LEBANESE AMERICAN UNIVERSITY

Targeting the MAP Kinase pathway in Human Acute Myeloid

Leukemia Cells using a recombinant Anthrax Lethal Toxin

By

Elias Kassab

This work has been published: Cytotoxicity of anthrax lethal toxin to human acute myeloid leukemia cells is nonapoptotic

and dependent on extracellular signal-regulated kinase 1/2 activity. Kassab E, Darwish M, Timsah Z, Liu S, Leppla SH, Frankel AE, Abi-Habib RJ.

Transl Oncol. 2013 Feb;6(1):25-32. Epub 2013 Feb

A thesis

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Molecular Biology

School of Arts and Sciences

May 2013

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Acknowledgment

I would like to express my deepest appreciation to all those who provided me the

patience and strength to complete this thesis. A special gratitude I give to my parents and

family whose encouragement has made this possible.

Furthermore I would also like to acknowledge with sincere gratitude the crucial role

of my supervisor, mentor and close friend Dr. Ralph Abi-Habib to whom I owe most of

what I achieved in my educational life and career.

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“To Pierre & Carmen Kassab I dedicate this thesis”

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Targeting the MAP Kinase pathway in Human Acute Myeloid

Leukemia Cells using a recombinant Anthrax Lethal Toxin

Elias Kassab

Abstract

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway

in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx).

Around 15,000 new case of Acute Myeloid Leukemia are diagnosed each year with a

fatality rate of 65%. The poor prognosis rate is due to the high proliferative and quick

progressive characteristics of AML. However, most AML patients eventually relapse due

to persistence of chemotherapy-resistant blasts in the bone marrow hence the need for

alternative approaches employing novel, more selective mechanisms for targeting AML

blasts. One such approach consists of targeting the mitogen-activated protein (MAP)

kinase (MAPK) pathway in AML cells. LeTx consists of protective antigen (PrAg) and

lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four

molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves

MAPK kinases, inhibiting the MAPK pathway. The MAKP pathway is a conserved

pathway between eukaryotes. Through a wide range of extracellular signals, the MAPK

pathway regulates growth, proliferation, differentiation and death. Its constitutive

activation, particularly the Ras/Raf/MEK1/2/ERK1/2 cascade promotes proliferation and

survival of most human cancer cells.

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We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines

showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific

mitogen-activated protein/extracellular signal–regulated kinase kinase 1/2 (MEK1/2)

inhibitor U0126, indicating that LeTx-induced cell death is mediated through the

MEK1/2–extracellular signal–regulated kinase (ERK1/2) branch of the MAPK pathway.

The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase

inhibitor LY294002. Flow cytometry analysis of MAPK pathway activation revealed

presence of phospho-ERK1/2 only in LeTx-sensitive cells. In this study, we have shown

that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the

MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in

AML cells is non-apoptotic and dependent on phospho-ERK1/2 levels.

Keywords: Mitogen-Activated Protein Kinase, Acute Myeloid Leukemia, Anthrax

Lethal Toxin, ERK1/2, MEK1/2, Cytotoxicity, Flow Cytometry, Western Blot.

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TABLE OF CONTENTS List of Tables…………………………………………………………………………...7

List of Figures………………………………………………………………………….8

Glossary……………………………………………………………………………….9-10

Chapter Page

I- Introduction

1.1. Overview of Acute Myeloid Leukemia 10

1.2. Targeting the MAPK pathway 12

1.3.The MAPK pathway 13

1.4. Anthrax Lethal Toxin (Letx) 15

1.5.Previous work 17

II- Materials and methods

2.1 Expression and purification of PrAg, LF and FP59 18

2.2 Cells and cell lines 19

2.3 Proliferation inhibition assay (cytotoxicity) 20

2.4 Cell cycle analysis 21

2.5 Inhibition Assays 21

2.6 Protein Extraction and Western Blotting 22

2.7 Intracellular Staining and Flow Cytometry Analysis 23

2.8 Analysis of Cell Cytotoxicity 24

III- Results

3.1 Cytotoxicity of Anthrax Lethal Toxin 25

3.2 Cell cycle effect of Anthrax Lethal Toxin 30

3.3 Inhibition of PI3 Kinase 35

3.4 Analysis of MAPK Activation 38

3.5 Analysis of Cell Death 42

IV- Discussion 45

V- Bibliography 50

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LIST OF TABLES

Table Table Title Page

Table 1. Sensitivity of human AML cell lines to LeTx (PrAg/LF), 26

U0126 and the protein synthesis inhibitor PrAg/FP59.

Table 2. Sensitivity of human AML cell lines to LeTx (PrAg/LF) 41

and activation level of the MAPK pathway illustrated by

the ratio of Phospho to total ERK1/2 levels.

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LIST OF FIGURES

Figure Figure Title Page

Figure 1: Schematic overview of the Mitogen-Activated Protein Kinase Pathway. 13

Figure 2: Mechanism of action of Anthrax toxin. 16

Figure 3: Non-Linear regression curves of LeTx (PrAg/LF)

and PrAg/FP59 on human AML cell lines. 29

Figure 4: Cell cycle analysis of AML cell lines following treatment with LeTx 34

Figure 5: Sensitivity of LeTx-resistant AML cell lines to LY294002 alone

and in combination with LeTx 38

Figure 6: Single cell, intracellular staining of Phospho-ERK1/2 in 4 AML cell

lines using flow cytometry. 39

Figure 7: Analysis of the mechanism of LeTx-mediated cytotoxicity in TF1-vRaf

cells using annexin V/PI and active caspase staining. 44

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GLOSSARY

AML Acute myeloid leukemia

LeTx Anthrax lethal toxin

PrAg Protective antigen

LF Lethal Factor

ANTXRs Anthrax toxin receptors

TEM-8 Tumor endothelium marker 8

CMG-2 Capillary Morphogenesis Gene 2

MAPK Mitogen-activated protein kinase

JNK c-Jun NH2-terminal kinase

Gab1 Grb2-associated binder 1

PH Plekstrin Homology

TNF-α Tumor necrosis factor- α

MEK Mitogen-activated protein kinase kinase

ERK Extracellular signal-regulated kinase

SEK1 SAPK-stress-activated protein kinase-/c-Jun NH2-terminal kinase /Erk

kinase

MLK Mixed-lineage kinase

ASK1 Apoptosis signal-regulating kinase 1

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PAI-1 Plasminogen activator inhibitor- 1

MMP Metalloprotease

FP59 Pseudomonas aeruginosa exotoxin A

EF-2 Elongation factor 2

Ras Rat-adeno-sarcoma

RTK Receptor tyrosine kinase

GRB2 Growth factor receptor-bound protein 2

SOS Son of Sevenless

GEF Guanine nucleotide exchange factor

PI3K Phosphatidylinositol 3-kinase

PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate

PDK1 3-phosphoinositide-dependent protein kinase-1

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CHAPTER ONE

INTRODUCTION

1.1 Overview of Acute Myeloid Leukemia

According to The American Cancer Society, the 2013 estimates for Leukemia of all kinds

in the United States are approximately 48,610 newly diagnosed cases with approximately

23,720 deaths. Acute Myeloid Leukemia (AML) is one of the most common leukemias in

adults with an estimated 14,590 newly diagnosed cases in 2013, mainly in adults, and an

estimated 10,370 deaths. AML is usually common in older individuals and rarely

diagnosed in people below the age of 40, with the mean age of 67 for patients diagnosed

with AML. The lifetime risk of acquiring AML in the average man is approximately 1 in

232 while in the average woman is 1 in 278, hence making AML slightly more frequent

in men than women (American Cancer Society, 2013). AML is highly proliferative and

its quick progression with the lack of effective treatment has a very poor prognosis.

