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8/10/2019 Studies of Targeted Therapies Against Htlv in Preclinical Animal Models
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STUDIES OF TARGETED THERAPIES AGAINSTHUMAN T LYMPHOTROPIC VIRUS TYPE-1 ADULT T-CELL LYMPHOMA
IN PRECLINICAL ANIMAL MODELS
DISSERTATION
Presented in Partial Fulfillment of the Requirement for the Doctor of PhilosophyDegree in the Graduate School of The Ohio State University
By
Bevin Zimmerman, B.A., D.V.M.
*****
The Ohio State University2009
Dissertation Committee:
Dr. Michael D. Lairmore, Advisor Approved by:
Dr. Stefan Niewiesk __________________________________
Dr. Lawrence Mathes Advisor
Dr. Paul Stromberg Veterinary Biosciences Graduate Program
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ABSTRACT
Human T lymphotropic virus type 1 (HTLV-1) infects 20 million people
worldwide. It is the causative agent of adult T-cell leukemia/lymphoma (ATL) and
HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). ATL is
a refractory T-cell malignancy with a poor prognosis due to its highly aggressive
nature and resistance of neoplastic cells to conventional chemotherapies.
Herein, the impact of multiple novel therapeutics was investigated in vitro in
multiple HTLV-1 cell lines, in an immunodeficient mouse model of ATL, and a
rabbit model of early infection.
The proteasome inhibitor bortezomib (PS-341 or Velcade) and the heat
shock protein inhibitor 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG)
exhibited efficacy against multiple cell lines in vitro. In Chapter 2, we document
the efficacy of PS-341 and 17-AAG alone and in combination in a preclinical
model of ATL. We demonstrate that the combination of PS-341 and 17-AAG
synergistically promoted cell death in ATL cell lines and reduced tumor burden in
mice during cycles of drug treatments. Positive effects of 17-AAG required
continuous drug treatments suggesting requirements for treatment protocols in
human subjects.
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Histone deacetylase inhibitors (HDACi) have shown efficacy against a
variety of cancers. In Chapter 3, we tested the histone deacetylase inhibitors
valproic acid and OSU-HDAC42 in our ATL mouse model. Both compounds
reduced proliferation of ATL cell lines by promoting apoptosis and histone
hyperacetylation. To further test the efficacy of this approach we evaluated
OSU-HDAC42 for survival in an ATL NOD/SCID mouse model. Our data
provide new directions for the treatment of ATL and support the further
development of this class of drug against HTLV-1-associated lymphoid
malignancies.
In Chapter 4, we tested the effects of the histone deacetylase inhibitor,
valproic acid (VPA), on HTLV-1 proviral load in the rabbit model of early infection.
Our data demonstrated that VPA can be safely administered in the rabbit model
and altered proviral copy numbers in infected rabbits when compared to controls.
This model will be useful to test other HDACi for their effects on HTLV-1 gene
expression.
In this thesis, we investigated targeted therapeutics in a preclinical,
NOD/SCID ATL mouse model and in a rabbit model of HTLV-1 infection. Our
data indicated new directions in the development of targeted therapies against
ATL and provide new directions for the design of treatement protocols for future
studies.
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Dedicated to my parents Michael and Margaret Zimmerman and my
husband, Raymond Kinney
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ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Michael Lairmore, for his guidance
and support. His integrity, leadership, and diligence have been an example
which I aspire to follow. In addition, I would like to thank my committee
members, Drs. Niewiesk, Mathes, and Stromberg for their mentorship and
support.
My colleagues in the laboratory, past and present, have been a constant
source of encouragement. This includes Drs.Chris Premanandan, Antara Datta,
Rashade Haynes, Laurie Millward, Nadine Bowden and Raj Anupam. You are
not just colleagues, you are friends. The camaraderie in our laboratory is unique.
In addition, I would like to add a special note of thanks to our laboratory
manager, Krissy Landes, for her technical support.
Drs. Steven Weisbrode and Paul Stromberg have shaped my pathology
training and have been influential to my development as both a pathologist and a
scientist. I would also like to thank Drs. Mary Carsillo and Gillian Beamer, my
good friends from the first day of the residency. Together, we’ve spent six years
growing, learning, and living.
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I would like to thank my family. My husband, Raymond Kinney, has stood
by me through my veterinary training, the pathology residency, and the PhD
training. His support never wavered. To my parents, Mike and Peggy
Zimmerman and my sister and brother, Brenna and Mike, thank you for always
pushing me to excel and supporting me through these years and years of school.
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VITA
November 10, 1976……………… Born- Buffalo, New York
1997……………………………….. B.A. (Biology)
State University of New York at Buffalo
2003……………………………….. Doctor of Veterinary Medicine
North Carolina State University, Raleigh
2003- present…………………….. Veterinary Anatomic Pathology Resident
Graduate Research Associate
Department of Veterinary Biosciences
The Ohio State University, Columbus, OH
PUBLICATIONS
1. Parrula C., Zimmerman B., Nadella P., Shu S., Rosol T., Fernandez S.,Lairmore M., Niewiesk S. Differential Expression of tumor invasion factors affectsystemic engraftment and induction of humoral hypercalcemia in mice by ATLcells. Veterinary Pathology, 2009 May 9 epub ahead of print
2. Arnold J, Zimmerman B., Li M., Lairmore M., Green P. HTLV-1 HBZ
Promotes Proliferation in Cell Culture and Tumor Growth in NOD/SCID
γchain-/-
(NOG) Mice, Blood 2008 Nov 1; 112(9): 3788-97
3. Knostman K., Venkateswaran A., Zimmerman B., Capen CC., Jhiang SM.Creation and Characterization of Doxycycline-Inducible Mouse Model of Thyroid
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Targeted RET/PTC1 Oncogene and Luciferase Reporter Gene Coexpression,Thyroid 2007 Dec; 17(12): 1181-8
4. Ratner L., Grant C., Zimmerman B., Fritz J., Weil G., Denes A., Rama S.,Campbell N., Jacobsen S., Lairmore M. Adult T-Cell Leukemia and StrongyloidesInfection, American Journal of Hematology 2007 Oct; 82(10):929-31
5. Zimmerman B., Yamaguchi M., Rush L. Immunoglobulin Crystals in ReactivePlasma Cells in a Dog, Veterinary Pathology. 2007 May; 44(3): 389-91
6. Zimmerman B., Rotstein D., Jones S. A Case of Large Granular Lymphoma
in a Mule, Vet Record 2004 Oct 9; 155(15):462-3
FIELD OF STUDY
Major Field: Veterinary Biosciences
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TABLE OF CONTENTS
Page
Abstract ..............................................................................................................ii
Dedication .........................................................................................................iv
Acknowledgements ........................................................................................... v
Vita ...................................................................................................................vii
List of Figures ...................................................................................................xii
List of Tables ....................................................................................................xiv
Chapters
1. Literature Review .................................................................................... 11.1 HTLV-1 Disease and Epidemiology ................................................ 1
1.2 The HTLV-1 Genome ......................................................................21.2.1 Structural Gene Products................................................... 41.2.2 Regulatory and Nonstructural Gene Products ...................5
1.3 HTLV-1 Associated Diseases ......................................................... 71.3.1 Adult T-cell Leukemia/Lymphoma ...................................... 71.3.2 Neurodegenerative and Other Diseases Associated with
HTLV-1 Infection .............................................................. 10
1.4 Animals Models of HTLV-1 Infection.............................................. 111.4.1 The Rabbit Model of HTLV-1 ............................................11
1.4.2 The Nonhuman Primate Model of HTLV-1........................131.4.3 The Rat Model of HTLV-1 Infection and Disease..............141.4.4 The Mouse Model of HTLV-1 Infection and Disease......... 15
1.4.4.1 Xenograft Mouse Models of ATL.............................. 161.4.4.2 Transgenic Mouse Models of HTLV-1...................... 20
1.5 Treatment of Adult T-cell Leukemia............................................... 24
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1.5.1 Proteasome Inhibitors....................................................... 261.5.1.1.Bortezomib (PS-341) ................................................... 27
1.5.2 Heat Shock Protein Inhibitors ........................................... 281.5.2.1.17-(Allylamino)-17-demethoxygeldanamycin (17-AAG)29
1.5.3 Histone Deacetylase Inhibitors ......................................... 301.5.3.1.Valproic Acid................................................................ 311.5.3.2.OSU HDAC42.............................................................. 32
1.6 Summary ...................................................................................... 33
1.7 References ................................................................................... 34
2. Combination Therapy of Bortezomib (PS-341) and 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) Decreases Tumor Burden in a MouseModel of Adult T-Cell Leukemia/Lymphoma………………………………………………………………...…….58
2.1 Abstract ............................................................................ 582.2 Introduction ...................................................................... 592.3 Materials and Methods .................................................... 632.4 Results ............................................................................ 69
2.5 Discussion .......................................................................732.6 References ...................................................................... 78
3. Efficacy of Histone Deacetylase Inhibitors in a Mouse Model of Human T-lymphotropic Virus Type 1 Adult T-cell Leukemia/Lymphoma ……………89
3.1 Abstract ............................................................................ 893.2 Introduction ...................................................................... 903.3 Materials and Methods .................................................... 933.4 Results ............................................................................ 983.5 Discussion .....................................................................1013.6 References .................................................................... 104
4. Effects of the Histone Deacetylase Inhibitor Valproic Acid on Human T-lymphotropic Virus Type 1 Replication in a Rabbit Model of Infection.117
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4.1 Abstract ..........................................................................1174.2 Introduction ................................................................... 1184.3 Materials and Methods .................................................. 1214.4 Results ..........................................................................1254.5 Discussion .....................................................................1274.6 References .................................................................... 131
5. Synopsis and Future Directions ............................................................. 1435.1 Animal Models of HTLV-1............................................... 1435.2 Targeted Therapies Against ATL.................................... 1455.3 The Need for Prolonged Dosing to Achieve Efficacy in
ATL.................................................................................1465.4 Evaluation of Combinational Therapy Against ATL ........ 1475.5 HDACi in ATL Therapy………………………..................... 1495.6 Control of Proviral Load in HTLV-1 Infection .................1525.7 Summary and Impact of Work........................................ 1555.8 References...................................................................... 156
Bibliography ...................................................................................................161
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LIST OF FIGURES
Figure Page
1.1 The HTLV-1 genome including all transcripts andprotein products ..........................................................................54
2.1 The addition of 17-AAG significantly inhibits growth in both the MT-2 and Jurkat cell lines …………………………………..……………84
2.2 Western blot of MT-2 cell whole cell lysate after 48hours...........................................................................…………..85
2.3 Histologic representation of mouse tissues from phase I…….….86
2.4 Serum IL-2Rα levels of mice and survival curves for phaseII………………………………………………………………………..87
3.1 Histone deacetylase inhibitors significantly inhibit growth in ATLcell lines……………………………………………..……………… .107
3.2 Western blot of whole cell lysates after 24 hours .................... ..109
3.3 Linear schematic of dosing regimen.......................................... 110
3.4 Engraftment of MET-1 cells in multiple organs.......................... 111
3.5 Oral gavage of OSU HDAC42 in the NOD/SCID ATLmouse model serum IL-2Rα levels of mice andsurvival curves ..........................................................................112
3.6 Mice fed formulated diets ad lib did not differ in intake.............. 113
3.7 In feed trial OSU-HDAC42 in the NOD/SCID ATLmouse model serum IL-2Rα levels of mice andsurvival curves. ......................................................................... 114
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4.1 Experimental design for rabbit inoculation andtreatment with valproic acid....................................................... 133
4.2 Linear timeline of valproic acid administration...........................134
4.3 Morphology of atypical lymphocytes. ........................................ 135
4.4 Hematologic values by differential cell count. ........................... 136
4.5 Rabbit serum responses to HTLV-1..........................................137
4.6 Grading of western blot strip intensity ....................................... 138
4.7 Proviral load varies with time………………………………………139
4.8 p19 production in the rabbit....................................................... 140
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LIST OF TABLES
Table Page
1.1 Engraftment of ATL cell lines in immunodeficient mice....................... 55
1.2 Transgenic mouse models of adult T-cell leukemia/lymphoma ........................................................................................... 56
1.3 Preclinical efficacy studies utilizing mouse models of ATL .................57
2.1 Summary of treatment groups and organ weights.............................. 82
2.2 Histologic ranking of tumor engraftment .............................................83
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CHAPTER 1
LITERATURE REVIEW
1.1 HTLV-1 Epidemiology and Disease Association
A clustering of T-cell leukemias and lymphomas in Japan and other parts
of the world with strikingly similar characteristics such as neoplastic T-cells
with multilobulated nuclei (also called “flower cells”) led to the discovery of the
first retrovirally induced human neoplasia. Poiesz et al.,detected retroviral
particles and reverse transcriptase activity in samples from a patient with
cutaneous T-cell lymphoma confirming the first human retrovirus, human T-
lymphotropic virus type 1 (HTLV-1) (1). Similar retroviral particles were
detected in Japanese patients with Adult T-cell Leukemia/Lymphoma (2).
Human T-lymphotropic virus type-1 (HTLV-1) is a member of the
deltaretroviridae, a family of retroviruses which includes both simian T
lymphotropic virus and bovine leukemia virus. Based on epidemiology
studies it has been estimated that approximately 15 to 20 million HTLV-1
carriers exist throughout the world, with endemic foci in Japan, the Caribbean,
and Africa (3). HTLV-1 is spread through contact with bodily fluids containing
infected cells most often from mother to child through breast milk and
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parenterally via blood transfusion. After prolonged latency periods (20-60
years), approximately 5% of HTLV-1 infected individuals will develop either
adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated
disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis
(HAM/TSP). ATL is an aggressive T-cell malignancy with a poor prognosis
and survival times typically less than one year (4). HAM/TSP is a chronic
inflammatory disease that results in progressive neurologic dysfunction and
damage to the thoracic spinal cord. The factors favoring the development of
ATL or HAM/TSP are not completely understood, but have been linked to
host (e.g., immune response) and virus factors (e.g., viral load). It has also
been demonstrated that the route of exposure to the virus may determine the
clinical outcome of HTLV-1 infection. Individuals subjected to mucosal
exposure are more likely to develop ATL while blood borne exposure favors
the development of HAM/TSP (5). In the following sections, HTLV-1 and its
disease outcomes will be detailed in context to therapeutic interventions and
animal models of ATL.
1.2 The HTLV-1 Genome
HTLV-1 is a complex retrovirus comprised of a diploid, single stranded,
positive sense, nine kilobase, RNA genome. Cell infection begins with the
viral protein envelope binding its cellular receptor. In some instances,
glucose transporter-1 (GLUT-1) appears to act as the cellular receptor (6).
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However, GLUT-1 is not necessary for infection of all cells as it is not present
on some target cells (7). Surface heparin proteoglycan and neuropilin have
been proposed as HTLV-1 receptors depending on the context of the infection
(8-11). Regardless of the receptor used, the virion fuses with the cell
membrane and releases the viral genome into the cytoplasm of the cell. The
viral genome is then reverse transcribed by the viral RNA dependent DNA
polymerase. The result is a double stranded DNA provirus that integrates
randomly into the host cell genome using virally encoded integrase (IN).
During the integration process, both the 5’ and 3’ ends of the retrovirus
are duplicated to form the 5’ and 3’ long terminal repeats (LTR). The 5’ and 3’
LTRs are identical, although the 5’LTR directs synthesis of the viral genome
while the 3’LTR extends transcription into the adjacent host DNA. The LTR is
comprised of U3, R, and U5 regions and acts as the viral promoter. In
addition, the LTR is responsible for polyadenylation of mRNAs, initiation and
termination of transcription and strand transfer. The U3 region of the LTR
contains the Tax Response Element (TRE). TRE binds multiple transcription
factors, including viral Tax and cellular cyclic adenosine monophosphate
response element binding proteins (CREB), CREB-2, ATF-1 and ATF-2, c-
Fos, c-Jun, p-300 and CBP binding protein (12).
The integrated provirus is transcribed into both unspliced and spliced
mRNA using the host cell’s DNA polymerase II. The mRNAs are used as
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both the genome of new virions and as templates for the translation of both
structural and nonstructural proteins.
1.2.1 Structural Gene Products
The HTLV-1 genome encodes structural, regulatory, and nonstructural
genes. The structural genes include gag, pol, and env typical of all
retroviruses. The Gag protein is translated from the unspliced full length
mRNA p55. It is post translationally modified by myristylation targeting Gag
to the inner plasma membrane. Gag is cleaved into three structural proteins,
matrix (MA or p19), capsid (CA or p24) and nucleocapsid (NC or p15), all are
components of the viral core. The matrix protein is localized to the inner
surface of the viral envelope and is critical to virion release and cell to cell
transmission.
Derived from the full length mRNA through ribosomal shifting, the pol
gene encodes the reverse transcriptase (RT), integrase (IN), protease (PR),
and RNase H proteins (13). Reverse transcriptase is an RNA dependent
DNA polymerase that transcribes the viral RNA into DNA that can be
integrated into the host genome. Integrase serves to integrate the DNA
provirus into the host cell chromosome. Protease is necessary for proteolytic
cleavage of primary translation products. The RNAse H removes the RNA
strand from the DNA-RNA hybrid that is synthesized from viral RNA using the
reverse transcriptase.
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Envelope protein is first synthesized as a 68 kDa precursor protein
from the singly spliced viral mRNA. It is then cleaved into surface unit (SU)
and transmembrane (TM) subunit; SU noncovalently associates with TM,
which anchors it to the cell surface. The envelope is necessary for
attachment and viral entry.
1.2.2 Regulatory and Nonstructural Gene Products
The pX region of the viral genome, located 3’ to env and 5’ to the 3’-
LTR is unique and contains genes coding for regulatory or accessory gene
products, which are generated by alternate splicing of RNA transcripts. This
region of the viral genome encodes the transactivating protein Tax and
regulatory protein Rex, as well as p30, p12, p13 and the newly discovered
antisense encoded HBZ (12).
Tax is a 353 amino acid, 40 kDa transactivator protein that acts a
dimer to promote cell transformation in vitro (14-18) and in transgenic mice
(19-21). It is also known to alter the expression of numerous cellular genes
known to contribute to malignant transformation and progression (22). Tax
regulates expression of cell cycle regulators, apoptosis regulating genes,
DNA repair genes, and tumor suppressor genes (including p53). NF-kB is a
well known transcription factor, induced by Tax, which is constitutively active
in ATL cells (23). Alteration of this transcription factor leads to alterations in
cell cycle, apoptosis, and cellular differentiation. Tax protein expression
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levels in ATL are low or undetectable. It is thought that the virus plays a role
in the early expansion of cells, but upon transformation, it is no longer
required for leukemic cell growth. Tax is also a primary antigenic target of
cytotoxic T-lymphocyte (CTL) responses against HTLV-1 (24). Rex is a 27
kDa phosphoprotein encoded by ORF III (25). It localizes to the nucleolus of
infected cells and is essential for viral replication. Rex is necessary for the
export of unspliced and singly spliced viral mRNAs from the nucleus to the
cytoplasm (25).
The pX region also encodes for several other accessory or
nonstructural gene products. Protein products p12, p30 and p13 are encoded
from open reading frames I and II (26). These proteins have several roles
including early viral spread and control of virus replication. Open reading
frame II encodes for both p13 and p30. p13 is encoded by singly spliced
RNA of ORF II and localized to the mitochondria, while p30 is encoded by a
doubly spliced mRNA and localizes to the nucleolus (12;27). p30 is a
nonstructural protein encoded by the open reading frame II (ORF II). It
serves as a negative regulator of viral expression (28) and may play a role in
the virus’ ability to evade the host immune response. Also of note, it acts as a
transcriptional regulator and, more importantly, is necessary for maintenance
of in vivo infection (29). Recently, we have shown that p30 alters cell cycle
regulation (30) and may play a role in viral integration through interactions
with ATM kinase and double stranded DNA breaks (Datta et al., submitted).
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Open reading frame I encodes p12. p12 is a 12 kDa protein that does
not appear necessary for replication in vitro (31). This viral protein
accumulates primarily in the golgi and endoplasmic reticulum (32). By
interacting with calcineurin, p12 can activates nuclear factor of activated T-
cells (NFAT) (33). In addition, p12 may interfere with the assembly of major
histocompatibility complex I (MHC-1) and its trafficking to the cell membrane
(34).
