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Role of integrin α4 in drug resistant acute lymphoblastic leukemia

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Role of integrin α4 in drug resistant acute lymphoblastic leukemia

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Content i

ROLE OF INTEGRIN α4 IN DRUG RESISTANT
ACUTE LYMPHOBLASTIC LEUKEMIA

by

Yao-Te (Stanley) Hsieh

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)

May 2013

Copyright 2013 Yao-Te (Stanley) Hsieh
ii

Dedication

I would like to dedicate this research work to my family; my father ( 謝祖佳
), mother ( 蔡淑芳), sister (謝惠雅), life partner ( 蘇曼菁), aunt (謝美瑜) and
other relatives, particularly

Aunt ( 蔡淑真), died of nasopharyngeal cancer in 2005
Aunt ( 謝洋子), died of breast cancer in 2005
Grandmother ( 吳秀燕), died of lung cancer in 2009
Aunt ( 莊櫻蘭), died of breast cancer in 2013
New-born nephew ( 張聿元), diagnosed as acute lymphoblastic leukemia

With great sorrow and lost, I gain the courage and motivation to complete
this project and dissertation.
I miss you all !

iii

Acknowledgments
This dissertation would not be possible without support of the following
people and labs:
 My mentor, Dr. Yong-Mi Kim, for wonderful guidance, support, trust
and encouragement.
 My co-workers, Dr. Enzi Jiang, Eugene Park, Brian De La Torre, Asha
Kadavallore, Cynthia Hinh and other volunteers for great helps and
amazing teamwork.
 My PhD committee members, Dr. Cheng-Ming Chuong and Dr.
Florence Hofman give me excellent input and guidance.
 Dr. Markus Mϋschen, Dr. Nora Heisterkamp, Dr. Steven Mittelman and
their lab members in Children Hospital Los Angeles (CHLA) for critical
supports of techniques, materials and knowledge.
 Dr. Halvard Böng in University of Washington and Dr. Peter
Vanderslice in St. Luke’s Episcopal Hospital for sharing Tysabri and
TBC3486 and their great advice.
 Dr. Mignon Loh’s lab in UCSF, Dr. Wolf-Karsten Hofmann’s lab in
University Hospital, and Dr. Eun-Suk Kang’s lab in Sungkyunkwan
University School of Medicine providing the invaluable ALL patient
samples.
 Mrs. Lisa Doumak in USC smoothly and kindly navigates me to
complete all requirements for the degree.

iv

Table of Contents
Dedication…………………………………………………………………..ii
Acknowledgments……………………………………………………….iii
List of Tables………………………………………………………………vi
List of Figures……………………………………………………………..vii
Abstract………………………………………………………………………xi
Chapter One: Introduction
1.1 Acute lymphoblastic leukemia (ALL)…………………………………….1
1.2 Drug resistance of leukemia ……………………………………………..2
1.3 Trafficking of normal leukocytes to the bone marrow………………….4
1.4 Trafficking of leukemia cells to the bone marrow ……………………...6
1.5 Bone marrow microenvironmental niches………………………………7
1.6 Other factors interact with leukemia in bone marrow………………….9
Chapter Two: Establishment of pre-clinical model to
evaluate alternative therapies for ALL
2.1 Introduction………………………………………………………………..11
2.2 Materials and methods…………………………………………………...14
2.3 Results……………………………………………………………………..18
2.4 Discussion…………………………………………………………………20
2.5 Figures……………………………………………………………………..23
Chapter Three: Targeting integrin α4 sensitizes drug
resistant human ALL to chemotherapy
3.1 Introduction………………………………………………………………..33
3.2 Materials and methods…………………………………………………...43
3.3 Results……………………………………………………………………..53
3.4 Discussion…………………………………………………………………66
3.5 Figures……………………………………………………………………..73

v

Chapter Four: Deletion of integrin α4 overcomes drug
resistance of mouse leukemia
4.1 Introduction………………………………………………………………131
4.2 Materials and methods …………………………………………………133
4.3 Results……………………………………………………………………136
4.4 Discussion………………………………………………………………..139
4.4 Figures……………………………………………………………………142
Chapter Five: Conclusion
5.1 Conclusions……………………………………………………………...161
5.2 Discussion and future directions….…………………………………...162

Reference List……………………………………………………………166

vi

List of Tables
Chapter Two
Table 1 Clinical information of xenograft samples…………………………23
Table 2 Immunophenotype of original patient and xenograft samples in
serial passages………………………………………………………………...24
Table 3 Antibodies used for flow cytometry…………………………………25
Chapter Three
Table 4 Sequences of oligonucleotide primers used..……………………..73
Table 5 List of antibodies for western blot analysis………………………..73

vii

List of Figures
Chapter Two
Figure 1 Schematics for xenograft mouse model and bioluminescent
imaging………………………………………………………………………….26
Figure 2 Morphology of xenograft samples…………………………………27
Figure 3 Transduction efficiency of luciferase by immunohistochemistry.28
Figure 4 Monitor leukemia progression by bioluminescence signal and
FACS (%) hCD45
+
in peripheral blood.…………………………………......29
Figure 5 Preclinically evaluate drugs of interests in xenograft model……30
Chapter Three
Figure 6 Roles of integrin.…………………………………..........................74
Figure 7 Structures of integrin α4 small molecule inhibitors, Bio1211 and
TBC3486………………………………………………………………………..75
Figure 8 Integrin α4 is overexpressed in ALL patients…………………….76
Figure 9 Correlation of integrin α4 with the survival outcome of ALL
patients………………………………………………………………………….77
Figure 10 Expression of integrin α4 in xenograft leukemia samples……..78
Figure 11 Dose determination of Tysabri for pre-B ALL, LAX7R…………80
Figure 12 Viability of normal pre-B cells is unaffected by Tysabri………..81
Figure 13 Tysabri does not induce antibody-dependent cell-mediated
cytoxicity (ADCC) on human leukemia cells………………………………..82
viii

Figure 14 Integrin α4 blockade inhibits the adhesion of leukemia cells to
human VCAM-1. ………………………………………………………………83
Figure 15 Inhibition of integrin α4 by Tysabri to hVCAM-1 affects the
integrin-mediated signaling.…………………………………………………..84
Figure 16 Targeting integrin α4 by Tysabri sensitizes drug resistant pre-B
ALL cells to chemotherapy.…………………………………………………..85
Figure 17 Blockade of integrin α4 delays the progression of leukemia and
prolongs survival of mice engrafted with pre-B ALL in vivo. ……………...86
Figure 18 Targeting integrin α4 by Tysabri in combination with
chemotherapy eradicates drug resistant primary pre-B ALL, LAX7R……88
Figure 19 Combined integrin α4 blockade and chemotherapy eradicates
primary ALL (LAX7R) in NSG recipient mice……………………………….90
Figure 20 Integrin α4 blockade prolongs survival of murine recipients of
primary pre-B ALL cells (ICN12).…………………………………………….91
Figure 21 Combined integrin α4 blockade and chemotherapy eradicate
pre-B ALL (RS4;11).…………………………………………………………..92
Figure 22 Engraftment of leukemia cells in vivo 3 days after injection…..94
Figure 23 Late α4 blockade (Day 6 post-injection of LAX7R) prolongs
survival.…………………………………………………………………………96
Figure 24 The cause of animal death by CBC analysis…………………...98
ix

Figure 25 Effect of integrin α4 blockade on homing of primary leukemia
cells in vivo……………………………………………………………………..99
Figure 26 Mobilization of primary leukemia cells by Tysabri…………….101
Figure 27 Integrin expression of three primary pre-B ALL samples…....103
Figure 28 Effects of TBC3486 treatment on integrin expression………..104
Figure 29 TBC3486 treatment inhibits adhesion of primary ALL to OP9
cells.……………………………………………………………………………105
Figure 30 Inhibition of integrin α4 by TBC3486 disrupts the adhesion of
primary ALL cells……………………………………………………………..106
Figure 31 Inhibition of α4 integrin sensitizes primary ALL cells to
chemotherapy………………………………………………………………...107
Figure 32 Inhibition of α4 integrin delays the progression of primary ALL in
vivo…………………………………………………………………………….108
Figure 33 Dose determination of Tysabri for T-ALL and AML…………..109
Figure 34 Integrin α4 blockade inhibits the adhesion of T-ALL and AML
cells to human VCAM-1.…………………………………………………….110
Figure 35 Targeting integrin α4 by Tysabri sensitizes drug resistant AML
cells to chemotherapy……………………………………………………….111
Figure 36 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary AML (AML-1) in vivo…………………………………….112
x

Figure 37 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary T-ALL (LAX1R) in vivo………………………………….113
Chapter Four
Figure 38 Retroviral vectors and transduction methods…………………142
Figure 39 Conditional knockout of Integrin α4 in murine (BCR-ABL1
+
)
leukemia……………………………………………………………………....143
Figure 40 Deletion of integrin α4 in B-ALL is confirmed by flow cytometry
and genomic PCR.…………………………………………………………...144
Figure 41 Adhesion of emptyER
T2
and creER
T2
cells on mVCAM-1……145
Figure 42 Deletion of integrin α4 sensitizes leukemia cells to
chemotherapy in vitro.……………………………………………………….146
Figure 43 Deletion of integrin α4 decrease the self-renewal of murine
leukemia.…………………………………………………………..................147
Figure 44 Deletion of integrin α4 affects signaling pathways of
leukemia……………………………………………………………………….148
Figure 45 Knockout of integrin α4 in vitro prolongs the survival time in
vivo…………………………………………………………………………….149
Figure 46 Incomplete in vivo deletion of integrin α4 in murine leukemia.150
Figure 47 In vivo deletion of integrin α4 eradicates murine leukemia…..151
Figure 48 Blood counts of recipient mice of murine pre-B ALL……...….153
Figure 49 Chemo-toxicity of Integrin α4 ablation………………………….154
xi

Abstract
Although cure rates for acute lymphoblastic leukemia (ALL) in children are
high, relapse of ALL leads to death in 50-95%. Adult ALL patients have a
survival rate of only 40%. Even survived, patients often suffer from late-
term secondary toxic effects of current treatments. Therefore,
chemotherapeutic drug resistance of ALL cells remains a major problem
and less toxic and more efficient therapies are needed. More than 80% of
first relapse of childhood and adult ALL occurs in the bone marrow.
Interaction of normal hematopoietic stem cells with the bone marrow (BM)
stromal cells has been shown to provide mechanical support and facilitate
proliferation and differentiation. BM stromal cells also provide protection of
ALL cells from chemotherapy, thus contributing to drug resistance due to
the lack of efficacy of current therapies. The exact mechanisms for
stroma-mediated chemoprotection and approaches to address this
problem remain elusive. In chapter two of this thesis, we summarize how
we established a xenograft model of primary ALL cells to evaluate novel
therapies. In chapter three, we use this preclinical model of primary ALL
and focus on integrin alpha 4 as a central adhesion molecule for stromal-
mediated chemoprotection and drug resistance of ALL. Integrin α4 is
known to mediate adhesion of normal and malignant B-cell precursors in
BM stromal cells. However, the functional modulation of integrin α4 and its
xii

consequences for drug resistance in ALL remains to be examined.
According to gene expression analyses, integrin α4 is overexpressed in
ALL patients and inversely correlated with the survival outcome.
Therefore, we tested whether interference with α4-mediated stromal
adhesion might be a new ALL treatment strategy. For this purpose, two
models of leukemia were used: one pharmacological using antibody, like
Tysabri, and small molecule inhibitor, like TBC3486, to target α4 of
primary pre-B ALL and later in chapter 4 a genetic model (conditional α4
ablation of BCR-ABL1-induced murine leukemia). Conditional deletion of
α4 sensitized murine leukemia cell to chemotherapy, Nilotinib. Adhesion of
primary pre-B ALL cells was α4-dependent and inhibiting α4 sensitized
primary ALL cells towards chemotherapy, VDL. Combination of
chemotherapy with Tysabri prolonged survival of NOD/SCID recipients of
primary ALL suggesting adjuvant integrin α4 inhibition as a novel strategy
for pre-B ALL. Taken together, our data demonstrate that integrin α4-
blockade with adjuvant chemotherapy can eradicate chemotherapy-
resistant leukemia.

1

Chapter One
Introduction

1.1 Acute lymphoblastic leukemia (ALL)
Leukemia is a type of cancer that initiates in blood-forming tissue such as
bone marrow and causes overproduction of immature lymphoid cells that
enter the bloodstream. Leukemia can be categorized as acute or chronic
by clinical course and (B or T) lymphoid or myeloid by lineage derivation.
In the United States, there are about 4000 new patients diagnosed with
acute lymphoblastic leukemia (ALL) every year and two third of which are
children and adolescents, making this disease the most common cancer in
this population (1). For adults, the survival rate of the disease is only
~40%(2). Even survive, patients often suffer from late-term secondary
toxic effects of current treatments, like heart damage, impaired fertility and
nerve system, and treatment-related secondary malignancy (1). Therefore,
less toxic and novel therapy modalities are needed to develop alternative
treatment strategies for ALL patients.

2

1.2 Drug resistance of leukemia
Although the remarkable advances have been achieved in the current
treatment for childhood acute lymphoblastic leukemia (ALL) over last four
decades, death from ALL still remains a problem for drug resistant
leukemia, which reoccurs in ~20% of children and ~60% of adults (1;2). Of
those children with recurrent leukemia, only 5-50% survive with current
treatments, depending on the sites of relapse (3). In fact, more than 80%
of the sites of first relapse in childhood and adult ALL is bone marrow with
only ~20% of 6-year survival rate for children and ~10% of 5-year survival
rate for adult. (4;5). The lack of efficacy to current chemotherapy can be
partly attributed to the fact that drugs cannot eradicate leukemia cells
protected by a specific “microenvironment” in bone marrow and leads
leukemia cells to drug resistance. Adhesion of leukemia cells to bone
marrow stromal cells has been reported to prevent B- and T-lineage ALL
(6) (7) (8) and acute myeloid leukemia (AML) (9) from chemotherapy-
induced apoptosis in vitro, which is described as cell adhesion-mediated
drug resistance (CAM-DR). Disruption of adhesive interactions between
leukemia cells and adhesive matrices, also named anoikis (Greek for
“homelessness”), may render cells more vulnerable to the induction of
death pathways (10). Astier and colleagues showed that ß1 integrin
3

stimulation inhibits caspase-3 and -7 induction in pre-B ALL cells (11). Bcl-
2 is the anti-apoptotic protein which was most exhaustively evaluated in
studies aimed at understanding programmed cell death. Modulation of the
biological activity of Bcl-2 by proteolytic cleavage, with a 23kDa cleaved
Bcl-2 product having pro-apoptotic activity, has been reported. Co-culture
of three pre-B ALL cell lines with bone marrow stromal cells during
chemotherapy exposure resulted in reduced levels of the 23kDa Bcl-2
protein indicating that the bone marrow microenvironment may contribute
to maintenance of residual leukemic disease during treatment by reducing
generation of pro-apoptotic 23kDa Bcl-2 (12). In another study regarding
the modulation of apoptosis mediators in chemotherapy-exposed, stroma-
co-cultured ALL cells, Wang et al assessed which molecular mediators of
apoptosis were involved (13). They reported reduced levels of PARP and
Bcl-2 cleavage products in ALL cells after chemotherapy when these were
cultured on stroma, and provided evidence that this effect was Akt-
mediated since stroma-induced attenuation of death pathways was not
observed in leukemia cells with blunted Akt activity. Whether different
integrin ligands can elicit different outside-in signals is not clear but seems
possible. Fortney et al reported that in ALL cells caspase 3 activity was
induced by Cytarabine or Etoposide (14). This effect was blunted in ALL
cells cultured with human bone marrow stroma cell layers. While this
4

observation does not explain the origin of the survival signal (stroma-
derived or leukemia cell-autonomous), it suggests that the level of the
leukemia cell survival may be due, at least in part, to modulation of the
caspase 3 pathway. Therefore, dislodging leukemia, like pre-B ALL and T-
ALL, from the protective bone marrow stromal cells may sensitize
leukemia cells to chemotherapy.

1.3 Trafficking of normal leukocytes to the bone marrow
Interaction with a specialized microenvironment has been shown to
provide mechanical support and facilitate the proliferation and
differentiation of normal hematopoietic stem cells (HSCs) (15). Similarly,
the bone marrow microenvironment plays a major role for leukemia cells
regarding homing, development and chemo-protection (7;16). Although
the knowledge and evidence about leukemia microenvironment are still
limited, the leukemia initiating cells have been assumed to share some
characteristics and locate in a similar bone marrow microenvironment with
HSCs (16). Hence, in order to understand leukemia microenvironment,
studying normal leukocytes trafficking and interaction with bone marrow
cells becomes necessary.
5

The normal leukocytes trafficking from the blood into peripheral tissues
which release inflammation signals occurs through four sequential steps,
including (i) rolling, (ii) integrin activation, (iii) stable arrest and (iv)
migration across endothelial cells (endothelium) (17). (i) Rolling is
primarily mediated by L-selectin on normal leukocytes to capture the
ligand, E-selectin, on endothelium (18). In addition, CD44 also facilitates
normal leukocytes to roll on endothelial surface by binding with hyaluronic
acid before arresting (19). (ii) As leukocytes roll on the endothelium and
encounter chemoattractants/chemokines on the apical endothelial surface,
the conformational changes of the integrin are triggered to alter its affinity
from low to high (inside-out signaling) (20). (iii) Activated integrins which
either contain β2 subunit, like αLβ2 (LFA-1) or α4 subunit, like α4β1
(VLA4), on leukocytes form a shear-resistant adhesion and a stable arrest
with endothelial cells (18). (iv) Once a firm adhesion is established,
leukocytes undergo a morphological change, called polarization, and
migrate across (or called diapedesis) the endothelial monolayer to
subendothelial tissues. In addition to inside-out signals for integrin
activation, integrins also integrate with mechanical signals to mediate
external forces from shear stress with endothelial contact and internal
forces from polymerized cytoskeletons (18). β2 integrin has been shown
6

to directly mediate migration of leukocytes and α4β1 plays an indirect role
by simulating β2 integrin (21).

