The ErbB signaling pathway in acute myeloid leukemiascripties.umcg.eldoc.ub.rug.nl/FILES/root/geneeskunde/2013/ScheepersE/... · The ErbB signaling pathway in Acute Myeloid Leukemia

The ErbB signaling pathway in acute myeloid leukemia

Name: Ellen Scheepers Student number: 1815407 Faculty supervisor: Prof. Dr. E.S.J.M. de Bont Research institute: University Medical Centre Groningen Beatrix Childrens Hospital Paediatric Oncology Research Laboratory Date: 01-03-2013

The ErbB signaling pathway in Acute Myeloid Leukemia

Ellen Scheepers 01-03-2013

2

CONTENT

Abstract 3

Samenvatting 4

1. Introduction 6

1.1. Cancer 6

1.2. Leukemia 6

1.3. Acute myeloid leukemia (AML) 7

1.4. Treatment options in pediatric acute myeloid leukemia 9

1.5. ErbB family transmembrane receptors and their role in treating cancer 9

1.6. ErbB signaling pathway in AML 11

2. Material & Methods 13

2.1. Cell lines 13

2.2. Used Antibodies 13

2.3. Flow cytometry analysis 13

2.4. Cell survival assays 14

2.5. Immunoprecipitation 14

2.6. Western blot analysis 14

2.7. Data analysis and statistics 15

3. Results 16

3.1. The effect of different ErbB inhibitors on AML cell survival 16

3.2. ErbB family member protein expression in AML 17

3.3. Downstream effects of ErbB stimulation and inhibition in AML 19

3.4. Influence of EGF stimulation on cell membrane ErbB1 expression 22

4. Discussion 23 4.1. Sensitivity towards ErbB inhibitors in AML 23

4.2. ErbB1 expression and functionality in THP-1 24

4.3. Conclusion 25

5. Acknowledgements 26

6. References 27

7. Appendices 31 7.1. Appendix 1 Flowcytometry analysis 31

7.2. Appendix 2 Cell survival assay 33

7.3. Appendix 3 Immunoprecipitation 35

7.4. Appendix 4 Protein analysis using western blot 38



3

ABSTRACT

Background: Acute myeloid leukemia (AML) is characterized by a defect of hematopoietic

stem and progenitor cells, leading to the accumulation of myeloid blast cells and cytopenia of

the remaining normal hematopoietic lineages. Among children and young adults up to 18

years, the 5-year event free survival of AML is 60-70%. Currently, cytarabine in combination

with antracycline, is the backbone of the chemotherapy. After achievement of a complete

remission with one or two chemotherapy courses, continuation of the chemotherapy is

necessary to reduce the relapse rate. Still, the relapse rate of the newly diagnosed children

with AML is 40-50%. To improve cure and relapse rates, quality of life and reduce additional

side effects for both patients and survivors, new treatment approaches are warranted. After the

success of tyrosine kinase inhibitors on the improved survival of cancer patients as for

example erlotinib and gefitinib for the treatment of lung and breast cancers, tyrosine kinase

inhibitors are the current focus of new therapeutic approaches in many forms of cancer

including AML. Recently, the epidermal growth factor receptor (EGFR) tyrosine kinase

inhibitor erlotinib has been shown to induce complete remission of AML in two patients with

concurrent non-small cellular lung cancer, suggesting a possible role for EGFR in AML.

However, EGFR expression analysis in AML is poorly defined and the role of EGFR and

other ErbB family members in AML is still unclear.

Aim: In this study, we studied the role of ErbB signaling pathway in AML by determining the

efficacy of ErbB inhibitors (canertinib, gefitinib, erlotinib and cetuximab) on cell survival and

cellular downstream targets, and analyzing the presence of ErbB family members in AML

cells.

Results: Using WST-1 assays, we have shown that especially the least selective ErbB

inhibitor canertinib induces cell death with a median LC50 of 7.0 µM (range: 2.6 - 15.2 µM

canertinib in n=8 AML cell lines). To determine whether this reduction of cell survival could

be induced via an ErbB dependent manner, we started with determining the cell membrane

protein expression levels of the ErbB family members using flowcytometry. ErbB1 (EGFR)

was only detectable in OCI-AML3 (23.2%) and in a lesser extent in EOL1, HL60 and THP1

cells (approximately 10%), whereas ErbB2 and ErbB4 were expressed in various degrees

among the different AML cell lines (with a range of respectively 8.8-63.9% for ErbB2 and

18.8-90.6% for ErbB4). ErbB3 was not detectable by flowcytometry. No correlation between

the cell membrane ErbB protein expression levels and their sensitivity towards ErbB

inhibitors in AML cell lines was observed. However, canertinib treatment and in a lesser

extent erlotinib treatment resulted in a reduction of phosphorylated Akt and Erk protein levels

using western blot analysis in THP-1 and EOL-1 cells. Moreover, total and phosphorylated

Akt and Erk levels are slightly increased in THP-1 cells after EGF stimulation. This suggests,

together with the ErbB1 cell membrane protein expression in THP-1 cells, that ErbB1 could

be present and functionally active in THP-1 cells. Focusing on ErbB1 in THP-1 cells, one

hour of EGF stimulation reduces the degree of total ErbB1 antibody expression in THP-1

cells, probably due to internalization of the activated ErbB1 receptor. Western blot analysis

did not show ErbB1 in THP-1 cells. Phosphorylated ErbB1in THP-1 was only detectable

using immunoprecipitation combined with western blot analysis. However, because of

antibody cross reactivity, further studies are needed to identify the observed receptor tyrosine

kinase.

Conclusion: In summary, these data suggest that ErbB family tyrosine kinase inhibitors is

suggested to be a therapeutic strategy in AML. However, further research is warranted to

determine the exact role of the ErbB signaling pathway in AML and how the presence of the

ErbB family members could contribute in relation to disease progression, relapse as well as

interaction with possible escape mechanisms in AML.



4

SAMENVATTING

Achtergrond: Acute myeloïde leukemie is gekenmerkt door een defect in de hematopoëitische

stamcel en zijn voorlopercellen die leiden tot een opeenstapeling van blasten en cytopenieën.

De 5-jaar recidief vrije overleving bij kinderen en jong volwassenen tot 18 jaar, is 60-70%.

Hedendaags is cytarabine in combinatie met antracycline de gouden standaard van de

chemotherapie. Na complete remissie na een of twee chemokuren, is het noodzakelijk de

chemotherapie te continueren om de kans op een recidief te reduceren. Desondanks is de

recidiefkans bij kinderen met AML nog steeds schrikbarend hoog, zo’n 40-50%. Om de

genezingskansen en recidief kansen, de kwaliteit van leven en de bijwerkingen van de

medicijnen voor AML patiënten en de overlevenden te verbeteren, zijn nieuwe

behandelingsstrategieën noodzakelijk. Nieuwe doelgerichte therapieën richtten zich op

tyrosine kinase inhibitors die intracellulaire signaal transductie routes kunnen beïnvloeden en

zo hun effect kunnen uit oefenen op de overleving van de leukemische cel. Onlangs liet

inhibitie van de epidermale groei factor receptor (EGFR) met behulp van de tyrosine kinase

inhibitor erlotinib, complete remissie zien bij twee AML patiënten met longkanker. Dit

gegeven suggereert een mogelijke rol voor de EGF receptor in AML. Echter blijkt de

expressie van EGFR in AML lastig aan te tonen en is de rol van EGFR in AML tot nog toe

onbekend.

Doel: Dit onderzoek bestudeert de rol van de ErbB signaal transductieroute in AML door de

invloed van ErbB inhibitoren (canertinib, gefitinib, erlotinib en cetuximab) op de cel

overleving en de betrokken intracellulaire proteine kinases te bepalen, én wordt de

aanwezigheid van de ErbB familie in AML cellen geanlyseerd.

Resultaten: WST-1 analyses lieten zien dat voornamelijk de minst selectieve ErbB inhibitor

canertinib aan afname van cel overleving induceert met een mediane LC50 van 7.0 µM

(range: 2.6 - 15.2 µM canertinib in n=8 AML cellijnen). Om te bepalen of dit verlies van cel

overleving wordt geïnduceerd via the ErbB signaal transductie route, is in deze studie gestart

met het bepalen van de celmembraan eiwitexpressie niveaus van de vier ErbB receptoren met

behulp van flowcytometrie. ErbB1 (EGFR) was alleen waarneembaar in OCI-AML3 (23.2%)

en in EOL1, HL60 en THP1 cellen (ongeveer 10%). Echter, ErbB2 en ErbB4 kwamen in

verschillende mate tot expressie onder de AML cellijnen (range ErbB2: 8.8-63.9% en range

ErbB4: 18.8-90.6%). ErbB3 was niet te detecteren met behulp van flowcytometrie. Er is geen

correlatie gevonden tussen celmembraan eiwitexpressie niveaus van ErbB receptoren en de

sensitiviteit tegen ErbB inhibitors in de bestudeerde AML cellijnen. Echter, behandeling met

canertinib en in mindere mate met erlotinib, resulteerde in een afname van de gefosforyleerde

Akt en Erk eiwit niveaus in THP-1 en EOL-1 cellen. Bovendien is de totale en

gefosforyleerde eiwit expressie van Akt en Erk na één uur EGF stimulatie licht gestegen in

THP-1 cellen. Dit effect in THP-1, tezamen met de ErbB1 celmembraan eiwitexpressie in

THP-1 cellen, suggereert de aanwezigheid en activiteit van ErbB1 in THP-1. Met de focus op

THP-1, één uur durende EGF stimulatie in THP-1 cellen reduceert het totale ErbB1

antilichaam eiwitexpressie op de celmembraan, waarschijnlijk als gevolg van internalisatie

van de receptor na activatie door ligand binding. Echter was ErbB1 in THP-1 cellen niet aan

te tonen met western blot analyse. Gefosforyleerde ErbB1 in THP-1 was alleen zichtbaar na

immunoprecipitatie en western blot analyse van de totale ErbB1 antilichaamexpressie. Alleen

zou er sprake kunnen zijn van antilichaam kruisreactiviteit van het gebruikte antilichaam voor

gefosforyleerde ErbB1. Daarom is verder onderzoek genoodzaakt om de aangetoonde

receptor tyrosine kinase te identificeren.

Conclusie: Dit onderzoek laat zien dat ErbB inhibitoren een therapeutische strategie in AML

kunnen zijn. Echter is verder onderzoek genoodzaakt om de exacte rol van de ErbB signaal



5

transductie route te bepalen in relatie tot ziekteprogressie, recidieven en mogelijke

ontsnappingsmechanismen van AML cellen.