According to the World Health Organization (WHO), AML is classified into four

different subtypes according to clinical and genetic factors. On the other hand, the

French-American-British (FAB) organization classifies AML into eight different

subtypes, M0 to M7, depending on the cell type and degree of maturity. Over the past

three decades, new therapeutic schemes have been developed that were successful in

increasing complete remission in patients following combination induction and

consolidation chemotherapy (American Cancer Society, 2013). The widely used

chemotherapeutic agents are “Cytarabine”, “Doxorubicin” and “Mitoxantrone”.

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Radiotherapy is an option for AML patients followed by bone marrow transplant from an

appropriate donor. However, most patients eventually relapse due to persistence of

chemotherapy-resistant blasts in the bone marrow (Bennett JM, 2002). Relapse rates

differ depending on the patient and the AML subtype, the rates range from 33% to 78%

with an average of 50%. Such patients with refractory and relapsed AML, unfortunately,

have no effective therapeutic options, with the disease remaining incurable in most

(Bennett JM, 2002). Hence, alternative approaches employing novel, more selective

mechanisms for targeting AML blasts are being sought. One such approach consists of

targeting the mitogen-activated protein (MAP) kinase (MAPK) pathway in AML cells

using a recombinant anthrax lethal toxin (LeTx) (Davies SP, 1999) (Koo HM, 2002)

(Abi-Habib RJ U. J., 2005).

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1.2 Targeting the MAPK pathway

The potential for targeting the MAPK pathway in AML has not been fully investigated

yet. A number of studies have been carried out in the past years investigating the role

played by the MAPK pathway in AML cell lines and in blasts from AML patient with

very few studies attempting to target the MAPK pathway in AML. In one study, Non-

steroidal anti-inflammatory drugs (NSAIDs), were analyzed and proved to induce

apoptosis in both AML cell lines and patient samples through the constitutive activation

of c-Jun N-terminal kinases (JNKs), which are part of the MAPK family (Singh R, 2011).

Another study conducted showed the important role of the MAPK pathway in blocking

the oxidative stress induced senescence in AML (Xiao Y, 2012). Nargenicin, a polyketide

antibiotic, was demonstrated as a possible treatment of neoplastic diseases, through its

role in enhancing leukemia cell differentiation by targeting the

PKCbeta1/MAPK pathways (Kim SH, 2009).

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1.3 The MAPK pathway

The MAPK pathway is a highly conserved signaling pathway among all eukaryotes; it

plays a key role in the transduction of a wide range of extracellular signals. Such signals

have a critical role in regulating growth, proliferation, differentiation and apoptosis in

eukaryotic cells. Constitutive activation of the MAPK pathway leads to cell proliferation

and survival in most human cancers (Abi-Habib RJ U. J., 2005) (Wellbrock C, 2004).

Figure 1. Schematic overview of the Mitogen-Activated Protein Kinase Pathway (AS

Dhillon, 2007).

As depicted in figure 1, a three tier kinase module makes up the MAPK pathway where

the phosphorylation of one (MAPKKK), is activated and in turn phosphorylates its

downstream processor, therefore activating the pathway. Specific abnormalities and

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deviations in the MAPK signaling pathway enhance the development and progression of

cancer cells through the acquirement of capabilities such as: liberty of proliferation

signals, apoptotic evasion, lack of sensitivity to anti-growth signals, infinite replication

potential, angiogenis, invasion, metastasis, drug resistance and resistance to oncogene

induced senescence. As seen in figure 1, activated Raf will, through phosphorylation,

activate MEK1 and MEK2. There exists three different isoforrms that differ in their

abilities to activate both MEK1/2. Raf-1 is one of the Raf isoforms that efficiently

activates both MEK1/2 equally. Once ERK1/2 is activated by MEK, it phosphorylates

several cytoplasmic and nuclear targets such as kinases, phosphatases, cytoskeletal

proteins and transcription factors. These ERK signals will in-turn regulate the processes

related to cell development and progression discussed earlier. Mutations leading to the

constitutive activation of the Ras-Raf-MEK1/2-ERK1/2 pathway are a hallmark of

several tumors such as melanoma and are found in a variety of cancers, including AML,

thus constituting an attractive target for novel AML therapies (Lee JT Jr, 2002) (Hilger

RA, 2002) (Wu, 2004) (Milella M K. S., 2001) (AS Dhillon, 2007).

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1.4 Anthrax Lethal Toxin

LeTx is a binary toxin produced by the gram positive bacterium Bacillus anthracis and

consisting of two separate proteins: the cell binding and internalization moiety protective

antigen (PrAg) and the catalytic moiety lethal factor (LF) (Bradley KA M. J., 2001)

(Scobie HM, 2003). PrAg binds to cells through its ubiquitously expressed cell surface

receptors tumor endothelial marker-8 (TEM-8) and capillary morphogenesis gene-2

(CMG-2), collectively referred to as anthrax toxin receptors (ANTXRs). Both TEM-8

and CMG-2 show a high degree of similarity; they are ubiquitously expressed type-1

transmembrane proteins, with each having an ectodomain (extracellular domain), a

single-pass transmembrane domain and a cytoplasmic domain. The gene Cmg2 is

expressed all over the human body with the exception of the human brain, however the

exact role Cmg2 is still largely unknown. TEM-8, being a tumor marker on one hand,

might also play a role in angiogenesis since both Tem8 and Cmg2 genes have been found

to be upregulated during angiogenesis (Deuquet J, 2012).

Following binding to its receptors, PrAg is cleaved at the sequence, 164RKKR167, by

cell surface furin-like proteases leading to the release of a 20-kDa fragment and the

generation of an active 63-kDa fragment (PrAg63) (Abi-Habib RJ U. J., 2005) (Abrami L

L. S., 2003). The latter then forms oligomers, bind 3-4 molecules of LF and undergo

receptor-mediated endocytosis (Abrami L L. S., 2006).Upon acidification of the

endosome, PrAg63 oligomers undergo a conformational change leading to a cation-

dependent pore formation and allowing LF to translocate into the cytosol (Melnyk PA,

2006) . LF is a zinc metalloprotease that cleaves and inactivates all mitogen-activated

protein/extracellular regulated kinase kinases (MEKs), leading to the inhibition of the

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MAPK pathway and subsequent growth inhibition and cell death. (Duesbery NS, 1998)

(Chopra AP, 2003) (Abi-Habib RJ S. R., 2006)

Figure 2. Mechanism of action of Anthrax Toxin (Moayeri, 2004)

We have previously shown that cytotoxicity of LeTx to human melanoma cell lines

carrying the V600E BRAF mutation is dependent on the activation status of the MAPK

pathway, particularly MEK1/2, allowing the use of this toxin for the selective targeting of

tumors with constitutive MAPK activation (Koo HM, 2002) (Abi-Habib RJ U. J., 2005)

(Wu, 2004) (Milella M K. S., 2001) (Abi-Habib RJ S. R., 2006)

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1.5 Previous work

We and others have previously demonstrated the potency and selectivity of LeTx to

melanoma cell lines in vitro and in an in vivo melanoma model (Abi-Habib RJ U. J.,

2005) (Koo HM, 2002). However, unlike melanoma, in which the importance of N-Ras

and BRAF mutations (found in up to 95% of cases) is well established, the importance of

MAPK pathway mutations in AML has been poorly investigated (Zaidi SK, 2009).

