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University of Southern California Dissertations and Theses

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Integrin mediated cellular adhesion may alter the cytokine profile in acute lymphoblastic leukemia

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Integrin mediated cellular adhesion may alter the cytokine profile in acute lymphoblastic leukemia

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Integrin
Mediated
Cellular
Adhesion
May
Alter
the
Cytokine
Profile
in
Acute

Lymphoblastic
Leukemia

by

Andrea
Osborne

A
Thesis
Presented
to
the

FACULTY
OF
THE
GRADUATE
SCHOOL

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

MASTER
OF
SCIENCE

(EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY)

August
2015

Copyright
2015

Andrea
Osborne

ii

TABLE OF CONTENTS
Acknowledgements: iv
List of Figures v
List of Tables vi
Abstract vii
Chapter 1: Introduction 1
1.1 Acute lymphoblastic leukemia 1
1.2 Bone Marrow Microenvironment: Hematopoietic Cell Niche 4
1.2.1 Endosteal/Osteoblastic Niche 5
1.2.2 Vascular Niche 6
1.2.3 Stromal Niche 7
1.3 Environment Mediated Drug Resistance 7
1.3.1 Cell Adhesion Mediated Drug Resistance 8
1.3.1.1 Integrins 10
1.3.1.2 Integrin and Chemoresistance 11
1.3.1.3 Integrin alpha4 12
1.3.1.4 Integrin alpha6 13
1.3.2 Soluble Factor Mediated Drug Resistance 14
1.3.2.1 Cytokines 15
1.3.2.2 Adipokines 17
1.3.2.3 Adiponectin 18
1.4 Basis For Thesis Project 19
1.5 Thesis Project Summary 20

iii

Chapter 2: Experimental Methods 22
2.1 Cell Culture 22
2.2 MACS Cell Sorting 23
2.3 Trypan Blue Cell Viability Test 23
2.4 Flow Cytometry 23
2.5 ELISA 24
2.6 Western Blot 25
2.7 Statistical Analysis 25
Chapter 3: Results 26
3.1 Leukemia Cell Expression of Integrin Alpha6 26
3.2 Integrin alpha6 blockade sensitizes ALL cells to chemotherapy 27
3.3 Determination of the cytokine expression profile in a
model of integrin alpha6 mediated drug resistance 32
Chapter 4: Discussion and Future Directions 36
References 41

iv

Acknowledgements
First, I would like to thank my advisor in the Hematology/Oncology Department at
Children’s Hospital Los Angeles, Dr. Yong-Mi Kim. Dr. Kim has continually guided me in my
development throughout this master’s thesis program. I truly appreciate the time I’ve had in her
lab and was able to learn so much in such a short period of time. I would like to thank Dr. Kim’s
NIH R01CA172896 grant for financially supporting my research within the lab, as well.
I would also like to thank all of the members in Dr. Kim’s lab for their help, support and
guidance: Enzi Jiang, Stephanie Shishido, Yann Duchartre, Hye-na Kim, Sandra Gonzalez,
Osanna Kosoyan, Sajad Khazal, and Alexa Velasquez. Completing my thesis would not have
been possible if it were not for everyone’s constant willingness to help educate me further. The
atmosphere in Dr. Kim’s lab was extremely supportive, fun and helpful. I want to sincerely thank
you all for answering all my questions, helping when you could and being overall remarkable
people to work with.
I would like to thank my committee members, Dr. Louis Dubeau and Dr. Florence
Hofman, for their guidance, time and support throughout my thesis defending process. I would
also like to thank all the people of the Department of Pathology, especially Lisa Doumak for all
her scheduling and procedural help.
Finally, I would like to thank my incredibly supportive family: specifically my parents,
Traci and Kenneth, and my two younger sisters, Hali and Ashlee. Thank you for all your
constant love, praise and encouragement. Without you all, I would not be the person I am today
and I strive to make you all very proud with all my current and future endeavors.

v

List of Figures
Figure 1: Hematopoietic stem cell niche in the bone marrow 5
Figure 2: Stromal cellular adhesion-mediated chemotherapy resistance (CAM-DR) 9
Figure 3: Soluble factor-mediated drug resistance (SFM-DR) model 15
Figure 4: Integrin alpha6 expression on patient-derived ALL cells
and ALL cell lines 27
Figure 5: Cell viability of Pre-B ALL cells treated short-term with P5G10
and/or chemotherapy 29
Figure 6: Identification of apoptosis of LAX7R cells treated short-term
with P5G10 and/or chemotherapy 30
Figure 7: Flow cytometry post-MACS sorting purity analysis 31
Figure 8: Long-term cell viability of LAX7R cells when sensitized to
chemotherapy through integrin alpha6 de-adhesion 32
Figure 9: Cytokine profile raw luminescent intensity ELISA array 34
Figure 10: Adiponectin and integrin alpha6 expression of Pre-B ALL cells
treated with integrin alpha6 antibody (P5G10) determined by western blot 35

vi

List of Tables
Table 1: Human Pre-B ALL Leukemia Cells Used In This Thesis 4
Table 2: Antibodies used for flow cytometry 24
Table 3: Signosis Multiplex Chemiluminescent Human Cytokine ELISA
Plate Array List of Applicable Pre-Coated Cytokine Capturing Antibodies 33

vii

Abstract
Although survival rates are promising in children with Acute Lymphoblastic Leukemia, in
relapsed childhood ALL, survival rates remain a problem. More than 80% of first relapse of
childhood and adult ALL occurs in the bone marrow. It has been shown that the bone marrow
can promote cell adhesion mediated drug resistance (CAM-DR) in leukemia cells by direct cell-
to-cell interaction. The adhesion of ALL cells to the bone marrow stromal cells can also
stimulate the secretion of soluble factors which leads to soluble factor mediated drug resistance
(SFM-DR). A combination of these two forms of drug resistance is also possible. Previous
studies indicate that by blocking leukemia integrin alpha4 and more recently alpha6 adhesion to
their supportive bone marrow stroma environment, leukemia cells can be sensitized to
chemotherapy. We hypothesized that SFM-DR is also involved in this alpha6-mediated drug
resistance. Here, we show that there is a distinct cytokine profile involved in alpha6-mediated
drug resistance indicating that particular cytokines could play a role in chemoresistance as well.
Taken together, this research attempts to understand the underlying mechanism of drug
resistance in ALL that could lead to novel, more appropriately developed treatments.

viii

1

Chapter 1: Introduction

1.1 Acute lymphoblastic leukemia
Leukemia is a is a cancer of the blood and bone marrow
(1;2)
. The disease can be
classified by how quickly it progresses (acute vs. chronic) and what cells the leukemia
arises from (myeloid vs. lymphoblastic). Myeloid leukemia rises from granulocytes or
monocytes while lymphoblastic leukemia affects lymphocytes. In acute leukemia, cells
that have not developed any immune function (blasts) replicate and their numbers
increases rapidly. This type of disease gets worse quickly, within months or even weeks.
Conversely, chronic leukemia cells become more mature but are not completely
“normal”, they increase less rapidly and they also remain in the bloodstream much longer
then normal white blood cells. This is an important distinction in formulating a prognosis
and treatment strategy for patients because these different types progress very differently
in the patient. In summary, leukemias can be categorized as Acute myeloid leukemia
(AML), Chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) and
Acute lymphocytic leukemia (ALL): with the focus of this study being on acute
lymphoblastic leukemia.
According to the American Cancer Society, in 2015 in the United States there will
be an estimated 6250 new diagnosed cases of ALL and 1450 deaths from ALL (including
children and adults). ALL can affect children and adults however adult patients have a
more negative overall survival
(3)
. ALL is the most common cancer for children and
adolescents. About two-thirds of new diagnoses are in these age groups with peak age
being children age 2-3 years
(4)
. Positively, overall survival in pediatric ALL has improved

2

to roughly 90% in developed countries
(5)
. However, for adults, the survival rate of the
disease is only ~40%
(6)
.
Increases in survival rates for pediatric patients with ALL have improved
significantly because ALL patients can be classified into different risk groups based on
clinical and biological features. Classifying patients based on the characteristics of their
type of leukemia plays a vital role in risk assessment, prognosis and successful therapy
development and implementation. Aside from the previously mentioned classifications of
leukemia, patients with ALL can also be classified based on their karyotype
(7)
. Abnormal
karyotypes that lead to the induction of ALL include aberrant expression of proto-
oncogenes, hyperdiploidy, or chromosomal translocations that create fusion genes
encoding active kinases and altered transcription factors
(2)
. These events alter key
regulatory processes by maintaining or enhancing an unlimited capacity of self renewal,
avoiding the controls of normal proliferation, blocking differentiation and promoting
resistance to death signals
(8)
. Molecular cytogenetic analysis in ALL has led to the
determination of specific nonrandom chromosomal abnormalities of prognostic
importance
(9)
(Table 1). For example, certain translocations such as t(4;11) and t(9;22)
are associated with treatment failure even when using intensive chemotherapy while the
t(12;21), t(1;19) and hyperdiploidy are associated with more favorable outcomes
(10-12)
.
Additionally, frequencies of common genetic lesions in ALL are different
between adult and childhood patients. Hyperdiploidy, more than 50 chromosomes per
leukemic cell and associated with favorable prognosis, occurs in 20% of childhood
patients
(13)
while it only occurs in 7% of adult patients
(2;9)
. The t(12;21) translocation that
leads to the TEL-AML1 fusion gene has been found to be the most common cytogenetic

3

abnormality in B-lineage childhood ALL
(14;15)
occurring in 22% of child patients and in
only 2% of adult patients
(2)
. Presence of the TEL-AML1 gene is also a strong
independent predictor of a favorable prognosis
(16)
. As another example, patients with
BCR-ABL t(9;22) translocation are identified as being Philadelphia chromosome positive
and usually demonstrate higher risk and poor prognosis even with intensive conventional
chemotherapy
(10)
. The BCR-ABL t(9;22) abnormality occurs in about 30% of adults
(10)
while it occurs in about 3% of child patients
(2)
. With respect to the outcome of infants, a
large percentage of infants have a t(4;11) translocation with rearrangement of the MLL
gene and this conveys a poor prognosis
(14)
. These differences demonstrate the importance
of being aware of a patients karyotype at the beginning of treatment in order to determine
the right treatment and get the best results. This is further solidified by a study that has
shown that tyrosine kinase (TK) inhibitors targeting the BCR-ABL1 fusion protein (such
as imatinib or nilotinib) have been used in combination with chemotherapy and result in
significantly higher rates of complete remission and overall survival
(10;17)
. But it is
imperative to uncover these genetic aberrations early on in order to grant the best
outcome possible.
Currently chemotherapy drug resistance remains a significant problem in ALL.
Unfortunately, ALL has been shown to reoccur in ~20% of children and 60% of
adults
(6;18)
. Of the children with recurrent leukemia, only 5-50% survive with current
treatments depending on the site of relapse
(3)
. Even with advances in identifying
reoccurring karyotypes and prognostic factors in ALL patients, about 20% of both child
and adult patients still remain with unknown karyotypes
(2)
and the role neighboring cells
and the leukemia cell environment remains to be fully understood. In order to better

4

understand leukemia, develop or advance techniques to overcome drug resistance and
improve survival and quality of life while reducing adverse effects later
(19)
it is important
that chromosomal alterations and their effect on patients disease progression and the
environment leukemia cells find themselves in be studied thoroughly.

Table 1: Human Pre-B ALL Leukemia Cells Used In This Thesis
1.2 Bone Marrow Microenvironment: Hematopoietic Cell Niche
The bone marrow microenvironment where ALL cells reside is very important for
understanding the mechanism of the cancer and successfully combating it. A thorough
understanding of the components that make up this environment is crucial for further
leukemia research. The primary function of the bone marrow is maintaining the
functional activity of differentiated and undifferentiated hematopoietic cells in the
peripheral blood
(20)
. At steady state, there are specific anatomical regions within the bone
marrow called “niches” for hematopoietic stem cell development as well as normal blood
cell production
(21)
. There are three niches that form the bone marrow microenvironment;
the endosteal niche, the stromal niche and the vascular niche
(22)
. (Figure 1) These niches

5

contribute to the maintenance, survival and fate specifications of normal and malignant
cells
(23)
.

Figure 1: Hematopoietic stem cell niche in the bone marrow: This shows the different
components present within the bone marrow microenvironment that can interact with
leukemia cells and possibly play a role in drug resistance.