HTLV-1 basic leucine zipper factor (HBZ) is a protein encoded by the
antisense strand of HTLV-1 provirus. It is conserved in ATL cells in which 5’
deletions and mutations often affect the structural proteins and prevent
production of infectious virions (35). In vitro, HBZ down regulates viral
transcription by its interaction with CREB-2 (36). It has also been shown that
HBZ is not necessary for cellular transformation in vitro, however, in the rabbit
model, HBZ enhanced infectivity and increased viral persistence (37). If HBZ
is abrogated using siRNA cells in culture have reduced proliferation and
HTLV-1-transformed cells have significantly reduced tumor size when
transplanted in mice (38).
1.3 HTLV-1 Associated Diseases
1.3.1 Adult T-cell Leukemia/Lymphoma
The acute form of ATL is an aggressive T-cell malignancy caused by
HTLV-1. It is characterized by several distinct clinical parameters including a
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leukemic phase of increased circulating CD4+ CD25+ T-cells (39). Patients
with the acute form of ATL have a poor prognosis independent of therapy with
mean survival time is less than one year (4). Adult T-cell leukemia/lymphoma
was first described in 1977 (4;40). A geographic clustering of patients with
particularly aggressive T-cell malignancies and several common
characteristics led the authors to coin the term “adult T-cell leukemia” to
describe the disease. Common characteristics of the original 16 patients
described include adult onset disease, a chronic leukemia with a rapidly fatal
progressive course, lymphadenopathy, hepatosplenomegaly, lobulated
neoplastic T-cells, and geographic distribution (4;40). Cells from patients from
these original cases were cocultured with human umbilical cord lymphocytes
and cell lines MT-1, MT-2, and MT-4 were generated (41). These cell lines
were instrumental in the discovery of HTLV-1 and are still used in HTLV-1
research.
The diagnostic criteria for ATL include the demonstration of a T-cell
malignancy, HTLV-1 antibodies in the patient’s sera, and demonstration of
monoclonal integration of HTLV-1 provirus in the leukemic cells by Southern
blot assay (42). Since the original description of ATL, lymphoproliferative
diseases associated with HTLV-1-infection has been classified into four
clinical subtypes. The two most aggressive forms are the acute and
lymphomatous forms (43). These forms are characterized by humoral
hypercalcemia of malignancy (HHM) with subsequent bone lysis,
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lymphadenopathy, visceral and skin involvement. The lymphomatous form is
distinguished by its normal peripheral white cell count, while in the acute form,
ATL cells are found in the peripheral blood. The smoldering and chronic
forms are the more indolent clinical subtypes. Criteria for diagnosing
smoldering ATL include a normal leukocyte count, lack of hypercalcemia, and
skin or lung involvement without evidence of other visceral disease. In
chronic ATL patients, their peripheral blood leukocyte count is typically
elevated, they lack hypercalcemia, but may have histologic evidence of
involvement of the liver, spleen or lungs (39;44).
The molecular pathogenesis from HTLV-1 infection to ATL
development has not been fully elucidated. The randomly integrated provirus
persists for many years and during this phase, infected subjects typically
remain asymptomatic. Effective replication of the virus relies on reverse
transcription and clonal expansion of infected cells (45). During the
premalignant phase, there is a progression from polyclonal to monoclonal T-
cells in circulation. Monoclonal T-cell expansion is believed to be promoted
by an increasing ability of malignant T-cells to escape immune elimination
and become less dependent on cell growth factors such as interleukin-2 (IL-
2). Also during this phase, there is an accumulation of somatic mutations
both within the provirus and within the flanking regions. Most often, the 5’
portions of the virus are deleted or mutated and functional infectious virions
may not be produced (45). Tax, in the initial phases of transformation, acts
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as a transcriptional transactivator and causes the simultaneous activation of
multiple cellular pathways that disrupt the normal cell cycle and death
pathways, leading to the transformation of the T-cells. Transformed T-cells
eventually grow autonomously and the expression of Tax appears to be no
longer required for maintenance of the malignancy.
1.3.2 Neurodegenerative and Other Diseases Associated with HTLV-1
The neurologic disease HAM/TSP is a degenerative neurologic condition
first described by Gessain et al. (46) and Osame et al. (47). The disease is
characterized by lymphocytic meningitis with demyelination and degeneration
of the spinal cord. It most commonly occurs in the lumbar spine (48) in
patients typically younger than those affected by ATL. Transfusion with
HTLV-1 contaminated blood has been associated with a more rapid onset of
development of HAM/TSP versus ATL (49;50). The pathogenesis of
HAM/TSP is thought to involve both molecular mimicry and autoantigens
(51;52). The autoantigen was identified from immunoglobulin G of HAM/TSP
patients as the heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1).
This immunoglobulin also cross reacted with HTLV-1 Tax, indicating
molecular mimicry (51). Cytotoxic T-cells (CTLs) directed against Tax are
found in high levels in the blood and within the central nervous system (53) .
As the disease progresses, the composition of the inflammatory component of
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the spinal cord lesions progresses from CD4+, CD8+ T-cells, B cells, and
macrophages to a lesion comprised predominantly of CD8+ T-cells (54).
1.4 Animal Models of HTLV-1 Infection
Multiple animal models are available to study the infection and
transmission of HTLV-1 (55). Rabbits (56;57), some nonhuman primates
(58;59) and rats (60;61) can all be infected with HTLV-1 and have been
utilized to monitor the virus spread, determine immune responses against the
infection and in the development of vaccines against the viral infection.
1.4.1 The Rabbit Model of HTLV-1
Rabbits are the model used most often to study early infection events,
viral transmission, and the host immune responses against the virus infection.
Rabbits can be consistently infected; however, they remain, in the majority of
cases, asymptomatic. The first reports of infectivity in rabbits were in the
1980s when MT-2 cells were inoculated into rabbits (56). The rabbit model
was used to confirm the modes of transmission in humans, as well as early
studies of the immune response against the infection or to develop vaccines
against HTLV-1 (62-67). Early studies utilizing the rabbit model of HTLV-1
provided important data on the number of cells capable of transmitting the
virus infection (64) and effective means to prevent the transmission of the
virus (64;68-71).
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Infection of rabbits with HTLV-1 parallels the asymptomatic infection of
the virus in humans. Rabbits inoculated with HTLV-1-infected cell lines
derived from patients with ATL or HAM/TSP demonstrate the heterogeneity in
the biological response to HTLV-1 infection (57). Rabbits vaccinated with
synthetic peptides verified immunodominant epitopes of the viral envelope
protein (Env) (72;73) and defined regions of Env important for antibody
dependent cell-mediated cytotoxicity (74). An infectious molecular clone of
HTLV-1 was used to immortalize human peripheral blood mononuclear cells
(PBMC) to create the ACH cell line, which was then used to infect rabbits
(75;76). Subsequently, ACH clones with mutations within the open reading
frames encoding the HTLV-1 accessory or regulatory proteins were
inoculated into rabbits to demonstrate the necessity of these accessory
proteins for establishment of infection and maintenance of proviral loads
(29;37;77-79) .
Sporadic reports of associated diseases in infected rabbits are
reported, but are of limited value due to the inconsistent expression of
disease in infected animals (80;81). An “ATL-like disease” following
intraperitoneal or intravenous injection of HTLV-1 transformed cells has been
reported, but this required a minimum of 1 x 108 cells in the inoculum, and
death occurred within the first few weeks of inoculation (82-84). These
studies failed to demonstrate if the leukemic cells were from the inoculum.
Other case reports of clinical disease in HTLV-1 infected rabbits include
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uveitis (80), cutaneous lymphoma (85;86), and thymoma (87). In each of
these, clinical disease developed after one to several years following
exposure to the virus limiting the use of rabbits as a disease model.
1.4.2 The Nonhuman Primate Model of HTLV-1
Cynomolgus macaques (Macaca fascicularis) and squirrel monkeys
(Saimiri sciureus) can be experimentally infected with HTLV-1. The monkeys
seroconvert, but typically do not demonstrate any clinical signs of disease
(88-90). The squirrel monkey has been used to demonstrate that reservoirs
of HTLV-1 include PBMCs, lymph nodes, and spleen (91). Generally, the
nonhuman primates remain asymptomatic, however, there are two reports of
malignant lymphoma in macaques that have HTLV-1 specific antibodies
(92;93) and a single report of arthritis, uveitis and polymyositis (94).
Pig-tailed macaques (Macaca nemestrina) inoculated with a pig-tailed
macaque cell line persistently infected with the ACH HTLV-1 molecular clone
developed clinical disease including rash and lymphadenopathy,
hypothermia, lethargy, lymphopenia, diarrhea, and arthropathy (95). The
nonhuman primate is a useful model for vaccine studies. Gag and Env
vaccines and naked DNA vaccines have all been tested in the nonhuman
primate model (96;97).
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1.4.3 The Rat Model of HTLV-1 Infection and Disease
The first reference to reproducible experimental infection of Rats with
HTLV-1 was in 1991 (61). Rats have not been used exclusively, in part, due
to considerable differences in the response of various rat strains to HTLV-1
infection (60;98;99). Wistar-King-Aptekman-Hokudai (WKAH) rats have
been used as a model of HAM/TSP. This rat strain, when infected by HTLV-
1, develops spastic paraparesis with degenerative thoracic spinal cord and
peripheral nerve lesions several months following inoculation (98;99). The
lesions (neurologic degeneration with a paucity of lymphocytes) are different
than those observed in human HAM/TSP (100). The use of the
immunodeficient rat has allowed for some studies of ATL (101). Adoptive
transfer of T-cells and Tax-specific peptide vaccines have been used to study
the role of immune responses against HTLV-1 infection in some rat models
(102;103). A protective effect was achieved with each of these systems.
Some protective effects have been reported against tumor formation in nude
rats with Tax-specific small interfering RNAs (siRNA) (104).
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1.4.4 The Mouse Model of HTLV-1 Infection and Disease
The need for useful animal models to study the histology,
tumorigenesis and to test preclinical efficacy of potential therapeutic agents
led to the development of several xenograft and transgenic mouse models.
When compared to other available animal models, mouse models are small,
comparatively inexpensive, and their genetics are easily manipulated. One
caveat is that genetically normal, immunocompetent murine cells are not
efficiently infected with HTLV-1 and do not develop a natural course of
disease (105;106). The mouse model, while not an exact replica of the human
course of disease, demonstrates multiple features in common with the human
disease. Xenograft mouse models display multiple organ engraftment with
ATL cell lines or patient samples (Table 1.1) (107-115). Large atypical
lymphocytes are evident histologically in peritoneum, liver, spleen, and
multiple organ types depending on the inoculum and the cell type. Xenograft
mouse models can also display biochemical characteristics similar to ATL
patients. These include PTHrP expression and increasing levels of serum IL-
2Rα and β-2 microglobulin that correspond to increasing tumor burden.
Transgenic mouse lines helped establish Tax as an oncoprotein and provided
new information about the pathogenesis of ATL (116).
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1.4.4.1 Xenograft Mouse Models of ATL
Transplantation of human cancer cells into immunodeficient mice has
been in practice since the late 1960’s (117) . Use of the xenograft provides
the ability to examine human cells in a more physiological relevant context
than in vitro systems allow.
The SCID mouse, developed in 1983 (118), is commonly used as a
model. This mouse contains a spontaneous nonsense mutation in the gene
for the protein kinase DNA activated catalytic polypeptide (Pkrdc) on
chromosome 16. The Pkrdc enzyme is necessary for the efficient
recombination of the B and T-cell receptors. Without this enzyme, no mature
B and T-cells develop. The SCID mouse retains normal macrophage, antigen
presenting cell, and natural killer cell function. SCID mice are used
extensively in human stem cell and tumor cell engraftment studies.
Engraftment of ATL cells and cell lines within the SCID mouse has been
shown to be cell line dependent (Table 1.1) (107;109). Cells that engraft and
are recovered, maintain the genotype and phenotype of the original inoculate
(107-109). Studies in the SCID mouse also demonstrated that the intact
HTLV-1 provirus does not appear necessary for neoplastic cell growth (119).
RV-ATL cells, an HTLV-1 positive leukemic cell line derived from a patient
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sample, were reported to establish tumors readily and this cell line was
propagated through mice(111). Whole body irradiation or administration of
antibodies to abrogate natural killer cell function proved necessary to
establish engraftment of non leukemic cells lines such as SLB-1 cells
(111;120;121). MT-2 cells developed tumors at the site of injection in SCID
mice treated with anti-asialo GM-1 antibody (122). Utilizing the knowledge of
the immune functions of the SCID mouse in combination with the engraftment
of various types of cell lines, Stewart et al., (123) demonstrated that the
natural killer cells of the SCID mouse mediate the lysis of HTLV-1 expressing
cell lines suggesting that the absence of HTLV-1 expression in the ATL lines
allows these cells to evade immune surveillance.
The SCID/beige mouse is a double mutant mouse in which the SCID
mutation is retained, but these mice have an additional beige/beige mutation
in the lysosomal trafficking gene found on chromosome 13. This results in
defective in B and T-cell function with lack of natural killer cell activity and
defective granulocyte function. The RV-ATL cell line was reported to engraft
in 75% of the SCID/beige mice while transformed cells (HT-1-RV, SLB-1, MT-
2, ACH, and ACH.p12) were unable to establish engraftment (111).
SCID mice used in xenograft studies have been found to display
“leakiness.” Leakiness allows for spontaneous rearrangement of antigen
receptors and development of functional lymphocytes in aged mice (124). To
combat the leakiness, the SCID mouse was crossed onto the NOD/Lt
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background. NOD (non obese diabetic) mice are a model used to study the
development of autoimmune mediated insulin dependent diabetes mellitus.
The resultant NOD/SCID mice lack functional B and T-cells, have low natural
killer cell activity, absence of complement activity (due to a lack of
complement component 5 from the NOD/Lt background), and impaired
macrophage and antigen presenting cell function. When compared to the
SCID mouse and the SCID/bg mouse, the NOD/SCID mouse was reported to
be more susceptible to engraftment with the HTLV-1 transformed cell lines
(111). Sublethal whole body irradiation of the NOD/SCID mice one day prior
to inoculation improved engraftment and tumorigenesis, as well as decreased
time to clinical signs. Tumor engraftment was described as a lymphoblastic
lymphoma with tumor development in the peritoneal cavity, spleen, and
mesenteric lymph nodes. Lymphoblasts had large irregular nuclei and large
prominent nucleoli. Abnormal mitotic figures were also observed. Tumor
cells invaded and displaced multiple abdominal organs (111).
Further immunodeficiency was produced with development of the
NOD/SCID mouse containing a targeted mutation in the Beta-2 microglobulin
gene. Beta-2 microglobulin is a protein necessary for the presentation of
antigens via major histocompatibility class (MHC) I. These mice lack all the
immune functions that their less immunodeficient NOD/SCID predecessors
lack with the addition of complete abolition of NK cell. ATL cells derived
directly from patients were able to engraft in these mice (113). The
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percentage of mice with tumor engraftment improves and time to clinical signs
decreases in mice with this modification when compared to NOD/SCID mice.
NOD/SCID gamma c null (NOG) mice are homozygous for the SCID
mutation and a targeted disruption of the interleukin (IL)-2Rγ gene (125). The
gamma chain is common to the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and
IL-21. NOG mice are easily transplanted with human cells that would not
normally transplant with the same efficiency in the more immunocompetent
mouse models (126). NOGs lack B and T-cell development as well as NK cell
function. NOG mice were found to have a severe reduction in IFN-γ
production from dendritic cells (126). Mice with this combined
immunodeficiency can be used to transplant cells directly from ATL patients
(127). These NOG mice can also be used in the proliferation of PBMCs from
asymptomatic carriers (128).
The degree of engraftment of ATL cells and their relationship to gene
expression has been studied in the NOG mouse. Tumor suppressor lung
cancer 1 (TSLC1) is aberrantly expressed in acute ATL cells and some cell
lines. The mice inoculated with TSLC1 expressing ED-40515 cells formed
larger tumors than their non TSLC1 expressing counterparts (115). The
immunodeficient mouse is also becoming a model of early infection in which
PBMCs and lethally irradiated MT-2 cells are inoculated in the NOG mouse
and infection of the human PBMCs ensues (114).
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Inoculation of MET-1 cells into NOD/SCID mice provide a model
system for slowly developing T-cell leukemia with multiple organ involvement
(129). In a comparative study by our group, leukemic mice had an increase in
serum calcium levels that correlated with expression of receptor activator of
nuclear factor κB (RANK) ligand on leukemic cells, and secretion of PTHrP
and IL-6 (Parrula et al., epub ahead of print 2009 May 9 ). MET-1 cells
expressed both the adhesion molecules CD11a (LFA-1α) and CD49d (VLA-
4α) and produced or induced expression of matrix metalloproteinases 1, 2, 3,
and 9.
In addition to investigating the early events, transformation, and tumor
behavior; paraneoplastic syndromes are an important component of disease
that can be addressed using mouse models. Humoral hypercalcemia of
malignancy (HHM) is an important paraneoplastic syndrome that occurs
frequently in ATL. It has clinical implications with regard to organ damage
and chemotherapy. Mouse models of ATL that demonstrate HHM include the
SCID mouse injected with human lymph node cells (130) and SCID/bg mice
injected with RV-ATL cells (131).
1.4.4.2 Transgenic Mouse Models of HTLV-1
Tax, the 40 kDa transactivating protein of HTLV-1, has been shown to
alter gene function. Tax has been shown to promote cell transformation in
vitro (14-18). Tax has a multitude of cellular targets, the most important of
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which are NF-κB and serum response factor. Through these signaling
pathways, Tax can cause alteration of expression in genes involved in
apoptosis, cell cycle, proliferation, and DNA repair. Tax in transgenic models
is sufficient to cause oncogenesis, however, leukemia and lymphoma are
rare. Serendipidously, the mouse models often recapitulate other chronic
conditions associated with HTLV-1.
Transgenic mice expressing the tax gene under the control of the LTR
promoter developed multicentric mesenchymal tumors of the nose, ear,
mouth, tail, and foot (Table 1.2) (20). These tumors were characterized by a
typical spindle cell component with infiltration of granulocytes. While this
transgenic system is not appropriate for the study of ATL, it proved that the
tax gene is, in fact, an oncogene. The close association of the mesenchymal
tumors with peripheral and cranial nerves lead to their identification as
neurofibromas and it was concluded that this model may be useful in the
study of neurofibromatosis, an inherited disorder of humans (19). In addition
to the mesenchymal tumors described above, these transgenic mice
developed a wasting disease characterized by degeneration of oxidative
muscle fibers. As such, this transgenic mouse may also serve to help
understand the aspects of HTLV-1 associated myopathies (132). LTR-tax
mice showed both gross and microscopic evidence of skeletal abnormalities.
Bones were grossly thicker and more fragile, while histologically they
exhibited high bone turnover characterized by increases in osteoclasts and
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osteoblasts. These lesions were without evidence of PTHrP expression
(133). The LTR-tax mice also develop a salivary and lacrimal exocrinopathy
with lesions similar to Sjögren syndrome of people (134). Lastly, tax
transgenic mice under the LTR promoter were shown to have ankylosing of
multiple joints (135) and have potential benefits as models of other human
diseases such as rheumatoid arthritis (136). Joint lesions similar to those
observed in the LTR-tax mice were observed in mice constructed with tax
under the control of the CD4 enhancer/promoter (135)
Combining the LTR-tax mice with LTR-βgal (β galactosidase) mice
generated a bitransgenic mouse in which the transactivator protein acts on
the LTR to increase expression of βgal. The enzyme was detected in bone,
muscle, cartilage, exocrine glands, and mesenchymal tumors (137). This
further supported the notion that the tax transgene was active in multiple
tissues that developed lesions in this mouse model.
Leukemia and lymphoma have been reported in tax transgenic mice in
which the transgene expression was restricted to the thymus by the Lck
promoter (138). This thymus derived, pre-T-cell tumor was associated with
constitutive NF-κB activation. This model verified that Tax expression alone
is sufficient to cause leukemia and lymphoma.
The development of the tax transgenic mouse under the control of the
granzyme B promoter restricted expression of Tax to CD4+, CD8+, Natural
Killer cells, and lymphokine-activated killer cells. These mice developed a
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large granular lymphocytic leukemia characterized by splenomegaly,
lymphadenopathy, and masses on the ears, legs, and tail (21). Similar to
ATL, these mice had humoral hypercalcemia of malignancy and osteolytic
bone lesions associated with metastasis (139). When these mice are crossed
with an interferon gamma knock out strain, there is enhanced rate of tumor
development (140).