Moreover, very few attempts have been made to target the MAPK pathway in AML, with

the exception of targeting the receptor tyrosine kinase FLT3 (fms-like tyrosine kinase) in

AML cells carrying FLT3 mutations (Ricciardi M, 2012) (Konopleva M, 2005). Hence, a

deeper investigation of the role played by the MAPK pathway in human AML cell lines

along with the possibility of selectively targeting AML through the inhibition of the

MAPK pathway are warranted.

In this study, we attempt to target the MAPK pathway in AML cell lines using a

recombinant anthrax lethal toxin and to characterize the response of AML cells to the

LeTx-mediated inhibition of the MAPK pathway.

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CHAPTER TWO

MATERIALS AND METHODS

Expression and purification of PrAg, LF and FP59:

Recombinant anthrax lethal toxin (LeTx) proteins PrAg and LF, as well as FP59 (fusion

of the PrAg binding domain of LF and the catalytic domain of Pseudomonas aeruginosa

exotoxin A) were expressed and purified in the laboratory of Stephen H. Leppla at the

National Institute of Allergies and Infectious Diseases (NIAID) of the National Institutes

of Health (NIH) in Bethesda, MD, as described previously (Ramirez DM, 2002) (Liu S,

2001). Briefly, fermentation was carried out by inoculating a 12 to 14 h old starter culture

grown from a frozen stock. Three to ten liter fermentations were carried out controlling

dissolved oxygen (DO) at 30% saturation, temperature at 37oC, and pH at 7.5. At harvest

time, 5 mM EDTA and 10 ug/ml PMSF (phenylmethyl sulfonyl fluoride) were added to

the culture. Purification was then carried out as follows: 1. Packed-bed hydrophobic

interaction chromatography during which the cell suspension was centrifuged and the

supernatant was passed through a 0.2 -µm hollow fiber filter (AGT, Needham, MA). The

filtered broth was then concentrated 20 fold using a 10K membrane. After sample

loading, the column was washed with 10 column volumes (CV) of equilibration buffer

and rPA was eluted with a 30 CV linear gradient of (NH4)2SO4 in 10 mM HEPES, 5

mM EDTA. The rPA-containing fractions were pooled for further purification. 2-

Expanded-bed hydrophobic interaction chromatography in which the cell suspension was

diluted 1:1 with buffer and the diluted cell suspension was loaded upward at 300 cm/ h.

The column was washed in expanded mode with 10 CV of equilibration buffer. Elution

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was performed in packed-bed mode with 8 CV of elution buffer at 100 cm/ h. 3- Anion

exchange chromatography in which fractions from HIC were dialyzed against 20 mM

Tris pH= 8.9/ 5 mM EDTA and loaded on a Q Sepharose Fast Flow (Amersham

Pharmacia Biotech) column. The protein was eluted using a 20 CV linear gradient of

NaCl in the same buffer. rPA-containing fractions were concentrated and dialyzed against

PBS. (Ramirez DM, 2002) (Liu S, 2001)

LY294002 was purchased from Cell signaling Technology (Danvers, MA).

Cells and cell lines:

Human AML cell lines HL60, U937, ML2, Mono-Mac-1, Mono-Mac-6, KG-1, SigM5,

TF1-vRaf, TF1-vSrc and TF1-HaRas were grown in RPMI 1640 medium with 10% heat-

inactivated fetal bovine serum supplemented with penicillin/streptomycin and l-

glutamine. ML1 was grown in Iscove’s modified Dulbecco’s medium with 10% heat-

inactivated fetal bovine serum (FBS) supplemented with penicillin/streptomycin and l-

glutamine (Ramage J, 2003).24

Cells were maintained in a water-jacketed CO2 incubator

at 37oC/5%CO2 and passaged every 2 to 3 days into new flasks containing fresh media.

Human CD34+ progenitor bone marrow blasts (PBMBs) were purchased from (Lonza,

Basel, Switzerland) and grown in StemlineIITM

hematopoietic stem cell expansion

medium (Sigma-Aldrich) supplemented with 10 ng/ml IL-3, 5 ng/ml granulocyte colony

stimulating factor (G-CSF), 5 ng/ml granulocyte macrophage colony stimulating factor

(GMCSF), 10 ng/ml stem cell factor (SCF), 10 µg/ml insulin and 100 µg/ml transferrin

(Sigma-Aldrich), as described previously (Blair A, 1998).

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Proliferation inhibition assay (cytotoxicity):

Sensitivity of AML cell lines and CD34+

Progenitor Bone Marrow Blasts (PBMBs) to

LeTx was determined using a proliferation inhibition assay as described previously (Abi-

Habib RJ U. J., 2005) We have also used a recombinant protein, termed FP59, consisting

of the PrAg binding domain of LF fused to the catalytic domain of Pseudomonas

aeruginosa exotoxin A. Binding of FP59 to PrAg and its translocation into the cytosol are

identical to those of LF, however, it does not target the MAPK pathway but rather ADP-

ribosylates elongation factor 2 (EF-2) leading to inhibition of protein synthesis and cell

death. PrAg/FP59 was used as a control for catalytic domain entry into the cytosol of

AML cells. Briefly, aliquots of 104 cells/well, in 100 l cell culture medium, containing a

fixed concentration of 10-9

M LF or FP59, were plated in a flat-bottom 96-well plate

(Corning Inc. Corning, NY). Then, 50 l PrAg in media were added to each well to yield

concentrations ranging from 10-8

to 10-13

M. When LY294002 or U0126 were used, they

were added as described above for PrAg but in concentrations ranging from 10-4

to 10-9

M. Following a 48 h incubation at 37oC/5% CO2, 50 l of XTT cell proliferation reagent

(Roche, Basel, Switzerland) were added to each well and the plates incubated for another

4 h. Absorbance was then read at 450 nm using a microplate reader (Thermo Fisher

Scientific, Waltham, MA). Nominal absorbance and percent maximal absorbance were

plotted against the log of concentration and a non-linear regression with a variable slope

sigmoidal dose-response curve was generated along with IC50 using GraphPad Prism 5

software (GraphPad Software, San Diego, CA). All assays were performed at least twice

with an inter-assay range of 30% or less for IC50.

