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MGMT in emergence of melanoma resistance to temozolomide

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MGMT in emergence of melanoma resistance to temozolomide

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Content 1

MGMT IN EMERGENCE OF MELANOMA
RESISTANCE TO TEMOZOLOMIDE

SUSHANT KARNIK

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
In
MOLECULAR MICROBIOLOGY & IMMUNOLOGY
AUGUST 2017

Copyright 2017 Sushant Karnik
2

CONTENTS
Contents ………………….…………………………………………………………… 2
List of Figures …………………………...…………………………………………… 4
Chapter 1 – Abstract …………………………………………………………………. 6
Chapter 2 – Introduction ……………………….…………………………………..... 8
2.1 – Melanoma ……………………………………………………...... 8
2.1.1 – Causes ……………………...……………...………..… 10
2.1.2 - Classification and Staging ………………...……...….. 12
2.1.3 – Epidemiology …………………………………….……. 13
2.1.4 - Current Treatment ………………………………......... 15
2.1.4.1 - Surgery and Radiation ………………….…. 15
2.1.4.2 - Immunotherapy ………………………..…… 15
2.1.4.3 - Checkpoint Inhibitors …………………..….. 16
2.1.4.4 - Cytotoxic Chemotherapy ………………..… 17
2.2 – Temozolomide ……………………………..……….………...... 18
2.2.1 - Resistance to Temozolomide ……………...….……… 20
2.3 - O6-Benzylguanine ………………………….……………...…… 24
2.4 - Temozolomide-POH ………………………..….……….…........ 25
2.5 - Hypothesis ………………………………………………………. 27
Chapter 3 - Materials & Methods …………………………..……………………..... 29
3.1 - Cell Lines and Maintenance …………………………..………... 29
3.2 - Western Blot ……………………………………………………… 29
3.3 - Colony Cloning …………………………………………………… 30
3.4 - Colony Formation Assay ………………………..……….…...... 31
3

3.5 - Chemotherapeutic Agents …………………………………….. 31
Chapter 4 – Results ………………………………...……………………………..... 32
4.1 - Expression of MGMT in various melanoma and glioblastoma
cell lines …………………………………………………………….... 32
4.2 - O6BG is a potent MGMT inhibitor ……….………………….. 33
4.3 - Variables responses to TMZ ± O6BG in MGMT- and MGMT+
cell lines ……………………………………………………………... 34
4.3.1 - A2058 ………………………………………...….…….. 34
4.3.2 - Clone 53 ……………………….……………………..... 35
4.3.3 - Clone 58 ………………………...……………….……. 36
4.4 - Presence of MGMT+ cells in a MGMT- cell line ……………. 37
4.5 - Response to TMZ ± O6BG in newly established clones ...... 38
4.6 - Response to TP ± O6BG in newly established clones ……... 41
4.7 - Response to cisplatin ± O6BG in newly established clones .. 44
Chapter 5 – Discussion ……………………………………………………………… 47
Chapter 6 – Bibliography ……………………………………………………………. 50
Acknowledgement ……………………………………………………………..…... 54

4

LIST OF FIGURES
Figure 1: A schematic diagram of normal skin with melanocyte ………………… 8
Figure 2: Malignant melanoma in a female .....................................………….….. 9
Figure 3: Formation of cyclobutane pyrimidine dimers …………………………. 10
Figure 4: Global incidence of melanoma per year ……………………………….. 14
Figure 5: Melanoma mortality rates in the US …………………………………….. 14
Figure 6: Chemical Structure of Temozolomide ……………………………….…. 18
Figure 7: Temozolomide mechanism of action …………..……………………….. 20
Figure 8: Cell fate after TMZ exposure in the presence of MGMT …………….. 22
Figure 9: Schematics of mode of action of MGMT ……………………………….. 22
Figure 10: Chemical Structure of NEO212/TP ……………………………………. 25
Figure 11: MGMT expression in various melanoma and glioblastoma multiforme
cell lines ……………………………………………………………………………….. 32
Figure 12: WB analysis of Clone 53 cells treated with TMZ, TP and O6BG ….. 33
Figure 13: A2058 vs TMZ ± O6BG .................................................................... 34
Figure 14: Clone 53 vs TMZ – O6BG……………………………………………… 35
Figure 15: Clone 53 vs TMZ + 15 uM O6BG ...…….…………………………….. 35
Figure 16: Clone 58 vs TMZ – O6BG ……………………………………………… 36
Figure 17: Clone 58 vs TMZ + 15 uM O6BG ………...…………………………… 36
Figure 18: WB analysis of A2058 Clones 1 through 8 …………………………… 37
Figure 19: WB analysis of A2058, Clones 9, 10, 2, 3 and 4 …………………….. 38
Figure 20: A2058 vs TMZ ± O6BG ………...…………………………………….… 39
Figure 21: Clone 2 vs TMZ ± O6BG ………...…………………………………….. 39
Figure 22: Clone 3 vs TMZ ± O6BG ……...………………………………………… 40
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Figure 23: A375 vs TMZ ± O6BG ……...…………………………………………... 40
Figure 24: A2058 vs TP ± O6BG …………...……………………………………… 41
Figure 25: Clone 2 vs TP ± O6BG ……………...…………………………………. 42
Figure 26: Clone 3 vs TP ± O6BG …………………………………………………. 42
Figure 27: A375 vs TP ± O6BG ………...………………………………………….. 43
Figure 28: A2058 vs Cisplatin ± O6BG …………...……………………………….. 44
Figure 29: Clone 2 vs Cisplatin ± O6BG ………...………………………………… 45
Figure 30: Clone 3 vs Cisplatin ± O6BG …………...……………………………... 45
Figure 31: A375 vs Cisplatin ± O6BG …………...………………………………… 46

6

1 – ABSTRACT
Melanoma incidence has been on the rise and alike many other
malignancies, major impediments in its treatment and management are the high
metastatic potential and frequent development of chemo-resistance. Use of
surgery and radiation are limited to local and regional tumors and current
therapeutic strategies provide a modest increase in overall survival.
Temozolomide (TMZ), a DNA alkylating agent, which is standard of care
for patients with glioblastoma multiforme and astrocytoma, is currently also being
evaluated for use against melanoma. Treatment with temozolomide (TMZ) has
also been met with development of resistance and is frequently linked to
overexpression of a DNA repair protein, MGMT (O6-methylguanine-DNA-
methyltransferase).
In this study, we attempt to characterize the emergence of MGMT driven
resistance to TMZ in vitro. Previous studies by our research group have shown
that treatment with TMZ can induce or select for cell populations that express
MGMT from a MGMT negative melanoma cell line. We hypothesized that, TMZ
treatment leads to selective propagation of cells which express MGMT, which are
likely already present in the culture.
Here, we show the presence of a pre-existing small number of MGMT
expressing cells in a culture of the MGMT-negative melanoma cell line (A2058).
These MGMT positive cells, also differ from the parental cells in response to
treatment with TMZ and NEO212, in the absence of O6-Benzylguanine (O6BG),
an MGMT inhibitor.
7

Inclusion of O6BG makes the MGMT positive cells sensitive to alkylating
agents. Emergence of MGMT resistance in vitro emphasizes further, the need for
assessing its expression in melanoma tumors, and development of novel
strategies such as a combination of NEO212 and O6BG to increase survival along
with improvement in quality of life.