6

1. INTRODUCTION

1.1. Cancer

Cancer is one of the most common diseases in the Western world and the most common cause

of death in the Netherlands, in 2011 44.038 people died of cancer (1). Tumorgenesis is a

multistep process which reflects genetic alterations that drives the transformation of normal

human cells into malignant cells. The genetic mutations, translocations and molecular

aberrations contribute to defects in mechanisms controlling cell division and cell death. These

genetic alterations may be inherited or acquired during life (e.g. viral agents, ionizing

radiation) and lead to the activation of oncogenes, or to a loss of tumor suppressor gene

function, which both lead to cancer progression. After these mutations, cells can become

malignant and will have at least one of the following characteristics. Cancer cells are often

able to produce growth factors and thereby stimulate their own growth (autocrine

stimulation). Malignant cells could also be insensitive to anti-growth factors and resistant to

apoptosis. Moreover, they have potential of uncontrolled proliferation, sustain angiogenesis

and metastasize (2). In addition, reprogramming of cellular metabolism in order to support

neoplastic proliferation and evading immunological destructions have also been proposed as

hallmarks of cancer (figure 1) (3).

Genetic alterations

Tissues invasion

and metastasis

Sustained

angiogenesis

Self sufficiency in

growth signals

Insensitive to anti-

growth factors

Evading

apoptosis

Reprogramming cel-

lular metabolism

Evading immunolo-

gical destruction

Limitless

replicative potential

Figure 1 - Hallmarks of cancer Basic characteristics of a cancer cell

Figure 1 – Hallmarks of cancer. The basic characteristics of a cancer cell. Modified from Hanahan et al. (2)

1.2. Leukemia

Leukemia is the type of cancer that is associated with disease of the blood and bone marrow.

Leukemia is characterized by a defect of hematopoietic stem and progenitor cells, leading to a

differentiation arrest of a certain hematopoietic lineage with subsequent suppression of the

other lineages leading to cytopenias (4). Worldwide, over 250 000 people are diagnosed with

leukemia each year, accounting for 2.5% of all cancers. There are four types of leukemia,

categorized by the type of cells involved and the state of maturity. Acute leukemia will arise

in the primitive hematopoietic compartments and the disease induction period is relatively



7

short, from days to weeks in comparison to chronic leukemias. The acute and chronic

leukemias can be further grouped making the distinction between the origin of the affected

cell; myeloid versus lymphoid cells (5, 6). The most common form is chronic lymphoid

leukemia (CLL) with an incidence rate of 50 patients per 100 000 citizens per year in the

Netherlands. The acute leukemias are diagnosed in four of 100 000 citizens each year in the

Netherlands and is mainly diagnosed in the elderly (6). Despite of a lower incidence rate of

leukemia in children, it is still the most common form of pediatric cancer. ALL is the most

frequently observed form of leukemia in children (7). Research from the last decades have

shown a remarkable improvement in acute lymphoid leukemia (ALL) with regard to treatment

strategies and improved the cure rates considerably up to 80% (4,8). In contrast to ALL,

survival of acute myeloid leukemia (AML) in children is less favorable.

1.3 Acute myeloid leukemia (AML)

This particular hematopoietic neoplasm involves cells committed to the myeloid lineage of

hematopoietic development in the bone marrow. AML is characterized by an accumulation of

myeloid stem and/or progenitor cells in the bone marrow and blood stream due to a

differentiation arrest. An accumulation of these blasts in the bone marrow decreases the

production of normal hematopoietic cells. As a consequence of the reduction of normal cells

in the peripheral blood, symptoms will arise. In a majority of the patients, general fatigue is

present, even as pallor and weakness due to the anemia. Moreover, a decrease of the amount

of granulocytes causes a higher risk of infections and a reduction of thrombocytes shows

more hemorrhagic events (9). To classify the different types of AML, the classification of the

French- American- British (FAB) is used and recently, the World Health Organization

(WHO) classification added more recent prognostic markers. These classifications focus on

chromosomal abnormalities, dysplasia and therapy related AML (table 1) (6, 8, 10).



8

A. WHO classification of AML

Genetic abnormalities t(8;21)(q22;q22), (AML1/ETO)

abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13;q22), (CBF/MYH11)

t(15;17)(q22;q12), (PML/RAR) and variants

11q23 (MLL) abnormalities

Multilineage dysplasia Following MDS or MDS/MPD

Without antecedent MDS or MDS/MPD, with dysplasia in at least 50% of cells ≥2 myeloid lineages

Therapy-related Alkylating agent/radiation-related type

Topoisomerase II inhibitor-related type (some may be lymphoid)

Others

Not otherwise categorized Acute myeloid leukemia without maturation (M0)

Acute myeloid leukemia, minimally differentiated (M1)

Acute myeloid leukemia with maturation (M2)

Acute myelomonocytic leukemia (M4)

Acute monoblastic/acute monocytic leukemia (M5)

Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia) (M6)

Acute megakaryoblastic leukemia (M7)

Acute basophilic leukemia

Acute panmyelosis with myelofibrosis

Myeloid sarcoma

B. FAB classification of AML

M0 AML with minimal differentiation

M1 AML without maturation

M2 AML with granulocytic maturation

M3 Acute promyelocytic leukemia

M4 Acute myelomonocytic leukemia

M4eo With bone marrow eosinophilia

M5A Acute monoblastic leukemia

M5B Acute monocytic leukemia

M6 Acute erytroid leukemia

M7 Acute megakaryoblastic leukemia Table 1 - Classification of AML. A) AML classified according to the FAB classification as used in earlier

years and B) the WHO classification for AML.

Over the years, cytogenetics and biomolecular markers became important determinants for

selecting the right therapy strategy and predicting the prognosis of patients with AML. Figure

2 displays an overview of the most frequent genetic lesions in childhood AML (11). The

frequency of many specific genetic lesions is less common in pediatric AML compared to

adult AML. Genetic abnormalities are able to influence common pathways involved in

proliferation, differentiation and cell survival. RUNX1-RUNX1T1 (15%) and CBFB-

MYH1(10%) are common and have a favorable prognosis, whereas FLT3-ITD (14%) is

associated with a poor prognosis. Moreover, it is well described that AML characterized by

t(15;17) , inv (16) or t(8;21) has a more favorable outcome compared to AML characterized

with a complex karyotype or EVI1 deletions (12). Mutations in the MLL gene show variable

clinical outcome (13, 14).

Despite of progression in predicting outcome by using cytogenetics and biomolecular

markers, AML still has a relative poor outcome. In the 1960’s the overall survival of AML

was less than 10%. Despite increasing cure rates due to the effectiveness of conventional

chemotherapy and intensive post remission therapies, still only 60% of the children will

belong to the long-term survivors (4, 8, 11). The current treatment strategies are limited as a

result of toxicity, which leads to treatment related morbidity and mortality (4). To reduce

these morbidity and mortality numbers in pediatric AML, new therapeutic strategies are

needed.



9

Figure 2 – Genetic abnormalities in childhood AML (11).

1.4. Treatment options in pediatric acute myeloid leukemia

Historically, cancer treatment is based on the mechanism that rapidly dividing cells are

particularly sensitive to the damage of their DNA synthesis. Based on this hypothesis,

chemotherapeutic agents were developed, which are able to destroy fast-dividing cells.

Chemotherapy is used very common in many types of cancer and was previously also used as

the backbone in treating pediatric AML (8, 15). To increase the efficacy, most of the time a

combination of cytotoxic chemotherapeutics was used. The most common remission

induction regimen used is cytarabine plus an antracyclin. Depending on patients’ response,

post-remission courses with high dose cytarabine were indicated to improve survival and

reduce relapse risks (8). Despite of a 5-year event free survival of 60-70% in children with

AML, over 40% of the children experience a relapse of their disease. In view of the fact that

the overall survival rates in pediatric AML patients are unsatisfactory, new approaches adding

anti-cancer drugs that involves targeting mechanisms on which cancer cells are more reliant,

will be essential to improve survival and relapse rates on one hand and reduce side effects of

treatment on the other hand. Targeted therapies are focusing on characteristics of the

cancerous cells, with the advantage that only tumor cells will be killed and that there is a

smaller chance of resistance to therapy. Targeted therapies mainly consist of small molecule

inhibitors and monoclonal antibodies. Both focus on the activation of cancer cell specialized

signal transduction pathways, including a large number of oncogenic tyrosine kinases. The

drugs are designed to specifically inhibit protein or receptor tyrosine kinases that result in an

intracellular transformation, which may lead to cell death (16).

1.5. ErbB family transmembrane receptors and their role in treating cancer

One of the commonly involved tyrosine kinase receptors in cancer biology are the ErbB

family members. The ErbB family consists of four transmembrane receptors (ErbB1-4),

which share a similar structure composed of an extracellular ligand binding domain, a

transmembrane lipophilic glycoprotein and an intracellular tyrosine kinase domain. These

receptors can bind different ligands (epidermal growth factor (EGF), heparin binding

epidermal growth factor (HB-EGF), epiregulin (EPR), tumor growth factor α (TGFα),

amphiregulin (AR), betacellulin (BTC), and neuroregulin 1-4 (NRG1-4)) to induce formation



10

of different homo- and heterodimers, and, as a consequence, the intrinsic kinase domain will

become activated, which results in phosphorylation and signal transduction pathways become

active (17). The RAS/RAF/MEK/ERK pathway, PI3K/AKT pathway and the JAK/STAT

pathway are the main signal transduction pathways which will be activated and are involved

in cell survival, proliferation and differentiation (18-20) (figure 3).

ErbB1 (EGFR) ErbB2 ErbB4

EGF TGFα

AR

JAK

STAT2/5

Cell survival

Akt

PI3K

HB-EGF EPR BTC

NRG-1 NRG-2

NRG-3 NRG-4

ErbB3

JAK

STAT2/5

RAS

RAF

MEK

Erk

P P

Erythroid diff.

Stem/prog. maintenance

Erythroid diff.

Stem/prog. maintenance Proliferation

Figure 3 – ErbB receptor tyrosine kinase family. The ErbB receptors, their ligands and their main

downstream protein pathways, all involved in cell proliferation, differentiation and apoptosis upon ligand

binding (15). The main transduction pathways are PI3K/Akt involved in cell survival, RAS/RAF/MEK/Erk

pathway involved in proliferation and the JAK/STAT pathway involved in erythroid differentiation and

maintenance of stem and progenitor cells (18-20).

