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Reactivation of the Epstein Barr virus lytic cycle in nasopharyngeal carcinoma by NEO212, a novel temozolomide-perillyl alcohol conjugate

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Reactivation of the Epstein Barr virus lytic cycle in nasopharyngeal carcinoma by NEO212, a novel temozolomide-perillyl alcohol conjugate

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Content 1

REACTIVATION OF THE EPSTEIN BARR VIRUS LYTIC CYCLE IN
NASOPHARYNGEAL CARCINOMA BY NEO212, A NOVEL TEMOZOLOMIDE-
PERILLYL ALCOHOL CONJUGATE

Hannah Hartman

A thesis presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2019

2

Acknowledgement
I would like to first thank my advisor, PI, Committee chair and friend Dr. Axel Schonthal
for his guidance and patience throughout my time here at USC. I would like to thank my thesis
committee members, Dr. Stanley Tahara and Dr. Martin Kast for contributing to my success as a
masters student. I would like to thank all of the members of the Dr. Chen Glioma group,
including Vahan Martirosian, Dr. Weijung Wang, Dr. Camelia Danilov, Krutika Deshpande, Thu
Zan Thein, Nagore Ramos, Robert Herrera and of course Dr. Thomas Chen. I would like to
extend a special thanks to Dr. Radu Minea, Dr. Josh Neman, Dr. Steve Swenson, Dr. Florence
Hoffman, and Dr. Heeyeon Cho for their advice, support and knowledge, as well as to my good
friends Catalina Silva, Frannie Ferri, and Samantha Stack for their unwavering support. Finally,
I would like to acknowledge my friends and family outside of the scientific community,
including my mom, dad, brothers, Kimiya Malek, Josh Call, Clark Mumaw, Jason Cox, Scott
Burch and Christian Johnston for their unwavering emotional and mental support and inspiration
throughout my scientific career.
I would like to thank Dr. Kwok-Wai Lo of the Chinese University of Hong Kong for
providing C666.1cells, and Dr. Chin-Tarng Lin of Academia Sinica for TW1 and TW4 cells.

3

Contents
Acknowledgement…………………………………………………………………..……………2
Contents……………………………………………………………………………….…..……3-5
List of Figures………………………………………………………………………………….6-7
List of Abbreviations…………………………………………………………………………..8-9
Abstract…………………………………………………………………………………………10
Chapter 1 – Introduction …………………………………………………………………..11-16
1.1. Nasopharyngeal carcinoma
1.1.1 Introduction
1.1.2 Nasopharyngeal carcinoma subtypes
1.2. Epstein-Barr Virus
1.2.1. Introduction
1.2.2. Virus lytic cycle
1.2.2.1.Immediate-early viral protein production/function
1.2.2.2.Early viral protein production/function
1.2.2.3.Glycoprotein and late viral protein production/function
1.2.3. Virus Latency
1.2.3.1.Viral latency and oncogenicity
1.2.3.2.Lytic reactivation and tumor treatment
1.3. NEO212
1.4. Hypothesis
Chapter 2 – Materials and Methods……………………………….………………………17-19
2.1. Cell lines and maintenance
2.2. Pharmacological agents
2.3. Colony formation assay (CFA)
2.4. Cell counting
4

2.5. Western blotting
Chapter 3 – Results………………………………………………………………………….20-36
3.1. Difference in cell death and MGMT depletion with NEO212 treatment among
NPC666.1 vs. other MGMT positive cell lines
3.1.1. Purpose of the study
3.1.2. C666.1 cells are more sensitive to NEO212 than all other MGMT-positive
NPCs – independent of MGMT
3.1.3. Verification of EBNA-1 and MGMT presence in TW1, TW4, C666.1 and
prNPC
3.2. Verification of EBV reactivation in NPC666.1 cell line
3.2.1. Purpose of the study
3.2.2. NEO212 treatment triggers IE and early protein production in C666.1 and
prNPC cells
3.3. Verification of EBV reactivated-NPC666.1 sensitivity to antiviral treatment
3.3.1. Purpose of study
3.3.2. C666.1 cells show lower cell counts when treated with GCV and NEO212
(short term)
3.3.3. NEO212 and ganciclovir work synergistically in cell survival prevention
3.3.4. NEO212 and ganciclovir work synergistically in apoptotic induction
3.4. EBV reactivation pathway and NEO212 specificity
3.4.1. NEO212 elements – TMZ, POH, TMZ+POH
3.4.1.2. Purpose of study
3.4.1.2. C666.1 cells are sensitive to pNEO2122 but not TMZ or POH
5

3.4.1.3. pNEO212 and POH can induce IE and early protein production in
C666.1 cells
3.4.2. Inhibition of PI3K pathway
3.4.2.1. Purpose of study
3.4.2.2. PI3K inhibition shields C666.1 cells from killing effects of
NEO212
3.4.2.3. PI3K inhibition prevents EBV IE and early protein production in
C666.1 cells
Chapter 4 – Discussion………………………………………………………………...……37-43
4.1 C666.1 cells indicate a novel killing mechanism for NEO212
4.2 NEO212 reactivates latent EBV in C666.1 cells and prNPC cells
4.3 Reactivation of EBV by NEO212 leaves C666.1 cells sensitive to anti-viral treatment
4.4 EBV reactivation pathway and NEO212 specificity
Bibliography……………………………………………………………………………..…..44-48

6

List of Figures
Figure 1. Epstein-Barr Virus Lytic Cycle
Figure 2. Depletion of MGMT with NEO212 treatment in endogenously MGMT-positive cell
lines
Figure 3. Cell survival analysis (CFA) of TW1, TW4, and C666.1 cell lines with TMZ and
NEO212 treatment, with and without the presence of 06BG.
Figure 4. Western Blot analysis of EBNA and MGMT in C666.1 and corresponding cell lines
Figure 5. Western Blot analysis of EBV immediate-early and early protein production in C666.1
cells treated with NEO212
Figure 6. NEO212 induces EBV IE and early protein production in novel prNPC cell line.
Figure 7. Western Blot analysis of EBV immediate-early and early protein production in TW4
cells treated with NEO212
Figure 8. Short term cell survival analysis (counting) of C666.1 cells treated with NEO212 in
combination with ganciclovir
Figure 9. Long term cell survival analysis (CFA) of C666.1 cells treated with NEO212 in
combination with ganciclovir
Figure 10. Western blot analysis of EBV early protein and apoptotic protein production in
C666.1 cells treated with NEO212 in combination with ganciclovir at 24, 48 and 72
hours.
Figure 11. Cell survival analysis (CFA) of C666.1 and TW4 cells treated with fresh and
pNEO212
Figure 12. Cell survival analysis (CFA) of C666.1 cells treated with TMZ, POH and TMZ in
combination with POH
Figure 13. Western Blot analysis of C666.1 cells treated with TMZ, POH, and TMZ in
combination with POH
Figure 14. Western Blot analysis of C666.1 cells treated with fresh NEO212, pNEO212 and POH
Figure 15. Cell survival analysis (CFA) of C666.1 cells treated with NEO212 in combination
with varying concentrations of idealilsib (PI3K inhibitor)
7

Figure 16. Western blot analysis of C666.1 cells treated with pNEO212 and fresh NEO212 in
combination with varying concentrations of idealilsib (PI3K inhibitor)

8

List of Abbreviations
O6BG – O6-benzylguanine
AIC – 5-aminoadozole-4-carboxamide
BMLF1 – EBV early gene sequence coding for protein Ea-D2
BRLF1 – EBV immediate early gene sequence coding for protein Rta (synonymous with
BRLF1)
BZLF1 – EBV immediate early gene sequence coding for protein ZEBRA (synonymous with
Zta, EB1, BZLF1)
C666.1 – nasopharyngeal carcinoma cell line C666-1 (clone of C666)
CFA – colony formation assay
cl. C-3/4/7/9 – cleaved caspase 3/4/7/9
CNS – central nervous system
DMSO – dimethyl sulfoxide
Ea-D2 – EBV Early D antigen (subunit 2)
EBER – Epstein-Barr virus-encoded small RNAs
EBNA-1 – Epstein-Barr Nuclear binding antigen 1
EBV – Epstein-Barr virus
FACS – Fluorescence activated cell sorting
FBS – fetal bovine serum
FITC-A – Fluorescein isothiocyanate - A
GC - gemcitabine
GCV - ganciclovir
gp - glycoprotein
IE – immediate early
LMP – Latent membrane protein
MAPK – mitogen-activated protein kinase
MGMT – O6-methylguanine-DNA methyltransferase
MTIC - methyltriazen-1-yl) imidazole-4-carboxamide
9