Transgenic mice using a tetracycline (Tet)-off system controlling wild
type Tax, a Tax mutant incapable of activating NF-κB, Tax incapable of
activating NF-κB or CREB were generated and crossed with EµSRα tTA mice
to drive Tax expression specifically in the lymphocyte compartment (141).
The Tax transgenic mice in which the ability to activate NF-κB was
maintained developed a fatal dermatologic disease characterized by
infiltration of Tax positive T-cells into the dermis and epidermis. Addition of
doxycycline (suppression of Tax expression) resulted in the resolution of
lesions. Although suggestive of cutaneous lymphoma observed in some ATL
patients, the infiltrates in the skin were oligoclonal or polyclonal not
monoclonal as in ATL (141).
Several transgenic mice strains were generated with Tax under the
regulatory control of CD-3ε promoter enhancer sequence. These mice
developed mesenchymal tumors at wound sites and mammary and salivary
adenomas (142).
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1.5 Treatment of Adult T-cell Leukemia
The strength of the mouse model lies in the ability to use it to test the
preclinical efficacy of therapeutics. This tool provides insight to clinicians
before the development of phase I clinical trials. Xenograft mouse models
have been used to test conventional and targeted therapeutics as well as
monoclonal antibodies (Table 1.3).
The finding that NF-κB is constitutively active in ATL led to several models
to investigate the outcome of therapy that utilizes the blockade of NF-κB
through multiple different mechanisms. These have proven especially
promising in reducing tumor size of subcutaneous xenografts. In vivo
preclinical efficacy has been studied with Arsenic trioxide (143), proteasome
inhibitors (144), all trans retinoic acid (ATRA) (145) , curcumin (146), and
ritonovir (147).
The proteasome inhibitor, PS-341, inhibits the degradation of the inhibitor
of NF-κB, IκBα. It was shown to inhibit NF-κB activation in ATL cells and Tax
transgenic tumor cells in culture and in mouse transplantation studies. In
several of these studies the majority of the cell death was found to be due to
apoptosis. An inhibitor NF-κB DNA binding activity, BAY 11-7082, was also
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shown to block NF-kB activity and resulted in tumor regression in ATL
transplanted NOG mice.
The expression of markers on the cell surface of ATL cells implanted in
mice has made them an excellent target for preclinical trials with monoclonal
antibodies directed against IL-2Rα (110;148), CD52 (149;150), and CD2
(150). The outcome of the mouse studies may be predictive of successful
therapy for human patients. A complete response has been reported in a
human with alemtuzumab (anti-CD52) (151).
Treatment of ATL-bearing mice with a humanized anti-CD2 monoclonal
antibody led to tumor regression (150). The activity of the mononclonal
antibody was most likely due to antibody-dependent cellular cytotoxicity, since
expression of Fcγ receptors on neutrophils and monocytes was required for
activity. Treatment of ATL bearing NOD-SCID mice with an alpha-emitting
radionuclide, bismuth 213, conjugated to an antibody to the IL-2 receptor
proved to be highly effective in inducing tumor regression (152). The activity
of bismuth 213 in this model was greater than that of unconjugate antibody or
radionuclide, or antibody conjugate to β-emitting radionuclide yttrium 90.
Flavopiridol, an inhibitor of cyclin-dependent kinases, was tested for its
therapeutic efficacy alone and in combination with humanized anti-Tac
antibody (HAT), which recognizes CD25, in a murine model of human ATL
using MET-1 leukemic cells (148). Either flavopiridol, given 2.5 mg/kg body
weight daily for 5 days, or HAT, given 100 μg weekly for 4 weeks, inhibited
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tumor growth and prolonged survival of the leukemia-bearing mice (148).
Collectively these studies provide hope that ATL transplant and Tax
transgenic models will provide new directions in the development of effective
therapies against HTLV-1-induced lymphoproliferative diseases including
ATL.
1.5.1 Proteasome Inhibitors
The 26S proteasome is a protease complex comprised of a 20S
catalytic subunit and multiple regulatory subunits. It functions in the
nonlysosomal breakdown of ubiquitinated proteins in an ATP dependent
manner. The catalytic domains, contained within the 20S subunit include six
active sites of tryptic, chymotryptic and caspase activity. At each end of the
proteasome, there is a 19S cap which functions in removal of ubiquitin and
unwinding of the target protein (153). Proteins targeted to the proteasome
are numerous and include Bcl-2 family members, cell cycle proteins, and
IκBα, to name a few.
Proteasome inhibition results in multiple effects that result in cancer
cell death. These pleiotrophic effects are reviewed in Nencioni et al. (154).
These effects include NF-κB inhibition, accumulation of p53,
antiangiogenesis, effects on cdc25, a shift to proapoptotic Bcl-2 family
members, JNK stabilization, and deregulation of cyclins, as well as increased
reactive oxygen species.
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1.5.1.1 Bortezomib (PS-341)
Bortezomib (PS-341 or Velcade) is a 26S proteasome inhibitor that
inhibits the rate limiting chymotryptic activity of the proteasome (154).
Inhibition of the proteasome confounds cellular signaling by the accumulation
of normally short lived proteins within the cell. These activities result in
increased cell death and decrease in tumor burden in multiple tumor types
including multiple myeloma (155;156) and mantle cell lymphoma (157). In
addition, the cellular transcription factor NF-κB, which is constitutively active
in ATL cells (23) is blocked through inhibition of the proteasome. HTLV-1
infected cells exhibit decreased cell viability, and increased apoptosis in the
presence of PS-341 (158). Tan et al., (129) reported an increase in
phosphorylated IκBα and ubiquitination when HTLV-1 cells were treated with
PS-341. Growth inhibition, increased apoptosis measured using Terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and evidence
of Poly ADP ribose polymerase (PARP) cleavage, G2/M cell cycle arrest, and
accumulation of p21 demonstrated were shown by Nasr et al. (159) in several
HTLV-1 cell lines. In vivo, in xenograft mouse models, administration of PS-
341 resulted in increased apoptosis in tumor cells (160). In a separate study,
PS-341 decreased tumor burden in ATL xenograft mice (158). However, PS-
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1.5.2.1 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG)
17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), a derivative of
the benzoquinone ansamycin geldanamycin, is a heat shock protein 90
inhibitor. 17-AAG binds to the ATP binding pocket at the amino terminus of
Hsp 90 (165). Disruption of the heat shock protein-client complex results in
ubiquitination of the client protein with direction to the proteasome for
degradation. Hsp 90 inhibition affects multiple signaling pathways, including
Akt and chimeric oncoproteins like Bcr/Abl and Her2Neu. It has been shown
that 17-AAG will sensitize cells to other therapies (166). Hsp90 is often
upregulated in malignancies, including ATL (163) and this makes 17-AAG an
attractive mechanism of pharmacologic disruption.
Administration of 17-AAG alone in a multiple myeloma model has
shown a decrease in tumor burden (166). Incubation of ATL cell lines and
PBMCs isolated from patient samples with 17-AAG resulted in a decrease in
cell survival and increase in apoptosis through the NF-kB, AP-1, and Akt
signaling pathways (163). 17-AAG is currently in clinical trials alone or in
combination in advanced solid malignancies (167). In ATL cell lines,
administration of 17-AAG resulted in inhibition of growth and increased
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apoptosis as well as decreases in survivin, cdk6, cdk 4, cyclin D1, Akt and
phosphorylated Akt (163).
1.5.3 Histone Deacetylase Inhibitors
Transcriptional regulation is controlled in part by the acetylation of
histones and non histone proteins. The acetylation of lysine residues on
histones was first discovered in 1968 (168) but was not linked to control of
DNA transcription until the mid 1990s (169). Acetylation of histones results in
the relaxation of chromatin and the promotion of transcription.
Histone deacetylases (HDACs) are enzymes that promote the removal
of the acetyl groups from the lysines. This results in the restoration of positive
charge, tightening of DNA (negatively charged) around the histone core, and
decrease in transcription of affected genes. Currently, there are at least 18
HDACs that are classified into six different groups. These HDACs control the
activation and repression of proteins and may affect non histone proteins as
well. Non histone proteins affected by HDACs include hormone receptors,
chaperones, viral proteins, and cytoskeletal proteins.
Inhibitors of the HDAC enzymes (HDACi) and their role in cancer
therapy have been recently reviewed in multiple journals (170-173). HDACi
promote the acetylated state of the histone and relax chromatin structure.
HDACi’s are divided into several classes including short chain fatty acids,
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hydroxamic acids, benzamides, and cyclic peptides. The classes differ in
their potency, but are generally not specific for HDAC isoenzymes (172).
Recently, HDACi have been used as targeted therapies in cancer
research. As reviewed in Xu et al., (171) HDACi have been shown to induce
apoptosis, induce cell cycle arrest, disrupt Hsp90 and the aggresome, inhibit
angiogenesis, and induce mitotic cell death, incuding autophagic cell death
and senescence. It is through these pleiotropic effects that HDACi have
shown efficacy in vitro against prostate cancer (174;175).
In addition, the HDACi suberoylanilide hydroxamic acid (SAHA or
Vorinostat) is approved for the treatment of cutaneous T-cell lymphoma.
Depsipeptide (FR901228) has been used in the treatment of peripheral and
cutaneous T-Cell lymphoma (176). Several other first generation HDACi are
currently in phase I and phase II clinical trials (177).
The role of the HDACi in viral gene expression is slowly being
unraveled. Trichostatin A (TSA) has been shown to increase bovine leukemia
virus (BLV) LTR expression in vitro. In sheep and cattle peripheral blood
mononuclear cells (PBMCs), BLV viral gene expression was increased in
response to TSA (178).
1.5.3.1 Valproic Acid
Valproic acid, an eight carbon branched chain fatty acid, has been
used in the treatment of epilepsy since 1978 (179). Recently, it has been
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shown to have efficacy in the treatment of cancer through blocking the
hyperacetylation of histones 3 and 4 (180). Valproic acid alone has been
shown to inhibit prostate cancer cell growth (181) , medulloblastoma (182) ,
and multiple myeloma (183). Valproic acid has been shown to enhance the
sensitivity of cells to chemotherapeutic agents (184;185).
In addition to the potential effects on neoplasia (ATL in the subsequent
chapters), valproic acid has effects on the expression of viral genes. Epstein
Barr Virus (EBV), a human herpesvirus, is often latent in the majority of
people infected. When exposed to valproic acid, EBV gene expression was
increased as was sensitivity of lytic gene expressing cells to chemotherapy
(184). In the deltaretroviruses, particularly BLV, valproic acid treatment of
isolated sheep PBMCs resulted in increased levels of BLV gene expression
and a decrease in leukocyte numbers in leukemic sheep (186).
1.5.3.2 OSU-HDAC42
OSU-HDAC42 is a phenylbutryrate derived HDACi that has been
included in the Rapid Access to Intervention Development Program through
the National Cancer Institute (175). The modifications to the phenylbutyrate
improved the potency over the original compound from the nanomolar range
to the submicromolar range (187). There are multiple non-histone related
affects of OSU-HDAC42. These include Akt dephosphorylation and disrupting
protein phosphatase 1 (188).
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In a prostate cancer model, OSU-HDAC42 was shown to decrease cell
viability, decreased phospho-Akt, Bcl-xL, and Survivin (174). In vivo, a
xenograft mouse model had decreased tumor burden (174) and decreased
tumor levels of phospho-Akt and Bcl-xL in response to the drug (175).
1.6 Summary
Despite repeared attempts to develop effective therapies against ATL,
the prognosis for the disease remains poor. Continued progress in
understanding the multiple genetic and cellular aberrations that occur in the
progression of HTLV-1 to ATL will provide many new therapeutic targets to
explore. Translating knowledge from the laboratory bench to appropriate
animal models and subsequently to cancer patients provides hope. The
thesis chapters herein document a series of studies using reproducible animal
models to test the efficacy of novel treatments against ATL. Our data
provides new directions in the design of targeted therapy against this lethal
viral-induced cancer.
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Figure 1.1 The HTLV-1 genome including all transcripts and theirprotein products. HTLV-1 full length provirus followed by all viral transcripts
and their corresponding proteins. This includes the HBZ protein which isencoded by the antisense RNA. Open reading frames are indicated byroman numerals and colored boxes indicate the protein coding region.
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Table 1.1 Engraftment of ATL cell lines in immunodeficient miceEngraftment is defined as having evidence of growth within the mouse line,using either serum biomarkers or histopathology.
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Table 1.2 Transgenic mouse models of adult T-cell leukemia/lymphoma
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Table 1.3 Preclinical efficacy studies utilizing mouse models of ATLEndpoints for studies included tumor burden (measured histologically,volumetrically or biochemically) or survival.
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CHAPTER 2
COMBINATION THERAPY OF BORTEZOMIB (PS-341) AND 17-
(ALLYLAMINO)-17-DEMETHOXYGELDANAMYCIN (17-AAG)
DECREASES TUMOR BURDEN IN A MOUSE MODEL OF ADULT T-CELL
LEUKEMIA/LYMPHOMA
2.1 ABSTRACT
The proteasome inhibitor bortezomib (PS-341 or Velcade) and the heat
shock protein inhibitor 17-(Allylamino)-17-demethoxygeldanamycin(17-AAG)
alone and in combination exhibit efficacy against multiple tumor types in vitro.
In this study, we document the efficacy of PS-341 and 17-AAG alone and in
combination in a preclinical model of adult T-cell leukemia/lymphoma (ATL).
ATL is a refractory CD4+/CD25+ T-cell malignancy with multiple clinical forms
caused by the retrovirus human T-lymphotrophic virus type 1 (HTLV-1). ATL
patients have a poor prognosis due to the highly aggressive nature of the
cancer and the resistance of neoplastic cells to conventional chemotherapies.
Consistent cellular alterations within these cells, such as NF-κB activation,
make targeted therapeutics a viable alternative to conventional
chemotherapeutic protocols. We utilized a NOD/SCID ATL mouse model to
test multiple dose regimens of PS-341 and 17-AAG alone and in combination.
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Tumor burden was quantified by measuring serum IL-2Rα as a biomarker and
by quantifying and grading tumor engraftment. Our data indicate that the
combination of PS-341 and 17-AAG synergistically promoted cell death in
ATL cell lines in vitro and reduced tumor burden during cycles of drug
treatments. Upon removal of drug therapy, the differences observed between
the treatment groups were minimized. Using this model, PS-341 and 17-AAG
targeted therapy offers promise to decrease tumor burden, but our data
indicates that the additive effects of 17-AAG are transient and require
continued presence of the drug.
2.2 INTRODUCTION
Adult T-cell leukemia/lymphoma (ATL) is a refractory CD4/CD25+
CD8- T-cell malignancy caused by human T-lymphotrophic virus type 1
(HTLV-1) (1). Persistent HTLV-1 infections are established following
transmission from infected mothers to their children, through breast feeding,
and horizontally, through transfer of contaminated whole blood products. In
approximately 1 to 5% of persistently infected individuals, clinical disease
may manifest as ATL or other lymphocyte mediated disorders including the
neurologic disease tropical spastic paraparesis (HAM/TSP) (2).
The mechanism of carcinogenesis induced by HTLV-1 infection is not
completely understood, but it is thought to occur following viral induced cell
dysregulation and accumulation of other transformation events. ATL is
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classified into four primary clinical subtypes. The two most aggressive forms
are the acute and lymphomatous (3). These forms are associated with
humoral hypercalcemia of malignancy (HHM), bone lysis, lymphadenopathy,
visceral and skin involvement. The lymphomatous form typically is
associated with a normal peripheral blood leukocyte cell count, whereas in
the acute form, ATL cells are found in high numbers in the peripheral blood.
The more indolent clinical subtypes are the smoldering and chronic forms.
HTLV-1 is a complex retrovirus that encodes viral structural and
enzymatic proteins from the gag, pol, and env genes as well as a pX region
that encodes four open reading frames (ORFs) immediately prior to the 3’
long terminal repeat (LTR) (4). The most well characterized product of these
ORFs is the transactivator oncoprotein Tax. Tax is a 40 kDa protein that acts
as a transcriptional transactivator to increase viral transcripts. Tax also acts
to upregulate several cellular gene products that result in dysregulation of cell
cycle and promotion of cell survival (3). These alterations include
upregulation of NF-κB through several mechanisms, including the
phosphorylation of IKKγ or IKKα and IKKβ, ultimately leading to an increase
in the phosphorylation of IκBα. Phosphorylation of IκBα targets it for
ubiquitination and degradation via the proteasome. Degradation of IκBα
exposes NF-κB for translocation to the nucleus where it acts to upregulate
transcription of several prosurvival genes. Tax can also act through the
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noncanonical pathway by binding to the p50 subunit of NF-κB and by
enhancing the phosphorylation of protein kinase B (Akt).
Conventional chemotherapies against ATL have produced inconsistent
or limited results in treatment outcome and prolongation of mean survival
times (1). A combination of Cyclophosphamide, Adriamycin, vincristine, and
prednisolone is typically the first line of therapy, but is of limited efficacy. The
use of nucleoside analogs, interferon, zidovudine, topoisomerase inhibitors,
and monoclonal antibodies has produced more encouraging results; however
the prognosis for ATL patients remains poor. Bone marrow transplant has
been reported to “cure” ATL (5). This is complicated by patient age,
condition, and likelihood of immunologic match and therefore is of limited use.
Bortezomib (PS-341 or Velcade) is a 26S proteasome inhibitor that
inhibits the rate limiting chymotryptic activity of the proteasome. Inhibition of
the proteasome confounds cellular signaling by the accumulation of normally
short lived proteins within the cell. These activities result in increased cell
death and decrease in tumor burden in multiple tumor types including multiple
myeloma (6;7) and mantle cell lymphoma (8). In addition, inhibition of the
proteasome blocks the function of the cellular transcription factor NF-κB,
which is constitutively active in ATL cells (9;10). HTLV-1 infected cells in
culture have decreased cell viability, and increased apoptosis in the presence
of PS-341 (11). Tan et al., (12) reported an increase in phosphorylated IκBα
and ubiquitination when HTLV-1 cells were treated with PS-341. In vivo, in a
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Tax transgenic mouse model, administration of PS-341 resulted in increased
apoptosis in tumor cells (13). In a separate study, PS-341 decreased tumor
burden in ATL xenograft mice (14). However, PS-341 exhibited enhanced
efficacy in combination with a humanized anti Tac (HAT) monoclonal antibody
(12).
A derivative of the benzoquinone ansamycin geldanamycin (17-AAG)
is a heat shock protein 90 inhibitor. 17-AAG binds to the ATP binding pocket
at the amino terminus of heat shock protein (Hsp) 90 (15). Disruption of the
heat shock protein-client complex results in ubiquitination of the client protein
with direction to the proteasome for degradation. Heat shock protein 90
inhibition affects multiple signaling pathways, including Akt and chimeric
oncoproteins such as Bcr/Abl and Her2Neu and sensitizes cells to other
therapies (16). In addition, Hsp90 is often over expressed in malignancies,
including ATL (17) and this makes 17-AAG an attractive mechanism of
pharmacologic disruption.
Administration of 17-AAG alone in a multiple myeloma model has
shown a decrease in tumor burden (16). Incubation of ATL cell lines and
peripheral blood derived mononuclear cells (PBMC) isolated from ATL patient
samples with 17-AAG resulted in a decrease in cell survival and increase in
apoptosis through the NF-kB, AP-1, and Akt signaling pathways (17). 17-AAG
is currently in clinical trials alone or in combination in advanced solid
malignancies (18;19). In ATL cell lines, administration of 17-AAG resulted in
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inhibition of growth, increased apoptosis and decreases amounts of Survivin,
cdk6, cdk 4, cyclin D1, Akt and phosphorylated Akt (17).
Herein, we demonstrate that the combination of PS-341 and 17-AAG
caused decreased growth of ATL cell lines and significantly diminished the
tumor burden in a xenograft NOD.Cg-PrkdcSCID (NOD/SCID) mouse model.
Our data indicate that targeted and synergistic therapy offers promise to
abate ATL, but indicates that additive effects of 17-AAG requires continued
treatment.