The oncogenic potential of ErbB receptors is intensively studied, especially the receptor

ErbB1 (epidermal growth factor receptor, EGFR) and ErbB2 (HER2). ErbB1 and ErbB2 are

mutated in many epithelial tumors, with the consequence of cancer development and

progression. EGFR activating mutations are commonly seen in non-small cellular lung cancer

(NSCLC) and ErbB2 mutations are frequently seen in breast cancer. ErbB3 and ErbB4

mutations are less frequently seen in cancer (21). During the years, these receptors were

studied for their implication as therapeutic targets in cancer (20, 22, 23). Selective tyrosine

kinase inhibitors have been designed to target the ErbB family members. Proposed to be the

least selective tyrosine kinase inhibitor is the pan-ErbB inhibitor canertinib, which is designed

to target all four receptors of the ErbB family. Erlotinib and gefitinib tyrosine kinase

inhibitors are both designed to target the phosphorylation sites of ErbB1. Unlike the ErbB

tyrosine kinase inhibitors cetuximab is a monoclonal antibody that is designed to target the

ErbB1 receptor at the extracellular domain of the receptor (20). However, evaluating their



11

selectivity, previous reports showed that the described ErbB inhibitors could also target other

protein tyrosin kinases (24-27). Figure 4 shows the different target sites of the discussed ErbB

inhibitors, including the current knowledge on their selectivity.

P P P P P

1

EGFR ErbB4ErbB3

Erlotinib

Gefitinib

Erlotinib

Gefitinib

CanertinibCanertinib

CetuximabCetuximab

ErbB2

PP

EGF

P

Drug Targets

Canertinib ErbB1-4 Src PDGF receptor FGF receptor Insulin receptor CDK1,2,4 PKC

Erlotinib ErbB1 ErbB3 Insulin receptor Src v-abl

Gefitinib ErbB1 ErbB2 BRK GAK RICK

Cetuximab ErbB1 VEGF-C IL-8

B

A

Figure 4 – ErbB receptor tyrosine kinase family included the pharmaceutically developed ErbB inhibitors

and their (A) designed targets and (B) characteristics of selectivity. Abbreviations: PDGF- platelet derived

growth factor; FGF- fibroblast growth factor; CDK – cyclin dependent kinase; BRK – breast tumor kinase;

GAK – G associated kinase; RICK - Receptor-interacting serine/threonine-protein kinase; VEGF-C –

Vascular endothelial growth factor C; IL-8 – interleukine 8.

1.6. ErbB signaling pathway in AML

In previous case reports it was shown that EGFR, the first member of the ErbB family might

play a role in AML. Two adult patients demonstrating non small cellular lung cancer in co-

occurrence with AML achieved complete remission of their AML after mono-therapy with

tyrosine kinase ErbB inhibitor erlotinib (28, 29). Moreover, in vitro studies showed that ErbB

inhibitors as canertinib, erlotinib and gefitinib can induce differentiation, cell cycle arrest and

apoptosis in various AML cell lines (30-32). In contrast, ErbB1 protein levels, as assessed by

immunochemistry, and mRNA levels of ErbB1 have been found to be doubtfully low in AML

blasts (32, 33). Altogether, in vitro studies in AML cell lines underscored the hypothesis that

the downstream effects of the ErbB inhibitors must be due to off-target effects (32, 34, 35).

However, a new indication for ErbB expression in AML arises. Data from our research group

have shown that ErbB1 protein is expressed and activated by phosphorylation in AML. The

ErbB1 protein was increasingly expressed in 13.5% of 511 AML patient samples and

increasingly phosphorylated in 8% of the 511 AML patient samples in comparison to normal

bone marrow CD34+ cells (submitted data). These data suggest that the ErbB signaling

pathway, especially ErbB1, could be actively involved in AML. Therefore, in this study we

evaluated the role of ErbB family members in AML by studying the efficacy of ErbB tyrosine

kinase inhibitors on cell survival and downstream targets, and we subsequently analyzed the

presence of ErbB family members in a broad panel of AML cell lines.



12

Objective:

What is the role of ErbB signaling pathway in AML?

a) What is the sensitivity of AML cell lines towards ErbB inhibitors?

b) Are ErbB family members functionally expressed in AML?



13

2. MATERIAL AND METHODS

2.1. Cell lines

Leukemic cell lines (HL-60, THP-1, NB4, TF-1, KG1a, OCI-AML3, MOLM13, EOL-1), the

lung cancer cell line A549, the pediatric ependymoma Res-196, and the breast cancer cell line

SkBr03 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Characteristics of the used AML cell lines are shown in table 2. All leukemic cell lines and

A549 cells were cultured in RPMI-1640 medium (Lonza). SkBr03 cells were cultured in

DMEM-high glucose and Res-196 cells were cultured in DMEM-F12. All media were

supplemented with 1% penicillin/streptomycin and 10% fetal calf serum (FCS; Hyclone).

Cells were grown at 37°C in a humidified incubator with 5% CO2.

Cell line Origin FAB Cytogenetics Mutations

TF-1 Acute promyelocytic leukemia M3 NRAS EGF INS ErbB3

EOL-1 Eosinophilic leukemia M2 ErbB4

HEL Erytroleukemia M6HL60 Acute promyelocytic leukemia M3 NRAS FLT-3

NB4 Acute promyelocytic leukemia M3 t(15;17)(p21; q23) KRAS

OCI-AML3 Acute myelomonocytic leukemia M4 NRAS NPM1

KG1A Myeloblastic relapse of

erytroleukemia

M6 FLT3

THP-1 Acute monocytic leukemia M5 t(9;11) NRASMOLM13 MDS M5a t(9;11); ins(11;9)(q23;p22p23) FLT3ITD EGF INS

Table 2 - Cell line characteristics from human acute myeloid leukemic cells (36-40).

2.2. Used antibodies

During this study the following antibodies are used for the different techniques: Target Antibody Abbreviation Company Technique

anti-EGFRantibody#231 ErbB1#231 Abcam FACS

total ErbB1 antibody #4267 ErbB1#4267 Cell Signaling

Technology

Immunoprecipitation

total ErbB1 antibody #2232 ErbB1#2232 Cell Signaling

Technology

Western blot

phosphorylated ErbB1 antibody #3777 pErbB1#3777 Cell Signaling

Technology

Immunoprecipitation

phosphorylated ErbB1 antibody #2234 pErbB1#2234 Cell Signaling

Technology

Western blot

ErbB2 total MAb#1129b ErbB2#1129b R&D systems FACS

ErbB3 total allophycocyanin #FAB3481A ErbB3# FAB3481A R&D systems FACS

ErbB4 total mAb#11311 ErbB4#11311 R&D systems FACS

total Erk antibody #9102 Erk#9102 Cell Signaling

Technology

Western blot

phosphorylated Erk#9106 pErk#9106 Cell Signaling

Technology

Western blot

total Akt #9272 Akt#9272 Cell Signaling

Technology

Western blot

phosphorylated Akt #4060s pAkt #4060 Cell Signaling

Technology

Western blot

ErbB1

Erk

Akt

Table 3 – Overview of the different antibodies used in this study

2.3. Flow cytometric analysis

Cell membrane protein expression levels of ErbB family members were measured by using

Fluorescence Activating Cell Sorting (FACS). 200uL of leukemic cells were incubated (0,5-

1,0*10^6 cells/ml) with 4uL of ErbB antibodies (ErbB1#231; ErbB2#1129b; ErbB3#

FAB3481A; ErbB4#11311) for 30 minutes at room temperature. Afterwards, 4uL of

secondary antibody was added. The fluorescent antibody of Alexafluor567 (Molecular

Probes) was used for ErbB1#231 and polyclonal rabbit anti mouse/APC (DAKO) was used

for ErbB4#11311. Eventually cells were washed with 2 ml phosphate-buffered saline (PBS)

with 1% BSA and the protein expression levels of ErbB1-4 were measured using FACS

(Calibur LSR-II (BD)). Cells stained with solely the secondary antibody and cells stained with

IgG-PE and IgG-APC conjugated antibodies were used as negative controls for the analysis.



14

Expression of negative controls (isotype controls and secondary antibody controls) was

subtracted from the expression levels of the ErbB family members. Beside, same

measurements were done for ErbB1 expression levels after treating the cells with EGF for 1

hour (200ng/ml). Expression levels below 5% are indicated as being undetectable. Data were

analyzed using FlowJo 7.6.5 (appendix 1).

2.4. Cell survival assays

For quantification of leukemia cell viability after drug inhibition, cell survival assays were

performed for the eight different leukemic cell lines. A WST-1 colorimetric viability assay

protocol was done following the procedures recommended by the manufacture (Roche). Cells

were seeded at a density of 25.000 cells per well (100ul) in medium supplemented with 10%

FCS. The cells were subjected to different concentrations (range 0-20uM) of canertinib (LC

laboratories), erlotinib (Axon Medchem 1128), gefitinib (Axon MedChem 1393) and

cetuximab (Erbitux, Merck Co supllied by the deparment of Pharmacy of University Medical

Centre Groningen) for 48 hours of incubation (5 replicates for each concentration). After

addition of 10µL WST-1 cell survival reagent (Roche) to each well, the absorbance was

measured at 450 nm in a microplate reader (imark; Bio-Rad, Veenendaal, The Netherlands).

The data was presented as the cell survival percentage relative to untreated cells (appendix 2).

The LC50 value (drug concentration needed to kill 50% of the leukemic cells) was calculated

according to the following equation:

%])50([%])50[%]50([*%)50[%%]50([%

)50%]50([%50 uMdruguMdruguMdrug

OSOS

OSLC

2.5. Immunoprecipitation

To identify the presence of the ErbB1 in AML cells, we performed an immunoprecipitation

combined with western blot analysis. After making cell lysates of 15 million THP-1 cells in

300 uL lysis buffer (R&D systems), proteins from the supernatant were incubated with total

or phosphorylated EGFR antibody (respectively ErbB1#4267 and pErbB1#3777) for 3 hours

at 4°C. Afterwards agarose beads (Protein A Agarose Beads #9863, Cell signaling

Technology) (40ul/cell sample) were added and incubated overnight at 4°C. After washing,

agarose beads were boiled in SDS sample buffer (Biorad) and proteins were separated on 10%

polyacrylamide gels as described under western blot section. Western blot analysis was

continued using another total ErbB1 antibody (ErbB1#2232) (appendix 3).