NEO212 – temozolomide-perillyl alcohol conjugate
NPC – nasopharyngeal carcinoma
OriLyt – lytic origin of replication
PARP – Poly (ADP-ribose) polymerase
PI3K – phosphoinositide 3-kinase
pNEO212 (pN) – pre-incubated NEO212
POH – perillyl alcohol
prNPC – novel primary nasopharyngeal carcinoma cell line isolated by Dr. Tom Chen
RRE – Rta response elements
TMZ - temozolomide
VCA – viral capsid antigen
Vh – vehicle treated
VPA – valproic acid
ZEBRA – EBV immediate-early protein product of BZLF-1 gene

10

Abstract
Nasopharyngeal carcinoma (NPC) is a malignant nasopharyngeal neoplasm arising from
the mucosal epithelium and is most prevalent in Southeast Asia. Nasopharyngeal carcinoma is
the most prevalent form of nose and neck cancer and is difficult to diagnose, with less than 15%
of patients diagnosed before stage III. A novel nasopharyngeal carcinoma cell line shows
hypersensitivity to the TMZ-perillyl alcohol conjugate NEO212, proposing a novel treatment for
certain nasopharyngeal carcinomas in the future. The objective of our study is to determine the
mechanism of killing and treatment, postulated to be by Epstein-Barr Virus reactivation by
NEO212 treatment in NPC cell lines. Analysis of EBV protein expression and cell death
determined the presence of IE and early EBV proteins, as well as synergy with anti-viral drugs
(ganciclovir) in C666.1 cells. NEO212 can provide a new and effective method of treatment for
EBV-positive nasopharyngeal carcinoma in patients.

11

Chapter 1 – Introduction
1.1. Nasopharyngeal carcinoma
1.1.1. Introduction
Nasopharyngeal carcinoma (NPC) is the most prominent cancer of the nasopharynx,
arising from the mucosal epithelium to form a malignant neoplasm. Although scarce in the
United States and most countries, NPCs are prominent in southern regions of China and
Southeast Asia [1-5]. Although many nasopharyngeal carcinomas present positive for latent
Epstein-Barr Virus (EBV) in the clinic, only one known immortalized cell line exists that
consistently harbors the virus. C666.1 cells (a clone of the C666 subtype) provide the only
model for studying EBV positive tumors, both in vitro and in vivo. Reactivating the EBV lytic
cycle in C666.1 cells not only reduces tumorigenicity, but also provides a novel mechanism for
tumor treatment, specifically in combination with anti-viral drug therapy.
1.1.2. Nasopharyngeal carcinoma subtypes
Three histopathological subtypes of nasopharyngeal carcinoma exist in the clinic. The
type I subtype is comprised of keratinizing squamous cells. Type I NPCs are the least
dangerous, showing highest survival rates and lowest metastatic capabilities. Type 2 NPCs are
significantly more tumorigenic, with low survival rates. Type 2 NPCs are subdivided into type
2a of non-keratinizing squamous cells, and type 2b of undifferentiated cells [6-8]. NPCs are most
common in southern regions of Southeast Asia, specifically type 2 nasopharyngeal carcinomas
[1-5]. Each subtype has unique genetic and epigenetic profiles, leading to varying levels of
carcinogenicity and mortality.

1.1.3. Nasopharyngeal carcinoma and EBV
Subtypes 2a and 2b are associated with Epstein Barr Virus infection [9-11]; however,
EBV is not exclusive to nasopharyngeal carcinoma. Many cancer types, including B cell
lymphomas, gastric carcinomas, and CNS lymphomas harbor latent EBV, with varying
histological subtypes. As previously mentioned, C666.1 subtype is the only nasopharyngeal cell
line that consistently harbors the EBV virus in cell culture through multiple passages (although
12

many EBV-positive cell lines exist in vivo). Although some cell lines have tested positive for
EBV, after multiple passages, the cells rapidly expel the virus in vitro [12].
Viral episome expulsion occurs through two primary mechanisms – whole episome
expulsion and gradual gene-by-gene removal [12]. Due to the highly mutative nature of the
virus, changes in DNA are frequent and determine not only the presentation, but the presence of
the virus within the cell. Multiple EBV genes, such as EBNA-1 (Epstein-Barr virus nuclear
antigen 1) and LMP-2 (latent membrane protein 2), anchor the EBV episome to the host cell
DNA [12-15]. Mutations in anchoring genes such as EBNA-1 lead to whole episome expulsion
where gradual mutation and expulsion of genes occurs with mutations in non-anchoring genes
(most common in lytic genes and late viral genes such as gp350 and VCAs [viral capsid
antigens])[15]. Due to the specificity and low number of genes that lead to whole episome
expulsion, this event occurs much less frequently in comparison to its gradual counterpart. This
tendency for the virus to mutate and expel its episome rationalizes variation in sensitivity to EBV
reactivation of individual cells within the same cell line over excessive passage as well as
variation in presentation and viral particle shedding.

1.2. Epstein-Barr Virus
1.2.1. Introduction
Although EBV (also known as human herpesvirus 4 [HHV4]) plays an active role in
multitude of cancers, such as B cell lymphomas, central nervous system lymphomas and gastric
carcinomas, it is predominantly responsible for the mononucleosis infection. Much of the
population carries some form of the latent Epstein-Barr virus [4, 5]. EBV contains an episomal
double-stranded DNA of approximately 172,000 base pairs and 85 genes [16]. Protein
nucleocapsid surrounds the genes, then tegument protein, followed by a lipid and glycoprotein
envelope, known as the viral capsid. Although classified as a lytic virus, as a driver of
tumorigenicity, EBV does not lyse tumor cell lines post infection, but instead resides within the
nucleus and buds out virion particles with a double membrane [9, 16, 17]. Many latent and lytic
genes have been linked to apoptotic and anti-apoptotic mechanisms, creating a delicate balance
of proteins that can help or hinder in cell survival, dependent on cell type and environmental
stimuli.
13

1.2.2. Virus lytic cycle
Reactivation of the EBV lytic cycle occurs via epigenetic modification through chromatin
remodeling, DNA methylation and histone deacetylation. This epigenetic modification triggers
transcription of two primary immediate early (IE) genes, BZLF1 and BRLF1. The products of
the transcription of these genes perform a variety of functions, including transactivation of early
gene production, inhibition of host gene production and direct binding and subsequent activation
of the EBV origin of replication (OriLyt) [1, 18]. BZLF1, also known as Zta, EB1, or by its gene
product ZEBRA, binds to Zta response elements (ZREs) on early gene promoters [ 19, 20].
BZLF1 reactivation can occur through many pathways, including AP-1, PI3K, p38 and
Ras/MEK/MAPK [21, 22]. Correspondingly, BRLF1 binds to Rta response elements (RREs)
[20, 23]. PI3K and Ras signaling pathways activate BRLF1 transcription and activation [24, 25].
Together, these two IE genes initiate transcription of six early viral gene products
necessary for lytic replication: BBLF2/3, BBLF4, BALF2, BSLF1, BMLF1 and BALF5 [26, 27].
ZEBRA also interacts directly with both the origin of replication (allowing binding of DNA
replication machinery such as BALF2) and the promoter for BRLF1 gene products [18]. Both
BZLF1 and BRLF1 contribute to immune evasion and suppression of latent EBV genes [28-32].
Most early gene products harbor more than one function, working both to aid in viral replication
as well as immune suppression/evasion, and host cell replicative inhibition.
After viral DNA is replicated, transcription of late viral gene products, such as
glycoproteins (gp350/220) and viral capsid antigens (VCA 1/2) proceeds. Late stage viral gene
products function as tegument and structural proteins for virion formation. Late stage viral
proteins have also been shown to deactivate BZLF-1 and BRLF-1 and to upregulate apoptotic
mechanisms, as well as functioning as immunosupressors (33, 34).
Virions leave the cell through budding of the nucleus, followed by budding of the cell
membrane, creating a unique double-membrane bound virion structure. In vivo, the virion does
not enter other cells, but activates cell surface receptors to induce the latent to lytic transition in
neighboring cells.

