The Pennsylvania State University The Graduate School College of Medicine MOLECULAR MECHANISMS OF NANOLIPOSOMAL C6-CERAMIDE-INDUCED CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA A Dissertation in Molecular Medicine by Ushma A. Doshi 2015 Ushma A. Doshi Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2015
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The Pennsylvania State University
The Graduate School
College of Medicine
MOLECULAR MECHANISMS OF NANOLIPOSOMAL
C6-CERAMIDE-INDUCED CELL DEATH IN CHRONIC
LYMPHOCYTIC LEUKEMIA
A Dissertation in
Molecular Medicine
by
Ushma A. Doshi
2015 Ushma A. Doshi
Submitted in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
December 2015
ii
The dissertation of Ushma A. Doshi was reviewed and approved* by the following: Charles Lang Director of the Graduate Program Professor of Cellular and Molecular Physiology Dissertation Co-Adviser Co-Chair of Committee
Mark Kester Professor of Pharmacology, University of Virginia School of Medicine Dissertation Co-Adviser Co-Chair of Committee
Thomas P. Loughran Professor of Medicine, University of Virginia Cancer Center Hong-Gang Wang Lois High Berstler Professor, Pediatrics and Pharmacology Jin-Ming Yang Professor of Pharmacology
David Claxton Special Member Professor of Medicine
Richard R. Young Professor of Supply Chain Management; Business Administration
*Signatures are on file in the Graduate School.
iii
ABSTRACT
Chronic lymphocytic leukemia (CLL) is the most prevalent form of adult
leukemia in Western countries. Despite a high incidence, its pathogenesis is still
poorly understood, hence limiting treatment strategies. Furthermore, since CLL is
predominantly a disease of the elderly, numerous therapeutic strategies are
unsuitable due to limited physical fitness of the patient. Therefore, the CLL
remains incurable for most patients. Further research is needed to develop novel
therapeutic strategies.
Ceramide is a ‘tumor suppressor’ sphingolipid known to regulate
differentiation, senescence and cell cycle arrest. While a large body of work
reveals the mechanism of nanoliposomal ceramide (CNL)-induced cell death in
several types of cancers, the effect in CLL remains unclear. This study
investigates the effect of CNL in CLL and deciphers the key signaling
mechanisms mediating CNL-induced cell death. We have shown that CNL
selectively induces cell death in CLL cells by targeting the Warburg effect
through reducing levels of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), with no detrimental effects on normal peripheral blood mononuclear
cells. Additionally, CNL treatment results in tumor regression in an in vivo murine
model of CLL. Several reports in the literature have shown that signal transducer
and activator of transcription 3 (STAT3) is constitutively phosphorylated on
serine-727 in CLL and that STAT3 might be a therapeutic target in this disease.
We demonstrate that CNL suppresses STAT3 phosphorylation at both tyrosine-
705 and serine-727 by inhibiting multiple upstream kinases that include Bruton’s
tyrosine kinase, mitogen-activated protein kinase kinase and protein kinase C.
iv
This suppression in STAT3 phosphorylation and the subsequent downregulation
of STAT3 transcriptional activity mediates CNL-induced cell death in CLL. Recent
work in the literature has uncovered that STAT3 phosphorylated at serine-727
associates with mitochondrial components and regulates the respiratory
chain. Overactivation of mitochondrial STAT3 phosphorylated at serine-727
confers viability and stress protection to CLL cells. Our initial results demonstrate
that CNL treatment reduces mitochondrial STAT3 levels, which might also be
critical to the cell death induction.
Taken together, our results suggest that inhibition of glycolytic respiration
and inhibition of STAT3 transcriptional activity are key signaling mechanisms of
CNL-induced cell death in CLL cells. Additionally, we also speculate that
inhibition of STAT3-dependent mitochondrial respiration is also critical for
induction of cell death by CNL treatment. We conclude that CNL could potentially
be an effective therapy for CLL. Overall, this work emphasizes targeting the
sphingolipid pathway and development of sphingolipids-based therapeutics for
cancer.
v
TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................................ vii
ABBREVIATIONS .......................................................................................................... ix
PREFACE ...................................................................................................................... xi
ACKNOWLEDGEMENTS .............................................................................................. xii
CHAPTER 1: Literature Review ................................................................................... 1
STAT3 Signal Transducer and Activator of Transcription 3
S1P Sphingosine 1-phosphate
TNF Tumor necrosis factor
TNFR1 Tumor necrosis factor receptor 1
TRAIL TNF-related apoptosis-inducing ligand
TRAILR1 TRAIL receptor 1
TRAILR2 TRAIL receptor 2
UGCG UDP-glucose ceramide glucosyltransferase
xi
PREFACE
I would like to recognize that Chapter 2 of my dissertation titled “C6-ceramide
nanoliposomses target the Warburg effect in chronic lymphocytic leukemia” is
derived from published data (PLoS One, 2013 Dec 19;8(12):e84648) for which I
am co-first author. This project was underway when I joined the Kester lab in
2011. Dr. Lindsay Ryland and I contributed towards the design of the
experiments within this particular chapter. Specifically, I was a major contributor
towards the following figures: Fig 2-1A, Fig. 2-3A, Fig. 2-4A and Fig 2-5. I am in
no way attempting to claim intellectual property over the design of all the
experiments within this particular chapter. Therefore, I would like to acknowledge
Dr. Lindsay Ryland and the other contributing authors for the work presented in
this chapter.
In addition, Dr. Lindsay Ryland performed experiments for Fig. 4-1 in
Chapter 4 of my dissertation titled “Effect of C6-ceramide Nanoliposomes on
mitochondrial bioenergetics and mitochondrial STAT3 in Chronic Lymphocytic
Leukemia”.
The remainder of my dissertation includes part of a published book
chapter for which I am first author (Chapter 1), manuscript for which I am first
author (Chapter 3), and manuscript in preparation for which I am first author
(Chapter 4).
xii
ACKNOWLEDGEMENTS
I would like to thank, first and foremost, my mentor and advisor Dr. Mark
Kester for giving me an opportunity to work in his laboratory and for guiding me
through this memorable journey. I owe my achievements to his incredible
support, guidance and his faith in my work. I would like to express my gratitude
towards him for encouraging me to pursue the MBA degree during the last two
years of my thesis work. Thank you for your patience and flexibility with my
unnatural working hours. I would also like to thank Dr. Thomas Loughran for his
mentorship throughout my graduate years. I would like to specifically thank Dr.
Charles Lang for his immense support and belief in my abilities as a graduate
student. I would not have been a part of Penn State College of Medicine, if it
were not for your conviction in my potential as a graduate student. I would like to
extend special thanks to Dr. HG Wang and Dr. Claxton for always being so
approachable and providing insightful comments on my work. In addition, I would
like to thank Dr. Jin-Ming Yang and Dr. Robert Young for being very supportive
committee members. All my professors at Penn State College of Medicine,
especially Dr. Kent Vrana, Dr. Ralph Keil and Dr. Xin Liu have extended their
support and guidance in my journey. I am indebted to past and present Kester
lab members for making the lab my second home. Special thanks to Dr. Todd
Fox and Dr. Su-Fern Tan for their valuable guidance in my work and their very
special friendship. Special thanks to Dr. Jeremy Haakenson, Dr. Jody Hankins
and Dr. Megan Young for their consistent support. I would like to thank Sriram
Shanmugavelandy for being a superb friend in lab. Thanks to Samuel Linton,
xiii
Tony Brown, Dr. Brian Barth and all other members in the Kester lab for this
spectacular journey. Special thanks to Taryn Dick, members of the Wang lab,
administrative staff of the Graduate Office at College of Medicine and the Penn
State Hershey Security.
I cannot imagine this journey without my friends and family. Vijay Kale,
Rameshwari Kale, Manmeet Raval and Darshan Trivedi – Hershey was indeed
the sweetest place on Earth for me because of your unfailing love and support.
Lastly, and most importantly, I am blessed with the best family. My best friend
and husband, Varun Prabhu, has been my guiding light and the best companion
in the last ten years. Thank you Varun, for your undying motivation, strength and
love. Special thanks to my sister, Kshama Doshi for being the source of my
strength, my best friend and my energy throughout. I heartily thank my parents
and my in laws for their love, constant support, motivation and their
understanding throughout my PhD years. I dedicate this work to my family.
Lastly, I would like to thank God for giving me the strength to dream big and
the grit to achieve them.
1
CHAPTER 1: Literature Review
Sphingolipids
Sphingolipids, a group of bioactive lipids, represent one of the eight major
classes of lipids [1]. This class of lipids are structurally characterized by a
sphingoid base backbone comprising of sphingosine most frequently, and the
presence of an amide-linked fatty acid and/or a headgroup attached to the
hydroxyl on carbon 1 (Fig. 1-1) [1].