8

2 - INTRODUCTION
2.1 MELANOMA:
Melanoma, also known as malignant melanoma, is a type of skin
cancer that develops from the melanocytes, and it is the most aggressive type of
cancers in humans. Melanocytes are the cells in the skin, specifically in the basal
layer of the epidermis, that make the pigment, melanin, and are derived from the
neural crest. Melanomas most commonly arise in the skin, but can also develop in
mucosal surfaces and the uveal tract. The incidence of melanoma has increased
in the past two decades, and the disease is extremely difficult to treat after the
onset of metastasis, with a high mortality rate. [1,2].

Figure 1: A schematic diagram of normal skin with melanocyte. (Adapted
Winslow, 2008, National Cancer Institute.)

The first accredited mention of melanoma was by Hippocrates in the fifth
century, B.C. Although melanoma is not a new disease, evidence for its occurrence
in ancient past seems to be low. [3]. ‘‘However, one example lies in a 1960’s
9

examination of nine Peruvian mummies, radiocarbon dated to be approximately
2,400 years old, which showed apparent signs of melanoma: melanotic masses in
the skin and diffuse metastases to the bones.’’ (Urtega, 1966)
‘‘John Hunter is reported to be the first to operate on metastatic melanoma
in 1787. Although not knowing precisely what it was, he described it as a cancerous
fungous excrescence.’’ (Bodenham, 1968). According to this study, it was not until
1968 that microscopic examination of the preserved specimen revealed it to be an
example of metastatic melanoma. [4]. An English general practitioner, William
Norris, presented the first report of melanoma in English from Stourbridge, in 1820.
‘‘In his later work in 1857, he remarked that there is a familial predisposition for
development of melanoma.’’ (Norris, 1820; Norris, 1857). Norris also suggested a
link between nevi and melanoma and a possible relationship between melanoma
and environmental exposures, based on his observation that a majority of his
patients had pale complexions. He likewise portrayed that melanomas could be
amelanotic and later demonstrated the metastatic potential of melanoma by
observing their dissemination to other organs. [5,6].

Figure 2: Malignant melanoma in a female (Adapted from Uhlen et al., 2015).
10

2.1.1 CAUSES:
Melanomas are usually caused by DNA damage resulting from exposure
to ultraviolet (UV) light from the sun. Heredity also assumes a role in the disease.
There is insurmountable evidence that exposure to UV radiation (UVA and UVB)
is one of the leading causes of melanoma. UVB light (wavelengths between 315 –
280 nm) from the sun is taken up by skin cell DNA and results in a type of direct
DNA damage called cyclobutane pyrimidine dimers (CPDs). [7]. The joining of two
adjacent pyrimidine bases within a DNA strand forms thymine-thymine, cytosine-
cytosine or cytosine-thymine dimers. UVA (wavelengths between 400 – 315 nm)
is somewhat similar to UVB, but is absorbed by the DNA at a lower efficiency than
UVB. Sun beds or tanning beds are sources of deep penetrating UVA rays, and
their regular use is a risk factor in the occurrence of skin cancers, including
melanoma. [7,8].

Figure 3: Schematic representation of formation of a cyclobutane pyrimidine
dimer by linkage of two adjacent pyrimidines (Adapted from Burkett, 2005).

Some rare mutations, which often run in families, greatly increase
melanoma susceptibility. Familial melanoma is thought to be genetically
11

heterogeneous, and loci for familial melanoma show up on the chromosome arms
1p, 9p, and 12q. Many hereditary events have been identified with melanoma's
pathogenesis. [9,10]. A family history of melanoma greatly increases a person's
risk, because mutations in several genes have been found in melanoma-prone
families. There is a high risk of developing a second primary tumor in people with
a history of one melanoma. [11,12].
According to a study, ‘‘one class of mutations affects the gene CDKN2A.
An alternative reading frame mutation in this gene leads to the destabilization
of p53. Another mutation in the same gene results in a nonfunctional inhibitor
of CDK4, a cyclin-dependent kinase that promotes cell division. Scattered
throughout the genome, these mutations reduce a cell's ability to repair DNA.’’ [9].
Mutations that cause the skin condition, Xeroderma Pigmentosum (XP), have also
been indicated in melanoma susceptibility. [13].
The MAPK pathway and the AKT pathway are two basic pathways taking
part in melanocyte propagation and melanoma development. [14]. BRAF is a
serine-threonine kinase, a member of the RAF kinase family, which is a part of the
RAF/MEK/ERK cascade, also referred to as the MAPK pathway. Thus pathway
regulates cell growth, survival and differentiation. It is activated by many different
membrane-bound receptors including receptor tyrosine kinases and G-protein
coupled receptors. Receptor activation by ligand binding, activates the small G
protein RAS, which is the upstream activator of RAF. RAF kinase family consists
of ARAF, BRAF and CRAF. MEK1/2, which is downstream of RAF can be activated
by all three RAF kinases. MEK1/2 then activate ERK1/2, which upon activation,
phosphorylate their target proteins, either in the cytoplasm or in the nucleus, into
which they translocate. Inside the nucleus, the targets of ERK1/2 are transcription
12

factors involved in the regulation of cell proliferation, differentiation or survival
related genes. [15].
B-RAF mutations are well established in many malignancies, but have the
highest frequency in malignant melanoma. The associated T-A nucleotide change
is not related to and distinct from the pyrimidine dimers formed by exposure to UV
radiation. [16]. There seem to be many identified amino acids as target sites for
BRAF protein mutations in melanoma, however, the single predominant site at
which the occurrence of mutation is greatest, is valine at position 600 (V600);
resulting in hyper-activation of the protein. It should be noted that BRAF mutations
have also been shown in benign nevi, implying that BRAF mutations are necessary
but not entirely sufficient in melanoma development. These mutations in BRAF can
make it oncogenic, resulting in constitutive ERK signaling, and stimulation of
survival and proliferation, which is evident from the fact that oncogenic BRAF
stimulates the expression of cell cycle progression genes such as CDK2 and
CDK4. [15].
The AKT pathway, which is hyper-activated in melanoma, has also been
shown to co-operate with oncogenic BRAF in tumor progression. Besides, many
studies have indicated the importance of BRAF not only in melanoma initiation and
progression, but also in maintaining essential tumor functions. Thus, there is much
emphasis on BRAF as a crucial therapeutic target in treatment of melanoma. [15].
2.1.2 CLASSIFICATION & STAGING:
Melanoma is divided into the following types -
 Lentigo maligna
 Lentigo maligna melanoma
 Superficial spreading melanoma
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 Acral lentiginous melanoma
 Mucosal melanoma
 Nodular melanoma
 Polypoid melanoma
 Desmoplastic melanoma. [17].
Melanoma stages with 5-year survival rates include:
 Stage 0: Melanoma in situ (Clark Level I), 99.9% survival
 Stage I / II: Invasive melanoma, 89–95% survival
 Stage II: High risk melanoma, 45–79% survival
 Stage III: Regional metastasis, 24–70% survival
 Stage IV: Distant metastasis, 7–19% survival. [18].