14

Figure 1. Epstein-Barr Virus Lytic Cycle. A brief overview of the genes, proteins and host
elements involved in the EBV lytic cycle. The lytic cycle can be activated with PI3k,
MEK/MAPK, P38/JNK or a combination of pathways. The two IE genes required to trigger the
lytic cycle, BZLF-1 and BRLF-1, make proteins ZEBRA (Zta) and Rta respectively, which are
responsible for multiple functions in triggering the lytic cycle, priming the viral promoter,
suppressing latent gene production, immune evasion and more. Ea-D2 is the first protein
activated of the early gene products and controls production of 11 primary proteins needed for
viral reproduction. Late viral proteins gp350, VCA1/2 and more are produced as structural,
scaffolding and budding proteins involved in exocytosis of the virion particle.

1.2.3. Virus Latency
1.2.3.1. Viral latency and oncogenicity
Major genetic contributors to NPC-related EBV, such as LMP2 and EBNA1/2, produce a
variety of viral gene products that, through DNA methylation, promote oncogenicity. LMP2 and
EBNA1/2 both help in development of tumorigenic environments, immune suppression/evasion,
and telomere stabilization [35-38].
EBV shows three distinct latency patterns, Latency I, II and III [39]. Variation in latency
pattern is determined by restriction and induction of RNA transcription. Latency I contains the
smallest gene expression pattern, with only EBNA-1 and EBER (Epstein-Barr virus small
RNAs). Latency I pattern is the least tumorigenic and most dormant. Latency II produces
15

EBNA-1 as well as LMP1, LMP2a/b and EBER, making it more tumorigenic than Latency
pattern I, as LMP2a has been indicated in cell survival, EMT and metastasis [40, 41]. Latency
pattern III produces all latent genes, including EBNA-1, EBNA-2, and EBNA-3, as well as
LMP1, LMP 2a/b, and EBERs. Latency pattern III is the most tumorigenic and is most common
in B lymphomas [42]. Nasopharyngeal (and all epithelial) tumors present latency II patterns
[43].
It is important to note that EBNA-1 is present in all forms of latency and is arguably the
most important latent viral protein. EBNA-1 is by far the most studied latent EBV protein. It is
responsible not only for latent gene replication and transcription, oncogenesis, EMT transition,
immune evasion, and lytic suppression. EBNA-1 also tethers the EBV episome directly to the
origin of replication (OriLyt) on host cell DNA, helping the episome to maintain in the nucleus
without constant replication. Episome copy number varies greatly between cell lines.
1.2.3.2. Reactivation of lytic cycle in tumor treatment
Recent in vitro and in vivo studies of EBV positive carcinomas and lymphomas focus on
reactivation of the lytic cycle through chemotherapeutic modification, specifically through
activation of the BZLF1 and BRLF1 promoters. Treatments with gemcitabine, valproic acid,
doxorubicin, cis-platinum, methotrexate, 5-fluorouracil, and radiation (often in combination)
reactivate the EBV lytic cycle in latent EBV positive cell lines [25, 44-47, 48-51]. Reactivation
provides a potential target for immunotherapy due to the production of viral kinases, all the while
making these cells more susceptible for antiviral treatment with drugs such as ganciclovir [43-
48]. In addition to directly aiding in treating infected cell lines, activating the lytic cycle inhibits
latent gene production, inhibiting viral oncogenic factors from promoting tumorgenicity and a
tumorigenic environment.
1.3. NEO212
NEO212 is a novel drug conjugate of temozolomide to perillyl alcohol (POH). The
naturally occurring monoterpene POH displays cytotoxic to a variety of tumor cell lines,
including those traditionally resistant to TMZ due to high levels of the DNA repair protein O6-
methyl-guanine-DNA-methyltransferase (MGMT) [52, 53]. Furthermore, POH improves the
lipophilicity of temozolomide, increasing its uptake into the brain [54]. Temozolomide degrades
16

into 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), a powerful alkylating agent,
which then degrades into 5-aminoadazole-4-carboxamide (AIC) [55]. MTIC methylates at
multiple locations in the DNA such as N3-methyladenine, N7-methyl-guanine and O6-methyl-
guanine positions [53]. This DNA methylation leads to incorrect base pairing and recruitment of
mismatch repair proteins (MMR) to remove the thymidine. MMR removes the thymidine, but
cannot repair the original lesion, leading to accumulation of double strand breaks and cell death
[56, 57]. High levels of base excision repair (BER) proteins indicate temozolomide resistance,
due to the ability to remove and repair damaged DNA bases [58-60]. PARP (Poly [ADP-ribose]
polymerase) activity enhances cellular resistance to TMZ, by further stabilizing the genome and
repairing TMZ-induced lesions [43-62]. Cells positive for the DNA repair protein MGMT repair
these legions, preventing cytotoxicity of temozolomide in gliomas and other MGMT-positive
cell lines [53].

1.4. Hypothesis
While running CFA and western blot analyses on a variety of MGMT-positive tumors, it
was noted that one specific cell line, C666.1, acted differently from all other MGMT-positive
tumors, including other MGMT-positive nasopharyngeal carcinoma cell lines. C666.1 cells
showed extreme sensitivity to NEO212, and no depletion of MGMT, indicating a novel
mechanism of cell killing in this cell line. We hypothesize that NEO212 reactivates the EBV
lytic cycle in C666.1 cells, leading to cell death through apoptosis.

17

Chapter 2 – Materials and Methods
2.1. Cell lines and maintenance
Human cell lines: NPC666.1 and TW4 were propagated in Dulbecco’s Modification of
Eagle’s Medium with 4.5 g/L glucose, L-glutamine & sodium pyruvate by Mediatech Inc.
(Manassas, VA) supplemented with 10% fetal bovine serum (FBS), 100 u/mL penicillin, and 0.1
mg/mL streptomycin in a humidified incubator at 37˚C and a 5% CO2 atmosphere. FBS was
obtained from Omega Scientific (Tarzana, CA) and from X&Y Cell Culture (Kansas City, MO).
NPC666.1 cells were obtained from Dr. Kwok-Wai Lo of the Chinese University of Hong Knog
(Shatin, Hong Kong) and these cells were passaged for up to four months at a time. TW1 and
TW4 cells were provided by Dr. Chin-Tarng Lin of Academia Sinica (Taipei, Taiwan) and were
passaged for up to ten months.

2.2. Pharmacological agents
TMZ was obtained from the pharmacy at the University of Southern California (USC) or
was purchased from Sigma Aldrich (St Louis, MO) and dissolved in DMSO (Santa Cruz
Biotechnology, Dallas, TX) to a concentration of 50 mM. 06-BG (Santa Cruz Biotechnology,
Santa Cruz, CA) was dissolved in DMSO to a concentration of 50 mM. NEO100 and NEO212
were provided by NeOnc Technologies (Los Angeles, CA) and diluted or dissolved in DMSO at
100 mM. In all cases of cell treatment, the final DMSO concentration in the culture medium
never exceeded 1% and was much lower in most cases. Stock solutions of NEO212 and TMZ
were stored at –80˚C. Stock solution of NEO100 (perillyl alcohol) was stored at –4˚C.
gemcitabine came from LC Laboratories (Woburn, MA). Ganciclovir was obtained from LKT
Labs (St. Paul, MN) and stored at -80˚C. Idealilsib (CAL-101, GS-1101) was obtained from
MedChemExpress (Princeton, NJ) and stored at -80˚C. Valproic acid was obtained from Sigma-
Aldrich and stored at -4˚C. All drugs were diluted in reduced pH medium to obtain final
treatment concentrations.
Pre-incubated NEO212 (pNEO212) was diluted from 100 mM stock solution to 100uM
in Dubecco’s Modification of Eagles Medium with 4.5 g/L glucose, L-glutamine & sodium
18

pyruvate. PNEO212 stock was then kept in humidified incubator at 37˚C and a 5% CO2
atmosphere in a 15 mL tube for 24 hours. Stock pNEO212 was stored at –80˚C.