Sphingolipid metabolism
Sphingolipid metabolism is a complex, compartmentalized and a highly
inter-connected system comprising of enzymes catalyzing the formation of
different classes of sphilgolipids. Ceramide is considered to be the central hub of
sphingolipid metabolism. The generic ‘ceramide’ is a family of more than 50
distinct molecular species with a base structure consisting of an acyl chain of
variable length attached to the sphingosine backbone [2]. Fig. 1-2 and Fig. 1-3
represent the sphingolipid metabolism pathway and the corresponding cellular
compartments.
Saturated bond forms
dihydroceramides
Figure 1-1 Fundamental structures of sphingolipids: Modified from Merrill et al. [1]. Sphingolipids are defined by having a sphingoid base (shown for sphingosine) that is often derivatized with an amide-linked fatty acid and/or headgroup of the general types shown.
2
Ceramide can be synthesized by an anabolic and a catabolic pathway. De
novo synthesis starts by serine palmitoyl transferase (SPT)-catalyzed
condensation of palmitate and serine to form 3-keto-dihydrosphingosine, which is
subsequently reduced to dihydrosphingosine followed by acylation by ceramide
synthases (CerS). Desaturases catalyze the formation of ceramide from
dihydroceramide. The endoplasmic reticulum (ER) and ER-associated
membranes are the site of de novo synthesis of ceramide [2].
The catabolic pathway of ceramide synthesis involves the conversion of
sphingomyelin to form ceramide by the action of acid sphingomyelinase (SMase)
residing in the outer membrane leaflet or neutral SMase in the inner leaflet of the
bilayer. Sphingomyelin transported to the lysosomes can also be converted to
ceramide by the action of lysosomal SMase. Ceramide can reversibly be
converted to sphingomyelin by the action of sphingomyelin synthase (SMS) in
the Golgi apparatus. The sphingomyelin produced in the Golgi can be
transported to the plasma membrane by vesicular transport where it can be
converted back to ceramide by the action of SMase as described earlier [2].
Ceramide can also be metabolized to glucosylceramide (GlcCer) in the
Golgi apparatus by the action of GlcCer synthase (GCS). GlcCer serves as the
precursor of complex glycosphingolipids in the Golgi. Reversibly, ceramide can
also be synthesized from GlcCer by the action of glucosyl ceramidase localized
in the lysosomal compartment [2]. Ceramide is also metabolized to ceramide-1-
phosphate by the action of ceramide kinase.
3
Another critical metabolism pathway is deacylation of ceramide to
sphingosine (Sph) by the action of ceramidases (CDase). There are five
ceramidases that are products of different genes: neutral CDase, acid CDase
and three forms of alkaline CDase [3]. These enzymes lie at a crucial juncture in
the sphingolipid pathway since, in conjunction with sphingosine kinases (SphK),
they balance the ceramide/sphingosine-1-phosphate (S1P) rheostat in cells.
Lysosomal acid CDase hydrolyses ceramide to form sphingosine (Sph), which is
favorably partitioned into the lysosomes due to its positive charge [2].
Furthermore, conversion of sphingosine to ceramide is mediated by CerS, which
forms a part of the de novo synthesis pathway of ceramide.
Sphingosine kinases 1 and 2 (SphK1 and SphK2) are the two sphingosine
kinase isozymes that catalyze the formation of S1P from sphingosine. It has
been postulated that SphK1 is present just outside the lysosomes ensuring
effective trapping of the Sph within the lysosomes by SphK1-mediated
phosphorylation. The presence of S1P phosphatases in the ER generates Sph
which can move among cellular biomembranes or which is eventually recycled to
form ceramide [2]. S1P lyase, which metabolizes S1P to ethanolamine
phosphate and hexadecenal, is the exit pathway in sphingolipid metabolism.
4
Figure 1-2 Sphingolipid metabolism pathways: Taken from Hannun et al. [2]
Figure 1-3 Compartmentalization of sphingolipid metabolism: Taken from Hannun et al. [2].
5
Functions of Sphingolipids
Sphingolipids function both as structural components of the cell and
mediators of cell signaling. As structural components of cellular biomembranes,
they play a critical role in regulating membrane fluidity and subdomain structure
of the lipid bilayer, especially lipid rafts [4]. Sphingolipids create lateral
differentiation of cellular membranes into a mosaic of structural domains with
unique molecular compositions. Lateral lipid assemblies are formed as a result of
varying miscibility of cell membrane-forming lipids like sphingolipids,
glycerolipids, and sterols. The protein content of such microdomains or rafts
characterize their function and serve as platforms for cellular events like signal
transduction, cell adhesion and protein sorting [5]. Sphingolipids like ceramides
affect the composition and properties of phospholipid bilayers by increasing the
order of the acyl chain in the bilayer, creating phase separation of ceramide-rich
and ceramide-poor domains and facilitating transition from bilayer to non-bilayer
structure [6]. In addition to cellular biomembranes, enzymatically-synthesized
ceramides also alter the properties of lipidic vesicles by inducing destabilization
of lipid bilayers through permeabilization and fusion [6]. Various biological
consequences follow ceramide-induced biophysical changes in cellular
biomembranes. For instance, the change in membrane fluidity after ceramide-
induced raft formation may result in modification of enzymatic activity of
membrane proteins, or may change the protein affinity for the membrane.
Ceramides also bind to specific sites in the target protein and alter enzymatic
activity. These target proteins are both membrane-bound proteins and other
6
cytoplasmic proteins transiently recruited to the bilayer where ceramides are
located [6].
In addition to structural roles, sphingolipids play a critical role in cellular
signaling. Extensive work has been done to elucidate the role of sphingolipids in
modulating cellular signaling. This section will broadly discuss the mechanisms
through which sphingolipids regulate cellular signaling mechanisms. Firstly,
sphingolipids act as ligands to receptors, initiating signaling pathways involved in
cellular processes like growth, adhesion, differentiation and migration. The
ligand-receptor interaction can be triggered in the same cell secreting the
sphingolipids or in neighboring cell types [7]. For instance, S1P is a ligand for a
family of G-protein coupled receptors called S1P receptors that regulate
biological processes such as cell proliferation, angiogenesis, migration, immune
cell trafficking and mitogenesis. Secondly, sphingolipids influence the properties
of receptors via specific lipid-protein interactions and regulating responsiveness
to external stimuli. For instance, it has been shown that the ligand-binding
capacity of human serotonin 1A receptors is impaired under glycosphingolipid-
depleted condition [8]. The authors speculate that this effect is due to a reduction
in the specific interaction of serotonin 1A receptors with membrane
glycosphingolipids [8]. Thirdly, as discussed earlier, sphingolipids alter the
biophysical properties of cellular biomembranes affecting the assembly of
membrane receptors and effector molecules in specific domains called rafts. This
sphingolipid-induced alteration in membrane biophysics regulates cellular
signaling [7]. Lipid rafts create a micro-environment suitable for effective receptor
7
activation by concentrating relevant kinases and protecting proteins from
phosphatases and other molecules that may diminish the signaling processes.
For example, lipid rafts are critical for immunoglobulin E signaling and T-cell
antigen receptor-mediated signaling [9]. Lastly, sphingolipids like ceramides and
S1P can act as direct signaling molecules in processes like proliferation,
differentiation, senescence, cell–cell interaction and transformation [7]. For
instance, intranuclear S1P has been shown to inhibit the enzymatic activities of
histone deacetylases, preventing the removal of acetyl groups from lysine
residues within histone tails. This work has shown the role of intranuclear S1P in
regulating gene expression [10].
Because sphingolipids are bioactive molecules and mediate several
cellular processes like proliferation, differentiation, migration, death and cell-cell
interaction, they have been implicated in the pathogenesis of several human
disorders. There is an abundance of literature reporting the role for sphingolipids
in inflammatory and immune responses, vascular function, neurodegeneration,
insulin signaling and diabetes, microbial pathogenesis and cancer pathogenesis
and therapy [11]. This dissertation is focused on studying the role of short-chain
ceramides as a potential therapy for chronic lymphocytic leukemia (CLL).
Furthermore, this dissertation also focuses on elucidating the key molecular
mechanisms which mediate the effect of short-chain ceramides in CLL cells.
8
Ceramide for cancer therapeutics
Sphingolipids have been implicated in cancer pathogenesis since they
mediate several cellular processes like proliferation, differentiation, migration,
death and cell-cell interaction. Numerous studies in the literature have reported a
dysregulation in sphingolipid metabolism in different types of cancers. The
dysregulated sphingolipid profile in cancer cells contributes to cancer progression
and metastasis, making it an ideal candidate for developing targeted
therapeutics. Moreover, sphingolipids may also serve as vital biomarkers for
cancer progression, as well as guide therapeutic regimens [12].