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Cell cycle analysis:

The impact of LeTx treatment on the cell cycle of AML cells was determined using

Propidium Iodide (PI)-staining on flow cytometry. Briefly, cells incubated with 3

different concentrations of LeTx (10,000, 300 and 4.5 pM) or media alone in flat-bottom

96-well plates (Corning Inc. Corning, NY) for 24 and 48 h at 37oC/5% CO2, were

harvested and fixed in 70% ethanol for a minimum of 24 h, at -20oC. Cells were then

incubated in 500 l PI staining solution (50 µg/ml) for 40 min at 37oC. Samples were

then read on a C6 flow cytometer (BD Accuri, Ann Arbor, MI) and total cell DNA

content was measured on FL2-A. Percent of cells in G0/G1, S and G2/M phase was

determined in control cells and in cells treated with the 3 different concentrations of LeTx

(10,000, 300 and 4.5 pM) following gating for the cell population on width versus

forward scatter.

Inhibition Assays

AML cells were incubated with either the small molecular weight PI3 kinase (PI3K)

inhibitor LY294002 (Cell Signaling Technology, Danvers, MA) alone and in

combination with LeTx or with the small molecular weight MEK1/2 inhibitor U0126

(Cell Signaling Technology, Danvers, MA). Briefly, 104 cells/well were plated in 100 l

of medium in a flat-bottom, 96-well plate. Then 100 l of either medium (control cells)

or medium containing LeTx (10-8

M PrAg/10-9

M LF), LY294002 (20 and 50 µM), U0126

(20 and 50 µM) or a combination of the above were added. Cells were then incubated for

48 h at 37oC/5% CO2 followed by the addition of 50 l of XTT cell proliferation reagent

(Roche, Basel, Switzerland). Cells were incubated for another 4 h and absorbance was

read at 450 nm using a 96-well plate reader (Thermo Fisher Scientific, Waltham, MA).

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Data was analyzed using GraphPad Prism V software (GraphPad Software, San Diego,

CA). The absorbance and the percent absorbance of controls were compared between the

different treatment groups.

Protein Extraction and Western Blotting

10 x 106

cells of each of the 11 cell lines were isolated, centrifuged at 1500 rpm for 5

min. Cells were then re-suspended in Cell Lytic™ Mamalian Cell Lysis Reagent (Sigma-

Aldrich) with 1% protease inhibitor (M221, Protease Inhibitor Cocktails, Amresco, USA)

then centrifuged at 1500 rpm for 5 min. The supernatant was collected and stored at -80

oC. All protein samples used for western blotting were prepared in the same way; 8 l of

each sample were added to 10 l of Protein Loading buffer (ProSieve®ProTrack™ Dual

Color, Lonza, Rockland USA) and 2 l DTT, for a total of 20 l. 10% polyacrylamide

precast 10-well gels (Bio-Rad) were used. 10 l of a Biotinylated protein ladder (Cell

Signaling Technology, Danvers, MA) was loaded in the first well. Bio-Rad Mini-

PROTEAN® Tetra System Electrophoresis apparatus was used for running at 120 Volts

and transfer at 0.25 mA on nitro-cellulose membrane. Running buffer used consisted of

60% glycine, 30% Tris-base and 10 % SDS at a pH of 8.4. The transfer buffer used was

prepared using 20% running buffer with 20% methanol and 60% distilled water. The

membranes were blocked overnight using 5% BSA and 0.1% Tween 20. The membranes

were then incubated for 1 hour with the primary anti-phospho-ERK1/2 (Ser217/221) or

anti-ERK1/2 (Thr202/Tyr204) rabbit monoclonal antibodies (Cell Signaling Technology,

Danvers, MA) in antibody binding buffer, and then washed using a washing buffer (TBS

+ 0.5% Tween 20) three times each for 15 min. After washing, the membranes were

incubated with the secondary FITC-conjugated mouse anti-rabbit polyclonal antibody

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(Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr and then washed three times for 15

min as described before. Development was done using Bio-Rad Molecular Imager®

(ChemiDoc™XRS+, bio-Rad USA) Imaging System. Band intensity was analyzed using

Quantity One® Software (Bio-Rad Laboratories, Inc., ©1998, version 4.6.9.30) and the

ratio of Phospho ERK to ERK was determined.

Intracellular Staining and Flow Cytometry Analysis

Activation of the MEK1/2-ERK1/2 pathway in AML cell lines [untreated and following a

10-hour incubation with LeTx (10-8M PrAg/10-9M LF)] was assessed by determining

the presence or absence of phospho-ERK1/2 using flow cytometry as described

previously.25

Approximately 3x106 cells were fixed in 70% ethanol for 15 min. Cells

were then incubated with a 1/100 dilution of anti-phospho-ERK1/2 (Ser 217/221) rabbit

monoclonal antibodies (Cell Signaling Technology, Danvers, MA) in antibody binding

buffer containing 0.05% Triton-X 100, for 1 h at 37oC, followed by a 30-minute

incubation with a 1/100 dilution of a FITC-conjugated mouse anti-rabbit polyclonal

antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Fixed cells stained only with

FITC-conjugated mouse anti-rabbit polyclonal antibody were used as isotypic control.

Samples were then analyzed using a C6 flow cytometer (BD Accuri, Ann Arbor, MI).

The presence of Phospho-ERK1/2 was analyzed and compared with that of the isotopic

control. Positivity for the presence of phospho-ERK1/2 was determined using the ratio of

fluorescence intensity (RFI) between the mean fluorescence intensity (MFI) of the

stained cells and the MFI of the isotypic control. RFI 2 was considered positive.

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Analysis of Cell Cytotoxicity:

Determination of apoptotic versus non-apoptotic cell death was carried out using an

Annexin V-fluorescin Isothiocyanate (Annexin V-FITC) and Propidium Iodide (PI)

labeled apoptosis/necrosis detection kit (Abcam, Cambridge, MA) and a FITC-

conjugated active caspase inhibitor (ApoStat Apoptosis Detection Kit, R&D Systems,

Abingdon, England) on flow cytometry. Briefly, 104 cells/well were plated in 100 l

media in a flat-bottom, 96-well plate and were incubated with either 100 l of medium

alone (control cells) or medium containing three different concentrations of LeTx (as

described above under cell cycle analysis) for 24 and 48 h at 37oC/5% CO2. Cells were

then harvested and incubated with a FITC-conjugated annexin V antibody (2.5 mg/ml

and PI (5 mg/ml) in antibody binding buffer for 45 min at 37oC or incubated with 0.5

µg/ml of apostat for 30 min then harvested. Cells were then read using a C6 flow

cytometer. Annexin V/PI data was analyzed on FL1-H versus FL2-H scatter plot and

active caspases were detected on FL1-H. Unstained cells were used as negative control.

Cells had to show positive annexin V staining, negative PI staining and positive active

caspase staining to be considered apoptotic, while cells positive for both annexin V and

PI staining and negative for active caspase staining were considered non-apoptotic.

