Development of a temozolomide-perillyl alcohol conjugate, NEO212, for the treatment of hematologic malignancies

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Request accessible transcript

Transcript (if available)

Content DEVELOPMENT OF A TEMOZOLOMIDE-PERILLYL ALCOHOL CONJUGATE, NEO212, FOR THE
TREATMENT OF HEMATOLOGIC MALIGNANCIES

Zhuoyue Yang

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
May 2023

Copyright 2023 Zhuoyue Yang
ii

Table of Contents

List of Figures ............................................................................................................................................... iv
Abbreviations ................................................................................................................................................ v
Abstract ....................................................................................................................................................... vii
Chapter 1 – Introduction ............................................................................................................................... 1
1.1 Hematologic Malignancies ……………………………………………………………………………………………………… 1
1.1.2 Canine hematologic malignancies ........................................................................................ 2
1.2 NEO212 ………………………………………………………………………………………………………………………………….. 3
1.2.1 TMZ ....................................................................................................................................... 3
1.1.1 POH ..................................................................................................................................... 11
1.1.2 NEO212 ............................................................................................................................... 12
1.2 Hypothesis ……………………………………………………………………………………………………………………………. 13
Chapter 2 – Materials and Methods ........................................................................................................... 14
2.1 Pharmacological agents ………………………………………………………………………………………………………… 14
2.2 Cell lines ……………………………………………………………………………………………………………………………….. 14
2.3 MTT assay …………………………………………………………………………………………………………………………….. 16
2.4 Cell counting ………………………………………………………………………………………………………………………….16
2.5 Isolation of RNA and Quantitative RT-PCR ……………………………………………………………………………. 16
2.6 Immunoblot …………………………………………………………………………………………………………………………. 17
2.7 In-Vivo … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … .. 19
2.8 Statistical analysis ………………………………………………………………………………………………………………….20
Chapter 3 – Results ..................................................................................................................................... 21
3.1 NEO212 inhibits proliferation of lymphoma and leukemia cells in vitro … … … … … … … … … … … … … 21
1. Effect of NEO212 on cell viability in the lymphoma cells measured by MTT assay .................... 21
2. Effect of NEO212 on cell viability in the leukemia cells measured by MTT assay ...................... 25
3. Effect of NEO212 on cell proliferation and death measured by cell counting ........................... 28
3.2 NEO212 overcomes MGMT-mediated drug resistances …………………………………………………………28
3.2.1 Multiple cell lines express different levels of MGMT ......................................................... 28
3.2.2 NEO212 treatment down-regulates MGMT protein levels. ............................................... 30
3.3 NEO212 induces macrophage differentiation, proliferation arrest and eventual apoptosis ….. 32
3.3.1 NEO212 increases CD11b (ITGAM) mRNA levels ................................................................ 32
3.3.2 NEO212 increases CDKN1A (p21) mRNA levels .................................................................. 35
iii

3.3.3 NEO212 upregulated γ-H2AX and cl. caspase7 protein level ............................................. 36
3.4 NEO212 contributes to mice survival in vivo … … … … … … … … … … … … … … … … … … … … … … … … … … … … . 39
Chapter 4 – Discussion ................................................................................................................................ 42
Bibliography ................................................................................................................................................ 46

iv

List of Figures

Figure 1. 1. Chemical structure of TMZ …………………………………………………………………………………………………………..……….4
Figure 1. 2. Chemical structure of MTIC …………………………………………………………………………………………………..………………5
Figure 1. 3. Chemical structure of AIC …………………………………………………………………………………………………………..…………5
Figure 1. 4. Chemical structure of POH …………………………………………………………………………………………………..……………..11
Figure 1. 5. Chemical structure of NEO212 ………………………………………………………………………………………………….………..12
Figure 3. 1 Toxicity of NEO212, TMZ, POH, and TMZ +POH on Raji lymphoma cell line at different cell density. ...... 23
Figure 3. 2 Toxicity NEO212, TMZ, POH, and TMZ +POH on lymphoma cell lines. ...................................................... 25
Figure 3. 3 Toxicity of NEO212 and TMZ on AML cell lines. ........................................................................................ 26
Figure 3. 4 Toxicity of DMSO on AML cell lines. .......................................................................................................... 27
Figure 3. 5 Effect of NEO212 on AML cell viability. ..................................................................................................... 28
Figure 3. 6 Hematologic malignancy cell lines express different levels of MGMT. ..................................................... 29
Figure 3. 7 Additional hematologic malignancy cell lines express different levels of MGMT. .................................... 30
Figure 3. 8 Treatment with NEO212 reduces MGMT protein levels............................................................................ 31
Figure 3. 9 Treatment with NEO212 induces the expression of markers for macrophage differentiation. ................ 33
Figure 3. 10 Treatment with NEO212 elevates the levels of CD11b mRNA................................................................. 34
Figure 3. 11 Treatment with NEO212 elevates the levels of CDKN1A mRNA. ............................................................. 36
Figure 3. 12 Treatment with NEO212 induces γ-H2AX and cl. caspase 7 protein levels. ............................................ 38
Figure 3. 13 Survival rates for HL60 mice treated with NEO212 versus control (vehicle). .......................................... 39
Figure 3. 14 Survival rates for 6D10 mice treated with NEO212 versus control (vehicle). .......................................... 40
Figure 3. 15 Survival rates for HL60 mice treated with NEO212, TMZ and control (vehicle). ..................................... 41
Figure 3. 16 Survival rates for HL60 mice treated with NEO212, TMZ and control (vehicle). ..................................... 41