The role of sphingolipids in cancer pathogenesis and treatment has been
of particular interest to the sphingolipid research community. First reports
describing the involvement of sphingolipids in mediating apoptosis in cancer cells
came in early 1990s. It was reported that synthetic short chain ceramide analogs
like C2-ceramide induced cell death in HL60 leukemic cells and caused
internucleosomal DNA fragmentation [13]. Tumor necrosis factor α (TNFα) and
ionizing radiation induced apoptotic cell death in cancer cells, which was
mediated by sphingomyelin hydrolysis and subsequent ceramide generation [14,
15]. Apoptotic cell death through CD95 crosslinking in U937 cells also utilized the
sphingomyelin pathway and depended on ceramide production [16]. The first
body of work demonstrating the role of sphingolipids in mediating chemotherapy-
induced apoptosis in cancer cells was published in 1995, wherein the authors
showed that daunorubicin, a chemotherapeutic drug, induced apoptosis in P388
and U937 leukemia cells by elevating intracellular ceramide levels. This increase
9
in the intracellular ceramide pool was not due to the action of SMase, but rather
via activation of CerS, which increased de novo ceramide synthesis within cells
[17]. This revelation of a potential role of sphingolipid metabolism in mediating
chemotherapy-induced cytotoxicity generated immense interest in the scientific
community to decipher the connection between chemotherapy and sphingolipid
metabolism. Since then, a large body of research has been conducted to
establish an in-depth understanding of how sphingolipid metabolism mediates,
enhances, or impedes chemotherapy-induced cytotoxicity, with the goal of
identifying critical therapeutic targets and better therapeutic regimens for
management and cure of cancer.
Among the major sphingolipids that play a role in regulating cancer cell
fate, ceramide is termed as a “tumor suppressor lipid” because of its ability to
potentiate signaling cascades that lead to cell death. By contrast, sphingosine-1-
phosphate (S1P) is considered as a pro-survival lipid. Thus, in the context of
sphingolipids, the ceramide-S1P rheostat dictates cancer cell fate. Efforts
directed at altering the sphingolipid balance in cancer cells to induce cell death or
to potentiate cytotoxicity of chemotherapeutic drugs would aim at either elevating
pro-apoptotic sphingolipids, especially ceramide, or down-regulating pro-survival
sphingolipids such as sphingosine-1-phosphate. This can be achieved by: (i)
chemotherapy-induced synthesis of pro-apoptotic ceramides or breakdown of
pro-survival sphingolipids; (ii) disruption of ceramide metabolism to enhance
ceramide accumulation; and (iii) delivery of exogenous ceramides to induce
apoptotic signaling (Fig. 1-4).
10
Figure 1-4 Strategies to alter the sphingolipid balance in cancer cells to potentiate
cytotoxicity of chemotherapeutic drugs. 1) Chemotherapy-induced synthesis of pro-apoptotic
sphingolipids or breakdown of pro-survival sphingolipids; 2) Disruption of metabolism of pro-
apoptotic sphingolipids to enhance accumulation; and 3) Nanoscale-based delivery of exogenous
pro-apoptotic sphingolipids in combination with standard chemotherapeutic drugs to induce
apoptotic signaling.
11
Ceramide and induction of cell death
Ample evidence in the literature exists that indicate that ceramide is a
tumor-suppressor lipid and halts tumor progression by inducing cell death and by
cell cycle arrest. Both stimulus-induced intracellular ceramide generation and
exogenous cell-permeable short-chain ceramides induce death in cancer cells by
apoptosis, necrosis or autophagy.
Ceramide and apoptosis
Ceramide generation has been linked to both, the extrinsic and the intrinsic
pathway of apoptosis. Ceramide is an important mediator of initiating cell death
by activation of the pro-apoptotic tumor necrosis factor (TNF) receptor
superfamily, including CD95, TNFR1 and the TNF-related apoptosis-inducing
ligand (TRAIL) receptors TRAILR1 and TRAILR2 [18]. Receptor activation leads
to ceramide synthesis at ceramide-enriched membrane platforms by the de novo
pathway or activation of SMases. The ceramide-enriched membrane platforms
act as scaffolds for localization of pro-apoptotic proteins that initiate intracellular
signaling for cell death, some of which possess ceramide-binding domains.
Ceramide-enriched membrane platforms assemble TRAILR2 and CD95, and act
as a scaffold for the formation of the death-inducing signaling complexes [19-21].
Activation of these cell death receptors induce intracellular ceramide generation
by activating specific CerS and SMases [18]. The importance of ceramide
generation in these cell death pathways has been demonstrated by reports that
inhibition of ceramide synthesis diminishes apoptosis. For instance, pretreatment
with dihydroceramide synthase inhibitor fumonisin B1 (FB1) has been shown to
12
diminish CD95-induced apoptosis in leukemia [22]. Similarly, low ceramide levels
have been correlated to resistance to CD-95 induced apoptosis, TNF-induced
and TRAIL-induced cell death in several cancer models [23-25].
Ceramide also mediates intrinsic pathway-driven apoptosis. Ceramide
accumulation affects mitochondrial bioenergetics and induces conformation of
mitochondrial pro-apoptotic proteins to initiate apoptosis. Intensive research on
the effects of ceramide generation in the mitochondria has convincingly
demonstrated that accumulation of ceramide macrodomains in mitochondria
cause formation of ceramide channels that induce mitochondrial outer membrane
permeabilization (MOMP) [26, 27]. Ceramide accumulation either through
exogenously delivered short chain ceramides or endogenous ceramide
generation synergizes with pro-apoptotic proteins like BAX and BID to
permeabilize mitochondrial membrane and the subsequent release of pro-
apoptotic proteins like cytochrome c and activation of the caspase cascade [28-
31]. Ceramide also initiates intrinsic pathway-driven apoptosis by activating
kinases like p38 MAPK, glycogen synthase kinase (GSK) 3β, JUN N-terminal
kinase (JNK), protein kinase C (PKC) δ or inactivating AKT, which eventually
perturb mitochondrial integrity and cause release of pro-apoptotic proteins [18].
Ceramide is linked to several signaling pathways in apoptosis. The role of
ceramide in inactivating the very crucial AKT pathway in cancer cells has been
widely studied. The downstream targets of ceramide to bring about this
inactivation include PP2A, PKCζ and p38 MAPK [18]. Furthermore, ceramide
activates apoptosis signal-regulating kinase 1 (ASK1) which eventually increases
13
p38 and JNK activation [32]. Additionally, ceramide promotes p53 activation in
certain cancer types which causes a reduction in BAX/BCL-2 ratio, eventually
leading to apoptotic cell death [33, 34]. Finally, ceramide has also been shown to
downregulate anti-apoptotic proteins like survivin to induce apoptotic cell death
[35].
Ceramide and necrosis
The role of ceramide in necrotic cell death is not as well characterized as
apoptosis. Treatment with short chain ceramides like C2 and C6-ceramide
induces necrotic cell death in A20 B-, Raji B- and Jurkat T cells. Cell death was
without caspase-3 activation, DNA fragmentation, cell shrinkage, or chromatin
condensation. FasL-dependent delayed elevation of ceramide promoted caspase
8-driven necrotic morphology after treatment. Inhibition of ceramide production
shifted the mechanism of cell death from necrosis to apoptosis [36]. Further
investigation of the necrotic mechanism has revealed that exogenous C6-
ceramide causes necrosis in lymphoid cells by rapid production of reactive
oxygen species (ROS), loss of mitochondrial membrane potential and ATP
depletion [37]. This is supported by data demonstrating that C2-ceramide-
induced oncotic necrosis in mouse epidermal tumor cells is modulated by a
decline in cellular glutathione and an elevation of ROS [38]. Our lab has also
shown that C6-ceramide delivered as a nanoliposomal formulation induces
necrotic cell death in chronic lymphocytic leukemia (CLL) cells by targeting the
Warburg effect through downregulation of glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) enzyme of the glycolysis pathway and ATP depletion,
14
supporting the report that synthetic ceramides induce non-apoptotic and necrotic
cell death in malignant B-lymphocytes [39, 40]. C2-ceramide has also been show
to predominantly induce necrotic cell death in NB16 neuroblastoma cells.