2.6. Western blot analysis

Cultured AML cells (1*10^6) were stimulated with EGF (200ng/ml) for one hour or treated

for two hours with canertinib (20µM) or erlotinib (20µM). Afterwards, cells were centrifuged,

and sample buffer (Biorad;100µL/sample) was added. After boiling the samples for 5

minutes, proteins were separated by gel electrophoresis (10% SDS polyacrylamide gel) and

transported to transfer membranes (Immobolin). Blots were blocked in 5% skim milk for one

hour and incubated overnight at 4ºC in TBST with 5% bovine serum albumin (BSA) (PAA

Cell culture company) containing a 1:1000 dilution of primary antibody: total or

phosphorylated ErbB1 (respectively ErbB1#2232 and pErbB1#2234), ERK(respectively

Erk#9102 and pErk#9106s) and Akt (respectively Akt#9272 and pAkt#4060s). After the

primary incubation was washed for three times in 30 mintues, we followed by incubating with

the appropriate secondary antibodies (1:2000 dilution, DAKO cytomation) in 5% skim milk

(BD) at room temperature for 1 hour. Beta actin was probed as a protein loading control

(Santa Cruz Biotechnology, 1:2000 dilution). Antibody binding was visualized by enhanced



15

chemiluminescence solution (2 mL Tris-HCl pH 8.8, 28 mL H2O, 10 µL H2O2 (Emsure) and

10 mg luminol (Sigma), enhancer) and developed on X-ray films (appendix 4).

2.7. Data analysis and statistics

Results were representative for at least three independent experiments unless stated otherwise.

Values were presented as the mean ± standard deviation (SD). Correlations were analyzed

using Pearson’s correlations test. A P value of ≤0.05 was considered significant in all

analysis. SPSS-20 was used for statistical analysis.



16

3. RESULTS

3.1. The effect of different ErbB inhibitors on AML cell survival

In order to evaluate whether ErbB inhibitors could influence the cell survival in AML cells,

we monitored the survival of AML derived cell lines upon exposure to different ErbB

inhibitors as canertinib, erlotinib, gefitinib and cetuximab using WST-survival assays. The

NSCLC cell line A549 was used as a control based upon the literature. Figure 5 shows a

heatmap of the LC50 values of the different AML cell lines 48 hours after adding one of the

ErbB inhibitors. First, evaluating the sensitivity towards the pan-ErbB inhibitor canertinib,

AML cell lines show different degrees in sensitivity with the LC50 in a range of 2.6-15.2µM.

MOLM13 cells are most sensitive towards canertinib, and TF-1 cells seem to be the most

resistant cells towards canertinib. Studying erlotinib, this drug inhibits cell survival most in

EOL-1 (LC50: 2.7µM) and in MOLM13 cells (LC50: 3µM). HEL and OCI-AML3 are less

sensitive to erlotinib, even in KG1A, TF1 and THP-1 cells a LC50 is not reached at 50 µM.

Gefitinib reduces cell survival also most in EOL-1 (LC50: 4.2µM) and MOLM13 cells

(LC50: 3µM), whereas THP-1, OCI-AML3 and KG1A are less sensitive. TF-1 and HEL cells

do not reach an LC50 after gefitinib treatment at 50µM. Finally, the monoclonal antibody

cetuximab solely affected THP-1 cells with a LC50 of 6.40µg/ml. Based on these results,

EOL-1 and MOLM13 cells are most sensitive AML cell lines towards the various ErbB

inhibitors, whereas TF1 is most resistant to all ErbB inhibitors. In the control cell line A549,

previous studies showed a LC50 of 8.9µM for erlotinib, 12.3µM for gefitinib, and 2.5µg/ml

for cetuximab (41). Comparing these LC50 values with LC50 values found in AML cell lines,

our data showed that three of the four AML cell lines which are sensitive towards erlotinib

(MOLM13, EOL-1, HEL) are even more sensitive towards erlotinib than A549 cells. THP-1,

OCI-AML3 and KG1A cells are less sensitive than A549 cells. The sensitivity towards

gefitinib in AML cell lines is also comparable or AML cells are even more sensitive than

A549 cells. KG1A cells show a similar LC50 value (12 µM), whereas MOLM13 and EOL-1

cells are even more sensitive. THP-1, OCI-AML3, TF-1 and HEL cells are less sensitive

towards gefitinib than A549 cells. Cetuximab do not reach a LC50 value in AML cell lines,

except for THP-1 cells, which show a higher LC50 value for cetuximab (6.4 µg/ml) in

comparison to A549 cells (2.5 µg/ml). In conclusion, canertinib, erlotinib, gefitinib and

cetuximab induce varying reduction of cell survival in AML cell lines. Moreover, there are

even AML cell lines which are as sensitive as A549 towards these ErbB inhibitors.

LC50s Canertinib(uM) Erlotinib(uM) Gefitinib(uM) Cetuximab (ug/ml)

THP-1 5,1 >50 17,8 6,4

OCI-AML3 6,8 13,3 24,8 >50

MOLM13 2,6 3 4,2 >50

KG1A 9 >50 12 >50

EOL-1 3,9 2,7 3 >50

HL60 8,4 N.A. N.A. N.A.

TF1 15,2 >50 >50 >50

NB4 5,6 N.A. N.A. N.A.

HEL N.A. 5,4 >50 >50

Low High

LC50

Figure 5 – The sensitivity of AML cells towards ErbB inhibitors of the different AML cell lines. LC50

values were determined 48 hours after treatment with the ErbB inhibitors canertinib, erlotinib, gefitinib and cetuximab. Dark bleu boxes stands for a lower LC50 value towards one of the ErbB inhibitors.

Abbreviation: N.A. – not applicable.



17

3.2. ErbB family member protein expression in AML

Previous studies in AML demonstrated no detectable ErbB protein expression levels in AML

and indicated that the effectiveness of ErbB inhibitors is due to off-target effects (30-35).

Nevertheless, data from our research group have shown that ErbB1 protein is expressed and

phosphorylated in a substantial amount of AML patient samples. To this extend we analyzed

the presence of ErbB family members in AML cells to determine whether the reduction of

cell survival by ErbB inhibitors could be induced via an ErbB dependent manner. We

measured cell membrane protein expression levels for several antibodies that recognize

different ErbB family members using flowcytometry. Figure 6 shows an overview of the

ErbB cell membrane protein expression levels for each AML cell line, together with the

elaborated data for the cell line with the highest ErbB 1-4 cell membrane protein expression

levels.

First, studying the ErbB1 cell membrane protein expression in AML cell lines, OCI-AML3

cells show the highest ErbB1 cell membrane protein expression level. Isotype control for

ErbB1 stains 2.34% of the OCI-AML3 cells, whereas isotype control together with ErbB1

antibody stains 25.5% of the viable cells (figure 6A). This shift from 2.34% to 25.5% gives an

ErbB1 cell membrane protein expression level of 23.2% in OCI-AML3 cells. Moreover,

ErbB1 cell membrane protein expression is also found in EOL-1, HL60 and THP-1 cells with

a protein expression level of respectively 9.8%, 8.4% and 10.7% (figure 6B). A549, used as a

positive control for ErbB1, shows a cell membrane protein expression level of 96.9%.

Flowcytometric analysis of the other ErbB family members revealed that total cell membrane

protein expression of ErbB2 was found in various degrees among the AML cells up to a

maximum in KG1A (63.1%) and MOLM13 cells (63.9%). SkBr03, the prototype cell line of

ErbB2 expression, shows a cell membrane protein expression level of 97.3%. All ErbB3 cell

membrane protein expression levels are less than 5%, so ErbB3 is indicated as being

undetectable in AML using flowcytometry. As a positive control for ErbB3 antibody, SkBr03

cells show a cell membrane protein expression level of 95.3%. Investigating ErbB4 cell

membrane protein expression in AML cells, flowcytometry shows that ErbB4 cell membrane

protein expression is found in a range of 18.8% in TF-1 cells up to 90.6% in THP-1 cells

(figure 6B). A negative control was used for ErbB4 antibody, Res-196 ependymoma cells

show a cell membrane protein expression level of 0%. Figure 6A shows also the elaborated

data of the AML cell line with the highest cell membrane protein expression for ErbB2,

ErbB3 and ErbB4. Altogether, these data showed that ErbB1 is only expressed in a low

percentage in some AML cell lines, whereas ErbB2 and ErbB4 seem to be frequently

expressed in various degrees in AML.

To gain more insights into the ErbB inhibitors in relation to the presence of ErbB family

members (ErbB1-4) in AML, we evaluated whether there is a relation between the ErbB

inhibitors and the cell membrane protein expression of the ErbB family members in different

AML cell lines. The LC50 values per drug per AML cell line was correlated with the cell

membrane protein expression of ErbB1, ErbB2, ErbB3 and ErbB4, using Pearsons correlation

tests. However, no significant correlation could be appreciated (data not shown).



18

-

-

-

-

-

-

Figure 6 – ErbB cell membrane protein expression in AML cell lines. (A) Example given of the AML cell

with the highest cell membrane protein expression level for each ErbB family member (ErbB1-4). (B)

Overview of the total levels of ErbB family cell membrane protein expression levels in percentages of the

viable cells. Expression of negative controls (isotype controls and secondary antibody controls) was

subtracted from the expression levels of the ErbB family members. Dark bleu boxes stands for a higher

level of ErbB antibody cell membrane protein expression. Levels below 5% are indicated as being

undetectable. Abbreviation: N.A. – not applicable.



19

3.3. Downstream effects of ErbB stimulation and inhibition in AML

Canertinib, erlotinib and gefitinib are able to reduce cell survival in most AML cell lines, to

determine whether this decrease in cell survival might be mediated by the ErbB-signaling

pathway, we studied the downstream effects of ErbB stimulation and inhibition in AML.

Based on the ErbB signaling pathway (figure 3), we only studied the cellular effects in total

and phosphorylated protein levels of the intracellular protein kinases Akt and Erk. Akt has a

molecular weight of 60 kDa and Erk has a molecular weight of 42 and 44kDa, so Erk

presence should show two bands on our western blot. Three AML cell lines were selected

based upon their difference in ErbB inhibitor sensitivity (figure 5). KG1A cells have a

relatively high LC50 towards canertinib, THP-1 cells have an intermediate LC50 towards

canertinib and EOL-1 cells were chosen as a very sensitive cell line towards canertinib.

KG1A and THP-1 cells did not reach an LC50 value after erlotinib treatment of 50µM,

whereas EOL-1 cells are very sensitive towards erlotinib.