2.3. Colony formation assay (CFA)
Depending on cell line and plating efficiency, 600-1000 cells were seeded into each well
of a 6-well plate and treated as described in detail previously [20]. In the case of co-treatment
with 06BG, TMZ, POH, NEO212, pNEO212, ganciclovir, gemcitabine, valproic acid, cisplatin
and idealilsib, all drug treatments were added within 30-60 minutes of previous drug treatment,
unless further noted (i.e. 24 hour time frames, etc.) Two days after the start of drug treatment
with idealilsib, the medium was removed and replaced with drug-free medium. All experiments
received fresh medium after 7 days of initiation, regardless of later changes in drug treatment
(i.e. 24 hour delayed treatments, idealilsib medium change, etc.) All cells received fresh, drug
free medium. After 14-17 days, formed colonies (groups of >50 cells) were stained with
methylene blue and counted. CFAs were always performed in duplicate or triplicate, and in most
cases were independently repeated once or twice.

2.4. Cell counting
C666.1 cells were seeded with approximately 50,000 cells into each well of a 6-well plate
and treated [20]. In the case of co-treatment with NEO212 and ganciclovir, all drug treatments
were added within 30-60 minutes of previous drug treatment. Cells were trypisinized at the
indicated time points and counted using a hematocytometer. All cell counts were performed in
duplicate, triplicate or quartet.

2.5. Western blotting
Total cell lysates were analyzed by Western blot analysis as described earlier [1]. The
following primary antibodies were used. For the detection of MGMT, we used a polyclonal
antibody (#2739) from Cell Signaling Technology (Danvers, MA) and a monoclonal antibody
(sc-5643: #F2512) from Santa Cruz Biotechnology. For the detection of cleaved caspase 3, we
19

used a monoclonal antibody (MAB10753) from Millipore Corp. (Darmstadt, Germany). For
detection of cleaved caspase 9, we used a monoclonal antibody (Asp175) from Cell Signaling
Technology. For the detection of EBNA-1, Ea-D2, ZEBRA, and b-Actin, we used monoclonal
antibodies (sc-81581:#J2417, sc-58121:#D2718, sc-53904:#L2717, sc-47778:#D1717) from
Santa Cruz Biotechnology. CleavedcCaspase 3 Horseradish peroxidase-antibody conjugates (i.e.
secondary antibodies) were obtained from Jackson ImmunoResearch laboratories (West Grove,
PA). All antibodies were used according to the supplier’s recommendations. All immunoblots
were repeated at least once to confirm results.
20

Chapter 3 – Results
3.1. Difference in cell death and MGMT depletion with NEO212 treatment among
NPC666.1 vs. other MGMT positive cell lines
3.1.1. Purpose of the study
The purpose of this study was to first determine the differences in efficacy of NEO212
and temozolomide among cell lines with varying MGMT expression. In previous investigations,
we have shown that all TW1 and TW4 cell lines show depletion of MGMT with NEO212
treatment; however, MGMT is not depleted in NPC666.1 cells when treated with NEO212,
indicating cell death not correlated with MTIC methylation of O6-methyl-guanine positions.

Figure 2. Concentration-dependent MGMT depletion in endogenously MGMT positive cell
lines by NEO212 treatment. Western blot analysis of MGMT protein levels with actin as the
loading control. C666.1, TW4, M249 and MCF7 cells were treated with indicated
concentrations of NEO212 for 24 hours before harvest of cellular lysates. Cells were treated
with 100 uM pNEO212. Cells were also treated with vehicle (Vhc, DMSO) or remained
untreated.

21

To test the cell killing efficacy of NEO212 on MGMT positive NPC cell lines, three cell
lines were set up as CFAs, at varying concentrations (figure 3). Although we see no depletion of
MGMT with NPC666.1 cell lines, we see significantly higher cell killing when treated with
NEO212, in comparison to TW1 and TW4 lines. When treated with TMZ, all cell lines showed
significantly increased IC50s and therefore significantly less cell death than NEO212 treatments.
Western blots were run to verify the presence of MGMT in all MGMT positive cell
(figure 5) . Cell lines were tested for EBNA-1, the only fully proliferate latent EBV gene, to
verify presence of EBV in various cell lines. From these results, we postulated a connection
between EBV and cell killing in NPC666.1 cells.
06BG, a well-known MGMT inhibitor was applied to cell lines in order to verify cell
killing through an MGMT independent mechanism. In TW1 and TW4 cells we see a significant
increase in cell killing ability when cells are treated with O6BG, verifying the necessity of
MGMT as a cellular repair mechanism in TW1 and TW4 cells when treated with TMZ and
NEO212. Only in C666.1 cells treated with NEO212 do we not see a significant difference
between cell killing with and without O6BG, indicating a novel mechanism for cell death.

3.1.2. C666.1 cells are more sensitive to NEO212 than all other MGMT-positive NPCs –
independent of MGMT
22

Figure 3. Differential sensitivity of MGMT-positive NPC cell lines to NEO212. Various
MGMT positive NPC cell lines were exposed to increasing concentrations of NEO212 with
(orange) and without (blue) 15 µM O6BG (MGMT inhibitor). The colony number from
untreated cells is set to 100%. The data shown is the average of duplicate or triplicate wells.
Comparison of cytotoxicity of NEO212 and TMZ shows a unique effect in C666.1 cells,
in comparison to other MGMT positive NPC cell lines. TW1 cells show resistance to TMZ, with
an IC50 above 100 µM, where NEO212 shows an IC50 of approximately 25 µM. When treated
with O6BG, TW1 cells are sensitized to TMZ, with an IC50 of approximately 10 µM (similar to
MGMT negative cell lines). TW1 cells show increased sensitivity to NEO212 as well, with an
IC50 similar to TMZ (10 µM). TW4 cells (which show even higher levels of MGMT – figure 4)
show resistance to TMZ with an IC50 higher than 100 µM (approx. 250 µM), and increased
resistance to NEO212 (as is expected with increased MGMT) with an IC50 around 40 µM
(similar to other MGMT positive cell lines, such as A375 and LN229M). With O6BG
application, cells show highly increased sensitivity to TMZ (IC50 of approximately 10 µM) and
increased sensitivity to NEO212 (IC50 of approximately 10 µM).
C666.1 cells are extremely resistant to TMZ, showing an IC50 >250 µM, but show extremely
low resistance to NEO212, with an IC50 <10µM (approx. 3 µM). Although cells are sensitized
23

to TMZ when treated with O6BG, there is no change in IC50 to NEO212 in C666.1 cells when
treated with O6BG.
All cell lines and treatments show statistically significantly higher cell death with
NEO212. All cell lines and treatments show a statistically significant increase in drug efficacy
with O6BG treatment, excluding NEO212 treated C666.1 cells.

3.1.3. Verification of EBNA-1 and MGMT presence in TW1, TW4, C666,1 and prNPC

Figure 4. Basal level expression of MGMT and EBNA-1 in various cell lines. Western blot
analysis of basal level MGMT and EBNA-1 protein levels with actin as the loading control.
Presence of EBNA-1 and MGMT was verified in TW1, TW4, C666.1 and prNPC cells. T98G
and U251 served as MGMT positive and negative controls respectively. Raji and U251 served
as EBNA-1 positive and negative controls respectively. Raji displays a known mutant variant of
EBNA-1.