v

Abbreviations

AIC: 4-amino-5-imidazolecarboxamide
AML: acute myeloid leukemia
AP: apurinic/apyrimidinic
APE-1: AP endonuclease
AraC: cytosine arabinoside
ATM: ataxia telangiectasia mutated
ATR: ATM-Rad3-related
AVMA: American Veterinary Medical
Association
BER: base excision repair
CAR-T cells: chimeric antigen receptor-T
cells
CDK: cyclin-dependent kinase
CDKN1A: cyclin-dependent kinase inhibitor
1A
cDNA: complementary DNA sequence
cGMP: current good manufacturing practice
CRC: colorectal cancer
DHPA: dihydroperillic acid
DMEM: Dulbecco’s Modified Eagle’s
Medium
DMSO: dimethyl sulfoxide
dRp: deoxyribose phosphate
dRpase: deoxyribophosphodiesterase
DSBs: Double stranded breaks
FBS: fetal bovine serum
FDA: Food and Drug Administration
FEN-1: flap endonuclease-1
GBM: glioblastoma multiforme
IACUC: Institutional Animal Care and Use
Committee
IC50: concentration of drug that reduces
cell proliferation by 50%
ITGAM/CD11b: integrin subunit alpha M
MEM: Minimum Essential Medium
MGMT: O6-methylguanine DNA
methyltransferase
MMR: mismatch repair
MTIC: metabolite 5-(3-methyl-1-triazen-1-
yl) imidazole-4-carboxamide
MTT: methylthiazoletetrazolium
N3-MeA: N3-methyladenine
N7-MeG: N7-methylguanine
NEO212: perillyl alcohol covalently linked to
temozolomide (TMZ-POH)
NETs: neuroendocrine tumors
NSCLC: tumors (NETs), non-small-cell lung
cancer
O6-MeG: O6-methylguanine
PA: perillic acid
PARP-1: Poly (ADP-ribose) polymerase-1
POH: perillyl alcohol
PVDF: polyvinylidene fluoride
RPMI: Roswell Park Memorial Institute
medium
vi

SDS: sodium dodecyl sulfate
TMZ: temozolomide

XRCC1: X-ray repair cross-complementing 1

vii

Abstract
Hematologic malignancy continues to claim the lives of many patients. The alkylating drug
temozolomide (TMZ) has been studied in the past to treat AML and has been found to be only
partially effective; however, tumor cells that contain the DNA repair enzyme O6-methylguanine
DNA methyltransferase (MGMT) confer profound therapeutic resistance to TMZ. The novel
anticancer compound NEO212 that we are creating has TMZ covalently linked to perillyl alcohol
(POH). In numerous preclinical cancer models, NEO212 has demonstrated strong therapeutic
activity. We examined its effects on human hematologic malignancy cell lines in the present
study and discovered that it exerts cytotoxic effects even on MGMT-positive cells that are
extremely resistant to TMZ. In addition, NEO212 significantly increased the expression of
numerous markers associated with macrophages, such as CD11b/ITGAM, as well as markers
associated with growth inhibition, such as CDKN1A/p21, and apoptosis, such as γ-H2AX and cl.
caspase 7. When cells were treated with equimolar mixtures of either TMZ or POH, the
anticancer effects of NEO212 could not be duplicated. Two 5-day cycles of 25 mg/kg NEO212
resulted in a significant cure in a mouse model implanted with TMZ-resistant, MGMT-positive
AML and lymphoma cells. In the AML model, mice survived for more than 300 days without
exhibiting any symptoms. In the lymphoma model, the number and time of survival of NEO212
treated mice is much greater than that of the non-administered group. The results indicate that
NEO212 affects hematologic malignancy cells in a variety of ways, including through
differentiation, proliferative arrest, and ultimately cell death. Also, the superior cytotoxic effects
of NEO212 appear to involve a downregulation of MGMT protein levels. NEO212 was well
tolerated in vivo even at doses significantly higher than those required for treatment, indicating
viii

a broad therapeutic window. These findings imply that NEO212 is a therapeutic agent for
hematologic malignancies that needs to be taken into consideration for development.

1

Chapter 1 – Introduction
1.1 Hematologic Malignancies
1.1.1 Human hematologic malignancies
Hematologic malignancies, also known as blood cancers, start in immune system cells or
blood-forming tissue, like the bone marrow. The first hematologic malignancy type was
described by Thomas Hodgkin in 1832, called Hodgkin’s lymphoma. Hematologic malignancies
are now recognized to include a wide range of genetically diverse diseases identified with the aid
of immunophenotyping, cytogenetic, and molecular genetic testing. Patients with hematologic
malignancies have an excess of aberrant blood cells, which push out normal blood cells and
impair their ability to function normally. These blood disorders are generally divided into three
main categories including leukemia, lymphoma, and myeloma. Leukemia is referred to as cancer
of the blood cells. The type of leukemia is determined by two variables. The first is the type of
blood cells, and the second is how quickly or slowly the cancer grows. While leukemia can affect
anyone at any age, it is the most frequent pediatric tumor. Lymphoma is a cancer that starts in
immune cells called lymphocytes (T or B lymphocytes) in the lymph nodes, spleen, thymus, and
bone marrow, but also in other organs of the body in the lymphatic system. Myeloma, also called
multiple myeloma, is a plasma cell cancer that develops in the bone marrow.
New hematologic malignancy cases are about 1.2 million each year worldwide, which are
around 7% of all newly diagnosed cancers (Auberger et al., 2020). About leukemia, 400,000 new
cases are diagnosed globally each year, accounting for 35.7% of all hematologic malignancy
cases. According to the Annual Report to the Nation on the Status of Cancer, based on cases from
2015 to 2019 and deaths from 2016 to 2020, the rate of new cases of leukemia was 14.1 per
2