2.1.3 EPIDEMIOLOGY:
Skin cancer is one of the most common malignancies diagnosed in the
United States. Melanoma represents less than 5% of skin cancers but results in
60% of skin neoplasia deaths. [1]. Melanoma was once a rare cancer. However in
the past 6-7 decades, its incidence in the developed world has risen faster than
any other cancer type. Cutaneous malignant melanoma (CMM) is among the top
ten commonly diagnosed cancers in the USA in both genders. A significant feature
of melanoma is that the incidence rate is extremely high in lighter skinned patients,
whereas darker skinned patients have a much lower incidence rate. Besides,
melanoma stands out among other skin cancers in terms of severity, it is estimated
that one person succumbs to melanoma every hour in the US. Lamentably,
individuals at their prime ages (median age: 52 years) are statistically most prone
to the disease. [19].
14

Figure 4: Global incidence of melanoma per year (Adapted from Holmes, 2014)

Figure 5: Melanoma mortality rates in the US. The highest rates are seen in the
northwestern US. (Adapted from Berrios-Colon & Williams, 2012).

Australia and New Zealand have the highest rates of melanoma in the
world. High incidence rates are also prevalent in Northern Europe and North
America, while it is less common in Asia, Africa, and Latin America. [2]. Overall,
15

melanoma seems to be more common in men than in women, and survival is worse
in men. Age is a well-recognized and important factor in the epidemiology of
melanoma. Though the disease is far more common in adults, it can also be found
in children. Older patients have poorer survival in comparison with younger
patients. [20].
2.1.4 CURRENT TREATMENT:
2.1.4.1 SURGERY & RADIATION:
Surgical excision is the most common and well-accepted treatment for
melanoma. Breslow thickness is often used as a substitute indicator for
invasiveness and prognosis of malignant melanoma. [19]. Radiation is regularly
used after surgical resection for patients with locally advanced melanoma or for
individuals with distant metastases that cannot be surgically excised. X-ray beams
in Kilovoltage appear to deliver the maximum radiation dose occurring close to the
skin surface and are used in radiation therapy [21].
2.1.4.2 IMMUNOTHERAPY:
Melanoma has been shown to be a particularly immunogenic tumor;
Hence, immunotherapy of melanoma has developed as a dynamic field for clinical
research. By far, ‘‘immunotherapies have been developed for malignant melanoma
patients with distant metastases (stage IV) and also for stage II–III patients with
micrometastatic disease among the fraction of patients having a microscopic
spread of tumor detected in lymph nodes.’’ (Erdei et al., 2010) Interrupting
immune-inhibitory mechanisms and adoptive immunotherapy using T-cells with
specific tumor reactivity for stimulating immune responses are among the
strategies that have been tested, but have shown varied success. [19].
16

With the exceptions of interleukin-2 and interferon-alpha, most other
studies about use of cytokines for melanoma treatment have failed. Interferon-
alpha has gained a relatively wide use in the adjuvant setting with evidence to
suggest its good effect upon overall survival, relapse-free survival and a 10%
decrease in mortality. However, there is uncertainty over its dosage, route of
administration and duration. [22].
There has been notable progress in research for melanoma treatment in
the past few years, involving immune checkpoint blocking antibodies and BRAF
mutation targeting agents. Antibodies against cytotoxic T-lymphocyte-associated
antigen 4 (CTLA-4) and programmed death 1 (PD-1) have been approved for used
by the FDA. Ipilimumab is an antibody against CTLA-4 and has been shown to
improve overall survival. Nivolumab and pembrolizumab are antibodies against
PD-1 and are used for treatment of metastatic melanoma. Both have shown
considerable efficacy over chemotherapy in clinical trials. [23].
2.1.4.3 CHECKPOINT INHIBITORS:
Vemurafenib and dabrafenib are two inhibitors targeting the BRAF
mutation and thus the MAPK pathway, which have been developed recently and
show improved progression-free and overall survival, as compared to
chemotherapy in patients with metastatic melanoma with BRAF V600 mutations.
Unfortunately, it has been observed that resistance develops in a majority of
patients treated with these inhibitors in a duration of few months, and is believed
to be predominantly due to reactivation or BRAF-inhibitor-induced paradoxical
activation of the MAPK pathway. Incidence of secondary cancers like squamous-
cell carcinoma, developing due to this paradoxical activation have also been
reported. [22,24].
17

Inhibition of MEK, downstream of RAF in the MAPK pathway, is another
option that has been explored as a target. Trametinib, a MEK inhibitor, has been
evaluated independently and has shown improved survival in patients with BRAF
mutation positive melanoma, without the paradoxical activation. Studies are now
being carried out to assess the efficacy of a combined BRAF and MEK inhibition,
against melanoma. [22,24]. C-KIT is another target that is being investigated in the
treatment of metastatic melanoma. It is a receptor tyrosine kinase that activates
MAPK signaling pathways, and studies have found mutations in c-kit in several
types of melanoma. Imatinib and sutinib are two inhibitors that have been
evaluated to target c-kit mutations and show some promise. [25].
2.1.4.4 CYTOTOXIC CHEMOTHERAPY:
Currently, multiple therapeutic approaches are used to treat patients with
metastatic melanoma, including chemotherapy and biologic therapies, both as
single treatments and in association. ‘‘The chemotherapeutic agents with
reproducible activity against melanoma include dacarbazine (DTIC), the platinum
analogs, various nitrosoureas, and tubular toxins, such as vinca alkaloids and
taxanes. Dacarbazine is the only chemotherapy drug currently approved for the
treatment of metastatic malignant melanoma. In large randomized trials, response
rates with dacarbazine ranged from 6% to 15%.’’ (Bajetta et al., 2002).
Given these disappointing overall results, the consensus among most
physicians is that patients with metastatic malignant melanoma should be given
more convenient treatment options or experimental treatment for these patients
seems appropriate. Because of oral dosing, Temozolomide is considered to be a
reasonable choice, particularly for patients who would have difficulty traveling to
treatment centers for intravenous chemotherapy. Although surgery is currently the
18

preferred option for patients with resectable or solitary brain metastases from a
primary melanoma, for individuals that require systemic treatment for brain
metastases, Temozolomide is the preferred chemotherapeutic agent. [26,27].

2.2 TEMOZOLOMIDE (TMZ):

Figure 6: Chemical Structure of Temozolomide (Adapted from drugs.com)
TMZ is a prodrug and an imidazotetrazine derivative of the alkylating agent,
dacarbazine. Presently, TMZ is a choice of treatment for some brain cancers like
glioblastoma multiforme (GBM) and astrocytoma. The antitumor activity of TMZ
depends on the formation of a reactive methyldiazonium cation, which is
responsible for DNA methylation. This species is found to produce methyl adducts
at the accessible nucleophilic atoms in DNA. The majority of the DNA methylation
caused by TMZ occurs at N-7 guanine, while the rest occurs at N-3 adenine and
O-6 guanine. Although the methylation at O-6 guanine accounts for only a small
percentage of the total, evidence suggests that this lesion can be correlated with
19

TMZ cytotoxicity, as these adducts are considered particularly mutagenic and
cytotoxic. [28].
As indicated in this study, ‘‘The cytotoxic effects generated by DNA O6 -
methylguanine (O6 -MeG) rely on the formation of O6 -MeG:T and O6 -MeG:C
mis-pairs during DNA duplication and on the subsequent engagement of the
mismatch repair (MMR) system. According to the futile repair model, the MMR
system recognizes and attempts to process O6-MeG:T, and O6-MeG:C mis-pairs.
However, since the modified base is in the template strand, and MMR targets the
newly synthesized strand, this repair event results in the degradation of the
pyrimidine-containing strand and the subsequent reinsertion of C or T opposite the
O6-MeG.’’ (Alvino et al., 2006).
Repeated futile attempts at repair actually lead to the formation of gaps in
the newly synthesized DNA, which are converted into DNA double strand breaks
in the following S-phase. O6-MeG:T and O6-MeG:C are processed unsuccessfully
and lead to DNA damage. As a result, a signaling cascade is activated that results
in cell-cycle arrest at the G2 phase in the next doubling event. This is then followed
by apoptosis, mitotic catastrophe, or a senescence-like state. [28,29].
20