An analysis of 7 cell lines (T98G, U251, TW1, TW4, C666.1, prNPC, Raji) indicated the
presence of latent EBV proteins in C666.1 cells, prNPC cells and Raji cells. Raji cells were used
as a positive control for EBNA-1. Raji cells contain a truncated form of EBNA, leading to
multiple bands at 88 kDa, and 3 bands between 100 kDa and 125 kDa. C666.1 and prNPC cells
presented traditional EBNA-1 presence at 115 kDa, where TW1 and TW4 cells showed
negativity, indicating latent EBV in C666.1 NPCs, but not in TW1 or TW4 NPCs, as expected.
prNPC cells are a primary NPC cell line newly isolated from patient tissue in fall 2018 from Dr.
Thomas Chen. These cells provide another possible EBV-positive cell line to be used for
research, as C666.1 cells are the only current endogenous EBV positive NPC cell line available.
24

These cells also verify that primary NPC tumor lines react similarly to immortalized cell lines
used for research.
High levels of MGMT were present in T98G, C666.1, and prNPC cells. TW1 and TW4
cells showed lower, but still present levels of MGMT. This correlates with TMZ and NEO212
resistance indicated in figure 3. T98G was included as a positive control, and U251 as a negative
MGMT control. From this data we can conclude that C666.1 cells, TW1 and TW4 cells are
positive for MGMT, despite C666.1’s novel behavior.

3.2. Verification of EBV reactivation in NPC666.1 cell line
3.2.1. Purpose of the study
Once a novel reaction to NEO212 was shown in NPC666.1 cells, independent of MGMT,
we decided to test cells for activation of the EBV lytic cycle, as the hypothetically cell death
mechanism. Cells were initially tested in conjunction with known EBV reactivators,
gemcitabine and valproic acid. NEO212 worked with both molecules to activate the cell cycle.
Due to strong activation capacity, NEO212 was tested alone at varying time points and
concentrations for EBV activation with NEO212 against a positive control of gemcitabine and
valproic acid. prNPC cells were also tested for EBV reactivation by verification of IE and early
gene products. TW4 cells were included as a negative control to EBV protein expression.
3.2.2. NEO212 treatment triggers IE and early protein production in C666.1 and prNPC cells
NEO212 was first administered at 10, 50, and 100 µM to determine the drugs ability to
trigger they lytic cycle. Untreated NPC666.1 cells were included as negative controls, with
NPC666.1 cells treated with GC and VPA as positive controls. A sample treated with 15µM
O6BG was included to test the importance of MGMT expression on lytic cycle activation.
NEO212 is capable of triggering expression of IE (ZEBRA) and early (Ea-D2) proteins in the
EBV lytic cycle at concentrations as low as 10µM. Depletion of MGMT in NPC666.1 cells did
not change lytic cycle activation.

25

Figure 5. IE and early EBV lytic gene expression in C666.1 cells after NEO212 treatment.
Western blot analysis of ZEBRA, Ea-D2 and cleaved caspase 3/7 protein levels with actin as the
loading control. A. C666.1 cells were treated with 30 µM NEO212 for indicated time frames
before harvest of cellular lysates. Cells were also treated with vehicle (Vhc, DMSO) or
remained untreated. B. C666.1 cells were treated with indicated concentrations of NEO212 for
48 hours before harvest of cellular lysates. Cells were also treated with vehicle (Vhc, DMSO) or
remained untreated.

To determine the lowest concentration (and verify results) at which NEO212 can activate
the lytic cycle, NPC666.1 cells were treated with 1, 3, 10, 30, and 50µM concentrations of
NEO212. Cells were compared to the negative control (LN229) and positive control (GC+VPA
treated NPC666.1) to determine protein level expression. ZEBRA protein (IE viral gene
product) showed concentration dependence, with expression as low as 1µM, where Ea-D2 (early
viral gene product) showed consistent activation at all levels of treatment, as low as 1µM.
Concentration dependence was verified by statistical analysis of density quantification. Although
some cleaved caspase 7 is present in these samples, we see thin, light bands at both 1 and 3 µM,
with significant bands forming at around 10µM and up.

NPC666.1 cells were then treated with 50 µM NEO212 and harvested at 24h, 48h, 72h
and 96h time points. Although NEO212 showed time dependent increase in cell death (cleaved
caspase 3), there was no significant change in ZEBRA and Ea-D2 expression at varying time
points, indicating a consistency in EBV reactivation. Later time points showing cell death
indicates that EBV reactivation must occur first to trigger apoptosis.
26

Figure 6. NEO212 induces EBV IE and early protein production in novel prNPC cell line.
Western blot analysis of ZEBRA, and Ea-D2 protein levels with actin as the loading control.
prNPC cells were treated with 10 µM and 50 µM NEO212 for 44 hours before harvest of cellular
lysates. Cells were also treated with vehicle (Vh, DMSO) or remained untreated.

prNPC cells were tested for EBV reactivation after EBNA-1 presence was confirmed.
Cells tested positive for IE and early EBV proteins ZEBRA and Ea-D2 with 10µM of NEO2112.
Both proteins showed concentration-dependent activation. prNPC cells were not further
analyzed (e.g. at varying concentrations, time points, with anti-viral drugs, etc.) due to low
viability after multiple passages.

Figure 7. Lack of EBV IE and early gene activation in TW4 cells after NEO212 treatment.
Western blot analysis of ZEBRA, Ea-D2 and cleaved caspase 4 protein levels with actin as the
loading control. TW4 cells were treated with 50 µM of NEO212 with and without gemcitabine
and valproic acid (shown to activate the lytic cycle in C666.1 cells in combination) for 24 hours.
Cells were also treated with vehicle (Vh, DMSO) or remained untreated. C666.1 cells treated
with 0.3mM valproic acid and 3µM gemcitabine were used as a positive control (+)

TW4 cells were tested with varying concentrations of NEO212, with and without verified
reactivators of the EBV lytic cycle. Gemcitabine and valproic acid were included to enhance the
27

chances for EBV activation. TW4 cells showed no activation of EBV at any level. NPC666.1
cells treated with gemcitabine and valproic acid were loaded as positive controls to confirm
efficacy of the assay.

3.3. Verification of EBV reactivated-NPC666.1 sensitivity to antiviral treatment
3.3.1. Purpose of study
To verify reactivation of the EBV lytic cycle, NPC666.1 cells were tested with
combination therapy of NEO212 and ganciclovir to verify viral action and anti-viral drug
sensitivity. Observed values were compared to calculated additive (expected) values and
determined to be statistically significant, showing significantly increased cell death when treated
with NEO212 and ganciclovir in CFAs. Figure 5 indicates that EBV reactivation occurs as early
as 24 hours, where NEO212 dependent cell killing (by EBV reactivation) does not begin until 48
hours. To verify and expand upon this western blot, a cell count experiment was performed with
NEO212 with and without ganciclovir to determine time points of cell killing as well as possible
cell cycle arrest. To verify cell death, apoptotic markers were measured by western blotting, and
showed significant increase with the addition of ganciclovir at 24, 48 and 72 hour time points.
3.3.2. C666.1 cells show lower cell counts when treated with GCV and NEO212 (short term
28

Figure 8. C666.1 cell number over 96 hours with NEO212 treatment. C666.1 cells were treated
with varying concentrations of NEO212 with and without ganciclovir and counted at 0, 24, 48,
72, and 96 hour time points. All cell counts were completed in duplicate or triplicate with
average number of cells reported.
C666.1 cells show differences in cell growth patterns starting at 20µM NEO212. 5 µM
and 10 µM of NEO212 show similar growth curves to untreated C666.1 cells, with only a
possible discrepancy at the 48-hour time point. At 0, 24, 72 and 96 hours there was no
significant difference between cell counts. Although we see a drop in untreated cell count at 72
hours, we do not infer significance in comparison to 5 µM or 10 µM values.
29

20 µM and 40 µM concentrations act similarly, showing no significant increase or
decrease in cell number for the first 72 hours. After 72 hours the cells recover at these
concentrations and begin to grow exponentially. At all time points after treatment, 20 µM and
40 µM of NEO212 is significantly different from untreated cells, with a p-value <0.01 at all time
points. 60 µM and 80 µM were included as controls to verify the efficacy of NEO212 and the
assay. The average seeded cell count across conditions was 54,000 cells.
With ganciclovir addition, cells show significant increased sensitivity to NEO212.
Although there was no significant difference in cell count prior to treatment, we see statistically
significant difference in untreated vs. 5 µM NEO212 with ganciclovir at 72 and 96 hours with a
p-value <0.01. At all time points post treatment 10 µM NEO212 treatment with ganciclovir is
significantly different from untreated. 20 µM, 40 µM and 60 µM with ganciclovir also show
statistically significantly different cell counts from untreated cell counts at all time points (p-
value <0.01).