Although combined treatment with TNFα and cycloheximide is mediated by
intracellular ceramide generation, this stimuli induces apoptosis instead of
necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic
ligands such as TNFα, which are thought to be mediated by an accumulation of
endogenous ceramide. C2-ceramide targets phosphatidylcholine in these cells
and elicits a mixture of cell death mechanisms, including necrosis and apoptosis,
the former being more predominant [41]. Ceramide also induces non-apoptotic,
caspase-independent cell death by inducing ROS generation in A172 human
glioma cells. NF-kappaB is involved in the regulation of ceramide-induced cell
death in human glioma cells [40]. Lastly, it has also been observed that certain
cellular parameters play an important role in determining the mechanism of cell
death after ceramide treatment. For instance, in Hep-G2 cells the mitochondrial
respiratory function determines the mechanism of cell death after treatment with
exogenous short chain ceramides. Herein, C2-ceramide induced necrosis which
was a result of 80% inhibition of the mitochondrial respiratory function leading to
ATP depletion and ROS generation. In contrast, C6-ceramide induced apoptotic
cell death in the same cells since mitochondrial function was not inhibited and
ATP production not diminished [42].
15
Ceramide and autophagy
Despite ceramide being a promoter of autophagy, its role in mediating
autophagic cell death is confounded by the role of autophagy in cancer cells, i.e.
lethal versus survival autophagy. Increased levels of long-chain ceramides
(C14:0 – C20:0 ceramides) and especially dihydroceramides have been
associated with both lethal and survival autophagy in different cancer cell types
[43, 44]. It is believed that the fate of the autolysosome dictates the function of
autophagy as lethal versus survival. Intracellular generation of sphingosine and
S1P in the autolysosomes by the hydrolysis of dihydroceramides and ceramides
promotes pro-survival autophagy. In contrast, accumulation of dihydroceramides
in the autolysosomes can enhance autolysosomal membrane permeability and
cause the release of cathepsins, thereby causing apoptotic cell death [45]. In this
case, ceramides promote lethal autophagy in cancer cells.
Thus, as discussed earlier sphingolipid-based therapeutics aim to alter the
sphingolipid balance to induce cell death by either elevating pro-apoptotic
sphingolipids, especially ceramide, or down-regulating pro-survival sphingolipids
such as S1P. This can be achieved by: (i) chemotherapy-induced synthesis of
pro-apoptotic ceramides or breakdown of pro-survival sphingolipids; (ii) disruption
of ceramide metabolism to enhance ceramide accumulation; and (iii) delivery of
exogenous ceramides to induce apoptotic signaling. The next section discusses
the first strategy as a proof-of-concept to demonstrate that the effectiveness of
current chemotherapies and investigational drugs is partially or completely
16
mediated by endogenous ceramide generation. The discussion will then focus on
the development of exogenous ceramide-based therapeutics for cancer.
Chemotherapy-induced ceramide generation by the de novo pathway
Many chemotherapeutics increase ceramide levels by upregulating the de
novo pathway of ceramide synthesis. This is accomplished by increasing the
activity of serine palmitoyl transferase (SPT), ceramide synthase (CerS), or both.
There are six CerS isoforms (CerS1-6), which are also known as longevity
assurance (LASS) genes, with each isoform corresponding to specific resulting
SKI II, 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole.
30
Chronic Lymphocytic Leukemia (CLL)
CLL is the most prevalent form of adult leukemia in Western countries. As
per 2015 statistics, CLL accounts for approximately 30% of total leukemia cases
diagnosed and it accounts for 20% of deaths from all kinds of leukemia [125].
CLL mainly affects older adults. The average age at the time of diagnosis is
around 71 years. It is rarely seen in people under age 40, and is extremely rare
in children.
CLL is a malignant lymphoproliferative disorder of mature B lymphocytes.
The disease is characterized by an accumulation of mature looking B
lymphocytes in the blood, bone marrow, lymph nodes or other lymphoid tissues.
Leukemic B cells express characteristic surface markers consisting of CD19,
CD20 (weak) and CD23, with co-expression of CD5. Most CLL cells are arrested
in the G0/G1 phase and are highly resistant to apoptosis, eventually leading to
an accumulation of malignant cells [126]. A large body of work has demonstrated
that several factors like microenvironmental stimuli, antigenic drive and
epigenetic and genetic deregulation dictate the pathogenesis of CLL.
CLL is classified into different sub-types that determine the prognosis of
the disease and the treatment strategy. One classification is based on the degree
of somatic hypermutation, i.e. whether the cells express mutated or unmutated
immunoglobulin heavy chain variable region (IGHV) genes. The two groups
follow a different clinical course, with poorer survival in patients exhibiting
unmutated IGHVs. In addition to this determinant, approximately 80% of CLL
cases also show chromosomal aberrations. These genetic aberrations are
31
observed in both IGHV mutated and unmutated CLL, the latter being associated
with higher incidence of high-risk aberrations [125]. Deletion in band 13q14 is the
most common genetic aberration in CLL and has a favorable prognosis. This part
of the chromosome contains mir-15a and mir-16-1 which have been implicated in
CLL pathogenesis [127]. Trisomy 12 is another frequent chromosomal
abnormality in CLL, however, the corresponding molecular implications on
pathogenesis remains unknown. Deletion in band 11q23 is not a very frequent
aberration in early stage disease, but is associated with rapid disease
progression and excessive lymphadenopathy [128, 129]. This deletion usually
corresponds with deletion of the ataxiatelangiectasia-mutated (ATM) gene, which
is an essential component of the cell cycle checkpoint system. Deletion in the
ATM gene is characterized by extreme sensitivity to irradiation, genomic
instability and a predisposition to lymphoid malignancies [125]. Lastly, deletion in
band 17p13, corresponding to p53 deletion and TP53 mutation is associated with
poor prognosis [125].
The microenvironment in the lymphoid organs plays a crucial role in the
pathogenesis of CLL. The microenvironment consists of T-cells, stromal cells and
soluble factors. Soluble factors from the microenvironment provide CLL cells with
a protective environment and provide anti-apoptotic and pro-proliferative stimuli
that are necessary for the progression of the disease. For instance, CLL cells
recruit CD3+ T cells which also express CD40L and CD4. CD40L from these
accessory cells initiate the indispensable B-cell receptor (BCR) signaling in CLL
cells after interaction with CD40 receptor. Such stimuli induce production of anti-
32
apoptotic proteins like survivin, Mcl-1 and Bcl-2 in CLL cells which are crucial for
cell viability [125, 130]. Furthermore, the stromal microenvironment promotes a
metabolic switch in CLL cells from mitochondrial respiration to aerobic glycolysis,
thus conferring growth advantage and conferring chemoresistance to CLL cells
[131]. In conclusion, ample evidence in the literature exists emphasizing the
crucial role of stromal microenvironment in the pathogenesis and
chemoresistance of CLL. A multitude of molecules including integrins, spleen
tyrosine kinase, stromal derived factor-1, Notch, CD44, and thioredoxin have
been identified to be part of the stromal cross talk [125].
The standard treatment regimen for physically fit CLL patients includes a
combination chemo-immunotherapy with fludarabine, cyclophosphamide and
rituximab with an overall response rate of approximately 90% and complete
remission of 72% [132, 133]. A combination of purine analogs, alkylating agents,
monoclonal antibodies (immunotherapy) and BCR pathway and tyrosine kinase
inhibitors is the standard treatment. Novel drugs recently incorporated in the
treatment regimen include ibrutinib, which targets an important component of
BCR signaling, Bruton’s tyrosine kinase (BTK). Another novel orally available
agent is idelalisib, a phosphatidylinositol-3-kinase (PI3K) δ inhibitor. Both of
these drugs have been for relapsed/refractory disease and first-line treatment of
patients with TP53 mutation/deletion [134]. Unfortunately these advances do not
benefit older CLL patients due to their frail health [135]. Overall, CLL is incurable
with the current therapies, with allogeneic stem cell transplantation as the only
potentially curative treatment option in CLL. However, this option is also limited to
33
young and relatively healthy patients. Despite these advances in therapeutics,
eventual drug resistance and relapse ultimately cause CLL to be an incurable
and chronic disease [136]. Further research is needed to develop therapeutic
strategies.
The role of sphingolipids in CLL pathogenesis or treatment has not been
explored yet. Synthetic ceramides have been shown to induce non-apoptotic and
necrotic cell death in malignant B-lymphocytes [137]. Additionally, a few reports
have also demonstrated that sphingolipids mediate cell death in CLL cells. It has
been reported that membrane microdomain sphingolipids are required for anti-
CD20-induced cell death in CLL cells [138]. The authors showed that resistance
to anti-CD20-induced cell death is associated with a defective recruitment of Csk-
binding protein, resulting in a lack of sphingomyelin and ganglioside M1 at the
outer leaflet of the plasma membrane of malignant B cells. Inducing P-
glycoprotein in resistant cells restored sensitivity to anti-CD20 antibody as the
inducer normalized the quantity of sphingomyelin within the membrane [138].