1.4 Trafficking of leukemia cells to the bone marrow.
The interactions of leukemia cells with bone microenvironment for homing,
diapedesis, and migration to other organs will be described as following.
Leukemia cells first home to femurs attracted via expressing VEGF,
CXCR4, and CD44 to bind with the respective ligands, like VEGFR-2,
SDF-1 (CXCL12), and hyaluronic acid, on vascular endothelial cells in
bone marrow shown in NOD/SCID mouse model (22;23). Using real-time
in vivo two-photon microscopy techniques and immunohistochemistry,
leukemia cells (pre-B ALL cell line, Nalm-6) have been reported to dock on
vascular endothelium through E-selectin and CXCR4/CXCL12
interactions, unlike HSCs majorly using E and L-selectins and
VLA4/VCAM-1interactions (24). Using a knockout model, Sipkin’s group
shows that the majority of the homing of leukemia in the model is
dependent on CXCR4/CXCL12 interaction. These results imply CXCR4
and CXCL12 interaction plays a critical role of leukemia homing to bone
marrow vascular niche (24). Like HSCs, leukemia cells first home to the
bone marrow through intramedullary vessels. Interacting with activated E-
7

selectins and integrins, like integrin α4β1 on vascular endothelial cells,
initiates rolling, arrest, adhesion and transmigration through vascular
endothelial cells to bone marrow cavity influenced by gradients of CXCL12
secreted from endothelial cells and Ca
2+
ion from bone marrow
osteoclasts during bone resorption (16). Furthermore, leukemia cells
invade to epiphysial trabecula region (25) and directly contact with
osteopontin positive (OPN
+
) osteoblast cells and adjuvant stromal cells on
inner bone membrane (26). Migration is the next step for egress of
leukemia blasts into the circulation through basement membrane and
sinusoidal endothelium. Integrin α4β1 and VCAM-1 interaction plays a
dominant role on the transmigration of ALL cells (27).

1.5 Bone marrow microenvironmental niches
Leukemia cells (including leukemia initiating cells) have been known to
interact with bone marrow microenvironment composed of 3 major niches:
(i) Vascular niche (ii) Osteoblast niche (iii) Stromal cell niche for homing,
development, mobilization and survival.
(i) Vascular niche: Besides homing to bone marrow as described above,
the consequence of leukemia cells binding with vascular niche elements,
including endothelium and CXCL12-abundant reticular cells (CAR), are
8

responsible for the proliferation and migration of leukemia cells to other
organs, like liver, spleen and central nervous system through
VEGF/VEGFR-2 signaling pathways and secretion of cytokines, like IL-6,
IL-3, G-CSF, and nitric oxide (NO) (28). In addition, endothelial cells in
bone marrow vascular niche promote the survival of ALL cells in vitro by
modulation of the anti-apoptotic Bcl-2 protein (29).
(ii) Osteoblast niche: Binding with osteoblast niche shows a higher
proliferation rate in primary leukemia, proven by BrdU staining in vivo (25).
Combined with chemotherapy, the residual leukemia is detected in
vascular niche as well as osteoblast niche. This result suggests that
leukemia cells receive antiapoptotic signals not only from vascular
endothelial cells but also osteoblasts (25).
(iii) Stromal cell niche: It consists of fibroblasts, macrophages, endothelial
cells and extracellular matrix (30). In serum-free tissue culture assay,
bone marrow stromal cells have shown to prevent B-lineage ALL cells
from apoptosis with reduced DNA fragmentation, chromatin condensation,
and nuclear fragmentation (31). Binding with bone marrow stromal cell
niche also provides B-lineage leukemia chemo-protection from drug-
induced apoptosis (7). In addition, adhesion of integrin α4 on leukemia
cells to fibronectin on bone marrow stromal cells leads to activation of the
9

PI-3K/AKT signaling pathway and upregulation of Bcl2 in AML cells, which
confers to drug resistance (32). For chronic myeloid leukemia (CML),
human bone marrow mesenchymal stromal cells (MSC) prevent CML
stem and progenitor cells from chemotherapy, TKI, through N-Cadherin-
mediated adhesion and Wnt-β-catenin signaling (33).

1.6 Other factors interact with leukemia in bone marrow
In addition to three major niches above, bone marrow microenvironment
also contains sympathetic nerve system (SNS) and mesenchymal stem
cells (MSC Nestin
+
) and their association leads to mobilization of
hematopoietic stem cells (HSC) (34). In contrast, bone marrow
macrophages secrete proteins to affect the production of CXCL12 by
MSCs and negatively regulate the mobilization of HSCs (35). Leukemia
cells also directly contact with cytotoxic effector cells, including nature
killer (NK) cells in bone marrow (36). NK cells bind to fibronectin and other
ECM in bone marrow mainly through VLA4 and VCAM-1 interaction. NK
(effector) cells first recognize and bind to the Fc region of the antibody,
such as IgG, which has bound to the surface of target cells, like the tumor
cells. The most common Fc receptor on the surface of NK cells is called
CD16 or FcγRIII. Once the Fc receptor binds to the Fc region, NK cells
10

release cytokines such as IFN-γ to promote cell death via apoptosis as
called the antibody-dependent cell-mediated cytotoxicity (ADCC) (37).
ADCC is involved in the binding between Fc region of the antibody on the
target cells and Fc receptor on the effector cells. Therefore, the cells
carrying Fc receptors may also generate ADCC effect, including
phagocytes (macrophages and monocytes), granulocytes (neutrophils and
eosinophils), and lymphocytes of the innate immune system. The
activated macrophages by various cytokines, like IL-4 and IFN-γ, have
been reported to perform ADCC effect as well as eosinophils on IgE-
coated parasites (38). Moreover, the interaction between leukemia cells
and adipocytes in bone marrow microenvironment has also been known to
prevent the chemotherapy-induced apoptosis (39).

11

Chapter Two
Establishment of pre-clinical model to evaluate alternative
therapies for ALL

2.1 Introduction

Xenograft model of primary ALL in NOD/SCID or NSG mice
Due to the limitations of the number of children eligible for the clinical trial
and more concerns about the safety, toxicity and efficiency of new drugs
prior to the clinical trial, a preclinical model for ALL is needed to evaluate
and prioritize novel therapies for future clinical trials (40). In most
experimental studies, ALL cell lines are first used but lack relevance to the
original patient cells. For example, a significantly higher frequency of
mutation or inactivation of p53, a tumor suppressor gene regulating cell
proliferation, in cell line has been observed compared to a lower frequency
in primary leukemia cases (41;42). Therefore, primary leukemia derived
from patients is the better choice because of the adequate reflection of
genotypic and biological characteristics to original patient sample.
Because only a small number of primary cells are withdrawal from patients,
primary cells were usually expanded in mice first, called xenograft mouse
12

model. Primary B-lineage leukemia cells were engrafted in immune-
deficient mice, but the engraftment efficiency has been problematic in the
beginning. Only 15.3% (104 out of 681 patient samples) of primary
leukemia samples was successfully engrafted in severe combined
immunodeficient (SCID) mice, which lack B and T cells (43). To increase
the successful rate, the nonobese diabetic/SCID (NOD/SCID) mice, which
had less natural killer cells and absence of circulating complement in
addition, have been used and the rate of engraftment raised to 69% (11
out of 16 patient samples) (44) and 75% (6 out of 8 patient samples) (45).
More importantly, this NOD/SCID xenograft model engrafted with primary
leukemia cells retains the leukemic morphology, sites of organ infiltration,
immunophenotype and frequency of p53 gene mutation of original patient
cells (46). On the other hand, to study hematopoietic stem cells (HSCs) in
NOD/SCID mice is restricted by the remaining NK cells and a short life
span due to development of thymic lymphomas (47). Therefore, new
mouse strain, NOD/SCID IL2Rγ
null
(NSG), has been developed, in which B
and T lymphocytes and NK cells are absent. These mice also have longer
life span (>16 months) with no mouse lymphoma developed from sublethal
irradiation (47). The engraftment of mobilized HSCs in NSG mice
generates 6-fold higher percentage than NOD/SCID mice (47). Due to the
13

high cost of NSG mice, I established xenograft model of primary ALL
using both NOD/SCID and NSG mice.

Prognostic features in xenograft model and preclinical evaluation of
alternative therapies
The group of Lock RB et. al. has reported the relationship between the
biologic characteristics of primary ALL cells in xenograft model and the
prognostic features of ALL patients (45;46). To evaluate novel or
alternative therapies, on the other hand, for ALL patients, the xenograft
NOD/SCID model has demonstrated the correlation between the in vivo
sensitivity to the drug of interests and the patient survival outcome. In this
chapter, I will first introduce establishment of the xenograft mouse model
and using this model to preclinically evaluate alternative therapies for
primary ALL. Ultimately, using this preclinical xenograft model may be
able to transfer the bench works of promising new treatments into clinical
trials for relapse and high-risk ALL patients.

14

2.2 Materials and methods:

Patient Samples
Bone marrow and peripheral blood samples from ALL patients were
provided by USC (Los Angeles, USA), UCSF (San Francisco, USA),
Samsung Medical Center (Seoul, South-Korea), and the University of
Bologna (Bologna, Italy) in compliance with the Institutional Review Board
regulations of each institution. Informed consent was obtained from all
human subjects.

Xenograft model of primary leukemia
Under IACUC approved protocols, NOD.Cg-Prkdc
scid
IL2rg
tm1Wjl
/SzJ (NSG)
or NOD/SCID mice of 5-7 weeks of age were conditioned with a single
sub-lethal dose of 250 cGy of whole body irradiation (250cGy), followed by
intravenous injection of about 1 – 5x10
6
pre-B ALL patient bone marrow or
peripheral blood cells per mouse (48).

Mice were monitored for weight
change and presence of human CD45
+
cells in the peripheral blood by
flow cytometry. Mice were sacrificed based on appearance, mobility, and
over 15% body weight loss of the animal. White blood cells (WBC) from
peripheral blood, spleen, and bone marrow were then isolated by gradient
separation (Ficoll) and stained with mouse and human CD45 antibodies to
15

confirm a full engraftment of primary ALL cells (>95% human CD45
+
).
Engrafted cells from mice directly receiving patient cells were considered
as primary passage and serially xenotransplanted into new mice up to
tertiary passage for cell expansion (Figure 1A).

Leniviral production, transduction, and bioluminescent imaging
pCCL-MNDU3-LUC was a third generation HIV-1 based, lentiviral vector
expressing the firefly luciferase gene (gift from Dr. Donald Kohn, UCLA).
Lentiviral supernatant was produced using the transfection reagent
polyetherimide (PEI; Sigma-Aldrich, St Louis, MO), for the triple
transfection of confluent HEK293FT cells with the 8.9 packaging plasmid,
pMDG-VSV-G, and the transfer plasmid. Plasmids were isolated using the
Qiagen Endotoxin Free Maxiprep (Qiagen, Hilden, Germany) grown from
stably transformed E.coli. Lentiviral supernatant was collected 72 hours
post-transfection, and concentrated by ultracentrifugation. Primary ALL
cells were transduced with pCCL-MNDU3-LUC lentivirus supernatant in
plates coated with retronectin (Takara, Shiga, Japan) containing serum-
free QBSF-60 medium (Quality Biologicals, Gaithersburg, MD) for 48
hours. Aliquots of all transduced cells were assayed to confirm
bioluminescent signal prior to transplantation (Figure 1B). The
transduction efficiency of luciferase was analyzed by immunofluorescence
16

(Figure 3) (48).

Monitoring of leukemia progression (LAX7R) by
determining bioluminescence signal development in mice was presented
in Figure 4A, B and compared with % of hCD45+ cells in peripheral blood
via FACS analysis (Figure 4C). A mouse with no leukemia injection and
treated only with luciferin at time of imaging was included as background
control mouse (Ctrl).

Immunohistochemistry
Tissues, including spleen, bone marrow, lung, and intestine from
experimental NOD/SCID or NSG mice were immersed in 10% formalin
(VWR, Radnor, PA) for 24 hours and transferred to PBS. Paraffin
embedding was done following standard procedures on a Tissue-TEK VIP
processor (Miles Scientific). 4 µm sections were mounted on Apex
superior adhesive slides (Leica Microsystems) and stained on a Ventana
BenchMark automated IHC stainer (Tucson, Arizona). The Bond
TM
ready-
to-use hCD45 (X 16/99) primary antibody (Leica Biosystems, Newcastle,
UK) was used and the antigen-antibody reaction were detected and
visualized by Ventana iView DAB detection kit (Tucson, Arizona), which
includes polymeric horseradish peroxidase (HRP) IgG secondary
antibody, substrate chromogen 3,3’-Diaminobenzidine tetrahydrochloride
(DAB), and other necessary reagents. Mounting medium (Pro-Long Gold
17

Antifade Reagent; Invitrogen) was applied and coverslips sealed, prior to
acquisition of fluorescent images at room temperature on a Zeiss Axiovert
200M inverted confocal microscope with a 40 Plan Neofluor objective
using IP Lab 4.0 software (Scanalytics). Photomicrographs were acquired
using a Hamamatsu ORCAER HAL100 digital camera (400x
magnification) and brightness was enhanced using Adobe Photoshop 6.0.

FACS antibodies
Cells were first stained with the antibody and the respective isotype
control for 30 minutes at 4 ْ C. After washed with washing buffer
(PBS+5%NGS +1%Sodium azide) once, cells were scanned by BD FACS
machine and the data was analyzed using FCS express software.

Mice
Under Childrens Hospital Los Angeles Institutional Animal Care and Use
Committee (IACUC) approved protocols, female NOD/SCID (NOD/LtSz-
scid/scid) mice of 5 -10 weeks of age were purchased from National
Cancer Institute at Frederick. NOD/SCID IL-2Rγ
-/-
(GKO), were purchased
from the Jackson Laboratory (ME, USA). All immunedeficient (ID) mice
were maintained in pathogen-free conditions and under a 12-hour light-
dark cycle and fed with irradiated food and autoclaved water.
18

2.3 Result

Establishment of a xenograft model of primary leukemia
To preclinically evaluate new therapy or improve the current treatment, it
is critical to establish a xenograft model using primary ALL and adequately
reflecting the biology of original patient leukemia. NOD/SCID or NSG mice
recipients of leukemia patients were sacrificed upon detection of human
CD45
+
cells in their peripheral blood as assessed by flow cytometry.
Xenograft spleens containing >95% human ALL cells were harvested as a
primary passage, characterized by flow cytometry and histology, and
expanded via serial transplantation up to three passages. So far we have
successfully engrafted 46 patient samples, including 40 pre-B ALL, 5 T
ALL and 1 AML, and developed a xenograft cell bank for worldwide
distribution (49;50). Among those, 14 xenograft and 1 cell line samples
were used for this study, and their characteristics and clinical information
were shown in Table 1. In order to non-invasively monitor the engraftment
of leukemia in vivo, primary leukemia cells were first transduced with
luciferase prior to the xeno-transplantation (Figure 1B). The transduction
efficiency of luciferase for a primary ALL with normal karyotype, LAX7R,
which is used through all experiments, was ~80%, calculated by
immunofluorescent staining (Figure 3). To compare monitoring techniques
19

for the growth of leukemia in vivo, mice engrafted with LAX7R cells were
continuously bled for FACS analysis and screened by bio-imaging. The
engraftment signal of leukemia was first detected on Day 11 post-injection
by bioimaging compared to Day 18 by FACS analysis (Figure 4 A,B).

Retention of phenotypic characteristics after serial passaging of
primary ALL in a xenograft mouse model
Phenotype and morphology studies were presented to determine
characteristics of xenograft samples over the series of passages were still
identical to the original patient cells. The immunophenotype data of 14
patient samples (1P) was compared with corresponding xenograft
samples in primary (1X), secondary (2X) and tertiary (3X) passages,
shown in Table 2. Patient cells and xenograft spleen cells were stained
with MHC (HLA-DR), B-lineage (CD10, CD19, CD24), T-lineage (CD2,
CD5, CD7), myeloid (CD13), and progenitor markers (CD34, CD38)
summarized in Table 3. The percentages (%) of cells staining positive for
expression of lineage-specific markers were analyzed by flow cytometry.
For morphologic feature of xenograft samples, Figure 2 demonstrated
immunohistochemistry of two selected examples, LAX7R and TXL2, after
serial passaging in NOD/SCID mice. Tissues, including bone marrow,
spleen, and liver, were stained with H&E (left panel) and human CD45
20

primary antibody (right panel) with 400X amplification. Comparison of the
immunophenotype and immunohistochemistry before and after passaging
in ALL xenograft mouse model shows that the original disease
characteristics were retained.

Preclinical evaluation of therapies for drug resistant ALL
To improve the current treatment regimen, several therapies, like
doxorubicin, cyclophosphamide, and methotrexate as examples, were
preclinically evaluated by xenograft mouse model. Different dose of
Doxorubicin (1 and 4mg/kg), Cyclophosphamide (100 and 150mg/kg) and
Methotrexate (6 and 8mg/kg) were given intraperitoneally to NOD/SCID
mice engrafted with luciferase labeled LAX7R cells. Bioimaging pictures
revealed capabilities of all three treatments to decrease the development
of primary leukemia at certain level in vivo (Figure 5A-C). Nevertheless,
the drugs needed further studies partly due to non-significant survival
benefit in the end or unbearable toxic side effect during the treatment.

2.4 Discussion
To preclinically evaluate drugs of interest, doxorubicin, methotrexate and
cyclophosphamide were taken as examples and investigated by xenograft
21

mouse model combined with bioluminescent imaging. Doxorubicin (Doxil)
was one of anthracycline antibiotics and used for cancer chemotherapy
due to its function on DNA intercalation. But, this drug had the most
serious adverse effect (>20%), including nausea, vomiting and life-
threatening heart damage, and thereby earned the nickname, like “red
devil” because of the drug color (Doxil product information, 2007).
Although both 1mg/kg and 4mg/kg Doxil treatments delayed the
engraftment of leukemia cells by bioimaging compared to the saline group,
the toxicity with significant weight loss (data not shown) caused the animal
death. Methotrexate (MTX) was the most widely used antimetabolite to
inhibit metabolism of folic acid and DNA synthesis for childhood cancers
(51). Cyclophosphamide (Cytoxan) was an alkylating agent to add an alkyl
group to DNA and to treat patients with cancer and some autoimmune
diseases (52). Although both MTX and Cytoxan treatments dramatically
decreased the progression of leukemia in vivo with less toxic side effects
compared to Doxil (data not shown), no survival benefits were observed in
xenograft model and some adverse effects such as nausea, vomiting and
hair loss were still reported in clinical trials. Therefore, novel drugs and
better therapeutic targets are still needed for ALL patients.
In summary, the original immunophenotype and morphology of the
patient’s disease can be retained after a serial of passages in mouse
22

xenograft model. Response to standard chemotherapy or other drug of
interests can be monitored by assessing peripheral blood of animals for
human CD45 and bioluminescent imaging and reflects the outcome of the
patient as a responder or non-responder to chemotherapy. This in vivo
model allows to study ALL as a systemic disease to understand the cause
for relapse of leukemia and to develop treatment improvements.