Studying the downstream intracellular protein kinases Akt and Erk in the AML cell lines, we

hypothesized that cell lines which showed decreased cell survival in the presence of an ErbB

inhibitor, will show a reduction of the phosphorylation levels of Akt and Erk protein kinases.

Figure 7A shows an overview of the protein kinases Akt and Erk protein levels in the studied

AML cell lines after one hour of EGF stimulation (200ng/ml), or two hours of canertinib or

erlotinib treatment (20µM).

Evaluating the effects in KG1A cells, we started off with the total Akt and total Erk levels,

which are both detectable using western blot analysis. However, in the steady state condition,

phosphorylated Akt is undetectable and the steady state protein level of phosphorylated Erk

showed a faint band. EGF stimulation does not show intracellular effects on phosphorylated

Akt and Erk levels. Canertinib and erlotinib treatment do not show a visible band of

phosphorylated Akt and Erk.

Secondly, we studied the cellular effects in THP-1 cells. THP-1 is more sensitive to canertinib

than KG1A cells, but is also insensitive towards erlotinib treatment. Total Akt and Erk protein

levels are detectable in all conditions. EGF stimulation shows a slight increase in the total and

phosphorylated protein levels of Akt and Erk protein kinases. Effects in Akt and Erk levels

after treatment with canertinib or erlotinib were only observed after canertinib treatment,

which shows a reduction in phosphorylated Akt protein expression levels, whereas the

phosphorylated Erk protein expression level increases. No effects in downstream targets were

observed after erlotinib treatment, which could be probably explained by the fact that THP-1

cells are not sensitive towards erlotinib according to our WST-1-survivalassays.

Finally, we analyzed the most sensitive cell line towards canertinib and erlotinib treatment,

EOL-1. EGF stimulation shows no effect on Akt and Erk levels, whereas canertinib as well as

erlotinib were able to reduce the phosphorylated Akt and Erk expression levels. Canertinib

shows the strongest effect (figure 7A).

Altogether, treatment with ErbB inhibitors, especially canertinib, shows a reduction in the

phosphorylated Akt and Erk levels in all studied AML cell lines. However, it is remarkable

that EGF stimulation shows no effect on intracellular protein kinases Akt and Erk, except of

an increase of those phosphorylated protein kinase levels in THP-1 cells. Because the EGF

ligand can only bind to ErbB1 receptor according to the literature, this might suggest that

ErbB1 could be involved in the THP-1 cells. Together with the flowcytometric data which

show ErbB1 expression of 10.7% in THP-1 cells, we therefore focused on ErbB1 expression

in THP-1 cells. Using western blot analysis, we first tried to detect ErbB1 protein expression

in the positive control cell line A549. Figure 7B shows that A549 cells do have total ErbB1

expression which is phosphorylated after 1 hour of EGF stimulation. However, ErbB1

expression in THP-1 cells was undetectable using western blot analysis, which might due to



20

the lower ErbB1 antibody cell membrane protein expression in THP-1. Therefore, we

performed immunoprecipitation followed by western blot analysis of total and phosphorylated

ErbB1 fraction in THP-1 cells. Immunoprecipitation was done using ErbB1#4267 for total

ErbB1 antibody expression and ErbB1#3777 for phosphorylated ErbB1 antibody expression.

Western blot analysis after immunoprecipitation shows that especially the

immunoprecipitation with phosphorylated antibody ErbB1#3777 was successful, based on the

observation of stronger bands from the light and heavy chain of the used antibody for

immunoprecipition (ErbB1#4267 for total ErbB1 expression and ErbB1#3777 for

phosphorylated ErbB1 expression) (figure 7C). Moreover, figure 7C showed a successful

identified ErbB1 phosphorylated antibody expression (pErbB1#3777) in THP-1 cells.

However, we have to consider that the used antibody pErbB1#3777 could also cross-react

with other phosphorylated proteins. Total ErbB1 antibody (ErbB1#4267) is not observed,

which probably due to unsuitability for immunoprecipitation analysis of total ErbB1

(ErbB1#4267) in THP-1 cells

In conclusion, downstream cellular effects of Akt and Erk are observed in a varying way

among the AML cells, especially after canertinib treatment. EGF stimulation showed an

increase of total and phosphorylated Akt and Erk levels only in THP-1 cells. Focusing on

ErbB1 in THP-1 cells, it is probably only possible to detect ErbB1 in THP-1 cells by

immunoprecipitation of the phosphorylated ErbB1 antibody (pErbB1#3777) combined with

western blot analysis of total ErbB1 antibody (ErbB1#2232). However because of cross

reactivity of the used phosphorylated ErbB1 antibody (pErbB1#3777), it is uncertain whether

the observed band at the level of ErbB1 stands for the presence of ErbB1 in THP-1 cells.



21

-

-

-

-

- -

- - -

- - -

- - -

- - -

- - -

- - -

- - -

- - -

- - -

-

-

-

- - -

- - -

- - -

- - -

- - -

- - -

To

tal E

GF

R C

S #

42

67

Pho

sp

ho

EG

FR

CS

#3

77

7

Heavy chain (55 kDa)

Light chain (25 kDa)

Actin

Total EGFR antibody#2232 (170 kDa)

Undefined RTK

THP-1

Figure 7 - Downstream effects of stimulation with EGF and treatment with ErbB inhibitors in AML cells.

(A) Akt and Erk activity and (B) EGF receptor expression after stimulation with EGF (200ng/ml) for one

hour, or treatment with canertinib (20µM) or erlotinib (20µM) for 2 hours. (C) ErbB1 receptor expression

level in THP-1 cells after immunoprecipitation with other ErbB1 antibodies (respectively ErbB1#4267 for total ErbB1 and pErbB1#3777 for phosphorylated ErbB1 receptor expression). The chosen cell lines were

based upon their difference in canertinib and erlotinib sensitivity.



22

3.4. Influence of EGF stimulation on cell membrane ErbB1 expression

To determine the presence and activity of ErbB1 in THP-1 cells in another way, we

stimulated THP-1 cells with 200ng/ml EGF for one hour, and measured the cell membrane

protein expression using FACS analysis. Based on the well-known phenomenon of

internalization of a transmembrane receptor after receptor activation by ligand binding, we

hypothesized that the cell membrane protein expression of ErbB1 will reduce after EGF

stimulation (16). The A549 control cells are highly stained with ErbB1 antibody (ErbB1#231)

on their cell membrane, after EGF stimulation, the ErbB1 cell membrane protein expression

decreases to 32.2%. Interestingly, to a lesser extent, the same effect is observed in THP-1

cells. The cell staining of total external ErbB1 antibody protein expression reduces with

almost 10% from 14.3% to 3.94% (figure 8). This effect of ErbB1 cell membrane protein

expression in THP-1 cells, together with the protein band showed after immunoprecipitaion

combined with western blot analysis, suggest a possible role for ErbB1 in THP-1 cells.

-

-

Figure 8 –ErbB1 cell membrane protein expression (ErbB1 #231) after one hour of EGF stimulation

(200ng/ml).



23

4. DISCUSSION

ErbB tyrosine kinase inhibitors are a clinically validated therapeutic option in solid tumors,

especially for those tumors that harbor sensitizing mutations for one of the ErbB family

members, like lung and breast cancer. Since erlotinib treatment induced complete remission

of AML founded in two NSCLC patients with concomitant AML (28, 29), some research was

done about the role of ErbB1 in AML. The effectiveness of ErbB inhibitors seems to be due

to off-target effects, since in these studies no detectable ErbB expression levels were found in

the used AML cells (MV4-11, U937, HL60, KG-1) (30-35). However, reverse protein profiler

assays (RPPA) of our research group have shown that ErbB1 seems to be expressed and

phosphorylated in a substantial amount of AML patient samples (submitted data). We used

this study as a new indication to evaluate the role ErbB signaling pathway in AML and

determined that AML cell lines are in a different degree sensitive towards canertinib,

erlotinib, gefitinib and cetxuimab. Moreover, except of ErbB3, ErbB family members are in a

various degree expressed among the studied AML cell lines, whereas ErbB1 seems to be

functionally active in THP-1 cells.

4.1. Sensitivity towards ErbB inhibitors in AML

This is the first study which screened the sensitivity of a broad panel of human derived AML

cell lines towards different ErbB inhibitors (canertinib, gefitinib, erlotinib and cetuximab).

We found that ErbB inhibitors induce varying reduction of cell survival in AML cell lines

with LC50 values which are comparable with LC50 values of A549 control cells. Evaluating

these sensitivities, our data seem to be in line with previous studies. Trinks et al showed also

apoptosis of canertinib treatment in human leukemic HL60 and U937 cells, even as in human

T-cell leukemia Jurkat cells (31, 42). Cytotoxic activity of the ErbB inhibitors gefitinib or

erlotinib was also found in previous studies in KG-1, HL60 and Kasumi (30, 32, 43).

This study showed that not all ErbB inhibitors do have the same effect in cell survival

reduction, the less selective inhibitor, the pan ErbB inhibitor canertinib, effect all AML cell

lines, whereas erlotinib and gefitinib, which are designed to target ErbB1, were not able to

reach the LC50 in every AML cell line. It seems interesting to determine which factors could

be involved in the sensitivity of AML cells towards ErbB inhibitors to gain more insights in

whether the ErbB signaling pathway is involved in the mechanism behind cell survival

reduction in AML.

First, the selectivity of the used drugs could play a role. That canertinib showed cell

survival reduction in all AML cells, is simply explainable by the fact that canertinib targets

the most receptors (figure 4). Erlotinib and gefitinib, and especially cetuximab seem to be

more specific to their original target which lead to cell survival reduction in only a few

studied AML cell lines. Despite of the fact that the cell membrane characteristics of AML

cells is not completely known, this suggest that the sensitivity towards an ErbB inhibitor

depends on which receptors are available at the AML cells.

Secondly, despite of the fact that, except of ErbB, all ErbB family members are

expressed in various degree among the AML cell lines, there is no correlation between the

sensitivity towards used ErbB inhibitors and the presence of ErbB family members. So in this

way we could not declare that the reduction of cell survival is mediated by the ErbB signaling

pathway. Nevertheless, what we do know is that the reduction of cell survival by canertinib or

erlotinib, is induced by the intracellular protein kinases Akt and Erk which are involved in

proliferation and cell survival. However, activation of Akt and Erk is also possible by

activation of G protein coupled receptors or other receptor tyrosine kinases, as the insulin

receptor (46). Together with the knowledge of the selectivity of these drugs, this suggests

again that other receptors could also be involved by the reduction of cell survival by ErbB



24

inhibitors in AML cells. To determine precisely whether the cell survival reduction is

mediated by the ErbB signaling pathway, a more specific tool like a short hairpin against

ErbB receptors, or mutations in the ErbB family members could be used.