1.2.1 Endosteal/Osteoblastic Niche
The endosteal (osteoblastic) niche is the area at the inner surface of the bone
between the bone and bone marrow
(24)
. It is a reservoir of osteoblasts (bone forming
cells) and long-term microenvironment for hematopoietic stem cells that can be
mobilized if there is tissue injury
(24)
. Hematopoietic cell development is tightly regulated
by bone marrow stromal cells, which produce cytokines, growth factors chemokines and
intracellular signals initiated by cellular adhesion to regulate hematopoietic stem cell
proliferation and differentiation
(25)
. Studies have shown that homeostatic interactions with
bone marrow stromal cells within the niche promote the growth and proliferation of

6

hematological malignancies
(26)
. The interaction between SDF-1α and its receptor CXCR4
contributes to their homing to the bone marrow microenvironment
(27)
. For example,
CXCR4 receptor on leukemia progenitor cells is significantly elevated in leukemic cells
of AML patients promoting increased attraction of leukemia cells to bone marrow
(28)
.
Ligation of leukemia cells to stromal cells through integrins within the microenvironment
triggers activation of pro-survival signaling cascades
(25)
, such as phosphorylation of Akt
in a PI3K-dependent manner promotes the survival of leukemia cells
(29)
.
1.2.2 Vascular Niche
The vascular niche is an area rich in blood vessels where the endothelial cells
create a microenvironment that affects the behavior of stem cell and progenitor cells
(23)
.
There is a functional relationship between hematopoiesis and vascular endothelium that
persists from early development into adult life
(30)
.The endothelial cells in the vascular
niche are important for regulating self renewal and the differentiation of lineage-
committed progenitors
(30)
. Endothelial cells can provide a fertile niche that allows for the
expansion of primitive and aggressive leukemic clones
(31)
. Activation of the blood vessel
endothelial cells by angiogenic factors and inflammatory cytokines in circulation or
released by immune cells or leukemia cells switch the vascular niche to promote tumor
growth
(23)
. For example, activation of endothelial cells by vascular endothelial growth
factor (VEGF-A) provides cues that enable leukemic cells to proliferate through an
autocrine mechanism
(32)
at higher rates as well as increasing the adhesion of leukemia
cells to endothelial cells
(31)
.

7

1.2.3 Stromal Niche
The stromal niche is located between the endosteal niche and the vascular niche.
Stromal cells form the bone marrow microenvironment that leukemia cells interact with
most. The stable balance of the stromal niche is influenced by activation of proliferative
signals, loss of anti-growth signals or infiltration of malignant cells
(33;34)
. The stromal
cells can regulate the remodeling and structure of the niche by expressing and secreting a
number of molecules and factors, such as expression of VEGFR-1 or secretion of GM-
CSF
(35)
. Signals such as wnt/B-catenin growth inducing pathway and BMP anti-growth
signal are also involved in regulating the balance between stasis and expansion of the
niche
(33;35)
. Additionally, growth factors, signaling molecules and secreted cytokines are
different in normal bone marrow environments compared to malignant environments
(34)
.
For example, IL-6 expression level is higher in leukemia presenting stromal cells
compared to normal bone marrow stromal cells, which leads to resistance to apoptosis
(26)
.
1.3 Environment Mediated Drug Resistance
Leukemia cell drug resistance can be attributed to decreased drug uptake,
increased drug efflux, repair of DNA damage, drug metabolism changes, or change of
downstream mediators of apoptosis pathway
(36)
. These events can be mediated by
leukemia cell contact to stromal cells within the microenvironment and by secretion of
soluble factors by stromal or immune cells in the microenvironment or leukemia cells
themselves
(37)
. This is referred to as environment-mediated drug resistance (EM-DR)
which is a de novo drug resistance that protects tumor cells from the effects of
chemotherapy, radiotherapy or receptor-mediated cell death
(38)
. Environment mediated-
drug resistance can be divided into two categories: cell adhesion-mediated drug

8

resistance (CAM-DR) and soluble factor-mediated drug resistance (SFM-DR)
(39)
. In both
forms of drug resistance, leukemia cells are protected from apoptosis by activation of
pro-survival pathways or deactivation of apoptosis pathways, allowing the leukemia cells
to proliferate
(40;41)
. For example, the binding of integrins leads to integrin clustering,
subsequently activating focal adhesion kinase which results in the activation of PI3K/Akt
signaling pathway reducing apoptosis and allowing proliferation
(42)
. Also, in epithelial
cells, integrin and EGFR signaling coordinate to prevent anoikis, detachment induced
apoptosis, by down regulating the BH3 domain-only pro-apoptotic protein Bim
(43)
.
1.3.1 Cell Adhesion Mediated Drug Resistance
It has been shown that cell adhesion-mediated drug resistance (CAM-DR) is one
of the most important mechanisms involved in tumor cell microenvironment
protection
(44;45)
. CAM-DR is facilitated largely, but not entirely, by non transcriptional
mechanisms including degradation of activators of apoptosis
(46)
, sub cellular
redistribution
(47)
and increased stability of suppressors of apoptosis and cell cycle
regulators
(48)
. CAM-DR is mediated by the adhesion of tumor cells to stromal fibroblasts
or extracellular matrix components such as laminin or fibronectin
(49;50)
. A possible
mechanism for stromal adhesion mediated chemotherapy resistance is presented in
Figure 2. Drug resistant leukemia cells reside next to bone marrow stromal cells because
research shows ALL cells maintain greatest viability when they are in direct contact with
stromal cell layers
(49)
.
Leukemia cells adhere to stromal cells via adhesion molecules and receptors
present on both cells. Cellular adhesion has the ability to alter the drug target and drug
response, conferring drug resistance. Adhesion molecules participate in the growth,

9

differentiation, survival and homing of hematopoietic cells
(50)
. When drug therapy is
introduced to the system, this adhesion can protect the leukemia cells from drug induced
programmed cell death pathways
(51;52)
. For example, adhesion of AML cells to
fibronectin via B-1 integrins
(46;53)
is associated with CAM-DR. Based on previous data
that integrin alpha4 has a role in drug resistance of leukemia
(54)
we think that integrins
play an important role in CAM-DR specifically in ALL.
Cell adhesion happens in both normal and malignant cell types. Stromal cell
fibronectin and VCAM-1 have been identified as critical molecules for adhesion of
progenitor cells to stromal cells
(55;56)
. Acting similarly to normal cells, B-lineage ALL
cells also express Very Late Antigen-4 (VLA-4) which is able to interact with VCAM-1
and fibronectin on the surface of stromal cells
(57)
. The adhesion molecules on the surface
of bone marrow stromal cells interact with cell surface receptors on the recipient cells by
initiating different signaling cascades within the recipient cells depending on which
pathway is activated
(58)
. For example, beta1 and beta2 integrins can cause activation of
ERK and PI-3K in a Syk-dependent manner as well as activation of NK-kB
(58)
.

Figure 2: Stromal cellular adhesion-mediated chemotherapy resistance (CAM-DR):
Leukemia cells can adhere to bone marrow stromal cells through integrins on the cell
surface. This adhesion then hides the malignant cells in a protective niche from the

10

chemotherapy treatments. Instead of all cells undergoing chemotherapy-induced
apoptosis, the adhesion promotes survival and proliferation of some of the malignant
cells. These successful protected malignant cells can then go on to cause MRD and
relapse.

1.3.1.1 Integrins
Linking cells to extracellular matrix requires transmembrane cell adhesion
proteins that act as matrix receptors
(59)
. The principal receptors on animal cells for
binding extracellular matrix proteins, such as collagens, fibronectin and laminin, and in
some cases cell-cell adhesion, are integrins
(59)
. Integrins are a super family of
heterodimeric trans-membrane glycoproteins consisting of alpha and beta subunits
(60)
.
Twenty-four distinct combinations of integrins can be formed in humans due to the
possible non-covalent linking of 18 alpha and 8 beta subunits
(60)
. Each different
combination preferentially interacts with a type of receptor based on the subunit
properties. For example, alpha4beta1 and alpha5beta1 integrins bind fibronectin, while
alpha3beta1 and alpha6beta1 bind more preferentiality to laminin
(61;62)
.
Roles of integrins include cell-cell or cell-extracellular matrix (ECM) adhesion,
inflammation, cell migration (i.e. lymphocyte trafficking), binding sites for some viruses,
embryonic development, and intracellular signaling regulation
(60;63;64)
. Upon extracellular
ligand binding, integrins function by transducing signals into the cells interior (outside-
in-signaling) or receiving intracellular signals that then regulate their ligand binding
affinity (inside-out-signaling)
(65)
. Variations in the particular structure or charge of the
ligand can strongly influence the binding affinity and the capacity of the integrin to
maintain a conformation that signals back into the cell
(65)
. This implies that ligand
binding can influence allosteric changes in the integrin that then dictate how the integrin
reports on the environment the cell is in. Therefore, integrins serve as sensors of their

11

molecular surroundings
(65)
and then act accordingly to carry out the functions deemed
necessary by the cell or its dynamic environment.
1.3.1.2 Integrins and Chemoresistance
Many types of cells, including leukemia cells, are dependent on proper ECM-
integrin ligation for their prolonged survival
(61;66)
. Conversely, integrins can also
modulate programmed cell death or apoptosis
(61)
. The fact that chemotherapy drugs are
thought to exert their cytotoxic effects partially via induction of apoptosis
(61;67)
yet
integrins are capable of providing survival advantage against death receptor mediated
apoptosis, suggests that integrins can promote cancer immune escape
(47;61;68)
. Recent
research focus has strived to understand integrin pro-survival function in the modulation
of the chemotherapeutic response in cancer cells. A current theory is that a cancer cells’
ability to resist apoptosis through integrins contributes to the development of drug
resistance
(69)
. This could potentially be an important factor in clinical relapse of cancer
patients treated with chemotherapy.
Studies have shown that integrins play a role in chemoresistance in various
hematological malignancies and to several different classes of chemotherapeutic
agents
(39;41;61;64)
. It has also been shown that integrin expression patterns are altered in
tumor cells
(41)
. Also, major constituents of bone marrow include Laminin (8 and 10) and
collagen 4
(70)
suggesting a possible phenomenon of homing and attraction of leukocytes
as well as leukemia cells to this bone marrow environment. Demonstrating this
possibility, the expression of a particular integrin, integrin alpha6 (ITGA6), is
significantly elevated in cells from AML cases
(71)
. Of additional importance, the integrin
dependent survival of leukemic cells in childhood ALL has been linked to the

12

aggressiveness of the disease
(66;72)
. It is therefore important to determine the mechanisms
of integrin mediated survival for potential clinical impact, as leukemia cell’s interaction
with ECM components through integrins could be an important determinant of drug
response
(37)
. Specifically two integrins, integrin alpha4 and integrin alpha6, may play a
key role in the resistance of leukemia cells to apoptosis and chemotherapy and are
therefore worth studying further.
1.3.1.3 Integrin alpha4
Many studies have pointed to integrin alpha4beta1 as a principal ECM receptor
involved in the survival and chemoresistance in myeloid and B lymphoid
malignancies
(61)
. Another name for integrin alpha4beta1 is very late antigen 4 (VLA4).
This integrin is capable of binding the counter receptors of extracellular matrix proteins
fibronectin, VCAM-1, and osteopontin (OPN)
(73-75)
. VLA4 has been shown to regulate
adhesion and homing as well as the engraftment of hematopoietic progenitors
(73;76)
and
ALL cells
(73;77)
in bone marrow. Specifically in AML cells, it has been suggested that the
expression of alpha4 may be an unfavorable risk factor because VLA4 has been shown to
be a dominant adhesion molecule
(45;73)
.
Previous work by my laboratory has shown that integrin alpha4 antagonism using
a humanized monoclonal antibody, clinically used against multiple sclerosis, de-adheres
leukemia cells from their supportive matrix and that this mono treatment leads to
prolongation of murine recipients of T-ALL
(73)
. Previous in vitro and molecular modeling
studies indicate that another non peptide small molecule acts a ligand mimic and
competes with VCAM-1 for the MIDAS site of integrin alpha4
(78)
. This small peptide
can lead to partial de-adhesion of pre-B-ALL cells from its counter receptor VCAM-1
(78)
.

13

Additionally, it has been previously shown that alpha4 is a central molecule for
chemoprotection of pre-B ALL and that alpha4 blockage can sensitize pre-B ALL cells to
chemotherapy
(78;79)
. In vivo, anti-alpha4 integrin treatment results in prolonged survival
of mice while combination treatment results in significantly prolonged survival time
(78;79)
.
Taken together, integrin alpha4 plays a chemoprotective role in both T-cell and B-cell
leukemia adhesion in vitro and in vivo and this suggests alpha4 blockade as a novel target
for therapeutic intervention in these leukemias.
1.3.1.4 Integrin alpha6
Less experimental discovery has been uncovered about integrin α6 and leukemia,
leaving large areas for potential novel ideas involving its role in leukemia and drug
resistance. Similarly to integrin alpha4, integrin alpha6 combines with integrin beta1 to
form VLA-6. The extracellular matrix molecule laminin (specifically LN511) is also an
integrin alpha6 ligand that is essential component of the hematopoietic stem cell niche
and supports their survival independent of cytokines and growth factors
(80)
. It has been
shown that AML cells express VLA-6 on their surface and that these receptors promote
the binding of leukemic blasts to bone marrow stromal cells
(81)
. Also compared to normal
bone marrow precursor cells where VLA-6 is absent, VLA-6 is expressed on greater then
70% of blasts in a subset of leukemias of varied phenotypes
(82)
. Specifically, VLA-6 is
responsible for the adhesion of AML blasts to laminin because antibodies to both the α
and β chains of this molecule were effective at de-adhering the cells from their extra
cellular matrix
(81)
.
The numerous similarities between VLA-6 and VLA-4, can lead us to implore
whether similar phenomenon and circumstances occur with VLA-6 like in VLA-4.