23

2.5 Figures
Table 1 Clinical information of xenograft samples

*Cell line

24

Table 2 Immunophenotype of original patient and xenograft samples in
serial passages

25

Table 3. Antibodies used for flow cytometry

26

Figure 1 Schematics for xenograft mouse model and bioluminescent
imaging.

27

Figure 2 Morphology of xenograft samples

28

Figure 3 Transduction efficiency of luciferase by immunohistochemistry

29

Figure 4 Monitor leukemia progression by bioluminescence signal and
FACS (%) hCD45
+
in peripheral blood

30

Figure 5 Preclinically evaluate drugs of interests in xenograft model

31

Figure legends:

Figure 1 Schematics for xenograft mouse model and bioluminescent
imaging.
(A) Patient cells were intravenously (I.V.) injected into the tail of
NOD/SCID or NSG mice with 250cGy sublethal-irradation. After a serial of
passaging from primary (1’) to tertiary (3’), xenograft cells were expanded
and harvested from mouse spleen. (B) Luciferase labeled xenograft ALL
cells were intravenously (I.V.) injected into mice. Bioluminescent
signalings from labeled ALL cells were detected and represented as the
engraftment of human leukemia.
Figure 2 Morphology of xenograft samples
Immunohistochemistry of LAX7R and TXL2 after serial passaging in
NOD/SCID. Tissues, including bone marrow, spleen, and liver, were
stained with H&E (left panel) and hCD45 primary antibody (right panel).
400X Amplification.

Figure 3 Transduction efficiency of luciferase by immunohistochemistry
Compared to the control cells with no transduction, luciferase-transduced
ALL cells showed red signal on the cell membrane and blue signal from
DAPI on nuclei by immunohistochemistry. The transduction efficiency (%)
32

was calculated by the number of transduced cells divided by the total
number of cells.

Figure 4 Monitor leukemia progression by bioluminescence signal and
FACS (%) hCD45
+
in peripheral blood
(A) The engraftment of LAX7R cells in vivo was monitored by
bioluminescence signal starting on Day11 post-cells injection. (B) The
signal at each time point was quantified by average radiance
(Photons/sec/cm
2
/sr). (C) % of human CD45+ LAX7R cells in peripheral
blood was analyzed by flow cytometry.

Figure 5 Preclinically evaluate drugs of interests in xenograft model
Luciferase-labeled LAX7R cells were injected into sublethally irradiated
NOD/SCID mice (2x10
5
cells/mouse). Recipient mice were treated with
Saline, (A) Doxorubicin (1 and 4mg/kg), (B) Cyclophosphamide (Cytoxan)
(100 and 150mg/kg) and (C) Methotrexate (MTX) (6 and 8mg/kg)
intraperitoneally starting on Day13 post-cell injection. Bioluminescent
imaging of experimental groups was used to monitor the engraftment of
primary leukemia in mice.

33

Chapter Three
Targeting integrin α4 sensitizes drug resistant human ALL
to chemotherapy

3.1 Introduction

Adhesion receptor, integrin α4
Integrins are heterodimeric glycoproteins and consist of non-covalently
linked subunits: 18 α and 8 β, which have been identified to assemble 24
distinct combinations of integrins in human up to date (30;53). Roles of
integrins include the cell-cell or cell-extracellular matrix (ECM) adhesion,
cell migration, like lymphocyte trafficking, embryonic development,
inflammation, binding sites for certain virus, and intracellular signaling
changes as shown in Figure 6 (53;54). Integrin α4 (ITGA4/CD49d) (MW
~155 kDa) forms a heterodimer of integrin α4β1 (VLA4) with integrin β1
(ITGB1/CD29) (MW ~150 kDa). Integrin α4 is expressed on most
mononuclear leukocytes (55) and human hematopoietic stem and
progenitor cells (HSPC) for regulation of cell homing and mobilization
(56;57). Its main ligand, VCAM-1 (CD106), is a surface membrane protein
expressed on many different types of cells, like activated endothelial cells,
34

bone marrow fibroblasts, and spleen stromal cells (58), and its expression
can be upregulated by cytokines such as IL-1, IL-4 and TNF-α (59).
VCAM-1 consists of 7 Ig-like domains (D1-D7) and two of domains, D1-D2
and D4-D5, bind to β-propeller on integrin α4 and β I/A-domain on integrin
β1 of VLA4 (60;61). Integrin α4 also binds to other ligands such as CS-1
region in the COOH-terminal heparin-binding domain of fibronectin (fn)
(62), which is a major component of extracellular matrix in bone marrow,
and mucosal addressin cell adhesion molecule-1 (MadCAM-1) expressed
on endothelium of venules (17). In addition to its dominant function in cell
adhesion and mobilization, integrin α4 also plays a role of signal receptor
bidirectionality across leukemia cells and the surrounding
microenvironment in order to regulate gene transcription, cell survival,
proliferation, differentiation, and apoptosis (54;63).

Integrin α4 in leukemia
Adhesion with bone marrow stromal cells has been reported to prevent B-
lineage ALL and chronic lymphoblastic leukemia (CLL) cells from
apoptotic cell death in vitro. (31;64). Disrupting the adhesion between
integrin α4 and stromal cells sensitizes AML cells to chemotherapy in vitro
and in vivo (32). In contrast, Philadelphia chromosome-positive CML
progenitor cells have defective binding capacity to stromal cells or
35

fibronectin to escape from growth control restraints and cause
extramedullary disease by the release of leukemia cells into the circulation
(65). Recently, the expression of integrin α4 has been proposed as a
prognostic marker for overall survival of ALL and CLL patients (66;67). For
T-ALL, the bindings between integrins, like integrin α1β1 and α2β1, and
their corresponding collagens, like collagen IV and I, protect T-ALL cell
line from apoptosis (8;68). However, our knowledge about integrin α4 in T-
ALL is limited. Furthermore, the functional modulation and therapeutic
translation of integrin α4 has not addressed in pre-B ALL. Therefore, I
hypothesized that integrin α4 plays a critical role in the regulation of cell
adhesion and bone marrow-mediated drug-resistance of ALL.

Targeting integrin α4 in primary leukemia by monoclonal antibody
To study the functionality and further mechnisms of integrins, antagonism
of targeting the binding site on integrins becomes the most common
approach. Integrin antagonism has been accomplished by the use of
monoclonal antibodies (mAb), peptide-based antagonists, and non-
peptide small molecules as described in details below. These integrin
antagonists have been developed in recent years and a large number of
clinical trials based on integrin inhibition for diverse inflammatory diseases
and late stage tumors are summarized by Hehlgans S. et al., 2007.
36

Monoclonal antibodies (mAb) are the most common integrin antagonists
due to their high specificities, long half-life, and lower immunogenicity.
High specificities to the target facilitate the efficiency of the treatment and
minimize the off-target effects of the drug (69). A longer half-life of the
drug allows infrequent dosing, and a lower immunogenicity improves the
risk-benefit ratio (70;71). These advantages lead to 20 mAbs approved by
FDA and more than 150 other mAbs in clinical trials up to now (72). The
disadvantages are the high cost of production, the infusion reaction,
including acute anaphylactic, serum sickness, tumor lysis syndrome
(TLS), cytokine release syndrome (CRS), and the infection, like PML as
described below (69;73).
Tysabri, (Natalizumab; Biogen Idec/Elan) is a recombinant monoclonal
antibody produced in mice but targeting human integrin α4 (humanized)
using IgG4 as its Fc region to reduce unwanted antibody-dependent cell-
mediated cytotoxicity (ADCC) (69). The drug is currently the only one α4
antagonist approved by FDA and used to treat patients with relapsing
multiple sclerosis (MS), the disease that T and B cells attack against the
white matter of the central nervous system (CNS) (74). It has also been
reported to be a potential therapy for inflammatory bowel disease (IBD)
such as Crohn’s disease, the attack against intestinal mucosa, and
rheumatoid arthritis, the attack against synovial lining of the joints, tested
37

in animal model and clinical trial phase II (75;76). The dose of Tysabri for
human studies has been designed and ranged from 3-6 mg/kg of body
weight (74). The half-life of Tysabri is approximately 11 days reported in
clinical trials and can be detected in serum 3 to 8 weeks post-treatment in
patients receiving 3mg/kg dose (77). After passing safety and
pharmacokinetic study in phase I in 1999 and the efficiency study in phase
II in 2002, Tysabri was approved by FDA for relapsed MS patients in 2004
(74). But after three patients (two fatal) taking Tysabri for more than two
years developed progressive multifocal leucoencaphalopathy (PML),
Tysabri was withdrawn from the market in 2006 (78). The following study
shows totally 3 patients getting PML out of 3116 patients receiving Tysabri
with a mean duration of 18 months that the estimated ratio of risk is 0.1%
for developing PML from Tysabri treatment (78). After this evaluation, FDA
re-approved Tysabri as a monotherapy for relapsing MS patients in 2006
(78). As of May 2010, a total of 49 cases of PML were reported among
67700 Tysabri-treated patients worldwide (79). The most frequent side
effects of this drug are headache (38%), fatigue (27%), and infusion
reaction such as urticarial or rash (24%) (77). The most severe side effect
is PML (0.1%) in patients receiving at average 18 months of Tysabri
treatment (80). PML is a fatal and rapidly progressive demyelinating
disease caused by reactivating the latent infection in the central nervous
38

system (CNS) with the polyoma virus John Cunningham virus (JCV) and
often seen in immunocompromised patients such as HIV patients. The
reason to increase the incidence of JCV infection in patient treated with
Tysabri is due to the inhibition of circulating immune cells, like T cells, in
CNS (81). To decrease the risk of the use of Tysabri, FDA has
recommended limiting the population to those refractory to other
treatments or with aggressive cases. Moreover, MRI imaging of the brain
and cerebrospinal fluid analysis for JCV DNA have been used to monitor
the occurrence of PML (82). In MS patients, Tysabri treatment has been
shown to increase the circulating hematopoietic and stem progenitor cells
(HSPC) in peripheral blood that theoretically could be a new mobilizing
agent for patients unresponsive to G-CSF (59).

Targeting integrin α4 in primary leukemia by small molecule
inhibitors
Due to the potential side effect of Tysabri treatment, the pharmaceutical
industry has been focused on other alternatives of integrin α4 antagonism
(78). Although this small molecular inhibitor generally has a shorter
plasma half-life and lack of specificity compared to mAbs (73), it can
decrease the chance to get PML symptoms and the costly plasma
39

exchange procedures for clearance of Tysabri and restoring the immune
response to the infection in the blood (83).
Peptide-based small molecular inhibitors are designed to contain peptide
sequences similar to ligand recognition sequence in endogenous integrin
ligands in order to compete with ligand to the integrin. This type of drugs
can be developed more easily since the ligand recognition sequence and
the paired integrin have been identified. Bio1211 is a peptide-based small
molecular inhibitor targeting two α4 integrins, α4β1 and α4β7 (Duel
antagonist). Peptides are short polymers of amino acids linked by peptide
bonds and usually contain less than 50 amino acids in sequence. Bio1211
contains a tri-peptide motif (LDV), including 3 amino acids: leucine (L),
aspartic acid (D) and valine (V) (Figure 7A), which is similar to ligand
recognition sequence to bind to fibronectin (84;85). Aspartic acid (D)
(negative charge amino acid) in Bio1211 is known to lie above the Mg
2+

ion in I-domain (~190 residues) near N terminus of β1 subunit of VLA4
integrin. This is similar to the binding interaction of VLA4 and its main
ligand, VCAM-1, which uses aspartic acid (Asp-40) on VCAM-1 to stabilize
divalent cation like Mg
2+
of β1 I-domain in VLA4 integrin (60). In Figure 7B,
it demonstrates the extrinsic ligand, like Bio1211, binds to β1 I-domain in
activated VLA4 integrin (86). Valine (V) and leucine (L) in LDV motif, on
the other hand, share hydrophobic interactions with L205 and P228
40

residues in β1 I-domain (87). Binding with Bio1211 has shown to activate
integrin VLA4 with the conformational changes (88). The mechanism of
integrin activation initiates from the binding of ligands (VCAM-1), ions
(Ca
2+
or Mg
2+
) or integrin inhibitors, like Bio1211. Through the cytoplasmic
domain of integrin β1, G-protein coupled receptors, adjacent to integrin
β1, trigger the intracellular signaling and induce the conformational
changes of the integrin, so called inside-out signaling. From inactive state
to active state, the conformation of integrin changes from a bent to an
extended form with a switchblade-like motion (89). Using FITC labeled
Bio1211 probe on U937 leukemia cell line monitors at real time the
conformational change of integrin VLA4 via a sequential reduction of the
disulfide bonds (-S-S- → -SH +HS-) (88). Bio1211, however, is unstable in
animal and human blood and plasma and converts rapidly to Bio1588, a
much less active molecule (17-18 fold) than Bio1211 (90). Therefore, the
compound is dropped in phase II trials of asthma due to disappointing
efficacy (78). In addition, this small molecule inhibitor is a high cost
product and requires intravenous administration which has potential to
cause the infusion reaction, as compared to non-peptide small molecule
inhibitor described following.
Non-peptide small molecule inhibitors are synthetic integrin antagonists
with low molecular weight that contributes to a faster synthesis and a less
41

cost of production, compared to antibody and peptide-based small
molecule inhibitor. In addition, this type of drug has better stability and can
be administered orally in many cases. This molecule still encounters
limitation in serum protein binding and low integrin specificity compared to
monoclonal antibodies (73). TBC3486 is a N,N-disubstituted amide and
non-peptidic small molecule inhibitor. It binds to Mg
2+
ion in β1 I-domain of
VLA4 integrin while maximizing the contact between its hydrophobic group
with L205 and P225 of β1 integrin. In addition, several hydrogen bonds
form between urea NH groups and nonurea carbonyl oxygen in TBC3486
and S227 and K208 in β1 integrin (Figure 7C) (87). TBC3486 inhibits only
the high affinity form of the integrin VLA4 to avoid the side-effects of the
treatment. In vitro toxicity studies and reproductive toxicology studies in
rats and rabbits have shown teratogenic results due to inhibition of
integrins with both high and low affinities (91;92). TBC3486 has shown a
20-fold selectivity to high affinity form of VLA4 in the presence of 1mM
Mn
2+
over the low affinity state with 1mM Mg
2+
and 1mM Ca
2+
(93). Unlike
Bio1211, TBC3486 has high selectivity to α4β1, not α4β7. The diseases
attenuated by inhibition of α4β1 or α4β7 integrins are different in many
cases (94). For examples, α4β1/VCAM-1 interaction plays an important
role in experimental autoimmune encephalomyelitis (EAE) model for
Multiple Sclerosis whereas α4β7/MAdCAM-1 interaction is dominant in
42

models of Inflammatory Bowel Disease (IBD) (95;96). With adhesion
assay in vitro, TBC3486 shows a 200-fold selectively inhibition to α4β1 on
activated T-cells compared to α4β7 (93). In addition, no activity of binding
is detected between TBC3486 and other integrins, like β2 and β3 integrins
(93). To determine the binding effect of TBC3486 on conformational
changes of VLA4 integrin, Vanderslice, P. et. al. group used a monoclonal
antibody B44 to recognize a ligand induced binding site (LIBS) on β1
integrin. This binding site was available for B44 only in the binding
interaction between VLA4 integrin and VCAM-1 ligand or the
conformational changes from inactivated form to activated form of integrin.
With the analysis of flow cytometry, TBC3486 and soluble VCAM-1
(sVCAM-1) both showed similar and increasing B44 binding results that
indicated TBC3486 not only induced activation and conformational
changes of VLA4 integrin, like Bio1211, but represented as a ligand
mimetic of VCAM-1 to act as a competitive antagonist against integrin
VLA4 (93). Blocking integrin α4β1 by TBC3486 has been reported to
inhibit inflammatory T-cell proliferation and activation in vitro (93).
Moreover, TBC3486 can suppress an eosinophil-mediated allergic
inflammation in lung using in vivo model by inhibit circulating leukocytes
from entering the lung (93). Due to the selectivity to the high affinity
conformation of integrin α4β1 and minimal activity to integrin α4β7,
43

TBC3486 can be a therapy for α4β1-dependent diseases with fewer side
effects. I plan to investigate the pre-clinical values of TBC3486 as
adjuvant treatments of pre-B ALL cells using our established models in
vitro and in vivo.

3.2 Materials and methods:

Drugs
Dr. Bonig from the University of Washington kindly provided Tysabri
(Natalizumab, Biogen Idec, Cambridge, MA, USA) (20mg/ml). Tysabri was
diluted with PBS (0.9% Sodium Chloride) for both in vitro and in vivo
treatment. TBC3486 is a non-peptidic small molecule inhibitor targeting
α4β1 integrin and synthesized in Dr. Vanderslice’s lab. The drug and its
enantiomer control THI0012 were dissolved in 100% sterile DMSO at a
concentration of 10 mM for in vitro assay but dissolved in PBS at a
concentration of 1 mg/ml for in vivo applications. Standard chemotherapy
for Ph- ALL, VDL, included the combination of V: Vincristine (2mg/2ml), D:
Dexamethasone (4mg/ml) and L: L-Asparaginase (2000IU/ml) in stock and
was administered 5 times a week for continuous 4 weeks (50). Vincristine
binds to Tubulin to disrupt the formation of microtubule and eventually
arrest metaphase in the cell mitosis. Dexamethasone is a steroid drug
44

usually for inflammatory and autoimmune diseases. L-Asparaginase is an
enzyme to catalyze the hydrolysis of asparagine to aspartic acid. VDL was
diluted with PBS (0.9% Sodium Chloride) for both in vitro and in vivo
treatment.

Microarray
Two independent gene expression datasets from the groups of James
Downing with St. Jude ALL microarrays (97) and Jacques van Dongen
with normal development arrays (98) were analyzed using BRB array tools
for integrin α4 expression. Integrin α4 overexpression was calculated by
the ratio of the averaged signal intensity of 15 patient samples in each
subtype of leukemia, including E2A-PBX1, TEL-AML1, BCR-ABL, MLL
rearrangements, and T-ALL, to the averaged 14 human umbilical cord
blood CD34
+
lin
-
isolated by MACS CD34 beads, precursor B cell subsets
from freshly bone marrow samples and CD3+/CD8+ thymocyte (T) cells of
healthy children (3-16 years of age) (98). The calculated signal ratios of
integrin α4 Gene Expression Probeset# 213416 using Affymetrix HG-U133
set with 39000 transcripts were visualized as a heatmap with Java
Treeview.