100,000 men and women per year. The rate of death from leukemia was 6.0 per 100,000 men
and women per year. These rates were age-adjusted. About lymphoma, over 500,000 people are
diagnosed with it worldwide, accounting for 44.6% of all hematologic malignancy cases.
Approximately 85% of all cases of lymphoma are non-Hodgkin lymphoma. According to
estimates, 120,000 new cases of lymphoma are diagnosed each year in Europe and 80,000 in the
USA (Islami et al., 2021).
Leukemia, lymphoma, and multiple myeloma treatments have come a long way including
immunotherapies, immune checkpoint inhibitors, and chimeric antigen receptor-T cells (CAR-T
cells)(Alard et al., 2020), but there are still many cases of treatment resistance brought on by
molecular and clinical relapse. Relapsed and refractory disease therapy is still incredibly
challenging, and failure to control the disease at this stage continues to be the main cause of
death in patients with hematologic malignancies. Leukemia and lymphoma are generally
characterized by resistance to all types of currently available therapies. Numerous efforts have
been made in recent years to pinpoint the genetic and epigenetic mechanisms that cause
hematologic malignancies to progress to the point at which they require clinical intervention.
There are different molecular and cellular mechanisms involved in these adaptations, including
the development of mutations and modulation of the signaling pathways. Affecting signaling
pathways often by affecting the regulation of apoptosis, autophagy, proliferation,
differentiation, metabolism, epigenetic changes, and oncogenes or tumor suppressors.
1.1.2 Canine hematologic malignancies
Dogs are human’s most loyal of friends and are also seen as our beloved family members.
According to the US Pet Ownership and Demographics Sourcebook (2022) by American
3

Veterinary Medical Association (AVMA), as of 2020, there were about 88 million dogs living in
45% of US households. Dog ownership increased by 6% from 38.4% (2016) to 44.6% (2020) and
keeps increasing. For all of 2020, U.S. households collectively spent about $17 billion annually on
veterinary care for dogs.
Many of the same cancers that occur in humans are also commonly developed in dogs
since dogs and humans are exposed to the same living environment and have highly similar
genetic, anatomical and physiological structures. Therefore, dogs with cancer have similar
symptoms to people with cancer. Studies of dogs can help collect clinical efficacy data for both
canine and human. Additionally, dogs have a shorter lifespan than humans, which permits them
to reach research endpoints faster. Thus, dogs can be used as a translational animal model which
may also help researchers to understand cancers and inform anticancer drug development for
both canine and human (Regan et al., 2018; Zandvliet, 2016).
1.2 NEO212
1.2.1 TMZ
Temozolomide (TMZ) was first synthesized at Aston University in the early 1980s as one
of a series of novel imidazotetrazinones (Friedman et al., 2000). Now, TMZ is the most common
chemotherapeutic drug used in conjunction with radiotherapy to treat glioblastoma multiforme
(GBM). In addition to being a first-line treatment for GBM, TMZ is also used as a second-line
treatment for anaplastic astrocytoma (Mason, 2009). Additionally, TMZ has also shown activity
in a variety of other cancers, such as breast cancer (Garza-Morales et al., 2018), ovarian cancer,
rectal cancer, neuroendocrine tumors (NETs) (Koumarianou et al., 2012), non-small-cell lung
cancer (NSCLC) (Pietanza et al., 2018), colorectal cancer (CRC) (Inno et al., 2014), and acute
4

myeloid leukemia (AML), per findings reported in the PubMed electronic database. In GBM,
although TMZ gives patients some hope, standard therapy involving TMZ, surgery, and radiation
in GBM only leads to a best 5-year survival rate of 9.8% and a median patient survival of 14.6
months (Stupp et al., 2005). Treatment failure has been associated with tumor drug resistance.
In addition to drug resistance, adjuvant TMZ therapy also leads to some side effects such as
hematologic toxic effect, neutropenia, and thrombocytopenia. Non-hematologic side effects are
dose-dependent such as nausea and vomiting.

Figure 1. 1. Chemical structure of TMZ
5

Figure 1. 2. Chemical structure of MTIC

Figure 1. 3. Chemical structure of AIC
TMZ, also named 3,4- dihydro-3-methyl-4-oxoimidazole, is a small, orally given alkylating
agent prodrug with a molecular weight of 194.154 g/mol and with lipophilic character. To
produce the active metabolite 5-(3-methyl-1-triazen-1-yl) imidazole-4-carboxamide (MTIC), TMZ
6