Figure 7: Temozolomide is converted to 3-methyl-(triazen-1-yl) Imidazole-4-
carboxamide (MTIC), which is broken down to the methyldiazonium cation, which
methylates DNA (Adapted from Saleem et al., 2003)

Temozolomide is currently in the advanced phase of evaluation and shows
the following ‘’potential advantages related to its pharmacokinetic and biochemical
properties – 1) high oral bioavailability, 2) spontaneous conversion at physiologic
pH, into an active metabolite, 3) ability to cross the blood-brain barrier at a
pharmacologically active concentration, 4) associated myelosuppression, and 5)
greater possibility of dose/schedule modulation.’’ (Bajetta et al., 2002). TMZ has
been shown to be advantageous in terms of improving the quality of life in patients
treated with it. [26].
2.2.1 RESISTANCE TO TEMOZOLOMIDE:
A major obstacle in the successful treatment of cancer is the rapid
development of resistance to chemotherapeutic agents. Many tumors are
intrinsically resistant to chemotherapy or may develop resistance after an initial
period of response. ‘‘Acquired chemoresistance can result from both drug-induced
21

selection of pre-existing resistant cell clones and drug-induced genetic and
epigenetic alterations of neoplastic cells.’’ (Alvino et al., 2006).
By increasing our understanding of the mechanisms underlying the
development of resistance, we may be able to develop more successful
therapeutic strategies. Metastatic melanoma has been found to exhibit primary or
acquired resistance to chemotherapy. As mentioned before, the cytotoxicity of
TMZ, like that of dacarbazine, is primarily due to its ability to methylate the O6
position of guanine in DNA. There appears to exist an inverse correlation between
the cytotoxicity of TMZ or in other words, the sensitivity of tumor cells to TMZ, and
the presence and activity of the DNA repair protein O6-methylguanine-DNA-
methyltransferase (MGMT). MGMT repairs this methyl lesion on O6-guanine in an
auto-inactivating manner, whereby; it takes up the methyl group via binding to an
internal cysteine residue, and is then degraded by the ubiquitin pathway. [29].
Previous studies have shown that primary resistance of melanoma cells to
TMZ is mainly dependent on high MGMT levels. Lack of MGMT has been shown
to make cells hypersensitive to both the cell death-inducing and mutagenic actions
of alkylating agents. In vivo studies have demonstrated that MGMT
-/-
mice have
LD
50
values about 20 times lower than MGMT
+/+
mice when treated with
dacarbazine. [29,30].
22

Figure 8: Cytotoxic methylating lesion set by Temozolomide at O6-guanine which
can be repaired in the presence of MGMT. In its absence, the mismatch repair
system makes futile attempts to repair the lesion but fails. (Adapted from
Wolfgang et al., 2014)

Figure 9: Schematics of mode of action of MGMT (Adapted from Stupp, 2010)

23

Despite MGMT expression being an important prognostic marker linked to
temozolomide resistance and patient survival, it is not the only DNA repair
mechanism that is associated with TMZ resistance. As seen earlier, ‘‘The
mismatch repair (MMR) pathway removes thymidine but is unable to repair the
original O6-meG lesion. This results in repeated unsuccessful MMR attempts to
repair the DNA, which ultimately leads to double-strand breaks, replication arrest,
and cell death. MMR thus plays a crucial role in temozolomide-induced cytotoxicity.
A deficiency in the MMR pathway would result in the failure to recognize and repair
at this position, thus resulting in continued DNA replication past the O6-meG block,
thereby producing no double-strand breaks, no cell death, and ultimately
resistance to temozolomide.’’ (Cho, et al., 2014).
There is another mechanism of temozolomide resistance that has been
implicated - the base excision repair (BER) pathway. This pathway involves a
number of DNA repair proteins, which cooperate in removing unwanted or harmful
DNA bases such as N7-MeG. Removal of these adducts ensures cellular
replication & survival. Also, low levels of BER proteins result in incomplete repair
and lead to cytotoxicity, but overexpression of these proteins is a protective
mechanism and induces temozolomide resistance. [31].
One protein that is important in successful DNA damage signalling and
BER is poly (ADP-ribose) polymerase-1 (PARP-1), that is activated in response to
DNA damage. It recruits BER in response to DNA damage, facilitating efficient
DNA repair and cell survival. PARP deficient cells have been shown to be more
sensitive to carcinogenic agents due to increase in the frequency of DNA strand
breaks. Studies also demonstrate increase in TMZ cytotoxicity in vitro and in vivo
24

by PARP inhibition followed by subsequent BER disruption and can be a means
of overcoming TMZ resistance. [32].
2.3 O6-BENZYLGUANINE (O6BG):
O6BG is a powerful inhibitor of MGMT, and it was developed based on its
restricted mechanism of action. The extent of repair by MGMT is dependent on the
initial amount of the protein. The level of this protein varies in cell types and tissues.
As mentioned earlier there is an inverse correlation between the presence of this
repair protein and the sensitivity of cells to the actions of the alkylating agents.
[33,34].
To counter this problem, in terms of chemotherapy with alkylating agents,
approaches were developed to modulate the activity of the alkyltransferase activity
and then examine the consequences of such modulation on carcinogenesis,
mutagenesis, and cytotoxicity. Another approach was to study the effects of
absence of alkyltransferase activity. Attempts were made to deplete this activity
with the use of inhibitors. It was thought that, these inhibitors, if potent and specific,
could have a considerable potential in chemotherapy besides being useful for
studies of the role of alkyltransferase in protection from alkylating agents. Previous
studies have demonstrated about 80% decrease in the amount of alkyltransferase
when cells were exposed to O6-methylguanine, which increased cell
responsiveness to chemotherapeutic agents. [34,35].
O6-methylguanine was found to act as a weak substrate for the
alkyltransferase protein. This function leaves less alkyltransferase available for
repairing cytotoxic lesions that are introduced by alkylating agents. [34]. However,
the clinical potential of O6-methylguanine was not enough due to the need for high
25

doses, long exposure periods and only a partial depletion in the target protein
activity. Benzyl groups are more easily displaced in biomolecular displacement
reactions than methyl groups, and it was predicted that the alkyltransferase could
have increased affinity for O6-benzylguanine (O6BG). After testing, it was found
that O6BG, even at low doses is a potent inactivator of the alkyltransferase and
that it can be useful to enhance the cytotoxicity of alkylating chemotherapeutic
agents. [34,36].
As mentioned in one study, O6BG reacts with MGMT by covalent transfer
of the benzyl group to the cysteine, which is the active site. This transfer causes
an irreversible inactivation of the enzyme. Long-lasting reduction in
alkyltransferase was observed at larger doses of O6BG, without any obvious
cytotoxic effects. O6BG at therapeutic levels, by itself, was found to be not toxic.
However, it was efficient in rendering the tumor cells 2-14 fold more sensitive to
alkylating agents in in vitro and in vivo settings. Thus, it was established that O6BG
is a potential therapeutic enhancer of alkylating drugs. [33].
2.4 TEMOZOLOMIDE-POH (NEO212) (TP):

