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The role of survivin in drug resistant pediatric acute lymphoblastic leukemia
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The role of survivin in drug resistant pediatric acute lymphoblastic leukemia
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Content
THE ROLE OF SURVIVIN IN DRUG RESISTANT
PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA
by
Eugene Park
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2010
Copyright 2010 Eugene Park
ii
Acknowledgements
Yong-Mi Kim, MD PhD
Markus Müschen, MD PhD
Michael Kahn, PhD
Zoltan Tokes, PhD
John Groffen, PhD
Nora Heisterkamp, PhD
Childrens Hospital Los Angeles/USC
Enzi Jiang, MD PhD
Yao-Te Hsieh
Asha Kadavallore
Lars Klemm
Cihangir Duy
University of Southern California
Cu Nguyen, PhD
Yi Zhao, MD PhD
Allen Yang, MD PhD
Samsung Medical Center, Sung Kyun Kwan University
Eun Suk Kang, MD PhD
Hong-Hoe Koo, MD
University Hospital Mannheim
Wolf-KarstenHofmann, MD PhD
University of California, Los Angeles
Gay Crooks, MD
University of California, San Francisco
Mignon Loh, MD
iii
Table of Contents
Acknowledgements …………………………………………………………………….……………ii
List of Tables ……………………………….………………………………………….……………. iv
List of Figures ……………………………...………………………………………………………...v
List of Abbreviations ...……………………….…………………………………………………...... vi
Abstract …………………………………….…………………………………………………………vii
Introduction: Acute lymphoblastic leukemia ………………………………………………………1
Chapter 1: Role of Survivin in drug resistance and self-renewal of drug resistant ALL
1.1 Introduction …….……………………………………………………………………... 3
1.2 Material and Methods…………………………………………………………..……. 6
1.3 Results…………………………………….…………………………..……………….11
1.4 Discussion…………………………………………….…………………………….…25
Chapter 2: Non-invasive bioluminescent imaging method to monitor leukemogenesis:
A preclinical platform for novel drug evaluation
2.1 Introduction …………………..……………………………………………………… 27
2.2 Material and Methods ………………………………………………………………..28
2.3 Results…………………………………………………………………..……………. 30
2.4 Discussion…………………………………………….…………………………...…..34
Chapter 3: Preclinical evaluation of survivin downregulation
3.1 Introduction ………………………………………………………………………..…. 36
3.2 Material and Methods……………………………………………………………….. 38
3.3 Results………………………………………………………………………………… 40
3.4 Discussion…………………………………………………………………………….. 43
References ..………………………………………………………………………………………… 46
iv
List of Tables
Table 1. ALL Patient Characteristics 12
Table 2. Complete Blood Counts for ICG-001 Evaluation 43
v
List of Figures
Figure 1. Survivin mRNA and Protein Expression in Pre-B ALL 13
Figure 2. Endogenous Survivin Characterization Using Human 14
Survivin Promoter GFP Reporter
Figure 3. Cell Cycle Analysis of Survivin Sorted Cells 15
Figure 4. Enrichment of Survivin Expressing Primary ALL with VDL Treatment 16
Figure 5. Survivin Over-expression in primary pre-B ALL 17
Figure 6. Functional studies of survivin overexpression in ALL 19
Figure 7. Survivin Overexpression Accelerates Leukemogenesis 20
Figure 8. Survivin shRNA Knockdown Vector Schematic, Western Blot, 21
and Quantitative PCR
Figure 9. Loss of Function Studies Using Survivin shRNA Knockdown Vector 22
Figure 10. Knockdown of Survivin Prolongs Survival in Combination 23
with Chemotherapy
Figure 11. Eradication of Survivin shRNA ALL 24
Figure 12. Bioluminescent imaging model for non-invasive monitoring 31
of primary ALL
Figure 13. Time Course Determination of Bioluminescent Intensity 32
Figure 14. Bioluminescent Tracking of Leukemic Engraftment 34
Figure 15. Differential co-activator usage in TCF/β-catenin transcription 37
and its role in differentiation vs. self-renewal
Figure 16. Pharmacological Downregulation of Survivin Using 40
CBP/β-Catenin Inhibitor, ICG-001
Figure 17. Preclinical evaluation of CBP/β-Catenin Inhibitor, ICG-001 42
vi
List of Abbreviations
ALL – Acute Lymphoblastic Leukemia
AML – Acute Myeloid Leukemia
APC – Allophycocyanin
BSD – Blasticidin
CFU – Colony Forming Units
cGY – Centigrays
CML – Chronic Myelogenous Leukemia
DAPI – 4',6-diamidino-2-phenylindole
Dox – Doxycycline
FACS – Fluorescence-Activated Cell Sorting
FITC – Fluorescein isothiocyanate
GFP – Green Fluorescent Protein
IRES – Intra Ribosomal Entry Site
IV – Intravenous
LUC – Luciferase
NOD/SCID – Non-Obese Diabetic/Severe Combined Immune Deficient
PB – Peripheral Blood
PE – Phycoerythrin
PEI – Polyetherimide
RFP – Red Fluorescent Protein
rtTA3 – Reverse Transactivator 3
shRNA – Short Hairpin Ribonucleic Acid
SSFV – Spleen-Focus Forming Virus
TRE – Tetracycline Responsive Element
VDL – Vincristine, Dexamethasone, and L-Asparaginase
VSV-G – Vesicular Stomatitis Virus G
vii
Abstract
Despite the recent advances in chemotherapy for acute lymphoblastic leukemia
(ALL), drug resistance resulting in relapse and long-term side effects of current
treatments warrant new treatment modalities. Survivin/BIRC5, an inhibitor of apoptosis
(IAP) protein, is critical for the survival and proliferation of cancerous cells and has
become the target of an increasing number of preclinical novel therapies against
primarily solid tumors. Survivin is expressed in AML and ALL cells and has been
implicated in leukemia relapse. We test the hypothesis that survivin is critical to the
pathway of self-renewal and maintenance of drug resistant ALL cells. To address this
hypothesis, we have developed a murine xenograft model of patient-derived ALL cells,
which are referred to here as primary ALL cells, allowing us to assess novel therapies
targeting survivin using non-invasive monitoring of leukemogenesis by bioluminescent
imaging. Our data suggest that overexpression of survivin increases self-renewal and
drug-resistance of patient-derived ALL cells in vitro and accelerates leukemogenesis in
vivo. In addition, in vitro inhibition of survivin using shRNA strongly synergizes with
conventional chemotherapy in patient-derived ALL cells and decreases self-renewal. In
vivo inhibition of survivin prolongs survival of mice engrafted with drug resistant
leukemia. Taken together, we show that survivin is a key component in drug-resistance
and stem cell self-renewal of drug resistant ALL cells.
1
Introduction
Drug resistant acute lymphoblastic leukemia.
Acute lymphoblastic leukemia (ALL) typically develops as the malignant
outgrowth of B-cell (and less frequently T-cell) precursors (Pui CH et al, 2008). Despite
substantial advances in the treatment of ALL during the past four decades, the present
rate of long-term survival remains at approximately 80 percent for children and 40
percent for adults
(Faderl S et al, 2010). Although cure rates in children are high,
resistant recurrent leukemia occurs in about 20% of all children and leads to death in 50-
95% depending on the site of recurrence (Gaynon P et al, 2005). The major challenge in
the therapy of ALL results from acquired drug resistance and relapse of leukemia
recently attributed to the persistence of leukemia stem cells in acute myeloid leukemia
(AML) and chronic myeloid leukemia (CML) (Huntly BJ et al, 2005; Jordan CT et al,
2005; Passegué E et al., 2003). Leukemia stem cells are defined by their ability to
reconstitute a heterogeneous full-blown leukemia in serial transplants into NOD/SCID
mice (Bonnet D et al., 1997). For these reasons, efficient targeting of leukemia stem
cells (LSCs) appears to be the key to further improvement of leukemia treatment. While
leukemia stem cells are well characterized in AML and CML, however this is not the
case for ALL. A specific phenotypic definition of leukemia stem cells in ALL is still
missing (Bernt KM et al., 2009). In addition, the existence of LSC or cancer stem
cells in general has been questioned recently. However, it is clear that drug resistant
leukemia-propagating/-initiating cells in leukemia are more resistant to drug treatment
than their progeny because they express significantly higher levels of multidrug
resistance proteins including ABCG2 transporters (Barnes DJ et al., 2005) and are
less sensitive to cell-cycle dependent drugs reflecting their quiescent phenotype
2
(Graham SM et al., 2002). Therefore, the challenge that will be addressed by this thesis
is the elimination of drug-resistant cells in ALL.