undergoes spontaneous hydrolysis. Water's impact on TMZ's highly electropositive C4 location
causes it to spontaneously transform into the reactive methylating agent MTIC. This pH-
dependent process activates the ring, emits CO 2, and produces MTIC. Target cell penetration is
hampered by MTIC's inherent characteristics, which limit an efficient contact with tumor cell
membranes. This could be one of the factors contributing to TMZ's less-than-expected efficacy
in malignancies. The last breakdown product, 4-amino-5-imidazolecarboxamide (AIC), is
produced when MTIC, which is unstable, transforms into a methyldiazonium ion, a reactive
molecule that transfers the methyl group to DNA. The organism eliminates AIC through the
kidney. Following methylation at the N3 position of adenine (N3-MeA; 9%), methylation at the
O6 position of guanine (O6-MeG; 6%), and methylation at the N7 position of guanine (N7-MeG;
70%), the methyldiazonium ion methylates purine bases of DNA (Li et al., 2022). The fatal kind of
methylation that occurs in the O6 position of guanine is thought to be what causes TMZ's
cytotoxicity. According to the hypothesis, long-lasting DNA nicks occur when this repair process
is directed at the DNA strand opposite the O6-MG and is unable to identify the right partner.
These nicks build up and continue into the following cell cycle, where they eventually prevent
the daughter cells' ability to begin replicating, halting the cell cycle at the G2-M boundary.
Previous studies have shown that TMZ sensitivity correlates with enhanced DNA fragmentation
and apoptotic cell death in human leukemia cells(Raúl Ortiz et al., 2021). In addition to DNA
methylation, the methyldiazonium cation can also interact with RNA and soluble and cellular
protein. However, it doesn't seem that methylation of RNA or protein, or carbamoylation of RNA,
plays a substantial role in the anticancer activity of TMZ.
7

The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by the suicide
enzyme O6-methylguanine-DNA methyltransferase (MGMT) in tumors expressing this protein,
which also called direct repair (Song et al., 2015). MGMT is a small protein (22kDa) present in
both the cytoplasm and nucleus. MGMT protects the cell genome by repairing damaged DNA
while eliminating methyl adducts, restoring guanine, but with diminishing efficiency as adduct
size increases. In a stoichiometric, auto-inactivating reaction, the O6-alkyl group is transferred
from guanine to the cysteine residue (Cys 145) of MGMT's active site, repairing DNA and
deactivating MGMT at the same time. MGMT binds damaged substrate DNA in the minor groove.
The target base is then flipped out of the helix and bound to MGMT, changing the conformation
of the DNA binding domain and allowing alkylated MGMT to be released from DNA and degraded
by the ubiquitin/proteasomal system (Pegg, 2011). Inactivation of MGMT caused by MGMT
mutations and protein phosphorylation has also been found in human tumors. But MGMT
promoter methylation is most frequently a result of MGMT activity loss. On the cytosine of CpG
islands, methylation occurs through the action of 5'-methylcytosine methyltransferase. The
MGMT promoter region contains CpG islands that are hypermethylated, which prevents
transcription factors from binding and silences the gene (McCabe et al., 2009). According to
clinical data, TMZ radiotherapy treatment benefits patients with MGMT promoter methylation
more than it does those without.
MGMT is not the only DNA repair mechanism involved in TMZ resistance. The DNA
mismatch repair (MMR) system is also related to TMZ resistance. During DNA replication in the
S phase, the MMR system recognizes the error that thymine is incorporated in the
complementary strand pair with O6-MeG instead of cytosine. MMR system corrects the error by
8

removing the thymine from the complementary strand, although the primary error at the
guanine O6 position of the main chain is still present (R. Ortiz et al., 2021). A number of subunits
make up the heterodimers that make up the MMR system. This DNA repair process involves
several linked steps. In the first step, MutSα (MSH2-MSH6) heterodimer or MutSβ (MSH2-MSH3)
heterodimer recognize and bind to the mismatch. Different heterodimers function differently
depending on the type of mismatch. Then the corresponding heterodimer draws in the MLH1-
PMS2 complex to form a tertiary complex, which gathers the proteins needed to create a gap. In
the second step, the EXO1 exonuclease does the excision and ligase L1G1 does the repair and
ligation. This is how the TMZ-induced DNA damage is processed by the MMR system(Shinsato et
al., 2013). When O6-MeG pairs with thymine in the absence of MGMT, the MMR system detects
the mispair and starts an ineffective repair process that results in DNA strand breaks. When the
number of double strand breaks in the DNA molecule rises, the cell stops in the G1/S or G2/M
phase to give the repair process more time to continue (Pawlowska et al., 2018). If the repair
ultimately fails, apoptosis, senescence, or autophagy can be induced in the cell. On the contrary,
if the MMR complex is altered or rendered inactive, the incorrect O6-MeG-thymine pairs may
not be recognized and then continuous DNA replication and resistance to TMZ treatment result
(Syro et al., 2018). As a result, tolerance to TMZ increases.
Besides MGMT+ and MMR-, disruption of the DNA base excision repair (BER) pathway is
also involved in TMZ resistance. Particularly when O6-MeG adducts are repaired by MGMT+ or
tolerated with MMR- (Thakur et al., 2022), persistence of potentially lethal N3- and N7- purine
lesions contribute to TMZ cytotoxicity. In contrast to O6-MeG methylation, N3-MeA and N7-MeG
methylations are much more frequent. More than 90% of the methylations caused by TMZ are
9