14

Specifically, if de-adhesion of integrin alpha6 expressing leukemia cells can be promoted
by an anti-integrin alpha6 antibody and if this de-adhesion can sensitize the leukemia
cells to chemotherapy. Therefore, research to determine the chemo protective effect of
integrin alpha6 and ways to combat this effect in order to overcome leukemia are
important to discovering possible alternative and more influential mechanisms behind
leukemia acquired drug resistance and relapse.
1.3.2 Soluble Factor Mediated Drug Resistance
Alternatively, soluble factor-mediated drug resistance (SFM-DR) is induced by
cytokines, chemokines and growth factors secreted by either the cancer cell or a cell
within its microenvironment. Interaction of malignant cells with bone marrow supportive
cells can enhance the secretion of growth factors by both cell types, which can increase
resistance to apoptosis
(41)
(Figure 3). The presence of some cytokines then can
significantly affect the result of disease progress
(83)
. Bone marrow stroma derived soluble
factors can provide critical survival cues to leukemia cells, mediate the expansion of
leukemia cells surface or prevent the terminal differentiation of these hematopoietic
cells
(84)
. However, direct contact of leukemia cells with stromal cells inhibits leukemia
cell apoptosis and can maintain viability and proliferation suggesting passage of soluble
factors alone is not enough to block drug resistance
(85)
. Therefore, a reasonable theory is
that bone marrow stroma derived factors could play a part along with cell adhesion in
granting leukemia cells drug resistance.

15

Figure 3: Soluble factor-mediated drug resistance (SFM-DR) model: When stromal
cells come in contact with leukemia cells soluble factors can be produced from either cell
type in a self stimulating cycle. Hematologic malignant cells could secrete these factors
in an attempt to promote their own survival or proliferation. These cytokines could then
stimulate the stromal cells to secrete additional soluble factors, which stimulate the
survival or proliferation of hematologic malignant cells as well. Reversibly, the stromal
cells could secrete soluble factors as a reaction to outside stimuli (including foreign
leukemia cells) present in their environment, which then cause the leukemia cells to
produce factors as well.

1.3.2.1 Cytokines
Cytokines affect nearly every biological process
(86)
. They are small proteins that
play a role in cell-to-cell communication and can be divided into multiple distinct classes
based on their function (for ex. Pro-/anti-inflammatory). While, many forms of
communication in the body occur through direct cell-cell interaction, the secretion of
cytokines enables the rapid propagation of immune signaling in a multifaceted and
efficient manner
(87)
. Cytokines can either be secreted or act as membrane bound proteins
to function as mediators regulating homeostasis
(87)
. Cytokines are an interesting topic of
research for multiple reasons, mainly their ability to exhibit different effects including
pleiotrophy, redundancy, synergy and antagonism. Pleiotrophy, whereby one cytokine

16

can have the ability to act on many different cell types while mediating diverse and
sometimes opposing effects and redundancy, meaning multiple cytokines have the same
and synergistic effect
(87)
renders the cytokine network to be one of the most complicated
regulatory networks to understand. Understanding how cytokines are released or
expressed by leukemia cells or cells within their environment can lead to better
comprehension of how these cells evade drug treatments. Then these properties can be
used to design new therapeutic regimens
(88-90)
.
Altered levels of cytokines and adhesion molecules have been found in many
pathological states and have been linked to many diseases
(91)
. The diverse presence of
cytokines, growth factors, and hormones has been shown to contribute to CAM-DR
phenotypes
(92)
. It is possible that soluble factor-mediated and cellular adhesion-mediated
drug resistance could be related to drug resistance of hematological malignancies in a
unique way for each type of cancer. For example, several growth factors including IL-6,
insulin like growth factor-1 and insulin and fibroblast growth factor are known to
promote myeloma survival as well as contribute to CAM-DR in myeloma
(92)
. It has also
been shown that granulocyte colony stimulating factor (G-CSF) and
granulocyte/macrophage colony stimulating factor (GM-CSF), angiopoietin-1, VEGF,
IL-8, and SDF-1 soluble factors play a role in attracting and sustaining life of leukemia
cells in the bone marrow
(34)
. Therefore, when studying the role of the bone marrow
microenvironment in disease progression and possible drug resistance the interplay
between CAM-DR and SFM-DR could be an important interaction worth uncovering
further.

17

1.3.2.2 Adipokines
A potentially interesting type of cytokine involved in leukemia is adipokines
(adipocytokines). Adipokines are peptides mostly secreted by adipose tissue that include
adiponectin, leptin, visfatin and resistin
(93)
. Adipokines function as circulating hormones
to communicate with other organs such as brain, liver, muscle, the immune system and
adipose tissue itself
(94)
. They are classified as pro- and anti-inflammatory adipokines
based on their effects on inflammatory responses in adipose tissue. Anti-inflammatory
adipokines include adiponectin, vaspin, omentin-1 and secreted frizzled related protein 5;
while pro-inflammatory adipokines include leptin, IL-6, TNF-a, resistin, and retinol
binding protein 4
(94)
. Altered adipokines levels have been observed in a variety of
inflammatory conditions but their pathogenic roles are not completely understood
(95)
.
Some adipokines play a major role in insulin resistance and cardiovascular complications
associated with (central or visceral) obesity
(96)
. Altered levels of these molecules may
underlie the association between obesity and hematological malignancies
(97)
because it
has been shown that obesity is considered to increase the risk of childhood AML
(97)
.
Similar to the bone marrow microenvironment, adipose tissue contains various
cell types such as immune cells, endothelial cells, fibroblasts and most importantly
adipocytes
(98)
. Adipocytes are a major component of the bone marrow microenvironment
and are the main neighboring but least studied cell type during hematological tumor
development
(99)
. They also become more prominent in the bone marrow following
induction of chemotherapy
(100)
. There is already evidence that pre-B ALL cells migrate to
adipose tissue in vivo and adipose tissue can protect leukemia cells from chemotherapy in
vitro
(101)
. Adipocytes have also been shown to contribute to chemoresistance in ALL

18

mice models due to up regulation of survival kinase Pim-2
(102;103)
. With this preceding
documentation it is possible that the cross-talk between adipocytes and cancer cells
through the secretion of different factors leading to SFM-DR could open novel
therapeutic options in leukemia.
1.3.2.3 Adiponectin
Adiponectin is a specific adipokine of interest in this study. Structurally,
adiponectin is composed of a globular and collagenous domain. Human adiponectin
contains 244 amino acid residues and consists of a 20-residue signal sequence
(104)
. The
molecule is similar in sequence to type VIII and type X collagens, complement protein
C1qm and hibernation regulation proteins. While, the 3D globular domain of adiponectin
is structurally similar to TNF-α
(95;105)
. Adiponectin is synthesized as a single polypeptide
of 30 kDa and then forms trimers which oligomerize into polymers of 4/6 trimers in the
endoplasmic reticulum
(106)
. The trimers and polymers are present in circulation and their
secretion from adipose tissue is normally regulated at least in part by SIRT1 (an enzyme
that deacetylates proteins that contribute to insulin sensitive cell regulation) activities
(106)

or insulin levels and the PI3K signaling pathway
(107;108)
.
Adiponectin is a protein hormone that modulates a number of normal and
diseased metabolic processes
(109)
and is produced mainly by fat cells (adipose tissue) of
the human body. Synthesis and secretion of adiponectin is regulated by several
mechanisms. For example, insulin and insulin like growth factor (IGF-1) increase
adiponectin synthesis in adipocytes from human visceral adipose tissue
(104;110)
. It is also
known that the peroxisome proliferator activated receptors (PPAR) are involved in the
regulation of adiponectin synthesis
(104)
. Also, TNF- α, produced by white adipose tissue,

19

is upregulated in obesity, contributes to insulin resistance by interfering with insulin
receptor signaling and suppresses adiponectin secretion in white adipose tissue
(111-113)
.
Additionally, two adiponectin specific receptors have been discovered AdipoR1 and
AdipoR2 that work to facilitate essential physiological functions
(114)
.
Adiponectin effect on human endothelial cells is partially mediated by cyclic
AMP and protein kinase A which suggests that adiponectin acts through cell surface
receptors coupled with adenylate cyclase
(115)
). Adiponectin can also partially exert its
effects through receptors for complement C1q components
(104)
. Additionally, and of
potential future study, adiponectin modulates the glycogen synthase kinase-3/beta-catenin
signaling pathway
(116)
. Adiponectin also plays an important role in the modulation of
glucose and lipid metabolism in insulin sensitive tissues in humans and animals
(105)
. It is
mostly known for its role in the regulation of insulin sensitivity at normally high
circulating concentrations in serum
(95;104)
. It also has anti-inflammatory properties by
reducing the production and activity of TNF-α, inhibiting IL-6 production and inducing
activity of IL-10 and IL-1ra
(95;117;118)
. This activity can be possibly explained by the fact
that adiponectin inhibits nuclear factor kB
(95;119)
. Adiponectin also reduces or down
regulates the expression of extracellular matrix adhesion molecules (ICAM-1 and
VCAM-1)
(95;120;121)
.
1.4 Basis for Thesis Project
To better understand the mechanism behind Acute Lymphoblastic Leukemia and
environment-mediated drug resistance, it is first important to understand the different
components that play a role in this phenomenon. Integrin alpha6 has been implicated in
minimal residual disease (MRD)
(122)
and we have functional data showing its role in drug

20

resistance of ALL in vitro and in vivo (data not shown). Therefore, we investigated SFM-
DR in alpha6-mediated CAM-DR and hypothesized that integrin alpha6 mediated cell
adhesion is also mediated by certain cytokines within the microenvironment. The main
aim of this project is to identify cytokines that are regulated by integrin alpha6. For this
purpose, we compared the cytokine profiles of untreated/control IgG-treated pre-B ALL
cells to P5G10 (anti-integrin alpha6 antibody) treated pre-B ALL cells. Future loss and
gain of function studies would then identify relevant soluble factors involved in survival
of leukemia cells.
This research is relevant because a greater understanding of the complex
mechanisms involved in leukemia onset and subsequent drug resistance is necessary to
develop innovative therapies. Chemotherapeutics need to be designed taking into account
the large impact components of the bone marrow microenvironment have on leukemia
progression and drug resistance
(91)
. Prognostic markers or immunotherapeutic approaches
can then be discovered in hopes to deliver more effective approaches to leukemia
therapy.
1.5 Thesis Project Summary
During my studies I: (1) Demonstrated that integrin alpha6 adhesion blockade
sensitizes acute lymphoblastic leukemia cells to chemotherapy, (2) Determined the
cytokine expression profile in integrin alpha6 mediated drug resistance model of acute
lymphoblastic leukemia and (3) determined the presence of a novel cytokine which may
be implicated in alpha6-regulated adhesion and can be probed for future study.
First, to demonstrate that integrin alpha6 adhesion blockade can sensitize acute
lymphoblastic leukemia cells to chemotherapy, leukemia cells were co-cultured with

21

growth supportive murine stromal cell layers in vitro. Then cells were exposed to either
anti-integrin alpha6 antibody and/or chemotherapy and subsequently cell viability was
determined by Trypan Blue exclusion of dead cells. Flow cytometry was also used to
determine percent apoptotic cells (Annexin V/7AAD) after each treatment. This was
carried out for short and long term experiments.
A chemiluminescent ELISA plate array was used to measure the presence of
specific cytokines present in each sample. As these studies were inconclusive, based on
literature search, a novel cytokine, adiponectin, was chosen and shown to be associated
with integrin alpha6 blockade using P5G10.

22

Chapter 2: Experimental Methods

2.1 Cell Culture
A murine calvaria-derived, adherent mesenchymal stromal cell line (OP9) was
cultured in Gibco
®
Minimum Essential Media (Life Technologies, Carlsbad, CA)
supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S).
Murine stromal cells were used because they have been shown to support patient derived
leukemia cell survival for extended period of time
(123)
. Human patient derived acute
lymphoblastic leukemia cells (LAX7R) collected after the patients’ disease developed
drug resistance and relapsed were maintained in Gibco
®
Minimum Essential Media (Life
Technologies, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS)
(Denville, Metuchen, NJ, USA) and 1% penicillin-streptomycin (P/S) (Invitrogen,
Carlsbad, CA, USA). The cell cultures were maintained in a humidified atmosphere at
5% CO
2
at 37°C.
OP9 stromal cell line was detached from tissue flask using Trypsin 1X 0.25%,
(Life Technologies), irradiated and then plated into 6 or 12 BD Falcon multiwall flat-
bottomed tissue culture plates (BD Biosciences, San Diego, CA). After allowing these
cells adhere, LAX7R ALL cells were added.
The leukemia cells were then treated with anti-integrin α6 antibody (P5G10) (20
mg/ml) its isotype control and/or chemotherapy drug, Vincristine (0.0005 µM).
Vincristine is known as a vinca alkaloid and works by binding to tubulin dimers and
inhibiting assembly of microtubule structures this interferes and arrests mitosis in
metaphase, which eventually leads to cell death. Because cancer cells divide faster than
normal cells, they are more likely than normal cells to be affected by Vincristine.