Finally, the question arises why for example MOLM13 and EOL-1 cells are very

sensitive towards all ErbB inhibitors, and TF-1 is almost resistant to all ErbB inhibitors, while

no correlation between the sensitivity towards ErbB inhibitors and the presence of ErbB

family members in AML is observed. Cytogenetic characteristics of AML cell lines could

play a role to answer this question (table 2). A previous study of Nordigarden et al

determined that sensitivity towards canertinib is associated with the presence of a FLT3

mutation in MV4 AML cells (47). According to the literature, HL60 and KG1A cells do have

a mutation of FLT3 in their stopcodon, whereas MOLM13 cells do have a FLT3-ITD

mutation (table 2). Evaluating the sensitivity of these cell lines towards ErbB inhibitors in

comparison to the other studied cell lines, we observed that especially MOLM13 cells are

sensitive to canertinib (LC50 of 3µM). Perhaps FLT3-ITD played a role in the degree of

sensitivity towards ErbB inhibitors. Another frequently seen mutation in AML cells is the

proto-oncogenic RAS mutation. Whether this mutation is associated with the sensitivity of

AML cells towards ErbB inhibitors is not studied yet. However, recently Hayes and Chaning

showed that RAS mutations are associated with higher levels of Erk which inhibits the

activity of phosphorylated ErbB1 (48). This suggests that AML cell lines with an RAS

mutation should express less ErbB1 with the consequence that these cells are more resistant

towards ErbB1 inhibitors. Evaluating the LC50 of the used AML cell lines, except of KG1A,

RAS mutated AML cells do indeed have a relatively high LC50 in comparison to other cell

lines.

Altogether, selectivity of the drugs, protein expression of ErbB family members in AML, and

cytogenetic characteristics of AML cells, could all be associated with the sensitivity of AML

cells towards ErbB inhibitors. However, based on only these results, there is still not

sufficient evidence that the cell survival reduction is mediated by the ErbB signaling pathway.

4.2. ErbB1 expression and functionality in THP-1

In the second part of this study we focused on the ErbB cell membrane protein expression

levels and tried to determine whether the expressed ErbB family members are functionally

active in AML. In comparison to previous studies (30, 32, 43), we were able to detect the

ErbB1 receptor in AML using flowcytometry. ErbB1 was slightly expressed in OCI-AML3,

EOL-1, HL-60 and THP1 cells. Moreover, we determined that ErbB2 and ErbB4 cell

membrane protein expression is also expressed in AML. Based on the clinical data which

described complete remission of AML in a patient with concomitant NSCLC after erlotinib

treatment, we focused on ErbB-1 expression in THP-1 cells (28,29).

ErbB1 cell membrane protein expression was only determined using flowcytometry ,

western blot analysis showed no visible band for ErbB1, perhaps due to the low level of

ErbB1 protein expression found in THP-1 (10.7%). After immunoprecipitation combined with

western blot analysis, a visible band of ErbB1 was shown. Remarkably, in comparison to the

total ErbB1 antibody, only phosphorylated ErbB1 antibody was successfully identified in

THP-1. The fact that heavy and the light chain of the total ErbB1 antibody can be less

observed indicates a poor immunoprecipitation for total ErbB1 expression which could have

resulted in an undetectable total ErbB1 antibody in THP-1 cells. Moreover, we have to

consider that the used phosphorylated ErbB1 antibody (pErbB1#3777) could, in comparison

to all other used antibodies, weakly cross react with other tyrosine-phosphorylated proteins.

Cross reactivity is more often observed among antibodies. Given the incredible complex



25

structure of each protein, antibodies are able to bind aberrantly to epitopes with identical or

similar structure in off target proteins, resulting in nonspecific cross-reactive signals.

Normally, this phenomenon is mainly seen in polyclonal antibodies, however it is also

observed in monoclonal antibodies. In previous reports, cross reactivity with ErbB1

antibodies was already noticed. Using the recently developed technique of multiplex epitope

mapping, they determined that ErbB1 antibodies can also bind towards integrins at the cell

surface receptor (44). Using autoassembly protein arrays, ErbB1 antibodies could also cross-

react with the epithelial cell adhesion molecule (EpCAM) and human trophoblast cell-surface

antigen (TROP2 also termed as GA733-1, M1S1 or EGP-1). Both proteins contain EGF-like

domains (45). These possibilities of cross-reactivity should be considered for the

pErbB1#3777 antibody used in this study. So despite of the fact the we used a specific

antibody for western blot analysis (ErbB1#2232) after immunoprecipitation which suggests

the presence of ErbB1 in THP-1, the observed protein is still an undefined receptor tyrosine

kinase. Mass spectrometry will be needed to determine which protein is observed and could

be involved in the mechanism of ErbB inhibition.

However, the fact that EGF stimulation lead to a reduction of ErbB1 antibody

expression in THP-1 cells using flowcytometry, together with a slightly increase of total and

phosphorylated Akt and Erk levels, indicate that there could be a role for ErbB1 in THP-1

cells. Unfortunately, we were not able to extensively investigate the ErbB1 functionality and

whether the other ErbB receptors are functionally active in AML. Based on the obtained data

about ErbB1 in AML, it might be valuable to determine the functionality of ErbB2 and

ErbB4 in AML by stimulation and inhibition experiments.

4.2. Conclusion

In summary, this study tried to investigate the role of ErbB signaling pathway in AML by

determining the sensitivity towards ErbB inhibitors and analyzing the expression and

functionality of ErbB family members in AML. In line with previous findings, ErbB

inhibition is suggested to be a potential therapeutic strategy in AML. However, the results

indicating that ErbB inhibitors function through a previously unrecognized ErbB dependent or

independent mechanism. Therefore, further research for analysing the exact mechanism of

cell survival reduction by ErbB inhibitors in AML cells is warranted to eventually determine

the contribution of the each expressed ErbB family member in AML, in relation to disease

progression, relapse as well as interaction with possible escape mechanisms of AML cells.



26

ACKNOWLEDGEMENTS

First of all, I am grateful for following my research internship at the department of pediatric

oncology UMCG. I would like to thank all the technicians of pediatric oncology. Tiny, Frank,

Harm Jan, your helpfulness and enthusiasm made that I followed my research internship with

pleasure. Besides, I would like to thanks Lindy and Mariska, especially for their mental

support. In particular, thanks towards Eveline and Kim for their intensive supervision for

experimental design. Kim, thank you for a well controlled supervision during the performing

of my experiments at the laboratory, and also thank you for your patience. Thank you all for a

very pleasant and instructive research internship.

Pediatric Oncology UMCG

Prof. Dr. E.S.J.M. de Bont

Dr. S. Diks

Dr. H. Mahmut

K.R. Kampen

H.J. Lourens

G.J. Meeuwsen

F.J.G. Scherpen

M. Sie

N. van der Sligte

L.T. Bosch



27

7. REFERENCES

1. CBS Statline. Doodsoorzaken; korte lijst (belangrijke doodsoorzaken), leeftijd,

geslacht 2011. Available at:

http://statline.cbs.nl/StatWeb/publication/default.aspx?VW=T&DM=SLNL&PA=705

2_95&D1=a&D2=a&D3=0&D4=31%2c38-l&HD=110413-

1513&HDR=G2%2cG1%2cG3&STB=T. Used on 10-12-2012.

2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000. Vol. 100(1): p. 57-70.

3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell, 2011. Vol.

144(5): p. 646-74.

4. Brown P, Smith FO. Molecularly targeted therapies for pediatric acute myeloid

leukemia progress to date. Pediatric Drugs, 2008. Vol. 10 (2): p. 85-92.

5. Rodriguez-Abreu D, Bordoni A, Zucca E. Epidemiology of hematological

malignancies. Ann Oncol, 2007. Vol. 18 Suppl 1: p. i3-i8.

6. UMCG Hematologie Groningen. Acute leukemie: diagnostiek en algemene

maatregelen van behandeling. Available at:

http://hematologiegroningen.nl/protocollen/ Used on 10-12-2012.

7. Davidoff AM. Pediatric oncology. Seminars in Pediatric Surgery, 2010. Vol 19: p.

225-233.

8. Shah M, Agarwal B. Recent advances in management of acute myeloid leukemia

(AML). Indian journal of pediatrics, 2008. Vol. 75: p. 831- 837.

9. Schiffer CA, Anastasi J. Clinical manifestations, pathologic features, and diagnosis of

acute myeloid leukemia. Available at: http://www.uptodate.com.proxy-

ub.rug.nl/contents/clinical-manifestations-pathologic-features-and-diagnosis-of-acute-

myeloidleukemia?source=search_result&search=Acute+myeloid+leukemia&selected

Title=1~150. Used on 28-12-2012

10. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO)

classification of the myeloid neoplasms. Blood, 2002. Vol. 100: p. 2292-2302

11. Ching- Hon Pui, Carrol WL, Meshinchi S, Arecci RJ. Biology, risk stratification, and

therapy of pediatric acute leukemias: an update. Journal of clinical oncology, 2011.

Vol. 29 (5): p. 551-565

12. Burnett AK, Kell J, Rowntree C. Acute myeloid leukemia: therapeutic indications.

Current Opinion in Hematology, 2000. Vol. 7(6): 333-8.

13. Rubnitz JE, Inaba H. Childhood acute myeloid leukaemia. British Journal of

Haematology, 2012. Vol. 159: p. 259–287.



28

14. Balgobind BV, Zwaan CM, Pieters R et al. The heterogenity of pediatric MLL-

rearranged acute myeloid leukemia. Leukemia, 2011. Vol. 25(8): p. 1239-48.

15. Bagnyukova T. et al. Chemotherapy and signaling, how can targeted therapies

supercharge cytotoxic agents? Cancer biology & Therapy, 2010. Vol. 10 (9): p. 839-

835.

16. Gerber DE. Targeted therapies: a new generation of cancer treatments. American

Family Physician, 2008. Vol. 77(3): p.311-9

17. Chen G, Kronenberger P, Teugels E, et al. Targeting the epidermal growth factor

receptor in non-small cell lung cancer cells: the effect of combining RNA interference

with tyrosine kinase inhibitors or cetuximab. BMC Medicine 2012. Vol. 10 (28): p.1-

15.

18. Sibilia M, Kroismayr R, Lichtenberger MB et al. The epidermal growth factor

receptor: from development to tumorigenesis. Differentiation, 2007. Vol. 75: p.770-

787.