45

Quantitative PCR
RNAs from ALL patient xenograft samples were isolated using RNeasy
Plus MiniKits (Qiagen, Hilden, Germany), with subsequent cDNA
synthesis using SuperScript III Reverse Transcriptase (Invitrogen,
Carlsbad, CA). Quantitative real-time PCR was performed by mixing
cDNA samples with the SYBR GreenER (Invitrogen) and primers listed in
Table 4. The samples were loaded into an optical 96-well reaction plate
(Applied Biosystems, Foster City, CA) and analyzed by ABI7900HT real-
time PCR system (Applied Biosystems, Foster City, CA). Human integrin
α4 mRNA expression (Accession Number: NM000885) as a percentage of
GAPDH mRNA was characterized in primary ALL xenografts by
quantitative PCR.

Western blotting
Cells were lysed in M-PER buffer (Thermo Scientific) supplemented with a
1% protease inhibitor cocktail (Pierce) and proteins were separated by 4%
to 12% SDS-PAGE and electro-transferred to PVDF membrane
(Invitrogen). For the detection of mouse and human proteins by Western
blot, primary antibodies listed in Table 5 were used together with the
WesternBreeze immunodetection system (Invitrogen). Antibodies against
β-actin were used as a loading control (AC-15, Santa Cruz
46

Biotechnology). Blots were visualized using Alkaline Phosphotase-
conjugated secondary antibody solution (Invitrogen) followed by
Chemiluminescent substrate detection (Invitrogen) and exposed to Blue
Lite Autorad film (GeneMate).

Correlation of integrin α4 gene expression on leukemic blasts with
clinical outcomes of pre-B ALL patients
Patient clinical and outcome data were obtained from the National Cancer
Institute TARGET Data Matrix
(http://target.nci.nih.gov/dataMatrix/TARGET_DataMatrix.html) of the
Children’s Oncology Group (COG) Clinical Trial P9906 (99).

COG

P9906
enrolled 272 eligible high-risk B-precursor ALL patients, which were
treated uniformly with a modified augmented Berlin-Frankfurt-Münster
Study Group (BFM) regimen, including an increasing intensity of
Vincristine, Prednisone, and Asparaginase (100). Patients with very high-
risk features (BCR-ABL1 or hypodiploidy) were excluded (99). The gene
expression microarray data were obtained from pretreated ALL patients in
Gene Expression Omnibus (http://www.ncbi.nih.gov/geo) with accession
number GSE11877. Kaplan-Meier survival analysis was used to estimate
overall survival (OS) and relapse-free survival (RFS). Log-rank test was
used to compare survival differences among patient groups. R package
47

"survival" version 2.35-8 was used for the survival analysis (R
Development Core Team, 2009). The majority of patients had MRD
assessed by flow cytometry, as previously described; cases were defined
as MRD positive or MRD negative at the end of induction therapy (day 29)
using a threshold of 0.01% (99).

Adhesion of primary leukemia cells
Primary leukemia cells (LAX7R, AML-1, LAX1R) were either pre-treated
with function-blocking integrin α4 antibody (Ab) Tysabri or control IgG4
(G17-4) Ab (BD Biosciences) for 30 minutes at 37 °C and washed once
with PBS. For TBC3486, primary ALL cells were activated by 1mM Mn
2+

for 45 minutes prior to TBC3486 or control treatments. Cells were then
loaded in triplicates on 12-well non-tissue culture treated plates coated
with or without human recombinant VCAM-1 (hVCAM-1; 10 μg/ml) (R&D
Systems, Minneapolis, MN) and BD BioCoat human fibronectin (Fn) (BD
Biosciences), plus 2% BSA (A2153, Sigma-Aldrich, St Louis, MO). Pre-
treated leukemia cells were also co-cultured with the mouse bone marrow
stromal cell line OP9, or the human bone marrow stromal cell line HS-5
(ATCC No. CRL11882) in 12-well tissue culture treated plates. After 2
hours incubation, suspension cells in the supernatant were removed and
the plate was washed once with PBS. Adherent cells were photographed
48

with an Olympus IX71 microscope and then detached by pipetting 20
times. The cell count for adherent cells was assessed by trypan blue
exclusion of dead cells.

Primary leukemia cells in vitro for drug testing
Leukemic cells harvested from the spleen were placed on irradiated OP9
feeder layers (1x10
6
cells/ml) in the presence of media (αMEM + 20% fetal
bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin) (101).
All primary leukemic cells established in this manner were analyzed by
FACS using human and mouse anti-CD45 antibodies to verify that they
were human leukemia cells. For drug testing, primary leukemia cells in
vitro were co-cultured with irradiated bone marrow stromal cells OP9 or
HS-5 and treated with anti-integrin α4 monoclonal antibodies as a single
agent or in combination with chemotherapies, VDL (0.0005 µM Vincristine,
0.005 nM Dexamethasone, 0.0005 IU/ml L-Asparaginase) for B or T-ALL
or AraC (1.0 µM) for AML. Experiments were performed in triplicates. Cell
viability was determined by trypan blue exclusion. Statistical significance
of differences between groups was determined by the unpaired t-test. All
cell cultures were kept at 37°C in a humidified incubator under a 5% CO
2

atmosphere.

49

Cell Starvation and Stimulation
LAX7R was washed with PBS and incubated in serum-free media for 4
hours prior to treatment. Cells were then treated with IgG4 or Tysabri for
30 minutes and washed with PBS once. LAX7R was stimulated by
seeding on VCAM-1+1%BSA coated plates with serum-free media for 48
hours (Figure 15A).

In vivo integrin α4 blockade of primary leukemia cells in combination
with chemotherapy
Luciferase-labeled leukemia cells were injected into sub-lethally irradiated
NSG or NOD/SCID mice as described above.

Mice were then treated
intraperitoneally with Immunoglobulin (Ig) control or anti-Integrin α4 mAb,
Tysabri (5 mg/kg) once per week, or combined with chemotherapy
consisting of VDL from Day 3 to Day 29 after cell transfer. For TBC3486,
mice were treated intraperitoneally with control THI0012 or TBC3486 for 2
weeks alone (Day3 to Day14), or in combination with chemotherapy
consisting of VDL. During the treatment, mice were weighed daily but
weekly after the treatment. Based on over 15% weight loss compared to
the initial weight, mice were sacrificed. White blood cells in peripheral
blood, spleen, and bone marrow were separated by gradient separation
using Ficoll and analyzed by flow cytometry and immunohistochemistry.
50

The survival time of mice is presented by a Kaplan-Meier survival curve
and analyzed by Log-rank test.

CBC counts
Peripheral blood was withdrawn via the tail vein for CBC analysis from
mice when they were sacrificed. Blood samples (~100 µl/sample) were
collected in BD microtainer tubes with EDTA (BD Biosciences) and
analyzed on a VetScan HM5 cell counter (Abaxis, Union City, CA).

CFU assays
CFU assay for primary ALL cells were performed in triplicates by plating
an equal number of viable white blood cells (WBC) isolated from femurs,
spleen, liver, and lung, pooled from 2 animals (1x10
5
cells/dish) as
described below, and from peripheral blood by red blood cell lysis using
ammonium chloride (1.2 ml/per dish). WBCs were cultured in MethoCult
GF
+
H4435 and incubated at 37
o
C in humidified 5% CO
2
for 18 days.
Colonies were captured under confocal fluorescent microscopy (Carl Zeiss
Microimaging, Thornwood, NY) equipped with an Orca C4742-80-12AG
camera (Hamamatsu Photonics, Bridgewater, NJ) at room temperature
under 2.5x magnification. The microscope and camera were controlled by
Micro-Manager 1.4 software. Micrographs were acquired using QCapture
51

software 2.98 (Quantitative Imaging Corp; Surrey, BC Canada) via a
QImaging QiCam, mounted on an Olympus IX71 microscope.

White blood cell isolation from mouse organs
For homing and mobilization assays, femurs, spleen, peripheral blood,
lung, and liver were harvested in cold PBS. The white blood cell isolation
from femurs, spleen, and peripheral blood was described similarly to the
xenograft model below. Lung and liver tissues were dissected into small
pieces, incubated with collagenase enzyme, 2 µl Liberase TM (2 U/ml)
(Roche, Mannheim, Germany)/mg of the tissue, to dissociate collagen in
organs at 37 ْ C for 45 minutes and finally then filtered through a 70µm cell
strainer (BD Bioscience). The resulting single cell suspension was kept in
cold IMDM media, plus 20% FBS for further analysis.

Isolation of NK cells and Calcein-AM release assay
ADCC (Antibody-dependent cell-mediated cytotoxicity) of primary ALL
cells was determined using a Calcein-AM release assay as previously
described
24
. NK cells (>94% CD56
+
CD3
-
) were isolated from fresh filtered
blood from healthy donors (CHLA Blood Bank) using Ficoll-plague plus
(GE Healthcare, Pasadena, CA) density gradient centrifugation and further
isolation with a MACS NK cell isolation kit (Miltenyi Biotec, Auburn, CA).
52

Primary ALL cells were labeled with 5µM Calcein-AM (Invitrogen) for 30
minutes at 37 ْ C and then treated with NZM or IgG4 control Ab and
washed with PBS once. Primary ALL cells as target cells (T) were mixed
with isolated NK cells as effectors cells (E) at E/T ratio from 5:1 to 15:1.
After 4 hours incubation, cells were transferred to a black ViewPlate-96
plate and read on Filter Max F3 Microplate reader (Molecular devices,
Sunnyvale, CA) using 485 nm excitation/535 nm emission filter set. %
specific lysis was calculated by the following equation: % specific lysis =
(mean experimental release–mean spontaneous release) / (mean
maximal release–mean spontaneous release) x 100% (102).

Immunohistochemistry
For Ki-67 staining, slides were prepared first as previously described
(103) and stained with the Bond
TM
ready-to-use human Ki-67 (K2) primary
antibody (Leica Biosystems) followed by polymeric alkaline phosphatase
(AP) IgG secondary antibody and substrate chromogen, Fast Red. Slides
were also counterstained with routine hematoxylin for cell nuclei.

53

3.3 Result

Correlation of integrin α4 gene expression on leukemic blasts with
clinical outcomes of ALL patients
I analyzed two publicly available gene expression datasets from the
groups of James Downing and Jacques van Dongen using BRB array
tools for integrin α4 expression (97;98). Compared to normal B and T-
cells, I determined pediatric ALL patients with different leukemia
subtypes, including E2A-PBX1, TEL-AML1, BCR-ABL1, MLL
rearrangements, and T-ALL, had 3 to 15 fold overexpression of integrin
α4 (Figure 8A, B). This comparison indicated that integrin α4 was
upregulated in ALL patients. To determine the role of integrin α4 in clinical
survival outcome, expression of integrin α4 mRNA (ITGA4) in 207 ALL
patients uniformly treated with the COG P9906 clinical trial (Kang H,
2010) was correlated with the survival outcome. Overall survival (OS) of
minimal residual disease positive patients (MRD
+
) (n=67) was analyzed
further by integrin α4 expression and could be separated into MRD
+
α4
high

(ITGA4 expression ≥ mean; n=34) and MRD
+
α4
low
expressing cases
(ITGA4 expression < mean, n=33) (Figure 9A,B). α4
high
leukemia was
associated with poor outcome indicating the potential use of α4 as a
54

therapeutic target, as it is expressed especially highly in the prognostically
poorest cases.

Integrin α4 is expressed in ALL.
Upon our mouse xenograft model, six patient-derived (primary) pre-B ALL
samples (Table 1) compared with two CD19
+
samples from peripheral
blood of healthy donors were screened by FACS for expression of human
CD49d antigen (integrin α4) and presented a s FACS dot plots on Figure
10A. Their mRNA and protein expression of human integrin α4 were
analyzed by quantitative real-time PCR and western blot in Figure 10D.
Our analysis concluded the up-regulated expression of human integrin α4
on primary ALL samples compared to normal pre-B cells. The expression
of integrin α4 for three primary T-ALL (Figure 10B) and one primary AML
samples (Figure 10C) was presented over 95% by FACS dot plot.

Blocking integrin α4 by Tysabri inhibits the adhesion of pre-B ALL.
To test the effect of integrin α4 on cell adhesion, I first performed a
saturation assay for Tysabri to determine the maximum dose of saturated
antibody binding. Primary relapsed pre-B ALL cells, LAX7R (1x10
6
cells)
were treated with different doses of Tysabri for 30 minutes. FACS
histogram reflected 20 µg/ml Tysabri as the saturating dose, compared
55

with uncoated control (Figure 11A). Viabilities of treated leukemia cells as
well as normal pre-B cells were unaffected by the same dose of α4 mAb
treatment (20 µg/ml) for 48 hours incubation (Figure 11B, Figure 11A, B).
Matched IgG4 Ab served as the control for all in vitro experiments. This
may not constitute an ideal control antibody, because it does not bind to
the ALL cells at a similar density and affinity as the anti-α4 antibody, but
has been routinely used for similar studies. α4 antibody-dependent cell-
mediated cytotoxicity (ADCC) was specifically excluded by demonstrating
that NK cells (CD56
+
CD3
-
) of healthy donors do not impact lysis of primary
ALL cells treated with IgG4 control or Tysabri as determined by a Calcein-
AM release assay (Figure 13A, B). For functional studies, LAX7R cells
were then pre-treated with Tysabri or corresponding isotype controls IgG4.
Cells were then loaded in triplicates to plates coated with 10µg/ml human
recombinant VCAM-1. After two hours incubation, the adhering cells were
photographed (Figure 14A). Integrin α4 blockade by Tysabri showed
significantly smaller % of adhesion to VCAM-1 compared to control IgG4-
treated cells. (Figure 14B). In addition, Tysabri treatment also inhibited
adhesion of LAX7R cells to human fibronectin (Fn), OP9 cells (mouse
bone marrow stromal cells) or HS-5 cells (human bone marrow stromal
cells) (Figure 14C). In summary, blocking Integrin α4 by Tysabri inhibits
adhesion of primary pre-B ALL cells to human VCAM-1.
56

Integrin α4 plays a critical role on integrin-mediated intracellular
signaling.
In addition to its main function on cell adhesion, integrin also regulates
intracellular signaling (Figure 6). After the ligand, like ECM, binds to the
extracellular domain of the integrin, the conformational changes of integrin
are induced, followed with integrin clustering for activation of signaling
cascades and recruitment of multiprotein kinases, like ILK and FAK (104).
To study the role of integrin α4 on signaling of human leukemia cells, I first
performed a short-term starvation assay by culturing LAX7R cells with
serum-free media and then stimulated leukemia cells by the ligand,
VCAM-1 (31;105) (Figure 15A). At different time points (less than 48
hours), Tysabri inhibited the adhesion of LAX7R cells to hVCAM-1 (Figure
15B) and downregulated expression of p-AKT and p-Erk1/2 protein by
western blot (Figure 15C). I concluded that blocking integrin α4 by Tysabri
treatment had a potential to reduce Akt-mediated integrin signaling in
human leukemia cells.

57

Targeting integrin α4 by Tysabri sensitizes drug resistant pre-B ALL
to chemotherapy.
Pre-B ALL (LAX7R) cells were first co-cultured with or without bone
marrow stromal cells OP9 and received with or without the chemotherapy
VDL (Vincristine, Dexamethasone, and L-Asparaginase). Compared with
the control groups with no OP9 cells, a significantly increasing viability in
OP9 co-culturing groups indicated chemo-protective effect from the bone
marrow stromal cells (Figure 16). But combining with Tysabri treatment
reduced the viability showing the fact that Tysabri sensitized primary ALL
cells to chemotherapy.

Blockade of integrin α4 delays the engraftment of leukemia in vivo.
The central hypothesis is that disruption of integrin α4/VCAM-1 binding will
sensitize ALL cells to conventional drug-treatment by dislodging them from
their bone marrow niche. To test this central hypothesis, I evaluated
integrin α4 blockade in vivo using our xenograft model of primary ALL as
described above. Six pre-B ALL cells labeled with luciferase and pre-
treated with Tysabri or Ig control were injected into 3 NOD/SCID mice in
each group. The engraftments of human leukemia were monitored by
bioimaging at different days post-injection presented in Figure 17A. Whole
body bioluminescent signals of six ALLs were quantified in unit of average
58

radiance in Figure 17B and demonstrated significant differences between
Tysabri and Ig-treated groups in all six pre-B ALL samples (p<0.05). The
survival time for individual and pooled ALLs were presented by Kaplan-
Meier survival curves in Figure 17C, D (p<0.05). I demonstrated that
integrin α4 blockade by Tysabri delayed the progression of pre-B ALL and
led to prolonged survival.

Pharmacologically targeting integrin α4 in combination with standard
chemotherapy prolongs survival of NOD/SCID recipients of human
leukemia cells in vivo
Luciferase-labeled primary drug resistant LAX7R cells were transplanted
into NOD/SCID mice, which were treated with either Ig alone, Tysabri
alone, Ig with chemotherapy VDL or the combination of Tysabri with VDL.
After one month of treatment, Tysabri treated group showed less
bioluminescent signal than the Ig group in Figure 18A. In addition to this,
in contrast to the relapse of leukemia in the Ig+chemotherapy group, no
occurrence of leukemia in the combination treatment group was observed
from day 34 to day 71 post cell injection (Figure 18A). This data shows
that by targeting integrin α4 in combination with chemotherapy we are able
to overcome relapse of primary drug resistant Pre-B ALL. The Kaplan-
Meier survival curve in Figure 18B indicated that Tysabri-treated group
59

had significantly prolonged survival time compared with the Ig control.
Soon after this, the Ig+chemotherapy treated group died rapidly. However,
the animals with the combination of Tysabri and chemotherapy survived
disease-free until we terminated the experiment. The absence of minimal
residual disease (MRD) in SPC and BM cells of the VDL+Tysabri group
was determined by staining with human CD45 APC and mouse CD45
FITC (Figure 18C). Through immunohistochemistry in Figure 18D, the
combination treatment group showed absence of human CD45
+
leukemia
cells in the bone marrow (BM), liver, spleen cells (SPC) and lung
compared with brown staining leukemia cells in the control group. Through
PCR on Figure 18E, I determined complete absence of human GAPDH
amplification in spleen and bone marrow of the combination treatment
group. I demonstrate that the combination treatment has a potential to
completely eradicate primary drug resistant Pre-B ALL. These data were
confirmed with a repeat experiment with NOD/SCID IL2Rγ
-/-
(NSG) hosts
carrying even less immune system than NOD/SCID (Figure 19A-C) as well
as with two additional primary leukemia cases. I further evaluated this
combination therapy on 2 other Pre-B ALL samples, ICN12 and RS4;11,
with different chromosomal translocations, E2A-PBX1 t(1;19) and MLL-
AF4 t(4;11). The second leukemia, ICN12, showed a delayed engraftment
of leukemia and a significantly prolonged survival of the combination
60

treatment group compared with the control group (Figure 20A,B). The
recipient mice of the third leukemia, RS4;11, treated with the combination
of Tysabri and chemotherapy survived completely until the termination of
the experiment (Figure 21A,B). By flow cytometry and PCR, I confirmed
again disease-free survival by showing no human leukemia cells in spleen
and bone marrow of the combination treatment group (Figure 21C,D).
Taken together, pharmacologically targeting integrin α4 in combination
with chemotherapy leads to prolonged survival in all 3 experiments, and to
a cure of leukemia in 2 out of 3 Pre-B ALL samples.
To confirm human leukemia cells have already engrafted in mice before
starting the treatment, LAX7R cells (5x10
4
cells/mouse) were injected into
NSG mice and two femurs were harvested on Day 3 post-injection. By
immunohistochemistry, human ALL cells were detected on Day 3 after
leukemia cell injection as compared to the control group with no leukemia
injection (Figure 22A,B). Detecting a chromosomal translocation by real-
time PCR (Figure 22C) evidenced that human leukemia mRNA already
existed in bone marrow prior to the treatment. Furthermore, I repeated the
experiment starting Tysabri ± chemotherapy only on Day 6 after leukemia
cell injection, to allow more leukemia cells expanding in mice prior to the
treatment (Figure 23A-D). Again, Tysabri ± chemotherapy markedly
prolonged survival of leukemia-bearing mice (MST=75 days vs. 147 days)
61

and two out of five mice under the combination therapy got cured with no
human CD45
+
leukemia cells in spleen and bone marrow (Figure 23D). By
blood count analysis (CBC), NSG mice highly engrafted with human
leukemia showed a significantly more white blood cells and lymophocytes
but less red blood cells and platelets in the peripheral blood compared to
the healthy NSG mice (Figure 24). This result revealed the death of the
animals was due to leukemia-associated anemia and thrombocytopenia.