23

Alternatively, tissue culture treated plates were coated in Laminin (25ug/ml) for at
least one hour at 37C. After incubation, laminin was removed from wells and 1mL
Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco) was used as a washing agent.
Leukemia cells were added to each well (12-well plate) and treatment procedures
followed as previously recorded.
2.2 MACS Cell Sorting
MACS Micro beads were used to separate CD19+ human ALL cells from murine
stromal cells after co-culture (MACS Miltenyl Biotec, San Diego, CA). In brief, cells in a
single cell suspension are magnetically labels with MACS Micro Beads. MACS micro
beads specifically bind to antigens on the cell surface. The sample is then applied to a
MACS column (matrix of ferromagnetic spheres covered in cell friendly coating) placed
in a MACS Separator for separation.
2.3 Trypan Blue Cell Viability Test
Cell viability and proliferation can be determined by Trypan blue (Thermo
scientific, Rockford, IL) cell counts. Statistical significance of differences between
groups is determined by an unpaired t-test.
2.4 Flow Cytometry
Anti-human CD19-FITC, anti-human CD49f-PE and anti-mouse CD106-PE
antibodies along with isotype controls (anti-rat IgG1a and anti-rat IgG2b) and Annexin
V, propidium iodide and 7-amino-actinomycin D (7-AAD) for apoptosis analyses were
obtained from BD Biosciences (San Jose, CA) (Table 2).

24

Table 2: Antibodies used for flow cytometry
2.5 ELISA
Cells were allowed to incubate with drug for either 24 or 72 hours. The cells were
then pipetted vigorously into 15 ml conical tubes and centrifuged at 500 rpc for 5min.
The cell pellet was saved for flow cytometry and western blot procedures. The
supernatant was transferred to a new 15 ml conical tube and centrifuged at 700 rpc for 10
min. Detection of cytokine release in the supernatants of co-cultured ALL cells were
analyzed by a Human Cytokine ELISA Chemiluminescent plate array. The ELISA used
was pre-coated with antibodies against each cytokine to quantitatively measure multiple
cytokines for a few samples through chemiluminescence. The assay utilizes a mouse anti-
human cytokine antibody for immobilization on the micro titer wells and goat anti-human
cytokine antibody along with streptavidin conjugated to horseradish peroxidase (HRP)
for detection. The test sample reacts with the two antibodies, which results in the
molecules of the cytokine being sandwiched between the solid phase and enzyme-linked
antibodies. The enzyme converts a substrate to a reaction product that emits light.
Protocol was followed according to manufacturer (Signosis, Innovative Plate Assay
Solutions).

25

2.6 Western Blot
Cells were lysed in M-PER (Thermo scientific, Rockford, IL) supplemented with
a 1% protease inhibitor cocktail (Pierce, Rockford, IL) and proteins were separated by 4-
12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and
electro transferred to polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA).
The following antibodies were used: anti-adiponectin (EPR3218) (Abcam Cambridge,
MA, USA); anti-β-Actin (AC-15), anti-integrin α6 (4F10) (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). All antibodies were diluted 1:1000 in 5% milk in TBST.
2.7 Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5.0 software or
Microsoft Excel. Statistical analysis of the differences among different treatment
conditions was performed using the unpaired student t-test. P<0.05 was considered
statistically significant.

26

Chapter 3: Results
3.1 Leukemia Cell Expression of Integrin Alpha6
Integrin expression has been shown to be present on human leukocytes and
human hematopoietic stem and progenitor cells
(60;73;79)
. In these cells, integrins regulate
cell homing, adhesion and mobilization. Critically, integrin alpha6 was included as a
recent new marker for detecting MRD+ ALL cells by flow cytometry
(122)
. Therefore, first
the expression of integrin alpha6 on leukemia patient derived cells or cell lines were
determined by flow cytometry. Human antibodies CD19 (FITC) and CD49f (PE) were
used to determine the integrin expression in three pre-B ALL leukemia cell lines (SUP-
B15, NALM-1, and REH) and one patient-derived pre-B ALL cell line (LAX7R) (Figure
4). While hCD19 is a general B-lymphocyte antigen found on the surface of B-cells,
CD49f is known to be an integrin alpha6 marker. This combination allows for the
determination of integrin alpha6 expressing B-cells based on double positive flow
cytometry analysis. Three out of four pre-B ALL cell lines expressed integrin alpha6
strongly (LAX7R: 93.92% - hCD19+, hCD49f+; SUP-B15: 96.10% - hCD19+,
hCD49f+; NALM-1: 68.95% - hCD19+, hCD49f+) but REH has a low expression of
integrin alpha6 (2.73% - hCD19+, hCD49f+).

27

Figure 4: Integrin alpha6 expression on patient-derived ALL cells and ALL cell
lines

3.2 Integrin alpha6 blockades sensitizes ALL cells to Chemotherapy
LAX7R cells were co-cultured with OP9 cells and were exposed to media only,
anti-integrin alpha6 antibody (P5G10) or its isotype control, and/or chemotherapy drugs
for various time points. In a short-term 72-hour assay, (Figure 5A) LAX7R cell viability
(determined by Trypan Blue exclusion staining) decreased when treated with anti-integrin
alpha6 antibody. The anti-integrin alpha6 antibody is capable of de-adhering the LAX7R
cells from their stromal support cells and causes the decreased viability of leukemia cells
compared to media control (85.13 +/- 4.35% vs. 100.51 +/- 1.05%; p=0.004).
Additionally compared to anti-integrin alpha6 antibody treatment alone, leukemia cell
viability significantly decreases when the antibody is combined with chemotherapy
(85.13 +/- 4.35% vs. 30.72 +/- 5.62%; p=0.00019). Also the Trypan blue cell viability
count decreases with combination treatment (30.72 +/- 5.62%) compared to
chemotherapy alone (44.61 +/- 3.33%)(p=0.02), this demonstrates the chemo sensitizing

28

capability of the anti-integrin alpha6 antibody on the leukemia cells in vitro. This
experiment was performed in triplicates and repeated 3 times.
SUP-B15 cells were plated on laminin extracellular matrix support only and were
exposed to media only, anti-integrin alpha6 antibody (P5G10) or its isotype control,
and/or chemotherapy drugs for various time points. In a short-term 72-hour assay,
(Figure 5B) SUP-B15 cell viability (determined by Trypan Blue exclusion staining) was
different then LAX7R cells. The anti-integrin alpha6 antibody is capable of de-adhering
the SUP-B15 cells from the extracellular matrix layer cells but does not cause significant
death of the SUP-B15 cells compared to media control (92.04 +/- 0.023% vs. 95.88 +/-
0.0093%;p=0.1651). However, compared to anti-integrin alpha6 antibody treatment
alone, leukemia cell viability significantly decreases when the antibody is combined with
chemotherapy (92.04 +/- 0.023% vs. 19.925 +/- 0.021%, p=0.00097). Also the Trypan
blue cell viability count decreases with combination treatment (19.925 +/- 0.021%)
compared to chemotherapy alone (38.91 +/- 0.0199%)(p=0.00075), this demonstrates the
chemo sensitizing capability of the anti-integrin alpha6 antibody on the Pre-B ALL cell
lines in vitro. This experiment was performed in triplicates three times.

29

Figure 5: Cell viability of Pre-B ALL cells treated short-term with P5G10 and/or
chemotherapy: patient derived LAX7R cells (A) or ALL Cell Line SUP-B15 cells (B)

Conducting Annexin V/7AAD Flow Cytometry on the cells from each treatment
group can confirm this effect (Figure 6). Annexin V/7AAD staining detects cells in
different stages of apoptosis. Annexin V binds to phosphatidylserine (PS) to identify
apoptotic cells. Upon initiation of apoptosis PS loses its asymmetric distribution on
cytosolic side of plasma membrane and translocates to the extracellular membrane where
it can be detected by the Annexin V label. Also, loss of cell membrane integrity in late
stage apoptosis allows the possible penetration and cellular uptake of 7AAD. Therefore
Annexin V staining paired with 7AAD allows the identification of early stage apoptosis
(Annexin V positive/ 7-AAD negative) and late stage apoptosis (Annexin V positive/7-
AAD positive) by flow cytometry. These results show that anti-integrin alpha6 antibody
is capable of producing an apoptotic effect, although insignificant, in the leukemia cells
after a period of 5 days compared to IgG1 control (18.53 +/- 3.72% vs. 17.18 +/- 1.73%;
p=0.599). More importantly, this FACS analysis also shows that chemotherapy reduces
the cell viability by inducing apoptosis in LAX7R cells compared to IgG1 control (39.94

30

+/- 0.18% vs. 17.18 +/- 1.73%, p=0.00002). The significant increased apoptosis
demonstrated in the combination group (45.54 +/- 1.75%) compared to the chemotherapy
only group (39.94 +/- 0.18%) (p=0.005) shows that the use of antibody preceding
chemotherapy sensitizes these cells to chemotherapy-induced apoptosis even further.
This experiment was performed in triplicates and repeated 3 times.

Figure 6: Identification of apoptosis of LAX7R cells treated short-term with P5G10
and/or chemotherapy. Representative FACS Plot is shown.

After co-culturing ALL cells with their stromal cell support (OP9) for 3 days,
these cells can be separated into two separate cell populations to do further analysis on
the specific protein and intracellular components that come from each cell group. This
study focuses on the changes within the leukemia cells that occur when these cells are
exposed to the different drug treatments, but similar studies in the future could focus on
the stromal components. MACS cell sorting was used to separate the human LAX7R
cells (hCD19+, mCD106-) from the murine stromal cells (hCD19-, mCD106+). The
purity of this separation (98.52 +/- 0.35) was determined by Flow cytometry analysis
using the antibodies previously mentioned (Figure 7).

31

Figure 7: Flow cytometry post-MACS sorting purity analysis
This co culture phenomenon can be expanded from short term to long-term in
vitro experiment when single agent (Vincristine only) chemotherapy is used (Figure 8).
The long term in vitro assay can be split into three different phases of killing, which
simulate the three phases seen within patients undergoing chemotherapy treatment. When
leukemia cells are exposed to anti-integrin alpha6 antibody treatment alone they are
initially killed to a small extent (~75%-80%). Leukemia cells in this antibody only
treatment group then regain viability similar to the media control group for the rest of the
assay (~90%). This regain of cell viability by cells treated only with anti-integrin alpha6
antibody confirms further that this drug functions to block integrin alpha6 adhesion and
de-adhere cells and doesn’t function primarily to kill them. Moreover, Figure 8 shows
leukemia cells are susceptible to chemotherapy treatment, reaching low viability (<20%)
within one week of treatment. This can be referred to as the “killing phase”. The cell
viability curve eventually hits a “plateau phase” when leukemia cells are treated with
chemotherapy. The “plateau phase” is where the leukemia cells retain constant a low
viability with no significant additional increase or decrease. When combination antibody
and chemotherapy are used, the plateau phase can be prolonged when compared to the
chemotherapy alone group. This demonstrates further that when leukemia cells are first
de-adhered from their stromal cell support layer, they are sensitized to the chemotherapy.

32

After cell viability plateau, the leukemia cells in both chemotherapy alone and
combination with antibody begin to regain their viability. This can be characterized as a
drug resistant or “relapse phase” where the leukemia cells have somehow gained an
advantage over the chemotherapy treatment and are resistant to apoptosis. In patients this
similar event can occur when a patient relapses because certain leukemia cells over time
can become resistant to chemotherapy and thrive.

Figure 8: Long-term cell viability of LAX7R cells when sensitized to chemotherapy
through integrin alpha6 de-adhesion: LAX7R cells co-cultured with OP9 stromal cells
demonstrate this 3-phase curve when treated with single chemotherapy agent Vincristine
alone.

3.3 Determination of the cytokine expression profile in a model of integrin alpha6
mediated drug resistance
In order to determine the effects of anti-integrin alpha6 antibody on Acute
Lymphoblastic Leukemia cells cytokine expression profile, RCH cells were plated on
with a laminin support layer as previously described and incubated for a period of 3 days
with isotype control or anti-integrin α6 antibody (P5G10)(20mg/ml). The cytokine
expression from the RCH cells was assessed using a human cytokine array ELISA, which
was previously coated with 32 antibodies (Table 3).