N3-MeA and N7-MeG methylations, which are quickly and effectively repaired by the BER
pathway (R. Ortiz et al., 2021). The BER pathway is a multistep procedure that replaces a single
nucleotide that contains a damaged base. It involves the cooperation of several DNA repair
proteins. Reactive oxygen species, ionizing radiation, and alkylating agents all produce these
damaged nucleotides. Besides repairing base modification, the BER pathway also functions on
single-strand DNA breaks, which are caused by different types of DNA damage, such as oxidation,
deamination, and spontaneous hydrolysis. Lesion-specific glycosylases that hydrolytically cleave
the N-glycosidic bond to create an abasic (apurinic/apyrimidinic; AP) site recognize damaged
bases. The 5’ side of the AP site’s phosphodiester backbone is then broken by AP endonuclease
(APE-1), leaving the 3’-OH and 5’-deoxyribose phosphate (dRp) termini at the DNA strand break
(Zhang et al., 2012). Exonuclease or DNA-deoxyribophosphodiesterase (dRpase) removes the
terminal 5’dRp residue, leaving a nucleotide gap. There are two ways to continue with the repair:
short-patch BER and long-patch BER (Kim & Wilson, 2012). In the short patch, replacement of
one nucleotide is involved. DNA polymerase fills single nucleotide gaps, and the DNA ligase III/X-
ray repair cross-complementing 1 (XRCC1) heterodimer seals nicks. Gap filling of 2 to 10
nucleotides is involved in the lengthy patch. It’s possible for the DNA strand to move and cause
a “flap”. The flap is cut by the enzyme flap endonuclease-1 (FEN-1), and DNA ligase I seals the
ends of the DNA. Poly (ADP-ribose) polymerase-1 (PARP-1) is a crucial protein (113 kDa) in the
BER pathway for effective DNA damage signaling. By converting NAD+ into poly (ADP-ribose),
PARP-1 is crucial for the recruitment of BER pathway proteins and subsequent DNA repair. PARP
deficient cells are more susceptible to TMZ because PARP inhibition increases the frequency of
DNA strand breaks, which increases the cytotoxicity of the lesions. Studies have revealed that
10

TMZ treatment of human tumor cells increased the activity of PARP, which is thought to be
involved in nucleotide excision repair, and it has been reported that PARP inhibition increases
the cytotoxicity of methylating agents(Muvarak et al., 2016).
By reason of the foregoing, the TMZ drug resistance has been mainly linked to the
expression of MGMT, MMR deficiency or the disruption of BER. MGMT and MMR seem to be
more significant pathways in TMZ resistance than BER for the following reasons. First, except in
tumors lacking in base excision repair, the significance of N3-MeA and N7-MeG adducts in the
drug's antitumor activity might be secondary to that of the O6-MG adduct. Furthermore, N3
lesions are fatal only if they are not repaired, and the cytotoxicity of N7 lesions does not seem to
be very high. Besides the above three triggers, the expression of MGMT, MMR deficiency or the
disruption of BER, the presence of cancer stem-like cell subpopulations in tumors and some
acquired drug resistance have also been related to TMZ resistance, such as Src tyrosine kinase
pathway. Additional studies are required to clarify the role of these targets in the biochemical
mechanism of action of TMZ.
11

1.1.1 POH

Figure 1. 4. Chemical structure of POH
POH, also called p-metha 1,7-diene-6-ol or perillyl alcohol, is a natural monoterpene
compound isolated through the mevalonate biosynthetic pathway from the essential oils of
plants such as lavender, citrus fruits, peppermint. POH is composed of both hydrophobic and
hydrophilic regions. POH exhibits a very high degree of oral bioavailability in mammals. POH has
two main metabolites in mammals, which are detectable within 10 min after oral administration
(Crowell, 1999). These two metabolites are perillic acid (PA) and dihydroperillic acid (DHPA).
Extensive preclinical studies provided strong evidence of POH's promotion phase anticancer
activity against liver cancer, mammary and pancreatic tumors. Although clinical studies have
shown the activity of POH as an oral anticancer chemotherapy drug (Chen et al., 2015), POH has
not shown a significant effect under the dose limitation of gastrointestinal toxicity (Morgan-
Meadows et al., 2003). Due to the shortcomings of oral use of POH, intranasal use of POH is
considered as an alternative administration route. The results proved that POH intranasal
12

administration showed a clinically well-tolerated therapeutic effect on glioma, and the side
effects of this mode of administration are very mild and negligible which includes tolerable local
irritation in the nasal mucosa (Schönthal et al., 2022). NEO100 is a modern good manufacturing
practice (cGMP)-manufactured, highly purified (>99%) variant of POH. An FDA-approved phase
IIa trial for recurrent GBM is currently taking place with NEO100. It has been given both fast-
track and orphan drug status (Marin-Ramos et al., 2019). Uncertainty surrounds the precise
mechanism by which POH fights cancer, but it is most likely the result of pleiotropic effects that
include inhibition of Ras oncoprotein, aggravation of endoplasmic reticulum stress, cell cycle
arrest, and induction of apoptosis (Chen et al., 2015).
1.1.2 NEO212

Figure 1. 5. Chemical structure of NEO212
NEO212 is a covalent conjugation of POH and TMZ. It is a novel small molecule anticancer
drug currently in preclinical development. NEO212 is expected to have both alkylation function
from TMZ and pleiotropic damage function to cancer from POH. In numerous in vitro and in vivo
13