Scope of the Dissertation
Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein, is critical for the survival
and proliferation of cancerous cells (Altieri, 2003). Survivin is expressed in ALL cells and
has been implicated in leukemia relapse (Bhojwhani, 2006). We hypothesize that
survivin is critical for self-renewal and drug resistance in ALL. To test the hypothesis, we
aimed to:
1. Determine the role of survivin in self-renewal and drug resistance is ALL.
Gain of function and loss of function studies of survivin were conducted to evaluate the
role of survivin for self-renewal and drug resistance in vitro and in vivo.
2. Establish a non-invasive bioluminescent imaging method as part of a
preclinical platform for real-time monitoring of leukemogenesis.
3. Preclinical evaluation of survivin downregulation: Determine if adjuvant
direct or indirect downregulation of survivin using shRNA or ICG-001 can eradicate drug
resistant leukemia.
3
Chapter 1. Role of Survivin in drug resistance and self-renewal
of drug resistant ALL
1.1 INTRODUCTION
Survivin, a member of the inhibitor of apoptosis protein (IAP) family.
Critically, resistance to apoptosis is the primary cause of treatment failure in
acute leukemias (Smolewski P et al., 2003). Apoptosis can be induced intrinsically by the
mitochondrial pathway and extrinsically by a death-receptor-mediated pathway (Altieri,
2003). The intrinsic pathway is involved in chemotherapeutic response of leukemia cells
(Liu et al., 2002). Both apoptotic pathways commonly result in activation of the
downstream caspases 3 and 7.
Survivin (BIRC5) belongs to the Inhibitor of Apoptosis (IAP) gene family
(Srinivasula SM et al., Ashwell JD et al., 2008), that counteracts cell death and controls
mitotic progression (Altieri DC et al., 2006). It is a 16 kDa protein that is present during
embryonic development, but is virtually undetectable in terminally differentiated adult
tissues and not required for normal cell viability (Velculescu et al., 1999). It is
alternatively spliced to generate several protein isoforms (Sampath and Pelus et al.,
2007), and is transcriptionally controlled in a cell cycle-dependent manner, with peak
expression at mitosis (Altieri et al., 2006 and Lens et al., 2006). Overexpression of
survivin is associated with inhibition of cell death, and targeting survivin function and/or
expression, especially in tumor cells, caused spontaneous cell death (Altieri DC, 2003).
However, survivin did not appear to function as a direct caspase inhibitor (Verdecia et
al., 2000), which was thought of as the main mechanism of IAP-mediated cytoprotection
(Salvesen and Duckett et al., 2002). Survivin-mediated anti-apoptotic mechanisms of
caspase inhibition have been reported in a variety of cancers. Inhibition and binding of
4
caspase 9 by the interaction of a survivin and hepatitis B X-interacting protein (HBXIP)
complex, but not either protein alone, has been reported (Marusawa et al., 2003). X-
linked inhibitor of apoptosis (XIAP)-mediated caspase 3 and 9 inhibition, was enhanced
when complexed with survivin. Also, proteasomal degradation of XIAP, was impaired
when bound to survivin (Dohi et al., 2007). Furthermore, direct association between
SMAC/DIABLO and survivin has been also been observed. XIAP binding by
SMAC/DIABLO, and the subsequent release of caspases, has been proposed to be
inhibited by the sequestration of SMAC/DIABLO by direct survivin binding (Song et al.,
2003). In addition, caspase-independent inhibition of apoptosis has been reported in
melanoma, through the inhibition of apoptosis-inducing factor (AIF) by survivin (Liu et al.,
2004).
Survivin in solid tumors and leukemia
Survivin has been found to be overexpressed in a number of different tumor
tissues and has been generally correlated with a poor outcome
(Altieri DC, 2003) and is
has become the target of an increasing number of novel therapies against solid tumors,
lymphomas and recently leukemia (Pennati M et al, 2008). Furthermore, initial evidence
in vitro suggests that targeting survivin may provide a viable approach to selectively
eliminate cancer cells (Guha M et al., Altieri DC et al., 2009). In leukemia,
overexpression of survivin is associated with lower survival in adult patients with acute
myeloid and adult T-cell leukemia as well as diffuse large B-cell lymphoma
(Nakayama K
et al., 2002; Adida C et al., 2000). In children, the overexpression of survivin in acute
myeloid leukemia
(Carter BZ et al., 2001) and acute lymphoblastic leukemia
(Bhojwani D,
2006; Troeger A et al., 2007), has been correlated with poor overall survival. Therefore,
5
determining the role and the function of survivin in drug resistant ALL is important not
only for therapy, but also for prognosis. Early identification of patients with a poor
prognosis could warrant an alternate therapeutic strategy.
Survivin in normal hematopoiesis.
To develop an ideal chemotherapeutic agent, its molecular target should be
preferentially required in tumor cells compared with normal tissues, and targeting it
should overcome the survival of drug resistant leukemic cells, alone or in combination
with existing chemotherapeutics. Physiologically, survivin is transiently expressed during
embryonic development but not in terminally differentiated adult tissues (Ambrosini G et
al., 1997). A growing number of studies indicate survivin is expressed in
normal adult
cells, particularly human CD34
+
hematopoietic stem
and progenitor cells
(Fukuda S et
al., 2001, 2002), where it may regulate their proliferation or
survival. It has also been
shown that survivin-deficient hematopoietic
progenitors failed to give rise to either
erythroid or megakaryocytic
colonies in vitro (Gurbuxani S et al., 2005) and that inducible
deletion of survivin leads to ablation
of the bone marrow, with widespread loss of
hematopoietic progenitors
and rapid mortality
(Leung CG et al., 2007). Nevertheless, it
remains to be clarified if pharmacological down-regulation of survivin is detrimental to
the viability of hematopoietic cells.
Therefore, we hypothesize that survivin is critical for self-renewal and drug
resistance in ALL. To test the hypothesis, gain of function and loss of function studies of
survivin were conducted to evaluate the role of survivin for self-renewal and drug
resistance in vitro and in vivo.
6
1.2 MATERIALS AND METHODS
Patient samples
IRB approved human leukemia samples were obtained from Childrens Hospital
Los Angeles and USC Norris Cancer Center. Patient derived ALL cell lines, were
obtained as kind gifts from the lab of Dr. Markus Müschen (Childrens Hospital Los
Angeles).
Establishment of xenografts in NOD/SCID mice
Under IACUC approved protocols, NOD/SCID mice of 5 -10 weeks of age were
conditioned with a single dose of 250 cGy of total body irradiation at a dose rate of 325
cGy/min, followed by tail vein injection of about 0.7 – 2 x10
6
fresh human bone marrow
cells per animal. Animals were monitored weekly for weight change and presence of
human CD45
+
cells in the peripheral blood, as assessed by flow cytometry. Mice were
scored as positive for human leukemia on the basis of greater than 1% detection of
human CD45
+
cells in peripheral blood. Our data indicate that human leukemia cells are
amplified to an average of 250 x 10
6
cells in one recipient spleen. For analyses of human
leukocyte populations in engrafted mice, peripheral blood, splenocytes, and thymocytes
of engrafted mice were gated on human CD45
+
cells. Mice exhibiting less than 1%
human CD45
+
cells in the bone marrow were considered nonengrafted and were
excluded from the final analysis.
7
Characterization of Survivin Expression in Primary ALL
RNAs from patient xenograft samples were isolated using RNeasy Plus MiniKits
(Qiagen, Hilden, Germany), with subsequent cDNA synthesis using SuperScript III
Reverse Transcriptase (Invitrogen, Carlsbad, CA). Survivin mRNA expression
(Accession Number: NM001168) as a percentage of GAPDH mRNA was characterized
in primary ALL xenografts by quantitative PCR using a
Survivin Forward Primer – 5’-CATCTCTACATTCAAGAACTGG-3’ and a
Survivin Reverse Primer– 5’-GGTTAATTCTTCAAACTGCTTC -3’. – (Carrol et al) and
GAPDH Forward Primer-5’-GTTGCCATCAATGACCCCTTCATTG-3’ and
GAPDH Reverse Primer-5’-GCTTCACCACCTTCTTGATGTCATC3’. (Müschen et al).
Immunoblotting of survivin and GAPDH, were conducted using polyclonal murine
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Alkaline phosphatase labeled
secondary antibody was used for detection, visualized by a chemiluminescent substrate
(Invitrogen, Carlsbad, CA).