Figure 10: Structure of NEO212 (Adapted from NeOnc Technologies, Inc.)
26

Perillyl alcohol (POH) is a naturally occurring monoterpene found in
lavender, liliac oil, cherries, spearmint, celery seeds and other plants that has been
used orally for the treatment of a variety of cancers, including breast cancer,
pancreas cancer, and lung carcinomas. It has been found to induce tumor
regression in rats with mammary carcinomas and inhibit growth of liver tumors by
apoptosis. POH has been shown to be a cytotoxic agent by functioning as a Ras
inhibitor, cell-cycle inhibitor, and upregulator of the pro-apoptotic protein Bax.
However, due to its adverse gastrointestinal side effects, it not well tolerated orally.
However, a Brazilian study found that intranasal administration of POH for
recurrent malignant glioblastoma multiforme is well tolerated with minimum
systemic side effects. Studies by our own research group have shown that POH
was cytotoxic to a variety of glioma cell lines, including the TMZ-resistant ones,
without significantly affecting normal cells. POH was also found to be a chemo-
sensitizing agent, by enhancing the cytopathic effect of TMZ. ‘‘POH decreased
pro-angiogenic growth factors and inhibited tumor cell invasion.’’ (Cho, et al.,
2012). The cytotoxic activity appeared to be due to ER-stress. In vivo, POH was
found to increase survival and reduce the growth of TMZ-resistant gliomas.
[37,38,39].
Temozolomide-POH (TP) or NEO212 is a novel drug, in which POH is
conjugated to temozolomide. Taking into consideration the drawbacks of
temozolomide such as deleterious myelosuppression and risk of developing
resistant tumors, a better-tolerated yet effective chemotherapeutic strategy is
needed. ‘‘The choice of POH as a linkage partner for temozolomide was based on
the knowledge that this agent has recognized anticancer activity and is cytotoxic
to temozolomide-resistant gliomas.’’ (Cho et al., 2014).
27

It was hypothesized that this covalently linked compound would yield an
inherently higher anticancer activity. Previous studies by our research group
demonstrated that NEO212 is cytotoxic to several types of temozolomide-resistant
glioma cells, including MGMT positive and MGMT negative ones. It possesses a
3-fold higher cytotoxic potency than that of TMZ in nasopharyngeal carcinoma cell
lines. They also show that the conjugated drug is much more active than the
mixture of its components, while showing no significant cytotoxicity to normal cells.
Furthermore, NEO212 is also able to downregulate MGMT protein levels in vitro.
[31,38].

2.5 HYPOTHESIS:
Taking the available knowledge into account, and with an objective of
improving chemotherapeutic outcome in melanoma treatment, we decided to
further investigate this emergence of resistance in vitro and its impact on the
efficacy of temozolomide and NEO212.
Our research group has previously seen that, following treatment of MGMT
negative melanoma cell lines with temozolomide, small populations of cells survive
and can be grown in culture while showing robust MGMT expression. This raised
a question as to whether, temozolomide induces the emergence of MGMT
overexpression by mutagenesis or epigenetic alterations or, the drug treatment
causes selection of MGMT positive cells.
We hypothesized that, there is in fact, a low number of cells that are MGMT
positive, already present in a melanoma cell line that appears to lack MGMT
expression on an immunoblot. We believe, this helps in answering the question
28

posed earlier and implies that TMZ treatment at an initial lower dose, in essence,
leads to selective survival of these MGMT positive cells in a MGMT negative
melanoma cell line.
With our studies, we have been able to show the presence of one MGMT
positive cell colony out of ten colonies, which were picked arbitrarily, from a low-
density culture of a MGMT negative melanoma cell line, untreated with any agent.
We also further demonstrate that this particular MGMT positive colony defers
substantially in response to temozolomide as compared to its parental cell line.
This finding, we anticipate, will be critical in terms of prognosis of patients with
metastatic melanoma and offers a new perspective on the emergence of
resistance.

29

3 - MATERIALS & METHODS
3.1 CELL LINES & MAINTENANCE:
Human melanoma cell lines, A2058, A375 and the A2058 derived Clone
53 & 58 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
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 5% CO
2
. All cell culture
reagents were provided by the Cell Culture Core Lab of the USC/Norris
Comprehensive Cancer Center and prepared with raw materials from
Cellgro/MediaTech (Manassas, VA), FBS was obtained from Omega Scientific
(Tarzana, CA). All cell culture techniques were carried out in an aseptic Biosafety
level 2 (BSL2) environment.
Cell Lines MGMT Status
A2058 -
Clone 53 +
Clone 58 +
A375 +
Clone 2 -
Clone 3 +

3.2 WESTERN BLOT:
The cells were harvested for Western Blot by trypsinisation or scraping.
Total cell lysates were prepared by lysis of cells with radioimmunoprecipitation
(RIPA) buffer by Pierce (Rockford, IL) and protein concentrations were determined
using the bicinchoninic acid (BCA) protein assay reagent from Pierce (Rockford,
IL). For analysis, 50 μg of each sample was processed as follows: The samples
30

were first run on a polyacrylamide gel and then transferred on a polyvinylidene
difluoride (PVDF) membrane, with the use of a Trans-Blot
®
SD Semi-Dry Transfer
Cell from Bio-rad Laboratories (Irvine, CA). After nonspecific blocking of the
antigens with SEA BLOCK Blocking Buffer from ThermoFisher Scientific
(Rockford,IL), the membrane was incubated with primary and then secondary
antibodies. The primary antibodies were purchased from Cell Signaling
Technologies (Beverly, MA), Cayman Chemical (Ann Arbor, MI), or from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA) and were used according to
manufacturer's recommendations. The secondary antibodies were coupled to
horseradish peroxidase, and were detected by chemiluminescence using the
SuperSignal™ West Pico Chemiluminescent Substrate from ThermoFisher
Scientific. Analysis of blots was carried out on an image analyser. All immunoblots
were repeated at least once to confirm the results.

3.3 COLONY CLONING:
A2058 (MGMT
-
) were seeded in 10 cm tissue culture plates at different
densities – 80 cells/plate, 200cells/plate and 800 cells/plate. The cells were
allowed to form colonies (defined as groups of >50 cells) large enough to be
pipetted out, which took about 10-16 days. Individual colonies were then picked
arbitrarily and seeded into 24 well plates, allowed to grow, transferred into 6 well
plates, then again allowed to grow and subsequently established for culture in 10
cm plates. Cells were then used for Western Blotting to assess the MGMT
expression as well as in Colony Formation Assays with temozolomide,
temozolomide-POH and O6-Benzylguanine.
31

3.4 COLONY FORMATION ASSAY:
Depending on the cell line (and plating efficiency), 600–800 cells were
seeded into each well of a 6-well plate a day before treatment and then treated
with different concentrations of the drugs temozolomide, temozolomide-POH and
cisplatin respectively and in combination with O6-Benzylguanine. The medium was
replaced with fresh medium without drugs after 48 hours, except O6BG, which was
added again. After 12–16 days, colonies (defined as groups of >50 cells) were
visualized by staining for 2-4 h with 1% methylene blue (in methanol), and were
then counted. Experiments were repeated at least once, but also under different
conditions.