Another recent report has uncovered a novel link between BCR signaling and
sphingolipid metabolism. It has been reported that BCR controls
chemoresistance of primary CLL cells by controlling glucosylation of ceramides
[139]. Specifically, BCR engagement increases levels of anti-apoptotic
glucosylated ceramides via upregulation of UDP-glucose ceramide
glucosyltransferase (UGCG), an enzyme which converts pro-apoptotic ceramide
to anti-apoptotic glucosylceramide. The authors have shown that inhibitors of
BCR signaling sensitize resistant CLL cells towards ABT-737 drug via UGCG
34
inhibition [139]. Another report has demonstrated that sphingolipid metabolism is
a potential novel mechanism of CLL [140]. Taken together, these reports provide
compelling evidence in support of the use of sphingolipid-modulating strategies,
and more specifically, ceramide-based strategies as novel therapeutics in CLL.
Conclusions
The role of nanoliposomal C6-ceramide in inducing cell death in several
types of solid and non-solid cancers is well understood. However, the effect of
nanoliposomal C6-ceramide treatment in CLL remains unclear. This dissertation
primarily focuses on delineating the molecular mechanisms of C6-ceramide-
induced cell death in CLL. Identifying the key signaling pathways inducing cell
death after ceramide treatment is also valuable to uncover additional targets for
potential combination therapies with ceramide nanoliposomes. Encapsulation of
chemotherapeutic drugs into nanoliposomes has been a largely successful
delivery formulation in cancer models in vitro and in vivo. Moreover, the ongoing
success of using ceramide nanoliposomes as a platform for combinatorial
therapy with other neoplastic agents presents a promising future to this endeavor
of developing more effective therapeutics for CLL.
35
CHAPTER 2: Nanoliposomal C6-ceramide target the Warburg effect in chronic lymphocytic leukemia
I would like to recognize that Chapter 2 of my dissertation titled “C6-ceramide
nanoliposomses target the Warburg effect in chronic lymphocytic leukemia” is
derived from the following published literature:
Ryland LK*, Doshi UA*, Shanmugavelandy SS, Fox TE, Aliaga C, Broeg K, Baab KT, Young M, Khan O, Haakenson JK, Jarbadan NR, Liao J, Wang HG, Feith DJ, Loughran TP Jr, Liu X, Kester M. C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia. PLoS One. 2013 Dec 19;8(12):e84648. I am the co-first author for this published work.
This project was underway when I joined the Kester lab in 2011. Dr. Lindsay
Ryland and I contributed towards the design of the experiments within this
particular chapter. Specifically, I was a major contributor towards the following
figures: Fig 2-1A, Fig. 2-3A, Fig. 2-4A and Fig 2-5. I am in no way attempting to
claim intellectual property over the design of all the experiments within this
particular chapter. Therefore, I would like to acknowledge Dr. Lindsay Ryland
and the other contributing authors for the work presented in this chapter.
36
Abstract
Ceramide is a sphingolipid metabolite that induces cancer cell death.
When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell
death is selectively induced in tumor models. However, the mechanism
underlying this selectivity is unknown. As most tumors exhibit a preferential
switch to glycolysis, as described in the “Warburg effect”, we hypothesize that
ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We
utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an
increased dependency on glycolysis. In CLL cells, we demonstrate that C6-
ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-
independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA
and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is
overexpressed in CLL. To confirm that ceramide targets GAPDH, we
demonstrate that downregulation of GAPDH potentiates the decrease in ATP
after ceramide treatment and exogenous pyruvate treatment as well as GAPDH
overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo
murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits
tumor regression, concomitant with GAPDH downregulation. We conclude that
selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-
ceramide could potentially be an effective therapy for leukemia by targeting the
Warburg effect.
37
Introduction
Sphingolipids are a class of complex cellular lipids that serve both a
structural role in the cellular membrane as well as an intracellular signaling role
within the cell. Several types of sphingolipid metabolites have been shown to
influence the balance between mitogenesis and apoptosis. Of particular interest
is the sphingolipid metabolite, ceramide, which regulates differentiation,
senescence and cell cycle arrest. Induction of cell death by this endogenous
lipid-derived second messenger occurs either via apoptotic, autophagic, or
were purchased from Sigma (St. Louis, MO). BTK inhibitor, ibrutinib was
purchased from MedChem Express (Monmouth Junction, NJ).
Patient characteristics and preparation of peripheral blood mononuclear
cells
All patients met the clinical criteria of CLL and were not on treatment at the time
of sample acquisition. Peripheral blood specimens from CLL patients were
obtained and informed consents signed for sample collection using a protocol
approved by the Institutional Review Board of Penn State Hershey Cancer
Institute. CLL PBMCs were chosen according to the following criteria: CD19+ >
80%, CD20+ > 80%, CD5+ > 90%. These criteria ensured that the PBMCs
isolated from CLL patient blood predominantly consisted of leukemic B-cells.
Buffy coats from normal donors were also obtained from the blood bank of the
Milton S. Hershey Medical Center, Pennsylvania State University, College of
Medicine. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-
Hypaque gradient separation, as described previously [160].
Cell culture
Culture of freshly isolated PBMCs and primary CLL patient cells was carried out
using RPMI-1640 medium supplemented with 10% fetal bovine serum (both from
Invitrogen). JVM-3 cells (DSMZ – German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany), a CLL cell line with wild type p53, were also
74
cultured in this same medium and cells were grown in 5% CO2 at 37˚C. Mec-2
cells (DSMZ – German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany), a CLL cell line with mutated p53 were cultured in
Iscove's MDM media supplemented with 10% FBS. HEK-293FT cells (Invitrogen,
Waltham, MA) were cultured in D-MEM media supplemented with 10% FBS and
1X Anti-anti antibiotic (Gibco, Waltham, MA).
Preparation of nanoliposomal ceramide
Preparation of nanoliposomal ceramide has been described in Chapter 2 of the
dissertation.
Preparation of lipid:BSA complexes
Lipids were dried under a stream of nitrogen and then resuspended in DMSO.
The lipid/DMSO solution was then added to a fatty acid-free BSA solution to
achieve a final concentration of 1 mM lipid, 1 mM BSA in 20 mM HEPES with
10% DMSO. Complexes were allowed to form by rocking at room temperature for
30 min, followed by sonication to clarity.
Cell viability assay
A set of experiments were conducted to determine the toxicity of the CNL and
Stattic in JVM-3 cells, Mec-2 cells, CLL patient cells and in normal donor PBMC.
Cell viability was performed using CellTiter 96® Aqueous One Solution assay kit
(Promega) and relative viable cell number was determined by reading the plates
at 490 nm wavelength in Synergy HT Multi-Detection Microplate Reader (Bio-
75
TEK). All samples were assayed in triplicate and each experiment was repeated
at least three times.
Cell death assays (Flow cytometry for AnnexinV/7AAD)
Apoptosis was determined in JVM-3 cells, Mec-2 cells and in CLL patient cells by
2-color flow cytometry with Annexin-V-PE and 7-amino-actinomycin D (BD
Pharmingen Transduction Laboratories) staining using 5 x 105 cells per sample.
Necrosis was quantidied in the Annexin V positive and 7AAD positive quadrant.
Data were collected by flow cytometry.
Western blot analysis
Western blot analysis was performed on whole-cell lysates collected using RIPA
buffer (Sigma). Blots were washed and developed with enhanced
chemiluminescence (Thermo Scientific, Waltham, MA) following the
manufacturer’s instructions. Densitometry analysis was performed using ImageJ
software.
shRNA Knockdown of STAT3
STAT3 shRNA plasmid clones (Human pTRIPZ vector) were purchased from
Open Biosystems (Huntsville, AL) and used to transfect JVM-3 cells.
Nucleofection was performed using the Amaxa Nucleofector I device. JVM-3
cells (3 × 106 ), resuspended in 100μl of Cell line Solution Kit V (Amaxa,
Cologne, Germany) with 6μg of shRNA were transfected with the Amaxa
Nucleofector I device (program X-001), cultured in six-well plates in complete
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medium for 24, 48, 72 and 96 hours and then examined for STAT3 knockdown
using Western blot analysis. Cells were also analyzed for percent viability using
flow cytometric analysis for AnnexinV and 7AAD at 24, 48, 72 and 96 hours after
nucleofection.