3.3.3. NEO212 and ganciclovir work synergistically in cell survival prevention
30

Figure 9. C666.1 cell survival after NEO212 treatment with and without ganciclovir. C666.1
cells were exposed to increasing concentrations of NEO212 with and without ganciclovir. Cell
survival was analyzed by CFA. The colony number from untreated cells is set to 100%. The
data shown is the average of duplicate or triplicate wells.
We see significant synergy when anti-viral drugs are used in combination with NEO212
chemotherapy, indicating the presence of viral particles, specifically early viral thymidine kinase
31

proteins (target to ganciclovir). In C666.1 cells, we see a 10 fold increase in cell death in CFA
with the addition of ganciclovir, most effectively at higher concentrations (20 µM) of ganciclovir
and moderate concentrations of NEO212 (1-10 µM).
Synergy was determined by comparison of actual drug combination colony values and
theoretical values determined by cell survival for each individual therapy combined. At all
concentrations in all experiments, we see significantly more cell death in actual values vs.
additive values. Ganciclovir does not have a cytotoxic effect on cells untreated with NEO212,
indicating the lack of spontaneous virion activation in this experiment.

3.3.4. NEO212 and ganciclovir work synergistically in apoptotic induction

Figure 10. Increase in apoptotic markers with GCV and NEO212 treatment in C666.1 cells.
Western blot analysis of Ea-D2 and various apoptotic protein levels with actin as the loading
control. C666.1 cells were treated with 1, 30 or 50 µM NEO212 with and without 20 µM GCV
at 24, 48 and 72 hours before harvest of cellular lysates Cells were also treated with vehicle
(Vhc, DMSO) or remained untreated. Full length (f.l.) and cleaved (cl.) forms of PARP and
cleaved caspases are indicated accordingly.

To verify cell death over only inhibition of cellular proliferation, western blots were
performed, testing for upregulation of apoptotic markers. Cells were treated with NEO212 with
and without ganciclovir at 24, 48 and 72 hours (after observation of halted cell growth in cell
count [3.3.2, figure 8]). At 48 hours, cells showed no difference in EBV activation (ZEBRA and
Ea-D2 levels showed no significant difference), but showed upregulation of apoptotic markers,
32

both in cleaved PARP/caspases and in uncleaved PARP/caspases. At 72 hours we see significant
upregulation of cleaved caspase 3 production. We also see the presence of a second (cleaved)
band forming and overall increase in protein levels for cleaved caspase 4 at 72 hours.

3.4. EBV Reactivation Pathway and NEO212 specificity
3.4.1. NEO212 elements – pNEO212, TMZ, POH, TMZ+POH
3.4.1.1. Purpose of study
Once NEO212’s ability to activate the EBV lytic cycle was confirmed, and anti-viral
synergy was shown at a cellular and subcellular level, determination of mechanism of action
became an important focus. Fresh NEO212 works through methylation of DNA but is unstable
in aqueous solution after 24 hours. To investigate the ability of NEO212 to kill independent of
methylation (of all DNA, not just MGMT lesions), we investigated pNEO212’s killing ability
and ability to activate the EBV lytic cycle.
To show NEO212’s unique capability to activate the EBV lytic cycle, we tested NEO212
against its individual components individually and in combination (TMZ, POH and TMZ &
POH). TMZ-related cell death has already been analyzed, showing little toxicity in C666.1 cells,
indicating it alone is not sufficient to trigger the lytic cycle or trigger apoptosis.

3.4.1.2. C666.1 cells are sensitive to pNEO212 but not TMZ or POH
33

Figure 11. C666.1 cell survival after TMZ, POH and TMZ &POH treatment. C666.1 and TW4
cells were exposed to pre-incubated (orange) and fresh (blue) NEO212 at varying concentrations.
The colony number from untreated cells is set to 100%. The data shown were the average of
duplicate or triplicate wells.
To determine the mechanism by which NEO212 activates the EBV lytic cycle, sensitivity
to fresh and pre-incubated NEO212 and TMZ was investigated in TW4 and C666.1 cells. TW4
cells showed significantly decreased sensitivity to pre-incubated NEO212 in comparison to fresh
NEO212, and virtually no sensitivity to pre-incubated TMZ, up to 500 µM. As with previous
experiments, TW4 cells show an IC50 of around 30 µM for fresh NEO212 and 150 µM for fresh
TMZ. TW4 cells show statistically significant differences in cell killing capacity between fresh
and pre-incubated NEO212 and TMZ.
C666.1 cells continued to show anomalous behavior to NEO212, showing no significant
difference in cell killing between pre-incubated and fresh NEO212, both with IC50s below 10
µM. As with previous experiments, C666.1 cells show an IC50 for TMZ above 250 µM (approx.
375 µM) C666.1 cells did show minor sensitivity to pre-incubated TMZ, but only at very high
concentrations, with an IC50 above 500 µM. C666.1 cells show statistically significant increase
in cell death when treated with fresh TMZ, but no significant difference between fresh and pre-
incubated NEO212.

34

Figure 12. C666.1 cell survival after TMZ, POH and TMZ &POH treatment. C666.1 cells
were exposed to TMZ, POH, or TMZ & POH at varying concentrations. The colony number
from untreated cells is set to 100%. The data shown were the average of duplicate or triplicate
wells. This experiment was verified again testing 50 µM, 150 µM and 250 µM concentrations
and showed conferring results.

To continue investigating the mechanism of action of NEO212, its various elements were
tested individually and in combination to determine their cell killing capacity. Neither TMZ nor
POH alone showed significant cell death at low concentrations, with an IC50 >250 µM. POH
and TMZ alone showed slightly increased cell killing capacity, but at relatively high
concentrations, with an IC50 at approximately 200 µM. NEO212 was also included as a control
for cell death, showing its normal IC50 around 3-10 µM.
3.4.1.3. pNEO212 and POH can induce IE and early protein production in C666.1 cells

35

Figure 13. EBV IE and early protein production in C666.1 cells with POH treatment. Western
blot analysis of ZEBRA, Ea-D2 and cl. C-3 protein levels with actin as the loading control.
C666.1 cells were treated with 50, 100 and 250 µM TMZ, POH or TMZ & POH for 48 hours
before harvest of cellular lysates. Cells were also treated with vehicle (Vh, DMSO) or remained
untreated. This experiment was verified again testing 25, 150 and 250 µM at 24h and showed
conferring results. All blots were performed in duplicate.

C666.1 cells were treated with TMZ, POH and TMZ in conjunction with POH to
determine which element of NEO212 is capable of activating the cell cycle. TMZ showed no
activation of the cell cycle, and no upregulation of apoptotic markers up to 250 µM. POH,
however, shows significant activation of the EBV IE and early proteins at as low as 50 µM
(warranting further investigation at lower concentrations), but not apoptotic markers (indicating
difference in cell death curves from NEO212). TMZ and POH together activate EBV IE and
early proteins at similar concentrations as POH, but show upregulation of cleaved caspase 3, an
apoptotic marker (synonymous with increased cell killing capacity in comparison to POH alone).
Statistical analysis of band density shows no difference in EBV IE and early gene activation
between POH alone and TMZ + POH.

Figure 14. EBV IE and early protein production in C666.1 cells with POH, pNEO212 and
fresh NEO212 treatment. Western blot analysis of ZEBRA, Ea-D2, and cleaved caspase 3/7
levels with actin as the loading control. C666.1 cells were treated with 5, 10 and 25µM of POH,
fresh NEO212 or pNEO212 (p.i.) for 48 hours before harvest of cellular lysates. Cells were also
treated with vehicle (Vhc, DMSO) or remained untreated.

C666.1 cells were treated with POH, pNEO212 and fresh NEO212 at lower
concentrations to determine apoptotic activation along with EBV IE and early protein
production. Pre-incubated NEO212 showed EBV IE and early activation similar to NEO212
36

treatment and produced significant apoptotic protein production. POH, shows lytic cycle
activation as low as 10 µM, but does not show increase of apoptotic markers at lower
concentrations.