3.5 CHEMOTHERAPEUTIC AGENTS:
Temozolomide (TMZ) was obtained from the pharmacy at University of
Southern California and temozolomide-POH (TP/NEO212) was obtained from
NeOnc Technologies, Inc. in powdered form and stored at 4°C. Prior to treatment,
TMZ was prepared as 50 mmol/L stock solution in DMSO (dimethyl sulfoxide) and
NEO212 was prepared as 100mmol/L stock solution in DMSO. Both drugs were
stored at -20°C. O6BG was obtained from Sigma Aldrich and prepared as 100
mmol/L stock solution in DMSO. Any subsequent dilutions for working solutions
were done in DMSO, keeping the final concentration of DMSO of less than 1% in
culture media. Cisplatin was obtained from Sigma Aldrich, prepared as 10mM
stock solution in 0.9% Nacl and stored at -20°C.

32

4 – RESULTS
4.1 EXPRESSION OF MGMT IN VARIOUS MELANOMA AND
GLIOBLASTOMA CELL LINES:
The purpose of this study was to ascertain the MGMT status of various
melanoma cell lines including A375, A2058, and the A2058 derived Clone 53 and
Clone 58 and compare it with previously established expression profiles, before
we proceeded with further experiments.

Figure 11: MGMT expression in various melanoma and glioblastoma multiforme
cell lines

The above figure shows the result of our Western Blot and is consistent
with previously characterized expression of the cell lines in the experiment, which
are as follows –
T98G – MGMT positive Glioblastoma Multiforme (GBM)
U251 – MGMT negative GBM
A375 – MGMT positive melanoma
27/59 – primary melanoma (brain metastasis)
A2058 – MGMT negative melanoma
Clone 53 & Clone 58 – MGMT positive clones derived from A2058
M249 – MGMT positive melanoma

T98G U251 A375 27/59 A2058 Clone 53 Clone 58 M249

MGMT 21kDa
Actin 42kDa
33

4.2 O6BG IS A POTENT INHIBITOR OF MGMT:

The purpose of this study was to establish MGMT as a key factor in
resistance of melanoma to TMZ, demonstrate the efficacy of O6BG as an inhibitor
of MGMT in a MGMT+ cell line and to demonstrate the previously mentioned
effects of TMZ and TP.

Figure 12: WB analysis of Clone 53 cells treated with TMZ, TP and O6BG.

Clone 53 is an MGMT+ cell line established from the A2058 (MGMT-)
melanoma cell line. Clone 53 cells were seeded into nine 10 cm plates at about
50-60% confluency one day before treatment. One plate was left untreated, and
one was treated with the vehicle (DMSO) as controls. The vehicle concentration
was the same as the highest amount of drug concentration (in micromolar) used.
The treatment duration was 24 hours, after which the cells where harvested from
each plate to perform Western Blot analysis. Figure 12 shows the results of this
experiment. As evident, O6BG works as a potent inhibitor of MGMT, TMZ has no
effect, and TP shows a small downregulation in the amount of MGMT.
1 2 3 4 5 6 7 8 9
O6BG (uM)
3 10 30 40 80
TMZ (uM) TP (uM)
40 80
MGMT
Actin
34

4.3 VARIABLE RESPONSES TO TMZ ± O6BG IN MGMT-
AND MGMT+ CELL LINES:
The purpose of this study was to demonstrate the varying cytotoxic effects
of TMZ alone and in combination with O6BG in A2058 and MGMT+ cell lines Clone
53 and Clone 58.
4.3.1 A2058 (MGMT-):

Figure 13: A2058 vs TMZ ± O6BG.

A2058 is an MGMT- melanoma cell line. Based upon the plating efficiency,
800 cells of A2058 were seeded in each well of two 6-well plates a day before
treatment. One plate was designated for treatment with TMZ alone, the other plate
was designated for treatment with TMZ in combination with O6BG. One well in
each plate was left untreated as control. 15 uM O6BG was added to the wells of
plate two, 30-45 minutes before treatment with TMZ. TMZ was added in increasing
concentrations (0 uM, 10 uM, 20 uM, 40 uM, 60 uM, 80 uM) in both plates. The
plates were then kept in the incubator at 37°C and 5% CO
2
, allowed to form
colonies, stained, and counted. Figure 13 shows the result of this experiment. As
0
20
40
60
80
100
0 204060 80
0uM O6BG 15uM O6BG
% Colonies
uM TMZ
35

can be seen, and supporting our hypothesis, inclusion of O6BG makes a small but
noticeable difference in the cytotoxicity of TMZ to A2058. The low percentage of
A2058 cells surviving at relatively higher concentrations of TMZ (without O6BG)
are what we believe to be the MGMT+ cells present in the A2058 culture. The
MGMT- cells die off at the initial low concentrations while the MGMT+ ones survive.
This can be supported by the outcome after inclusion of O6BG which kills of all the
cells at the initial lower TMZ concentration (20 uM).
4.3.2 CLONE 53 (MGMT+):

Figure 14: Clone 53 vs TMZ – O6BG.

Figure 15: Clone 53 vs TMZ + 15 uM O6BG.

Figures 14 and 15 show results of the experiment with Clone 53. The
experiment was performed as described for A2058 with certain differences which
0
20
40
60
80
100
0 100 200 300 400 500
% Colonies
uM TMZ
0
20
40
60
80
100
0 20406080
% Colonies
uM TMZ
36

are as follows - The number of cells seeded into each well of the 6-well plates was
600, owing to the high plating efficiency and higher duplication rate. The
concentration of TMZ used alone was much higher (0 uM, 100 uM, 200 uM, 300
uM, 400 uM, 500 uM) than that used for A2058 based on previous studies by our
lab and the MGMT
+
status. Clearly, inclusion of O6BG made a striking difference
in the outcome and consistent with our experiment with A2058, whereby, all cells
are killed off at 20 uM TMZ. TMZ alone is much less effective, requiring a
concentration of 300 uM to achieve the same.
4.3.3 CLONE 58 (MGMT+):

Figure 16: Clone 58 vs TMZ – O6BG.

Figure 17: Clone 58 vs TMZ + 15 uM O6BG.
0
20
40
60
80
100
0 100 200 300 400 500
% Colonies
uM TMZ
0
20
40
60
80
100
0 20406080
% Colonies
uM TMZ
37

Figures 16 and 17 show our results for the experiment with Clone 58. The
experiment was performed as described for Clone 53. Clone 58, as can be seen
in Figure 10 above, expresses high levels of MGMT. Consistent with this fact,
Clone 58 shows high resistance to TMZ alone, requiring concentrations exceeding
400 uM to kill off all the cells. Inclusion of O6BG again, as can be expected makes
the cells highly sensitive to TMZ treatment by inhibiting MGMT, although, bringing
about complete cell death at a concentration of 40 uM TMZ, twice that of what is
required for A2058 and Clone 53.

4.4 PRESENCE OF MGMT+ CELLS IN A MGMT- CELL LINE:
The purpose of this experiment was to characterize the expression of
MGMT in the clones established from A2058 culture. This was a critical experiment
to test our hypothesis, that there might be MGMT+ cells already present or evolving
in A2058 culture and their subsequent involvement in developing chemo-
resistance in melanoma cell lines.

Figure 18: WB analysis of A2058 Clones 1 through 8.
1 2 3 4 5 6 7 8 + Ctrl
A2058 Clones MCF7
MGMT
Actin
38

Figure 19: WB analysis of A2058, Clones 9, 10, 2, 3 and 4.

