell Res, 2003. 284(1): p. 66-77.

75 REFERENCES

[149] Naresh A., et al., The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells. Cancer Res, 2006. 66(12): p. 6412-20. [150] Paatero I. and K. Elenius, ErbB4 and its isoforms: patentable drug targets? Recent Pat DNA Gene Seq, 2008. 2(1): p. 27-33. [151] Heidel F., et al., Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood, 2006. 107(1): p. 293-300.

Acti laboris iocundi….. Efter avslutat arbete är vilan skön……

(Cicero)