19. Jorissen RN, Walker F, Pouliot N et al. Epidermal growth factor receptor: mechanisms

of activation and signaling. Experimental Cell Research, 2003. Vol. 248: p.31-53.

20. Sebastian S, Settleman J, Reshkin SJ et al. The complexity of targeting EGFR

signaling in cancer: from expression to turnover. Biochemica et Biophysica Acta,

2006. Vol. 1766: p. 120-139.

21. Holbro T, Hynes NE. ErbB receptors directing key signaling networks throughout life.

Annual Review of Pharmacology and Toxicology, 2004. Vol. 44: p.195-217.

22. Wells A. Molecules in focus EGF receptor. The international Journal of Biochemistry

& Cell Biology, 1999. Vol. 31: p. 637 – 643.

23. Hynes N, MacDonald G. ErbB receptors and signaling pathways in cancer. Current

opinion in cell biology, 2009. Vol. 21: 177-184.

24. Allen LF, Eiseman IA, Fry DW et al. CI-1033, an irreversible pan-ErbB receptor

inhibitor and its potential application for treatment of breast cancer. Seminars in

Oncology, 2003. Vol. 30: p.65-78.

25. Brehmer D, Greff Z, Godl K et al. Cellular targets of gefitinib. Cancer research, 2005.

Vol. 65(2): p. 379-82.

26. EMA Europe. Scientific discussion. Available at:

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-

_Scientific_Discussion/human/000618/WC500033991.pdf. Used on 20-02-2013.

27. Buck E, Eyzaguirre A, Haley JD et al. Inactivation of Akt by the epidermal growth

factor receptor inhibitor erlotinib is mediated by HER-3 in pancreatic and colorectal



29

tumor cell lines and contributes to erlotinib sensitivity. Molecular Cancer

Therapeutics, 2006. Vol. 5(8): p. 2051-9.

28. Pitini et al. Erlotinib in a patient with acute myelogenous leukemia and concomitant

non-small-cell lung cancer. Journal of oncology, 2008. p 3645-3646.

29. Chan G, Pillchowska M. Complete remission in a patient with acute myelogenous

leukemia treated with erlotinib for non-small-cell lung cancer. Blood, 2007. Vol.

110(3). p. 1079-1080.

30. Boehrer S, Adès L, Galluzzi L et al. Erlotinib and gefitinib for the treatment of

myelodysplastic syndrome and acute myeloid leukemia: a preclinical comparison.

Biochemical pharmacology, 2008. Vol. 76: p.1417-1425.

31. Trinks C, Djerf EA, Hallbeck AL et al. 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, 2010. Vol.

393: p. 6-10.

32. Lindhagen E, Eriksson A, Wickström M et al. Significant cytotoxic activity in vitro of

the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia.

European Journal of Haematology, 2008. Vol. 81: p. 344-353

33. Sun J, Ying L, Xu Y et al. Epidermal growth factor receptor expression in acute

myelogenous leukemia is associated with clinical prognosis. Hematological Oncology

2012. Vol. 30: p. 89-97.

34. Hahn CK, Berchuck JE, Ross KN et al. Proteomic and genetic approaches identify Syk

as an AML target. Cancer Cell, 2009. Vol. 16(4): p. 281-94.

35. Boehrer S, Galluzi L, Lainey E et al. Erlotinib antagonizes constitutive activation of

SRC family kinases and mTOR in acute myeloid leukemia. Cell cycle, 2011. Vol.

10(18): p. 3168-75.

36. Collins SJ. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation

and cellular oncogene expression. Blood 1987. 70: p. 1233-1244

37. Gallagher R, Collins S, Truijllo J et al. Characterization of the continuous,

differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic

leukemia. Bood 1979. 54: p. 713-733.

38. Furley AJ, Reeves BR, Mizutani S et al. Divergent molecular phenotypes of KG1 and

KG1a myeloid cell lines. Blood 1986. 68: p.1101-1107.

39. Odero MD, Zeleznik-Le NJ, Chinwalla V, Rowley JD. Cytogenetic and molecular

analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9

transloctaion. Genes, chromosomes & cancer, 2000/ 29: p. 333-338.



30

40. Mayumi M. EoL-1, a human eosinophilic cell line. Leukemia and Lymphoma. Vol. 7:

p. 243-250.

41. Xu F, Tian Y, Huang Y et al. EGFR inhibitors sensitize non-small cell lung cancer

cells to TRAIL-induced apoptosis. Chinese journal of cancer, 2011. Vol. 30(10): p.

701-11.

42. Trinks C, Severinsson EA, Holmlund B et al. The pan-ErbB tyrosine kinase inhibitor

canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat cells.

Biochemical and Biophysical research communications, 2011. Vol. 410(3), 422-7.

43. Stegmaier K, Corsello SM, Ross KN et al. Gefitinib induces myeloid differentiation of

acute myeloid leukemia. Blood, 2005. Vol. 106(8): p.2841-8.

44. Hudson EP, Uhlen M, Rockberg J. Multiplex epitope mapping using bacterial surface

display reveals both linear and conformational epitopes. Scientific reports, 2012. Vol.

2: p. 706

45. Gaster RS, Hall DA, Wang SX. Autoassembly protein arrays for analysing antibody

cross-reactivity. Nano Letters, 2011. Vol. 11(7): p. 2579-83.

46. Cheung M, Testa JR. Diverse mechanisms of AKT pathway activation in human

malignancy. Current Cancer Drug Targets, 2013.

47. Nordigarden A, Zetterblad J, Trinks C et al. Irreversible pan-ErbB inhibitor canertinib

elicits anti-leukaemic effects and induces the regression of FLT3-ITD transformed

cells in mice. British Journal of Haematology, 2011. Vol. 155(2): p. 198-208

48. Hayes TK, Channing D. Mutant and wild-type Ras: co-conspirators in cancer. Cancer

discovery, 2013. Vol 3(1): p.24-6.



31

7. APPENDICES

7.1 Appendix 1 Flowcytometric analysis

FACS analysis

Cell membrane receptor expression

Negative control: blanco

1. Take samples of 200uL cells (0,5-1,0*10^6 cells/mL).

2. Ready for FACS.

Isotype control: IgG-FITC


2. Add 4uL secondary antibody (Swine F(ab`)2 FITC DAKO A/S, Denmark, or

Alexafluor488, Molecular Probes) and incubate for 30 minutes at room temperature in the

dark.

3. Wash with 2mL PBS 1%BSA.

4. Spin down (5 min 1500 rpm).

5. Remove supernatant.

6. Ready for FACS.

Samples for experiment


2. Add 2,5 uL of primary antibody:

- Total EGFR antibody – ErbB1#4267

- Total EGFR antibody – ErbB1#231

- Phosphorylated EGFR antibody – pErbB1#3777

- Total ErbB2 antibody – RD#Fab1129b

- Total ErbB4 antibody – RD#Mab11311

3. Incubate for 30 minutes at room temperature.

4. Add 4uL secondary antibody (Swine F(ab`)2 FITC DAKO A/S, Denmark, or

Alexafluor488, Molecular Probes) and incubate for 30 minutes at room temperature in the

dark.

5. Wash with 2mL PBS/1%BSA.

6. Spin 5 min 1500 rpm.


8. Ready for FACS.

NB. Tick the cells after every step.

For intracellular receptor staining fix the cells before adding primary antibody by

adding 1ml methanol while vortexing and incubate for 20 minutes in the freeze (-

20°C). Afterwards, wash twice with PBS/1%BSAand remove supernatant before

adding primary antibody.

Apoptosis

1. Take a sample of 200 uL cells (0,5-1,0*10^6 cells/mL).

2. Add 1 mL PBS.

3. Spin down (5min 1500 rpm).


5. Label with annexin PI (20uL in 1 mL 100uL/sample).



32

Cell cycle arrest (DNA staining)

1. Take a sample of 500uL-1mL cells (+/- 1 million).

2. Spin down (5min 1500 rpm).


4. Add 1 mL PBS.

5. Add 2,5 mL ethanol.

6. Incubate for 15 min in the freezer.

7. Wash the cells in PBS 1% BSA.

Cell staining


9. Add 200 uL PBS 1% BSA.

10. Add 2,5uL phospho-histone H3-Alexa 488 (Cell signaling) and resuspend.

11. Incubate for 20 min at room temperature in the dark.

12. Add 3mL PBS.





33

7.2 Appendix 2 Cell survival assay

WST- Cell proliferation reagent 0-10uM Canertinib

1. Count the cells of the different cell lines.

2. Calculate the amount of concentrations of drug you need.

.. cellines, in … plo and blanco/plate= .. wells/concentration. ..*50uL/well = .. ul

3. Make dilutions of the drug for the right concentrations. Keep in mind that concentrations

will be diluted when you add them in wells.

Canertinib 10,3mM

Canertinib RPMI DMSO

Final amount of solution (min 1 mL

needed to fill all the wells)

20 uM 10 uL 10,3mM 5140 uL RPMI 4 ml

15 uM 1,5 ml 20uM 0,5 ml 4 ml

10 uM 1,5 ml 20uM 1,5 ml 3 ml

5 uM 1 ml 10 uM 1 ml 3 ml

2 uM 0,5 ml 10 uM 2 ml 5 ml

0 uM 0 1,5 ml 3 ml

Total 13 ml

4. Make RPMI DMSO at the same concentration as in the highest concentration of

canertinib.

Canertinib is solved in 100% DMSO. DMSO is cytotoxic in increased concentrations, 1:1000

DMSO is recommended. The highest concentration made is 20uM. So DMSO is 10300/20=

515x diluted and Canertinib is solved in 1:515 DMSO. If you need 515 mL RPMI DMSO,

you will add 1 mL DMSO to get this ratio. However, 13 mL RPMI DMSO is needed. Let’s

make a small excess, e.g. 15 mL. Then we will need 15/515= 29,1ul DMSO and 14,97mL

RPMI for the ratio of 1:515. After adding the DMSO to the wells, the concentration of DMSO

will decrease to an concentration of ~1:1000 DMSO/well.

5. Calculate how many cells you need to fill all the wells (40.000cells/well).

alcellenhuidigaant

talcellengewenstaandedmLcellsnee

Concentration cells/well: 40.000 cells/50uL = 800.000/mL

Per celline: 6 concentration, in … plo so ... well present per celline.

..*40.000 cellen/..*50uL = … /… mL

If we are going to make 2 mL cells in a concentration of 800.000 cells/ml, we will need

1.600.000 cells.