Preparation of pervandate
1mM pervanadate stock was prepared by adding 10µL of 100mM sodium
orthovanate, 0.3% hydrogen peroxide diluted in 20mM HEPES and 940µL of
water. After 5 minutes of incubation, a small amount of catalase was mixed in the
pervanadate stock to remove excess hydrogen peroxide. The pervanadate was
used within 2 hours of preparation.
Luciferase reporter assay
Cignal reporter assay kit from Qiagen (Hilden, Germany) was used for obtaining
plasmids for the luciferase reporter assay. JVM-3 cells (2x106 cells) were
transfected with 4µg of either reporter construct, negative control construct or
positive control construct using the Amaxa Nucleofector I device (X-001
program). Cell were allowed to be in culture for 24 hours post transfection and
then treated for 12 hours with CNL or ghost liposomes. Dual-Glo luciferase assay
system from Promega (Madison, WI) was used to obtain luciferase
luminescence. The assay and quantification was done following the
manufacturer’s instructions.
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Lentiviral transduction for STAT3C overexpression
Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA) was used
for expressing STAT3-C in JVM-3 cells [32]. Briefly, viral particles were produced
in HEK293-FT cells using pLOC, VSVG, tat and DR8.2 plasmids and JVM-3
cells were transduced thrice with the viral media according to the manufacturer’s
protocol. JVM-3 cells were grown for 72 hours after the last transduction. The
STAT3-C overexpression vector has an EGFP sequence as a selectable marker
and the transduced cells were sorted for EGFP and grown as a pure population
(JVM3-STAT3C cells). The cells were then harvested for experiments. Human
pLOC overexpression vector (Open Biosystems) containing a RFP sequence
was used as a negative control. Cells were treated with CNL or ghost liposomes.
JVM-3 cells were transduced thrice with media containing the negative control
virus. FACS was not performed for the control JVM3-RFP cells since we
obtained approximately 70-80% transduction efficiency. 72 hours after the last
transduction, JVM3-RFP cells were harvested for experiments. JVM3-STAT3C
cells and JVM3-RFP cells were treated with 20µM and 40µM CNL and ghost
liposomes for 24 hours. 24 hours after treatment, cells death was analyzed using
flow cytometric analysis using AnnexinV-V450 and 7AAD staining.
Statistical analysis
All data are expressed as mean plus or minus SEM. Paired Student t test (2-tail
paired) and 2-way analysis of variance test were used to determine the statistical
78
significance and P value of 0.05 or less was considered statistically significant.
All results are a mean of three independent biological triplicates unless specified.
Results
STAT3 is a potential therapeutic target in CLL
Several reports suggest that STAT3 might play a role in the pathogenesis of
CLL [189, 190, 197]. We compared levels of total STAT3 between normal cells
and CLL cells. STAT3 was highly overexpressed in both, CLL cell lines and
patient cells in comparison to peripheral blood mononuclear cells (PBMC)
obtained from normal blood donors (Fig. 3-1A). Next, we evaluated if inhibiting
STAT3 signaling in CLL cells would induce cell death. Knock down of STAT3 in
JVM3 cells by using an inducible lentiviral STAT3-shRNA significantly increased
the number of Annexin V positive cells 24 hours after doxycycline induction (Fig.
1B). An average of 57% knockdown of STAT3 protein was observed 24 hours
after induction (Fig. 3-1B). Doxycycline was non-toxic to JVM3 cells at doses
used for induction. STAT3 knockdown caused a 108% increase in cell death 24
hours after doxycycline induction as compared to control cells. As the
knockdown weakened at later time points, cell death induction reduced to 42%
48 hours after doxycycline induction, and cell death was further reduced as the
protein levels resumed to normal levels 72 and 96 hours post doxycycline
induction (Fig. 3-1B). Reduction in STAT3 protein levels also corresponded with
a simultaneous reduction in downstream proteins like Mcl-1 that are under the
transcriptional control of STAT3. We also confirmed the role of STAT3 signaling
in CLL cell viability by using a pharmacological inhibitor of STAT3 called Stattic.
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Stattic is a non-peptidic small molecule selective inhibitor of STAT3 [198]. It
inhibits STAT3 signaling by selectively inhibits activation, dimerization, and
nuclear translocation of STAT3 [198]. We observed that treatment with Stattic for
24 hours caused a dose-dependent reduction in cell viability in two different CLL
cell lines (Fig. 3-1C (i) and (ii)). JVM-3 is a CLL cell line with wild-type p53, while
Mec-2 cells have mutated p53. Furthermore, we tested three different CLL
patient samples and observed a similar reduction in cell viability after treatment
with Stattic, whereas PMBCs from normal blood donors were resistant to
treatment with Stattic (Fig. 3-1C (iii)). Taken together, these results demonstrate
that STAT3 is essential for CLL cell survival. STAT3 signaling inhibited by either
molecular or pharmacological interventions reduced cell viability and induced
death in CLL cells.
Figure 3-1A. STAT3 is overexpressed in CLL cell lines and patient cells. JVM-3 cells, Mec-2
cells, PBMCs from normal blood donors (n=2) and PBMCs from CLL patients (n=4) were lysed
and protein extracted. Western blot analysis was one to determine the level of total STAT3 in the
samples`
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Figure 3-1B. Knockdown of STAT3 induces cell death in CLL cells. JVM-3 cells
were transfected with several clones of STAT3 shRNA and flow cytometric analysis was
performed to determine the % dead cells 24-96 hours after induction. Western blot
analysis was done at the same time points to determine the knockdown of STAT3 and
the levels of Mcl-1, a protein regulated by the transcriptional activity of STAT3. Cells
nucleofected with TE buffer containing no plasmid were used as a control. 1µg/mL
doxycycline was used to induce the expression of STAT3 shRNA 24 hours after
nucleofection. This concentration was non-toxic to cells. The results are a mean of two
independent triplicates. Students t-test was used for statistical analysis, *** p < 0.0001,
** p < 0.05.
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Figure 3-1C. STAT3 inhibition reduces viability of CLL cell lines and patient cells.
(i) JVM-3 cells were treated with increasing concentrations of Stattic for 24 hours and
cell viability was determine after 24 hours treatment. Western blotting analysis for p-
STAT3-Y705, p-STAT3-S727 and total STAT3 was performed to confirm the
effectiveness of 24 hours treatment with Stattic. (ii) Mec-2 cells and (iii) PBMCs from
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normal donors (PBMC n=2) and CLL patients (CLL n=3) were treated with Stattic for 24
hours and cell viability was determined after treatment.
CNL suppresses the phosphorylation of STAT3 at both tyrosine-705 (p-
STAT3-Y705) and serine-727 (p-STAT3-S727) residues in CLL cells
To identify the mechanism of CNL-induced cell death in CLL, we examined
the effect on STAT3 phosphorylation. JVM-3 cells were treated with CNL or
ghost nanoliposomes (negative control) for 24 hours and Western blotting was
performed to detect changes in protein phosphorylation. While the levels of total
STAT3 protein did not change significantly with CNL treatment, phosphorylation
of STAT3 was significantly down regulated. p-STAT3-S727 and p-STAT3-Y705
were suppressed by 45% and 67% respectively after treatment with 40µM CNL
for 24 hours (Fig. 3-2A (i)). Several studies have reported that only S727 residue
of STAT3 is constitutively phosphorylated in CLL [188, 189]. However, we
observed constitutive phosphorylation at both S727 and Y705 sites in both CLL
cell lines and primary cells. This can be attributed to increased activity of
upstream kinases like cAbl that contribute to STAT3 phosphorylation [199]. We
observed a similar trend on STAT3 phosphorylation suppression after CNL
treatment in Mec-2 cells, although after longer treatment with CNL (Fig. 3-2A (ii)).
Similarly, 7 CLL patient cells were treated with 40µM CNL or ghost
nanoliposomes for 24 hours and evaluated for STAT3 phosphorylation.
Consistent with the above results, we observed suppression in STAT3
phosphorylation at both the residues in 6 out of 7 patient samples (Fig. 3-2B).
Although we observed a slight reduction in total STAT3 levels in some patient
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cells after CNL treatment, the overall change in total STAT3 did not reach
statistical significance in most patient cells. We also tested the effect on CNL
treatment on HEK293 cells. CNL treatment did not affect the viability of HEK293
cells (Fig. 3-2C (i)), nor did it affect the phosphorylation of STAT3 (Fig 3-2C(ii)),
thereby demonstrating that this phenomenon is specific to cancer cells.
Figure 3-2A. CNL suppresses the phosphorylation of STAT3 in CLL cell lines. (i)
JVM3 cells were treated with 20µM and 40µM of ghost nanoliposomes and CNL for 24
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hours. Western blotting analysis was performed to determine protein levels before and after
treatment. The graphs represent the quantification of Western blotting. Statistical analysis
was performed using Student’s t-test, * p < 0.05, ** p < 0.01(ii) Mec-2 cells were treated
with 40µM nanoliposomes for 48hours and 72 hours and protein levels were determined
using Western blotting analysis.