Endogenous survivin characterization was investigated using a human survivin
promoter (-1887/+17, 1904 bp) driven GFP reporter (Kind gift from Dr. Michael Kahn
Lab, USC). The generation of pLenti6 R4R2 V5-DEST phSurvivin GFP, was as
previously described (Ma H et al., 2005). Briefly, the human survivin promoter PCR
product was subcloned from XhoI–BamHI subcloning into the pTOPO vector. Both the
pTOPO – human survivin promoter and the pENTR 5’ – eGFP plasmids were introduced
in the Gateway destination vector using LR II Clonase (Invitrogen, Carlsbad, CA), with
subsequent Ampicillin selection. Lentiviral transduction of primary ALL cells was as
previously described, with subsequent selection of transduced cells using blasticidin
(Invitrogen, Carlsbad, CA) Upon complete selection confirmed by DAPI stained FACS
8
analysis, transduced cells were analyzed and sorted by flow cytometry. Sorted GFP
hi
and GFP
lo
cell populations were analyzed by quantitative real-time PCR. In addition, flow
cytometric analysis of the cell cycle using propidium iodine (PI) staining was performed.
Cell cycle analysis was performed using FlowJo software (Treestar, Ashland, Oregon).
Survivin Gain of Function and Loss of Function
Generation of the pCL6 SSFV Survivin IRES GFP (pCL6 IEGwo Survivin)
The human wild-type survivin gene was amplified from pENTR201 Survivin
(MGC Clone ID, Image:100002035) using primers 5’-
GTCATACTCGAGGCCACCATGGGTGCCCCGACGTTGCC–3’ and 5’–
TACGCCGAATTCTCAATCCATGGCAGCCAGCTGCT–3’. Subcloning of the survivin
gene into the lentiviral backbone pCL6IEGwo (kind gift from the Dr. Markus Müschen
lab) at XhoI and EcoRI was confirmed through restriction enzyme digestion and DNA
sequencing. Transfection into Stbl3 chemically competent E. Coli, was performed per
manufacturer’s protocol (Invitrogen, Carlsbad, CA). Production of pCL6 IEGwo and
pCL6 IEGwo survivin lentiviral supernatant was as described previously. Stable ectopic
survivin expression was achieved using lentiviral transduction of ALL with pCL6 IEGwo
Survivin, in parallel with pCL6 IEGwo GFP control, prior to enrichment of cells
expressing GFP by FACS cell sort. Survivin overexpression levels were analyzed by
qPCR and western blot. An in vitro MTT assay was used to assess cell proliferation after
treatment by VDL treatment for various concentrations per manufacturer’s protocol
(Trevigen, Gaithersburg, MD). Cell viability was determined 48 hours post-treatment.
Significance of triplicates measured determined by a two-tail paired t-test (Graphpad
Software, La Jolla, CA).
9
Generation of the pCL6 rtTA3 tRFP Survivin shRNA IE Puro (pCL6 Survivin shRNA)
Survivin knockdown was achieved via lentiviral delivery of targeting shRNA or
non-silencing shRNA. Survivin targeting sense sequence 5’ –
ACCTTAGCAATGTCTTAGGAAA – 3’ and anti-sense 5’ –
TTTCCTAAGACATTGCTAAGGG – 3’ (Open Biosystems pTRIPZ V2THS262484,
Huntsville, AL), as well as non-silencing sense sequence of 5’ –
ACCTCCACCCTCACTCTGCCAT – 3’ sequence and anti-sense 5’ –
ATGGCAGAGTGAGGGTGGAGGG – 3’ (Open Biosystems pGIPZ RHS4346,
Huntsville, AL) were used. Respective XhoI - EcoRI non-silencing and survivin shRNA
fragments were subcloned (New England Biolabs, Ipswich, MA) into the pCL6 rtTA3
tRFP shRNA IE Puro vector (Kind gift from the Muschen Lab). Restriction enzymatically
digested fragments were gel purified and ligated into the pCL6 rtTA3 tRFP IE Puro
vector using the Quick DNA Ligase system (New England Biolabs, Ipswich, MA).
Recombinant plasmids were transduced into Stbl3, with antibiotic selection of single-
colony transformants. Clones were analyzed for presence of insert via restriction
enzymatic digestion. Lentiviral supernatant produced using purified transfer plasmid,
was as described previously. Antibiotic selection of transduced cells used 2 ug/ml
puromycin for 3 days, with comparison to complete selection of non-transduced controls.
Doxycycline induction (500 ng/ml) of both cell types 48 hours before quantification of
survivin knockdown by qPCR and western blot.
In Vitro Drug Testing
An in vitro MTT assay used to assess cell viablilty after treatment by VDL
treatment of various concentrations. Various cells types were treated with vincristine,
10
dexamethasone, or L-asparaginase, as single agents as a combination of all treatments.
Cell viability determined 48 hours post-treatment, by addition of MTT reagent per
manufacturer’s protocol (R&D Systems, Minneapolis, MN). Significance of triplicates
measured determined by paired t-test. Similarly before the survivin shRNA knockdown in
vitro MTT assay, cells were induced 48 hours before VDL treatment. Viability was
determined 48 hours post-treatment.
CFU Assays
Primary ALL cells transduced with survivin IRES GFP and GFP-only control cells
were plated (10,000 cells per plate) in triplicate on a murine OP-9 feeder layer (50,000
cells per plate) in MethoCult GF+ H4435 (StemCell Tech, Vancouver, BC). GFP colonies
counted under UV fluorescence. Primary ALL cells transduced with non-silencing and
surviving shRNA were similarly plated (10,000 cells per plate) in triplicate 48 hours, post-
induction with doxycycline (0.5 ug/ml) on a murine OP-9 feeder layer. RFP colonies were
counted under UV fluorescence. For inducible survivin knockdown, induction by
doxycycline of both cell types was initiated 48 hours before plating in MethoCult GF+
H4435 (StemCell Tech, Vancouver, BC) in triplicate. RFP colonies counted under UV
fluorescence, representative colonies shown.
In Vivo Survivin Overexpression
Transduced primary relapse cells were transplanted into NOD/SCID IL-2R γ-
Chain Knockout mice via intravenous injection (1.0 x 10
6
cells per mouse). Weights were
monitored weekly to assess overall health.
11
In Vivo Survivin Knockdown
Transduced primary relapse cells were induced with doxycycline (0.5 ug/ml) 48
hours prior to FACS enrichment of RFP positive non-silencing and survivin shRNA
expressing cells. Induced cells were transplanted into NOD/SCID IL-2R γ-Chain
Knockout mice via intravenous injection (0.2 x 10
6
cells per mouse). VDL treatment (0.5
mg/kg vincristine, 10.5 mg/kg dexamethasone, and 1500 IU/kg of L-asparaginase)
began on day 4 post-xenotransplantation. Doxycycline (200 ug/ml) was administered
P.O. via drinking water, refreshed twice weekly.
1.3 RESULTS
Survivin Expression in Pre-B ALL
Initially we compared survivin mRNA expression in normal pre-B cells derived
from healthy volunteers versus primary patient pre-B ALL and pre-B ALL cell lines. ALL
cells encompassing various cytogenetic subgroups (Table 1) showed significantly
greater survivin expression versus MNCs derived from healthy volunteers, ranging from
2 to greater than 20-fold higher, thereby showing that survivin is upregulated in leukemic
versus non-leukemic pre-B cells across various samples paneled (Figure 1).
Overexpression of survivin protein in ALL was also confirmed by western blot (Figure 1).
12
Sample ID Sample Type Karyotype
LAX-2 Relapse BCR-ABL p210
TXL-2 Diagnosis BCR-ABL p210
TXL-3 Diagnosis BCR-ABL p210
SFO-2 Relapse BCR-ABL p210
ICN-6 Diagnosis TEL-AML1
ICN-12 Diagnosis Hyperdiploid
LAX-3 Refractory Refractory
SFO-1 Relapse Normal
SFO-3 Relapse Normal
LAX-6 Diagnosis Normal
LAX-7 Diagnosis Normal
LAX-7R Relapsed Matched Pair of US-7 Normal
Table 1. ALL Patient Characteristics.
Primary human ALL sample information regarding sample origin and karyotype.
13
Survivin mRNA of GAPDH [%]
0
5
10
TEL-AML1 Normal Karyotype
BCR-ABL Hyperdiploid
Survivin
β β β β-Actin
A
1 2 3 4 5 6 7 8 9 10 11 12
0
5
1 0
B
1 Normal MNC
2 LAX-2
3 TXL-2
4 TXL-3
9 SFO-3
10 LAX-6
11 LAX-7
12 LAX-7R
5 SFO-2
6 ICN-6
7 ICN-12
8 SFO-1
To further characterize cells based on endogenous expression a survivin
promoter-driven GFP lentiviral reporter, was employed. Figure 2A, shows a simplified
schematic of a vector kindly provided by the Dr. Michael Kahn Lab.
Figure 1. Survivin mRNA and Protein Expression in Pre-B ALL.
(A) Primary ALL across various cytogenetic subgroups were assayed for survivin
mRNA and protein expression. (B) Corresponding primary sample identifiers.
14
A
Transduction
Control
(Blasticidin)
Untransduced
Control
Blasticidin-
Selected
phSurvivin GFP
FSC
DAPI
DAPI Negative
GFP
B
GFP
Low
GFP
High
0 %
GFP
Low
GFP
High
GFP
Low
GFP
High 0 %
DAPI -
0 256 512 768 1024
DAPI -
0 256 512 768 1024
DAPI -
DAPI - DAPI -
0.5%
EM7 GFP HuSurvivin BSD
Upon transduction, variable survivin expression within a primary pre-B ALL
sample was observed. A sorted subpopulation of GFP high ALL exhibited an
approximately 7-fold higher level of survivin mRNA than the GFP low subpopulation
assayed by real-time PCR, thus showing heterogeneity in survivin expression within an
ALL sample (Figure 2A).
Figure 2. Endogenous Survivin Characterization Using Human Survivin
Promoter GFP Reporter.
(A) Schematic of human survivin promoter-driven GFP reporter characterization
of primary ALL (B) FAC Sorting of survivin expressing ALL.
15
A B
Unsorted
Bulk
GFP Low
GFP High
PI
Pre-B Cell
Unsorted Bulk
GFP Low
GFP High
0
20
40
60
80
100
Survivin mRNA of GAPDH [%]
6.9 Fold
Pre-B Cell
Unsorted Bulk
GFP Low
GFP High
0
20
40
60
80
100
Survivin mRNA of GAPDH [%]
6.9 Fold
G2/M = 56%
G2/M = 13.7%
G2/M = 6.7%
Cell cycle analysis of GFP high and low sorted populations, further showed the
utility of the survivin expression reporter. Observed was a 4-fold enrichment of cells in
G2/M within the GFP high population versus untransduced control. Conversely, reporter
based depletion of cells within the GFP low population yielded half the numbers of cells
in G2/M phase, as compared to control (Figure 3B).
Figure 3. Cell Cycle Analysis of Survivin Sorted Cells.
(A) Survivin mRNA characterization of survivin sorted cells. (B) Cell cycle analysis of
survivin enriched cells.
16
phSurvivinGFP cells were treated with either media control or a combination of
Vincristine (1 nM), Dexamethasone (0.1 nM), and L-Asparaginase (0.01 IU) (VDL) for 48
hours. A representative histogram for experiments performed in triplicate, show enriched
survivin promoter-driven GFP in viable, annexin V
-
/7-AAD
-,
, VDL treated ALL than
control (Figure 4B).
Gain of function studies of survivin in ALL cells demonstrate increased self-
renewal and decreased cytotoxicity of chemotherapeutic drugs.
To determine whether overexpression of survivin confers self-renewal and drug
resistance in ALL cells, we transduced primary pre-B ALL and ALL cell lines with a
Figure 4. Enrichment of Survivin Expressing Primary ALL with VDL Treatment.
(A) LAX-7R phSurvivinGFP transduced cells were analyzed by FACS, 48 hours post
VDL treatment and control. Enrichment of GFP expressing cells was observed in the
Annexin V – and 7-AAD –, compartment of VDL treated cells, versus control.
A
phSurvivin GFP
Annexin V
7-AAD
Control
VDL
Annexin V - , 7-AAD -
89
67
45
22
0
19%
49%
Control
VDL
17
bicistronic lentiviral survivin IRES GFP reporter construct (Figure 5A). To serve as a
control, ALL cells were infected with a lentivirus containing a GFP reporter construct
only. Cells were subsequently sorted by FACS. Real-time PCR, shown in Figure 5B of
survivin IE GFP cells showed over 3-fold higher survivin expression than GFP-only
controls (Figure 5B). Protein over-expression of survivin IRES GFP versus GFP-only
controls was confirmed by western blot (Figure 5C).
Figure 5. Survivin Over-expression in primary pre-B ALL.
(A) A lentiviral bicistronic Survivin IRES GFP reporter was used to sort ALL cells
ectopically expressing Survivin by flow cytometry. Survivin expression was confirmed
by qPCR (B) and Western Blot (C).
18
Next, colony formation assay (CFU) using OP-9 cells as a feeder layer was
performed in primary pre-B ALL and cell lines subsequently to ascertain the functional
relevance of survivin overexpression with representative colonies shown of a pre-B ALL
of an adult, with normal karyotype, who relapsed after therapy, is shown in figure
6A. We observed greater self-renewal of survivin overexpressing cells, which
significantly yielded over 3-fold more primary colonies than GFP-only controls in both the
primary and secondary CFU platings (p=0.015 and p=0.01, respectively). Subsequently,
an MTT assay was performed to determine the role of survivin overexpression in drug
resistance (Figure 6C). Overexpression of survivin attenuated the effect of vincristine on
ALL cell proliferation when compared to ALL cells transduced with empty vector controls
(Figure 6C). Vincristine IC
50
value determination for the control was 0.1 nM, whereas
survivin overexpression resulted in an approximately 10-fold higher IC
50
value of 10 nM
(p<0.01). Similarly, significantly higher concentrations of L-asparaginase (>0.01 I/U vs
>0.1 I/U; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed
to achieve drug cytotoxicity (data not shown). We conclude that survivin overexpression
decreases sensitivity of ALL cells to chemotherapy and increases self-renewal in vitro.
19
0
20
40
60
80
100
Paired T-Test 0.009
*
0.011
*
C
A
B
Empty IRES GFP Survivin IRES GFP
No Tx
Vincristine (mol/L)
10
-9
10
-7
Empty IRES GFP
Survivin IRES GFP
Viability [%]
OP-9
0
20
40
60
80
100
Control Survivin Control Survivin
GFP GFP GFP GFP
Primary Plating Secondary Plating
P=0.01
OP-9
Control Survivin Overexpresion
0
20
40
60
80
100
P=0.015
Colonies
0
20
40
60
80
100
OP-9
Control Survivin Overexpresion
0
20
40
60
80
100
P=0.015
Colonies
0
20
40
60
80
100
Survivin overexpression contributed to accelerated leukemogenesis after
xenotransplantation with a median survival time (MST) of 43 days observed for survivin
GFP recipients (n=4) versus a 51.5 day MST for GFP controls recipients (n=4). Gehan
Breslow Wilcoxon Test performed for statistical significance analysis of survival.
Statistical analyses for colony count comparisons and in vitro drug testing performed
using a two-tail, type 1 paired T-test (Figure 7).
Figure 6. Functional studies of survivin overexpression in ALL.
Overexpression of survivin confers increased self-renewal as detected by primary and
secondary platings of CFU assays (A,B) and decreased chemosensitivity as
measured by MTT assay (C )
20
0 10 20 30 40 50
A
P=0.011
Survivin GFP GFP Control
0
20
40
60
80
100
0
20
40
60
80
100
Survival[%]
10
Days Post-Injection
20 30 40 50
Loss of function of survivin sensitizes drug resistant ALL cells to chemotherapy
and decreases self-renewal in vitro.
Next loss-of-function studies were carried out using an inducible lentiviral shRNA
vector (Figure 8A). Doxycycline induction of a downstream tRFP was confirmed by flow
cytometry. Subsequently, induced scrambled and survivin knockdown cells were
assayed for survivin mRNA expression by RT-PCR shown in Figure 8B. 72 hrs post-
induction yielded in over a 60% reduction in survivin expression. Knockdown of survivin
on the protein level was confirmed by western blot (Figure 8C).
Figure 7. Survivin Overexpression Accelerates Leukemogenesis.
(A) Survivin overexpression contributed to accelerated leukemogenesis after
xenotransplantation with a median survival time (MST) of 43 days observed for
surviving GFP recipients (n=4) versus a 51.5 day MST for GFP controls recipients
(n=4).
21
Pre-B Cells
HuESCs
Untransduced
Non-Silencing Control
Survivin shRNA
0
5
10
15
Survivin mRNA of GAPDH
mRNA [%]
A
B
tRFP
7-AAD
Control shRNA
SSFV tRFP IRES TRE Survivin shRNA rtTA3 Puro SSFV tRFP IRES TRE Survivin shRNA rtTA3 Puro
Survivin
β β β β-Actin
+ +
Dox
shRNA Control
C
Next, we performed a loss of function studies using an ALL cell line (697) and
primary human US7R cells. RFP colonies were counted under UV fluorescence, in a
CFC assay of survivin knockdown with representative colonies shown (Figure 9A). The
numbers of colonies were markedly decreased after survivin knockdown (Figure 9B)
indicating the role of survivin for self-renewal in vitro (p<0.05). Primary plating show the
Figure 8. Loss of function studies.
Knockdown of survivin was performed using an inducible lentiviral shRNA vector
(A). Knockdown was quantified by qPCR (B) and Western Blot (C).
22
diminished self-renewal potential of survivin knockdown cells versus the non-silencing
control which significantly yielded over four-fold more colonies than knockdown colonies.
Lentivirally-mediated survivin shRNA knockdown of the same cell type significantly
sensitized a greater percentage of the leukemia to 10 nM vincristine versus non-
silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected
population than controls, assayed using MTT (p<0.003) (Figure 9C). This demonstrates
that in vitro self-renewal is impaired by survivin down-regulation versus non-silencing
control, in addition to increased sensitization to vincristine treatment.
0
20
40
60
80
100
Paired T-Test 0.003
*
0.02
*
Viability [%]
Control Survivin Knockdown
0
100
200
300
400
500
Colonies
C
A
B
Paired T-Test = 0.001
Non-Silencing Control Survivin shRNA
Non-Silencing Control Survivin shRNA
No Tx
Vincristine (mol/L)
10
-10
10
-9
10
-7
Non-Silencing Control
Survivin shRNA
Figure 9. Functional studies of Survivin knockdown.
Knockdown of Survivin confers decreased self-renewal as detected by CFU assay
(A, B) and increased chemosensitivity as measured by MTT assay (C )
23
Loss of function of survivin sensitizes drug resistant ALL cells to
chemotherapy and decreases self-renewal in vivo.
LAX-7R cells transduced with either non-silencing or survivin shRNA were
xenotransplanted into NOD/SCID IL-2R γ-chain knockout mice (200,000 cells / recipient).
Doxycycline was administered P.O. via drinking water started on Day 1. An MST of 42
days was observed for both non-silencing and survivin shRNA mice. Mice receiving VDL
treatment had an observed MST of 117 days for non-silencing shRNA, while survivin
shRNA mice receiving VDL were sacrificed on day 213 to assess leukemic burden
(Figures 10 and 11).
A
0
20
40
60
80
100
VDL+
Non-
silencing
Control
VDL+
Survivin
shRNA
Survivin
shRNA
Non-
silencing
Control
28 Day
VDL Tx
Survival [%]
0
60
100
4
Days Post-Injection
30 50 100 150 200
P=0.025
20
40
80
Figure 10. Survivin Knockdown In Vivo.
(A) LAX-7R recipients transduced with either non-silencing or surviving shRNA
(200,000 cells/mouse; n = 3 per cohort), were administered Doxycycline P.O. via
drinking water from Day 1. An MST of 42 days was observed for both non-silencing
and survivin shRNA mice. Mice receiving VDL treatment had an observed MST of
117 days for non-silencing shRNA, while survivin shRNA mice receiving VDL were
sacrificed on day 213 to assess leukemic burden.
24
Immunohistology of representative mice are shown, with secondary staining of
huCD45 shown in brown. Leukemic cells were present in all stained tissues of the non-
silencing control, conversely to the absence of huCD45 stained cells in the survivin
shRNA and VDL-treated mouse (Figure 11A). Murine and human CD45 FACS staining
of bone marrow and splenic cells are shown, with values displayed as the isotype-
normalized percentages, further showed the absence of survivin shRNA leukemic cells
versus the non-silencing controls (Figure 11B).
A B
muCD45
huCD45
Non- Survivin
Silencing shRNA
94%
4%
99%
0.3%
SPC
BM
93%
0%
0%
94%
SPC
BM
muCD45
huCD45
Non- Survivin
Silencing shRNA
94%
4%
99%
0.3%
SPC
BM
93%
0%
0%
94%
SPC
BM
Non- Survivin
Silencing shRNA
Bone Marrow
Spleen
Liver
Lung
Figure 11. Confirmation of Survivin shRNA Transduced ALL Eradication.
(A) Immunohistologial staining of non-silencing and survivin shRNA transduced ALL
xenograft recipients (B) FACS staining of non-silencing and survivin shRNA
transduced ALL. (Magnification 400x)
25
1.4 DISCUSSION
Our data confirms that survivin is expressed in ALL regardless of the cytogenetic
abnormality. Further confirmation of survivin overexpression across cytogenetically
distinct primary human ALL samples, highlights the differential contribution of the onco-
fetal protein to a variety of cancer pathologies. We hypothesized, that survivin is critical
for self-renewal and drug resistance in ALL. To test the hypothesis, we performed gain
of function and loss of function studies and determined the effect on self-renewal
and drug resistance of ALL.
We observed that overexpression of survivin increases self-renewal in vitro and
confers increased drug resistance. Next, in vivo evaluation demonstrated that
leukemogenesis is accelerated in recipient mice of leukemia cells overexpressing
survivin versus control. Loss of function studies demonstrate that survivin decreases
self-renewal capability and sensitizes drug resistant ALL cells to chemotherapy in vitro.
Direct inhibition of survivin through mRNA knockdown by lentivirally-mediated targeting
in a relapse model of B-ALL, highlighted the importance of survivin in leukemic
progression. In vivo eradication of leukemia in the shRNA and VDL treated cohort
confirm the dual role in self-renewal and in apoptotic inhibition, observed in in vitro CFU
assays and drug sensitivity assays.
It has been shown that survivin is required for FLT3-mediated AML in mice
(Fukuda et al., 2009) arguing for an intrinsic requirement for survivin in proliferation.
Here, we have now shown for the first time that survivin has a role for self-renewal,
using CFU assays, and drug resistance. However, we have not further investigated
survivin’s possible link with leukemia stem cell maintenance and senescence, as for now
a defined phenotype of leukemia stem cells in ALL remains elusive (Bernt et al.,
26
2009). Future studies should address whether there is a defined leukemia stem cells
in ALL like in AML/CML, and if survivin can further distinguish LSC from normal
mature hematopoietic cells.
Overexpression of survivin in MCF-7 breast carcinoma cell-line, was shown to
upregulate fibronectin expression determined through microarray analysis (Mehrotra et
al., 2010). Similar microarray analysis on ALL transduced with ectopic survivin and
survivin knockdown are planned to determine if the NF-kB mediated transcription of focal
adhesion molecules are also functionally relevant in ALL. Determination of NF-kB
nuclear translocation and iKKb expression are to be characterized within the gain-of-
function and loss-of-function models. Mediation of NF-kB transcription was shown to
require the interaction of survivin and XIAP. The binding of survivin with XIAP, in the
inhibition of caspases 3 and 9, as well as survivin interaction with other proteins have
been previously described (Altieri, 2003). Future experiments to determine whether
downstream transcriptional events are also associated with the interaction of survivin
with these molecules will be experimentally determined. Survivin has been shown to co-
immunoprecipitate with another IAP, BIRC6, also known as BRUCE (Pohl et al, 2008).
Taken together, as the critical role of survivin for self-renewal of drug resistant
ALL has been determined, we next established a preclinical xenograft platform of
primary leukemia to preclinically evaluate survivin downregulation as an adjuvant to
chemotherapy to eradicate drug resistant leukemia.
27
Chapter 2. Real-Time Bioluminescent Tracking of ALL
2.1 INTRODUCTION
Preclinical models are critical for evaluation of new drug treatments against
leukemia. The usefulness of human ALL cell lines is hampered by the fact that most
such lines have been propagated in culture for extended periods of time and may
therefore have acquired mutations that select for growth in vitro (Drexler HG et al.,
2000). A suitable model, which allows us to include the complex interactions of leukemia
cells with the host environment, is the murine xenograft model (Kung AL et al., 2007). It
has been described, that phenotypic and characteristics have been shown to be
preserved after serial passaging of engrafted human leukemia cells into secondary and
tertiary hosts (Baersch G et al., 1997; Borgmann A et al., 2000). Using this model for
preclinical testing has been shown that the leukemia cells’ response to chemotherapy
can mirror the outcome of the patient’s therapy (Lock RB et al., 2002). There are though
two disadvantages of the widely used xenograft model which have yet to be overcome:
First, this xenograft model has in the past relied on using a CD45 marker on leukemia
cells detected by flow cytometry as a surrogate to monitor leukemia engraftment,
progression and therapeutic success. Since bleeding restrictions of mice limit frequent
sampling of blood for flow cytometry, early and serial monitoring of leukemia is difficult.
Secondly, the tumor burden may be underestimated considering that it may take a while
until leukemic blasts present in the peripheral blood while they may already exist in the
bone marrow.
28
A way to overcome these disadvantages is the application of non-invasive
bioluminescent imaging. Bioluminescent imaging uses labeled cells of interest with an
enzyme, firefly luciferase, which are injected via tail-vein into immunocompromised mice.
Once the luciferase enzyme reacts with its substrate, luciferin, visible light gets released
and becomes detectable. It has been used for in vivo monitoring of growth of normal
hematopoietic cells (Wang X et al, 2003), leukemia cell lines (Armstrong SA et al, 2003)
or murine transduced bone marrow cells (Stubbs MC et al, 2008), but not yet widely for
primary human leukemia cells, as their fragile viability in vitro is an obstacle. We have
engrafted childhood leukemia samples, which retain the phenotypic characteristics of the
original leukemia after serial passaging to secondary and tertiary xenografts. Using
these well-characterized xenografts, we have now developed a more sensitive method
to determine engraftment of leukemia using bioluminescent imaging. This method to
monitor primary human leukemia is a new platform for preclinical drug testing.
2.2 MATERIALS AND METHODS
Lentiviral vector expressing the luciferase reporter gene
pCCL-MNDU3-LUC is a third generation HIV-1 based, lentiviral vector
transducing the firefly luciferase gene. The vector has the U3 region from the MND
oncoretroviral vector as an internal promoter driving expression of the firefly luciferase
gene from SP-LUC+ (Promega no. E178A; Promega, Madison,WI). The self-inactivating
backbone vector pCCL contains the cPPT/CTS sequence from HIV-1, 3 copies of the
UES polyadenylation enhancement element from SV40, has deletions of enhancers and
promoters of the HIV-1 long terminal repeat (LTR; SIN), and a minimal HIV-1 RRE (kind
gift from Dr. Donald Kohn, Childrens Hospital Los Angeles). Lentiviral supernatant was
29
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 hours post-
transfection, and concentrated by ultracentrifugation.
Transduction of xenografted ALL with luciferase vector
Primary ALL cells were transduced with pCCL-MNDU3-LUC virus supernatant in
plates coated with recombinant fibronectin (Takara, Shiga, Japan) containing serum-free
QBSF-60 medium (Quality Biologicals, Gaithersburg, MD). Twenty-four hours after the
initial transduction, cells were thoroughly washed 2 times with serum-free Iscove’s
Modified Dulbecco’s Medium (IMDM). Wild-type NOD/SCID splenocytes transduced with
same vector were used as a vector control. Aliquots of all transduced cell types were
assayed for confirmed bioluminescence prior to transplantation.
Bioluminescent imaging
5 - 7 weeks old NOD/SCID mice, obtained from Jackson Laboratories
(Sacramento, CA), were sublethally irradiated with 250 cGy prior to infusion of 1 of 3
separate luciferase - transduced cell types. Refractory pre-B ALL cells (1.5 x 10
6
cells/mouse), relapse T-ALL cells (0.3 x 10
6
cells/mouse) or Wild-type NOD/SCID
splenocytes (0.5 x 10
6
cells/mouse) were infused via tail vein. In vivo optical imaging
was conducted using a Xenogen IVIS 100 Imaging System (Alameda, CA) at various
timepoints after transplantation. Intraperitoneal injections of 125 mg/kg luciferin doses
30
(Biosynth AG, Switzerland), were given 15 minutes prior to image acquisition. Optimal
timing of image acquisition was previously established in a kinetic study of peak
bioluminescent signal acquiring images every minute post luciferin injection. Anesthesia
was induced using 5% isoflurane, and then maintained at 2% isoflurane prior to and
during image acquisition. Visible light photographs and luminescent images were
acquired via a cooled charge-coupled device (CCD) camera, over exposure times
between 1-3 minutes per acquisition. Images of both posterior and then anterior aspects,
respectively, were taken for up to 5 mice per acquisition. Display and analysis of optical
images were performed with the Igor (Wavemetrics, Lake Oswego, OR) and IVIS Living
Image (Xenogen, Hopkinton, MA) software packages. Regions of interest (ROIs) were
manually gated around the mice to assess signal emission intensity. Optical signals
were expressed as average radiance, in units of photons/s/cm
2
/steradian, to normalize
values obtained from differently sized ROIs.
2.3 RESULTS
In Vivo Bioluminescent Tracking of ALL
Frozen and fresh white blood cells of ALL patients were engrafted in NOD/SCID
mice. As previously described (Teachey DT et al., 2006). Our data indicate that human
leukemia cells are amplified and retain phenotypic characteristics of the patients
determined by flow cytometry (Dr. Yong-mi Kim lab, data not shown). To demonstrate
that engraftment and growth of ALL cells can be monitored in vivo at high sensitivity,
leukemic blasts were transduced with a lentiviral construct encoding firefly luciferase
prior to injection. Luciferase expression in primary human ALL cells was accomplished
using a lentiviral (HIV-1)-based vector. The proportion of ALL cells expressing the
31
luciferase transgene was assessed in vitro
by analyzing pre-B- and T-ALL cells after 3
days transduction. Luciferase labeling of primary ALL samples achieved by lentiviral
transduction, was determined using immunohistochemical detection of luciferase protein
and was found to have a mean transduction efficiency of 79% ± 9% (Dr. Yong-mi Kim
lab, data not shown).
Serial Bioluminescent
CCD Imaging
Luciferase Labeling
of Primary Human
Leukemia
IV Xenotransplantation
250 cGY WBI
Luciferin I.P.
A
The development of a bioluminescent signal is critical in quantitative studies. As
described previously (Wang et al., 2003), we studied first the kinetics of the bioimaging
signal in luciferase-expressing ALL cells. Mice injected with 0.3 x10
6
cells of T- ALL cells
of a patient, who relapsed despite chemotherapeutic treatment (relapse ALL), were
administered luciferin intraperitoneally (i.p.). Images of the mice were acquired serially
starting 6 minutes post injection, each with a 3-minute exposure. Peak signals were
detected 15 minutes after i.p. injection and decreased rapidly over the next few minutes
Figure 12. Bioluminescent imaging model for non-invasive monitoring of primary ALL
Primary leukemia cells were luciferase labeled and injected into sublethally conditioned
recipient mice. Upon intraperitoneal injection of the luciferin substrate serial bioluminescent
imaging was performed.
32
(Figure 13A) and the signal was further quantified (Figure 13B). Therefore, the
standard of 3-minute image acquisitions in our experiments was set to 12 minutes after
intraperitoneal injection of luciferin.
B
0 10 20 30
1.0× × × ×10
6
2.1× × × ×10
6
4.2× × × ×10
6
6 12 15 18 24
Minutes Post Luciferin Injection
Prone Total Flux (Photons/sec)
Figure 13. Time Course Determination of Bioluminescent Intensity.
Bioluminescent images (A) and quantification of bioluminescent signal (B)of recipient
animals of luciferase labeled T-ALL.Peak signal intensity for bioluminescense of
luciferase-labeled leukemia was determined by serial acquisitions of images of three-
minute durations starting continually 6 minutes post luciferin injection (i.p) for a total
of 24 minutes, 13 days post-xenotransplantation.
33
We measured luciferase activity in xenografted mice 8, 12 and 15 days after
injection by bioluminescent imaging. Whereas engraftment as judged by FACS analysis
of peripheral blood is not seen with the tested T-ALL xenograft (Figure 14B), the
recipient mice already showed engraftment after 8 days and 12 days via the
bioluminescent analysis (Figure 14A). The bioluminescent signal can be quantified,
distinguishing regional signal intensity, from that originating from whole body (Figure
14C).
34
Day 8 Day 15 Day 12
A B
1 6 13 15 20
0
20
40
60
80
100
Start of VDL Tx
Days Post Xenograft Injection
Peripheral Blood % huCD45
1 6 8 12 15
8.2× × × × 10
3
1.6× × × × 10
4
3.3× × × × 10
4
6.6× × × × 10
4
1.3× × × × 10
5
2.6× × × × 10
5
Days Post Xenograft Injection
Photons/sec/cm2
C
2.4 DISCUSSION
The utility of xenograft models for pre-clinical testing of investigational
compounds targeting leukemia have been widely reported (Teachey DT et al, 2006).
Bioluminescent imaging of solid tumor models, and more recently the dynamic tracking
of hematopoietic progenitors have also been shown (Wang et al., 2003). By combining
the two models, the use of an ALL luciferase-based reporter is being shown as an
Figure 14.
Bioluminescent
Tracking of
Leukemic
Engraftment
Bioluminescent
imaging of two
mice followed
(A), flow cytometry
detection of
human leukemia
cells ( B) and
quantification of
the
bioluminescent
signal post-
injection of 3 x 10
5
cells (C).
35
integral in vivo component of the pre-clinical platform. Bioluminescent imaging allows for
the dynamic tracking of leukemic burden and modeling of ALL relapse in vivo. Drug
dosing optimization in real-time, was possible without the reliance on frequent peripheral
blood interrogation, or the more rudimentary binary outcome of survival data. With real-
time imaging the MTD dose finding studies were more informative, as efficient
therapeutic targeting could be modeled with tracking of leukemic progression. The
transduction efficiency of primary leukemia cells was reliably 80% as determined by
histochemistry (Dr. Yong-mi Kim lab, data not shown) and has been reported with
similar success in normal hematopoietic cells (Wang et al, 2003). It is conceivable
that selection of luciferase positive leukemia cells prior to injection into recipient
animals will further enhance the sensitivity of this non-invasive monitoring method
of leukemogenesis.
In summary, our xenograft model using primary leukemia cells offers a valuable
platform for understanding leukemia in its microenvironment and for preclinical testing
of novel drugs. Therefore, next we used this model to determine in vivo the effect
of survivin downregulation to eradicate relapse leukemia.
36
Chapter 3. Preclinical evaluation of survivin downregulation
3.1 INTRODUCTION
Acute lymphoblastic leukemia and WNT/β-catenin signaling.
A major signaling pathway critical in survivin regulation is the WNT signaling
cascade. WNT signaling is critical in numerous events in development including both the
proliferation and differentiation of stem cells
(Cadigan KM et al., 1997). Recently, using a
selective antagonist of the β-catenin/CBP interaction ICG-001
(Emami KH et al., 2004),
our collaborator at USC, Dr. Michael Kahn, has developed a model to explain the
divergent activities of WNT/β-catenin signaling (Figure 15). Our model highlights the
distinct roles of the coactivators CBP and p300 in the WNT/β-catenin signaling pathway
(Ma H et al., 2005; Teo JL et al., 2005; McMillan M et al., 2005). The critical feature of
the model is that T-cell factor (TCF)/β-catenin/CBP mediated transcription is critical for
stem cell/progenitor cell proliferation. However, a switch to TCF/β-catenin/p300
mediated transcription, whether induced pharmacologically with ICG-001
or naturally, is
critical to initiate a differentiative program with a more limited proliferative capacity.
Aberrant regulation of the balance between these two related transcriptional programs
may be associated with a wide array of diseases including cancer.
37
TCF
CBP
p300
CCND2
AXIN2
HNKD
SURVIVIN
S100A4
ICG-001
JUN
FRA1
Nucleus
Self-renewal Differentiation
β β β β-catenin
β β β β-catenin
β β β β-catenin
TCF
TCF
β β β β-catenin
TCF TCF
CBP
p300
CCND2
AXIN2
HNKD
SURVIVIN
S100A4
ICG-001
JUN
FRA1
Nucleus
Self-renewal Differentiation
β β β β-catenin
β β β β-catenin
β β β β-catenin
TCF TCF
TCF TCF
β β β β-catenin β β β β-catenin
(Müschen 2009;Teo et al, PNAS 2005)
CBP/ β-catenin regulation of Survivin in drug resistant ALL.
While survivin appears to play an important role in the initiation and progression
of various human cancers, its mechanism is not explained fully. A better understanding
of both the physiologic and pathophysiologic roles of survivin is critical for further clinical
application of reagents that inhibit survivin expression. The hypothesis that inhibiting
the transcription of the survivin gene, via disrupting the CBP/β-catenin
interaction, will lead to selective elimination of drug-resistant ALL cells without
adversely affecting endogenous hematopoiesis providing a novel therapy for ALL
patients. There is evidence in solid tumors linking survivin to the WNT/ β-catenin
pathway. Earlier studies in colon cancer cells suggested that the regulation of survivin
expression is at least partially WNT/β-catenin dependent. Dr. Kahn (USC) recently
Figure 15. Differential co-activator
usage in TCF/β-catenin
transcription and its role in
differentiation vs. self-renewal.
Depending on usage of p300 or CBP
co-activators, β-catenin will lead to
transcriptional activation of sets of
genes that are either implicated in
differentiation (p300) or self-renewal
(CBP). ICG-001 can selectively block
the interaction between β-catenin and
CBP and therefore initiates
differentiation and loss of self-renewal
capacity. The main concept for the use
of ICG-001 is to deplete leukemia
stem cells by selective induction of
differentiation at the expense of their
self-renewal capacity.
38
demonstrated utilizing both pharmacologic inhibition with the CBP/β-catenin specific
inhibitor ICG-001
(Emami et al, 2004) and also with CBP and p300 specific siRNA that
survivin transcription via WNT/β-catenin signaling is critically dependent on the
coactivator CBP. Furthermore, utilizing chromatin immunoprecipitation assay (ChIP), it
was demonstrated that coactivator switching from CBP to p300 at the survivin promoter
is associated with the recruitment of transcriptionally repressive elements
(Ma
H et al.,
2005). Microarray analysis demonstrated that survivin was upregulated at relapse
compared to initial diagnosis in childhood ALL (Bhojwani D et al., 2006). Recently,
Morrison et al. demonstrated that targeting survivin with short hairpin RNA constructs
increased apoptosis in MOLT4 cells and in particular in combination with
chemotherapeutics (e.g. doxorubicin, and etoposide) (Morrison et al ASCO abstract
#9522). Therefore, targeting survivin expression by blocking WNT/CBP/β-catenin driven
gene expression with ICG-001, in conjunction with standard chemotherapies, would
appear to be a very attractive approach to eradicate leukemia stem cells. Based on our
preliminary data confirming the role of Survivin for self-renewal and drug resistance
in ALL, we next evaluated preclinically survivin down regulation using our xenograft
model and determined if adjuvant direct or indirect downregulation of survivin using
shRNA or ICG-001 can attenuate development of leukemia.
3.2 MATERIALS AND METHODS
In Vitro Drug Testing
As previously described, an in vitro MTT assay used to assess cell viablilty after
treatment by Imatinib (STI 571) treatment of various concentrations. SUP B15 cells were
39
treated with STI 571 as a single agent and in combination of with ICG-001 cotreatment.
Cell viability determined 48 hours post-treatment, by addition of MTT reagent per
manufacturer’s protocol (R&D Systems, Minneapolis, MN). Significance of triplicates
measured determined by paired t-test. Viability determined 48 hours post-treatment.
In Vivo Pharmacologic Survivin Targeting Using ICG-001
Lentivirally labeled relapse T-ALL, and pre-B ALL LAX-3, were transduced as
previously described. Four-eight hours post-transduction, cells were confirmed for
luciferase activity by addition of luciferin to aliquoted cell suspension before a one-
minute acquisition using the Xenogen IVIS system. T-ALL labeled cells, 1.9 x 10
5
cells,
were xenotansplanted via intravenous injection, into sublethally irradiated NOD/SCID
recipients. Model 100D osmotic pumps (Alzet, Cupertino, CA), were filled with an
equivalent of 28 doses of ICG-001 to individually deliver pre-weighed mice with either,
25 mg/kg/day or 50 mg/kg/day. Osmotic pumps were primed for 24 hours in sterile PBS,
at 37
o
C. Control mice received PBS filled osmotic pumps, primed in parallel to ICG-001
pumps. Osmotic pumps were subcutaneously implanted under isoflurane induced
anesthesia. Post-operative care and analgesia was administered per IAUAC approved
protocols. VDL chemotherapy induction was delivered i.p. 48 hours post-implantation.
Similarly, LAX-3 pre-B ALL cells, 1.5 x 10
6
cells were xenotransplanted into sublethally
irradiated NOD/SCID IL-2R γ-Chain Knockout mice. Osmotic pumps were filled with an
equivalent of 28 doses of ICG-001 to individually deliver 50 mg/kg/day, primed and
implanted subcutaneously. Upon completion of VDL induction, osmotic pumps were
surgically removed.
40
3.3 RESULTS
Pharmacological downregulation of Survivin.
Pharmacological targeting of survivin using a CBP/B-Catenin inhibitor (Dr.
Michael Kahn) was used to sensitize drug resistant leukemia. Downregulation of survivin
expression in a primary Philadelphia chromosome positive ALL of a drug resistant adult
patient, is clearly shown, in Figure 16A, in combination with chemotherapy. In Figure
16B, an MTT assay, subsequently shows greater sensitivity to chemotherapy (STI571)
with ICG-001 cotreatment versus single-agent alone, which for a few dose combinations
show half the number of viable cells versus single-agent treatment.
STI 571 (uM)
0 0.1 1 10
STI 571 (uM)
+ ICG 10uM
0.1 1 10
A
Survivin
β β β β-Actin
B
No Tx 0.1 uM 1 uM 10 uM
0
20
40
60
80
100 DMSO
ICG-001 10uM
STI-571
Viability [%]
No Tx 0.1 uM 1 uM 10 uM
0
20
40
60
80
100 DMSO
ICG-001 10uM
STI-571
Viability [%]
Preclinical in vivo model to test of survivin downregulation.
We have established a preclinical platform to test novel drugs for drug resistant
ALL. This platform includes a murine NOD/SCID and NOD/SCID IL2R gamma
-/-
xenograft model, which has been used to expand primary human ALL of drug resistant
Figure 16. Pharmacological downregulation of Survivin using ICG-001.
ICG-001 is a CBP/β–catenin inhibitor which downregulates survivin as detected
by WB (A) and decreases viability of ALL cells in a MTT assay (B).
41
patients with various karyotypes. In addition, leukemia progression can be monitored
and quantified using non-invasive lentiviral bioluminescent imaging. Figure 17A shows
significantly attenuated leukemogenesis by bioimaging and prolonged survival (p<0.05)
(Figure 17B) after treatment with combined ICG-001 vincristine, dexamethasone and L-
asparaginase (VDL) compared to VDL only. Gehan Breslow Wilcoxon Test was
performed for statistical significance analysis of survival. Cohorts were treated with VDL
(Vincristine 0.5 mg/kg/day, Dexamethasone 10.5 mg/kg/day and L-Asparaginase 1500
IU/kg/day) as well a cohort combined with ICG-001 (50mg /kg/day) for 28 days. Median
survival time (MST) of the saline group (n=6) was 55.5.days and 88.5 days for VDL only
group (n=6). The 98.5 day MST of VDL + ICG-001 treated cohort (n=8) was significantly
longer than that the 88.5 day MST of the combined VDL cohort. Blood count analysis
revealed no impact of combined ICG-001+VDL treatment on WBC, erythrocyte, and
thrombocyte counts compared to VDL only treatment (Table 2)
42
Day 57 Day 40 Day20 Day15
A B
VDL
VDL+
ICG-001
0 10 20 30 40
0
20
40
60
80
100
50 60 80 90 100
Saline
VDL
% Survival
ICG-001 50mg/kg/d
Vincristine 0.5 mg/kg/d
Dexamethasone 10.5mg/kg/d
L-Asparaginase 1500 IU/kg/d ICG-001+VDL
p<0.0001
95 85 103
(n=6), MST = 55.5 Days
(n=6), MST = 88.5 Days
(n=8), MST = 98.5 Days
Saline VDL ICG-001+VDL
VDL for
28 days
Days Post Injection
Figure 17. Targeting Survivin In Vivo.
Representative bioimaging for VDL and ICG-001+VDL cohorts post-
xenotransplantation, shown in Figure 17A. (B) Kaplan-Meier curve indicating ICG-
001+VDL prolongs event-free median survival time by 10 days, over the VDL cohort.
43
Day 21 Post-Treatment Day 29 Post-Treatment
Saline VDL VDL+ICG-001 Saline VDL VDL+ICG-001
(n=2) (n=2) (n=3) (n=4) (n=2) (n=2)
WBCs
Lym (10
3
/ul) 2.6 ±1.3 3.9 ±1.1 3.5 ±0.1 7.3 ±9.4 2.1 ±0.5 2.0 ±1.6
Neu (10
3
/ul) 1.6 ±1.1 3.3 ±0.8 3.0 ±0.8 5.5 ±7.1 1.9 ±0.3 1.7 ±1.4
Lym (%) 34.1 ±4.4 7.4 ±4.5 5.9 ±2.5 21.0 ±4.5 2.6 ±0.1 5.3 ±1.9
Mon (%) 6.9 ±8.5 7.7 ±5.9 8.4 ±1.8 5.4 ±3.6 6.2 ±7.6 11.8 ±2.3
Neu (%) 59.0 ±12.9 85.0 ±1.4 85.7 ±3.0 73.6 ±6.4 91.3 ±7.7 83.0 ±4.2
Erythrocytes
RBC (10
3
/ul) 12.7 ±2.1 0.0 ±1.69 8.53 ±0.8 5.6 ±1.5 4.0 ±0.4 3.7 ±1.1
Hgb (g/dl) 20.9 ±2.9 15.0 ±3.61 13.9 ±0.8 7.8 ±2.5 5.5 ±0.4 4.8 ±2.0
HCT(%) 61.1 ±10.3 39.9 ±7.69 39.1 ±3.3 25.6 ±6.9 19.1 ±1.6 17.3 ±5.6
Thrombocytes
PLT (10
3
/ul) 452.5 ±19.1 745.5 ±245 664.3 ±361 229.5 ±101 424.5 ±123 439.5 ±129
Taken together, knockdown of survivin using shRNA and pharmacological
downregulation using ICG-001 may sensitize drug resistant ALL cells in vivo and in
vitro to chemotherapy.
3.4 DISCUSSION
Indirect targeting of survivin, via disruption between direct upstream
transcriptional WNT pathway elements CBP and β-Catenin, further show the therapeutic
benefit in combining survivin antagonism with the existing standard of care.
Pharmacological downregulation of survivin by ICG-001 in combination with
chemotherapy is observed, while not achieved with single agent alone. Subsequently
Table 2. Complete Blood Counts (CBCs) for ICG-001 In Vivo Evaluation.
CBCs for Saline Control, VDL, and ICG-001+VDL cohorts post-treatment. Counts for
White Blood Cell (WBCs), Lymphocytes (Lym), Neutrophils (Neu), Monocyte (Mon),
Red Blood Cells (RBC), Hemoglobin (Hgb), Hematocrit (HCT), and Platlets (PLT)
shown. VDL+ICG-001 CBCs were comparable to those of the VDL treated cohort.
44
greater sensitivity to chemotherapy with ICG-001 cotreatment versus single-agent alone,
sensitized ALL to chemotherapy across multiple dose combinations. In vivo, leukemia
progression was attenuated with ICG-001 cotreatment in marked contrast to VDL only
treatment with survival, significantly prolonged after combined treatment with ICG-001
combination versus VDL only treatment. Pharmacological downregulation of survivin
using ICG-001 sensitizes primary drug resistant ALL cells to chemotherapy both in vitro
and in vivo. Off-target effects of ICG-001 have not been evaluated. It has been
described to downregulate in addition to survivin also other β-Catenin-regulated
genes such as CCND2 and S100A4 in colon cancer cell lines (Teo JL et al., 2005).
Leung et al have shown decreased erythopoiesis after knockout of survivin in a murine
mouse model. However, we did not observe such effects after downregulation of
survivin.
Taken together our data suggests that targeting survivin in pre B-ALL may
sensitize to chemotherapeutic treatment, thus highlighting the role of survivin as a target
for novel therapies in pre B-ALL drug resistance.
Future Experiments
Targeting of survivin becomes an attractive therapeutic target, as differential
expression in cancer reduces the chances for cytotoxicity to normal cells. Anti-sense
oligonucleotides (ASO), with chemical modifications to the nuclei acid to improve
pharmacokinetics, targeting survivin expression have entered pre-clinical studies for
various malignancies. Future experiments targeting surviving in ALL would involve
characterization of both the in vitro and in vivo response of primary human leukemias to
ASO therapy.
45
While the knockdown achieved using shRNA targeting of survivin mRNA yielded
silencing of over half the mRNA transcript, complete genomic ablation of survivin
expression using a conditional survivin knockout mouse model. Such a model would
allow insight into the role of survivin in the leukemogenesis of ALL, and subsequently the
importance of surviving in the relapse of drug-resistant ALL.
Taken together, our data highlights the role of survivin in recalcitrant pre-B-ALL
as a target for novel therapies. Further preclinical studies, in particular addressing the
mechanism downstream survivin and the safety of survivin downregulation for normal
cells, are warranted to understand the pathway-specific eradication of drug resistant
leukemia and to translate our findings into clinical trial.
46
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Abstract (if available)
Abstract
Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), drug resistance resulting in relapse and long-term side effects of current treatments warrant new treatment modalities. Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein, is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicated in leukemia relapse. We test the hypothesis that survivin is critical to the pathway of self-renewal and maintenance of drug resistant ALL cells. To address this hypothesis, we have developed a murine xenograft model of patient-derived ALL cells, which are referred to here as primary ALL cells, allowing us to assess novel therapies targeting survivin using non-invasive monitoring of leukemogenesis by bioluminescent imaging. Our data suggest that overexpression of survivin increases self-renewal and drug-resistance of patient-derived ALL cells in vitro and accelerates leukemogenesis in vivo. In addition, in vitro inhibition of survivin using shRNA strongly synergizes with conventional chemotherapy in patient-derived ALL cells and decreases self-renewal. In vivo inhibition of survivin prolongs survival of mice engrafted with drug resistant leukemia. Taken together, we show that survivin is a key component in drug-resistance and stem cell self-renewal of drug resistant ALL cells.
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Asset Metadata
Creator
Park, Eugene
(author)
Core Title
The role of survivin in drug resistant pediatric acute lymphoblastic leukemia
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2010-08
Publication Date
08/04/2010
Defense Date
06/30/2010
Publisher
University of Southern California
(original),
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Tag
acute lymphoblastic,BIRC5,IAP,leukemia,OAI-PMH Harvest,survivin
Language
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Electronically uploaded by the author
(provenance)
Advisor
Kahn, Michael (
committee chair
), Kim, Yong-Mi (
committee chair
), Müschen, Markus (
committee member
), Tokes, Zoltan A. (
committee member
)
Creator Email
eugenehpark@gmail.com,eupark@chla.usc.edu
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Tags
acute lymphoblastic
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