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CHAPTER THREE

RESULTS

Cytotoxicity of Anthrax Lethal Toxin:

We tested the cytotoxicity of anthrax lethal toxin (LeTx) on a panel of 11 human acute

myeloid leukemia (AML) cell lines using an XTT proliferation inhibition assay. Seven

out of eleven AML cell lines (64%) were sensitive to the LF-mediated inhibition of the

MAPK pathway with IC50 values ranging from 13 to94 pM and percent cell kill at highest

concentration > 75%. The remaining 4 cell lines were not sensitive to the LF-mediated

inhibition of the MAPK pathway with IC50 > 10,000 pM and percent cell kill at highest

concentration ≤ 40% (Table 1, Fig. 3F). Human CD34+ Progenitor Bone Marrow Blasts

(PBMBs), on the other hand, were not sensitive to the LeTx-mediated inhibition of the

MAPK pathways indicating that LeTx selectively targets AML cells while sparing

normal progenitor blasts. To demonstrate that resistance to LeTx was due to resistance to

the LF-mediated inhibition of the MAPK pathway and not to the inability of LF to

translocate into the cytosol of targeted cells, we tested the cytotoxicity of a combination

of PrAg and FP59 to AML cell lines. FP59 is a fusion of the PrAg binding domain of LF

and the catalytic domain of Pseudomonas aeruginosa exotoxin A. Binding to PrAg and

translocation of FP59 into the cytosol are identical to those of LF, however, FP59 does

not target the MAPK pathway but rather ADP-ribosylates elongation factor 2 (EF-2)

leading to the inhibition of protein synthesis and subsequent cell death. The combination

of PrAg and FP59, therefore, induces MAPK-independent cytotoxicity to all cells that

express the anthrax toxin receptors. PrAg/FP59 was cytotoxic to all the AML cell lines

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tested with IC50 = 0.7-18 pM and percent cell death > 90% indicating that differential

sensitivity of AML cells to LeTx is independent of the ability of LF to translocate into the

cytosol (Table 1, Figure 3A-D) and dependent on the addiction of cells to the MAPK

pathway.

Table 1. Sensitivity of human AML cell lines to LeTx (PrAg/LF), U0126 and the protein

synthesis inhibitor PrAg/FP59.

Cells and Cell

lines

LeTx (PrAg/LF)

(IC50;pmol/L)

PrAg/FP59

(IC50;pmol/L)

U0126

(IC50;µmol/L)

AML Cell lines

HL60 13.0 0.7 2.6

TF1-VSrc 15.0 3.0 2.0

TF1-VRaf 16.0 4.0 1.3

Mono-Mac-6 39.0 13.0 2.1

SigM5 40.0 17.0 1.9

ML-2 81.0 7.0 1.5

TF1-HaRas 94.0 0.4 2.7

ML-1 > 10000 6.0 > 1000

U937 > 10000 1.0 > 1000

KG-1 > 10000 1.0 > 1000

Mono-Mac-1 > 10000 2.0 > 1000

Normal Cells

CD34+ BMPBs > 10000 10.0 N/A

N/A: Not available

18

In addition, intracellular staining and flow cytometry analysis of phospho-ERK1/2 in

sensitive cell lines before and after a10h incubation with LeTx revealed the absence of

phospho-ERK1/2 in cells treated with LeTx, compared to non-treated cells, thus

indicating that cytotoxicity of LeTx to AML cells is mediated through the proteolytic

cleavage of MEKs by LF and subsequent inhibition of the MAPK pathway as evidenced

by the absence on phospho-ERK1/2 in LeTx-treated cells (Data not shown). Moreover,

since anthrax lethal toxin inhibits all branches of the MAPK pathway, it is possible that

cytotoxicity of LeTx to AML cell lines is due to the simultaneous inhibition of all three

branches of the MAPK pathway and not only the MEK1/2-ERK1/2 branch. Therefore, in

order to determine the contribution of the MEK1/2-ERK1/2 branch to the cytotoxicity of

LeTx, we tested the sensitivity of our panel of AML cell lines to U0126, the specific

MEK1/2 inhibitor. Cytotoxicity of U0126 mimicked that of LeTx with the same pattern

seen with both LeTx and U0126 across the panel of AML cell lines (Table 1, Figure 3G).

A)

HL60

-14 -13 -12 -11 -10 -9 -8 -70.0

0.5

1.0

1.5

2.0

2.5

PrAg/LF

PrAg/FP59

Log [M]

Ab

so

rba

nc

e

19

B)

TF1-vRaf

-14 -13 -12 -11 -10 -9 -8 -70.0

0.5

1.0

1.5

2.0

2.5

PrAg/FP59

PrAg/LF

Log [M]

Ab

so

rba

nc

e

C)

Mono-Mac-1

-14 -13 -12 -11 -10 -9 -8 -70.0

0.2

0.4

0.6

0.8

1.0

PrAg/FP59

PrAg/LF

Log [M]

Ab

so

rba

nc

e

D)

U937

-14 -13 -12 -11 -10 -9 -8 -70.0

0.5

1.0

1.5

2.0

2.5

PrAg/LF

PrAg/FP59

Log [M]

Ab

so

rba

nc

e

20

E)

CD34+ PBMBs

-14 -13 -12 -11 -10 -9 -8 -70.4

0.6

0.8

1.0

PrAg/FP59

PrAg/LF

Log [M]

Ab

so

rba

nce

F)

LeTx

-14 -13 -12 -11 -10 -9 -8 -70

20

40

60

80

100

120

TF1-vRaf

TF1-HaRas

TF1-vSrc

MonoMac-1

MonoMac-6

U937

HL60

KG-1

ML-2

ML-1

Sig-M5

Log [M]

Perc

en

t C

on

trol

G)

U0126

-10 -9 -8 -7 -6 -5 -4 -30

20

40

60

80

100 TF1-vRaf

HL60

Mono-Mac-1

U937

Log [M]

Pe

rce

nt

Co

ntr

ol

21

Figure 3. Non-Linear regression curves of LeTx (PrAg/LF) (square) and PrAg/FP59

(triangle) on human AML cell lines and normal CD34+ BMPBs. HL60 (A) and TF1-vRaf

(B) cell lines are sensitive to both LeTx and PrAg/FP59. Mono-Mac-1 (C), U937 (D) and

CD34+ BMPBs (E) are only sensitive to PrAg/FP59 but not to PrAg/LF indicating

resistance to the LF-mediated inhibition of the MAPK pathway. F) Compilation of LeTx

non-linear regression curves on all AML cell lines tested. G) Non-linear regression

curves of the specific MEK1/2 inhibitor U0126 on TF1-vRaf (square), HL60 (triangle),

Mono-Mac-1 (inverted triangle) and U937 (diamond) cell lines. The LeTx-sensitive cell

lines TF1-vRaf and HL60 are sensitive to U0126 while the LeTx-resistant cell lines

Mono-Mac-1 and U937 are resistant to U0126.