Figure 3-2B. CNL suppresses phosphorylation of STAT3 in CLL patient cells. CLL
patient cells (n=7) were treated with 40µM of ghost nanoliposomes and CNL for 24 hours.
Western blotting analysis was performed to determine protein levels before and after
treatment. The graphs represent the quantification of Western blotting. Statistical analysis
was performed using Student’s t-test, * p < 0.05.
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Figure 3-2C. CNL does affect cellular viability & STAT3 phosphorylation in HEK293
cells. (i) Cell viability of HEK293 cells was determined after 24 hour treatment with ghost
nanoliposomes and CNL. (ii) Western blotting analysis was performed to determine levels
of STAT3 phosphorylation in HEK293 cells after 24 hours treatment with ghost
nanoliposomes and CNL.
Suppression of STAT3 phosphorylation is specific to STAT3 and C6-
ceramide
We next evaluated the specificity of CNL-induced suppression of STAT3
phosphorylation. In addition to STAT3, STAT1 is also constitutively
phosphorylated on S727 in CLL patient cells [188]. Although strong evidence
supporting the role of STAT1 in CLL is lacking, some investigations have
reported that fludarabine and JAK kinase inhibitors induce apoptosis in CLL cells
and inhibit STAT1 signaling, thereby suppressing the ability of the cells to
respond to growth signals, interferons and cytokines [200, 201]. STAT1
activation is also critical for differentiation of CLL cells in response to Byrostatin 1
[202]. These reports raise a possibility that STAT1 might play a role in
pathogenesis of CLL by impacting the anti-apoptotic signals in CLL. We
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observed that treatment with CNL does not significantly affect the
phosphorylation of STAT1 (Fig. 3-3A). The other STATs were either not
constitutively active (STAT2 and STAT5) or were not expressed in JVM-3 cells
(STAT4). We next examined if suppression of STAT3 phosphorylation was
specific to C6-ceramide sphingolipid. We tested three other sphingolipids:
dihydro-C6-ceramide, an inactive analog of C6-ceramide; sphingosine and
sphingosine-1-phosphate. None of the other sphingolipids tested decreased
STAT3 phosphorylation (Fig. 3-3B), thereby demonstrating the specificity of C6-
ceramide. All together, these results prove the specificity of CNL-induced
suppression of STAT3 phosphorylation in CLL.
Figure 3-3A. CNL-induced suppression of phosphorylation is specific to STAT3. JVM-3
cells were treated with 40µM ghost nanoliposomes and CNL for 24 hours and Western blotting
analysis was done. A positive control of STAT2 phosphorylation was also used.
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Figure 3-3B. Suppression of STAT3 phosphorylation is specifically an effect of CNL and
not other sphingolipids. JVM-3 cells were treated with dihrdro-C6-ceramide nanoliposomes or
BSA:sphingosine complex or BSA:S1P complex for 24 hours. Western blotting analysis was
performed.
CNL induces necrotic cell death in CLL cells
We have previously demonstrated that CNL selectively induces caspase
3/7-independent cell death in CLL cells [39]. Using phase contrast microscopy
we had shown that CLL cells treated with CNL resembled a necrotic morphology
[39]. Here, we confirm these findings using flow cytometric analysis for Annexin V
and a viability dye, 7-AAD. Several reports suggest that necrotic cell death is
quantified in the Annexin V-7AAD double positive quadrant [203, 204]. Necrotic
cell death was induced in both cell lines in a dose-dependent and time-
dependent manner after treatment with CNL (Fig. 3-4A (i) and (ii)). Under the
same conditions, ghost nanoliposomes did not have effect on cell death. Recent
evidence suggests that CLL patients with p53 pathway dysfuntion have poor
prognosis due to reduced response to conventional chemotherapies [133, 205].
We observed that cell death in p53mutated Mec-2 cells was induced after a longer
88
treatment with CNL as compared to p53wild-type JVM-3 cells (Fig. 3-4A). This
preliminary evidence demonstrating the effectiveness of CNL treatment to induce
cell death in Mec-2 cells presents a potential treatment strategy for p53mutated B
malignancies that are in urgent need of better therapeutic approaches. We also
evaluated the effect of CNL on CLL patient cells obtained from 7 different
patients. Treatment with 40µM CNL for 24 hours induced cell death in 5 out of
the 7 patients tested (Fig. 3-4B). Taken together, these results indicate that CNL
increases cell death in both p53mutated and p53wild-type CLL cells. To determine if
suppression of phosphorylation preceded induction of cell death, we examined
phosphorylation levels at early time points after CNL treatment. Significant
necrotic cell death in JVM3 cells was observed 12 hours after treatment with CNL
(Fig 3-4C (i)). However, suppression of STAT3 phosphorylation at both the
residues started about 6 hours after CNL treatment and thus preceded induction
of cell death (Fig. 3-4C (ii)). This suggests that reduction in STAT3
phosphorylation might mediate CNL-induced cell death. Consistent with this, we
also observed suppression of STAT3 phosphorylation in 3 CLL patient cells after
12 hours of treatment with CNL (Fig. 3-4D). Overall, these results indicate that
CNL suppresses STAT3 phosphorylation and this might mediate cell death in
CLL cells.
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Figure 3-4A. CNL induces necrotic cell death in CLL cell lines (p53 wt and p53 mutated). (i)
JVM-3 cells that have wild type p53 and (ii) Mec-2 cells that have mutated p53 were treated with
20µM and 40 µM ghost nanoliposomes and CNL for indicated time periods. Flow cytometric
analysis for Annexin V and 7AAD stating was performed to determine effect on cell death. Two-
way ANOVA was used to perform statistical analysis * p < 0.01.
Figure 3-4B. CNL induces necrotic cell death in CLL patient cells. CLL patient cells (n=7)
were treated with 40 µM ghost nanoliposomes and CNL for 24 hours. Flow cytometric analysis for
Annexin V and 7AAD stating was performed to determine effect on cell death. Student’s t test
was used to perform statistical analysis ** p < 0.01.
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Figure 3-4C. CNL-induced suppression of p-STAT3 precedes induction of cell death (i)
JVM-3 cells were treated with ghost nanoliposomes and CNL for indicated time periods and flow
cytometric analysis was performed to determine % cell death. (ii) JVM-3 cells were treated with
40µM ghost nanoliposomes and CNL for indicated time periods and Western blotting was
performed for to determine levels of p-STAT3. Graphical representation of protein levels is also
shown. Statistical analysis was done using Student’s t-test * p < 0.05.
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Figure 3-4D. CNL-induces early time point suppression of p-STAT3 in CLL patient cells.
CLL patient cells (n=3) were treated for 12 hours with 40µM ghost nanoliposomes or CNL and
Western blotting was done.
CNL suppresses Bruton’s tyrosine kinase (BTK), mitogen-activated protein
kinase kinase (MEK) and protein kinase C (PKC) activity to suppress STAT3
phosphorylation
Suppression in STAT3 phosphorylation can be a result of CNL-induced
activation of downstream phosphatases and/or CNL-induced suppression in
upstream kinases. We studied the effect of CNL on downstream phosphatases
and upstream kinases to determine which enzymes predominantly mediate CNL-
induced suppression in STAT3 phosphorylation. Okadaic acid (OA) was used as
an inhibitor of serine/threonine phosphatases PP1 and PP2A. As shown in Fig.
3-5A (i), pretreatment with OA did not rescue CNL-induced-suppression of p-
STAT3-S727, indicating that suppression of phosphorylation is not a result of
CNL-induced activation of serine/threonine phosphatases. Pretreatment with OA
rescued p-Akt-S473 after CNL treatment, suggesting that the inhibitor was
functional in inhibiting PP2A and PP1. Similarly, we observed that CNL treatment
92
had no effect on the activity of tyrosine phosphatases. Pervanadate (PV) was
used as a functional inhibitor of tyrosine phosphatases. As demonstrated in Fig.
3-5A (ii), pretreatment with PV did not rescue CNL-induced-suppression of p-
STAT3-Y705, indicating that this event is independent of the action of tyrosine
phosphatases. The basal levels of p-STAT3-Y705 increased after pretreatment
with PV confirming that PV was effective in inhibiting tyrosine phosphatases.
Figure 3-5A. CNL does not activate phosphatases. (i) JVM-3 cells were pretreated with 5nM
okadaic acid (OA) for 2 hours, followed by 12 hours of treatment with ghost nanoliposomes and
CNL. (ii) JVM-3 cells were pretreated with 50µM pervanadate (PV) for 2 hours, followed by 24
hours treatment with 40µM ghost nanoliposomes and CNL. Both the inhibitors were non-toxic to
cells at the specific concentration. Western blotting was performed.