tumor models, NEO212 has demonstrated strong anti-cancer therapeutic activity (Cho et al.,
2014; Song et al., 2019). NEO212's physicochemical characteristics, composition, structural
features, and passive and active targeting to the tumor tissue are advantages in the treatment
of cancer. Such characteristics lengthen their bloodstream half-life and increase their specificity
for tumor tissues, making it possible to overcome drug resistance mechanisms as well. The TMZ
analogs, NEO212, maintain the TMZ mechanism of action while offering some benefits, such as
avoiding MGMT overexpression in tumor cells or boosting its concentration in the brain while
lowering it in the plasma.
1.2 Hypothesis
NEO212 is a promising anticancer drug for the treatment of hematologic malignancies for
both humans and canines. Given the striking similarities between hematological malignancies in
humans and dogs, it is of interest to investigate both the effect and mechanism of action of
NEO212 on human and canine hematological malignancies. Based on NEO212’s effect on other
cancer cells in prior studies. NEO212 is supposed to have a greater cytotoxic effect on canine and
human hematological malignancy cell lines than TMZ alone, POH alone, or a non-covalently
bound mixture of TMZ + POH. Therefore, when the high concentration of TMZ required for
treatment leads to resistance in blood cancer cell lines, this is expected not to happen with
NEO212 because it acts on cancer cells at a lower concentration. Due to the aforementioned, the
main causes of TMZ drug resistance have been identified as MGMT expression, MMR deficiency,
and/or BER disruption, it is reasonable to assume that perhaps NEO212 can overcome some of
these mechanisms and thus not develop drug resistance.

14

Chapter 2 – Materials and Methods
2.1 Pharmacological agents
NEO212 was manufactured by Norac Pharma (Azusa, CA) under current good
manufacturing practice (cGMP) conditions and was provided by NeOnc Technologies (Los
Angeles, CA). NEO212 was dissolved in dimethyl sulfoxide (DMSO) (Santa Cruz Biotechnology,
Dallas, TX) at 100 mM for in vitro experiment and at 500 mM for in vivo experiment. TMZ was
obtained from Sigma Aldrich (St. Louis, MO), and dissolved in DMSO to a concentration of 50
mM. Stock solutions of NEO212 and TMZ were stored at –80˚C. POH was provided by NeOnc
Technologies (Norac Pharma) and diluted with DMSO at 100 mM. It is an oily liquid that was
stored in a brown bottle at 4˚C. Phosphate buffered saline (PBS) was provided by the Cell Culture
Core Laboratory at the USC/Norris Comprehensive Cancer Center at the Keck School of Medicine
of the University of Southern California (Los Angeles, CA). Drugs were further diluted in tissue
culture medium when investigated for their antiproliferative effects in vitro.
2.2 Cell lines
We used nine human cell lines including U937, HL60, 6D10, THP1, KG1, Raji, LN229,
LN229M, and T98G cell lines. In addition, we use two canine cell lines, CLBL1 and CLL1390. The
cell line details were listed in the table below.

15

Number Cell line Type Source
1. U937 human myeloid leukemia ATCC
2. HL60 human myeloid leukemia ATCC
3. 6D10 human myeloid leukemia David Largaespada, U. Minnesota
4. THP1 human myeloid leukemia ATCC
5. KG1 human myeloid leukemia ATCC
6. Raji human Burkitt’s lymphoma ATCC
7. LN229 human glioblastoma ATCC
8. LN229M human glioblastoma ATCC
9. T98G human glioblastoma ATCC
10. CLBL1 canine lymphoma Peter Moore, UC Davis
11. CLL1390 canine T cell leukemia Peter Moore, UC Davis

U937, HL60, 6D10, THP1, KG1, Raji, LN229, LN229M, and T98G cell lines were maintained
in RPMI 1640 culture. CLBL1 and CLL1390 were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) culture medium. Medium was provided by the Cell Culture Core lab of the
USC/Norris Comprehensive Cancer Center. RPMI and MEM medium was supplemented with 10%
fetal bovine serum (FBS) (Omega Scientific (Tarzana, CA), 100 U/mL penicillin and 0.1 mg/mL
streptomycin (Cellgro/MediaTech, Manassas, VA). Cells were grown at 37°C in humidified air
containing 5% CO2.
U937, HL60, 6D10, THP1, KG1, Raji, LN229, LN229M, T98G, CLBL1, and CLL1390 cell lines
were passaged for less than 6 months after receipt.
16

2.3 MTT assay
Cells (8000, 4000, 2000, 1000) were seeded in 96-well plates in a volume of 50 µL per
well. 50 µL POH, TMZ or NEO212 with medium were added at 2 X drug concentrations and cells
were incubated for 5 days. 12 µL MTT dye was added at the end of incubation time. Then plates
were put back in the incubator for 3-4 hours. 100 µL solubilization solution was added into each
well. Then plates were incubated overnight in the incubator. The following day, absorbance was
assessed at 570 nm with a microtiter plate reader. Relative to the control cells that were not
treated, the percentage viability was calculated. Every experiment was carried out in duplicates
at each of more than three different cell densities.
2.4 Cell counting
10, 000 cells were seeded in 6-well plates in a volume of 3 mL per well. NEO212 with
medium were added at increasing drug concentrations. Fresh medium was added every day to
make sure that the volume of each well was 3 mL in total. Cell numbers were counted under a
microscope every day for 7 days.
2.5 Isolation of RNA and Quantitative RT-PCR
Total RNA from cell lines was isolated using Quick-RNA
TM
Miniprep Kit (Cat. # R1054;
ZYMO RESEARCH) according to the manufacturer’s protocol. RNA concentration was tested by
NanoDrop™ 2000/2000c Spectrophotometers (Thermo Scientific™). DNase/RNase-free water
was used as a negative control while measuring RNA concentration.
Quantitative RT-PCR was performed with iTaq Universal SYBR® Green One-Step Kit (Cat.
#1725150, BIO-RAD). The human ITGAM (sense, 5’-CAGCATCAATATCAGGTCAGCA-3’; antisense,
5’-GAAGCTCAGCCAGAAAGTCG-3’; 5 nmol; Manufacturing ID: 445118616; Integrated DNA
17