37

Chapter 4 – Discussion
4.1. C666.1 cells indicate a novel killing mechanism for NEO212
Previous studies indicate a connection between NEO212/TMZ resistance and the
presence of MGMT (06-methylguanine methyltransferase) in nasopharyngeal carcinoma,
melanoma and glioblastoma cell lines, in vitro and in vivo. Temozolomide (and TMZ conjugates
such as NEO212) breakdown into MTIC that alkylates DNA at multiple positions, such as N3-
methyladenine, N-7-methyl-guanine, and O6-methyl-guanine [55]. Traditional treatment of
MGMT positive cells with NEO212 leads to concentration dependent MGMT depletion (with the
surviving cells resistant to TMZ treatment)[ [52]. MGMT is a DNA repair protein, responsible
for removing methyl groups from mutagenic O6-methylguanine. If left unrepaired, O6-
methylguanine will cause a mismatch pair to thymine, leading to transition mutation through
replication and possible cell death.
High levels of MGMT correlate with extreme TMZ resistance (with an approximate IC50
of 250 µM) and with moderate NEO212 resistance (approximate IC50 of 40 µM). In contrast,
MGMT-negative cell lines show much lower IC50s, only requiring around 20 µM TMZ and 2-3
µM NEO212 to reach 50% cell death value [52,53]. Correlation of MGMT levels was confirmed
as the indicator of these drug potencies when treated with an MGMT inhibitor, O6BG (raising
the IC50s to levels similar to MGMT-negative cell lines). When treated with pre-incubated
NEO212, all cell lines showed increased resistance, due to the instability of NEO212 in aqueous
solutions [55]. While investigating MGMT-positive cell lines, an outlier, NPC666.1 cells,
showed high resistance to TMZ (as expected), but surprisingly low resistance to NEO212 (2-
3µM), and high sensitivity to pre-incubated NEO212. These cells also did not show NEO212-
dependent MGMT depletion or change in sensitivity with O6BG treatment.
The nasopharyngeal carcinoma cell line C666.1 display endogenous MGMT expression,
as does NPC cell lines TW4 and TW1 (which do not display hypersensitivity to NEO212 or
preincubated NEO212). NPC666.1 cells differ from these other NPC cell lines due to the
presence of EBV proteins [9-11].
The Epstein-Barr virus, although primarily responsible for mononucleosis, is present in
multiple types of cancers, including B cell lymphomas, CNS lymphomas and gastric carcinomas
[4,5]. EBV latent proteins play a distinct role in carcinogenesis, through transformation of cells,
38

immune evasion, EMT transition, apoptotic avoidance and increase in cell growth factors.
Primary latent EBV genes include EBNA-1/2/3, LMP 1/2a/2b and EBERs.
EBV shows 3 primary forms of latency – Latency I, II and III respectively, with latency I
showing the lowest tumorigenicity and latency III showing the highest tumorigenicity. EBNA-1
was chosen as the target in NPC666.1 cells and prNPC cells as it is the only protein present in all
forms of EBV latency. C666.1 and prNPC cells showed EBNA-1 presence at 79kDa, the most
common size for EBNA-1 in tumor cell lines. Raji cells presented a truncated form of EBNA-1,
as expected 49 kDa. These differences in size do not correlate to any inhibition or regulation, as
both forms of the protein show similar function and efficiency.
This confirmation of latent EBV provides a novel option for cell killing, unrelated to
methylation at the O6-methylguanine position, and may offer new anti-viral treatment options
for nasopharyngeal carcinoma if latent EBV can be reactivated by NEO212.

4.2. NEO212 reactivates latent EBV in C666.1 cells and prNPC cells
Once latent EBV presence was confirmed in C666.1 and prNPC cells, reactivation levels
were tested through traditional methods (application of gemcitabine and valproic acid) to verify
the ability to reactivate the lytic cycle in this cell line [25, 44-47]. C666.1 cells were tested in
combination with gemcitabine and valproic acid but showed no synergy of EBV reactivation.
Once reactivation was shown by gemcitabine and valproic acid, with and without NEO212,
reactivation was tested with treatment with NEO212 in both cell lines at varying concentrations
and time points. NEO212 showed concentration dependent activation of the lytic cycle, with
EBV lytic protein production as 1 µM in C666.1 cells and 10 µM in prNPC cells. The inclusion
of prNPC cells verifies the ability of NEO212 to reactivate the EBV lytic cycle in primary tumor
cell lines, as well as immortalized cells, such as C666.1. C666.1 cells were tested at various time
points, showing synonymous EBV reactivation at 1-4 days, with significant increase in cell death
markers at 3 and 4 days. C666.1 cells did not begin to express apoptotic markers until day 2,
indicating a lag between EBV reactivation and apoptosis induction. Experiments on prNPC cells
were halted at this point, due to their inability to survive long term in cell culture (lasting only a
few passages), making them more difficult to study, in comparison to C666.1 cells. To confirm
that NEO212 was reactivating latent EBV as a cell death mechanism, TW4 cells (EBV negative)
39

were treated with NEO212, proving lack of any EBV reactivation, and continuing to show
decreased cell death. This further confirms the importance of latent EBV in NPC treatment by
NEO212.

4.3. Reactivation of EBV by NEO212 leaves C666.1 cells sensitive to anti-viral treatment
Once viral proteins were confirmed in C666.1 cells, experiments were performed to
determine the activity and targetability of these viral proteins. An anti-viral drug, previously
used in reactivated NPC treatment, ganciclovir, targets EBV viral thymidine kinase, a protein
kinase triggered by Ea-D2 transcription (early gene product) [48-50, 63]. When treated in
combination with NEO212, ganciclovir was shown to significantly increase cell killing in CFAs,
increasing cell death up to 10 times in comparison to NEO212 alone. These values were
compared with expected additive values to confirm synergy over additive effects.
Although CFA provides an effective tool for long term killing analysis, it does not verify
the difference between cell death and inhibition of cellular proliferation (cell cycle arrest), nor
short term killing effects (or short term cell cycle arrest effects). To clarify the difference and
verify the efficacy/cell death mechanism of NEO212 treatment, with and without ganciclovir,
cells were treated with NEO212 (with and without ganciclovir) and counted every 24 hours
(CFA data collected at approximately 17 days post seeding) for the first 5 days.
In the cell viability experiments, it is expected and shown that we see concentration
dependent decrease in cell count over time with the addition of NEO212. That being said, even
at concentrations that show full recovery of cells (20 µM and 40 µM) there is an initial halt in the
cell number for 48 hours. This could indicate arrest of the cell cycle, as indicated to occur in
concurrence with EBV reactivation in cell lines. With the addition of ganciclovir, we see this
similar cell proliferation arrest, but often for 1-2 more days, and at higher NEO212
concentrations, cells do not recover (indicating ganciclovir is able to kill the cells by EBV
targeting). Statistical comparison of NEO212 alone vs. with the addition of ganciclovir shows
that ganciclovir works synergistically with NEO212 to enhance cell killing at all concentrations
from 5 µM to 60 µM.
When comparing the two treatments, it should be noted we see no significant difference
between the starting cell counts (pre-treatment) (p-value >0.1). Cells treated with ganciclovir
40