33

Table 3: Signosis Multiplex Chemiluminescent Human Cytokine ELISA Plate Array
List of Applicable Pre-Coated Cytokine Capturing Antibodies

Each experimental sample was loaded into its respective well at the same volume
(100µL) therefore the intensity of the ELISA signal varies for each different cytokine
target based on the concentration of cytokine present in that sample (i.e. more cytokines
produced by the leukemia cells then a more intense/higher signal output). These signals
were then reported in relative light units, which could be quantitatively analyzed for their
up- or down-regulation when exposed to integrin alpha6 antibody. Figure 9 shows the
expression level of the cytokines when Pre-B ALL cells are exposed to integrin alpha6
antibody. From this ELISA data it can be seen that some cytokines increase expression in
the presence of P5G10 compared to the two controls (ex. TGF-b, IL-12, VEGF, IL-17a
and Adipo) while others decrease expression (ex. IFNy, IL-6, Leptin and IL-8). Further
study would be required to determine the exact role of each cytokine.
Then, from this experiment it could be concluded that the increase or decrease of
certain cytokines promotes apoptosis or survival of the leukemia cells. However, the
ELISA results show that this method for detecting secreted cytokines is not sensitive or a
perfect model. This is confirmed by the fact that standard deviations are very large
between each of the four assays and also there are large differences between media and
IgG control conditions in some cases. From these ELISA results, a cytokine of interest

34

could not conclusively be determined to study further. Possible explanations include; the
time frame (3 days) was insufficient to lead to necessary cytokine production, the ELISA
plates are not designed for triplicate set up of multiple conditions, or that 4 plates of 4
different experiments were used.
A B
C D
E
Figure 9: Cytokine profile raw luminescent intensity ELISA array (A) Experiment
#1, (B) Experiment #2, (C) Experiment #3, (D) Experiment #4, (E) Summarized Mean
Values of 3 conditions Media, antibody control IgG and P5G10 treated RCH cells

35

Based on a literature search, we became interested in the cytokine adiponectin, as
it has been implicated in drug resistance
(124;125)
and in integrin regulated drug
resistance
(107;120)
. To further explore the role of adiponectin protein when ALL cells are
treated with integrin alpha6 antibody, western blots were preformed using protein
samples from the same conditions as previously mentioned in the ELISA section.
Treatment with integrin alpha6 antibody demonstrated a down regulation of Adiponectin
protein by western blotting in LAX7R and ICN24 cells (Figure 10). It is possible that
there is a difference in secreted vs. intracellular adiponectin levels. Therefore, further
research into the mechanism by which adiponectin functions is needed.

Figure 10: Adiponectin and integrin alpha6 expression of Pre-B ALL cells treated
with integrin alpha6 antibody (P5G10) determined by western blot

36

Chapter 4: Discussion and Future Directions
Chemotherapeutic drug resistance in ALL remains a significant problem.
Currently, studies about leukemia cells niche have highlighted the importance of
therapeutically targeting the bone marrow microenvironment
(126)
. This area has been
shown to provide chemoprotection for leukemia cells and sheltered cells in the bone
marrow microenvironment are also a frequent source of relapse
(3;5)
.
We have shown that integrin alpha6 mediates cell adhesion to stromal cells and
promotes their survival and drug resistance. Of potential clinical importance, we have
shown integrin alpha6 blockade can sensitize human Pre-B ALL cells to chemotherapy
treatment in vitro. To investigate if SFM-DR is also involved in CAM-DR, we have
found that ELISA is not a method of choice for the experimental assays we have chosen.
There are alternative ways to detect cytokines of in vitro cultured leukemia cells
including real-time PCR and flow cytometry (CyTOF), which can be performed instead
of ELISA. However, it remains to be determined if the sensitivity of these is better than
the ELISA.
Adiponectin has been implicated in integrin-controlled drug resistance of human
prostate cancer
(127)
. Western blot data, which was done only one time for two ALL cases,
for adiponectin shows a decrease in adiponectin protein after the blockade of integrin
alpha6 by the integrin alpha6 antibody (P5G10). Little information has been published
with regard to the relationship between Acute Lymphoblastic Leukemia, adiponectin and
drug resistance and published research to date have proposed conflicting ideas. On one
hand, adiponectin has been shown to be a negative regulator of the immune system and
hematopoiesis
(128;129)
but the ability of adiponectin to inhibit proliferation and induce

37

apoptosis in lymphoid cell lines has not been confirmed
(130)
. Conversely, adipocytes,
which secrete adiponectin, may interact with cancer cells to promote their invasion,
proliferation or drug resistance
(124;131)
. Additionally, serum levels of adiponectin are
increased in obese patients and obesity has been shown to worsen prognosis of child high
risk ALL patients
(132)
. These conflicting reports make adiponectin an innovative research
topic to study further.
The up-regulation or down-regulation of certain cytokines most likely reflects
their function to either promote survival of leukemia cells or cause their death. More in
depth studies on the roles of each cytokine would help to better understand why certain
ones are up-regulated in the presence of P5G10 while others are down-regulated. Western
blot was conducted in order to measure intracellular levels of adiponectin protein as well.
Measurement of extracellular and intracellular cytokine levels has yielded useful
information previously on the pathological process in multiple diseases
(133)
. Therefore, it
is important to confirm the presence extracellular and intracellular adiponectin before any
further conclusive mechanistic studies can be done. Taken together, further studies into
adiponectin and its’ involvement in integrin alpha6 mediated cell adhesion of Acute
Lymphoblastic Leukemia are needed.
As stated previously, adiponectin has been previously related to leukemia
(134)
but
further research is needed to uncover its’ exact role within Acute Lymphoblastic
Leukemia. Adiponectin beneficial effects include its action to increase insulin sensitivity
and its normally anti-inflammatory properties which we now know could possibly be a
negative influence in leukemia
(114)
. Adiponectin has been shown to modulate cell
proliferation and apoptosis in leukemia cells
(135)
. It has been published that adipocytes

38

may interact with cancer cells to promote invasion, proliferation or drug resistance
(124;131)
.
These interactions could be regulated by the release of cytokines, such as adiponectin,
secreted by cancer cells or their environment to stimulate these events. Adiponectin has
previously been reported to be a negative regulator in the immune system and
hematopoiesis
(128;129)
. This is confirmed by the fact that patients with CLL
(136)
and
AML
(125)
have lower adiponectin levels. And correlation studies have revealed that there
are significant negative associations between serum adiponectin levels and bone marrow
blast cells and serum lactic dehydrogenase in acute leukemia patients
(134)
. Also as said
previously, it has been shown that obesity worsens treatment outcome in childhood
patients with high risk ALL
(132)
showing an important over-arching correlation between
adiponectin, adipocytes, leukemia and potential drug resistance. However the relation
between adipocytes, obesity and leukemia still remains elusive and the precise
mechanism of how adipocytes contribute to ALL relapse is completely unknown at this
time. For these reasons, adiponectin has been chosen to probe further for relevance within
integrin alpha6 cell adhesion in ALL.
By determining the mechanism behind why adiponectin as well as other cytokines
are produced and secreted normally and in the presence of integrin alpha6 antibody
treatment it might be possible to further sensitizes leukemia cells to chemotherapy.
Continuing future research projects within the lab will follow up to determine the most
relevant cytokines altered in the presence of integrin alpha6 antibody. This could be done
by alternative detection methods including flow cytometry, RT-PCR, RNA sequencing
and gene expression analysis. From these studies, more consistent and conclusive results
could possibly be drawn to uncover the mechanism by which soluble factors affect ALL

39

cells and their survival. Additionally, once adiponectin or another cytokine are confirmed
to be relevant, gain-of and loss-of function studies can be done in order to expose the
mechanism by which the protein of interest functions. Examples of possible experiments
include knock-out mice, integrin alpha6 deleted mice experiments, protein of interest
over expression studies, or the use of shRNA for gene silencing. Once the actual role of
soluble factors and their reason for increasing or decreasing in the presence of P5G10 are
understood, they could possibly be targeted for therapeutic use. From this research, given
the overall tumor-supporting role of adipocytes and adipokines, targeting these cells and
their products in combination with conventional chemotherapies could be a promising
approach.
Currently, there are already two types of cytokines that have FDA approval for
use as single agents to treat patients with cancer: interferons (IFN) for secondary therapy
of stage 3 melanoma and interleukin-2 (IL-2) for metastatic melanoma and renal cell
carcinoma. It has been shown that IFN can enhance a patient’s immune response to
cancer cells by activating certain white blood cells (natural killer cells and dendritic
cells)
(137)
. IFN-alpha can also inhibit the growth of cancer cells or promote their
death
(138)
. Interleukins can be used to enhance the body’s normal immune response and
its ability to respond to cancer. Of particular note, these cytokines have been approved for
treatment of several hematologic cancers as well
(139-141)
.
Additionally, research is continually being conducted on the benefit of other
cytokines and hematopoietic growth factors in cancer therapy. Some hematopoietic
growth factors have been approved for clinical use in combination with other biological
therapies to strengthen the patient’s own anti-cancer immune response. For example,

40

granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony
stimulating factor (G-CSF) can enhance the immune systems anticancer responses by
increasing the number of white blood cells or by increasing the number of cancer fighting
T cells
(142)
. Additionally, several IL-6 antibodies have been developed in recent years and
evaluated in clinical trials
(143;144)
. These new IL-6 target treatments are exciting because
they not only target the malignant cancer cells but also target the interaction of cancer
cells with their environment
(143)
.
In conclusion, current treatment regimens for leukemia have been developed with
little attention to the role of the microenvironment, bone marrow stromal cells and
soluble factors, in leukaemogenesis. We have found in a pilot assay that blockade of
integrin alpha6-mediated cell adhesion is associated with a decrease in adiponectin.
Further studies are necessary to address if adiponectin is actually involved in SFM-DR.

41

References

(1)

Pui
CH,
Robison
LL,
Look
AT.
Acute
lymphoblastic
leukaemia.
Lancet

2008
Mar
22;371(9617):1030-‐43.

(2)

Pui
CH,
Relling
MV,
Downing
JR.
Acute
lymphoblastic
leukemia.
N
Engl
J

Med
2004
Apr
8;350(15):1535-‐48.

(3)

Gaynon
PS.
Childhood
acute
lymphoblastic
leukaemia
and
relapse.
Br
J

Haematol
2005
Dec;131(5):579-‐87.

(4)

Pui
CH,
Evans
WE.
Acute
lymphoblastic
leukemia.
N
Engl
J
Med
1998
Aug

27;339(9):605-‐15.

(5)

Pulte
D,
Gondos
A,
Brenner
H.
Trends
in
5-‐
and
10-‐year
survival
after

diagnosis
with
childhood
hematologic
malignancies
in
the
United

States,
1990-‐2004.
J
Natl
Cancer
Inst
2008
Sep
17;100(18):1301-‐9.

(6)

Faderl
S,
O'Brien
S,
Pui
CH,
Stock
W,
Wetzler
M,
Hoelzer
D,
et
al.
Adult

acute
lymphoblastic
leukemia:
concepts
and
strategies.
Cancer
2010

Mar
1;116(5):1165-‐76.

(7)

Olde
NL,
Mellink
C,
van
der
SE,
van
den
BH.
Karyotyping,
FISH,
and
PCR

in
acute
lymphoblastic
leukemia:
competing
or
complementary

diagnostics?
J
Pediatr
Hematol
Oncol
2009
Dec;31(12):930-‐5.

(8)

Hanahan
D,
Weinberg
RA.
The
hallmarks
of
cancer.
Cell
2000
Jan

7;100(1):57-‐70.

(9)

Jarosova
M,
Holzerova
M,
Mihal
V,
Lakoma
I,
Divoky
V,
Blazek
B,
et
al.

Complex
karyotypes
in
childhood
acute
lymphoblastic
leukemia:

cytogenetic
and
molecular
cytogenetic
study
of
21
cases.
Cancer
Genet

Cytogenet
2003
Sep;145(2):161-‐8.

(10)

Zhang
Y,
Le
Beau
MM.
Cytogenetics
and
Molecular
genetics
of

myelodysplastic
Syndromes.

7-‐28-‐2015.

Ref
Type:
Generic

(11)

Ribeiro
RC,
Abromowitch
M,
Raimondi
SC,
Murphy
SB,
Behm
F,
Williams

DL.
Clinical
and
biologic
hallmarks
of
the
Philadelphia
chromosome
in

childhood
acute
lymphoblastic
leukemia.
Blood
1987
Oct;70(4):948-‐53.

(12)

Moorman
AV,
Ensor
HM,
Richards
SM,
Chilton
L,
Schwab
C,
Kinsey
SE,
et

al.
Prognostic
effect
of
chromosomal
abnormalities
in
childhood
B-‐cell

precursor
acute
lymphoblastic
leukaemia:
results
from
the
UK
Medical

Research
Council
ALL97/99
randomised
trial.
Lancet
Oncol
2010

May;11(5):429-‐38.

42

(13)

Mullighan
CG.
Molecular
genetics
of
B-‐precursor
acute
lymphoblastic

leukemia.
J
Clin
Invest
2012
Oct;122(10):3407-‐15.

(14)

Friedmann
AM,
Weinstein
HJ.
The
role
of
prognostic
features
in
the

treatment
of
childhood
acute
lymphoblastic
leukemia.
Oncologist

2000;5(4):321-‐8.

(15)

Romana
SP,
Poirel
H,
Leconiat
M,
Flexor
MA,
Mauchauffe
M,
Jonveaux
P,

et
al.
High
frequency
of
t(12;21)
in
childhood
B-‐lineage
acute

lymphoblastic
leukemia.
Blood
1995
Dec
1;86(11):4263-‐9.