Technologies) gene, the human CDKN1A (sense, 5’-GCATGACAGATTTCTACCACTCC-3’; antisense,
5’-CGGCCAGGGTATGTACATGA-3’; 5 nmol; Manufacturing ID: 445118617; Integrated DNA
Technologies) gene, and human GAPDH (sense, 5’-TGACTTCAACAGCGACACCCA-3’; 5’-
CACCCTGTTGCTGTAGCCAAA-3’; 10 nmol; Manufacturing ID: 445118615; Integrated DNA
Technologies) gene, as an internal control, were amplified according to the manufacturer’s
protocol. In short, the RxnReady®oligos primers were 2 oligos premixed in a single tube shipped
dry and were then resuspended in DNase/RNase-free water to get 100 µM final concentration
and then diluted 1:20 to get the working concentration 5 µM. The reaction mix was 6.13 µL
containing 0.13 µL reverse transcriptase, 5 µL SYBR Green, and 1 µL of 5 µM random primers.
The isolated cell RNA was diluted with DNase/RNase-free water to make 200 ng/3.9 µL. The 6.13
µL reaction mix was mixed with 3.9 µL RNA, and then added as 10 µL/well to the 96-well PCR
plate to analyze.
RT-PCR conditions were as follows: 50 °C for 2 min, then 95°C for 10 min followed by 40
cycles at 95°C for 15 s, 60°C for 1 min. Quantitative RT-PCR was analyzed on Applied Biosystems
7500 Fast Real-Time PCR System (ThermoFisher) using Standard 7500 Mode.
Different samples' relative expression levels were calculated by: 2ΔΔCt (Livak Relative
Quantification Model).
2.6 Immunoblot
Cell samples were lysed in ice-cold Pierce RIPA Buffer (Cat. #89900; Thermo scientific)
supplemented with both protease and phosphatase inhibitors. Lysates were centrifuged at
18

14,000 rpm for 15 min in cold room (4°C). Supernatant was transferred to new prechilled 1.5 mL
Eppendorf tubes.
Each protein sample was in total 24 µL made of 20 µL protein (50 µg) plus RIPA buffer and
4 µL 6X sample loading buffer. These samples were incubated at 100°C for 5 min and stored in -
20°C.
SDS/PAGE running gel was made of 30% acrylamide/bis-acrylamide, 37.5:1 (2.7%
crosslinker) solution. 50 µg protein was loaded per well in the gel and ran at constant voltage.
The voltage was started with 90 V at the beginning for 10 min to let the samples run through the
stacking gel and line up, and then the voltage was increased to 100 V for 80 min while samples
were running in the resolving gel.
A small container of Semi-dry transfer buffer was used to immediately equilibrate the
SDS/PAGE gel after it had finished running for 10 minutes. The membrane used for semi-dry
transfer was polyvinylidene fluoride (PVDF) (Genesee Scientific) and its pore size is 0.45 µm.
PVDF membrane was pre-activated in pure methanol for 1 min and then equilibrated in the
transfer buffer. The components of the transfer buffer included 25 mM Tris, 192 mM glycine,
and 10% methanol. The whole Semi-dry transfer was run at 150 mA for 80 min.
Membranes were incubated in blocking buffer for 1 hour at room temperature. Blocking
buffer was made of 100 mL 1X TBST (TBS with Tween 20) with 5% nonfat dry milk. After blocking,
membranes were incubated overnight at 4 °C with primary antibody in the blocking buffer. After
washing 3 times with TBST for each time 10 min under fast shaking, the membranes were
incubated at room temperature for 45 min with 1:2500 peroxidase-conjugated AffiniPure Goat
19

Anti-Rabbit IgG (H+L) secondary antibody (Cat. #111-035-003; Jackson ImmnoResearch) in
blocking buffer with mild shaking.
After secondary antibody binding, the membranes were washed three times with TBST
for each time 10 min at room temperature under fast shaking. The membranes were detected
by the HRP system using ProSignal™ Pico (Cat. #: 20-300; Genesee Scientific). The membranes
were exposed in the chemiluminescence imager iBrightCL1000 (ThermoFisher).
2.7 In-Vivo
The Institutional Animal Care and Use Committee (IACUC) at the University of Southern
California reviewed and approved all animal research (USC). Female, immune-deficient NOD-
SCID mice were obtained from the Jackson Laboratory at 6–8 weeks of age for the
transplantation of human tumor cells into mice (Bar Harbor, ME). Female Fischer 344 rats that
were 29 weeks old and weighed around 200 grams were used to study the hazardous side effects
of high doses of NEO212 and temozolomide (Wilmington, MA). The USC Medical Center Animal
Facility, which is AAALAC and AALAS certified and has a written assurance of animal welfare with
the NIH-OLAW (Office of Laboratory Animal Welfare) committing the institution to uphold the
standards set forth by the Animal Welfare Act, served as the facility's home for all of the animals.
5x10
4
tumor cells in 50 L of 0.9% NaCl were injected into the peritoneum of mice to implant
AML cells. A few days later, mice were treated orally with 25 mg/kg NEO212 or just the vehicle.
The course of treatment was two or three cycles, with each cycle consisting of five days of once-
daily dose followed by a period of time without medication. Every day, animals were watched
after and taken care of.
20

2.8 Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.0.