alone vs. untreated cells showed higher growth after 96 hours, indicating that ganciclovir has no
cell killing capacity; therefore, all additive effects can be regarded as synergistic.
At 5 µM, we see significantly more cell death with the addition of ganciclovir at 24, 48,
72 and 96 hours (all p-values <0.01). At 120 hours (not pictured) cells eventually make a full
recovery, showing no statistically significant growth between treatments with or without
ganciclovir (p-value >0.1). At 10 µM, we do not see significantly increased cell death at 24
hours (likely due to high standard deviation values), but we do see significant cell death at 48. At
72 hours we see a 4 fold increase in cell death. At 96 hours the two points diverge even more,
increasing to a difference of 5 fold.
With 20 µM NEO212 alone, cell counts seem to stall (or even decrease) for the first 48
hours, then make a full recovery. With ganciclovir treatment added, cell counts increase slightly,
but stall again until the 96 hour time point, finally recovering at 120 hours. We do not see
significantly more cell death with the addition of ganciclovir at 24, 48, or 72 hours (both
treatments hover around the initial seeded cell count). At 96 hours the NEO212 only cells begin
to recover, with significantly more cell death with the addition of ganciclovir at 96 and 120
hours.
We see a similar trend in 40 µM NEO212 alone, with cell counts hovering around 50,000
cells for the first 48 hours. In comparison, we observe a steady decrease in cell count with the
addition of ganciclovir (although not as staunch as 60 µM or 80 µM cell death curves). There is
significantly lower cell counts with the addition of ganciclovir at 24, 48, 72, and 96 hour time
points. With 60 µM NEO212, with ganciclovir we see 99% cell death of cells were killed over
120 hours. Without ganciclovir, we see 95% cell death at 96 hours. With ganciclovir, there are
significantly lower cell count values at 48 hours as well as 96 hours.
In long term cell count studies (CFAs), we see significantly more colony formation
without ganciclovir, in comparison to both raw values and additive values. Synergy was
confirmed by verifying statistical significance between additive and observed values at NEO212
concentrations between 0 and 30 µM. At all values with all levels of ganciclovir, there is
significantly less colony formation, with 20 µM showing the greatest difference in cell curves.
No values above 20 µM ganciclovir have been tested.
41

Increase of cell death over inhibition of cellular proliferation was confirmed through
measurement of cell death (apoptotic markers), such as cleaved caspase 3, 4, and PARP. At 24
hours, 48 hours and 72 hours, cells treated with ganciclovir in conjunction with NEO212 showed
higher quantities of apoptotic proteins than with NEO212 treatment alone. Both NEO212 and
NEO212 with ganciclovir showed higher apoptotic protein expression than untreated or vehicle
treated C666.1 cells. Two concentrations of NEO212 were used at all time points, but did not
show any difference in synergy with ganciclovir. This verifies not only the ability of NEO212 to
kill cells, but the ability of NEO212 to trigger the EBV lytic cycle and work synergistically with
anti-viral drugs, such as ganciclovir. This also verifies that we are seeing an increase of cell
death with the addition of ganciclovir at 24, 48 and 72 hours. It should be noted that NEO212
alone does not show high levels of apoptotic proteins at 24 hours (although we do see low levels
of expression, not significant in comparison to 48 or 72 hours) and shows more uncleaved
versions of apoptotic proteins than at 48 or 72 hour time points. This comparison could indicate
cell cycle arrest for the first 24-48 hours, coupled with low level cell killing that increases over
time. Further experimentation is required to verify or negate cell cycle arrest.

4.4. EBV Reactivation Pathway and NEO212 specificity
Once EBV reactivation was confirmed, studies were performed to examine the potential
mechanisms by which NEO212 is reactivating the cell cycle. NEO212 traditionally acts on cells
by inducing DNA lesions through alkylation of various DNA positions, such as N3-adenine, N7-
guanine and O6-guanine positions [53]. With 24 hour pre-incubation in culture medium,
NEO212 (and TMZ respectively) loses alkylating abilities, therefore losing ability to inflict DNA
damage, and losing the ability to kill cells through an MGMT-dependent mechanism.
Due to the irrelevancy of MGMT presence in NPC666.1 cells in terms of cell killing, it
was postulated that cell death is triggered through an alkylation-independent mechanism. This
was verified by testing the effects of pre-incubated NEO212 on C666.1 cells. C666.1 cells
showed the same sensitivity to pre-incubated NEO212 in comparison to fresh NEO212 in terms
of cell killing capacity. Pre-incubated NEO212 was tested for ability to reactivate the EBV lytic
cycle, and successfully reactivated the production of EBV IE and early proteins, indicating EBV
reactivation. Pre-incubated NEO212 was shown ineffective in EBV reactivation or cell killing in
EBV-negative NPC cells (TW4). This information provides insight into the ability of NEO212
42

to reactivate the cell cycle, by indicating an alkylation-independent mechanism. Moreover, it is
known that TMZ (and NEO212) breakdown into the inactive AIC molecule after 24 hours of
incubation. This unknown molecule should contain a conjugate of POH, implying that POH
action is semi-responsible for this EBV reactivation.
To confirm the individuality of this NEO212 molecule, in comparison to its components,
TMZ and POH, TMZ and POH were tested for their ability to activate the EBV lytic cycle, as
individual components and in conjunction. NEO212 was shown far superior to its component,
requiring less than 1/100
th
the quantity of drug to show the same cell killing capacity. This being
said, POH was capable of reactivating transcription of IE and early gene products (ZEBRA and
Ea-D2). This ability to reactivate the EBV lytic cycle was verified at lower concentrations, but
not as low as NEO212. This indicates that, although POH may be partly responsible for cell
cycle reactivation, ONLY NEO212, fresh or pre-incubated, can create EBV reactivation that
leads to increase in cell death.
Over the past 10 years, reactivation of the EBV lytic cycle has proven a formidable and
effective method for treatment of various cancer cell types, including nasopharyngeal carcinoma
[44-49]. Until now, no direct connection has been made between NEO212’s killing capacity and
EBV reactivation. With this new information, we open a gateway to new discovery and
treatment options for patients of all kinds.
Current studies are focusing on in vivo application of NEO212 to subcutaneous injections
of C666.1 cells, as well as FACS analysis of cell cycle regulation by EBV reactivation.
Knockout of EBV in C666.1 cells is also of great interest to our lab, to test the ability of
NEO212 to kill cells, independent of EBV reactivation.
Once more is known on this pathway and NEO212’s reactivation of EBV, immunological
studies towards the role of B and T cells can further treatment options, through upregulation of
viral targeting. Exploration of EBV’s evasion of the immune system in nasopharyngeal
carcinoma can help to sensitize these cancer cells to the immune system, allowing the body to
kill cancer cells without the need of excessive exogenous drug treatment beyond NEO212.
NEO212’s ability to reactivate the lytic cycle also opens a slew of options for further
experimentation in other EBV latent cell lines, such as Burkitt’s lymphoma, gastric carcinomas
and CNS lymphomas. Due to the low cytotoxicity of NEO212 (and POH) in normal healthy
cells, this could provide an effective treatment with little to no negative side effects for patients.
43

NEO212 has been shown to be a novel and superior treatment for many cancer cell lines
already. with further knowledge of its mechanistic capabilities, the future holds many new
avenues of research, and many new therapeutic options to help extend (and hopefully save) the
lives of many patients to come.

44

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Abstract (if available)

Abstract Nasopharyngeal carcinoma (NPC) is a malignant nasopharyngeal neoplasm arising from the mucosal epithelium and is most prevalent in Southeast Asia. Nasopharyngeal carcinoma is the most prevalent form of nose and neck cancer and is difficult to diagnose, with less than 15% of patients diagnosed before stage III. A novel nasopharyngeal carcinoma cell line shows hypersensitivity to the TMZ-perillyl alcohol conjugate NEO212, proposing a novel treatment for certain nasopharyngeal carcinomas in the future. The objective of our study is to determine the mechanism of killing and treatment, postulated to be by Epstein-Barr Virus reactivation by NEO212 treatment in NPC cell lines. Analysis of EBV protein expression and cell death determined the presence of IE and early EBV proteins, as well as synergy with anti-viral drugs (ganciclovir) in C666.1 cells. NEO212 can provide a new and effective method of treatment for EBV-positive nasopharyngeal carcinoma in patients.

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Creator Hartman, Hannah (author)

Core Title Reactivation of the Epstein Barr virus lytic cycle in nasopharyngeal carcinoma by NEO212, a novel temozolomide-perillyl alcohol conjugate

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 08/15/2019

Defense Date 03/08/2019

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

Tag cancer biology,EBV,Epstein Barr virus,MMI,Molecular Biology,Molecular Microbiology and Immunology,nasopharyngeal carcinoma,NEO212,NPC,OAI-PMH Harvest,perillyl alcohol,temo plomo de,virus,viruses,western blot

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