2.3 MATERIALS AND METHODS
Cell lines
The HTLV-1 infected cell lines C8166-45 (20), MT-2 (21), and HTLV-1
negative Jurkat cells (clone E6-1; American Type Culture Collection catalog
number TIB-152) were maintained in RPMI 1640 supplemented with 10%
fetal bovine serum, 10% penicillin/streptomycin (100 μg/mL), and 10%
glutamine (0.03 mg/mL) at 37°C in 5% carbon dioxide. MET-1 cells are an
HTLV-1 positive cell line derived from a patient with ATL (22). These cells,
like patient ATL cells, are unable to proliferate in vitro, but can be expanded
by passage through NOD/SCID mice. MET-1 cells were expanded by
inoculating 2 x 107 cells intraperitoneally (IP) into each mouse and harvesting
at optimal tumor growth, typically at 5-6 weeks post inoculation. Spleen,
lymph nodes and inoculation site masses were harvested from the mice at
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necropsy. Tissues were minced and passed through a 100 μm cell strainer to
create a single cell suspension. Isolated cells were then frozen in 90% fetal
bovine serum and 10% DMSO or propagated in NOD/SCID mice to produce
tumors for experimental procedures.
Animals
Female immunodeficient NOD/SCID (NOD.CB17-Prkdc /J), (scid Jackson
Laboratory, Bar Harbor, ME), were maintained under specific pathogen-free
conditions in animal facilities in the College of
Veterinary Medicine at The
Ohio State University. Mice were kept in individually ventilated cages at 20 ±
2o C in accordance with the Guide for Care and Use of Laboratory Animals.
This includes 12 hour light/dark cycle, free choice standard rodent chow, and
water ad lib. All procedures were approved by The Ohio State University
Institutional Laboratory Animal Care and Use Committee.
Mice were injected IP or subcutaneously with 2 x 107 MET-1 cells in 1
ml calcium/magnesium free phosphate buffered saline. Mice were monitored
visually daily and weighed regularly. At 19 days post inoculation, serum was
drawn and tested for serum IL-2Rα levels (R&D Systems). On Day 20, mice
began a treatment regimen (Table 2.1). In the first phase of the experimental
protocol mice were divided into four groups. The first group received vehicle
only (100 µl of normal saline and 200 µl of Intralipid/10%DMSO/2.5% Tween
20). The second group received 0.43 mg/kg PS-341 in normal saline in a
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total volume of 100 µl. The third group received 40 mg/kg 17-AAG in
Intralipid/10% DMSO/2.5% Tween 20. The fourth group received
combination therapy of PS-341 and 17-AAG . All mice received injections IP
on days 20, 23, 27, 30, 41, 44, 48, and 51. Serum was drawn via the anterior
facial vein every two weeks. Mice were euthanized using carbon dioxide
inhalation on day 68.
In phase II, the mice were divided into three groups, eliminating the 17-
AAG alone treatment group. To ascertain differences in survival between
treatment groups, mice were continually monitored after completion of
treatment regimen until such time as euthanasia criteria were met. Mice were
euthanized by CO2 inhalation upon developing signs of morbidity (rapid
weight loss, rough hair coat, lethargy or persistent recumbence, labored
breathing, difficulty with ambulation, or obtaining food and water).
Compounds
PS-341 (bortezomib) was provided by Millennium Pharmaceuticals.
17-AAG (Kosan Biosciences) was provided by the Drug Synthesis and
Chemistry Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute. PS-341 was diluted in
normal saline. 17-AAG was diluted in Intralipid/10% DMSO/2.5% Tween 20.
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Growth Inhibition Assays
Forty thousand cells were plated in each well of a 96 well plate and
incubated with media only, 1, 2.5, 5 nM PS-341, 1000 nM 17-AAG, or a
combination of the increasing doses of PS-341 and 1000nm 17-AAG. After
24, 48, and 72 hours, 20 µl of MTS reagent (Cell Titer 96® AQueous assay kit
from Promega) was added to each well. The plate was incubated for two
hours at 37° C. After this incubation period, the absorbance of the formazan
(resulting from the chemical reduction of the MTS) was measured at 490 nm.
Color change within the wells correlate to the number of metabolically active
cells.
Western Blot Assays
Cells (2 x 106) were incubated with 5 nM PS-341, 1000 nM 17-AAG or
both 5nM PS-341 and 1000 nM 17-AAG for 48 hours. Cells were washed
once in PBS and lysed. Aliquots of protein (20 µg) were run on SDS
polyacrylamide gels. Protein was transferred to a nitrocellulose membrane
and blocked overnight at 4° C in 1% BSA in 5% nonfat dry milk in 0.1%
Tween-20 in Tris buffered Saline (TBST). Membranes were incubated with
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primary antibodies in 5% milk in TBST. Poly (ADP-ribose) polymerase
(PARP) antibody (Oncogene Catalog #AM30-100UG) was diluted at 1:100.
Akt (Stressgen BioReagents cat# KAP-PK004) and phosphorylated Akt (Cell
Signaling cat# 9271) were both dilute 1:1000. Ubiquitin (BD Pharmingen cat#
550944) was diluted to 1:10,000. Mouse Monoclonal antibody to beta-actin
AC-74 (Sigma) was diluted 1:20,000. Membranes were then incubated with
1:1000 dilutions of corresponding secondary antibodies. Bound antibodies
were detected with a chemiluminescent detection system (Cell Signaling
Technologies #7003: 20X LumiGLO and 20X peroxide).
Histology and Immunohistochemistry
In phase I, all mice were euthanized on day 68. A routine necropsy
was performed on all mice and organs were weighed and fixed in 10% neutral
buffered formalin for 24 hours. Representative sections of tissues of interest
were embedded in paraffin, sectioned, and stained with hematoxylin and
eosin or stained with Ki67 (KiS5 clone) monoclonal antibody with ARK kit.
Serum IL-2Rα
Whole blood was collected from the anterior facial vein prior to the start
of treatment and periodically throughout the study (minimum time between
samples of 14 days). Immediately post collection, samples were centrifuged
to separate the serum. Aliquots of serum were frozen at -80° C. To assay for
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the presence of the human serum interleukin 2-receptor alpha chain (sIL-
2Rα), a specific proliferation/tumor marker of human lymphocytes, the R&D
systems IL-2sRα Duo Set ELISA kit was used. Samples were diluted 1:500 in
reagent diluents and assayed according to manufacturer’s protocol.
Statistical Analysis
Two way ANOVA models were used for the data analyses of cell
growth, body and organ weights, cell counts and sIL-2Rα. Treatment group,
time and their interaction were included in the models. Contrasts of treatment
group means of interests, such as the combination group versus no treatment
group, combination group versus PS-341 group at each time point were
calculated and p-values were adjusted for multiplicity by Holm’s step down
(23) or Dunnet’s method (24). Engraftment as measured by histologic grading
was analyzed using Fisher’s exact test. Kaplan-Meier curves were compared
using the log-rank test for survival analyses. Cox regression models were
performed to compare the effects of combination therapy PS-341 and sIL-2Rα
on survival time.
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2.4 RESULTS
17-AAG and PS-341 synergistically inhibit growth of Jurkat T-cell and
MT-2 ATL cell lines
PS-341 and 17-AAG alone or in combination were first tested for their
ability to inhibit the growth of ATL cell lines, HTLV-1 positive T-cell lines MT-2
and C8166 or the HTLV-1 negative Jurkat T-cell line (Fig. 2.1).
Concentrations of PS-341 and 17-AAG were chosen based on previous
literature on the effects in ATL cells (17;25). The combination of 1000 nm of
17-AAG and 2.5 nM PS-341 produced a statistically significant decrease in
growth in the MT-2 cell line and Jurkat T-cell lines (p<0.001). Similar results
were seen at 48 and 72 hours. Using these concentrations, we were not able
to cause a statistically significant decrease in growth in C8166 cells. At 24
hours, in all three cell lines, a significant decrease in growth was not achieved
with 1000 nM of 17-AAG alone or with increasing concentrations of PS-341
alone.
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The combination of PS-341 and 17-AAG diminishes T-cell signaling
through inhibition of Akt and the proteasome
To test the influence of PS-341 and 17-AAG alone or in combination
on the signaling pathways important for T-cell proliferation, cell lysates of MT-
2 cells treated with 5 nM PS-341 and 1000 nM 17-AAG for 48 hours were
probed with multiple antibodies to test for inhibition of the proteasome and
Hsp90 signaling pathway (Fig. 2.2). Our data indicated a decrease in PARP,
phosphorylated Akt (p-Akt), Akt, as well as evidence of proteasome inhibition
with increased ubiquitination of proteins. Thus, the combination of these two
drugs appeared to target critical components of Tax-mediated cell
proliferation and further justified in vivo studies in our mouse model of ATL.
Treatment with PS-341 alone and in combination with 17-AAG reduced
tumor burden in organs
An established NOD/SCID mouse model of ATL (22) was used to test
the in vivo efficacy of PS-341 alone and in combination with 17-AAG. In
phase I, 20 mice were divided into four groups, and treated with different
regimens of drugs or control vehicle (Table 2.1). Body weights of the mice
did not differ between any treatment groups when compared to vehicle only
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treatment. However, the liver and spleen weights in the animals receiving
both PS-341 and 17-AAG were significantly lower than those in the vehicle
only group (p=0.0013, 0.0354 respectively). In this ATL animal model, the
liver and the spleen are typically heavily infiltrated with tumor, thus a
significantly reduced spleen or liver weights suggested less tumor cell
infiltration of these organs (confirmed by histologic examination below).
Neoplastic infiltrates in organs were reduced in mice treated with PS-
341 alone and in combination with 17-AAG
To visualize and quantify the tumor burden in mice at the end of our
study, routine hematoxylin and eosin staining was performed on paraffin
embedded sections of collected tissues. Large neoplastic lymphocytes with
characteristic MET-1 cell morphology were present in the liver, spleen,
pancreas, brain, kidney, and heart (summarized in Table 2.2). The
engraftment was widespread throughout the sections, but the size of the
individual foci of neoplastic accumulations varied (Fig. 2.3). Statistical
comparison indicated that tumor-bearing mice treated with PS-341 alone or in
combination with 17-AAG had reduced tumor engraftment in systemic organs.
Tumor-bearing mice treated with 17-AAG alone did not have significant
differences in organ infiltration compared to vehicle treated control mice.
To compare the number of mitotically active cells in neoplastic foci
between groups, we stained paraffin embedded sections of lung, liver, and
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spleen with an antibody specific for the human proliferation marker Ki67.
Liver and lung sections from tumor-bearing mice treated with PS-341 in
combination with 17-AAG had reduced number of Ki67 positive foci compared
to vehicle control mice (Fig. 2.3). There was no difference in the number of
Ki67 positive foci between the liver and lung section of PS-341 only or 17-
AAG only when compared to the vehicle treatment. No statistically significant
difference was observed in the spleen tissue from any of the treatment
groups.
Serum biomarker IL-2Rα was reduced mice treated with PS-341 alone
and in combination with 17-AAG
To monitor tumor burden noninvasively in mice, we measured serum
IL-2Rα, a biomarker that has been effectively used to correlate with ATL
tumor proliferation (26). Tumor-bearing mice treated with PS-341 and 17-
AAG in combination had significantly lower sIL-2Rα levels than the vehicle
only group (p<0.0001). The PS-341 alone treatment group also had
significantly lower sIL-2Rα when compared to the vehicle only group
(p=0.046). Tumor-bearing mice treated with 17-AAG alone exhibited a trend
toward a difference in the sIL-2Rα levels (p=0.0571), but were not
significantly different compared to the vehicle control group (Fig. 2.4A).
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Additive effects of 17-AAG on survival required continuous treatment
To establish if combinational therapy of PS-341 with 17-AAG prolonged the
survival of tumor-bearing mice, additional groups of mice were inoculated with
ATL cells and allowed to survive post drug treatments until they met
euthanasia criteria. A Kaplan Meier curve was generated and compared
between groups (Fig. 2.4B). Median survival times were 64, 85, and 105
days respectively for the vehicle control, PS-341 and 17-AAG combination,
and PS-341 alone groups. A statistically significant difference was found
between treatment groups (p=0.03). However, the median survival time for
the mice treated with the combination therapy was not different than the mice
treated with the PS-341 alone.
2.5 DISCUSSION
Our data from both in vitro analysis and in an established ATL mouse
model indicated that the combination therapy of PS-341 and 17-AAG is
effective to abate the growth of ATL cell lines and reduce tumor burden in
vivo. The addition of 17-AAG to PS-341 resulted in growth inhibition and
induction of apoptosis of ATL cells indicated by decrease in PARP, Akt, p-Akt,
and evidence of both Hsp90 and protesome inhibition with increased
ubiquitination. We are the first to demonstrate that treatment with PS-341 in
combination with 17-AAG reduced the tumor burden of established ATL cells
in a mouse model. Our histologic examination of mice on drug treatment
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revealed significant difference in engraftment when both drugs were
combined. Reductions in the expression of the proliferation marker, Ki67,
confirmed these data in both the liver and the lung in the combination therapy
group when compared to the vehicle only group. Importantly, our survival
studies in ATL engrafted mice indicated that the differences observed
between the treatment groups were minimized upon removal of drug therapy,
Thus, while this combination of targeted therapy offers promise to decrease
tumor burden, our results indicate that additive effects of 17-AAG are
transient and require continued presence of the drug.
The acute form of ATL is a refractory malignancy with a median
survival time of less than one year regardless of therapy (27). Adult T-cell
leukemia/lymphoma-derived cells have multiple ways of surviving and
persisting in presence of chemotherapy. While, HTLV-1 is clearly linked to
the development of ATL, active virus replication is not required to maintain
tumor growth. The outgrowth of ATL cells is believed to be initiated by key
viral proteins such as Tax, but is more dependent upon genetic or epigenetic
changes of the host cell for eventual selection of clonally expanded malignant
lymphocytes. Recently, the expression of HBZ, a unique antisense encoded
viral protein or its mRNA has been correlated with ATL cell survival (28;29).
Over expression of the multidrug resistance protein has been reported in ATL
(30;31) further complicating treatment options.
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Various therapeutic approaches have been attempted against ATL, but
with limited sustained success. These include chemotherapy regimens, a
combination of zidovudine and interferon alpha-2a, antibody or antibody-
radioconjugate therapy, stem cell transplantation, or targeted approaches with
bortezomib or arsenic trioxide [reviewed in (1)]. Conventional
chemotherapeutics have thus far failed to significantly increase the median
survival time of ATL patients. As a result, new therapeutic approaches to
abate this aggressive malignancy have focused on targeted therapies. In
vitro and in vivo studies with targeted therapies have included monoclonal
antibodies (alemtuzumab (32), Anti-Tac (33)), histone deacetylase inhibitors
(34), and NF-κB inhibitors (13;35). While these studies have shown promise
in experimental models, single agent therapies are rarely effective when
translated to ATL patients (1).
Proteasome inhibition has been demonstrated to result in increased
cell death and decrease in tumor burden in multiple tumor types including
multiple myeloma (36;37) and mantle cell lymphoma (38). Myeloma cells
respond to administration of PS-341, by upregulating heat shock proteins to
assist in the folding and function of the proteins that would normally be
targeted to the proteasome for degradation (16). Administration of 17-AAG
alone in a multiple myeloma animal model decreased tumor burden (16).
The combination of PS-341 and 17-AAG has been shown to produce
synergistic effects, including increased chymotryptic activity in multiple
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myeloma (16). In other cell lines, combination treatment has been shown to
enhance protein ubquitination and enhance antitumor activity (39)
The cellular transcription factor NF-κB, which is constitutively active in
ATL cells (9) is blocked through inhibition of the proteasome (40). HTLV-1
infected cells in culture have decreased cell viability, and increased apoptosis
in the presence of PS-341 (41). Tan et al., (12) reported an increase in
phosphorylated IκBα and ubiquitination when HTLV-1 cells were treated with
PS-341. Nasr et al., (42) reported growth inhibition, increased apoptosis
measured by terminal deoxynucleotidyl transferase biotin-dUTP nick end
labeling (TUNEL) and evidence of PARP cleavage, G2/M cell cycle arrest,
and accumulation of p21 in several HTLV-1 cell lines treated with PS-341.
The effect of PS-341 on the growth of tumors in a Tax transgenic mouse
model revealed no consistent inhibition of NF-κB activation in vivo, however
transplanted Tax tumors in Rag-1 mice showed consistent inhibition of tumor
growth and prolonged survival (13). In a separate study, PS-341 decreased
tumor burden in ATL xenograft mice (43). In the MET-1 ATL mouse model,
PS-341 was not beneficial alone, however enhanced efficacy was noted in
combination with humanized anti-Tac (CD25) monoclonal antibody (12).
Our studies reveal that PS-341 and 17-AAG, while effective at
reducing established tumor burdens in short term studies, indicated a need
for continuous drug pressure to maintain the additive benefits of both drugs in
longer term survival studies. Mice were treated only eight times in both the
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phase I and phase II studies. In phase I, the mice were euthanized shortly
after the final treatment. Our design allowed us to test the effects of the
continued pressure of the combination therapy. In phase II, the animals
continued without treatment until they met euthanasia criteria. The removal of
drug pressure for a prolonged time appeared to allow tumor cells to regain
growth capacity and thus minimize the affects of the combination therapy
when compared to the single agent therapy. The mouse model, while
providing an excellent preclinical model in which to assay the effects of the
compounds on the ATL cell growth, does not assay the effects of prolonged
therapy in context to ATL patients or in the presence of a functional immune
system. Despite the limitations of the model system, our results have
implications for the design of human trials using this combination of drugs or
when considering 17-AAG or derivatives as adjunct therapy to standardized
protocols. Our data support further investigations to establish therapeutic
dosing regimen and drug cycles of PS-341 and 17-AAG in both animal and
human clinical trials.
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(8) Fisher RI, Bernstein SH, Kahl BS, Djulbegovic B, Robertson MJ, deVos S, et al. Multicenter Phase II Study of Bortezomib in Patients WithRelapsed or Refractory Mantle Cell Lymphoma. J Clin Oncol 2006 Oct20;24(30):4867-74.
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(10) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
(11) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
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1;104(3):802-9.
(14) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
(15) Chiosis G, Huezo H, Rosen N, Mimnaugh E, Whitesell L, Neckers L.17AAG: Low Target Binding Affinity and PotenT-cell Activity--Findingan Explanation. Mol Cancer Ther 2003 Feb 1;2(2):123-9.
(16) Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Kung AL, DaviesFE, et al. Antimyeloma activity of heat shock protein-90 inhibition.Blood 2006 Feb 1;107(3):1092-100.
(17) Kawakami H, Tomita M, Okudaira T, Ishikawa C, Matsuda T, TanakaY, et al. Inhibition of heat shock protein-90 modulates multiplefunctions required for survival of human T-cell leukemia virus type I-infected T-cell lines and adult T-cell leukemia cells. Int J Cancer 2007
Apr 15;120(8):1811-20.
(18) Ramalingam SS, Egorin MJ, Ramanathan RK, Remick SC, SikorskiRP, Lagattuta TF, et al. A Phase I Study of 17-Allylamino-17-Demethoxygeldanamycin Combined with Paclitaxel in Patients with
Advanced Solid Malignancies. Clin Cancer Res 2008 Jun1;14(11):3456-61.
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(19) Ramalingam SS, Egorin MJ, Ramanathan RK, Remick SC, SikorskiRP, Lagattuta TF, et al. A Phase I Study of 17-Allylamino-17-Demethoxygeldanamycin Combined with Paclitaxel in Patients with
Advanced Solid Malignancies. Clin Cancer Res 2008 Jun1;14(11):3456-61.
(20) Salahuddin SZ, Markham PD, Wong-Staal F, Franchini G,Kalyanaraman VS, Gallo RC. Restricted expression of human T-cellleukemia--lymphoma virus (HTLV) in transformed human umbilicalcord blood lymphocytes. Virology 1983 Aug;129(1):51-64.
(21) Yamamoto N, Okada M, Koyanagi Y, Kannagi M, Hinuma Y.Transformation of human Leukocytes by cocultivation with an adult T-cell leukemia virus producer cell line. Science 1982;217(20):737-9.
(22) Phillips KE, Herring B, Wilson LA, Rickford MS, Zhang M, GoldmanCK, et al. IL-2Ralpha-Directed monoclonal antibodies provide effective
therapy in a murine model of adult T-cell leukemia by a mechanismother than blockade of IL-2/IL-2Ralpha interaction. Cancer Res 2000Dec 15;60(24):6977-84.