1.6/cellcount = ml cells needed and complement with RPMI untill 2mL.

sfcatorverdunning

rugeveelheiddgewenstehougneededaantalmldr

][

][

gewenst

huidigsfactorverdunning



34

6. Culture cells in microplate in a final volume of 100uL/well.

- 50uL Cells.

- 50uL Canertinib.

Blanco: 50uL RPMI en 50uL RPMI DMSO.

7. Put the 96 wells plaat in the incubator at 37C for 48h.

8. Add after 48h WST reagent: 10uL/well.

9. Incubate the cells for 0,5h-4h (0,5-1-2-4h).

10. Measure the absorbance of the samples against a background control as blank using a

ELISA reader at 420-480nm. The reference wave length should be more than 600nm.

Blanco:

Medium and WST

50uL cells 50uL drug

50uL RPMI DMSO 50uL RPMI DMSO

1 uM

2 uM

5 uM

8 uM

10 uM

0 uM

Canertinib

concentration



35

7.3 Appendix 3 Immunoprecipitation

Protocol in-gel trypsin digest for MALDI-TOF peptide mapping

Cell lysates

1. Take a sample of 10,15,25 million cells.

2. Spin down for 5 min 1500 rpm at room temperature.

3. Add 1 ml PBS.

4. Pipette the solution in an eppendorf tube.



7. Put the pallets in the fridge at -80ºC when they will not directly used.

Immunoprecipitation

1. Take samples from the freezer and keep the samples on ice.

2. Add 300uL of lysis buffer, resuspend and rotate the cells for 20 minutes at 4 ºC.

3. Spin down and use supernatant for continuing the experiment.

4. Incubate supernatant with EGFR antibody (EGF Receptor Antibody #2232, Cell Signaling

Technology) for 3 hours at 4°C.

5. Add 20uL protein G plus agarose beads (Protein A Agarose Beads #9863, Cell signaling

Technology) and incubate overnight while rotating.

6. Spin down (5 minutes 10.000rpm) and remove supernatant.

7. Wash three times with lysis buffer and solve the pellet in 30uL sample buffer.

8. Boil them for 5 minutes and spin down the beads.

9. Use the supernatant to run the samples at a 10% SDS polyacrylamide gel.

Colloidal Coomassie Brilliant Blue staining

1. Add 16 mL ortho-phosphoric acid in 768 mL milliQ.

2. Add 80 gram ammoniumsulphate

3. Prepare a solution of 5% Coomassie Brilliant Bleu – G250 (CBB; Merck, Darmstadt

Germany)

4. Add 16 mL 5% CBB solution to the solution made at step 1.

5. Directly before usage: Slowly add 200 mL of methanol to the solution to give a final

concentration of 0.08% CBB, 1.6% ortho-phosphoric acid, 8% ammoniumsulphate,

20% methanol.

Staining with colloidal CBB and destaining

1. Wash the gel 3x 5 minutes in 200 ml of milli-Q water.

2. Remove the water.

3. Add 50 ml of Coomassie Stain (or enough to completely cover gel)

4. Gently shake for 1 hour. Refresh the Coomassie stain after 1 hour and incubate overnight.

5. Destain the gel with milli-Q water for 2 hours.

Cutting of bands

1. Cut the selected band with a clean (alcohol rinsed) knife/scalpel, as small a piece as

possible. It is better to cut inside the stained edges than outside (visibly stained bands

contain more than enough protein). Large pieces also make it more difficult for washing

solutions and trypsin to enter.



36

2. A piece 5 mm long (lane width), 0.5-1 mm thick (gel thickness) and 0.5-1 mm wide

(“band width”) is transferred to an Eppendorf tube. Large bands can be cut into smaller

pieces.

3. The gel pieces can be stored at –20°C until the protocol is continued. It is a good idea to

divide the gel piece in half and store one half in case something goes wrong in the rest of

the procedure. Again, half a gel piece still contains plenty of protein.

Important: always wear gloves during handling of samples and solutions. Make sure

pipette tips, Eppendorf tubes and other disposables come from a clean box or bag.

Solutions and chemicals stored in fridge:

- 100 mM Ammonium Bicarbonate (NH4HCO3) in water (pH ~7.6, do not set)

- water

- acetonitrile

- DTT (dithiothreitol), also as pre-weighed aliquots in Eppendorf tubes in freezer

- Iodoacetamide, also as pre-weighed aliquots in Eppendorf tubes in freezer

In freezer:

- Porcine trypsin (Promega), 20x stock aliquots

- -cyano-4-hydroxycinnamic acid, recystallised (Laser Biolabs)

Washing

1. Add 200 µl of 70 % 25 mM NH4HCO3 in water + 30 % acetonitrile.

2. Shake for about 30 minutes.

3. Remove solution with pipette.




7. Add 200 µl of 100 % acetonitrile.



10. Dry the gel pieces in the oven.

Reduction and alkylation

1. Add 15 µl 10 mM DTT in 100 mM NH4HCO3 (Freshly prepared, e.g. to 1.0 mg DTT add

648 µl 100 mM NH4HCO3).

2. Incubate at 56°C for 30 minutes (in Bischoff’s lab or in oven).

3. Add 10 µl 55 mM iodoacetamide in 100 mM NH4HCO3 (Freshly prepared, e.g. to 1.0 mg

iodoacetamide add 98 µl 100 mM NH4HCO3).

4. Incubate at room temperature in the dark for 30 minutes (in a drawer).




8. Dry the gel pieces in the SpeedVac vacuum centrifuge.

Trypsin digestion

1. Add 20 µl 10 ng/µl trypsin (porcine, Promega) to the gel pieces (dilute the stock 20x in

100 mM NH4HCO3).

2. Incubate overnight at 37°C.



37

3. Remove the residual liquid from the tubes (usually very little, combine with extract from

next step).

Peptide extraction

1. Add 10 µl of 75 % acetonitrile + 25 % (5% formic acid in water).


3. Briefly centrifuge tubes and collect the fluid.

4. Before optional ZipTip purification:

5. Dry the extracted peptides in the SpeedVac vacuum centrifuge.

6. Resuspend the dry peptides in 10 µl 0.1 % formic acid in water.

Peptides were measured using Biomedical Orbitrap.



38

7.4 Appendix 4 Protein analysis using western blot

Samples

1. Take a sample of 1 million cells.


3. Add 1 ml PBS.

4. Pipette the solution in an eppendorf tube.



7. Put the pallets in the fridge at -80ºC when they will not directly use for western blot.

Sample adaptation

1. Take samples from the fridge and keep the samples on ice! Add 100uLsamplebuffer (add

800ul demiwater before use). Location: -20ºC freeze hematology.

2. Vortex the eppendorf tubes

3. Boil the samples in water for 5 min.

4. Put them directly on ice.

Making 10% SDS polyacrylamide gel

1. For small gels for 8 samples, use 7,5mm glass slides.

2. Clean the glass slides and degrease with ethanol.

3. Pipet the running gel (2mL Tris – HCl pH 8.8, 2mL Acrylamide/Bisacrylamide(Biorad),

3.9 ml H2O, 80µL SDS 10%, 80 uL APS, 4uL TEMED(Biorad); add TEMED totally in

the end because of the coagulation process) Pipet approximately 3,5 mL running gel.

4. Pipet a small layer of isobutanol (80uL) above the running gel.

5. Remove the isobutanol after polymerisation of the running gel.

6. Pipet stacking gel (0.5mL Tris – HCl pH 8.8, 0.5mL Acrylamide/Bisacrylamide(Biorad),

3 ml H2O, 40µL SDS 10%, 40 uL APS, 4uL TEMED(Biorad); add TEMED totally in the

end because of the coagulation process)Wait untill the stacking gel is polymerized.

Electrophoresis

1. After polymerization the gel could be placed in the electrophoresis system.

2. Add 1x electrophoresis buffer in the system.

3. Add ~ 20ul protein lysate (200.000 cells)/ sample.

4. Add page ruler at the outside (7ul). Location: Fridge HJ (-20 ºC).

5. Run the electrophoresis at 100V for approximately 2 hours.

Blotting

1. Place three filter papers in transfer buffer and put them at the bottem of the transfer

device.

2. Activate the nitrocellulose membrane by rinsing it in methanol for one minute (in the

hood!). After use, put the methanol in the recycle bottle in the hood.

3. Rinse the nitrocellulose membrane in demiwater.

4. Put the nitrocellulose membrane in transferbuffer.

5. Place the nitrocellulose membrane at the filter papers.

6. Remove the gel from the glass slides and place the gel in the right position at the

nitrocellulose membrane (keep position 1 in mind!).

7. Place three filter papers in transferbuffer and close the system by placing these papers

above the gel.



39

8. Connect the transfer device and set the mA and V at a maximum for 1,5 hours.

9. Remove the gel and clip the membrane at the right kDa if necessary.

Blocken

1. Put the blots in 1x TBST.

2. Block with 5% skim milk in TBST for 1 hour.

Primary antibody

1. Wash blots in TBST for 10 min.

2. Prepare primary antibody solution: 1:1000 antibody in 5% BSA in TBST.

3. Incubate the blots with primary antibody overnight at 4°C.

Secondary antibody

1. Retain primary antibody solution in 15 ml tubes and keep them in -20°C.

2. Wash blots three times in half an hour in 1xTBST.

3. Prepare secondary antibody solution: 1:2000 antibody in 5% skim milk

4. Add secondary antibody and rotate blots for 1 hour.

Developing films

Take care of the levels of liquid in the machine!

1. Wash blots 3x in TBST in 30min.

2. Prepare chemiluminescence solution(5 mg luminol (sigma), 1mL Tris-HCl pH 8.8, 14 mL

H2O, 5µL H2O2 (Emsure), enhancer); add enhancer in the end.

3. Put plastic at the developer sheet and glue them.

4. Put the blots in chemiluminescence solution for 1 minute.

5. Put blots between the plastic layers, and remove air bubbles.

DOKA:

1. Put films (3x) at the blots.

2. Wait for 15 min.

3. Develop with X-ray films .

4. Wash blots in TBST en store them in TBST in the fridge.

Stripping

Only necessary for protein measurements which have the same kDa as proteins detected

before. Wash with stripiing buffer for one hour.

NB. Keep the upper side of the blots up during washing and antibody incubations!

Documents

The ErbB signaling pathway in acute myeloid leukemiascripties.umcg.eldoc.ub.rug.nl/FILES/root/geneeskunde/2013/ScheepersE/... · The ErbB signaling pathway in Acute Myeloid Leukemia