The four cell lines that were resistant to LeTx-induced cytotoxicity were also resistant to

U0126 (IC50 > 1000 µM and percent cell kill at the highest concentration ≤ 35%), thus

confirming that these cell lines do not rely on the MAPK pathway for survival and are

resistant to both the specific inhibition of MEK1/2 and the inhibition of the entire MAPK

pathway. The seven AML cell lines that were sensitive to LeTx-induced cytotoxicity

were also sensitive to the specific MEK1/2 inhibitor U0126 (IC50 = 1.3-2.7 µM, percent

cell kill at the highest concentration > 60%), thus indicating that cytotoxicity of LeTx is

mediated through the inhibition of the MEK1/2-ERK1/2 branch of the MAPK pathway.

Cell cycle effect of Anthrax Lethal Toxin:

In order to determine whether, in addition to its cytotoxicity, LeTx induces cell cycle

arrest in AML cells, we determined the cell cycle status of our panel of AML cell lines

following 24 and 48 h incubation with three different concentrations of the LeTx. One of

the four AML cell lines that were not sensitive to the cytotoxicity of LeTx (ML1) showed

dose-dependent cell cycle arrest at 24 h (data not shown) and 48 h following treatment

with LeTx, while the other three (U937, Mono-Mac-1 and KG-1) did not show any effect

22

of LeTx treatment on cell cycle (Fig. 4A and B). The fraction of cells in the G0/G1 phase

of ML1 increased from approximately 39% and 34% of the total cell population in

control cells to approximately 74% and 60% of the total cell population following

incubation with 10,000 pM of LeTx for 24 and 48 h, respectively. This was associated

with a corresponding decrease in the percentage of cells in the G2/M phase from

approximately 10% and 19% of the cell population in control cells to approximately 7%

and 8% of the cell population in treated cells at 24 and 48 h, respectively (Fig. 4A).

A)

23

B)

C)

24

D)

E)

25

F)

Figure 4. Cell cycle analysis of AML cell lines following treatment with LeTx. Control

cells are represented in the left panels and cells treated with 10 nM LeTx for 48 h in the

right panels. Cells are gated on width versus forward scatter (R1/R2). Cells in G0/G1 are

gated M2, G2/M are gated M3 and pre-G0/G1 (dead) are gated M4 or M5. One of the cell

lines that did not show a cytotoxic response to LeTx, ML1 (A), did show cell cycle arrest

while the other, U937 (B), did not. Similarly, three of the LeTx-sensitive cell lines ML2

(C), HL60 (D) and TF1-HaRas (E) showed cell cycle arrest in addition to cell death while

another, TF1-vRaf (F), showed complete cell death following treatment.

Furthermore, cell cycle arrest was observed in four out of the seven cell lines that showed

a cytotoxic response to the LF-mediated inhibition of the MAPK pathway (ML2, HL60,

TF1-HaRas and TF1-vSrc) at 24 h (data not shown) and 48 h following treatment with

LeTx. A dose-dependent increase in the percentage of surviving cells in the G0/G1 phase

was observed (42%, 58%, 41% and 40% in control cells versus 54%, 70%, 67% and 54%

in treated cells) at 48 h, in ML2, HL60, TF1-HaRas and TF1-vSrc cells, respectively

(Fig. 4C, D and ). This was associated with a corresponding decrease in the percentage of

cells in the G2/M phase, by approximately 10%, in treated versus control cells in all four

cell lines. The remaining three LeTx-sensitive cell lines [TF1-vRaf (Fig. 4D), SigM5 and

26

Mono-Mac-6], showed complete cell death with more than 90% of cells in the pre-G0/G1

peak and no difference in the percentage of cells in the G0/G1 phase between control

cells and surviving cells at the highest concentration of LeTx at both 24 h (data not

shown) and 48-h incubation. This indicates that, in addition to significant cytotoxicity to

the majority of AML cell lines, LeTx induces cell cycle arrest in a subset of AML cells

(approximately 45%) irrespective of their cytotoxic response to the LF-mediated

inhibition of the MAPK pathway.

Inhibition of PI3 Kinase:

In order to determine whether AML cell lines that are resistant to the LF-mediated

inhibition of the MAPK pathway are susceptible to the inhibition of the PI3K/Akt

pathway, we tested the sensitivity of the LeTx-resistant cell lines (ML1, Mono-Mac-1,

U937 and KG-1) to the small molecular weight PI3K inhibitor LY294002. Incubation

with LY294002 at 20 and 50 µM for 48 h induced significant cytotoxicity in all four cell

lines with cell viability decreasing by approximately 40 to 50% at the 20 M

concentration and 60 to 80% at the 50 M concentration compared to untreated cells (p <

0.05) (Fig. 5A). Co-incubation of these cells with both LeTx (10 nM) and LY294002 (20

and 50 M) led to only a slight decrease in cell viability, approximately 10%, compared

to LY294002 alone. Although the decreases in cell viability observed following co-

incubation with both LeTx and LY294002 were statistically significant for the U937 cell

line at the 20 M concentration, the ML1 cell line at the 50 M concentration and the

Mono-Mac-1 and KG-1 cell lines at both the 20 and 50 M concentrations (P < 0.05),

compared to LY294002 alone, they were not considered functionally significant since co-

27

incubation only accounted for an additional decrease in cell viability of no more than

10% compared to LY294002 alone (Fig. 5A). Moreover, cytotoxicity of the combination

of LY294002 and LeTx was similar to the cytotoxicity of either LeTx alone or LY294002

alone as evidenced by similar non-linear regression curves and similar IC50 values, at 48

h, in a proliferation inhibition assay (Fig. 5 B, C and D).

A)

U937

Contr

ol

LeTx

(10n

M)

M)

LY2940

02 (2

0M

)

LeTx

(10n

M) +

LY (2

0M

)

LY2940

02 (5

0M

)

LeTx

(10n

M) +

LY (5

0

0

20

40

60

80

100

Pe

rce

nt

Con

tro

l

Mono-Mac-1

Contr

ol

LeTx

(10n

M)

M)

LY2940

02 (2

0M

)

LeTx

(10n

M) +

LY (2

0M

)

LY2940

02 (5

0M

)

LeTx

(10n

M) +

LY (5

0

0

20

40

60

80

100P

erc

en

t C

on

tro

l

ML1

Contr

ol

LeTx

(10n

M)

M)

LY2940

02 (2

0M

)

LeTx

(10n

M) +

LY (2

0M

)

LY2940

02 (5

0M

)

LeTx

(10n

M) +

LY (5

0

0

20

40

60

80

100

Pe

rce

nt

Con

tro

l

KG-1

Contr

ol

LeTx

(10n

M)

M)

LY2940

02 (2

0M

)

LeTx

(10n

M) +

LY (2

0M

)

LY2940

02 (5

0M

)

LeTx

(10n

M) +

LY (5

0

0

20

40

60

80

100

Pe

rce

nt

Co

ntr

ol

28

B)

U937

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -30.0

0.5

1.0

1.5

2.0PrAg/LF

LY294002

PrAg/LF+LY294002

Log [M]

Ab

so

rba

nc

e

C)

Mono-Mac-1

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -30.0

0.2

0.4

0.6

0.8

1.0

PrAg/LF

LY294002

PrAg/LF+LY294002

Log [M]

Ab

so

rba

nc

e

D)

TF1-vSrc

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -30.0

0.5

1.0

1.5

2.0

2.5

PrAg/LF

LY294002

PrAg/LF+LY294002

Log [M]

Abs

orb

anc

e

29

Figure 5. Sensitivity of LeTx-resistant AML cell lines to LY294002 alone and in

combination with LeTx. A) LeTx resistant cell lines incubated with LY294002 at 20 and

50 µM, for 48 h were sensitive to both concentrations of LY294002 while co-incubation

with LeTx (10 nM) did not lead to any significant increase in sensitivity cell sensitivity.