(23) Holm S. A Simple Sequentially Rejective Multiple Test Procedure.Scandinavian Journal of Statistics 1979;6(2):65-70.
(24) Dunnett CW. A Multiple Comparison Procedure for Comparing SeveralTreatments with a Control. Journal of the American Statistical
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(25) Nasr R, El-Sabban ME, Karam JA, Dbaibo G, Kfoury Y, Arnulf B, et al.Efficacy and mechanism of action of the proteasome inhibitor PS-341in T-cell lymphomas and HTLV-I associated adult T-cellleukemia//lymphoma. Oncogene 2004 Nov 15;24(3):419-30.
(26) Kondo A, Imada K, Hattori T, Yamabe H, Tanaka T, Miyasaka M, et al. A model of in vivo cell proliferation of adult T-cell leukemia. Blood1993;82:2501-9.
(27) Siegel R, Gartenhaus R, Kuzel T. HTLV-I associatedleukemia/lymphoma: epidemiology, biology, and treatment. CancerTreat Res 2001;104:75-88.
(28) Mesnard JM, Barbeau B, Devaux C. HBZ, a new important player inthe mystery of adult T-cell leukemia. Blood 2006 Dec 15;108(13):3979-82.
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(29) Arnold J, Zimmerman B, Li M, Lairmore MD, Green PL. Human T-cellLeukemia Virus Type-1 Antisense-encoded Gene, Hbz, Promotes TLymphocyte Proliferation. Blood 2008 Aug 8.
(30) Ikeda K, Oka M, Yamada Y, Soda H, Fukuda M, Kinoshita A, et al.
Adult T-cell leukemia cells over-express the multidrug-resistance-protein (MRP) and lung-resistance-protein (LRP) genes. Int J Cancer1999 Aug 12;82(4):599-604.
(31) Yasunami T, Wang Yh, Tsuji K, Takanashi M, Yamada Y, Motoji T.Multidrug resistance protein expression of adult T-cellleukemia/lymphoma. Leuk Res 2007 Apr;31(4):465-70.
(32) Zhang Z, Zhang M, Goldman CK, Ravetch JV, Waldmann TA. Effectivetherapy for a murine model of adult T-cell leukemia with the humanizedanti-CD52 monoclonal antibody, Campath-1H. Cancer Res 2003 Oct1;63(19):6453-7.
(33) Zhang M, Zhang Z, Goldman CK, Janik J, Waldmann TA. Combinationtherapy for adult T-cell leukemia-xenografted mice: flavopiridol andanti-CD25 monoclonal antibody. Blood 2005 Feb 1;105(3):1231-6.
(34) Mori N, Matsuda T, Tadano M, Kinjo T, Yamada Y, Tsukasaki K, et al. Apoptosis induced by the histone deacetylase inhibitor FR901228 inhuman T-cell leukemia virus type 1-infected T-cell lines and primaryadult T-cell leukemia cells. J Virol 2004 May;78(9):4582-90.
(35) Mori N, Yamada Y, Ikeda S, Yamasaki Y, Tsukasaki K, Tanaka Y, et
al. Bay 11-7082 inhibits transcription factor NF-kappaB and inducesapoptosis of HTLV-I-infected T-cell lines and primary adult T-cellleukemia cells. Blood 2002 Sep 1;100(5):1828-34.
(36) Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Fanourakis G, GuX, et al. Molecular sequelae of proteasome inhibition in human multiplemyeloma cells. Proceedings of the National Academy of Sciences ofthe United States of America 2002 Oct 29;99(22):14374-9.
(37) Cavo M. Proteasome inhibitor bortezomib for the treatment of multiplemyeloma. Leukemia 2006 Jun 29;20(8):1341-52.
(38) Fisher RI, Bernstein SH, Kahl BS, Djulbegovic B, Robertson MJ, deVos S, et al. Multicenter Phase II Study of Bortezomib in Patients WithRelapsed or Refractory Mantle Cell Lymphoma. J Clin Oncol 2006 Oct20;24(30):4867-74.
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(39) Mimnaugh EG, Xu W, Vos M, Yuan X, Isaacs JS, Bisht KS, et al.Simultaneous inhibition of hsp 90 and the proteasome promotesprotein ubiquitination, causes endoplasmic reticulum-derived cytosolicvacuolization, and enhances antitumor activity. Mol Cancer Ther 2004May;3(5):551-66.
(40) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
(41) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
(42) Nasr R, El-Sabban ME, Karam JA, Dbaibo G, Kfoury Y, Arnulf B, et al.
Efficacy and mechanism of action of the proteasome inhibitor PS-341in T-cell lymphomas and HTLV-I associated adult T-cellleukemia/lymphoma. Oncogene 2005 Jan 13;24(3):419-30.
(43) Satou Y, Nosaka K, Koya Y, Yasunaga J, Toyokuni S, Matsuoka M.Proteasome inhibitor, bortezomib, potently inhibits the growth of adultT-cell leukemia cells both in vivo and in vitro. Leukemia 2004 Jun10;18(8):1357-63.
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ND= not determined- terminal study
Table 2.1 Summary of treatment groups and organ weights
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Table 2.2 Histologic ranking of tumor engraftment
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Figure 2.1 The addition of 17-AAG significantly inhibits growth in both theMT-2 and Jurkat cell lines. MT-2, C8166, and Jurkat T-cells were grown inmedia only, 1, 2.5, or 5nM PS-341 with or without the addition of 1000nM 17-
AAG. At 24, 48, and 72 hours, growth was measured by the addition of MTS
reagent and absorbance at 490 nM
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Figure 2.2 Western blot of MT-2 cell whole cell lysate after 48 hours. Cellswere treated with media only, 5nM PS-341 or 1000 nM 17-AAG or thecombination of PS-341 and 17-AAG. Cells show decreased PARP, Akt and pAktwith increased ubiquitination.
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Figure 2.3 Histologic representation of mouse tissues from phase I. Panels A and B are sections of lung stained with routine H&E. Histologic findingsinclude fewer neoplastic round cells in the PS-341 and 17-AAG group whencompared to the no treatment group. Panels C and D are Ki67 stained sectionsof lung from Phase I. Numbers of positive nuclei (brown staining) were countedin ten randomly selected high powered fields (400X magnification) for eachtissue.
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A
BFigure 2.4 Serum IL-2Rα levels and survival curves of mice. A. Serum IL-2Rα levels of mice at the end of Phase I study. There is a statistically significantdecrease in serum IL-2Rα levels in both the PS-341 and combination therapygroups while there is a trend toward a significant decrease in the 17-AAG
treatment group. B. Kaplan Meier curve for Phase II in vivo experiments. Whiletreatment with either PS-341 or combination therapy proved better than vehicleonly treatment, treating with combination therapy did not improve survival overPS-341 alone.
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CHAPTER 3
EFFICACY OF HISTONE DEACETYLASE INHIBITORS IN A MOUSE MODEL
OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 ADULT T-CELL LYMPHOMA
3.1 ABSTRACT
Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell
leukemia/lymphoma (ATL) a refractory CD4+/CD25+ T-cell malignancy. ATL
patients have a poor prognosis due to the highly aggressive nature of the cancer
and the resistance to conventional chemotherapies. The low penetrance and
prolonged latency period of ATL suggests that genetic and epigenetic alterations
precede the development of ATL. Histone deacetylase inhibitors (HDACi), which
promote the relaxation of chromatin and the promotion of transcription have
shown efficacy against a variety of cancers and are in phase I and phase II
clinical trials. Herein, we tested if the histone deacetylase inhibitors valproic
acid and the novel OSU-HDAC42 reduced the proliferation of ATL cell lines by
promoting apoptosis and histone hyperacetylation. Both compounds reduced
cell growth and elicited a dose dependent increase in cytochrome C and cleaved
Poly (ADP-ribose) polymerase (PARP) indicating the induction of cell death by
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apoptosis. To further test of the efficacy of this approach we evaluated a novel
HDACi, OSU-HDAC42, for survival in an ATL NOD/SCID mouse model. A
dietary formulation of OSU-HDAC42 prolonged survival of ATL engrafted mice
compared to controls. Our data provide new directions for the treatment of ATL
and support the further development of this class of drug against HTLV-1-
associated lymphoid malignancies.
3.2 INTRODUCTION
Human T-lymphotropic virus type 1 (HTLV-1) infection is associated with
an array of devastating malignancies and inflammatory disorders. Patients with
the acute form of adult T-cell leukemia/lymphoma (ATL) present with a refractory
CD4+/CD25+ T-cell malignancy. These patients have a poor prognosis due to
the highly aggressive nature of the cancer and the resistance of neoplastic cells
to conventional chemotherapies. HTLV-1 is estimated to have infected 15 to 20
million people worldwide (1). The virus is endemic in southern Japan (2), the
Caribbean basin (3), central Africa (4), Central and South America (5;6), the
Melanesian Islands in the Pacific basin, and in the aboriginal population in
Australia (7). Typically, 1-5 % of HTLV-1-infected individuals develop ATL after a
latent period of 20-30 years (8;9). ATL patients typically present with malaise,
fever, lymphadenopathy, hepatosplenomegaly, jaundice, weight loss, and various
opportunistic infections, such as Pneumocystis carinii (10). Based on the clinical
course of the disease, four classifications have been used to categorize ATL
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patients as acute, smoldering, chronic and lymphoma stages (11-13). Acute ATL
is highly refractory to standard chemotherapeutic approaches (14) and patients
have a higher incidence of hypercalcemia of malignancy, in association with lytic
bone lesions, elevated lactate dehydrogenase (LDH), and the presence of
soluble form of interleukin-2 (IL-2) receptor in their serum (15).
Due to the low penetrance and prolonged latency period (up to 70 years),
the hypothesis that genetic and epigenetic alterations precede the development
of ATL has been postulated. Transcriptional regulation at the chromosomal level
is controlled in part by the acetylation of histones and non-histone proteins
(16;17). Acetylation of histones results in the relaxation of chromatin and the
promotion of transcription. This is controlled, in part, by histone deacetylases
(HDACs) that promote the removal of the acetyl groups from lysines on histones.
This results in the restoration of positive charges, tightening of DNA (negatively
charged) around the histone core, and decreases in transcription of affected
genes. Currently, there are eighteen HDACs classified into six different groups
(18). These HDACs control the activation and repression of proteins and may
also functionally influence non-histone proteins such as hormone receptors,
chaperones (heat shock proteins), viral proteins, and cytoskeletal proteins (18).
The role of inhibitors of HDACs (HDACi) in cancer therapy has recently
been reviewed (18-23). HDACi promote the acetylated state of the histone and
relaxed chromatin structure. They are divided into several classes including
short chain fatty acids, hydroxamic acids, benzamides, and cyclic peptides.
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These classes differ in their potency, but are generally not specific for HDAC
isoenzymes (19).
Recently, HDACi have been used as targeted therapies in cancer
research. As reviewed in Xu et al. (18), HDACi have been shown to induce
apoptosis, induce cell cycle arrest, disrupt Hsp90 and the aggresome, inhibit
angiogenesis, and induces mitotic and autophagic cell death and promote
senescence. It is through these pleiotropic effects that HDACi have shown
efficacy in vitro against prostate cancer (24;25). In addition, the HDACi
suberoylanilide hydroxamic acid (SAHA or Vorinostat) is approved for the
treatment of cutaneous T-cell lymphoma. Depsipeptide (FR901228) has also
been used in the treatment of peripheral and cutaneous T-cell lymphoma (26).
Several other first generation HDACi are currently in phase I and phase II clinical
trials (27).
Herein, we evaluated the histone deacetylase inhibitors valproic acid
(VPA) and the novel OSU-HDAC42 for their ability to reduce the proliferation of
ATL cell lines through apoptosis and histone hyperacetylation. Our data
indicated that both compounds reduced cell growth and promoted a dose
dependent increase in cytochrome C and cleaved Poly (ADP-ribose) polymerase
(PARP). Finally, we tested the efficacy of a novel HDACi, OSU-HDAC42 for its
ability to enhance the survival of NOD/SCID mouse model engrafted with ATL
cells. We are the first to demonstrate that a dietary formulation of OSU-HDAC42
prolongs the survival of mice engrafted with ATL cells. Our findings support
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further development of this class of drug against HTLV-1-associated lymphoid
malignancies.
3.3 MATERIALS AND METHODS
Cell lines
The HTLV-1 infected cell lines C8166-45(28), MT-2(29), and HTLV-1
negative Jurkat cells (clone E6-1; American Type Culture Collection catalog
number TIB-152) were maintained in RPMI 1640 supplemented with 10% fetal
bovine serum, 10% penicillin/streptomycin (100 μg/mL), and 10% glutamine (0.03
mg/mL) at 37°C in 5% carbon dioxide. MET-1 cells are an HTLV-1 positive cell
line derived from a patient with ATL (30). These cells, like patient ATL cells, are
unable to proliferate in vitro, but can be expanded by passage through
NOD/SCID mice. MET-1 cells were expanded by inoculating 2 x 107 cells
intraperitoneally (IP) into each mouse and harvesting at optimal tumor growth,
typically at 5-6 weeks post inoculation. Spleen, lymph nodes and inoculation site
masses were harvested from the mice at necropsy. Tissues were minced and
passed through a 100 μm cell strainer to create a single cell suspension.
Isolated cells were then frozen in 90% fetal bovine serum and 10% DMSO or
passaged as above to produce tumors for experimental procedures.
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Animals
Female immunodeficient NOD/SCID (NOD.CB17-Prkdc /J), (scid Jackson
Laboratory, Bar Harbor, ME), were maintained under specific pathogen-free
conditions in animal facilities in the College of Veterinary Medicine at The Ohio
State University. Mice were kept in individually ventilated cages at 20 ± 2oC in
accordance with the Guide for Care and Use of Laboratory Animals. This
includes 12 hour light/dark cycle, free choice standard rodent chow, and water ad
lib. All procedures were approved by The Ohio State University Institutional
Laboratory Animal Care and Use Committee.
Mice were injected IP with 2 x 107 MET-1 cells in 1 ml calcium/magnesium
free phosphate buffered saline (PBS). Mice were monitored visually daily and
weighed regularly. At 20 days post inoculation and every two weeks throughout
the study, serum was drawn and tested for serum IL-2Rα levels (R&D Systems).
On Day 21, mice began a treatment regimen. In the first phase of the
experimental protocol mice were divided into two groups. The first group
received vehicle only (methylcellulose/Tween 80). The second group received
50 mg/kg (S)-HDAC42 orally in methylcellulose/Tween 80 in a total volume of 10
µl/gram body weight by gavage.
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In phase II, the mice were again divided into two groups. The mice were
injected IP with 2 x 107 MET-1 cells. After the engraftment period of 21 days, the
mice were fed a diet formulated by Research Diets, Inc (New Brunswick, NJ).
The AIN-76A rodent diet with and without 208 ppm OSU-HDAC42 was fed to the
treatment groups. Dietary intake was monitored by weight of diet consumed
(24).
To ascertain differences in survival between treatment groups, mice were
continually monitored until such time as euthanasia criteria were met. Mice were
euthanized by CO2 inhalation upon developing signs of morbidity (rapid weight
loss, rough hair coat, lethargy or persistent recumbence, labored breathing,
difficulty with ambulation, or obtaining food and water).
Compounds
OSU-HDAC42 was provided by The College of Pharmacy at The Ohio
State University. It was diluted in DMSO to obtain a stock solution of 10 mg/ml
then diluted in 10% FBS containing culture media for in vitro studies. For in vivo
studies, the OSU-HDAC42 was prepared as a suspension in 0.5%
methylcellulose and 0.1% Tween 80 in sterile water. The OSU-HDAC42
containing diet and control diet were prepared by Research Diets, Inc (New
Brunswick, NJ). Valproic acid was obtained from Sigma (Saint Louis, MO) and
prepared in PBS stock solution prior to dilution in 10% FBS containing media for
in vitro studies.
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Growth Inhibition Assays
Forty thousand cells were plated in each well of a 96 well plate and
incubated with media only, 1, 2.5, 5 mM VPA or 0.5, 1, or 2.5 μM OSU-HDAC 42.
Concentrations of OSU-HDAC42 and VPA were chosen based on the previous
literature (24;25;31). After 24, 48, and 72 hours, 20 µl of MTS reagent (Cell Titer
96® AQueous, Promega) was added to each well prior to incubation for two hours
at 37°C. Absorbance was measured at 490 nm indicating the metabolically
active cells.
Western blot assays
Cells (2 x 106) were incubated with media only, 1, 2.5, 5 mM valproic acid
or 0.5, 1, or 2.5 µM OSU-HDAC 42 for 24 hours. Cells were washed once in
PBS and lysed. Aliquots of protein (20 µg) were run on SDS polyacrylamide
gels. Protein was transferred to a nitrocellulose membrane and blocked
overnight at 4° C in 1% BSA in 5% nonfat dry milk in 0.1% Tween-20 in Tris
buffered saline (TBST). Membranes were incubated with primary antibodies in
5% milk in TBST. The following antibodies were used: Poly (ADP-ribose)
polymerase (PARP) (Oncogene Catalog #AM30-100UG) diluted at 1:100,
cleaved PARP (AbCam #ab4830) diluted 1:1000, acetylated histone H3
(Millipore #06-599) diluted 1:3000, cytochrome C (AbCam #ab28137) diluted
1:500, and beta-actin AC-74 (Sigma) diluted 1:20,000. Membranes were then
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incubated with 1:1000 dilutions of corresponding secondary antibodies. Bound
antibodies were detected with a chemiluminescent detection system (Cell
Signaling Technologies #7003: 20X LumiGLO and 20X Peroxide).
Histology
Standard necropsies were performed on all mice and organs fixed in 10%
neutral buffered formalin for 24 hours. Representative sections of liver, lung,
kidney, heart, spleen, and brain were embedded in paraffin, sectioned, and
stained with hematoxylin and eosin (Fig 3.4).
Serum IL-2Rα
Whole blood was collected from the anterior facial vein prior to the start of
treatment and periodically throughout the study (minimum time between samples
of 14 days). Immediately post collection, samples were centrifuged to separate
the serum. Aliquots of serum were frozen at -80°C. To assay for the presence of
the human serum interleukin 2-alpha chain (sIL-2Rα), a specific proliferation/
tumor marker of human lymphocytes, the R&D systems IL-2sRα Duo Set ELISA
kit was used. Samples were diluted 1:500 in reagent diluents and assayed
according to manufacturer’s protocol.
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Statistical Analysis
ANOVA models were used to test if there were significant differences
between concentrations of compounds and if differences existed between
different time points. The numbers of multiple comparisons were adjusted by
Tukey’s method.
3.4 RESULTS
OSU-HDAC42 and valproic acid significantly inhibit growth of ATL cell lines
The histone deacetylase inhibitors OSU-HDAC42 and VPA were tested for
their ability to inhibit growth of HTLV-1 positive cell lines prior to in vivo testing in
our animal model of ATL. Direct testing of ATL cells in cell culture is difficult as
these cells typically do not grow in culture and are unavailable. To provide
surrogate models to test HDACi, we tested the HTLV-1 positive cell lines MT-2
and C8166, which were originally derived from ATL patients. The Jurkat cell line
was used as the HTLV-1 negative T-cell control.
Valproic acid significantly inhibited growth in C8166 cells at 2.5 mM
concentration (p<0.0001). In this cell line, we also demonstrated that increased
duration of exposure significantly inhibited growth (Fig. 3.1A). When compared
to 24 hours of exposure, 48 and 72 hour exposures significantly inhibited growth
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at all concentrations (p<0.0001). Valproic acid inhibited growth in MT-2 cells at a
concentration of 1mM (p<0.0001). Increasing the duration of exposure did not
further inhibit growth until 72 hours (p=0.0005). Growth of Jurkat cells was
inhibited with 2.5 mM VPA (p<0.0001). Further significant decreases in Jurkat T-
cell growth were observed at 72 hours of exposure (p<0.0001).
OSU-HDAC42 inhibited growth in all three cell lines tested. In the C8166
cell line, there was a statistically significant decrease in growth with 0.5 μM
concentration and 48 hours duration (p=0.0011). In MT-2 cells, a statistically
significant decrease in growth was observed with 0.5 μM OSU-HDAC42
(p<0.0001). Jurkat T-cells were significantly inhibited in growth at the 2.5 µM
OSU-HDAC42 concentration (p=0.0001)(Fig 3.1B).