Targeting integrin α4 impairs the homing and affects the mobilization
of primary leukemia in vivo.
To determine how integrin α4 blockade decreases the engraftment of
leukemia in vivo, I performed the homing assay by injecting Tysabri or Ig
pre-treated primary leukemia cells (LAX7R) into mice and analyzing the
distribution of leukemia cells in each organ 18 hours post-injection. Anti-α4
blockade inhibits homing of primary ALL cells to bone marrow, spleen, and
liver; instead, cells were increased in lung compared to control Ig as
assessed by CFU counts (Figure 25A, B). Bone marrow has been
considered as the initiation site of pre-B leukemia cells. Impaired homing
may explain the result about the delayed engraftment by Tysabri
treatment. To understand why Tysabri treatment has this remarkable
survival benefit, on the other hand, the mobilization experiment was setup
62

by injecting primary ALL cells first into mice, which then received one time
of Tysabri or Ig treatment on Day14 post-injection. Figure 26A-G showed
human CD45
+
and CD19
+
leukemia cells were mobilized out of the
femurs, spleen and liver and moved into the lung and the peripheral blood
while the cell viability and proliferation were unaffected in femurs and
spleen. In femurs, bone marrow stromal cells have been shown to provide
chemoprotection to human leukemia cells as depicted above. Mobilizing
leukemia cells away from femurs may allow the chemotherapy to
completely eliminate leukemia cells and overcome drug resistance.

Small molecule inhibitor, TBC3486, downregulates the expression of
integrin α4 in primary ALL cells
Due to the potential side effect of Tysabri treatment, a small molecule
inhibitor, TBC3486, to inhibit integrin α4 was also investigated. By FACS,
three primary pre-B ALL samples, LAX7R, ICN03 and SF03, first showed
high % of integrin α4, α5, α6, and β1 (Figure 27). Activated LAX7R cells
were then treated with different doses of TBC3486 and its control,
THI0012 on OP9 cells for 4 days. TBC3486 treatment only downregulated
the intensity (MFI) of integrin α4 and β1; but futher evidence is required to
confirm on-target effect. (Figure 28). In addition, 25 µM TBC3486 as the
63

saturating dose reduced the adhesion of LAX7R cells to bone marrow
stromal cells, OP9 compared to the control group (Figure 29A, B) .

TBC3486 inhibits adhesion of primary ALLs and sensitizes leukemia
cells to standard chemotherapy in vitro.
Like Tysabri treatment, adhesion assay was performed by culturing three
primary ALL samples treated with TBC3486 or THI0012 in hVCAM-1
coated plate. Compared to the control group, TBC3486 treated ALL cells
showed a significantly decreasing adhesion with hVCAM-1 but no
difference on cell viability indicating no toxic side effect from the treatment
(Figure 30A-F). To study the drug resistance of human leukemia, primary
ALL (LAX7R) cells were co-cultured with OP9 stroma cells and treated
with or without chemotherapy, VDL, for 4 days. Regardless of co-culturing
with OP9 cells, TBC3486 with VDL treatment reduced the cell viability of
human leukemia, indicating this combination therapy sensitized leukemia
cells to chemotherapy which induced cell apoptosis (Figure 31A,B).

Inhibition of α4 integrin by TBC3486 delays the progression of
primary ALL in vivo.
After these encouraging observations in vitro, I next evaluated the benefit
of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R
64

cells were lentivirally labelled with luciferase for in vivo tracking and
injected into NOD/SCID hosts. Three days after leukemia cell transfer,
mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2
weeks (intraperitoneally), with or without VDL chemotherapy (Figure 32A).
Bioluminescence imaging (Figure 32B) revealed a marked delay of
leukemia cell dissemination (p<0.0001) in TBC3486 treated mice that
resulted in a prolonged survival time compared to THI0012 control mice
(Figure 32C). In combination with chemotherapy, the Kaplan-Meier
survival curve indicated that TBC3486+VDL treated group had significantly
prolonged survival time (MST = 82 days) compared with the control (MST
= 58 days).

Blocking integrin α4 by Tysabri inhibits the adhesion of T-ALL and
AML and sensitizes AML cells to chemotreatment.
Like pre-B ALL, I first performed a saturation assay for Tysabri to
determine the maximum dose of saturated antibody binding to primary T-
ALL and AML cells. FACS histogram reflected 20 µg/ml Tysabri as a
saturating dose for LAX1R and AML-1 samples (Figure 33A, B). For
functional studies, LAX1R and AML-1 cells were then pre-treated with
Tysabri or corresponding isotype controls IgG4. Cells were then loaded in
triplicates to plates coated with 10µg/ml human recombinant VCAM-1.
65

After two hours incubation, the adhering cells were photographed (Figure
34A, B). Integrin α4 blockade by Tysabri showed significantly smaller % of
cell adhesion to VCAM-1 compared to control IgG4-treated cells. (Figure
34C, D). In summary, blocking Integrin α4 by Tysabri inhibits adhesion of
primary T-ALL and AML cells to human VCAM-1. For drug resistance,
primary LAX1R and AML-1 cells were first co-cultured with or without bone
marrow stromal cells HS-5 and received with or without the chemotherapy
VDL for LAX1R and 1µM AraC for AML-1. Unlike pre-B ALL, bone marrow
stromal cells did not provide chemoprotection to LAX1R cells and no
difference on viability in combination with Tysabri treatment (Figure 35A).
In contrast, HS-5 cells protected AML-1 cells from AraC but Tysabri
treatment sensitized leukemia cells to chemotherapy (Figure 35B).

Blocking integrin α4 integrin by Tysabri delays the progression of
primary T-ALL and AML and prolongs the survival time in vivo.
Like pre-B ALL, similar experiments were setup for T-ALL and AML by
treating primary LAX1R and AML-1 mice with Tysabri or Ig control for one
month. Figure 36(AML-1) and Figure 37 (LAX1R) demonstrated the result
that Tysabri treatment delayed the leukemia engraftment and prolongs the
survival time in vivo.

66

3.4 Discussion

The biologic and genetic heterogeneity of ALL has been characterized by
genetic abnormalities and chromosomal translocation, like hyperdiploidy,
hypodiploidy, and t[9;22] in in BCR-ABL1. Moreover, patient information,
such as age, sex, white blood cell count before treatment,
immunophenotype of the leukemia cells and measures of minimal residual
disease (MRD) at the end of the induction therapy have been exploited to
categorize the risk of the disease as low, standard/intermediate, high or
very high and adjust the following treatment (99;106). Although relapses
are more frequent in patients classified as very high-risk disease, like
BCR-ABL1 or hypodiploidy, relapses still occur in all currently defined risk
groups (1). Thus, the risk classification for ALL has to be improved by
identifying new prognostic factors and therapeutic targets. Kang H et al.
analyzed the gene expression profiles from 207 pretreated children with
high-risk ALL and build a new classifier containing 38 unique genes to
predict relapse free survival (RFS) of high-risk patients (99). Shalapour S
et al. also investigated 56 childhood pre-BLL with first relapse and
reported integrin α4 as a clinical and prognostic factor for this disease. In
this study, I first identified high expression of integrin α4 gene was
associated to a poor survival outcome of high-risk ALL patients.
67

The bone marrow niche has also been found to provide chemoprotection
for leukemia cells and residual leukemia cells in bone marrow are a
frequent source of relapse (7). As to the molecular mechanisms
responsible for stroma-derived chemo-protection, different hypotheses can
be entertained. Thus, integrins could facilitate migration into the
extracellular matrix, providing physical protection from chemotherapy,
could provide approximation of leukemia cells to anti-apoptotic factors
secreted from stromal cells which themselves are relatively chemotherapy
resistant, or could elicit pro-survival/anti-apoptotic signaling through
outside-in signaling through activated integrin molecules. To maximize
changes on cell signaling, culturing cells in serum-free media (starving)
was commonly used to quiescence the signaling first and be stimulated by
the binding of hVCAM-1 as an example in this project. To study the role of
integrin α4 in the cell signaling, primary ALL cells were pretreated with
anti-α4 antibody, Tysabri, and showed a downregulated expression of p-
Akt and p-Erk proteins after 6 to 48 hours-incubation (Figure 15C). In
consequence, completely inhibiting the binding or contact between
primary ALL cells and human VCAM-1 by Tysabri treatment (Figure 14B)
resulted to attenuate Akt and Erk involved pathways, which have been
known to regulate cell survival and proliferation in integrin-mediated
signaling (54) (Figure 6). Since no survival and proliferation differences
68

were observed after treating leukemia cells with Tysabri in vitro, further
experiments would be needed to confirm this result. In addition, co-
culturing with bone marrow stromal cells, OP9, presented an up-regulated
expression of p-Akt and p-Erk in primary ALL cells compared to the group
without OP9 cells by western blot analysis (data not shown). To study the
role of integrin α4 on bone marrow stromal cells, primary ALL cells were
treated with Tysabri or control IgG4 and all co-cultured with OP9 cells in a
static condition using only tissue culture plate. No significantly signaling
changes were measured by this method (data not shown) probably due to
(i) soluble factors secreted by OP9 cells and (ii) incomplete blocking
between ALL cells and OP9 cells. (i) Bone marrow stromal cells have
been shown to prevent majority of pre-B ALL cases from apoptosis and
required direct contact with stromal cells in ~50% of ALL cases (31). As
culturing human ALL cells on microporous membranes above bone
marrow stromal layer (indirect contact), no significant differences on
viability are observed in 6 of 8 ALL cases compared to direct contact
(107). The data suggests the soluble factors, like IL-1, IL-6, IL-7, SCF and
TNF-α, derived from stromal cells, like OP9 cells, play a critical role in cell
survival at least in some ALL cases. On the other hand, direct contact is
absolutely necessary for cell proliferation in all cases of human ALL
compared to the indirect contact (107). (ii) Compared to hVCAM-1 in
69

Figure 14B, Tysabri was unable to completely inhibit the cell adhesion to
bone marrow stromal cells whether OP9 or HS-5 (Figure 14C), due to the
presence of other adhesion molecules, like VLA5 and CD44, on ALL cells.
Bonig H et al. has reported the mobilization effect on normal
hematopoietic stem and progenitor cells in MS patients by Tysabri
treatment (59). Therefore, in order to move primary ALL cells away from
the condition with soluble factors and minimize the binding with OP9 cells,
the flow condition should be utilized instead of the static condition to
determine the effect of blocking integrin α4 on long-term intracellular cell
signaling.
We used both NOD/SCID and NSG mice as recipients of leukemia
because often we were limited by availability of NSG mice. According
to our unpublished data, primary ALL cells can engraft in NSG mice with
no sublethal irradiation, which we assume would avoid damaging the bone
marrow microenvironment for further homing or mobilization studies. For
comparison, primary ALL cells were engrafted in NOD/SCID as well as
NSG mice and treated with Tysabri or/and chemotherapy VDL. Blocking
integrin α4 by Tysabri in combination with chemotherapy eradicated drug
resistant primary ALL cells in NOD/SCID or NSG mice (Figure 18 and
Figure 19).
70

Mice with engrafted primary leukemia were received an early treatment on
Day 3 post-leukemia injection. The reason I do not address longer
engrafted leukemia and a higher leukemia burden is that my preliminary
data show that highly engrafted leukemia is not curable in the xenograft
model but just leads to a prolonged survival (Figure 23). This has
important clinical implications. Therefore, it is critical to define the time slot
either for Tysabri or TBC3486 treatments to inhibit integrin α4. Leukemia
burden needs to be reduced by chemotherapy first, and then adjuvant
treatments be administered along with chemotherapy. In other words, I
would propose that high-risk ALL patients in clinical trials could receive
standard chemotherapy in combination with Tysabri treatment during
periods of low leukemia cell burden during consolidation, re-induction,
maintenance therapy, or as part of a conditioning regime prior to
allogeneic transplantation, all situations where leukemia load is small like
in a minimal residual disease (MRD) situation. Standard-risk patients
unable to tolerate standard-dose chemotherapy, a frequent problem in
adult ALL, might achieve a remission with reduced-intensity chemotherapy
in combination with Tysabri treatment.
Due to the potential side effect from Tysabri treatment, a small molecule
inhibitor, TBC3486, was used to target integrin α4. After comparing with
the use of Tysabri in vivo, TBC3486 in combination with chemotherapy
71

only prolonged the survival time but not completely eliminate leukemia
cells in mice. This difference may result from a lower binding specificity
and a shorter duration time of TBC3486 treatment compared to
humanized antibody, Tysabri. According to Dr. Vanderslice’s information,
TBC3486 is very highly protein bound (>99%) that means a higher dose of
the drug is used to provide for in vitro and in vivo experiments. Besides, in
pharmacokinetic study, the level of TBC3486 in plasma starts dropping 6
hours post-treatment (data not shown) compared to 11±4 days as a half-
life of Tysabri treatment in human (108). Therefore, a higher dose and
more frequency of TBC3486 should be administered to maximize the
effect of the treatment on ALL in vivo.
By analyzing gene expression profiles of T-ALL patients, integrin α4 was
overexpressed up to 15-fold compared to normal T-cells. For AML,
blocking integrin α4 by the commercial antibody has shown to sensitize
AML cells to chemotherapy through PI-3K/AKT/Bcl-2 signaling pathway
(32). Our data using the xenograft samples, LAX1R and AML-1, points to
the possibility that Tysabri can inhibit adhesion of both primary T-ALL and
AML cells to hVCAM-1 in vitro, although clearly more cases are to be
studied and in depth functional analysis is needed. For drug resistance,
only AML-1, but not LAX1R, was chemo-protected by stromal cells and
sensitized by Tysabri treatment to chemotherapy (Figure 35A, B),
72

probably due to relatively less IgG4-treated LAX1R cells adhering to
hVCAM-1 compared to AML-1 cells (Figure 34A, B). In xenograft mouse
model, Tysabri treatment alone already prolonged the survival time of
mice receiving LAX1R and AML-1 cells, but chemotherapy has to be
included in the future experiment to determine if targeting α4 can
overcome drug resistance of T-ALL and AML in vivo. After all, the
conclusion needs to be consolidated by using other primary T-ALL and
AML samples than LAX1R and AML-1.
Current treatment regimens for leukemia have been developed with little
attention to the role of the microenvironment, like bone marrow stromal
cells, in leukemogenesis. Mobilizing leukemia cells from their
chemoprotective niche by inhibiting adhesion molecules, like integrin α4,
may make standard chemotherapy more efficient. In this study, I have pre-
clinically evaluated that targeting integrin α4 by Tysabri or TBC3486 in
combination with chemotherapy has a potential to attenuate progressions
of ALL, AML, and T-ALL in xenograft mouse model. These encouraging
findings can be immediately translated into clinical trials since clinical-
grade α4 antibody, Tysabri, is commercially available. In addition, my
studies may change current concepts of ALL treatments to less toxic but
more efficacious and lead to elimination of drug resistance of leukemia.

73

3.5 Figures

Table 4 Sequences of oligonucleotide primers used

Table 5 List of antibodies for western blot analysis

74

Figure 6 Roles of integrin

(Guo W, 2004)

75

Leu. (L) Asp. (D) Val. (V)
Bio1211
(Luo BH, 2007)

Extrinsic ligand, like Bio1211

Figure 7 Structures of integrin α4 small molecule inhibitors, Bio1211 and
TBC3486.

A

B

C

(Yang GX, 2003)

Bind to Mg
2+

Hydrophobic
group
Form H-bonds
Form H-bonds
TBC3486

(You TJ, 2002)

76

Figure 8 Integrin α4 is overexpressed in ALL patients.

77

Figure 9 Correlation of integrin α4 with the survival outcome of ALL
patients

78

Figure 10 Expression of integrin α4 in xenograft leukemia samples

79

80

Figure 11 Dose determination of Tysabri for pre-B ALL, LAX7R.

81

Figure 12 Viability of normal pre-B cells is unaffected by Tysabri

82

Figure 13 Tysabri does not induce antibody-dependent cell-mediated
cytoxicity (ADCC) on human leukemia cells.

83

Figure 14 Integrin α4 blockade inhibits the adhesion of leukemia cells to
human VCAM-1.

84

Figure 15 Inhibition of integrin α4 by Tysabri to hVCAM-1 affects the
integrin-mediated signaling.

85

Figure 16 Targeting integrin α4 by Tysabri sensitizes drug resistant pre-B
ALL cells to chemotherapy.

86

Figure 17 Blockade of integrin α4 delays the progression of leukemia and
prolongs survival of mice engrafted with pre-B ALL in vivo.

87

88

Figure 18 Targeting integrin α4 by Tysabri in combination with
chemotherapy eradicates drug resistant primary pre-B ALL, LAX7R.

89

90

Figure 19 Combined integrin α4 blockade and chemotherapy eradicates
primary ALL (LAX7R) in NSG recipient mice.

91

Figure 20 Integrin α4 blockade prolongs survival of murine recipients of
primary pre-B ALL cells (ICN12).

92

Figure 21 Combined integrin α4 blockade and chemotherapy eradicate
pre-B ALL (RS4;11).