To test our hypothesis that A2058 melanoma cell line has a subpopulation
of cells that are MGMT+ present inherently or evolving in culture, we picked
individual colonies from a low density culture allowed to grow without any
treatment. We then cultured these colonies, allowing them to grow sufficiently to
be harvested and analysed by Western Blot for their MGMT expression. We were
able to pick 24 colonies from the initial low density culture, out of which 10
remained viable in culture. In Figures 18 and 19, we obtained results consistent
with our expectation and demonstrated the presence of 10% MGMT positive cells
in the parental A2058 (MGMT-) population. Clone 3 as evident from above, was
positive for MGMT. We repeated the experiment to confirm the result. MCF7
(Human Breast Carcinoma) and A375 cells were used as positive controls.

4.5 RESPONSE TO TMZ ± O6BG IN NEWLY ESTABLISHED
CLONES:
To further assess the impact of emerging MGMT activity in cells in culture,
we challenged the newly established clones with TMZ ± O6BG and compared their
dose-response graph to those of A2058 and MGMT positive A375.
+ Ctrl A2058 9 10 2 3 4
A2058 Clones
MGMT
Actin
A375
39

Figure 20: A2058 vs TMZ ± O6BG.

Figure 21: Clone 2 vs TMZ ± O6BG.
0
20
40
60
80
100
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25
A2058 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM TMZ
0
20
40
60
80
100
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25
Clone 2 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM TMZ
40

Figure 22: Clone 3 vs TMZ ± O6BG.

Figure 23: A375 (MGMT+) vs TMZ ± O6BG.

The assay was performed as previously described while using a different
range of TMZ concentrations to more correctly identify the IC
50
. Figures 20, 21, 22
and 23 show the results of the study. In Figure 20, the dose-response graph of
A2058 is consistent with our previous studies. In Figure 21, however, our results
deviate from our expectation. Keeping in mind that Clone 2 lacks MGMT, we
expected to find no difference in TMZ cytotoxicity with or without O6BG and yet,
0
20
40
60
80
100
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25
Clone 3 (MGMT+)
0 uM O6BG 15 uM O6BG
% Colonies
uM TMZ
0
20
40
60
80
100
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25
A375 (MGMT+)
0 uM O6BG 15 uM O6BG
% Colonies
uM TMZ
41

here we see a considerable difference. Clone 2 also has an IC
50
significantly higher
than that of A2058, in contrast to what we predicted. Clone 3 (MGMT+) was in line
with our expectation, besides showing a dose-response line similar to that of the
MGMT positive melanoma cell line A375.

4.6 RESPONSE TO TP ± O6BG IN NEWLY ESTABLISHED
CLONES:
The purpose of this experiment was to help support our understanding of
the behavior of MGMT activity in vitro and in response to a more potent drug.

Figure 24: A2058 vs TP ± O6BG.

0
20
40
60
80
100
02.557.5 10 12.5 15
A2058 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM TP
42

Figure 25: Clone 2 vs TP ± O6BG.

Figure 26: Clone 3 vs TP ± O6BG.
0
20
40
60
80
100
0 2.5 5 7.5 10 12.5 15
Clone 2 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM TP
0
20
40
60
80
100
02.557.5 10 12.5 15
Clone 3 (MGMT+)
0 uM O6BG 15 uM O6BG
% Colonies
uM TP
43

Figure 27: A375 vs TP ± O6BG.

Temozolomide-POH (TP), as mentioned before, is a much more potent
cytotoxin than TMZ by itself. We believed challenging the cells and clones with TP
would be beneficial in developing a better foundation for understanding the
emergence and impact of MGMT expression in culture. Figures 24, 25, 26 and 27
are the results of our colony formation assay. These were performed as previously
described and the TP concentrations used were 0 uM, 2.5 uM, 5 uM, 7.5 uM, 10
uM and 12.5 uM. While it should be mentioned that A2058, Clone 3 and A375 all
show responses consistent with the literature, our studies, and our hypothesis,
Clone 2, again has a response that is peculiar and deviating from our line of
thinking. Surprisingly, a higher concentration of TP is required for significant cell
death in Clone 2, when compared to A2058. Assuming that all cells in a Clone 2
culture are negative for MGMT, we expected it to be noticeably sensitive to the
drugs, relative to A2058, while also showing no difference in its response with
inclusion of O6BG.

0
20
40
60
80
100
02.557.5 10 12.5 15
A375 (MGMT+)
0 uM O6BG 15 uM O6BG
% Colonies
uM TP
44

4.7 RESPONSE TO CISPLATIN ± O6BG IN NEWLY
ESTABLISHED CLONES:
The purpose of this study was to test a part of our hypothesis, that the
emergence of MGMT in vitro implicates its importance in treatment of malignant
melanoma with alkylating agents like temozolomide and temozolomide-POH and
establishes the demethylating repair mechanism as a major player in developing
chemoresistance to these drugs. It would also help in determining whether the
A2058 Clones vary in response to other chemotherapeutic agents. Cisplatin was
chosen as a drug of choice for this experiment, since it is one of the most widely
used chemotherapeutic agents against many types of cancers.

Figure 28: A2058 vs Cisplatin ± O6BG.

0
20
40
60
80
100
00.5 11.522.533.5 44.55
A2058 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM Cisplatin
45

Figure 29: Clone 2 vs Cisplatin ± O6BG.

Figure 30: Clone 3 vs Cisplatin ± O6BG.

0
20
40
60
80
100
00.5 11.522.5 33.544.5 5
Clone 2 (MGMT‐)
0 uM O6BG 15 uM O6BG
% Colonies
uM Cisplatin
0
20
40
60
80
100
00.5 11.5 22.5 33.5 44.55
Clone 3 (MGMT+)
0 uM O6BG 15 uM O6BG
uM Cisplatin
% Colonies
46

Figure 31: A375 vs Cisplatin ± O6BG.

Figure 28, 29, 30 and 31 show the results of this study. The CFA was
performed as previously described. In this case as well, O6BG was added 30-45
minutes prior to treatment with cisplatin. The following concentrations were used
for cisplatin – 0 uM, 1 uM, 2 uM, 3 uM and 5 uM. It is evident from the above figures
that all melanoma cell lines and clones used had similar responses to Cisplatin,
even with inclusion of O6BG. We anticipated that since the DNA lesions set by
cisplatin differ from those set by TMZ, MGMT expression would not play a role in
determining the sensitivity of these cells to cisplatin and we were able to confirm
our hypothesis.