OSU-HDAC42 and valproic acid induced apoptosis and hyperacetylation of
histones
Whole cell lysates of C8166 and MT-2 cells treated with VPA and OSU-
HDAC42 for 24 hours were tested for hyperacetylation of histones and HDACi
affects on non-histone target proteins by western immunoblot assay (Fig. 3.2).
Our data indicated dose dependent increases in acetylated histone H3 in both
MT-2 and C8166. In addition, dose dependent increases in both cytochrome C
and cleaved PARP were observed, each indicative of apoptosis induced by each
compound in both cell lines (Fig. 3.2A and B). Collectively our western
immunoblot data indicated that both VPA and OSU-HDAC42 targeted the
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appropriate pro-apoptotic pathways to limit cell proliferation and justified in vivo
studies with our NOD/SCID ATL mouse model.
OSU-HDAC42 prolongs survival in the ATL NOD/SCID mouse model with
continued drug administration
We utilized a previously established NOD/SCID ATL mouse model (30) to
test the efficacy of OSU-HDAC42 in vivo (Fig 3.3). Following engraftment with
MET-1 cells, we first divided 10 mice into two groups of 5 each and treated them
by oral gavage with 50 mg/kg OSU-HDAC42 or vehicle alone respectively for
three times per week for four weeks. Serum was obtained every 14 days to
measure the serum biomarker for tumor progression (serum IL-2Rα). After one
month, OSU-HDAC42 was discontinued and mice were monitored for
development of removal criteria. Mice treated with OSU-HDAC42 tended to have
lower serum IL-2Rα levels (Fig. 3.5A), but did not differ in time to removal (Fig.
3.5B).
During phase II of our study, we used a dietary formulation of OSU-
HDAC42 and compared it to a commercially prepared vehicle only diet (Table
3.1). Mice were inoculated intraperitoneally with 2 x 107 MET-1 cells and allowed
three weeks to engraft. At three weeks post inoculation, mice were given free
choice access to either the control diet or the OSU-HDAC42 diet. Food
consumption was measured by weight of food remaining in cage every other day
and divided by the number of animals in the cage. The intake of food did not
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differ between groups (Fig. 3.6). Mice treated with OSU-HDAC42 overall
exhibited a trend to survive longer than the vehicle only treated group (Fig. 3.7).
3.5 DISCUSSION
Our data indicate that HDACi are an effective treatment for ATL. In vitro,
both VPA and OSU-HDAC42 abate growth of ATL cell lines. Exposure of ATL
cell lines to either HDACi resulted in hyperacetylation of histones. In addition, we
demonstrated that both VPA and OSU-HDAC42 induced apoptosis. This was
indicated by accumulation of cytochrome C and increased cleaved PARP. We
are the first to demonstrate that OSU-HDAC42 was safe and efficacious in the
ATL NOD/SCID mouse model. In addition, our feed formulation design
demonstrated that oral dosing of OSU-HDAC42 appeared to be more effective
compared to oral gavage in improving survival of the engrafted mice.
Acute ATL is a CD4+ CD25+ T-cell malignancy with a universally poor
prognosis. HTLV-1 is linked to the development of ATL; however, active virus
replication is not required to maintain tumor growth. In part, the outgrowth of ATL
is dependent on genetic or epigenetic changes of the host cell for selection of
clonally expanded malignant lymphocytes. Thus, novel treatments are needed
against ATL to more selectively target key cancer cell pathways to induce cell
death. Conventional chemotherapies against ATL have produced inconsistent or
limited results against ATL (32). New therapeutic approaches have focused on
targeted therapies. In vitro and in vivo studies with targeted therapies have
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included monoclonal antibodies (alemtuzumab (33), Anti-Tac (34)), NF-κB
inhibitors (35;36), and HDACi (37).
Histone deacetylase inhibitors promote the acetylated state of histones
and relax chromatin structure leading to induction of apoptosis, cell cycle arrest,
disruption of Hsp90 and the aggresome, inhibition of angiogenesis, mitotic cell
death, and senescence (reviewed in (18)). In ATL cell lines, the histone
deacetylase inhibitor FR901228 induced apoptosis and reduced tumor size in
mice (37). In our study, we compared two HDACi (VPA and OSU-HDAC42) for
their ability to abate ATL cell growth and reduce tumor burdens in a mouse
model. Valproic acid (VPA) is an eight carbon branched chain fatty acid that has
been used in the treatment of epilepsy since 1978 (21). Recently, it has been
shown to have efficacy in the treatment of cancer through the hyperacetylation of
histones 3 and 4 (38). VPA inhibits prostate cancer cell growth (39)
medulloblastoma (40) , and multiple myeloma (41) in vitro. OSU-HDAC42 is a
phenylbutryrate derived HDACi included in the Rapid Access to Intervention
Development (RAID) program through the National Cancer Institute (25). In a
prostate cancer model, OSU-HDAC42 was shown to decrease cell viability,
decreased phospho-Akt, Bcl-xL, and Survivin (24). In vivo, a xenograft mouse
model had decreased tumor burden (24) and decreased tumor levels of
phospho-Akt and Bcl-xL in response to the drug (25).
In our study, we demonstrated that OSU-HDAC42 while not improving
survival in short term tumor engraftment studies did prolong survival when
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provided as a continuous feed supplement. Thus, OSU-HDAC42 was required
on a more continuous basis to demonstrate antitumor activity. Mice were only
treated 12 times in phase I of the study. In phase I, the mice were allowed to
progress until they met euthanasia criteria. Our phase II design allowed us to
test the effects of the continued pressure of OSU-HDAC42. In phase II, the
animals continued with in feed treatment until they met euthanasia criteria. The
addition of continued drug pressure for a prolonged time appeared to prolong
survival and prevent tumor cells from regaining growth capacity.
The mouse model, while providing an excellent preclinical model in which
to assay the effects of the compounds on the ATL cell growth, does not assay
the effects of prolonged therapy in context to ATL patients or in the presence of a
functional immune system. Our results have implications for the design of
human trials using OSU-HDAC42 as adjunct therapy to standardized protocols
and support further investigations to establish more effective therapeutic dosing
regimens and drug cycles of OSU-HDAC42 in both animal and human clinical
trials.
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3.6 REFERENCES
(1) Mahieux R, Gessain A. HTLV-1 and associated adult T-cellleukemia/lymphoma. Rev Clin Exp Hematol 2003 Dec;7(4):336-61.
(2) Hinuma Y, Komoda H, Chosa T, Kondo T, Kohakura M, Takenaka T, et al. Antibodies to adult T-cell leukemia virus-associated antigen (ATLA) insera from patients with ATL and controls in Japan: a nation-wideseroepidemiological study. Int J Cancer 1982;29:631.
(3) Blattner W, Kalyanaraman V, Robert-Guroff M, Lister T, Galton D, SarinPS, et al. The human type-C retrovirus, HTLV, in blacks from theCaribbean region and relationchip to adult T-cell leukemia/lymphoma. Int JCancer 1982;30:257.
(4) Saxinger WC, Blattner W, Levine P, Clark J, Biggar RJ, Hoh M, et al.Human T-cell leukemia virus (HTLV-I) antibodies in Africa. Science1984;225:1473.
(5) Reeves W, Saxinger C, Brenes M, Quinoz E, Hoh M, Clark J, et al.Human T-cell lymphotropic virus type I (HTLV-I) and risk factors inmetropolitan Panama. Am J Epidemiol 1988;127:539.
(6) Merino F, Robert-Guroff M, Clark J, Biondo-Bracho M, Blattner W, GalloRC. Natural antibodies to human T-cell leukemia/lymphoma virus inhealthy Venezuelan populations. Int J Cancer 1984;34:501.
(7) Asher DM, Goudsmit J, Pomeroy K, Garruto RM, Bakker M, Ono S, et al. Antibodies to HTLV-I in populations of the southwestern Pacific. J MedVirol 1988;26:339.
(8) Cleghorn FR, Manns A, Falk R, Hartge P, Hanchard B, Jack N, et al.
Effect of human T-lymphotropic virus type I infection on non- Hodgkin'slymphoma incidence. J Nat Cancer Inst 1995;87:1009-14.
(9) Yamaguchi K, Takatsuki K. Adult T-cell leukaemia-lymphoma. ClinHaematol 1993;6(4):899-915.
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(10) White JD, Zaknoen SL, Kastensportes C, Top LE, Navarroroman L,Nelson DL, et al. Infectious complications and immunodeficiency inpatients with human T-cell lymphotropic virus I-associated adult T- cellleukemia/lymphoma. Cancer 1995;75:1598-607.
(11) Shimoyama M. Diagnostic criteria and classification of clinical subtypes ofadult T- cell leukaemia-lymphoma. A report from the Lymphoma StudyGroup (1984- 87)5222. Br J Haematol 1991 Nov;79(3):428-37.
(12) Yamaguchi K, Nishimura H, Kohrogi H, Jono M, Miyamoto Y, Takatsuki K. A proposal for smoldering adult T-cell leukemia: a clinicopathologic studyof five cases. Blood 1983 Oct;62(4):758-66.
(13) Kawano F, Yamaguchi K, Nishimura H, Tsuda H, Takatsuki K. Variation inthe clinical courses of adult T-cell leukemia. Cancer 1985 Feb15;55(4):851-6.
(14) Ratner L, Harrington W, Feng X, Grant C, Jacobson S, Noy A, et al.Human T-cell leukemia virus reactivation with progression of adult T-cellleukemia-lymphoma. PLoS ONE 2009;4(2):e4420.
(15) Ishitsuka K, Tamura K. Treatment of adult T-cell leukemia/lymphoma:past, present, and future. Eur J Haematol 2008 Mar;80(3):185-96.
(16) Gershey EL, Vidali G, Allfrey VG. Chemical studies of histone acetylation.The occurrence of epsilon-N-acetyllysine in the f2a1 histone. J Biol Chem1968 Oct 10;243(19):5018-22.
(17) Brownell JE, Zhou J, Ranalli T, Kobayashi R, Edmondson DG, Roth SY, etal. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5plinking histone acetylation to gene activation. Cell 1996 Mar 22;84(6):843-51.
(18) Xu WS, Parmigiani RB, Marks PA. Histone deacetylase inhibitors:molecular mechanisms of action. Oncogene 2007 Aug 13;26(37):5541-52.
(19) Lin HY, Chen CS, Lin SP, Weng JR, Chen CS. Targeting histonedeacetylase in cancer therapy. Med Res Rev 2006 Jul;26(4):397-413.
(20) Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise ofepigenetic (and more) treatments for cancer. Nat Rev Cancer 2006Jan;6(1):38-51.
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(21) Eadie MJ, Hooper WD, Dickinson RG. Valproate-associated hepatotoxicityand its biochemical mechanisms. Med Toxicol Adverse Drug Exp 1988Mar;3(2):85-106.
(22) Smith KT, Workman JL. Histone deacetylase inhibitors: anticancer
compounds. Int J Biochem Cell Biol 2009 Jan;41(1):21-5.
(23) Epping MT, Bernards R. Molecular basis of the anti-cancer effects ofhistone deacetylase inhibitors. Int J Biochem Cell Biol 2009 Jan;41(1):16-20.
(24) Sargeant AM, Rengel RC, Kulp SK, Klein RD, Clinton SK, Wang YC, et al.OSU-HDAC42, a histone deacetylase inhibitor, blocks prostate tumorprogression in the transgenic adenocarcinoma of the mouse prostatemodel. Cancer Res 2008 May 15;68(10):3999-4009.
(25) Kulp SK, Chen CS, Wang DS, Chen CY, Chen CS. Antitumor effects of a
novel phenylbutyrate-based histone deacetylase inhibitor, (S)-HDAC-42,in prostate cancer. Clin Cancer Res 2006 Sep 1;12(17):5199-206.
(26) Piekarz RL, Robey R, Sandor V, Bakke S, Wilson WH, Dahmoush L, et al.Inhibitor of histone deacetylation, depsipeptide (FR901228), in thetreatment of peripheral and cutaneous T-cell lymphoma: a case report.Blood 2001 Nov 1;98(9):2865-8.
(27) Marsoni S, Damia G, Camboni G. A work in progress: the clinicaldevelopment of histone deacetylase inhibitors. Epigenetics 2008May;3(3):164-71.
(28) Salahuddin SZ, Markham PD, Wong-Staal F, Franchini G, KalyanaramanVS, Gallo RC. Restricted expression of human T-cell leukemia--lymphomavirus (HTLV) in transformed human umbilical cord blood lymphocytes.Virology 1983 Aug;129(1):51-64.
(29) Yamamoto N, Okada M, Koyanagi Y, Kannagi M, Hinuma Y.Transformation of human Leukocytes by cocultivation with an adult T-cellleukemia virus producer cell line. Science 1982;217(20):737-9.
(30) Phillips KE, Herring B, Wilson LA, Rickford MS, Zhang M, Goldman CK, etal. IL-2Ralpha-Directed monoclonal antibodies provide effective therapy in
a murine model of adult T-cell leukemia by a mechanism other thanblockade of IL-2/IL-2Ralpha interaction. Cancer Res 2000 Dec15;60(24):6977-84.
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(41) Kaiser M, Zavrski I, Sterz J, Jakob C, Fleissner C, Kloetzel PM, et al. Theeffects of the histone deacetylase inhibitor valproic acid on cell cycle,growth suppression and apoptosis in multiple myeloma. Haematologica2006 Feb;91(2):248-51.
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Figure 3.1. Histone deacetylase inhibitors significantly inhibit growth inATL cell lines. A. MT-2, C8166, and Jurkat T-cells were grown in media only, 1,2.5, 5 mM valproic acid. At 24, 48, and 72 hours, growth was measured by theaddition of MTS reagent and absorbance at 490 nM. B. MT-2, C8166, andJurkat T-cells were grown in media only, 0.5, 1, 2.5 μM OSU-HDAC42. At 24,48, and 72 hours, growth was measured by the addition of MTS reagent andabsorbance at 490 nm.
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Figure 3.1
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Figure 3.2 Western blot of whole cell lysate after 24 hours. MT-2 cells weretreated with media only, 1, 2.5, 5 mM valproic acid or 0.5, 1, 2.5 μM OSU-HDAC42. Cells showed a dose dependent increase in cleaved PARP,cytochrome C, and acetylated histone H3. C8166 cells were treated with mediaonly, 1, 2.5, 5 mM valproic acid or 0.5, 1, 2.5 μM OSU-HDAC42. Cells showed adose dependent increase in cleaved PARP, cytochrome C, and acetylatedhistone H3.
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2 x 10 MET-1Cells IP
Gavage 50mg/kgOSU-HDAC42 orvehicle 3x week
0 1 2 5 6 7 83 4
0 1 2 5 6 7 83 4
2 x 10 MET-1Cells IP
Week
Week
In feed OSU-HDAC42or control diet
Phase I
Phase II
Figure 3.3 Linear schematic of dosing regimen. In phase I, mice weretreated by gavage with 50mg/kg OSU-HDAC42 of vehicle three times per weekfor two weeks, rested one week and treated in a similar manner for two weeks.In phase II, the mice were started on OSU-HDAC42 or vehicle in diet at day 21.In both instances, the mice were allowed to survive until removal criteria weremet.
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Figure 3.4 Engraftment of MET-1 cells in multiple organs. Examples ofNOD/SCID mice inoculated intraperitoneally with 2x107 MET-1 cells. Neoplasticround cells are present in the brain (meninges),stomach, heart, lung, kidney, andliver. All tissues magnified 200X, bar denotes 50 µm.
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Figure 3.5 Oral gavage of OSU-HDAC42 in the NOD/SCID ATL mousemodel. A. Serum IL-2Rα levels of mice during the Phase I study. Solid squaresindicate values for mice administered vehicle only. Open circles indicate valuesfor mice administered OSU-HDAC42. Serum levels of this biomarker do not
differ significantly between the groups at any time. B. Kaplan Meier curve forPhase I in vivo experiments. The solid line represents control mice. Dotted linerepresents the OSU-HDAC42 treated. Treatment of OSU-HDAC42 by oralgavage did not significantly improve survival in mice.
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Figure 3.6 Mice fed ad lib formulated diets did not differ significantly inintake. Mice were fed a diet containing OSU-HDAC42 or control diet ad lib.
Average intake per mouse per day did not differ between the groups
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Figure 3.7 In feed OSU-HDAC42 in the NOD/SCID ATL mouse model. A.Serum IL-2Rα levels of mice during the Phase II study. Solid squares indicatevalues for mice administered vehicle only. Open circles indicate values for miceadministered OSU-HDAC42. Serum levels of this biomarker do not differsignificantly between the groups at any time. B. Kaplan Meier curve for Phase IIin vivo experiments. The solid line represents control mice. Dotted linerepresents the OSU-HDAC42 treated. There is a trend toward prolongedsurvival in the treatment group.
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CHAPTER 4
EFFECTS OF THE HISTONE DEACETYLASE INHIBITOR VALPROIC ACID
ON HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 REPLICATION IN A RABBIT
MODEL OF INFECTION
4.1 ABSTRACT
Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult
T-cell leukemia/lymphoma (ATL) and a number of lymphocyte mediated
disorders including HTLV-1-associated myelopathy/tropical spastic paraparesis
(HAM/TSP). The pathogenesis of HTLV-1-mediated disease is unclear. The
virus establishes a lifelong infection with approximately 5% of infected subjects
developing disease. It is known that disease outcomes are controlled by multiple
viral and host determinants of gene expression. Following integration, the
expression of the HTLV-1 provirus is dependent upon the local chromatin
environment and influences upon the cell that mediated gene expression.
Histone deacetylase inhibitors (HDACi), which promote the relaxation of
chromatin and promote gene transcription have been shown to promote
transcription of viral genes in vitro and offer new therapeutic strategies against
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ATL. Herein, we tested the effects of the histone deacetylase inhibitor, valproic
acid (VPA), on HTLV-1 proviral load in the rabbit model of early infection. We
inoculated three groups of six rabbits intravenously with HTLV-1 infected cells.
The first group of rabbits was treated with 70 mg/kg valproic acid subcutaneously
three times per week during the first four weeks of infection. The second group
of rabbits was treated with valproic acid during weeks four through eight of
infection. The third group was treated as a control and given subcutaneous
saline injections three times per week during weeks zero through four. Our data
demonstrate that VPA can be safely administered in the rabbit model of HTLV-1
infection, but did not significantly alter viral parameters of infection. Our data
indicated that VPA treatment caused a decrease in proviral load when
administered during the first four weeks of infection and altered virus infection-
associated lymphocytosis in treated rabbits. This model will be useful to test
other combinations of HDACi for their effects on HTLV-1 gene expression in the
rabbit model.
4.2 INTRODUCTION
Human T-lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus that
causes adult T-cell leukemia/lymphoma (ATL) and a variety of lymphocyte-
mediated diseases (1). Disease outcomes associated with HTLV-1 infection
appear to be controlled by both viral and host determinants of gene expression.
After infection and integration into the host cell chromatin the HTLV-1 provirus is
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dependent upon the local chromatin environment and epigenetic influences on
local gene expression. Histone deacetylase inhibitors (HDACi), which promote
the relaxation of chromatin and promote gene transcription, provide new
therapeutic strategies against ATL, but their influence on virus replication in vivo
is largely unknown. It is clear that HTLV-1 infected patients with high proviral
load are more likely to develop HAM/TSP when compared to patients that
develop ATL or remain asymptomatic (2). To full understand how HDACi may be
used therapeutically in infected patients, a more complete understanding of this
class of drugs on HTLV-1 replication in vivo is needed.
Increased viral expression and corresponding proviral copy numbers in
circulating lymphocytes has been associated with lymphocyte-mediated disease
syndromes associated with HTLV-1 infection. HTLV-1 expression is controlled
by both methylation and histone acetylation (3-7). Both methylation and histone
acetylation cause transcriptional silencing of HTLV-1 at the 5’LTR (8). It has
been estimated that approximately 9 % of the HTLV-1 proviral load may be
influenced by acetylation of histones (9). In addition, histone deacetylases such
as HDAC1 decreased Tax mediated gene expression (10) and modulate HTLV-1
p30 transcriptional effects (4).