93

94

Figure 22 Engraftment of leukemia cells in vivo 3 days after injection

95

96

Figure 23 Late α4 blockade (Day 6 post-injection of LAX7R) prolongs
survival.

97

98

Figure 24 The cause of animal death by CBC analysis

99

Figure 25 Effect of integrin α4 blockade on homing of primary leukemia
cells in vivo

100

101

Figure 26 Mobilization of primary leukemia cells by Tysabri.

102

103

Figure 27 Integrin expression of three primary pre-B ALL samples.

104

Figure 28 Effects of TBC3486 treatment on integrin expression.

105

Figure 29 TBC3486 treatment inhibits adhesion of primary ALL to OP9
cells.

106

Figure 30 Inhibition of integrin α4 by TBC3486 disrupts the adhesion of
primary ALL cells.

107

Figure 31 Inhibition of α4 integrin sensitizes primary ALL cells to
chemotherapy

108

Figure 32 Inhibition of α4 integrin delays the progression of primary ALL in
vivo

109

Figure 33 Dose determination of Tysabri for T-ALL and AML.

110

Figure 34 Integrin α4 blockade inhibits the adhesion of T-ALL and AML
cells to human VCAM-1.

111

Figure 35 Targeting integrin α4 by Tysabri sensitizes drug resistant AML
cells to chemotherapy

112

Figure 36 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary AML (AML-1) in vivo.

113

Figure 37 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary T-ALL (LAX1R) in vivo.

114

Figure legends:

Figure 6 Roles of integrin
Roles of integrins, a heterodimer of α and β subunits, include the cell-
extracellular matrix (ECM) adhesion, cell migration, survival, proliferation
and intracellular signaling changes.

Figure 7 Structures of integrin α4 small molecule inhibitors, Bio1211 and
TBC3486.
(A) Bio1211 contains a tri-peptide motif (LDV), including 3 amino acids:
leucine (L), aspartic acid (D) and valine (V). (B) The extrinsic ligand, like
Bio1211, binds to β I-domain in activated and extended VLA4 integrin. (C)
The structure of TBC3486 and the binding to integrin β1.

Figure 8 Integrin α4 is overexpressed in ALL patients.
(A) 15 samples from each subtype of ALL patients, including BCR-ABl1,
E2A-PBX1, MLL rearrangement, TEL-AML1, and T-ALL, were analyzed
and showed overexpression of integrin α4 as compared to normal B or T
samples by heatmap.(B) The summary of the fold of overexpression to
normal cells.

115

Figure 9 Correlation of integrin α4 with the survival outcome of ALL
patients
(A) Kaplan-Meier estimates of overall survival (OS) for ALL patients
negative (black) or positive (red) with minimal residual disease (MRD) at
the end of the induction chemotherapy cycle of flow cytometry (day 29).
(B) Analysis of the overall survival (OS) of minimal residual disease
positive patients (MRD
+
) (n=67) and α4 expression (205885_at) separates
MRD
+
Integrin α4
high
(α4 expression ≥ mean; n=34) and MRD
+
α4
low

expressing cases (α4 expression < mean, n=33) (p=0.0175, logrank test).

Figure 10 Expression of integrin α4 in xenograft leukemia samples
(A) FACS analysis of 2 normal donors, 6 pre-B ALL, (B) 3 T-ALL, and (C)
1 AML xenograft samples stained with anti-human CD19 or CD45 FITC
and anti-human CD49d PE. Percentages of CD19 or CD45 and CD49d
double positive populations are given. (D) Human integrin α4 mRNA and
protein expression in normal CD19
+
pre-B cells and 6 Pre-B ALL samples
by the analysis of quantitative real-time PCR and western blot.

Figure 11 Dose determination of Tysabri for different subtypes of human
leukemia cells.
116

To determine the saturating dose of anti-functional anti-α4 Ab for pre-B
ALL cells, (A) LAX7R were treated with different doses Tysabri (0-20
μg/ml) for 30 minutes and washed with PBS once, subsequently stained
with Alexa Fluor 488 labeled Tysabri for 30 minutes and again washed
once with PBS. Histograms show unstained control (Black dashed line),
non-saturating doses of Tysabri (black), the saturating dose of Tysabri
(blue line) and untreated control (green). (B) LAX7R cells treated with
IgG4 and Tysabri for 48 hours in vitro showed no difference in viability as
compared to the control IgG4-treated cells. NS= non-significant (p>0.05).
This experiment was performed in triplicates.

Figure 12 Viability of normal pre-B cells is unaffected by Tysabri
Normal pre-B cells were sorted by magnetic separation using human
CD19 microbeads (Miltenyi Biotec, Auburn, CA). (A) The saturating dose
of Tysabri for normal pre-B cells was determined as 20 μg/ml by staining
cells with Alexa Fluor 488 labeled Tysabri, similarly to primary ALL cells in
Figure xx. (B) 48 hours in vitro incubation with Tysabri showed no
difference in viability as compared to the control IgG4-treated cells. NS=
non-significant (p>0.05). This experiment was performed in triplicates.

117

Figure 13 Tysabri does not induce antibody-dependent cell-mediated
cytoxicity (ADCC) on human leukemia cells.
(A) NK cells were >94% CD56
+
CD3
-
as determined by flow cytometry. (B)
Calcein-AM release assay was performed in triplicates by using Calcein-
AM labeled human leukemia cells (target cells, T) with isolated NK cells
(effector cells, E). Cells were incubated for 4 hours and % of specific lysis
was calculated as described in method section. NS = non-significant
(p>0.05), mean± s.d., unpaired t-test.

Figure 14 Integrin α4 blockade inhibits the adhesion of leukemia cells to
human VCAM-1.
LAX7R were pre-incubated with Tysabri or control IgG4 Ab and
subsequently seeded on plates coated with or without human (h)VCAM-1
for 2 hours. (A) Adhering cells on hVCAM-1 coated plates was
photographed under microscope with 200X magnification. (B) Number of
adhering cells was counted by trypan blue exclusion of dead cells. (C)
Tysabri treatment also significantly decreased the binding of primary ALL
(LAX7R) cells to Fibronectin (Fn), and mouse (OP9) and human (HS-5)
stromal cell lines. *p<0.05., performed in triplicates, mean± s.d., unpaired
t-test.

118

Figure 15 Inhibition of integrin α4 by Tysabri to hVCAM-1 affects the
integrin-mediated signaling.
(A) Schematic for the assay of cell starvation and stimulation. Tx:
Treatment. (B) At 6, 24, and 48 hours post-treatment, IgG4 or Tysabri pre-
treated LAX7R adhering in hVCAM-1 coated plates was photographed
under microscope. (C) Western blot analysis of untreated, IgG4 or Tysabri
pre-treated LAX7R proteins at 6, 24, 48 hours post-treatment. *Untreated
LAX7R in 1%BSA coated plate.

Figure 16 Targeting integrin α4 by Tysabri sensitizes drug resistant pre-B
ALL cells to chemotherapy
LAX7R cells were co-cultured with or without bone marrow stromal cells
OP9. LAX7R cells were then treated with standard chemotherapy VDL
(0.0005 µM Vincristine, 0.005 nM Dexamethasone, 0.0005 IU/ml L-
Asparaginase) for 4 days. Cell viability was assessed by trypan blue
exclusion of dead cells. *p<0.05, performed in triplicates, mean± s.d.,
unpaired t-test.

Figure 17 Blockade of integrin α4 delays the progression of leukemia and
prolongs survival of mice engrafted with pre-B ALL in vivo.
119

(A) Six pre-B ALLs were pre-treated with either 20 µg/ml Tysabri (n=3 per
sample) or Ig control (n=3 per sample) and monitored in vivo by
bioimaging at 3 different days post-cell injection. (B) Whole body
bioluminescent signals of six ALLs were quantified in unit of average
radiance (photons/sec/cm
2
/sr) at the last time point of bioimaging. *p<
0.05, between Tysabri and Ig pre-treated groups, mean±s.d., unpaired t-
test. (C) Kaplan-Meier survival curves of six pre-B ALL samples pre-
treated with Tysabri (Red) or control Ig (black) (n=3/per leukemia case;
n=18 /group) were analyzed for significance using the Log-rank Test.
*p<0.05. (D) Pooled Kaplan-Meier survival curve with median survival
time: Tysabri (102 days) vs. Ig (67 days). *p= 0.03, Log-rank Test.

Figure 18 Targeting integrin α4 by Tysabri in combination with
chemotherapy eradicates drug resistant primary pre-B ALL, LAX7R.
(A) Bioluminescent imaging of mice transplanted with LAX7R cells and
treated with Ig (n=4), Tysabri (n=4), VDL+Ig (n=9) or VDL+Tysabri (n=9)
on Day 34, Day 57, and Day 71 after leukemia cell transfer. A mouse with
no leukemia injection treated only with luciferin at time of imaging was
included as background control (Ctrl). (B) Kaplan-Meier survival curve was
analyzed and MST was calculated for each group: Ig (MST = 38 days),
Tysabri (MST = 52 days), VDL+Ig (MST = 74 days), VDL+Tysabri
120

(sacrificed at the end of follow-up, Day 151 post leukemia injection). (C)
The absence of human LAX7R cells in spleen (SPC) and bone marrow
(BM) of the VDL+Tysabri group was determined by flow cytometry using
an anti-human CD45 Ab. (D) Tissues, including SPC, BM, liver, and lung
from two groups were stained with anti-human CD45 antibody by
immunohistochemistry (brown). (E) The presence of murine and human
DNA in spleen (SPC) and bone marrow (BM) was evaluated using
genomic PCR for murine HPRT (Hypoxanthine
phosphoribosyltransferase) and human GAPDH, respectively.

Figure 19 Combined integrin α4 blockade and chemotherapy eradicates
primary ALL (LAX7R) in NSG recipient mice.
(A) Luciferase-labeled LAX7R cells were injected into sublethally
irradiated NSG mice (5x10
4
cells/mouse). Bioluminescent imaging of
experimental groups on Day 29 and 57 post-cell injection and background
control mouse (Ctrl) with no leukemia injection were presented. (B)
Kaplan-Meier Survival analysis: The Median Survival time (MST) for each
group (n=4) is: Ig 30 days, Tysabri 37 days, VDL+Ig 62 days. The animals
treated with combined VDL+Tysabri survived until the experiment was
terminated on Day 151 post-cell injection. *p=0.03 between Ig and Tysabri
treated groups, **p=0.007 between VDL+Ig and VDL+Tysabri treated
121

groups, Log-rank Test. (C) The absence of human leukemia cells in the
VDL+Tysabri group determined by flow cytometry (human CD45) in
spleen cells (SPC) and bone marrow (BM) cells on the day of sacrificing
mice.

Figure 20 Integrin α4 blockade prolongs survival of murine recipients of
primary pre-B ALL cells (ICN12).
(A) Monitor the engraftment of ICN12 treated with Ig (n=4), Tysabri (n=4),
VDL+Ig (n=4) or VDL+Tysabri (n=4) by bioImaging on Day43, 60 and 81
post-cell injection. (B) Kaplan-Meier survival curves were analyzed and
median survival time (MST) was calculated for each group: Ig:54 days
(n=3); NZM:66 days (n=3) , VDL+Ig: 80 days (n=4); VDL+NZM:91 days
(n=4). *p=0.03; **p=0.007, Log-rank Test.

Figure 21 Combined integrin α4 blockade and chemotherapy eradicate
pre-B ALL (RS4;11).
(A) Bioluminescent imaging of mice receiving Ig (n=3), Tysabri (n=3),
chemotherapy treatment (Tx) VDL (V: Vincristine 0.8 mg/kg, once per
week; D: Dexamethasone 10.5 mg/kg, 5 days/week; L: L-Asparaginase
800 IU/kg, 5 days/week)+Ig (n=5) and VDL+Tysabri (n=5) treatments on
Day 60 and Day 102 after leukemia cell transfer. Bio-imaging control
122

mouse (Ctrl) with no leukemia injection. (B) Kaplan-Meier survival curve
was analyzed and MST was calculated for each group: Ig: 60 days,
Tysabri: 83 days, VDL+Ig: 109 days. VDL+Tysabri was sacrificed at end
of follow-up, Day 143 post leukemia injection. *p=0.02, **p=0.002, Log-
rank Test. (C) The absence of human RS4;11 cells in spleen cells (SPC)
and bone marrow (BM) of the VDL+Tysabri group was determined by flow
cytometry using an anti-human CD45 Ab on the day of sacrificing mice.
(D) The presence of murine and human cells in spleen (SPC) and bone
marrow (BM) was evaluated using PCR for murine HPRT and human
GAPDH, respectively on the day sacrificing mice. The decreasing mouse
HPRT expression indicates less mouse cells remaining in bone marrow of
the mice treated with VDL+Ig. Pre-B ALL cells are known to initiate
leukemia in the bone marrow replacing resident hematopoiesis, and then
spread to other organs.

Figure 22 Engraftment of leukemia cells in vivo 3 days after injection
(A) Primary leukemia cells (LAX7R) were injected into NSG mice (5x10
4
cells/mouse) (n=3) and sacrificed on Day 3 post-cell injection. Femoral
bones were immunostained with human CD45 antibody (Brown) to detect
the injected primary leukemia cells (Red arrows) with counterstain by
hematoxylin. (B) Control mouse did not receive primary leukemia cells
123

(n=3). 400x magnification. To determine the engraftment of injected
leukemia cells in animals, (C) human pre-B ALL RS4;11 (MLL-AF4) cells
were injected into three NSG mice. Mice were sacrificed on Day 3 post-
injection and mouse bone marrow (BM) and spleen cells (SPC) were
studied for MLL-AF4 mRNA levels by quantitative RT-PCR. Healthy NSG
mice with no injection of leukemia cells were used as normal BM and SPC
for negative (-) control. RS4;11 leukemia cells were used as positive (+)
control.

Figure 23 Late α4 blockade (Day 6 post-injection of LAX7R) prolongs
survival.
(A) (B) LAX7R NOD/SCID recipient mice (5x10
4
cells/mouse) were treated
from Day 6 to Day 32 with Ig (n=3), Tysabri (n=3), VDL (V: Vincristine 0.8
mg/kg/d, once per week; D: Dexamethasone 10.5 mg/kg/d, 5 days/week;
L: L-Asparaginase 800 IU/kg/d, 5 days/week) +Ig (n=5) or VDL+Tysabri
(n=5) and the engraftment of leukemia cells was monitored by bioImaging
on Day 6, 32, 75 and 160 post-cell injection. A mouse with no leukemia
injection was used as background control (Ctrl). (C) Kaplan-Meier survival
curve was analyzed and median survival time (MST) determined for each
group: Ig: 34 days, Tysabri: 45 days, VDL+Ig: 75 days, VDL+Tysabri: 147
days. * p<0.05, Log-rank Test. (D) Spleen cells (SPC) and bone marrow
124

(BM) cells were stained with human CD45 (hCD45) antibody for FACS
analysis from the group treated with VDL+Ig (sacrificed day 75 post-
leukemia injection) and VDL+Tysabri (Mice survived to day 160 and were
sacrificed on day 160 post-cell injection).

Figure 24 The cause of animal death by CBC analysis
To determine the cause of death in mice, blood samples NSG mice
engrafted with human pre-B ALL (LAX7R) were collected via tail vein
bleeding for blood count analysis when mice were sacrificed. Blood
samples (~100µl /mouse, 3 mice/group) were collected in BD microtainer
tubes with EDTA (BD Biosciences) and analyzed by VetScan HM5 cell
counter (Abaxis, Union City, CA). WBC = White blood cell, LYM =
Lymphocyte, RBC = Red blood cell, PLT = Platelet. *p<0.05, mean±s.d.
(unpaired two-tailed t-test). Experiments were performed in triplicates.

Figure 25 Effect of Integrin α4 blockade on homing of primary leukemia
cells in vivo
To determine the effect of α4 blockade on homing of pre-B ALL cells in
vivo, RS4;11, cells were first pre-treated with Tysabri or Ig control in vitro
and injected into NOD/SCID mice (40x10
6
cells/mouse, n=6/group). 18
hours post-injection, mouse organs, including femurs, spleen, lung, liver,
125

kidney, and peripheral blood, were harvested for CFU assay (A) Colony
forming assays were performed in triplicate by plating an equal number of
viable cells (mouse + human) per dish (1x10
5
cells/dish) with MethoCult
GF
+
H4435. Images were taken with 2.5x magnification on Day 18 after
tissues harvesting. (B) The number of colonies was qualified. Scale bar:
0.5 mm. *p<0.05, mean±s.d. (unpaired two-tailed t-test). NS= non-
significant (p>0.05).

Figure 26 Mobilization of primary leukemia cells by Tysabri.
To determine the mobilization effect, primary ALL (LAX7R) cells were
injected into NOD/SCID mice (5x10
4
cells/mouse; n=6/group). Mice were
treated one time with Tysabri or Ig on Day 14 post-leukemia injection. (A)
White blood cells from femurs, spleen, lung, liver and peripheral blood
were analyzed at 60 hours post-Tysabri treatment and stained with human
CD19 and CD45 antibodies to distinguish human pre-B ALL cells from
mouse recipient cells. Percentages of hCD45
+
/hCD19
+
cells were
analyzed by flow cytometry. (B) Viability of human pre-B ALL cells
(hCD45
+
/hCD19
+
) in femurs and spleen was determined by PI staining
and flow cytometry. (C) Spleens in Ig and NZM groups were photographed
and the weights were measured (n=6/group). (D) Femur section with
immunohistochemistry staining showed more hCD45
+
human leukemia
126

cells (brown) in the Ig-treated group. Background control was the
secondary antibody for hCD45 staining. 400x magnification (E) Numbers
of hCD45
+
cells in each section was quantified. (F) hCD45
+
/Ki-67
+
double
staining was performed to visualize proliferative (Ki-67
+
) human
hCD45
+
leukemia cells in femurs. (G) The % Ki-67 was defined by the
percent of Ki-67
+
cells (red nucleus) per 500 human leukemia cells
(hCD45
+
). 20 sections /group. * p<0.05, mean±s.d. (unpaired two-tailed t-
test). NS= non-significant (p>0.05)

Figure 27 Integrin expression of three primary pre-B ALL samples.
Dot plots for integrin expression of pre-B ALL. Number in the quadrants
indicates percentage of cell population.

Figure 28 Effects of TBC3486 treatment on integrin expression.
Quantified mean fluorescence intensity (MFI) of α4, α5, α6, and β1 integrin
in LAX7R cells treated with different doses of TBC3486 and THI0012
control for 4 days with OP9 cells.