(16)

Rubnitz
JE,
Downing
JR,
Pui
CH,
Shurtleff
SA,
Raimondi
SC,
Evans
WE,
et

al.
TEL
gene
rearrangement
in
acute
lymphoblastic
leukemia:
a
new

genetic
marker
with
prognostic
significance.
J
Clin
Oncol
1997

Mar;15(3):1150-‐7.

(17)

Jeha
S,
Coustan-‐Smith
E,
Pei
D,
Sandlund
JT,
Rubnitz
JE,
Howard
SC,
et
al.

Impact
of
tyrosine
kinase
inhibitors
on
minimal
residual
disease
and

outcome
in
childhood
Philadelphia
chromosome-‐positive
acute

lymphoblastic
leukemia.
Cancer
2014
May
15;120(10):1514-‐9.

(18)

Pui
CH,
Evans
WE.
Treatment
of
acute
lymphoblastic
leukemia.
N
Engl
J

Med
2006
Jan
12;354(2):166-‐78.

(19)

Pui
CH,
Mullighan
CG,
Evans
WE,
Relling
MV.
Pediatric
acute

lymphoblastic
leukemia:
where
are
we
going
and
how
do
we
get
there?

Blood
2012
Aug
9;120(6):1165-‐74.

(20)

Schepers
K,
Campbell
TB,
Passegue
E.
Normal
and
Leukemic
Stem
Cell

Niches:
Insights
and
Therapeutic
Opportunities.
Cell
Stem
Cell
2015

Mar
5;16(3):254-‐67.

(21)

Morrison
SJ,
Scadden
DT.
The
bone
marrow
niche
for
haematopoietic

stem
cells.
Nature
2014
Jan
16;505(7483):327-‐34.

(22)

Wilson
A,
Trumpp
A.
Bone-‐marrow
haematopoietic-‐stem-‐cell
niches.

Nat
Rev
Immunol
2006
Feb;6(2):93-‐106.

(23)

Ribatti
D.
Bone
marrow
vascular
niche
and
the
control
of
tumor
growth

in
hematological
malignancies.
Leukemia
2010
Jul;24(7):1247-‐8.

(24)

Kiel
MJ,
Morrison
SJ.
Uncertainty
in
the
niches
that
maintain

haematopoietic
stem
cells.
Nat
Rev
Immunol
2008
Apr;8(4):290-‐301.

(25)

Konopleva
MY,
Jordan
CT.
Leukemia
stem
cells
and
microenvironment:

biology
and
therapeutic
targeting.
J
Clin
Oncol
2011
Feb
10;29(5):591-‐
9.

43

(26)

Nwajei
F,
Konopleva
M.
The
bone
marrow
microenvironment
as
niche

retreats
for
hematopoietic
and
leukemic
stem
cells.
Adv
Hematol

2013;2013:953982.

(27)

Colmone
A,
Amorim
M,
Pontier
AL,
Wang
S,
Jablonski
E,
Sipkins
DA.

Leukemic
cells
create
bone
marrow
niches
that
disrupt
the
behavior
of

normal
hematopoietic
progenitor
cells.
Science
2008
Dec

19;322(5909):1861-‐5.

(28)

Mohle
R,
Schittenhelm
M,
Failenschmid
C,
Bautz
F,
Kratz-‐Albers
K,
Serve

H,
et
al.
Functional
response
of
leukaemic
blasts
to
stromal
cell-‐derived

factor-‐1
correlates
with
preferential
expression
of
the
chemokine

receptor
CXCR4
in
acute
myelomonocytic
and
lymphoblastic
leukaemia.

Br
J
Haematol
2000
Sep;110(3):563-‐72.

(29)

Tabe
Y,
Jin
L,
Tsutsumi-‐Ishii
Y,
Xu
Y,
McQueen
T,
Priebe
W,
et
al.

Activation
of
integrin-‐linked
kinase
is
a
critical
prosurvival
pathway

induced
in
leukemic
cells
by
bone
marrow-‐derived
stromal
cells.

Cancer
Res
2007
Jan
15;67(2):684-‐94.

(30)

Cogle
CR,
Goldman
DC,
Madlambayan
GJ,
Leon
RP,
Al
MA,
Clark
HA,
et
al.

Functional
integration
of
acute
myeloid
leukemia
into
the
vascular

niche.
Leukemia
2014
Oct;28(10):1978-‐87.

(31)

Poulos
MG,
Gars
EJ,
Gutkin
MC,
Kloss
CC,
Ginsberg
M,
Scandura
JM,
et
al.

Activation
of
the
vascular
niche
supports
leukemic
progression
and

resistance
to
chemotherapy.
Exp
Hematol
2014
Nov;42(11):976-‐86.

(32)

Dias
S,
Hattori
K,
Zhu
Z,
Heissig
B,
Choy
M,
Lane
W,
et
al.
Autocrine

stimulation
of
VEGFR-‐2
activates
human
leukemic
cell
growth
and

migration.
J
Clin
Invest
2000
Aug;106(4):511-‐21.

(33)

Li
L,
Neaves
WB.
Normal
stem
cells
and
cancer
stem
cells:
the
niche

matters.
Cancer
Res
2006
May
1;66(9):4553-‐7.

(34)

Azizidoost
S,
Babashah
S,
Rahim
F,
Shahjahani
M,
Saki
N.
Bone
marrow

neoplastic
niche
in
leukemia.
Hematology
2014
Jun;19(4):232-‐8.

(35)

Saki
N,
Abroun
S,
Farshdousti
HM,
Asgharei
F.
Neoplastic
bone
marrow

niche:
hematopoietic
and
mesenchymal
stem
cells.
Cell
J

2011;13(3):131-‐6.

(36)

Hazlehurst
LA,
Landowski
TH,
Dalton
WS.
Role
of
the
tumor

microenvironment
in
mediating
de
novo
resistance
to
drugs
and

physiological
mediators
of
cell
death.
Oncogene
2003
Oct

20;22(47):7396-‐402.

44

(37)

Feldhahn
N,
Arutyunyan
A,
Stoddart
S,
Zhang
B,
Schmidhuber
S,
Yi
SJ,
et

al.
Environment-‐mediated
drug
resistance
in
Bcr/Abl-‐positive
acute

lymphoblastic
leukemia.
Oncoimmunology
2012
Aug
1;1(5):618-‐29.

(38)

Shain
KH,
Dalton
WS.
Environmental-‐mediated
drug
resistance:
a
target

for
multiple
myeloma
therapy.
Expert
Rev
Hematol
2009
Dec;2(6):649-‐
62.

(39)

Meads
MB,
Gatenby
RA,
Dalton
WS.
Environment-‐mediated
drug

resistance:
a
major
contributor
to
minimal
residual
disease.
Nat
Rev

Cancer
2009
Sep;9(9):665-‐74.

(40)

Li
ZW,
Dalton
WS.
Tumor
microenvironment
and
drug
resistance
in

hematologic
malignancies.
Blood
Rev
2006
Nov;20(6):333-‐42.

(41)

Meads
MB,
Hazlehurst
LA,
Dalton
WS.
The
bone
marrow

microenvironment
as
a
tumor
sanctuary
and
contributor
to
drug

resistance.
Clin
Cancer
Res
2008
May
1;14(9):2519-‐26.

(42)

Khwaja
A,
Rodriguez-‐Viciana
P,
Wennstrom
S,
Warne
PH,
Downward
J.

Matrix
adhesion
and
Ras
transformation
both
activate
a

phosphoinositide
3-‐OH
kinase
and
protein
kinase
B/Akt
cellular

survival
pathway.
EMBO
J
1997
May
15;16(10):2783-‐93.

(43)

Reginato
MJ,
Mills
KR,
Paulus
JK,
Lynch
DK,
Sgroi
DC,
Debnath
J,
et
al.

Integrins
and
EGFR
coordinately
regulate
the
pro-‐apoptotic
protein
Bim

to
prevent
anoikis.
Nat
Cell
Biol
2003
Aug;5(8):733-‐40.

(44)

Damiano
JS,
Hazlehurst
LA,
Dalton
WS.
Cell
adhesion-‐mediated
drug

resistance
(CAM-‐DR)
protects
the
K562
chronic
myelogenous
leukemia

cell
line
from
apoptosis
induced
by
BCR/ABL
inhibition,
cytotoxic

drugs,
and
gamma-‐irradiation.
Leukemia
2001
Aug;15(8):1232-‐9.

(45)

Matsunaga
T,
Takemoto
N,
Sato
T,
Takimoto
R,
Tanaka
I,
Fujimi
A,
et
al.

Interaction
between
leukemic-‐cell
VLA-‐4
and
stromal
fibronectin
is
a

decisive
factor
for
minimal
residual
disease
of
acute
myelogenous

leukemia.
Nat
Med
2003
Sep;9(9):1158-‐65.

(46)

Hazlehurst
LA,
Argilagos
RF,
Dalton
WS.
Beta1
integrin
mediated

adhesion
increases
Bim
protein
degradation
and
contributes
to
drug

resistance
in
leukaemia
cells.
Br
J
Haematol
2007
Jan;136(2):269-‐75.

(47)

Shain
KH,
Landowski
TH,
Dalton
WS.
Adhesion-‐mediated
intracellular

redistribution
of
c-‐Fas-‐associated
death
domain-‐like
IL-‐1-‐converting

enzyme-‐like
inhibitory
protein-‐long
confers
resistance
to
CD95-‐induced

apoptosis
in
hematopoietic
cancer
cell
lines.
J
Immunol
2002
Mar

1;168(5):2544-‐53.

45

(48)

Lwin
T,
Hazlehurst
LA,
Dessureault
S,
Lai
R,
Bai
W,
Sotomayor
E,
et
al.

Cell
adhesion
induces
p27Kip1-‐associated
cell-‐cycle
arrest
through

down-‐regulation
of
the
SCFSkp2
ubiquitin
ligase
pathway
in
mantle-‐cell

and
other
non-‐Hodgkin
B-‐cell
lymphomas.
Blood
2007
Sep

1;110(5):1631-‐8.

(49)

Mudry
RE,
Fortney
JE,
York
T,
Hall
BM,
Gibson
LF.
Stromal
cells
regulate

survival
of
B-‐lineage
leukemic
cells
during
chemotherapy.
Blood
2000

Sep
1;96(5):1926-‐32.

(50)

Hazlehurst
LA,
Dalton
WS.
Mechanisms
associated
with
cell
adhesion

mediated
drug
resistance
(CAM-‐DR)
in
hematopoietic
malignancies.

Cancer
Metastasis
Rev
2001;20(1-‐2):43-‐50.

(51)

Iyoda
T,
Fukai
F.
Modulation
of
Tumor
Cell
Survival,
Proliferation,
and

Differentiation
by
the
Peptide
Derived
from
Tenascin-‐C:
Implication
of

beta1-‐Integrin
Activation.
Int
J
Cell
Biol
2012;2012:647594.

(52)

Shain
KH,
Dalton
WS.
Cell
adhesion
is
a
key
determinant
in
de
novo

multidrug
resistance
(MDR):
new
targets
for
the
prevention
of
acquired

MDR.
Mol
Cancer
Ther
2001
Nov;1(1):69-‐78.

(53)

Hazlehurst
LA,
Argilagos
RF,
Emmons
M,
Boulware
D,
Beam
CA,
Sullivan

DM,
et
al.
Cell
adhesion
to
fibronectin
(CAM-‐DR)
influences
acquired

mitoxantrone
resistance
in
U937
cells.
Cancer
Res
2006
Feb

15;66(4):2338-‐45.

(54)

Shishido
S,
Bonig
H,
Kim
YM.
Role
of
integrin
alpha4
in
drug
resistance

of
leukemia.
Front
Oncol
2014;4:99.

(55)

Garcia-‐Gila
M,
Lopez-‐Martin
EM,
Garcia-‐Pardo
A.
Adhesion
to

fibronectin
via
alpha4
integrin
(CD49d)
protects
B
cells
from
apoptosis

induced
by
serum
deprivation
but
not
via
IgM
or
Fas/Apo-‐1
receptors.

Clin
Exp
Immunol
2002
Mar;127(3):455-‐62.

(56)

Wang
L,
Fortney
JE,
Gibson
LF.
Stromal
cell
protection
of
B-‐lineage
acute

lymphoblastic
leukemic
cells
during
chemotherapy
requires
active
Akt.

Leuk
Res
2004
Jul;28(7):733-‐42.

(57)

Sbaa-‐Ketata
E,
Vasse
M,
Lenormand
B,
Schneider
P,
Soria
C,
Vannier
JP.

Fibronectin
increases
the
migration
induced
by
stromal
cell-‐derived

factor-‐1
alpha
(SDF-‐1
alpha)
in
pre-‐B
acute
lymphoblastic
leukemia

cells.
Eur
Cytokine
Netw
2001
Apr;12(2):223-‐30.