Valproic acid (VPA) is an eight carbon branched chain fatty acid that has
been used in the treatment of epilepsy since 1978 (11). Recently, it has been
shown to have efficacy in the treatment of cancer through the hyperacetylation of
histones 3 and 4 (12). VPA inhibits prostate cancer cell growth (13)
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medulloblastoma (14), and multiple myeloma (15) in vitro. During two months of
treatment with the valproic acid (VPA), HAM/TSP patients exhibited a transient
increase in proviral load followed by decreased proviral load by undetermined
mechanism (16).
Herein, we evaluated the effects of VPA on HTLV-1 proviral load in the
rabbit model of infection. We inoculated three groups of six rabbits intravenously
with HTLV-1 infected cells. The first group of rabbits was treated with 70 mg/kg
valproic acid subcutaneously three times per week during the first four weeks of
infection. The second group of rabbits was treated with valproic acid as in group
1 during weeks four through eight of infection. The third group was treated as a
control and given subcutaneous saline injections three times per week during
weeks zero through four . Our data indicated that VPA treatment caused a
decrease in proviral load when administered during the first four weeks of
infection and altered virus infection-associated lymphocytosis in treated rabbits.
Our study demonstrates that VPA can be safely administered in rabbits and
establishes parameters to test HDACi for their effects on HTLV-1 gene
expression in the rabbit model.
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4.3 MATERIALS AND METHODS
Rabbit and human cell lines
A CD4+ rabbit lymphocyte line (R49) was used to establish HTLV-1
infection in New Zealand white rabbits as described (17). The derivation and
infectious properties of the full-length, wild-type HTLV-1 proviral clone (ACH) and
generation of the R49 cell line has been previously reported (17). Jurkat T-cells
(clone E6-1; American Type Culture Collection catalog number TIB-152) were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 10%
penicillin/streptomycin (100μg/mL), and 10% glutamine (0.03 mg/mL) at 37°C in
5% carbon dioxide. Peripheral blood mononuclear cells (PBMC) were isolated
from up to 10 ml of whole blood from the cental auricular artery at each time point
indicated. Percoll (Sigma-Aldrich Corp., St. Louis, MO) at 1.083 g/ml was used
to isolate rabbit lymphocytes. Rabbit lymphocytes were cultured in RPMI 1640
(GIBCO BRL), 15% fetal bovine serum (FBS) (GIBCO BRL) , 1% penicillin-
streptomycin (GIBCO BRL), 1% sodium pyruvate (GIBCO BRL), 2% L glutamine
(GIBCO BRL), and 250 µl of 2 mercaptoethanol.
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Rabbit inoculation
Twenty, 12 week old, specific pathogen free, female New Zealand White
rabbits were purchased from Harlan Laboratories (Indianapolis, IN) and housed
and maintained individually. All procedures were approved by the Institutional
Laboratory Care and Use Committee. After an acclimation period of one week,
Rabbits were inoculated via the lateral auricular vein with either 1 x 10
7
R49 cells
or 1 x 107 Jurkat cells (Fig. 4.1). The rabbits were regularly evaluated for overt
signs of clinical disease. Seven to ten milliliters of whole blood was drawn from
the median auricular artery for analysis at week 0, 1, 2, 4, 6, and 8. A complete
blood count (CBC) and blood smear were performed on the whole blood prior to
isolation of peripheral blood mononuclear cells (PBMCs). At the conclusion of
the study (week 8), rabbits were humanely euthanized and necropsied.
Compound and VPA treatment
Valproic Acid was obtained from Sigma (St. Louis, MO) and diluted in
0.9% sodium chloride (normal saline). Rabbits were treated with 70 mg/kg
valproic acid subcutaneously three times per week. Rabbit serum levels of
valproic acid were measured using Beckman Coulter SYNCHRON LX® Systems
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particle enhanced turbidimetric immunoassay method in the clinical laboratories
at the Ohio State University.
Hematology
White blood cell differential cell counts were obtained by counting 100
cells from blood films in a blinded manner by two independent examiners.
Morphologic examination was used to classify blood leukocytes as normal
lymphocytes, atypical lymphocytes (large lymphocytes with at least two nuclear
indentations), heterophils, monocytes, basophils and eosinophils.
Serological response to HTLV-1 infection
Reactivity to specific viral antigenic determinants was detected using a
commercial HTLV-1 western immunoblot assay (GeneLabs Diagnostics,
Singapore) adapted for rabbit plasma by use of alkaline phosphatase-conjugated
goat anti-rabbit immunoglobulin G (1:1,000 dilution; BioMerieux, Inc.). Plasma
(diluted 1:2000) showing reactivity to HTLV-1 Gag (p24 or p19) and Env (p21 or
gp46) antigens was classified as positive for HTLV-1 seroreactivity.
Detection of p19 (MA) matrix antigen from cultured rabbit PBMC
To test for active HTLV-1 infection in rabbit PBMCs p19 matrix antigen
(MA) enzyme-linked immunosorbent assays (ZeptoMetrix, Buffalo, NY) were
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preformed on supernatants from one day PBMC cultures (1.0 x 10 6 per well) as
described (18).
Detection of proviral copy number by PCR
To quantify the proviral copy number genomic DNA was isolated from
rabbit PBMCs using the manufacturer’s protocol (Qiagen). DNA concentration
and quality was determined (NanoDrop Technologies). HTLV-1 polymerase
gene (pol) primers were used at a final concentration of 200 nM of both forward
and reverse primers. The forward pol primer 5’-CCC TAC AAT CCA ACC AGC
TCA G-3’ and the reverse primer 3’-TTT TGG GCT ACC GTC GAA GTG GTG-5’
were used to amplify genomic DNA. Standard curves were generated using
DNA from a molecular clone of HTLV-1, the ACH plasmid and assayed
concurrently with test samples. Forward rabbit GAPDH (rGAPDH) primers 5’-
TGC CCA GTG TCA AGT CTG TT-3’ and reverse primers 3’-TCT TCG TCG
TGT GTC GTG TC-5’ were used at a final concentration of 250 nM to amplify
genomic DNA. The standard curves for rGAPDH were generated by subcloning a
780 base pair fragment from the rGAPDH gene into the pCR®4-Topo vector
(Invitrogen) and run concurrently with test samples. For each run, a standard
curve was generated from triplicate samples of log 10 dilutions of plasmid DNA in
DNAse/RNAse free water. The HTLV-1 proviral load was expressed as infected
cells per 10,000 PBMCs and was calculated using the following formula: (HTLV-1
pol copy number) / (rabbit GAPDH copy number / 2) x 10000.
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lymphocytes. Atypical lymphocytes, defined as large lymphocytes with at least
two nuclear indentation of 1/3 nuclear width (Fig 4.2), were visualized at each
time point. A decrease in lymphocytes was observed at week 4 in Group 1. This
decrease was not observed in either Group 2 or Group 3 (Fig. 4.3A). In both
Group 2 and Group 3 there was an increase in the atypical lymphocyte count at
week 1, which was not present in the VPA treated Group 1 (Fig 4.3B).
At each time point, serum was analyzed by western blot for antibody
response to HTLV-1. Western blot strips were evaluated for reactivity to four
major viral proteins p19, p24, p21, and gp46 at defined intervals throughout the
infection. Responses were rated 0-3 based on band intensity (Fig 4.4). The
negative control rabbits remained negative throughout the study. At week one,
antibodies to p19 were present in the serum at detectable levels in all infected
rabbits. By week eight, the intensity of most bands had equilibrated (Fig 4.5).
Valproic acid treatment did not appear to change the antibody response during
the first eight weeks of infection.
HTLV-1 p19 production from ex vivo cultured PBMC
To monitor active virus replication in rabbits, ex vivo cultures of PBMC
samples were obtained and supernatants tested for HTLV-1 p19 MA. In all three
groups, there is a decrease in p19 production from week 1 to week 2. At week
four and week six, Group 1 p19 levels increased while Group 2 and Group 3
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remained static. Valproic acid treatment for one month resulted in increased p19
production (Fig. 4.6).
Proviral copy numbers
To measure proviral copy number in the PBMCs from each rabbit,
genomic DNA was isolated from each sample of rabbit PBMCs. Fifty nanograms
of DNA was probed for integrated provirus and normalized to rabbit GAPDH
concentration for each sample. Proviral copy numbers of saline treated animals
varied minimally during the eight week study. Group 1 rabbits proviral copy
numbers were increased at week 1, 2, and 4, but decreased at week 6. The
proviral load levels in this group rebounded at week 8 (one month removed from
treatment). The proviral load set point in group 2 was greater than group 1 or
group 3, but remained static after week 1.
4.5 DISCUSSION
Rabbits are the model used most often to study early HTLV-1 infection
events, test viral transmission routes, and evaluate host immune responses
against the virus infection. The rabbit model was used to confirm the modes of
transmission in humans, as well as early studies of the immune response against
the infection or to develop vaccines against HTLV-1 (19-24). Early studies
utilizing the rabbit model of HTLV-1 helped confirm routes of viral transmission
(21) and effective means to prevent the transmission of the virus (21;25-28).
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Herein, we utilized the rabbit model to test the influence of VPA, a known
modulator of histone acetylation, to test its influence on HTLV-1 expression in
vivo. HTLV-1 expression is controlled by both methylation and histone
acetylation (3-7). Both methylation and histone acetylation cause transcriptional
silencing of HTLV-1 at the 5’LTR (8). In vitro, Mosley et al. estimated acetylation
of histones controlled approximately 9 % of the HTLV-1 proviral load (9). In
addition, histone deacetylases such as HDAC1 decreased Tax mediated gene
expression (10). Our data using the rabbit model of HTLV-1 infection indicate
that VPA treatment at 70 mg/kg for four weeks caused a decrease in proviral
load when administered during the first four weeks of infection. We also noted
an increase in atypical lymphocytes in both group 2 and group 3 rabbits at one
week after virus exposure. This increase was not observed in the VPA treated
rabbits. In HTLV-1 infection, the presence of atypical lymphocytes is usually
indicative of transformed T cells and is one of the diagnostic criteria for ATL (29).
These cells have marked chromosomal abnormalities and aneuploidy linked to
the expression of Tax (30). Atypical lymphocytes may also be present in the
cerebrospinal fluid (CSF) of HAM/TSP patients (31). While the presence of
atypical lymphocytes in rabbit blood smears has not been documented with
HTLV-1 infection before, it can be inferred that the increase in atypical
lymphocytes with HTLV-1 infection is expected due to increased Tax expression
and viral replication. The static nature of the atypical lymphocytes in group1 may
correlate to the subsequent decrease in proviral load observed at week 6.
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Our findings parallel and support previous studies of bovine leukemia virus
infection in sheep (32). In BLV-infected sheep administered 80 mg/kg VPA three
times per week for one month, all sheep seroconverted at similar times and had
similar hematologic values. The authors did recognize a slight reduction in
proviral load, although the sample size was too small to draw a statistical
comparison (32).
In parallel to human clinical trials, our data indicated an early increase of
proviral copy number in PBMC in response to VPA followed by reductions.
Patients administered VPA demonstrated a transient increase in proviral load,
which lasted approximately 45 days prior to reductions (16). In addition, proviral
loads of patients in this study that were treated with VPA fluctuated more than
untreated controls. Consistent with this study, our group 1 rabbit’s proviral load
fluctuated more than either group 2 or group 3.
HTLV-1 expression is controlled by both methylation and histone
acetylation (3-7). Both methylation and histone acetylation cause transcriptional
silencing of HTLV-1 at the 5’LTR (8). Acetylation of histones has been estimated
to control approximately 9 % of the HTLV-1 proviral load in vitro (9). In addition,
histone deacetylases such as HDAC1 decreased Tax mediated gene expression
(10).
Controlling viral expression is critical to the outcome of HTLV-1 infection.
Increased viral expression and corresponding proviral copy numbers in
circulating lymphocytes has been associated with lymphocyte-mediated disease
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syndromes associated with HTLV-1 infection. The goal of VPA therapy in early
HTLV-1 infection and in HAM/TSP is to force the virus out of latency and expose
it to the immune response. Expression of viral proteins causes an increase in
immune surveillance and destruction of HTLV-1 infected cells by cytotoxic T-
cells. During two months of treatment with VPA, HAM/TSP patients exhibited a
transient increase in proviral load followed by decreased proviral load (16).
Histone deacetylase inhibitors are not specific and have widespread
cellular effects. Concurrent immune response to HTLV-1 infected cells
complicate interpretation of the effects of HDACi on proviral copy numbers in
patients and in our infected animals. In vitro, Mosley et al., (9) demonstrated that
HDACi decreased the efficiency of CTL killing of HTLV-1 infected cells, possibly
by inhibition of HDAC-6 in mediating cell-to-cell contact. Because of the effect on
the CTL, they concluded that HDACi might not be efficient in controlling proviral
load and a poor therapy.
Our experiments provide data to assist in the design of future studies to
test the effects of HDACi on HTLV-1 early infection. Future studies to optimize
the duration of treatment in the rabbit model, as well as the response to removal
of VPA are warranted. Our data indicate early and prolonged intervention with
HDACi may be needed to control proviral load.
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4.6 REFERENCES
(1) Lairmore M, Franchini G. Human T-cell Leukemia Virus Types 1 and 2. In:David M.KNIPE, editor. Fields Virology. Fifth ed. Wolters Kluwer/lippincottWilliams & Wilkins; 2007. p. 2071-105.
(2) Nagai M, Usuku K, Matsumoto W, Kodama D, Takenouchi N, Moritoyo T,et al. Analysis of HTLV-I proviral load in 202 HAM/TSP patients and 243asymptomatic HTLV-I carriers: high proviral load strongly predisposes toHAM/TSP. J Neurovirol 1998 Dec;4(6):586-93.
(3) Lu H, Pise-Masison CA, Fletcher TM, Schiltz RL, Nagaich AK, RadonovichM, et al. Acetylation of nucleosomal histones by p300 facilitatestranscription from tax-responsive human T-cell leukemia virus type 1chromatin template. Mol Cell Biol 2002 Jul;22(13):4450-62.
(4) Michael B, Nair AM, Datta A, Hiraragi H, Ratner L, Lairmore MD. Histoneacetyltransferase (HAT) activity of p300 modulates human T lymphotropicvirus type 1 p30(II)-mediated repression of LTR transcriptional activity.Virology 2006 Oct 25;354(2):225-39.
(5) Sharma N, Nyborg JK. The coactivators CBP/p300 and the histonechaperone NAP1 promote transcription-independent nucleosome evictionat the HTLV-1 promoter. Proc Natl Acad Sci U S A 2008 Jun
10;105(23):7959-63.
(6) Lodewick J, Lamsoul I, Polania A, Lebrun S, Burny A, Ratner L, et al. Acetylation of the human T-cell leukemia virus type 1 Tax oncoprotein byp300 promotes activation of the NF-kappaB pathway. Virology 2009 Mar30;386(1):68-78.
(7) Chen J, Zhang M, Ju W, Waldmann TA. Effective treatment of a murinemodel of adult T-cell leukemia using depsipeptide and its combination withunmodified daclizumab directed toward CD25. Blood 2009 Feb5;113(6):1287-93.
(8) Takeda S, Maeda M, Morikawa S, Taniguchi Y, Yasunaga J, Nosaka K, etal. Genetic and epigenetic inactivation of tax gene in adult T-cell leukemiacells. Int J Cancer 2004 Apr 20;109(4):559-67.
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(9) Mosley AJ, Meekings KN, McCarthy C, Shepherd D, Cerundolo V,Mazitschek R, et al. Histone deacetylase inhibitors increase virus geneexpression but decrease CD8+ cell anti-viral function in HTLV-I infection.Blood 2006 Aug 15.
(10) Ego T, Ariumi Y, Shimotohno K. The interaction of HTLV-1 Tax withHDAC1 negatively regulates the viral gene expression. Oncogene 2002Oct 17;21(47):7241-6.
(11) Eadie MJ, Hooper WD, Dickinson RG. Valproate-associated hepatotoxicityand its biochemical mechanisms. Med Toxicol Adverse Drug Exp 1988Mar;3(2):85-106.
(12) Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histonedeacetylase is a direct target of valproic acid, a potent anticonvulsant,mood stabilizer, and teratogen. J Biol Chem 2001 Sep 28;276(39):36734-41.
(13) Xia Q, Sung J, Chowdhury W, Chen CL, Hoti N, Shabbeer S, et al.Chronic administration of valproic acid inhibits prostate cancer cell growthin vitro and in vivo. Cancer Res 2006 Jul 15;66(14):7237-44.
(14) Shu Q, Antalffy B, Su JM, Adesina A, Ou CN, Pietsch T, et al. Valproic Acid prolongs survival time of severe combined immunodeficient micebearing intracerebellar orthotopic medulloblastoma xenografts. ClinCancer Res 2006 Aug 1;12(15):4687-94.
(15) Kaiser M, Zavrski I, Sterz J, Jakob C, Fleissner C, Kloetzel PM, et al. The
effects of the histone deacetylase inhibitor valproic acid on cell cycle,growth suppression and apoptosis in multiple myeloma. Haematologica2006 Feb;91(2):248-51.
(16) Lezin A, Gillet N, Olindo S, Signate A, Grandvaux N, Verlaeten O, et al.Histone deacetylase mediated transcriptional activation reduces proviralloads in HTLV-1 associated myelopathy/tropical spastic paraparesispatients. Blood 2007 Nov 15;110(10):3722-8.
(17) Collins ND, Newbound GC, Ratner L, Lairmore MD. In vitro CD4(+)lymphocyte transformation and infection in a rabbit model with a molecularclone of human T-cell lymphotropic virus type 1. J Virol 1996;70(10):7241-6.
(18) Hiraragi H, Kim SJ, Phipps AJ, Silic-Benussi M, Ciminale V, Ratner L, etal. Human T-Lymphotropic Virus Type 1 Mitochondrion-Localizing Protein
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p13II Is Required for Viral Infectivity In Vivo. J Virol 2006 Apr;80(7):3469-76.
(19) Hirose S, Kotani S, Uemura Y, Fujishita M, Taguchi H, Ohtsuki Y, et al.Milk-borne transmission of human T-cell leukemia virus type I in rabbits.
Virology 1988 Feb;162(2):487-9.
(20) Iwahara Y, Takehara N, Kataoka R, Sawada T, Ohtsuki Y, Nakachi H, etal. Transmission of HTLV-1 to Rabbits via Semen and Breast Milk fromSeropositive Healthy Persons. Int J Cancer 1990;45:980-3.
(21) Kataoka R, Takehara N, Iwahara Y, Sawada T, Ohtsuki Y, Dawei Y, et al.Transmission of HTLV-I by blood transfusion and its prevention by passiveimmunization in rabbits. Blood 1990 Oct 15;76(8):1657-61.
(22) Kotani S, Yoshimoto S, Yamato K, Fujishita M, Yamashita M, Ohtsuki Y,et al. Serial Transmission of Human T-Cell Leukemia Virus Type 1 by
Blood Transfusion in Rabbits and its Prevention by use of X-IrradiatedStored Blood. Int J Cancer 1986;37:843-7.
(23) Uemura Y, Kotani S, Yoshimoto S, Fujishita M, Yamashita M, Ohtsuki Y,et al. Mother-to-offspring transmission of human T cell leukemia virus typeI in rabbits. Blood 1987 Apr;69(4):1255-8.
(24) Uemura Y, Kotani S, Yoshimoto S, Fujishita M, Yano S, Ohtsuki Y, et al.Oral Transmission of Human T-Cell Leukemia Virus Type-1 in the Rabbit.Jpn J Cancer Res 1986;77:970-3.
(25) Takehara N, Iwahara Y, Uemura Y, Sawada T, Ohtsuki Y, Iwai H, et al.Effect of Immunization on HTLV-1 Infection in Rabbits. Int J Cancer1989;44:332-6.
(26) Sawada T, Iwahara Y, Ishii K, Taguchi H, Hoshino H, Miyoshi I.Immunoglobulin Prophylaxis against Milkborne Transmission of Human TCell Leukemia Virus type 1 in Rabbits. J Infect Dis 1991;164:1193-6.
(27) Miyoshi I, Takehara N, Sawada T, Iwahara Y, Kataoka R, Yang D, et al.Immunoglobulin prophylaxis against HTLV-I in a rabbit model. Leukemia1992;6 Suppl 1:24-6.:24-6.