Figure 29 TBC3486 treatment inhibits adhesion of primary ALL to OP9
cells.
127

(A) Microscopic images for adhering LAX7R cells treated with different
doses of TBC3486 and THI0012 control on OP9 cells for 4 days. 400x
magnification. (B) Percentage of adhesion of TBC3486 (red) and THI0012
(white) treated cells on OP9. *p <0.05, mean ± s.d., performed in
triplicates.

Figure 30 Inhibition of integrin α4 by TBC3486 disrupts the adhesion of
primary ALL cells.
Primary ALLs, (A) LAX7R, (C) ICN3, and (E) SF03,were treated with
THI0012 control or TBC3486 on plates coated with human VCAM-1 or 2%
BSA as control for two days. Adhesion of ALL cells (400x magnification)
and % of adhering cells was shown. Viability of (B) LAX7R, (D) ICN3 and
(F) SF03 cells treated with TBC3486 or THI0012 control was determined
by trypan blue exclusion. *p <0.05, mean ± s.d., performed in triplicates.

Figure 31 Inhibition of α4 integrin sensitizes primary ALL cells to
chemotherapy
(A) Viability of primary ALL LAX7R treated with THI0012 control or
TBC3486 in combination with chemotherapy VDL was assayed by trypan
blue exclusion. *p <0.05, mean ± s.d., performed in triplicates. (B)
128

Apoptosis was presented by Annexin V and 7AAD using flow cytometry in
LAX7R.

Figure 32 Inhibition of α4 integrin delays the progression of primary ALL in
vivo.
(A) The treatment regime for VDL (V: Vincristine 0.8mg/kg; D:
Dexamethasone 10.5mg/kg; L: L-Asparaginase 800IU/kg) and TBC3486
or THI0012 (10 mg/kg/day with i.p. injection) (Vanderslice P, 2010;
Cannella B, 2003). (B) Luciferase-labeled primary ALL cells (LAX7R) were
injected into NSG mice (5x10
4
cells/mouse). Mice were then treated with
TBC3486 or THI0012 control for 2 weeks. The engraftments of ALL cells
were monitored by bioluminescent imaging on Day22 post-cell injection.
(C) Kaplan-Meier survival curve was analyzed and median survival time
(MST) for each group: THI0012 (MST=33 days), TBC3486 (MST=41
days), THI0012+VDL (MST=58 days) and TBC3486+VDL (MST=82 days).
*p <0.05, Log-rank test.

Figure 33 Dose determination of Tysabri for T-ALL and AML.
To determine the saturating dose of anti-functional anti-α4 Ab for T-ALL
and AML cells, (A) LAX1R and (B) AML-1 cells were treated with different
doses Tysabri (0-20 μg/ml) for 30 minutes and washed with PBS once,
129

subsequently stained with Alexa Fluor 488 labeled Tysabri for 30 minutes
and again washed once with PBS. Histograms show unstained control
(Black dashed line), non-saturating doses of Tysabri (black), the saturating
dose of Tysabri (blue line) and untreated control (green).

Figure 34 Integrin α4 blockade inhibits the adhesion of T-ALL and AML
cells to human VCAM-1.
LAX1R and AML-1 were pre-incubated with Tysabri or control IgG4 Ab
and subsequently seeded on plates coated with or without human
(h)VCAM-1 for 2 hours. Adhering of (A) LAX1R and (B) AML-1 cells on
hVCAM-1 coated plates was photographed under microscope with 200X
magnification. (C) (D) Number of adhering cells was counted by trypan
blue exclusion of dead cells. *p<0.05., performed in triplicates, mean± s.d.,
unpaired t-test.

Figure 35 Targeting integrin α4 by Tysabri sensitizes drug resistant AML
cells to chemotherapy
(A) LAX1R and (B) AML-1 cells were co-cultured with or without bone
marrow stromal cells HS-5. LAX1R cells were then treated with standard
chemotherapy VDL (0.0005 µM Vincristine, 0.005 nM Dexamethasone,
0.0005 IU/ml L-Asparaginase) for 4 days. AML-1 cells were treated with
130

chemotherapy, 1µM AraC, for two days. Cell viability was assessed by
trypan blue exclusion of dead cells. *p<0.05, performed in triplicates,
mean± s.d., unpaired t-test.

Figure 36 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary AML (AML-1) in vivo.
(A) Luciferase-labeled primary AML cells were intrafemorally injected into
NSG mice (1x10
5
cells/mouse). The engraftments of AML cells in Ig
control or Tysabri treated mice were presented by bioimaging on Day56
post-cell injection. (B) Kaplan-Meier survival curve was analyzed and
median survival time (MST) for each group: Ig (MST=76 days) and Tysabri
(MST=107 days). *p<0.05, Log-rank Test.

Figure 37 Inhibiting integrin α4 by Tysabri delays the progression of drug
resistant primary T-ALL (LAX1R) in vivo.
(A) Luciferase-labeled primary LAX1R cells were intravenously injected
into NSG mice (5x10
4
cells/mouse). The engraftments of T-ALL cells in Ig
control or Tysabri treated mice were presented by bioimaging on Day16
post-cell injection. FD= Found dead (B) Kaplan-Meier survival curve was
analyzed and median survival time (MST) for each group: Ig (MST=18
days) and Tysabri (MST=28 days). *p<0.05, Log-rank Test.
131

Chapter Four
Deletion of integrin α4 overcomes drug resistance of
mouse leukemia

4.1 Introduction

Deletion of integrin α4 in normal hematopoietic stem cells.
Integrin α4 antagonism has also been reported to involve in fetal and
embryonic development, such as fertilization, implantation, placental
formation and cardiac development (109). In vivo studies, integrin α4
knockout mice as well as VCAM-1 knockout mice have shown to develop
defects on embryonic development, disruption of placentation, cardiac
abnormalities and to cause embryonic lethality (73). Therefore, it is difficult
to study specific roles of Integrin α4 in hematopoiesis between fetal and
adult stages (15). As a result, Papayannopoulou T et al. and others’ labs
have generated integrin α4 gene “conditional” knockout model in adult
mice summarized in Rettig, MP et al., 2011 (84). First, a 13.9 kb clone
containing mouse integrin α4 gene (α4
+
) was isolated from genomic library
(15). The targeting vector included the a PGK-neo-p(A) cassette (positive
selection) flanked by two loxP in the promoter, two exons, the third loxP
132

distal to the second exon, and the DT-A cassette for negative selection.
After homologous recombination, clones with a floxed (α4
flox
) were
selected by G418 (neomycin), injected into C57BL/6 blastocysts (3.5 days
postcoitum) and transferred into pseudopregnant female mice to generate
offspring carrying α4
flox
gene (15). For the knockout model, α4
flox/flox
mice
were then mated with Mx.cre
+
mice and the offspring were cross-mated to
eventually generate Mx.cre
+
α4
flox/flox
mice compared to the control
Mx.cre
+
α4
+/+
mice (15). The genetic ablation by Cre recombinase can be
induced by the treatment of poly(I)-poly(C), a synthetic RNA molecule to
stimulate endogenous interferon expression (15). Only ~3 % of bone
marrow cells in Mx.cre
+
α4
flox/flox
mice expressed Integrin α4 compared to
93% in control mice. No changes were detected in other integrins by flow
cytometry except the reduced fluorescent intensity on integrin β1, which
usually formed a heterodimer with Integrin α4 (15). Deletion of Integrin α4
in adult hematopoietic cells has revealed the impaired homing and the
delayed short-term engraftment in bone marrow but enhancement in
peripheral blood (15). For long-term repopulating study, lack of VLA4
expression in hematopoietic stem cells (HSCs) restricts the competitive
function in hematopoietic reconstitution and self-renewal activity compared
to non-deleted normal HSCs (110).

133

Conditional knockout model of integrin α4 in mouse leukemia
To directly study the role of integrin α4 on leukemia, I established a
conditional knockout model in mouse leukemia. In the first place, integrin
α4
flox/flox
cells were retrovirally transduced with BCR-ABL1 p210 and p190
(111;112). Oncogenically transformed cells are subsequently transduced
with emptyER
T2
or creER
T2
retrovirus for conditional deletion (113).
Integrin α4 deletion can be induced in vitro and in vivo by addition of
tamoxifen and described in Figure 39 and 47. Using this genetic ablation
mouse model, I can determine the role of integrin α4 on cell adhesion,
self-renewal, intracellular signaling, and drug resistance of leukemia in
vitro and in vivo.

4.2 Materials and methods:

Studies with Integrin α4
fl/fl
bone marrow cells in vitro and in vivo
Integrin α4
flox/flox
(α4
fl/fl
) mice from Thalia Pappayannopoulou (University of
Washington, Seattle, USA) were first mated with C57BL/6 wild-type mice
to generate heterozygous mice. The homozygous mice were obtained
eventually by cross-mating heterozygous mice and confirmed the genome
(α4
flox/flox
) by genotyping the piece of mouse ear with qPCR (Transneyx
134

provided the genotyping service). Bone marrow cells from integrin α4
fl/fl

mice were cultured with IMDM and 20% FBS media supplemented with 10
ng/ml murine IL-7 (Peprotech, Rocky Hill, NJ) and retrovirally transduced
with BCR-ABL1 p210 (Figure 38A) and cultured under lymphoid
conditions. Oncogenically transformed B220
+
CD19
+
murine leukemia cells
were selected by withdrawal of IL-7 and subsequently transduced with
EmptyER
T2
or CreER
T2
retrovirus (Figure 38B,C). After 1 µg/ml puromycin
selection, integrin α4 deletion was induced in vitro by addition of 1.0 µM
Tamoxifen (Sigma-Aldrich) dissolved in 100% sterile ethanol. The
adhesion assays for mouse cells were performed on immobilized
recombinant mouse VCAM-1 (10 µg/ml, R&D Systems) as described for
primary ALL cells. To test the effect of integrin α4 ablation on p210
+

leukemia cells in an in vivo model, non-deleted CreER
T2
cells and
EmptyER
T2
cells were injected either via tail vein or via intrafemoral
injection into C57BL/6 Ly5.1
+
(CD45.1
+
) recipient mice (5x10
5
cells/mouse),
which had been irradiated with 525 cGy prior to cell transfer. To induce
deletion in vivo, mice were treated with 100-125 mg/kg/day Tamoxifen
(Sigma-Aldrich) by oral plastic feeding tubes (Instech Solomon, Plymouth
Meeting, PA) for 5 days at indicated times (Figure 47). Mice were treated
daily for 4 weeks with 50 mg/kg/day Nilotinib (Novartis, Stein, Switzerland)
by oral plastic feeding tubes.
135

Gene expression profiles of Empty and CreER
T2
cells
Total RNA from cells used for microarray was isolated by RNeasy
(Qiagen, Valencia, CA) purification. Measuring RNA quality and scanning
GeneChip (Affymetrix, GeneChip Mouse Genome 1.0) were performed by
USC/CHLA Microarray Core. The raw CEL files were normalized using the
JustRMA function on unfiltered probeset data with BRB array tools 4.2.0
(Bethesda, MD). Relative signal intensities of probesets were determined
by comparing the signal intensity from emptyER
T2
(n=3) and creER
T2
cells
(n=3) to the mean signal value of all probesets. A class comparison using
a nominal significance threshold of p < 0.001, with a two-sample univariate
T-test (random variance model), yielded 1532 genes and a Kolmogorov-
Smirnov statistic = 0.0162. Hierarchical clustered tree diagram was
generated using Partek Genomics Software Suite (Madison, WI), from a
genelist of 59 genes that passed significance threshold of 0.000001. The
calculated signal ratios of upregulated and downregulated probesets were
visualized as a heatmap with Java Treeview, with table of corresponding
p-values and false discovery rates (FDR) listed for genelists.

136

4.3 Result

Conditional knockout mouse model of integrin α4
First, I compared the course of α4 competent and α4 deficient murine
leukemia. I used bone marrow cells (BMC) from α4
fl/fl
mice

(Figure 39A)
and retrovirally transduced them in vitro using BCR-ABL1 (p210)

(Figure
38A) to generate B220
+
/CD19
+
murine leukemia (Figure 40A).
Subsequently, leukemia cells were transduced with either Empty-ER
T2

control or Cre-ER
T2
vector to delete α4 (Figure 38B,C and Figure 39B).
Deletion of α4 in transduced cells was efficient, as demonstrated by flow
cytometric analyses (Figure 40B) and genomic PCR (Figure 40C).

Deletion of integrin α4 inhibits cell adhesion, enhances chemo-
sensitization and decreases self-renewal of murine leukemia
α4-deficient murine leukemia cells (CreER
T2
) adhered to mVCAM-1-
coated plates with markedly reduced efficiency compared to EmptyER
T2

(α4 competent) control cells (p<0.05) (Figure 41A, B). In addition, α4-
ablation sensitized murine leukemia cells co-culturing with OP9 cells to
chemotherapy, VDL (Figure 42). Moreover, α4-deletion was associated
with loss of colony forming units (CFU) of BCR/ABL1
+
cells in primary and
secondary platings (Figure 43A, B) (Cooperated with Enzi Jang).
137

Deletion of integrin α4 in vitro affects the signaling pathways and
prolongs the survival in vivo.
Due to the effects on drug resistance and self-renewal, the gene
expression profiles of α4-deleted cells (CreER
T2
) and non-deleted
(EmptyER
T2
) cells were performed via microarray and the part of
downregulated genes in α4-deleted cells were presented by the heatmap
(Figure 44A). The expression of integrin α4 (Itga4) gene was 3.8-fold
decrease in CreER
T2
cells (p= 3x10
-6
). Frizzled homolog 6 (Fzd6), one of
surface receptors of the Wnt-signaling pathway (114), showed a
downregulated expression (3.4-fold) in CreER
T2
cells (p= 3x10
-5
). The
differential expression of Fzd6 gene has also been confirmed in protein
level by western blot analysis (Figure 44B). Not only Fz6, but p-Akt and p-
Erk all showed reduced expression in α4-deleted cells. When these in vitro
α4 pre-deleted (CreER
T2
) and undeleted (EmptyER
T2
) murine leukemia
cells were injected into sublethally irradiated mice, Nilotinib treatment led
to prolonged survival of α4-deleted recipients as opposed to recipients of
α4-competent B220
+
/CD19
+
murine leukemia cells (Figure 45A, B).

138

In vivo deletion of integrin α4 eradicates murine leukemia
To account for potential differences in mobilization and progression of
leukemia cells with α4 deletion, non-deleted EmptyER
T2
and CreER
T2
cells
(Luciferase
+
/GFP
+
) were first injected into C57/BL6 Ly5.1
+
recipient mice
(1.0x10
6
cells/mouse) which were then intraperitoneally treated with one
time of Tamoxifen on Day7 for α4 deletion in vivo (Figure 46) (Cooperated
with Eugene Park). According to bioimaging (Figure 46B), injected
CreER
T2
leukemia cells were partially mobilized from bone marrow to
spleen 5 days post-Tamoxifen induction (Day12). But no significant
differences on the engraftment of leukemia and survival time were
observed, majorly due to the incomplete and inefficient deletion of α4 in
spleen and bone marrow (~20% deletion) (Figure 46C). To improve the
efficiency of in vivo deletion, half number of leukemia cells were injected
into mice (5x10
5
cells/mouse) which then received two times of 5-days
Tamoxifen treatment by oral gavage starting on Day3 (Figure 47A).
Kaplan-Meier survival analysis revealed prolonged survival of the group
receiving α4-CreER
T2
in vivo ablated leukemia cells compared with the
EmptyER
T2
group (MST = 31 days vs. MST = 15 days; p=0.0008; Figure
47B). Flow cytometric analyses confirmed complete in vivo α4-deletion in
sacrificed animals (Figure 47C). Importantly, animals receiving α4-
CreER
T2
murine leukemia cells plus Nilotinib (NTB) survived until the end
139

of follow-up, compared to animals receiving α4-EmptyER
T2
(i.e. non-
ablated) murine leukemia cells plus Nilotinib (MST= Undefined vs. MST=
45 days; p=0.002) (Figure 47B). Similar results were obtained with
intrafemoral injection of leukemia cells (Figure 47D, E). I furthermore
determined that mice died of leukemia-associated anemia and
thromobocytopenia as assessed by blood count analysis (Figure 48), and
also determined that chemotherapy treatment of α4-deficient mice did not
result in excessive hematopoietic toxicity against normal cells (Figure 49A-
D) (Cooperated with Dr. Bonig).

4.4 Discussion

To directly study the function of the integrin receptor in a genetic
experiment, I developed a BCR-ABL1
+
mouse model (B220
+
CD19
+
) for
loss of function of integrin α4. The α4
fl/fl
cells were first oncogenically
transformed using BCR-ABL1 (p210) and cultured under lymphoid
conditions. Subsequent transduction with CreER
T2
or EmptyER
T2

generated leukemia cells in which α4 ablation could be induced (CreER
T2
)
by in vitro and in vivo treatment of Tamoxifen confirmed by FACS
analysis.
140

Gene expression profiles of integrin α4 deleted CreER
T2
cells compared to
non-deleted EmptyER
T2
control cells showed downregulated expression of
several interesting genes, including Fzd6 (Figure 44A). Frizzled homolog 6
(Fzd6) is one of known surface receptors of the Wnt-signaling pathway
(114). Wnt signaling regulates self-renewal of hematopoietic stem cells
(HSCs) and leukemia stem cells (LSCs) (115). Constitutively active
nuclear β-catenin signaling reduces HSC quiescence and blocks HSC
differentiation (116). Wnt pathway activation in the niche limits HSC
proliferation and preserves self-renewal (117). Other studies have shown
that β-catenin plays an important role in the long-term maintenance of
HSC (118). These observations suggests an association of Wnt/β-catenin
activity with the microenvironment which contributes to maintainance of
stem cell quiescence (119). In a mouse model of chronic lymphocytic
leukemia (CLL), the expression of Fzd6 was shown to be highly up-
regulated in transformed CD5
+
B cells of these mice (120). Elimination of
Fzd6 expression by crossing this CLL mouse model into Fzd6(-/-) mice
significantly delayed development of CLL. These observations point to the
possible association of Wnt signaling and integrin expression, however
this is preliminary and warrants further investigations. In addition to gene
expression analysis, the downregulated expression of Fzd6 protein in
CreER
T2
cells has also been confirmed by western blot analysis (Figure 44
141

B). But more experiments, like loss of functional studies of Fzd6, have to
be performed to prove this observation. Moreover, other 5-10 most
interesting candidate genes with differential expression (Figure 44A) will
be selected manually based on literature searches to identify correlations
with promotion or inhibition of survival, proliferation and adhesion of ALL
cells. I will also determine if there is a network of interconnected between
these selected genes and integrin α4. Identified genes will be validated by
Western Blot, quantitative real-time PCR, immunohistochemistry and gain
and loss of functional studies, as well as by knockout mice with the
respective identified genes if available.
Using this novel tool of oncogenically-transformed knockout mouse model
of integrin α4, I have determined leukemia cells upon integrin α4 deletion
inhibits cell adhesion, induces chemosensitization, decreases self-
renewal, affects integrin-mediated signaling pathways, and eventually
eliminates mouse leukemia cells in combination with chemotherapy in
vivo. These studies will further elucidate the biological and molecular
mechanisms controlled by integrin α4 of ALL cells and tremendously
enhance our understanding of the interaction between pre-B ALL and the
binding microenvironment.