(58)

Reyes-‐Reyes
M,
Mora
N,
Gonzalez
G,
Rosales
C.
beta1
and
beta2

integrins
activate
different
signalling
pathways
in
monocytes.
Biochem

J
2002
Apr
15;363(Pt
2):273-‐80.

46

(59)

Alberts
B,
Johnson
A,
Lewis
J,
et
al.
Integrins.
Molecular
Biology
of
the

Cell.
4th
ed.
New
York:
Garland
Science;
2002.

(60)

Hsieh
YT.
ROLE
OF
INTEGRIN
a4
IN
DRUG
RESISTANT

ACUTE
LYMPHOBLASTIC
LEUKEMIA
2013.

(61)

Aoudjit
F,
Vuori
K.
Integrin
signaling
in
cancer
cell
survival
and

chemoresistance.
Chemother
Res
Pract
2012;2012:283181.

(62)

Hynes
RO.
Integrins:
bidirectional,
allosteric
signaling
machines.
Cell

2002
Sep
20;110(6):673-‐87.

(63)

Guo
W,
Giancotti
FG.
Integrin
signalling
during
tumour
progression.
Nat

Rev
Mol
Cell
Biol
2004
Oct;5(10):816-‐26.

(64)

Hehlgans
S,
Haase
M,
Cordes
N.
Signalling
via
integrins:
implications
for

cell
survival
and
anticancer
strategies.
Biochim
Biophys
Acta
2007

Jan;1775(1):163-‐80.

(65)

Takada
Y,
Ye
X,
Simon
S.
The
integrins.
Genome
Biol
2007;8(5):215.

(66)

Astier
AL,
Svoboda
M,
Hinds
E,
De
BR,
Munoz
O,
Freedman
AS.
Integrins

regulate
survival
of
pre-‐B-‐ALL
cells
through
differential
IAP
and

caspase-‐7
ubiquitination
and
degradation.
Leukemia
2004

Apr;18(4):873-‐5.

(67)

Kaufmann
SH,
Earnshaw
WC.
Induction
of
apoptosis
by
cancer

chemotherapy.
Exp
Cell
Res
2000
Apr
10;256(1):42-‐9.

(68)

Gendron
S,
Couture
J,
Aoudjit
F.
Integrin
alpha2beta1
inhibits
Fas-‐
mediated
apoptosis
in
T
lymphocytes
by
protein
phosphatase
2A-‐
dependent
activation
of
the
MAPK/ERK
pathway.
J
Biol
Chem
2003
Dec

5;278(49):48633-‐43.

(69)

Vinogradov
S,
Wei
X.
Cancer
stem
cells
and
drug
resistance:
the

potential
of
nanomedicine.
Nanomedicine
(Lond)
2012
Apr;7(4):597-‐
615.

(70)

Timpl
R.
Macromolecular
organization
of
basement
membranes.
Curr

Opin
Cell
Biol
1996
Oct;8(5):618-‐24.

(71)

Yamakawa
N,
Kaneda
K,
Saito
Y,
Ichihara
E,
Morishita
K.
The
increased

expression
of
integrin
alpha6
(ITGA6)
enhances
drug
resistance
in

EVI1(high)
leukemia.
PLoS
One
2012;7(1):e30706.

(72)

Kumagai
M,
Manabe
A,
Pui
CH,
Behm
FG,
Raimondi
SC,
Hancock
ML,
et

al.
Stroma-‐supported
culture
in
childhood
B-‐lineage
acute

47

lymphoblastic
leukemia
cells
predicts
treatment
outcome.
J
Clin
Invest

1996
Feb
1;97(3):755-‐60.

(73)

Hsieh
YT,
Shishido
S,
Osborne
A,
Garvey
C,
Gang
EJ,
Bonig
H,
et
al.

Blockade
of
Integrin
a4
Mediated
Adhesion
of
T-‐ALL
cells.
Annals
of

Pediatrics
&
Child
Health
2014
May
29;2(2).

(74)

Imai
Y,
Shimaoka
M,
Kurokawa
M.
Essential
roles
of
VLA-‐4
in
the

hematopoietic
system.
Int
J
Hematol
2010
May;91(4):569-‐75.

(75)

Harima
A,
Nakaseko
C,
Yokota
A,
Kitagawa
M,
Morimoto
C,
Harigaya
K,

et
al.
Fibronectin
promotes
cell
proliferation
of
human
pre-‐B
cell
line

via
its
interactions
with
VLA-‐4
and
VLA-‐5.
Hematology
2008

Aug;13(4):236-‐43.

(76)

Scott
LM,
Priestley
GV,
Papayannopoulou
T.
Deletion
of
alpha4
integrins

from
adult
hematopoietic
cells
reveals
roles
in
homeostasis,

regeneration,
and
homing.
Mol
Cell
Biol
2003
Dec;23(24):9349-‐60.

(77)

Filshie
R,
Gottlieb
D,
Bradstock
K.
VLA-‐4
is
involved
in
the
engraftment

of
the
human
pre-‐B
acute
lymphoblastic
leukaemia
cell
line
NALM-‐6
in

SCID
mice.
Br
J
Haematol
1998
Sep;102(5):1292-‐300.

(78)

Hsieh
YT,
Gang
EJ,
Shishido
SN,
Kim
HN,
Pham
J,
Khazal
S,
et
al.
Effects
of

the
small-‐molecule
inhibitor
of
integrin
alpha4,
TBC3486,
on
pre-‐B-‐ALL

cells.
Leukemia
2014
Oct;28(10):2101-‐4.

(79)

Hsieh
YT,
Gang
EJ,
Geng
H,
Park
E,
Huantes
S,
Chudziak
D,
et
al.
Integrin

alpha4
blockade
sensitizes
drug
resistant
pre-‐B
acute
lymphoblastic

leukemia
to
chemotherapy.
Blood
2013
Mar
7;121(10):1814-‐8.

(80)

Landowski
TH,
Gard
J,
Pond
E,
Pond
GD,
Nagle
RB,
Geffre
CP,
et
al.

Targeting
integrin
alpha6
stimulates
curative-‐type
bone
metastasis

lesions
in
a
xenograft
model.
Mol
Cancer
Ther
2014
Jun;13(6):1558-‐66.

(81)

Bendall
LJ,
Kortlepel
K,
Gottlieb
DJ.
Human
acute
myeloid
leukemia

cells
bind
to
bone
marrow
stroma
via
a
combination
of
beta-‐1
and
beta-‐
2
integrin
mechanisms.
Blood
1993
Nov
15;82(10):3125-‐32.

(82)

Reuss-‐Borst
MA,
Klein
G,
Waller
HD,
Muller
CA.
Differential
expression

of
adhesion
molecules
in
acute
leukemia.
Leukemia
1995
May;9(5):869-‐
74.

(83)

Sepehrizadeh
Z,
Mohammadi
M,
Emami
A,
Yazdi
MT,
Bozchlou
SH,

Khorramizadeh
MR,
et
al.
Assessment
of
cytokine
expression
profile
in

acute
myeloid
leukemia
patients
before
and
after
chemotherapy.
Turk
J

Haematol
2014
Jun;31(2):149-‐54.

48

(84)

Weisberg
E,
Nonami
A,
Griffin
JD.
Combination
therapy
with
nilotinib

for
drug-‐sensitive
and
drug-‐resistant
BCR-‐ABL-‐positive
leukemia
and

other
malignancies.
Arch
Toxicol
2014
Dec;88(12):2233-‐42.

(85)

Garrido
SM,
Appelbaum
FR,
Willman
CL,
Banker
DE.
Acute
myeloid

leukemia
cells
are
protected
from
spontaneous
and
drug-‐induced

apoptosis
by
direct
contact
with
a
human
bone
marrow
stromal
cell
line

(HS-‐5).
Exp
Hematol
2001
Apr;29(4):448-‐57.

(86)

Dinarello
CA.
Historical
insights
into
cytokines.
Eur
J
Immunol
2007

Nov;37
Suppl
1:S34-‐S45.

(87)

Lee
S,
Margolin
K.
Cytokines
in
cancer
immunotherapy.
Cancers
(Basel)

2011;3(4):3856-‐93.

(88)

Lotem
J,
Sachs
L.
Cytokine
control
of
developmental
programs
in

normal
hematopoiesis
and
leukemia.
Oncogene
2002
May

13;21(21):3284-‐94.

(89)

Moqattash
S,
Lutton
JD.
Leukemia
cells
and
the
cytokine
network.
Proc

Soc
Exp
Biol
Med
1998
Oct;219(1):8-‐27.

(90)

Moqattash
S,
Lutton
JD.
Leukemia
cells
and
the
cytokine
network:

therapeutic
prospects.
Exp
Biol
Med
(Maywood
)
2004
Feb;229(2):121-‐
37.

(91)

Tabe
Y,
Konopleva
M.
Advances
in
understanding
the
leukaemia

microenvironment.
Br
J
Haematol
2014
Mar;164(6):767-‐78.

(92)

Lwin
T,
Crespo
LA,
Wu
A,
Dessureault
S,
Shu
HB,
Moscinski
LC,
et
al.

Lymphoma
cell
adhesion-‐induced
expression
of
B
cell-‐activating
factor

of
the
TNF
family
in
bone
marrow
stromal
cells
protects
non-‐Hodgkin's

B
lymphoma
cells
from
apoptosis.
Leukemia
2009
Jan;23(1):170-‐7.

(93)

Trayhurn
P,
Wood
IS.
Adipokines:
inflammation
and
the
pleiotropic

role
of
white
adipose
tissue.
Br
J
Nutr
2004
Sep;92(3):347-‐55.

(94)

Kwon
H,
Pessin
JE.
Adipokines
mediate
inflammation
and
insulin

resistance.
Front
Endocrinol
(Lausanne)
2013;4:71.

(95)

Fantuzzi
G.
Adipose
tissue,
adipokines,
and
inflammation.
J
Allergy
Clin

Immunol
2005
May;115(5):911-‐9.

(96)

ntuna-‐Puente
B,
Feve
B,
Fellahi
S,
Bastard
JP.
Adipokines:
the
missing

link
between
insulin
resistance
and
obesity.
Diabetes
Metab
2008

Feb;34(1):2-‐11.

49

(97)

Dalamaga
M,
Crotty
BH,
Fargnoli
J,
Papadavid
E,
Lekka
A,
Triantafilli
M,

et
al.
B-‐cell
chronic
lymphocytic
leukemia
risk
in
association
with

serum
leptin
and
adiponectin:
a
case-‐control
study
in
Greece.
Cancer

Causes
Control
2010
Sep;21(9):1451-‐9.

(98)

Huh
JY,
Park
YJ,
Ham
M,
Kim
JB.
Crosstalk
between
adipocytes
and

immune
cells
in
adipose
tissue
inflammation
and
metabolic

dysregulation
in
obesity.
Mol
Cells
2014
May;37(5):365-‐71.

(99)

Johrer
K,
Ploner
C,
Thangavadivel
S,
Wuggenig
P,
Greil
R.
Adipocyte-‐
derived
players
in
hematologic
tumors:
useful
novel
targets?
Expert

Opin
Biol
Ther
2015
Jan;15(1):61-‐77.

(100)

Orgel
E,
Tucci
J,
Alhushki
W,
Malvar
J,
Sposto
R,
Fu
CH,
et
al.
Obesity
is

associated
with
residual
leukemia
following
induction
therapy
for

childhood
B-‐precursor
acute
lymphoblastic
leukemia.
Blood
2014
Dec

18;124(26):3932-‐8.

(101)

Pramanik
R,
Sheng
X,
Ichihara
B,
Heisterkamp
N,
Mittelman
SD.
Adipose

tissue
attracts
and
protects
acute
lymphoblastic
leukemia
cells
from

chemotherapy.
Leuk
Res
2013
May;37(5):503-‐9.

(102)

Behan
JW,
Yun
JP,
Proektor
MP,
Ehsanipour
EA,
Arutyunyan
A,
Moses

AS,
et
al.
Adipocytes
impair
leukemia
treatment
in
mice.
Cancer
Res

2009
Oct
1;69(19):7867-‐74.

(103)

Asano
J,
Nakano
A,
Oda
A,
Amou
H,
Hiasa
M,
Takeuchi
K,
et
al.
The

serine/threonine
kinase
Pim-‐2
is
a
novel
anti-‐apoptotic
mediator
in

myeloma
cells.
Leukemia
2011
Jul;25(7):1182-‐8.

(104)

Beltowski
J.
Adiponectin
and
resistin-‐-‐new
hormones
of
white
adipose

tissue.
Med
Sci
Monit
2003
Feb;9(2):RA55-‐RA61.

(105)

Chandran
M,
Phillips
SA,
Ciaraldi
T,
Henry
RR.
Adiponectin:
more
than

just
another
fat
cell
hormone?
Diabetes
Care
2003
Aug;26(8):2442-‐50.

(106)

Qiang
L,
Wang
H,
Farmer
SR.
Adiponectin
secretion
is
regulated
by

SIRT1
and
the
endoplasmic
reticulum
oxidoreductase
Ero1-‐L
alpha.
Mol

Cell
Biol
2007
Jul;27(13):4698-‐707.