Non-linear regression curves of U937 (B), Mono-Mac-1 (C) and TF1-vSrc (D) cells

incubated with LeTx (square), LY294002 (triangle) or a combination of both (inverted

triangle). The combination of LeTx and LY294002 did not lead to increased cytotoxicity

compared to either LeTx or LY294002 alone.

Hence, cell lines that are not sensitive to LeTx are sensitive to the inhibition of the

PI3K/Akt pathway with the simultaneous inhibition of both pathways not resulting in

increased cell cytotoxicity. This shows the existence of two distinct populations of AML

cell lines sensitive to the inhibition of either the MAPK pathway or the PI3K/Akt

pathway with no additive or synergistic effect observed when both pathways are inhibited

simultaneously.

Analysis of MAPK Activation:

We examined the activation level of the MAPK pathway in AML cell lines by

determining the phosphorylation status of phospho-ERK1/2, using both single-cell

intracellular staining on flow cytometry and western blotting. A representative sample of

four AML cell lines was tested using single-cell intracellular staining, while lysates of the

entire panel of AML cell lines were tested by western blot to determine the ratio of

phosphor/total ERK1/2 levels.The presence or absence of phospho-ERK1/2 was linked to

the cells response to the LF-mediated inhibition of the MAPK pathway.

Both of the cell lines that showed a cytotoxic response to the LF-mediated inhibition of

the MAPK pathway (ML-2 and TF1-vRaf) had an active MEK1/2-ERK1/2 pathway as

30

evidenced by the presence of phospho-ERK1/2 using single-cell intracellular staining

(RFI = 2.14 and 2.32 for ML-2 and TF1-vRaf, respectively, compared to isotype control)

(Fig. 6 A and B).

A) B)

C) D)

Figure 6. Single-cell, intracellular staining of Phospho-ERK1/2 in 4 AML cell lines

using flow cytometry. TF1-vRaf (A) and ML-2 (B) cells, which showed a cytotoxic

response to the LF-mediated inhibition of the MAPK pathway, were positive for the

31

presence of phospho-ERK1/2 as evidenced by a ratio of fluorescence intensity (RFI) > 2

between cells stained for phospho-ERK1/2 (grey) and cells incubated with an isotype

control (black). Cells are gated on width versus forward scatter (R2 and R3). ML-1 (C)

and U937 (D) cells, which were resistant to the cytotoxicity of LeTx, were negative for

phospho-ERK1/2 with RFI < 1.5.

On the other hand, the two cell lines that did not show a cytotoxic response to the LF-

mediated inhibition of the MAPK pathway (U937 and ML-1) were not positive for the

presence of phospho-ERK1/2, hence had an inactive MEK1/2-ERK1/2 pathway (RFI =

1.41 and 1.51 for U937 and ML-1, respectively, compared to isotype control) (Fig. 6 C

and D).

Analysis of the ratio of phospho to total ERK1/2 in cell lysates of AML cell lines, an

indicator of the activation level of the Ras-Raf-MEK1/2-ERK1/2 branch of the MAPK

pathway, revealed the presence of a higher ratio of phospho to total ERK1/2 in AML cell

lines sensitive to the LeTx-induced inhibition of the MAPK pathway (mean ratio 0.74)

compared to cells resistant to the inhibition of this pathway (mean ratio 0.49) (Table 2).

32

Table 2. Sensitivity of human AML cell lines to LeTx (PrAg/LF) and activation level of

the MAPK pathway illustrated by the ratio of phospho to total ERK1/2 levels.

Cell line LeTx (PrAg/LF)

(IC50;pmol/L)

Ratio of Phospho to total

Erk1/2

HL60 13.0 1.00

TF1-VSrc 15.0 0.74

TF1-VRaf 16.0 0.73

Mono-Mac-6 39.0 0.70

SigM5 40.0 N/A

ML-2 81.0 0.27

TF1-HaRas 94.0 1.00

ML-1 > 10000 0.31

U937 > 10000 0.54

KG-1 > 10000 N/A

Mono-Mac-1 > 10000 0.62

N/A: Not Available

As illustrated in Table 2, all the LeTX-sensitive AML cell lines, except ML-2, had a ratio

of phospho to total ERK1/2 higher than 0.7 indicating that more than 70% of total

ERK1/2 in those cells is in the phosphorylated form, demonstrating a high level of

activity of the MEK1/2-ERK1/2 branch of the MAPK pathway which correlates with the

sensitivity of those cells to the inhibition of the MAPK pathway. On the other hand,

33

LeTx-resistant cell lines had a relatively lower activation level of the MEK1/2-ERK1/2

branch of the MAPK pathway, as illustrated by lower ratios of phospho to total ERK1/2,

which correlates with their resistance to the inhibition of that pathway.

This indicates that phospho-ERK1/2 levels, which reflect the activation level of the

MAPK pathway, may serve as a useful marker for predicting the sensitivity of AML cells

to the inhibition of the MAPK pathway. However, as illustrated by the low ratio of

phospho to total ERK1/2 in one of the LeTx-sensitive cell lines (ML2, 0.27) and the

relatively high ratio in one of the LeTx-resistant cell lines (Mono-Mac-1, 0.6), the

addiction of a tumor cell to a particular signaling pathway, i.e. its inability to survive its

inhibition, is much more complex than its simple activation level in that cell.

Analysis of Cell Death:

To determine the mechanism of the cell death observed following the LF-mediated

inhibition of the MAPK pathway in AML cells, we tested for caspase activation and

annexin V/PI staining in our panel of AML cell lines following treatment with three

different concentrations of LeTx (10,000, 300 and 4.5 pM) for 24 and 48 h. The four cell

lines that did not show a cytotoxic response following treatment with LeTx in the

proliferation inhibition assay, stained negatively with both PI and Annexin V, confirming

the absence of cell death following LF-mediated inhibition of the MAPK pathway in

these cell lines. On the other hand, in all seven LeTx-sensitive cell lines (TF1-vRaf, TF1-

vSrc, TF1-HaRas, HL60, SigM5, Mono-Mac-6 and ML-2), an increase in the percentage

of cells stained with both annexin V and PI was observed, at both 24 and 48 h, in cells

treated at the highest concentration of LeTx (10 nM) compared to controls, indicating

34

either necrotic or late-stage apoptotic cell death (Fig,7). However, staining for active

caspases revealed a total absence of caspase activation in all AML cell lines tested,

following treatment with LeTx for 24 and 48 h (Fig. 7). The absence of caspase

activation, in addition to the loss of membrane integrity as evidenced by positive PI

staining, indicate that LeTx-induced cytotoxicity in AML cells is mediated through

caspase-independent, non-apoptotic mechanisms.

A)

35

B)

C)

Figure 7. Analysis of the mechanism of LeTx-mediated cytotoxicity in TF1-vRaf cells

using annexin V/PI (A) and active caspase staining (B) and in HL60 cells using annexin

V/PI (C). TF1-vRaf and HL60 cells incubated with 10 nM LeTx for 24 or 48 h (right

panel) stained positively with both annexin V (FL1-H) and PI (FL2-H). Incubation of

LeTx-treated TF1-vRaf cells with a cell permeable, FITC-conjugated active caspase

inhibitor revealed the absence of active caspases following incubation with LeTx (grey).

36

CHAPTER FOUR

DISCUSSION

In this study, we have shown that anthrax lethal toxin (LeTx) is highly cytotoxic to a

majority of human AML cell lines through the LF-mediated inhibition of the MAP kinase

pathway. Limited information exists regarding the importance of the MAPK pathway in

AML cells and the potential targeting of AML through the inhibition of the MAPK

pathway has not been sufficiently investigated so far (Zaidi SK, 2009) (Ricciardi M,

2012) (Konopleva M, 2005). We have demonstrated that differential sensitivity of AML

cell lines to anthrax lethal toxin was not due to the inability of the LF moiety to

translocate into the cytosol, but rather to the differential sensitivity of AML cell lines to

the LF-mediated inhibition of the MAP kinase pathway. When we replaced the catalytic

domain of LF with the catalytic domain of Pseudomonas aeruginosa exotoxin A, which

inhibits protein synthesis rather than the MAPK pathway, differential sensitivity was lost

and all AML cell lines tested were sensitive to the combination of PrAg and FP59,

demonstrating that differential sensitivity of AML cells to LeTx was due to the

differential response of AML cell lines to the LF-mediated inhibition of the MAPK

pathway. In addition, we have shown that the pattern of cytotoxicity of LeTx on our

panel of AML cell lines was mimicked by the specific, small molecular weight MEK1/2

inhibitor U0126, indicating that cytotoxicity of LeTx is mainly mediated through the

inhibition of the MEK1/2-ERK1/2 branch of the MAPK pathway. Moreover, we have

shown that incubation of AML cells with LeTx leads to a marked inhibition of the

MEK1/2-ERK/12 branch of the MAPK pathway as evidenced by the absence of

37

phosphorylated ERK1/2 in cells incubated with LeTx for 10h as compared to non-treated

cells.

The four AML cell lines that proved to be resistant to the inhibition of the MAPK

pathway by LeTx were sensitive to the inhibition of the PI3K/AKT pathway by

LY294002, a small molecular weight PI3 kinase inhibitor. Interestingly, co-incubating

AML cell lines with both LeTx and LY294002 did not show any significant increase in

cytotoxicity compared to LY294002 alone. Therefore, the panel of AML cell lines tested

consisted of two distinct populations, one sensitive to the inhibition of the MAPK

pathway, the other sensitive to the inhibition of the PI3/AKT pathway with the

simultaneous inhibition of both pathways not leading to any additive or synergistic

effects. These findings are surprising since the inhibition of both the MEK1/2-ERK1/2

pathway and the PI3K/Akt pathway has been shown to have additive or synergistic

effects in a number of tumor types, including hepatocellular carcinoma, colorectal cancer,

pancreatic adenocarcinoma and melanoma (Shimizu T, 2012) (Gedaly R, 2012)

(Haagensen EJ, 2012) (Williams TM, 2012) Moreover, it has been shown that resistance

to MEK inhibitors is mediated through the activation of the PI3k/Akt pathway in a

number of tumors (Wee S, 2009).30

Inhibition of both the MEK1/2-ERK1/2 pathway and

the PI3K/Akt pathway in AML cells has not been investigated to date. Our results

demonstrate that AML cells are sensitive to the inhibition of either the MEK1/2-ERK1/2

pathway or the PI3K/Akt pathway with no additive or synergistic effect for the

simultaneous inhibition of both pathways.

In addition to its cytotoxic effect on a majority of AML cell lines, we have shown that

LeTx also induces cell cycle arrest in a large subset of cell lines, independently of their

38

sensitivity to the cytotoxic effects of the LF-mediated inhibition of the MAPK pathway.

One of the four cell lines that did not show a cytotoxic response to LeTx, in addition to

four of the seven cell lines that did show a cytotoxic response had an increase in the

fraction of cells in the G0/G1 phase along with a decrease in the fraction of cells in the

G2/M and S phases in surviving cells at the highest concentration compared to controls.

We then determined the activity of the Ras-Raf-MEK1/2-ERK1/2 pathway in AML cell

lines by assessing phospho-ERK1/2 levels using single cell intracellular staining and

comparing it to the cytotoxic response of these cells to the LF-mediated inhibition of the

MAPK pathway. Cell lines in which the LF-mediated inhibition of the MAPK pathway

induced cytotoxicity were positive for phospho-ERK1/2 indicating an active Ras-Raf-

MEK1/2-ERK1/2 pathway while cell lines in which the inhibition of this pathway did not

induce cytotoxicity were negative for phospho-ERK1/2. This indicates that sensitivity of

AML cells to the LF-mediated inhibition of the MAPK pathway is determined by the

activity of the MAPK pathway and that phospho-ERK1/2 levels may be used as a marker

to predict sensitivity of AML cells to the inhibition of the MAPK pathway. Predicting a

cells response to the inhibition of a signaling pathway, such as the MAPK pathway, may

be more complicated than a simple determination of the activity of the pathway, however,

in the absence of alternatives, this remains the only available means of assessing the

importance of a particular pathway to the survival of cells.

Analysis of the mechanism of cell death in AML cell lines following LF-mediated

inhibition of the MAPK pathway revealed that LeTx induces caspase-independent, non-

apoptotic cell death in AML cells. Cells treated with LeTx for 24 h were positive for both

Annexin V and PI while being negative for the presence of active caspases. Anthrax

39

lethal toxin has been shown to induce caspase-dependent, apoptotic cell death in a

number of tumor types, including melanoma (Koo HM, 2002) However, LeTx-mediated

inhibition of the MAPK pathway has not been investigated in AML cells and the type of

cell death induced by the LF-mediated inhibition of this pathway may be dependent on

the tumor type. Furthermore, though some studies have shown that Ras inhibitors and

MEK inhibitors do induce apoptosis in AML cell lines, the non-apoptotic cell death

induced by LeTx in AML cells is not surprising knowing the different mechanism of

action of LeTx which catalytically cleaves all MEKs leading to the complete inhibition of

all branches of the MAPK pathway (James JA, 2003) (Milella M E. Z., 2002) (Morgan

MA, 2001).

In this study we have shown that a majority of AML cell lines are sensitive to the LF-

mediated inhibition of the MAPK pathway, hence confirming the potential for selectively

targeting this pathway in AML. Furthermore, we have demonstrated that LeTx-induced

cytotoxicity in AML cells, following inhibition of the MAPK pathway, is non-apoptotic

and is dependent on phospho-ERK1/2 levels in targeted cells.

40

Grant Support:

This work was supported in part by the Intramural Research Program of the National

Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA.

41

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