0
20
40
60
80
100
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
A375 (MGMT+)
0 uM O6BG 15 uM O6BG
% Colonies
uM Cisplatin
47

5 – DISCUSSION
Malignant melanoma, one of the leading fatal cancers is on the rise in
Americans and around the world. It is well known to be highly aggressive and
metastatic, however, the cause for greater concern is its ability to frequently
develop chemo-resistance and should be addressed. [1,29]. Considerable
research has been done to improve strategies to combat melanoma. While
surgery, early detection and adjuvant therapy has shown improved outcomes, the
prognosis still remains very poor, especially after onset of metastasis. At the
advanced stage, melanoma has one of the most disappointing median survival, a
five survival rate of only about 5%, and considered to be extremely treatment-
refractory. Treatment options remain limited despite extensive investigations. [40].
Presently, the only chemotherapeutic agent with reproducible activity
against metastatic melanoma and approved for use is dacarbazine, which also has
shown low response rates in large randomized trials. [26]. With that being said,
there is a need for novel, effective, and convenient strategies to combat this
debilitating disease. Temozolomide, as previously stated, is currently being
evaluated given its potential uses and benefits. Development of resistance is
common in treatment with temozolomide in patients with glioblastoma multiforme
and often linked to the overexpression of MGMT protein. [26,29].
Our hypothesis was based on previous work by our research group that
demonstrated the survival of small populations of MGMT negative melanoma cell
line when treated with temozolomide. These survivors, when established in culture
and analyzed, where found to strongly express MGMT. We anticipated the
presence of these MGMT expressing cells in the existing melanoma culture or that
they may be emerging in vitro, and that treatment with temozolomide, is in
48

essence, selecting for these particular cells, which then survive and show robust
resistance to the alkylating agent. This has previously been demonstrated in GBM
tumors, but had not been seen to happen in melanoma cell lines. [31].
With our experiments, we were able to demonstrate the presence of about
10% MGMT positive cells in a MGMT negative melanoma cell line (A2058) as seen
in Figures 18 and 19. Upon further investigation with colony formation assays, we
showed that these cells respond quite differently to temozolomide compared to the
parental cell line. To assess these results, we decided to include O6BG in our
assays. O6BG, being a MGMT inhibitor, helped us establish MGMT as the
underpinning behind the resistance. Inclusion of O6BG with temozolomide, made
the MGMT positive cells significantly more sensitive to the alkylating agents while
making a smaller difference in the sensitivity of A2058, owing to the presence of
only a small subpopulation of MGMT positive cells. [Figures 20-23].
NEO212 or TP, as mentioned earlier, has been shown to be significantly
more toxic to temozolomide-resistant glioma cells and we demonstrate this effect
in melanoma cell lines as well. [Figures 24-27]. To show that the emergence of
MGMT expression and activity is critical to treatment-resistance with alkylating
agents, we used cisplatin, a chemotherapeutic agent commonly used against the
melanoma cell lines. We were able to show that the MGMT positive and negative
melanoma cell lines respond very similarly to cisplatin, in the presence as well as
in the absence of O6BG. [Figures 28-31].
However, it remains unclear how Clone 2, despite being negative for
MGMT, has a dose-response graph in contrast to our initial expectations, by being
less sensitive than A2058, its parental cell line. We think it is likely, that Clone 2
itself has evolved some MGMT positive cells in vitro, but that fails to explain why it
49

also responds poorly to O6BG as well. Perhaps, it is possible that some other DNA
repair mechanism such as the MMR or BER, plays a role by their respective under
or over expression, which remains to be investigated.
Further studies are warranted with Clone 2 as well as Clone 3 to further
advance our understanding of MGMT-mediated resistance. Clone 3, the MGMT
positive cell line, derived from A2058 should be studied in vivo, in comparison with
A2058 and A375. The efficiency of O6BG as a potential therapeutic adjuvant in
treatment of MGMT positive melanomas should be evaluated in mouse models of
the disease. Fully comprehending the development of resistance in melanoma will
help in targeting the molecular effectors of the disease and provide novel
therapeutic strategies aimed at combating malignant melanoma.

50

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54

ACKNOWLEDGEMENT
‘All scientists must communicate their work, for what is the point of learning new
things about how the world works if you don’t tell anyone about them?’
- Jim Al-Khalili

First and foremost, I would like to thank Dr. Axel Schonthal for giving me the
opportunity to be a part of his laboratory and allowing me to develop my research
skills. I express my deepest gratitude to Dr. Schonthal for the continuous support,
for his patience, motivation and exuberance of immense knowledge. He has been
an excellent mentor and contributed to my advance in technical ability with his
exceptional teaching. His guidance in times of difficulty has proved to be of
immense help in my research study.
Furthermore, I would like to acknowledge the support given to me by the
entire research group, especially, Dr. Thomas Chen, Dr. Florence Hofman, Dr.
Josh Neman, Dr. Steve Swenson and Dr. Weijun Wang. Their critique,
encouragement and insightful comments have incented me to widen the horizons
of my scientific inquiry. It has been a tremendous learning curve for me since the
day I joined.
My sincere thanks goes to the members of my thesis committee Dr. Axel
Schonthal, Dr. Florence Hofman and Dr. Joseph Landolph for aiding me in the
successful completion of my thesis.
I would also like to state and appreciate the precious support and help given
to me by my fellow lab-mates, especially, Dr. Nymph Chan and former lab member
Jiali Yu. The engaging conversations and stimulating discussions with them have
55

made my experience all the more exciting and one that I’ll always look back on as
an example to excel in a professional environment.
I am truly humbled by the opportunity given to me by the Graduate School
of the University of Southern California to be a part of a world-class institution and
providing an environment for me to inculcate the qualities of a Trojan. I would like
to express my deepest gratefulness for the same.
Finally, I express profound gratitude and acknowledgement to my parents
and friends for their unfailing support throughout my years of study and research,
and their constant encouragement to strive harder. This accomplishment would
not have been possible without them.

Abstract (if available)

Abstract Melanoma incidence has been on the rise and alike many other malignancies, major impediments in its treatment and management are the high metastatic potential and frequent development of chemo-resistance. Use of surgery and radiation are limited to local and regional tumors and current therapeutic strategies provide a modest increase in overall survival. Temozolomide (TMZ), a DNA alkylating agent, which is standard of care for patients with glioblastoma multiforme and astrocytoma, is currently also being evaluated for use against melanoma. Treatment with temozolomide (TMZ) has also been met with development of resistance and is frequently linked to overexpression of a DNA repair protein, MGMT (O6-methylguanine-DNA-methyltransferase). In this study, we attempt to characterize the emergence of MGMT driven resistance to TMZ in vitro. Previous studies by our research group have shown that treatment with TMZ can induce or select for cell populations that express MGMT from a MGMT negative melanoma cell line. We hypothesized that, TMZ treatment leads to selective propagation of cells which express MGMT, which are likely already present in the culture. Here, we show the presence of a pre-existing small number of MGMT expressing cells in a culture of the MGMT-negative melanoma cell line (A2058). These MGMT positive cells, also differ from the parental cells in response to treatment with TMZ and NEO212, in the absence of O6-Benzylguanine (O6BG), an MGMT inhibitor. Inclusion of O6BG makes the MGMT positive cells sensitive to alkylating agents. Emergence of MGMT resistance in vitro emphasizes further, the need for assessing its expression in melanoma tumors, and development of novel strategies such as a combination of NEO212 and O6BG to increase survival along with improvement in quality of life.

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Asset Metadata

Creator Karnik, Sushant (author)

Core Title MGMT in emergence of melanoma resistance to temozolomide

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/17/2017

Defense Date 06/20/2017

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

Tag Melanoma,MGMT,O6-benzylguanine, perillyl alcohol,OAI-PMH Harvest,resistance,temozolomide

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Schönthal, Axel (committee chair), Hofman, Florence (committee member), Landolph, Joseph (committee member)

Creator Email sshntkarnik@yahoo.co.in,sskarnik@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-399463

Unique identifier UC11264367

Identifier etd-KarnikSush-5524.pdf (filename),usctheses-c40-399463 (legacy record id)

Legacy Identifier etd-KarnikSush-5524.pdf

Dmrecord 399463

Document Type Thesis

Rights Karnik, Sushant

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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