(107)

Cong
L,
Chen
K,
Li
J,
Gao
P,
Li
Q,
Mi
S,
et
al.
Regulation
of
adiponectin
and

leptin
secretion
and
expression
by
insulin
through
a
PI3K-‐PDE3B

dependent
mechanism
in
rat
primary
adipocytes.
Biochem
J
2007
May

1;403(3):519-‐25.

(108)

Blumer
RM,
van
Roomen
CP,
Meijer
AJ,
Houben-‐Weerts
JH,
Sauerwein

HP,
Dubbelhuis
PF.
Regulation
of
adiponectin
secretion
by
insulin
and

50

amino
acids
in
3T3-‐L1
adipocytes.
Metabolism
2008
Dec;57(12):1655-‐
62.

(109)

Diez
JJ,
Iglesias
P.
The
role
of
the
novel
adipocyte-‐derived
hormone

adiponectin
in
human
disease.
Eur
J
Endocrinol
2003
Mar;148(3):293-‐
300.

(110)

Halleux
CM,
Takahashi
M,
Delporte
ML,
Detry
R,
Funahashi
T,

Matsuzawa
Y,
et
al.
Secretion
of
adiponectin
and
regulation
of
apM1

gene
expression
in
human
visceral
adipose
tissue.
Biochem
Biophys
Res

Commun
2001
Nov
16;288(5):1102-‐7.

(111)

Maeda
N,
Takahashi
M,
Funahashi
T,
Kihara
S,
Nishizawa
H,
Kishida
K,
et

al.
PPARgamma
ligands
increase
expression
and
plasma
concentrations

of
adiponectin,
an
adipose-‐derived
protein.
Diabetes
2001

Sep;50(9):2094-‐9.

(112)

Hotamisligil
GS,
Spiegelman
BM.
Tumor
necrosis
factor
alpha:
a
key

component
of
the
obesity-‐diabetes
link.
Diabetes
1994

Nov;43(11):1271-‐8.

(113)

Kappes
A,
Loffler
G.
Influences
of
ionomycin,
dibutyryl-‐cycloAMP
and

tumour
necrosis
factor-‐alpha
on
intracellular
amount
and
secretion
of

apM1
in
differentiating
primary
human
preadipocytes.
Horm
Metab
Res

2000
Nov;32(11-‐12):548-‐54.

(114)

Jung
TW,
Youn
BS,
Choi
HY,
Lee
SY,
Hong
HC,
Yang
SJ,
et
al.
Salsalate
and

adiponectin
ameliorate
hepatic
steatosis
by
inhibition
of
the

hepatokine
fetuin-‐A.
Biochem
Pharmacol
2013
Oct
1;86(7):960-‐9.

(115)

Ouchi
N,
Kihara
S,
Arita
Y,
Okamoto
Y,
Maeda
K,
Kuriyama
H,
et
al.

Adiponectin,
an
adipocyte-‐derived
plasma
protein,
inhibits
endothelial

NF-‐kappaB
signaling
through
a
cAMP-‐dependent
pathway.
Circulation

2000
Sep
12;102(11):1296-‐301.

(116)

Liu
J,
Lam
JB,
Chow
KH,
Xu
A,
Lam
KS,
Moon
RT,
et
al.
Adiponectin

stimulates
Wnt
inhibitory
factor-‐1
expression
through
epigenetic

regulations
involving
the
transcription
factor
specificity
protein
1.

Carcinogenesis
2008
Nov;29(11):2195-‐202.

(117)

Kumada
M,
Kihara
S,
Ouchi
N,
Kobayashi
H,
Okamoto
Y,
Ohashi
K,
et
al.

Adiponectin
specifically
increased
tissue
inhibitor
of

metalloproteinase-‐1
through
interleukin-‐10
expression
in
human

macrophages.
Circulation
2004
May
4;109(17):2046-‐9.

51

(118)

Okamoto
Y,
Kihara
S,
Ouchi
N,
Nishida
M,
Arita
Y,
Kumada
M,
et
al.

Adiponectin
reduces
atherosclerosis
in
apolipoprotein
E-‐deficient
mice.

Circulation
2002
Nov
26;106(22):2767-‐70.

(119)

Wulster-‐Radcliffe
MC,
Ajuwon
KM,
Wang
J,
Christian
JA,
Spurlock
ME.

Adiponectin
differentially
regulates
cytokines
in
porcine
macrophages.

Biochem
Biophys
Res
Commun
2004
Apr
9;316(3):924-‐9.

(120)

Ouchi
N,
Kihara
S,
Arita
Y,
Maeda
K,
Kuriyama
H,
Okamoto
Y,
et
al.
Novel

modulator
for
endothelial
adhesion
molecules:
adipocyte-‐derived

plasma
protein
adiponectin.
Circulation
1999
Dec
21;100(25):2473-‐6.

(121)

Kawanami
D,
Maemura
K,
Takeda
N,
Harada
T,
Nojiri
T,
Imai
Y,
et
al.

Direct
reciprocal
effects
of
resistin
and
adiponectin
on
vascular

endothelial
cells:
a
new
insight
into
adipocytokine-‐endothelial
cell

interactions.
Biochem
Biophys
Res
Commun
2004
Feb
6;314(2):415-‐9.

(122)

Flotho
C,
Coustan-‐Smith
E,
Pei
D,
Iwamoto
S,
Song
G,
Cheng
C,
et
al.

Genes
contributing
to
minimal
residual
disease
in
childhood
acute

lymphoblastic
leukemia:
prognostic
significance
of
CASP8AP2.
Blood

2006
Aug
1;108(3):1050-‐7.

(123)

Gang
EJ,
Hsieh
YT,
Pham
J,
Zhao
Y,
Nguyen
C,
Huantes
S,
et
al.
Small-‐
molecule
inhibition
of
CBP/catenin
interactions
eliminates
drug-‐
resistant
clones
in
acute
lymphoblastic
leukemia.
Oncogene
2014
Apr

24;33(17):2169-‐78.

(124)

Sheng
X,
Mittelman
SD.
The
role
of
adipose
tissue
and
obesity
in
causing

treatment
resistance
of
acute
lymphoblastic
leukemia.
Front
Pediatr

2014;2:53.

(125)

Petridou
E,
Mantzoros
CS,
Dessypris
N,
Dikalioti
SK,
Trichopoulos
D.

Adiponectin
in
relation
to
childhood
myeloblastic
leukaemia.
Br
J

Cancer
2006
Jan
16;94(1):156-‐60.

(126)

Iwamoto
S,
Mihara
K,
Downing
JR,
Pui
CH,
Campana
D.
Mesenchymal

cells
regulate
the
response
of
acute
lymphoblastic
leukemia
cells
to

asparaginase.
J
Clin
Invest
2007
Apr;117(4):1049-‐57.

(127)

Lang
K,
Ratke
J.
Leptin
and
Adiponectin:
new
players
in
the
field
of

tumor
cell
and
leukocyte
migration.
Cell
Commun
Signal
2009;7:27.

(128)

Crawford
LJ,
Peake
R,
Price
S,
Morris
TC,
Irvine
AE.
Adiponectin
is

produced
by
lymphocytes
and
is
a
negative
regulator
of
granulopoiesis.

J
Leukoc
Biol
2010
Oct;88(4):807-‐11.

52

(129)

Yokota
T,
Oritani
K,
Takahashi
I,
Ishikawa
J,
Matsuyama
A,
Ouchi
N,
et

al.
Adiponectin,
a
new
member
of
the
family
of
soluble
defense

collagens,
negatively
regulates
the
growth
of
myelomonocytic

progenitors
and
the
functions
of
macrophages.
Blood
2000
Sep

1;96(5):1723-‐32.

(130)

Ozturk
K,
Avcu
F,
Ural
AU.
Aberrant
expressions
of
leptin
and

adiponectin
receptor
isoforms
in
chronic
myeloid
leukemia
patients.

Cytokine
2012
Jan;57(1):61-‐7.

(131)

Castells
M,
Thibault
B,
Delord
JP,
Couderc
B.
Implication
of
tumor

microenvironment
in
chemoresistance:
tumor-‐associated
stromal
cells

protect
tumor
cells
from
cell
death.
Int
J
Mol
Sci
2012;13(8):9545-‐71.

(132)

Ehsanipour
EA,
Sheng
X,
Behan
JW,
Wang
X,
Butturini
A,
Avramis
VI,
et

al.
Adipocytes
cause
leukemia
cell
resistance
to
L-‐asparaginase
via

release
of
glutamine.
Cancer
Res
2013
May
15;73(10):2998-‐3006.

(133)

Sullivan
KE,
Cutilli
J,
Piliero
LM,
Ghavimi-‐Alagha
D,
Starr
SE,
Campbell

DE,
et
al.
Measurement
of
cytokine
secretion,
intracellular
protein

expression,
and
mRNA
in
resting
and
stimulated
peripheral
blood

mononuclear
cells.
Clin
Diagn
Lab
Immunol
2000
Nov;7(6):920-‐4.

(134)

Aref
S,
Ibrahim
L,
Azmy
E,
Al
AR.
Impact
of
serum
adiponectin
and
leptin

levels
in
acute
leukemia.
Hematology
2013
Jul;18(4):198-‐203.

(135)

Barb
D,
Williams
CJ,
Neuwirth
AK,
Mantzoros
CS.
Adiponectin
in
relation

to
malignancies:
a
review
of
existing
basic
research
and
clinical

evidence.
Am
J
Clin
Nutr
2007
Sep;86(3):s858-‐s866.

(136)

Avcu
F,
Ural
AU,
Yilmaz
MI,
Bingol
N,
Nevruz
O,
Caglar
K.
Association
of

plasma
adiponectin
concentrations
with
chronic
lymphocytic
leukemia

and
myeloproliferative
diseases.
Int
J
Hematol
2006
Apr;83(3):254-‐8.

(137)

Sutlu
T,
Alici
E.
Natural
killer
cell-‐based
immunotherapy
in
cancer:

current
insights
and
future
prospects.
J
Intern
Med
2009

Aug;266(2):154-‐81.

(138)

Chronic
Myeloid
Leukemia
Trialists'
Collaborative
Group.
Interferon

alpha
versus
chemotherapy
for
chroni
myeloid
leukemia:
a
meta-‐
analysis
of
seven
randomized
trials.
J
Natl
Cancer
Inst

1997;89(21):1616-‐20.

(139)

Joshi
S,
Kaur
S,
Redig
AJ,
Goldsborough
K,
David
K,
Ueda
T,
et
al.
Type
I

interferon
(IFN)-‐dependent
activation
of
Mnk1
and
its
role
in
the

generation
of
growth
inhibitory
responses.
Proc
Natl
Acad
Sci
U
S
A

2009
Jul
21;106(29):12097-‐102.

53

(140)

Jonasch
E,
Haluska
FG.
Interferon
in
oncological
practice:
review
of

interferon
biology,
clinical
applications,
and
toxicities.
Oncologist

2001;6(1):34-‐55.

(141)

Tagawa
M.
Cytokine
therapy
for
cancer.
Curr
Pharm
Des
2000

Apr;6(6):681-‐99.

(142)

Finn
OJ.
Cancer
immunology.
N
Engl
J
Med
2008
Jun
19;358(25):2704-‐
15.

(143)

Guo
Y,
Xu
F,
Lu
T,
Duan
Z,
Zhang
Z.
Interleukin-‐6
signaling
pathway
in

targeted
therapy
for
cancer.
Cancer
Treat
Rev
2012
Nov;38(7):904-‐10.

(144)

Trikha
M,
Corringham
R,
Klein
B,
Rossi
JF.
Targeted
anti-‐interleukin-‐6

monoclonal
antibody
therapy
for
cancer:
a
review
of
the
rationale
and

clinical
evidence.
Clin
Cancer
Res
2003
Oct
15;9(13):4653-‐65.

Abstract (if available)

Abstract Although survival rates are promising in children with Acute Lymphoblastic Leukemia, in relapsed childhood ALL, survival rates remain a problem. More than 80% of first relapse of childhood and adult ALL occurs in the bone marrow. It has been shown that the bone marrow can promote cell adhesion mediated drug resistance (CAM‐DR) in leukemia cells by direct cell‐to‐cell interaction. The adhesion of ALL cells to the bone marrow stromal cells can also stimulate the secretion of soluble factors which leads to soluble factor mediated drug resistance (SFM-DR). A combination of these two forms of drug resistance is also possible. Previous studies indicate that by blocking leukemia integrin alpha4 and more recently alpha6 adhesion to their supportive bone marrow stroma environment, leukemia cells can be sensitized to chemotherapy. We hypothesized that SFM‐DR is also involved in this alpha6‐mediated drug resistance. Here, we show that there is a distinct cytokine profile involved in alpha6‐mediated drug resistance indicating that particular cytokines could play a role in chemoresistance as well. Taken together, this research attempts to understand the underlying mechanism of drug resistance in ALL that could lead to novel, more appropriately developed treatments.

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