(28) Tanaka Y, Tanaka R, Terada E, Koyanagi Y, Miyanokurosaki N,Yamamoto N, et al. Induction of antibody responses that neutralize humanT- cell leukemia virus type I infection in vitro and in vivo by peptideimmunization. J Virol 1994;68:6323-31.
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(29) Shimoyama M. Diagnostic criteria and classification of clinical subtypes ofadult T- cell leukaemia-lymphoma. A report from the Lymphoma StudyGroup (1984- 87) Br J Haematol 1991 Nov;79(3):428-37.
(30) de La FC, Gupta MV, Klase Z, Strouss K, Cahan P, McCaffery T, et al.
Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformaticsapproach. Retrovirology 2006;3:43.
(31) Araujo A, Lima MA, Silva MT. Human T-lymphotropic virus 1 neurologicdisease. Curr Treat Options Neurol 2008 May;10(3):193-200.
(32) Achachi A, Florins A, Gillet N, Debacq C, Urbain P, Foutsop GM, et al.Valproate activates bovine leukemia virus gene expression, triggersapoptosis, and induces leukemia/lymphoma regression in vivo. Proc Natl
Acad Sci U S A 2005 Jul 19;102(29):10309-14.
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Jurkat
PBMC
PBMC genomic DNA
Real time PCR
p19 gag ELISA
Western blot
Blood collection at weeks 0.1, 2. 4. 6. 8
R49 cells
ex vivo culture
Humoral response
Viral load
Serum
or
Rabbits 1-20
IV inoculation
Group 1 Group 2 Group 3
R15-20Saline txt
R9-14VPA SQ70 mg/kg
Weeks 4-8
R1-8VPA SQ70 mg/kg
Weeks 0-4
Viral Production
Hematology
Blood Smears
Valproic Acid Levels
Figure 4.1 Experimental design for rabbit inoculation and treatment withVPA. Twenty New Zealand white rabbits were inoculated with either R49 orJurkat cells. At 0,1,2,4,6,8 weeks, whole blood was collected from the centralauricular artery and processed for hematology, VPA levels, humoral response,viral load, and viral production.
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Figure 4.2 Linear timeline of valproic acid administration. All rabbits wereinoculated with R49 or control cells at week 0. Group 1 received VPA starting at
week zero, three times per week ending at week four. Group 2 received VPAbeginning at week four of infection through week eight. Group 3 received salinebeginning at week zero through week four.
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Figure 4.3 Morphology of atypical lymphocytes. Atypical lymphocytes arelarger than normal and had at least two nuclear indentations that were greaterthan 1/3 of the nuclear diameter. Panel A is a normal small lymphocyte. Panel B
represents a reactive lymphocyte. Panel C is a representative atypicallymphocyte
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A
BFigure 4.4 Hematologic values by differential cell count. A: Percent of whiteblood cells that are lymphocytes. There is a marked variation in the percentlymphocytes in group 1. This fluctuation is not observed in group 2 or group3.
Grey bars denote duration of VPA treatment. B. Percent Atypical lymphocytes. Atypical lymphocyte percentages increase at week 1 in both group 2 and group3. This is not observed in group 1.
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Figure 4.5 Rabbit serum response to HTLV-1. The antibody response to
HTLV-1 infection in the rabbits at eight weeks is similar among the groups.Rabbits 4 and 8 were inoculated with Jurkats and remained uninfectedthroughout the study.
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Figure 4.6 Grading of western blot strip intensity. Four serum antibody
responses were measured at each timepoint in each infected rabbit. Responseswere rated 0-3 based on intensity. Solid lines represent group 1. Dotted linesrepresent group 2. Dashed lines represent group 3.
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Figure 4.7: Proviral load varies with time. Proviral load in group 1 fluctuatesmore than proviral load in group 2 or group 3
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Figure 4.8 p19 production in the rabbits. Ex vivo viral production, as
measured by p19 ELISA. There is a general decrease in p19 production withtime in all groups.
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CHAPTER 5
SYNOPSIS AND FUTURE DIRECTIONS
5.1 Animal Models of ATL
Adult T-cell leukemia/lymphoma (ATL) is a refractory CD4/CD25+ CD8- T-
cell malignancy caused by human T-lymphotrophic virus type 1 (HTLV-1) (1).
Persistent HTLV-1 infections are established following transmission from infected
mothers to their children, through breast feeding, and horizontally, through
transfer of contaminated whole blood products. In approximately 1 to 5% of
persistently infected individuals, clinical disease may manifest as ATL or other
lymphocyte mediated disorders including the neurologic disease tropical spastic
paraparesis (HAM/TSP) (2).
The mechanism of carcinogenesis induced by HTLV-1 infection is not
completely understood, but it is thought to occur following viral induced cell
dysregulation and accumulation of other transformation events. ATL is classified
into four primary clinical subtypes. The two most aggressive forms are the acute
and lymphomatous (3). These forms are associated with humoral hypercalcemia
of malignancy (HHM), bone lysis, lymphadenopathy, visceral and skin
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involvement. The lymphomatous form typically is associated with a normal
peripheral blood leukocyte cell count, whereas in the acute form, ATL cells are
found in high numbers in the peripheral blood. The more indolent clinical
subtypes are the smoldering and chronic forms.
In this thesis we have investigated the use of targeted therapeutics in a
preclinical, NOD/SCID ATL mouse model and in a rabbit model of HTLV-1
infection. The number of available mouse models of ATL is limited, in part, due
to the lack of ATL cell lines obtained directly from patients. HTLV-1 cell lines
transformed in cell culture systems often fail to engraft in immunodeficient mice
(4-7). The mouse model chosen (MET-1 cells in NOD/SCID mice, (8) has as
aggressive but consistent phenotype with all mice succumbing to ATL-like
disease within 9 -12 weeks. A distinct advantage of this animal model is the use
of a biomarker to noninvasively measure tumor burden by measuring the human
specific serum IL-2Rα levels. Unfortunately, the MET-1 cell line does not grow in
vitro so compounds cannot be tested in this cell line prior to administration in the
mouse. An ideal mouse model would be one in which we could test compounds
in vitro and explore mechanisms of action in vitro prior to the use of the same cell
line in the immunodeficient mouse. This would help predict behavior and would
also confirm in vitro that the in vivo effects are due to the expected targeting.
Generation of new mouse models of ATL is dependent on procuring leukemic
cells from infected patients and inoculating them into immunodeficient mice. In
our experience, not every patient sample will grow and engraft in the
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immunodeficient mouse. If the ATL cells do engraft in the immunodeficient
mouse, they may not engraft on subsequent passage through additional mice
and they may not grow in vitro. This is why the development of additional ATL
cell lines is paramount.
5.2 Targeted Therapies Against ATL
Adult T-cell leukemia/lymphoma is a refractory malignancy with a
uniformly poor prognosis regardless of therapy in its acute form(9-13). The acute
form of ATL is a refractory malignancy with a median survival time of less than
one year regardless of therapy (14). Adult T-cell leukemia/lymphoma-derived
cells have multiple ways of surviving and persisting in presence of
chemotherapy. While, HTLV-1 is clearly linked to the development of ATL, active
virus replication is not required to maintain tumor growth. The outgrowth of ATL
cells is believed to be initiated by key viral proteins such as Tax, but is more
dependent upon genetic or epigenetic changes of the host cell for eventual
selection of clonally expanded malignant lymphocytes. Over expression of the
multidrug resistance protein has been reported in ATL (15;16) further
complicating treatment options.
Conventional chemotherapies against ATL have produced inconsistent or
limited results in treatment outcome and prolongation of mean survival times
(reviewed in (1;13;17). A combination of Cyclophosphamide, Adriamycin,
vincristine, and prednisolone is typically the first line of therapy, but is of limited
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efficacy. The use of nucleoside analogs, interferon, zidovudine, topoisomerase
inhibitors, and monoclonal antibodies has produced more encouraging results;
however the prognosis for ATL patients remains poor. Bone marrow transplant
has been reported to “cure” ATL (18). This is complicated by patient age,
condition, and likelihood of immunologic match and therefore is of limited use.
With the development of the ATL mouse model, preclinical efficacy studies
in the mouse have provided insight into the use of targeted chemotherapy.
These compounds take advantage of the genetic or epigenetic alterations that
are consistently found in the neoplastic cells but not the normal cells of the body.
This provides specificity for the neoplastic population that is not found with the
standard cytotoxic chemotherapies.
5.3 The Need for Prolonged Dosing to Achieve Efficacy in ATL
In the previous chapters, we demonstrated a marked in vitro efficacy of
preclinical compounds. The compounds tested in vitro were tested in the
NOD/SCID mouse model of ATL with varied success. In Chapter 2, we showed
that the combination of PS-341 and 17-AAG decreased tumor burden in the mice
when measured immediately after treatments. Phase II studies in which PS-341
and 17-AAG were withdrawn and mice were allowed to survive until removal
criteria were met. Extending the study resulted in a dampening of the benefits of
combination therapy.
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In chapter 3, we tested the novel histone deacetylase inhibitor, OSU-
HDAC42, in vitro in ATL cell lines and in the NOD/SCID ATL mouse model. In
the in vitro experiments, cell death and histone hyperacetylation was evident.
Translating this to the in vivo model was less clear. In phase I of this study, mice
dosed by gavage and removed from therapy did not have a significantly different
change in survival when compared to the vehicle only treated mice. In phase II,
we used daily in feed dosing to provide a more consistent and longer term dosing
of the mice. While the change in survival was not statistically significant, this was
likely due to sample size. One might infer from the Kaplan Meier curve of the in
feed study that with an expanded sample size there would be a significant effect
on survival.
Based on the information provided by the previous chapters, investigation
of long term therapy for ATL is warranted. Our model indicates that removal of
compounds diminishes the benefits of the targeted therapy.
Several other neoplastic diseases require long term adjuvant therapy to
prolong survival. As early at 1974, long term chemotherapy was suggested in
breast cancer (19) and is now one of the mainstays of estrogen receptor positive
breast cancer (20).
5.4 Evaluation of Combinational Therapy Against ATL
In chapter 2 of this thesis we present data to indicate that the combination
therapy of PS-341 and 17-AAG when tested both in vitro and in an established
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ATL mouse model is effective to block the growth of ATL cell lines and reduce
tumor burdens mice. Using HTLV-1 transformed cell lines, we found that the
addition of 17-AAG to PS-341 induced apoptosis. Our data is the first to
demonstrate that treatment with PS-341 in combination with 17-AAG reduced the
tumor burden of established ATL cells in a mouse model. Of significance, our
survival studies in ATL engrafted mice indicated that the differences observed
between the treatment groups were minimized upon removal of drug therapy,
Thus, while this combination of targeted therapy offers promise to decrease
tumor burden, our results indicate that additive effects of 17-AAG are transient
and require continued presence of the drug.
The effect of PS-341 on the growth of tumors in a Tax transgenic mouse
model revealed no consistent inhibition of NF-κB activation in vivo, however
transplanted Tax tumors in Rag-1 mice showed consistent inhibition of tumor
growth and prolonged survival (21). In a separate study, PS-341 decreased
tumor burden in ATL xenograft mice (22). In the MET-1 ATL mouse model, PS-
341 was not beneficial alone, however enhanced efficacy was noted in
combination with humanized anti-Tac (CD25) monoclonal antibody(23). Our
studies reveal that PS341 and 17-AAG, while effective at reducing established
turmor burdens in short term studies, our survival study indicated a need for
continuous drug pressure to maintain the additive benefits of both drugs. Our
design allowed us to test the effects of the continued pressure of the combination
therapy. Our mouse model, while providing an excellent preclinical model in
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which to assay the effects of the compounds on the ATL cells, does not assay
the effects of prolonged therapy in context to ATL patients or in the presence of a
functional immune system. Despite the limitations of the model system, our
results have implications for the design of human trials using this combination of
drugs or when considering 17-AAG or derivatives as adjunct therapy to
standardized protocols. Our data support further investigations to test various
dosing regimen and drug cycles of PS-341 and 17-AAG in both animal and
human clinical trials. The website http://www.clinicaltrials.gov shows, current
clinical trials using this combination include phase I dose escalation studies in
leukemia, lymphoma, and small intestinal cancer.
5.5 HDACi in ATL Therapy
Our chapter 3 data presented in this thesis indicate that the use of HDACi
is a potential effective treatment strategic against ATL. In vitro, both VPA and
OSU-HDAC42 abate growth of ATL cell lines. Exposure of ATL cell lines to both
VPA and OSU-HDAC42 resulted in hyperacetylation of histones and induction of
apoptosis. Our data are the first to demonstrate that OSU-HDAC42 is safe and
efficacious in the ATL NOD/SCID mouse model. In addition, our feed formulation
design demonstrated that oral dosing of OSU-HDAC42 appeared to be more
effective compared to oral gavage in improving survival of the engrafted mice.
Human T-lymphotropic virus type 1 is linked to the development of ATL;
however, active virus replication is not required to maintain tumor growth. The
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outgrowth ATL cells appear to be dependent on genetic and epigenetic changes
of the host cell for selection of clonally expanded malignant lymphocytes. The
refractory nature of ATL indicates that novel treatments are needed against the
malignancy to more selectively target key cancer cell pathways to block tumor
cell growth. Conventional chemotherapies against ATL have produced
inconsistent or limited results against ATL (1). New therapeutic approaches have
focused on targeted therapies.
Histone deacetylase inhibitors promote the acetylated state of histones
and relax chromatin structure leading to induction of apoptosis, cell cycle arrest,
disruption of Hsp90 and the aggresome, inhibition of angiogenesis, mitotic cell
death, and senescence (reviewed in (24). In ATL cell lines, the histone
deacetylase inhibitor FR901228 induced apoptosis and reduced tumor size in
mice (25). In chapter 3 of this thesis, we compared two HDACi (VPA and OSU-
HDAC42) for their ability to abate ATL cell growth and reduce tumor burdens in a
mouse model. Recently, VPA has been shown to have efficacy in the treatment
of cancer through the hyperacetylation of histones 3 and 4 (26). OSU-HDAC42
is a phenylbutryrate derived HDACi included in the Rapid Access to Intervention
Development (RAID) program through the National Cancer Institute (27). In a
prostate cancer model, OSU-HDAC42 was shown to decrease cell viability,
decreased phospho-Akt, Bcl-xL, and Survivin and decreased tumor burden in a
mouse model (28).
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In our study, we demonstrated that OSU-HDAC42 while not improving
survival in short term tumor engraftment studies did prolong survival when
provided as a continuous feed supplement. Thus, OSU-HDAC42 was required
on a more continuous basis to demonstrate antitumor activity. The addition of
continued drug pressure for a prolonged time appeared to prolong survival and
prevent tumor cells from regaining growth capacity. Expanding the numbers of
mice to provide for statistically significant sample size for a second survival study
is one logical continuation. It would also be valuable to design a study similar to
the phase I study of chapter 2 in which the mice are euthanized at a single
timepoint and the tumor burden is quantitated using histology and
immunohistochemistry.
The mouse model, while providing an excellent preclinical model in which
to assay the effects of the compounds on the ATL cell growth, does not assay
the effects of prolonged therapy in context to ATL patients or in the presence of a
functional immune system. Our results have implications for the design of
human trials using OSU-HDAC42 as adjunct therapy to standardized protocols
and support further investigations to establish more effective therapeutic dosing
regimens and drug cycles of OSU-HDAC42 in both animal and human clinical
trials. We have shown that shorter duration dosing may be more effective than
every other day dosing.
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5.6 Control of Proviral Load in HTLV-1 Infection
Rabbits are the model used most often to study early HTLV-1 infection
events, test viral transmission routes, and evaluate host immune responses
against the virus infection. The rabbit model was used to confirm the modes of
transmission in humans, as well as early studies of the immune response against
the infection or to develop vaccines against HTLV-1 (29-34). Early studies
utilizing the rabbit model of HTLV-1 helped confirm routes of viral transmission
(31) and effective means to prevent the transmission of the virus (31;35-38).
Herein, we utilized the rabbit model to test the influence of VPA, a known
modulator of histone acetylation, to test its influence on HTLV-1 expression in
vivo. HTLV-1 expression is controlled by both methylation and histone
acetylation (39-43). Both methylation and histone acetylation cause
transcriptional silencing of HTLV-1 at the 5’LTR (44). Acetylation of histones has
been estimated to control approximately 9 % of the HTLV-1 proviral load in vitro
(45). In addition, histone deacetylases such as HDAC1 decreased Tax
mediated gene expression (46). Our data using the rabbit model of HTLV-1
infection indicate that VPA treatment at 70 mg/kg for four weeks caused a
decrease in proviral load when administered during the first four weeks of
infection. We also noted an increase in atypical lymphocytes in both group 2 and
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group 3 rabbits at one week after virus exposure. This increase was not
observed in the VPA treated rabbits. In HTLV-1 infection, the presence of
atypical lymphocytes is usually indicative of transformed T cells and is one of the
diagnostic criteria for ATL (47). These cells have marked chromosomal
abnormalities and aneuploidy linked to the expression of Tax (48). Atypical
lymphocytes may also be present in the cerebrospinal fluid (CSF) of HAM/TSP
patients (49). While the presence of atypical lymphocytes in rabbit blood smears
has not been documented with HTLV-1 infection before, it can be inferred that
the increase in atypical lymphocytes with HTLV-1 infection is expected due to
increased Tax expression and viral replication. The static nature of the atypical
lymphocytes in group1 may correlate to the subsequent decrease in proviral load
observed at week 6.
Our findings parallel and support previous studies of bovine leukemia virus
infection in sheep (50). In BLV-infected sheep administered 80 mg/kg VPA three
times per week for one month. All sheep seroconverted at similar times and had
similar hematologic values. The authors did recognize a slight reduction in
proviral load, although the sample size was too small to draw a statistical
comparison (50).
In parallel to human clinical trials, our data indicated an early increase of
proviral copy number in PBMC in response to VPA followed by reductions.
Patients administered VPA demonstrated a transient increase in proviral load,
which lasted approximately 45 days prior to reductions (51). In addition, proviral
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loads of patients in this study that were treated with VPA fluctuated more than
untreated controls. Consistent with this study, our group 1 rabbit’s proviral load
fluctuated more than either group 2 or group 3.
HTLV-1 expression is controlled by both methylation and histone
acetylation (39-43). Both methylation and histone acetylation cause
transcriptional silencing of HTLV-1 at the 5’LTR (44). Acetylation of histones has
been estimated to control approximately 9 % of the HTLV-1 proviral load (45). In
addition, histone deacetylases such as HDAC1 decreased Tax mediated gene
expression (46).
Controlling viral expression is critical to the outcome of HTLV-1 infection.
Increased viral expression and corresponding proviral copy numbers in
circulating lymphocytes has been associated with lymphocyte-mediated disease
syndromes associated with HTLV-1 infection. The goal of VPA therapy in early
HTLV-1 infection and in HAM/TSP is to force the virus out of latency and expose
it to the immune response. Expression of viral proteins causes an increase in
immune surveillance and destruction of HTLV-1 infected cells by cytotoxic T-
cells. During two months of treatment with VPA, HAM/TSP patients exhibited a
transient increase in proviral load followed by decreased proviral load (51).
Histone deacetylase inhibitors are not specific and have widespread
cellular effects. Concurrent immune response to HTLV-1 infected cells
complicate interpretation of the effects of HDACi on proviral copy numbers in
patients and in our infected animals. In vitro, Mosley et al., (45) demonstrated
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that HDACi decreased the efficiency of CTL killing of HTLV-1 infected cells,
possibly by inhibition of HDAC-6 in mediating cell-to-cell contact. Because of the
effect on the CTL, they concluded that HDACi might not be efficient in controlling
proviral load and a poor therapy.
Our experiments provide data to assist in the design of future studies to
test the effects of HDACi on HTLV-1 early infection. Future studies to optimize
the duration of treatment in the rabbit model, as well as the response to removal
of VPA are warranted. Our data indicate early and prolonged intervention with
HDACi may be needed to control proviral load.
5.7 Summary and impact of work
In the preceding four chapters, we have studied the effects of targeted
therapies on HTLV-1 infection and disease. Conventional chemotherapy has
shown little improvement over no treatment in ATL and HAM/TSP. The
development of targeted therapeutics is necessary to combat these diseases.
Our work serves as a stepping stone to larger scale animal studies and supports
the ongoing Phase I clinical trials for several therapeutic regimens.
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