142

4.5 Figures
Figure 38 Retroviral vectors and transduction methods

143

Figure 39 Conditional knockout of Integrin α4 in murine (BCR-ABL1
+
)
leukemia

144

Figure 40 Deletion of integrin α4 in B-ALL is confirmed by flow cytometry
and genomic PCR.

145

Figure 41 Adhesion of emptyER
T2
and creER
T2
cells on mVCAM-1

146

Figure 42 Deletion of integrin α4 sensitizes leukemia cells to
chemotherapy in vitro

147

Figure 43 Deletion of integrin α4 decrease the self-renewal of murine
leukemia

148

Figure 44 Deletion of integrin α4 affects signaling pathways of leukemia

149

Figure 45 Knockout of integrin α4 in vitro prolongs the survival time in vivo.

150

Figure 46 Incomplete in vivo deletion of integrin α4 in murine leukemia

151

Figure 47 In vivo deletion of integrin α4 eradicates murine leukemia

152

153

Figure 48 Blood counts of recipient mice of murine pre-B ALL

154

Figure 49 Chemo-toxicity of Integrin α4 ablation

155

Figure legends:

Figure 38 Retroviral vectors and transduction methods
Schematic of the MSCV-based retroviral vectors used for oncogenetic
tranduction of (A) BCR-ABL1 p210
+
; for conditional deletion of α4 (B)
CreER
T2
and (C) EmptyER
T2
(Control) transduction. Transfections of the
above MSCV-based retroviral constructs encoding BCR-ABL1,
EmptyER
T2
and Cre-ER
T2
were performed using Lipofectamine 2000
(Invitrogen, Carlsbad, CA) with Opti-MEM media (Invitrogen) similarly to
lentiviral production as described previously.

Figure 39 Conditional knockout of Integrin α4 in murine (BCR-ABL1
+
)
leukemia
(A) Schematic of oncogenic transformation of α4
fl/fl
cells with BCR/ABL1
p210
+
. (B) After viral transduction, integrin α4 EmptyER
T2
BCR-ABL
+
(Black) and CreER
T2
BCR-ABL
+
(red) cells were selected by puromycin
(1µg/ml) for 72hours (Vector map). Viability of selected cells was
assessed by trypan blue exclusion of dead cells and compared to control
untransduced BCR-ABL1
+
cells (Black circle).

156

Figure 40 Deletion of integrin α4 in B-ALL is confirmed by flow cytometry
and genomic PCR.
(A) α4 EmptyER
T2
BCR-ABL
+
and CreER
T2
BCR-ABL
+
cells were stained
with mouse B220 and CD19 for FACS analysis. Deletion of α4 induced by
Tamoxifen was confirmed by (B) flow cytometry and (C) PCR of genomic
DNA.

Figure 41 Adhesion of emptyER
T2
and creER
T2
cells on mVCAM-1
(A) After 2 hours of incubation with mVCAM-1, the adherent cells were
photographed by microscope. 400X magnification (B) Adhesion of α4
deleted CreER
T2
and non-deleted EmptyER
T2
cells on mVCAM-1 coated
plates.

Figure 42 Deletion of integrin α4 sensitizes leukemia cells to
chemotherapy in vitro
α4 deleted cells (CreER
T2
:Red) and non-deleted control cells (EmptyER
T2
:
Black) were cultured with OP9 or without OP9 (Control). Cells were then
treated with standard chemotherapy VDL (0.005 µM Vincristine, 0.05 nM
Dexamethasone, 0.005 IU/ml L-Asparaginase) for 4 days. Cell viability of
initial viability on Day 0 was assessed by trypan blue exclusion of dead
157

cells. *p<0.05, performed in triplicates, mean± s.d., unpaired t-test. NS=
non-significant (p>0.05).

Figure 43 Deletion of integrin α4 decrease the self-renewal of murine
leukemia
Colony forming assays (CFU) in primary and secondary platings were
performed and the number of colonies were counted on Day 15. *p<0.05,
mean± s.d., unpaired t-test, three independent experiments performed in
triplicate.

Figure 44 Deletion of integrin α4 affects signaling pathways of leukemia
(A) Down-regulated genes between integrin α4 and Fzd6 after deleting
integrin α4 in creER
T2
cells. (B) Western blot analysis of creER
T2
and
emptyER
T2
proteins The experiment was performed in triplicates in each
group.

Figure 45 Knockout of integrin α4 in vitro prolongs the survival time in vivo.
(A) Kaplan-Meier survival curve of α4-deleted CreER
T2
and non-deleted
EmptyER
T2
. In vitro deleted cells (5x10
5
cells/mouse) were injected into
NSG mice intravenously and mice were treated with or without 50mg/kg
Nilotinib (NTB). MST was calculated for each group by Log-rank Test
158

(MST for EmptyER
T2
= 37 days; MST for CreER
T2
= undefined; MST for
EmptyER
T2
+NTB = 64 days MST for CreER
T2
+NTB = undefined). *
p<0.0001, n=6 /group. (B) Bone marrow cells from EmptyER
T2
and
EmptyER
T2
+NTB mice at time of death were stained with mouse
B220
+/
CD19
+
antibodies and analyzed by flow cytometry.

Figure 46 Incomplete in vivo deletion of integrin α4 in murine leukemia
(A) Schematic of in vivo inducible deletion of integrin α4. (B) Non-deleted
luciferase/GFP-labeled α4 CreER
T2
and EmptyER
T2
cells were sorted by
FACS Aria II cell sorter through GFP expression. Cells were then injected
into NSG mice (1x10
6
cells/mouse). The engraftment of injected cells was
first detected by bioimaging on Day7 post-cell injection. Tamoxifen (20
mg/kg, weight of mice) was then given intraperitoneally to induce α4
deletion in vivo. (C) The injected cells were distinguished by GFP
expression via FACS analysis. The incomplete and inefficient in vivo
deletion of α4 in spleen cells (SPC) and bone marrow (BM) cells was
determined by flow cytometric analysis with mouse CD49d (mCD49d)
antibody (Day 12 post-cell injection).

Figure 47 In vivo deletion of integrin α4 eradicates murine leukemia
159

(A) Schematic for α4 in vivo deletion induced by Tamoxifen treatment
(100-125 mg/kg/day). BCR-ABL1
+
mouse cells were intravenously (i.v.)
injected into C57/BL6 Ly5.1
+
mice. (B) Kaplan-Meier survival curve of
CreER
T2
and EmptyER
T2
cells injected C57/BL6 Ly5.1+ recipient mice
treated with or without nilotinib (NTB). MST was calculated for each group
by Log-rank Test. (C) EmptyER
T2
and CreER
T2
mice of the in vivo
experiment were sacrificed on Day 15 and Day 32 post-injection, based on
the sacrificing criteria. The injected murine leukemia cells were
distinguished from endogenous mouse cells (CD45.1
+
) by mouse CD45.2
expression by FACS analysis. The FACS dot plot shows complete in vivo
deletion of mouse CD49d in spleen cells (SPC) and bone marrow (BM)
cells. (D) Schematic for α4 in vivo deletion induced by Tamoxifen
treatment (100-125 mg/kg/day). BCR-ABL1
+
mouse cells (1x10
5
cells/mouse) were intrafemorally (i.f.) injected into C57/BL6 Ly5.1
+
mice.
(E) Kaplan-Meier survival curve. Mice were treated with or without
50mg/kg Nilotinib (NTB). MST was calculated for each group by Log-rank
Test (MST for EmptyER
T2
= 17 days; MST for CreER
T2
= 20.5 days; MST
for EmptyER
T2
+NTB = 36 days MST for CreER
T2
+NTB = 44.5 days). *
p=0.001, n=6 /group.

Figure 48 Blood counts of recipient mice of murine pre-B ALL
160

To determine the cause of death in mice, blood samples from C57BL/6
mice engrafted with BCR-ABL1+ murine leukemia were collected via tail
vein bleeding for blood count analysis when mice were sacrificed. Blood
samples (~100µl /mouse, 3 mice/group) were collected in BD microtainer
tubes with EDTA (BD Biosciences) and analyzed by VetScan HM5 cell
counter (Abaxis, Union City, CA). WBC = White blood cell, LYM =
Lymphocyte, RBC = Red blood cell, PLT = Platelet. *p<0.05, mean±s.d.
(unpaired two-tailed t-test). Experiments were performed in triplicates.

Figure 49 Chemo-toxicity of Integrin α4 ablation
Mice with hematopoietic-specific ablation (Tie2cre-driven) of α4 were used
here. WT or α4-/- mice received sublethal irradiation followed by a 28-day
course of VDL (0.55 mg/kg Vincristine, 11.5 mg/kg Dexamethasone, 880
IU/kg L-Asparaginase). Blood counts (CBC) were regularly monitored
using a Hemavet hemacytometer (Drew Scientific, Barrow in Furness,
UK). Indicated are total white blood cells (WBC) (A), neutrophils (B),
hemoglobin (C) and platelets (D) after VDL treatment. n=5/group,
mean±s.d. The chemotherapy was only modestly hematotoxic; the kinetics
of leukocyte and erythrocyte recovery were indistinguishable.

161

Chapter Five

5.1 Conclusions

Current treatment regimens for ALL have been developed with little attention to
the role of the bone marrow microenvironment, including bone marrow stromal
cells, in drug resistance. Mobilizing leukemia cells from their chemoprotective
niche by blocking adhesion molecules, like integrin α4, may make standard
chemotherapy more efficient. In this dissertation, I have introduced a pre-clinical
platform using xenograft mouse model to evaluate new treatment strategy for
drug resistance of primary ALL. Correlation between the expression of integrin α4
in ALL patients with minimal residual disease and their survival outcome has
been demonstrated. Targeting integrin α4 by the humanized anti-α4 antibody,
Tysabri, in combination with standard chemotherapy has the potential to
eradicate drug-resistant primary leukemia. This finding can be immediately
translated into clinical trials since Tysabri has been approved by the FDA for use
of in the other disease. Small molecule inhibitor, TBC3486, is another antagonist
that targets integrin α4 and delays the progression of human leukemia thereby
prolonging survival time. As shown in the mouse knockout model, genetic
ablation of integrin α4 inhibits cell adhesion, affects integrin-associated signaling
and self-renewal capability and overcomes drug resistance of leukemia. Our
studies provide proof-of-concept of targeting integrins as a novel strategy to
162

eliminate drug resistance. However, there are several limitations of our study,
which are discussed in this chapter.

5.2 Discussions and future directions

We mentioned that we labeled primary leukemia cells with firefly luciferase
and used bioluminescence imaging to monitor the engraftment of human
leukemia cells in mice. Even though the transduction efficiency of
luciferase-labeled primary ALL was high (~80%), a portion of unlabeled
leukemia cells cannot be detected by bioimaging in vivo. To improve this,
FACS analysis was routinely used and combined with
immunohistochemistry and PCR to detect minimal residual disease and
confirm the real presence of human leukemia cells in each organ after
sacrificing animals.
We would also like to note that integrin α4 expression is not restricted to
leukemia cells; it is also expressed on many normal cells, like
hematopoietic stem cells (56) and CD19
+
pre-B cells (Figure 10A). In fact,
integrin α4 was expressed at relatively low level in normal pre-B cells
compared to leukemia cells (Figure 8A and Figure 10D), indicating that
less binding of Tysabri to normal cells could be expected. Moreover, no
163

toxic side effects were observed in leukemia or normal cells in vitro
(Figure 11B and Figure 12B) and in vivo (Data not shown). In the knockout
mouse model, I also provided evidence that integrin α4 deletion is not
associated with excessive hematopoietic toxicity (Figure 49). However, the
possibility of protracted immune modulation still needs to be considered
(as in MS patients), but this was generally not associated with clinical
evidence of impaired immunity, such as increased risk of infection.
Reactivation of the John-Cunningham virus (JC-virus) was observed in a
small cohort of MS patients treated with Tysabri (~0.1%); however, this
was only observed after prolonged treatment of about 3 years (121) and
would likely not be expected in leukemia patients who will only receive a
few doses over one month, similar to our preclinical xenograft model. The
risk of JC-encephalitis appears remote compared to the risk of death from
leukemia. Moreover, serological assays have been developed for JC virus,
and exclusion or specific vigilance programs for the JC-positive patients
can be anticipated.
What clearly remains to be analyzed in depth is the underlying mechanism
for integrin-mediated chemoprotection of ALL cells. Each integrin has a
large extracellular, a short transmembrane and a small intracellular
domains (54). The ligand binding to the extracellular integrin domain
induces conformational changes in the integrin and activates signaling
164

cascades and recruitment of multiple protein kinases, like ILK, FAK, talin,
paxillin and GTPases. In addition, integrin-receptor tyrosine kinases (RTK)
act as receptors for cytokines and growth factors and cooperates with
integrins to mediate cell signaling, known to determine cell survival,
proliferation, differentiation and apoptosis and called outside-in signaling
(Figure 6) (54). Importantly, we did not observe changes in proliferation
or apoptosis after deleting or blocking integrin α4, suggesting the
possibility that the major advantage of this strategy is the physical
dislodgement of ALL cells from the niche, but further studies would be
needed to explain the underlying mechanism of adhesion-mediated
chemoprotection independent of apoptosis. To study and identify the
target protein in integrin-mediated signaling, a comparative gene
expression analysis, which we have only begun, can be utilized to select
several gene candidates, like Fzd6 in Figure 44. The selected
genes/proteins need to be validated by gain- or loss-of-function studies.
The three methods commonly used to stop the function of targets include
the use of receptor antagonists/antibodies, small molecule inhibitors, and
microRNAs (mRNA)-based gene silencing. For gain-of function studies,
the target gene/protein can be amplified or overexpressed by viral
transduction (48).
165

In addition to LAX7R with normal karyotype (Figure 18), I also investigated
the combination therapy of Tysabri and standard chemotherapy VDL on
three other pre-B ALL samples, including ICN12 (Figure 20), RS4;11
(Figure 21), and TXL2 (Data not shown) with the respective different
chromosomal translocations, E2A-PBX1 t(1;19), MLL-AF4 t(4;11) and
BCR-ABL1 t(9;22). The results showed a survival prolongation in all three
ALL samples under the combination therapy but a cure of leukemia was
achieved only in the experiment using RS4;11 (Figure 21). As our gene
expression analysis for different subtypes of ALL patients, including pre-B
ALL and T-ALL, has indicated a variety of integrin α4 overexpression
ranging from 3-fold to 15-fold (Figure 8), it is imperative to evaluate
whether integrins play different roles in subtypes of leukemia. It would be
critical to pursue targeting integrins for all subtypes of leukemia.
Significantly more samples with different subtypes of B or T-ALL are
required to further conclude the relationship between the treatment
outcome and the expression levels of integrin α4. These additional studies
would provide further the basis for the integration of targeting integrin α4
into leukemia therapy to benefit leukemia patients with all different
karyotypes.

166

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Abstract (if available)

Abstract Although cure rates for acute lymphoblastic leukemia (ALL) in children are high, relapse of ALL leads to death in 50-95%. Adult ALL patients have a survival rate of only 40%. Even survived, patients often suffer from late-term secondary toxic effects of current treatments. Therefore, chemotherapeutic drug resistance of ALL cells remains a major problem and less toxic and more efficient therapies are needed. More than 80% of first relapse of childhood and adult ALL occurs in the bone marrow. Interaction of normal hematopoietic stem cells with the bone marrow (BM) stromal cells has been shown to provide mechanical support and facilitate proliferation and differentiation. BM stromal cells also provide protection of ALL cells from chemotherapy, thus contributing to drug resistance due to the lack of efficacy of current therapies. The exact mechanisms for stroma-mediated chemoprotection and approaches to address this problem remain elusive. In chapter two of this thesis, we summarize how we established a xenograft model of primary ALL cells to evaluate novel therapies. In chapter three, we use this preclinical model of primary ALL and focus on integrin alpha 4 as a central adhesion molecule for stromal-mediated chemoprotection and drug resistance of ALL. Integrin α4 is known to mediate adhesion of normal and malignant B-cell precursors in BM stromal cells. However, the functional modulation of integrin α4 and its consequences for drug resistance in ALL remains to be examined. According to gene expression analyses, integrin α4 is overexpressed in ALL patients and inversely correlated with the survival outcome. Therefore, we tested whether interference with α4-mediated stromal adhesion might be a new ALL treatment strategy. For this purpose, two models of leukemia were used: one pharmacological using antibody, like Tysabri, and small molecule inhibitor, like TBC3486, to target α4 of primary pre-B ALL and later in chapter 4 a genetic model (conditional α4 ablation of BCR-ABL1-induced murine leukemia). Conditional deletion of α4 sensitized murine leukemia cell to chemotherapy, Nilotinib. Adhesion of primary pre-B ALL cells was α4-dependent and inhibiting α4 sensitized primary ALL cells towards chemotherapy, VDL. Combination of chemotherapy with Tysabri prolonged survival of NOD/SCID recipients of primary ALL suggesting adjuvant integrin α4 inhibition as a novel strategy for pre-B ALL. Taken together, our data demonstrate that integrin α4-blockade with adjuvant chemotherapy can eradicate chemotherapy-resistant leukemia.

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Asset Metadata

Creator Hsieh, Yao-Te (Stanley) (author)

Core Title Role of integrin α4 in drug resistant acute lymphoblastic leukemia

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Pathobiology

Publication Date 05/02/2013

Defense Date 05/02/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag acute lymphoblastic leukemia,All,alpha4,drug resistant,integrin,Natalizumab,OAI-PMH Harvest,TBC3486,Tysabri

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kim, Yong-Mi (committee chair), Chuong, Cheng-Ming (committee member), Heisterkamp, Nora (committee member), Hofman, Florence M. (committee member), Mittelman, Steven D. (committee member)

Creator Email yhsieh@chla.usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-250789

Unique identifier UC11287896

Identifier etd-HsiehYaoTe-1644.pdf (filename),usctheses-c3-250789 (legacy record id)

Legacy Identifier etd-HsiehYaoTe-1644.pdf

Dmrecord 250789

Document Type Dissertation

Rights Hsieh, Yao-Te (Stanley)

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA