PRIMA-1met Induces Apoptosis in Waldenström's Macroglobulinemia Independent of p53, alone ...

77 Pages·2012·0.93 MB·English

PRIMA-1met Induces Apoptosis in Waldenström's
MacroglobulinemiaIndependent of p53, alone and in
Combination with Bortezomib
By:
Mona Sobhani
A thesis submitted in conformity with the requirements for the degree of
Masters of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Mona Sobhani 2015
PRIMA-1met Induces Apoptosis in Waldenström's
Macroglobulinemia Independent of p53, alone and in
Combination with Bortezomib
Mona Sobhani
Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2015
Abstract:
PRIMA-1met has shown promising preclinical activity in various cancer types. However, its
effect on Waldenström’s Macroglobulinemia (WM) as well as its exact mechanism of action is
still elusive. In this study, we evaluated theanti-tumor activity of PRIMA-1met alone and in
combination with dexamethasone or bortezomib in WM cell line and primarysamples.
Treatment of WM cells with PRIMA-1met resulted in induction of apoptosis, inhibition of
migration and colony formation. Upon PRIMA-1met treatment, p73 was upregulated and Bcl-xL
was downregulated while no significant change in expression of p53 was observed. siRNA
knockdown of p53 in WM cell line did not influence the PRIMA-1met-induced apoptotic
response whereas silencingofp73 inhibited latter response in WM cells. Combined treatment
with PRIMA-1met and dexamethasone or bortezomib induced synergistic reduction in cell
survival in WM cells. Our study provides the rationale for PRIMA-1met’s clinical evaluation in
patients with WM.
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Acknowledgements
I would like to extend my sincerest thanks and regards to all those who supported and
encouraged me during my Master’s study.
First, I would like to express my special gratitude to my supervisors Dr. Hong Chang whose
expert guidance, support, patience and encouragement made my Master’s studies a productive
experience. I am grateful to have had the opportunity to train in a diverse learning environment
with admirable individuals, particularly Manujhandra Saha, Yijun Yang and Yan Chen, whose
proficiency and expertise in the lab have been immeasurably helpful to me. I would also like to
express my special thanks to my committee members Dr. Donald Branch and Dr Chen Wang for
their valuable and constructive comments.
My deep gratitude is expressed to my mother and father whose love, inspiration and endless
encouragement made my years ofstudies an enjoyable and unforgettable experience. They
deserve special and heartfelt thanks.
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Table of Contents
Abstract…………………………………………………………………………………………….i
Acknowledgments...........................................................................................................................ii
List of Tables ..................................................................................................................................v
List of Figures ................................................................................................................................vi
List of Abbreviations ................................................................................................................... vii
Chapter 1:Introduction ...............................................................................................................1
1.1. Waldenström's Macroglobulinemia..........................................................................................1
1.1.1Waldenström's Macroglobulinemia.................................................................................1
1.1.2. Incidences, Demographics, and Etiology........................................................................2
1.1.3.Diagnosis……...…………………...................................................................................3
1.1.4. Clinical Features………………………………………………………………...……...4
1.1.5. Laboratory and Pathological Findings……………………………………..…………...7
1.1.6. Molecular Pathology.......…………….............................................................................9
1.1.6.1. Genetics…………..…………………………………………………………....9
1.1.6.2. Epigenetics……………………...…………………………………………….13
1.1.6.3. Microenvironment…...………………………………………………………..14
1.1.7.WM Current Treatments………………….…………….……………………………...15
1.2. PRIMA-1met……………………………………………………………………………....21
1.2.1. P53 and Apoptosis……………………………………………………………………...21
1.2.2. PRIMA-1met………...………………………………………………………………...25
1.3. Rationale, Hypothesis, and Experimental Aims…………………………………………….30
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Chapter 2: PRIMA-1metInduces Apoptosis inWaldenström’s Macroglobulinemia
Independent of p53:.……………………………………………………………………………32
2.2. Introduction ...........................................................................................................................32
2.3. Results …................................................................................................................................34
2.4. Discussion… ..........................................................................................................................42
2.5. Materials and Method……………………………………………………………………….44
2.6. References…………………………………………………………………………………...46
Chapter3: Discussion……………….………………………………………………………….50
Chapter 4:Conclusions and Future Directions………………………………………………56
References .....................................................................................................................................61
v
List of Table
Table 1: p53-activating small molecule drugs utilized in hematological malignancies………………………24
vi
List of Figures
Figure1: B cell maturation in WM……………………………………………………………...5
Figure2: Clinical features of WM……………………………………………………………….7
Figure3:MYD88L265 activation of NF-κB pathway…………………………………………11
Figure4:Mechanismof p53 driven intrinsic apoptoticpathway…………………………….22
Figure5: PRIMA-1met structure and mode of action……………………………………...28
Figure 6: Proposed mechanism linking PRIMA-1metinduced P73 and ROS production..60
Paper Figures:
Figure1:The effect of PRIMA-1meton viability of WM cell lines and patient samples..…36
Figure 2:Theapoptoticeffect of PRIMA-1met in WM cell line.............................................37
Figure3:The effect of PRIMA-1meton apoptotic signaling in BCWM-1 cells………………….37
Figure 4: Anti-tumoractivities of PRIMA-1metin WM cells…………………………………………..38
Figure5:Effects of PRIMA-1metin combination with current WM therapeutics……………39
Figure 6: PRIMA-1met cytotoxicity is P53-independent……………………………………………………40
Figure7: PRIMA-1met effect on BCWM-1 survival is P73-dependent…….………………………..41
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List of Abbreviations
AML Acute Myeloid Leukemia
aCGH Array-based Genomic Hybridization
Apaf-1 Apoptotic protease activating factor 1
ASCT Autologous Stem Cell Transplants
AS-PCR Allele Specific Polymerase Chain Reaction
Bax Bcl-2 associated x protein
Bcl-xL B-cell lymphoma-extra Large
Bcl-2 B-cell lymphoma 2
BMNC Blood Mononuclear Cell
BMSC Bone Marrow Stromal Cells
BTK Bruton's Tyrosine Kinase
CAD Caspase-Activated DNase
CAN Chromosomal Numerical Abnormalities
CXCR4 C-X-C Receptor type 4
CR Complete Remission
GLS2 Glutaminase
G6PD Glucose-6-Phosphate Dehydrogenase
HSP70 Heat Shock Protein 70
IAP Inhibitors of Apoptosis Protein
Ig Immunoglobulin
International Prognostic Staging System for Waldenstrom’s
IPSSWM Macroglobulinemia
IRAK Interleukin-1 Receptor-Associated Kinase
MDM2 Mouse Double Minute 2
MGUS Monoclonal Gammopathy of Undetermined Significance
MM Multiple Myeloma
MPT Mitochondrial Permeability Transition
MQ Methylene quinuclidinone
viii
MR Minor remission
MYD88 Myeloid Differentiation Primaryresponse gene 88
NADPH Nicotinamide Adenine Dinucleotide Phosphate
ORR Overall Response Rate
PBMNC Peripheral Blood Mononuclear Cell
PRIMA-1 P53-dependent reactivation and induction of massive apoptosis
PRDM1 PR Domain Zinc Finger Protein 1
Puma P53 upregulated modulator of apoptosis
REAL Revised European-American Lymphoma
ROS Reactive Oxygen Species
SDF-1 Stromal Cell-Derived Factor-1
SCID Sever Combine Immunodeficient
Smac/DIABLO Second Mitochondria-derived Activator of Caspases/ Direct
IAP- Binding protein with Low PI
TNF Tumor Necrosis Factor
TNFAIP3 Tumor Necrosis Factor Alpha-Induced Protein 3
VGPR Very Good Partial Remissions
WM Waldenström's Macroglobulinemia
WHO World Health Organization
XBP1 X-box Binding Protein 1
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Chapter 1
Introduction
1.1. Waldenström's Macroglobulinemia
1.1.1. What is Waldenström's Macroglobulinemia?
Waldenström's Macroglobulinemia (WM) is a chronic B-cell lymphoproliferative
malignancy (Gertz,2012).It was first described by Dr. Jan Gösta Waldenström, a
Swedish internist, in two patients who presented oronasal bleeding, anemia,
lymphadenopathy, hypergammaglobulinemia, an elevated sedimentation rate,
hyperviscosity, normal bone survey, cytopenias, and a predominantly lymphoid
involvement of the bone marrow (Shaheen et al. , 2012). Today, the World Health
Organization (WHO) defines WM as a lymphoplasmacytic lymphoma
characterized by plasmacytic infiltration of bone marrow and immunoglobulin M
(IgM) monoclonal gammopathy (Shaheen et al., 2012). Malignant cells in WM
are quite various cytologically ranging from small lymphocyte to plasmacytoid
lymphocytes and plasma cells (Naderi and Yang, 2013). These cells originate late
in B cell development after somatic hypermutation but before final differentiation
to plasma cells (Jenz, 2013). Common symptoms of WM includes: fatigue due to
anemia, thrombocytopenia, hyperviscosity symptoms and in more severe cases of
the disease; organomegaly, neuropathy and symptoms associated with Ig
deposition (Treon, 2013).
Historically, any type of lymphoma with high levels of Igs was associated with
WM; therefore, it was popular to consider WM as a clinical syndrome that is
associated with various lymphoma types instead of a separate disease (Shaheen et
al., 2012). Until a few years ago, differential diagnosis of WM was quite difficult
for hematopathologists both due to inefficient definitions of the disease and lack
of proper diagnostic tools. In 1994, the Revised European-American Lymphoma
(REAL) classification defined Lymphoplasmacytic Lymphoma (LPL) as “a
1