After investigating the effect of CNL on downstream phosphatases, we
studied the effect of CNL on upstream kinases. We used two strategies to
determine the effect of CNL on upstream kinases. Firstly, we performed western
blot analysis to study the effect of CNL on the activating phosphorylation of the
kinase or to look at the phosphorylation pattern of the immediate downstream
target of the kinase. These results act as a surrogate for determining the effect
93
on the kinase activity of the enzyme. Additionally, we also treated cells with an
inhibitor of the kinase to determine the direct effect of kinase inhibition on STAT3
phosphorylation. One of our criteria for screening was to obtain inhibition of
kinase activation at early time points after treatment with CNL, which would
thereby indicate that kinase activity suppression preceded suppression in STAT3
phosphorylation. Taken together, these two strategies answer the question of the
effect of CNL on upstream kinases.
We observed that CNL suppresses the activity of BTK, a tyrosine kinase
critical in mediating BCR signaling in CLL cells. As shown in Fig. 3-5B, we
observed a significant reduction in phosphorylation of BTK at Y223 in JVM-3
cells as early as 4-6 hours after treatment with CNL but not ghost liposomes. The
levels of total BTK remained unchanged after treatment. BTK phosphorylation at
Y223 is an activating phosphorylation and is necessary of full activation of BTK
[206, 207]. CNL-induced inhibition of BTK is an exciting observation since BTK is
a promising target in CLL and a BTK inhibitor, ibrutinib, is currently used in the
clinic for CLL therapy [208]. Furthermore, we also observed that treatment with
ibrutinib significantly reduced p-STAT3-Y705, but not p-STAT3-S727 (Fig. 3-5C).
Taken together, these results imply that CNL-induced BTK inhibition mediates
suppression of p-STAT3-Y705.
94
Figure 3-5B. CNL suppresses the activity
of BTK. JVM-3 cells were treated with 40µM
ghost liposomes and CNL for indicated time
periods and Western blotting was performed.
Figure 3-5C. BTK inhibitors suppress phosphorylation of STAT3. JVM-3 cells were treated
with BTK inhibitor, ibrutinib for 6 hours and Western blotting was performed to determine protein
levels. Graphical representation of the Western blot is also shown. Student’s t-test was used to
perform statistical analysis, * p < 0.05.
We also observed that CNL suppresses MEK activity, a serine/threonine
kinase in CLL cells. As shown in Fig. 3-5D, only CNL treatment significantly
suppressed phosphorylation of Erk, a direct downstream target of MEK at early
time points after addition of the liposomes. Furthermore, treatment with U0126, a
MEK inhibitor reduced p-STAT3-S727 and p-STAT3-Y705 in both JVM-3 (Fig. 3-
95
5E (i)) cells and CLL patient cells (Fig. 3-5E (ii)). We also observed that CNL
suppresses PKC activity. CNL treatment, but not ghost liposomes, significantly
suppressed phosphorylation of MARCKS, a direct downstream target of PKC at
early time points, while total MARCKS levels remained unchanged (Fig. 3-5F).
Additionally, treatment with BisI, a PKC inhibitor also suppressed p-STAT3-S727
and p-STAT3-Y705 levels in both JVM-3 cells and CLL patient cells (Fig. 3-5G (i)
and (ii)).It was confounding to obtain inhibition of p-STAT3-Y705 after treatment
with U0126 and BisI, both of which are inhibitors of serine/threonine kinases.
However, some results in the literature have demonstrated that MEK inhibitors
like U0126 and PKC inhibitors like BisI inhibit p-STAT1-Y701 [202]. Since STAT1
and STAT3 pathways share similar upstream signaling, we speculate that MEK
and PKC might also play a role in phosphorylating STAT3-Y705. We conclude
that CNL-induced suppression in phosphorylation of STAT3 is not mediated by
downstream phosphatases, but instead by inhibition of upstream kinases that
include BTK, MEK and PKC.
Figure 3-5D. CNL suppresses the activity of MEK1/2 kinase. JVM-3 and Mec-2 cells were treated with 40µM ghost liposomes and CNL for indicated time periods and Western blotting was performed.
96
Figure 3-5E. MEK1/2 inhibitors suppress phosphorylation of STAT3. (i) JVM-3 cells were
treated for 6 hours and 12 hours with 10µM U0126 and Western blotting was performed to
evaluate protein levels. Blots were probed for p-Erk to confirm that U0126 suppressed MEK
activity. Graphical representation of the blots is also shown. (ii) CLL cells (n=3) were treated for
12 hours with 10µM U0126 and Western blotting was performed to evaluate protein levels.
Graphical representation of the blots is also shown. Student’s t-test was used for statistical
analysis, * p < 0.05.
97
Figure 3-5F. CNL suppresses the
activity of PKC. JVM-3 cells were
treated with 40µM ghost liposomes
and CNL for indicated time periods
and Western blotting was
performed.
Figure 3-5G.
PKC inhibitor
suppress
phosphorylati
on of STAT3
(i) JVM-3 cells
were treated
for 6 hours and
12 hours with
5µM Bis-I and
Western
blotting was
performed to
evaluate
protein levels.
Graphical
representation
of the blots is
also shown.
(ii) CLL cells
(n=3) were
treated for 12
hours with 5µM
Bis-I and Western blotting was performed to evaluate protein levels. Graphical representation of
the blots is also shown. Student’s t-test was used for statistical analysis, * p < 0.05.
98
CNL suppresses the transcriptional activity of STAT3
Having established that CNL suppresses STAT3 phosphorylation, we next
sought to determine if CNL suppressed the transcriptional activity of STAT3.
CNL treatment caused a significant downregulation of STAT3-regulated proteins
like Mcl-1, survivin, XIAP, cyclin D1 and p21 in both JVM-3 and Mec-2 cells (Fig
3-6A). CNL-induced downregulation of critical anti-apoptotic proteins like Mcl-1,
survivin and XIAP was encouraging since ample evidence in the literature has
established that these anti-apoptotic proteins are critical mediators of CLL cell
survival. Mcl-1 levels correlate with poor disease prognosis and chemoresistance
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Appendix: Letters of Permission
Chapter 1 and Chapter 2 has material that has been previously published. This
material is used with permission from the respective sources. Copies of the letter
granting permission for use in this dissertation are as follows.
154
Permission letter for Chapter 1
Reprinted (adapted) with permission from the chapter:
Doshi UA, Haakenson JK, Linton SL, Kelly K, Kester M (2015). Chemotherapy
and sphingolipid metabolism. In Bioactive Sphingolipids in Cancer Biology and
2015 Ph.D. Molecular Medicine, Pennsylvania State University 2015 MBA Pennsylvania State University 2012 M.S. Biotechnology, University at Buffalo, New York 2008 B.S. Pharmaceutical Sc., Institute of Chemical Technology, Mumbai, India
Publications Doshi UA, Haakenson JK, Linton SL, Kelly K, Kester M (2015). Chemotherapy and
sphingolipid metabolism. In Bioactive Sphingolipids in Cancer Biology and Therapy
(pp. 401-436), New York, NY: Springer.
Ryland LK*, Doshi UA*, Shanmugavelandy SS, Fox TE, Aliaga C, Broeg K, Baab KT, Young M, Khan O, Haakenson JK, Jarbadan NR, Liao J, Wang HG, Feith DJ, Loughran TP Jr, Liu X, Kester M. C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia. PLoS One. 2013 Dec 19;8(12):e84648. *Co-first authorship
Hankins JL, Doshi UA, Haakenson JK, Young MM, Barth BM, Kester M. The therapeutic potential of nanoscale sphingolipid technologies. Handb Exp Pharmacol. 2013;(215):197-210.
Manuscripts in preparation:
Doshi UA, Fox TE, Loughran TP, Kester M. STAT3 mediates nanoliposomal C6-ceramide-induced cell death in chronic lymphocytic leukemia.
Doshi UA, Fox TE, Loughran TP, Kester M. Nanoliposomal C6-ceramide targets cellular bioenergetics to induce cell death in chronic lymphocytic leukemia.
Awards College of Medicine Class of 1971 Endowed Scholarship, Pennsylvania State
University, 2014
Beta Gamma Sigma Inductee, The Penn State Capital College Chapter of Beta Gamma Sigma scholastic honor society in Business Administration, 2014
Outstanding Graduate Student Award, 7th International Ceramide Conference , 2013
University Graduate Fellowship, Pennsylvania State University, 2010