21

Chapter 3 – Results
3.1 NEO212 inhibits proliferation of lymphoma and leukemia cells in vitro
The anticancer effects of NEO212 were characterized by MTT cytotoxicity assays and cell
countinig.
1. Effect of NEO212 on cell viability in the lymphoma cells measured by MTT assay
To start with, Raji cells were used to perform standard in vitro MTT cytotoxicity assays.
Four different cell densities were used, which are 8000, 4000, 2000 and 1000. The highest
concentration used in these assays was 100 µM. As shown in Figure 3.1, cell viability in response
to drug treatment in different cell densities were shown similar.
22

23

Figure 3. 1 Toxicity of NEO212, TMZ, POH, and TMZ +POH on Raji lymphoma cell line at different
cell density.
Raji cells were treated with increasing concentrations of NEO212, TMZ, POH, and TMZ +POH
separately after being seeded at four different cell densities (8000, 4000, 2000, and 1000). Cells
were treated for 72 hours, and the results were determined using an MTT assay. The percentage
of survival was calculated using untreated cells set at 100% as the baseline. (n = 2)

Cell viability in response to NEO212 treatment was compared to that after TMZ
treatment, POH treatment, and TMZ + POH treatment. It is shown that NEO212, as the
24

conjugated drug, is composed of two units, which are TMZ and POH. TMZ or POH individually has
its own anticancer capacity. In order to test if a simple mix of these two molecules, as individual
agents, would be able to mimic the potency of the conjugated NEO212 product, the TMZ + POH
treatment group was set up. In TMZ + POH group, TMZ and POH were added to cells at equimolar
concentrations.
As shown in Figure 3.2 (A), 2000 cells were used. CLBL1 cells were effectively killed by
NEO212, with an IC50 of about 10 µM or below. Compared to that of TMZ alone, the cytotoxic
IC50 of the TMZ + POH group was not decreased as the NEO212 group. For example, when cells
were treated with drug concentration at 50 µM for 72 hours, the cell viability of the TMZ + POH
group was similar to the TMZ group, which is around 50%. There was no enhancing cytotoxic
effect showing that simple mixed TMZ and POH cannot increase TMZ’s anticancer effect.
However, after treatment with 50 µM NEO212 for 72 hours, cell viability, which is around 15%,
was much lower than the TMZ group. According to the previous studies, to show the anticancer
effect, a very high concentration of POH needs to be used. Figure 3.2 (B) confirmed again that
the mixture of the two drugs is not able to mimic the greater toxicity of the conjugated NEO212
molecule. POH did not exhibit any cytotoxic effect when the drug concentration was below 100
µM. In conclusion, these results demonstrate that NEO212 was strikingly more potent than the
sum of its parts.

25

Figure 3. 2 Toxicity NEO212, TMZ, POH, and TMZ +POH on lymphoma cell lines.
A. CLBL1 cell line was used. Cells were treated with increasing concentrations of drugs for 3
days. Graph prepared by Dr. Axel Schönthal. (n = 3) B. Raji cell line was used. Cells were treated
with increasing concentrations of drugs for 3 days. (n = 2) The results were determined using an
MTT assay. The percentage of survival was calculated using untreated cells set at 100% as the
baseline.

2. Effect of NEO212 on cell viability in the leukemia cells measured by MTT assay
As shown in Figure 3.3, the comparison of observed IC50 between NEO212 and TMZ
differed greatly in different leukemia cell lines. For U937 and 6D10 leukemia cell lines, at the
26

same drug concentration, NEO212 and TMZ had similar toxic effects. However, for HL60, THP1,
KG1 and CLL1390 cell lines, the IC50 of NEO212 was much lower than TMZ.

0 50 100 150 200
0
50
100
CLL1390
Concentration ( μM)
Viability (%)
NEO212
TMZ

Figure 3. 3 Toxicity of NEO212 and TMZ on AML cell lines.
U937, 6D10, HL60, THP1, KG1, and CLL1390 cell lines were used. Cells were treated with
increasing concentrations of drugs for 5 days, and the results were determined using an MTT
assay. The percentage of survival was calculated using untreated cells set at 100% as the baseline.

27

Figure 3. 4 Toxicity of DMSO on AML cell lines.
HL60, U937 and THP1 cell lines were used. Cells were treated with increasing concentrations of
DMSO for 5 days, and the results were determined using an MTT assay. The percentage of
survival was calculated using untreated cells set at 100% as the baseline. Graph prepared by Dr.
Axel Schönthal.

For obtaining different drug concentrations to treat cancer cells, we dissolved the drug in
DMSO solvent. DMSO is an inert vehicle. There is no evidence that DMSO can treat cancer in
humans and no clinical studies have not been performed. However, some laboratory studies
have shown that DMSO may slow down the progression of cancer (Wang et al., 2012). For this
reason, to determine whether DMSO alone might exert any effects in our system, U937, HL60,
and THP1 cells were treated with increasing concentrations of DMSO, and cell viability was
determined by MTT assay as above. As shown in Figure 3.4, DMSO reduced cellular viability
starting at around 0.8% concentration, and at 1.5% it killed most of the cells. However, at
concentrations up to 0.4% it did not show a measurable negative impact on cell viability. Because
the DMSO concentration range used in our experiments was from 0.02 to 0.2%, we deemed the
potential impact of this vehicle as negligible.
28

3. Effect of NEO212 on cell proliferation and death measured by cell counting
The growth rate of KG1 cells was dramatically reduced under 10 µM of NEO212
treatment.
0 2 4 6 8
0
100
200
300
KG1
Time (Day)
Cell density (cell/ml x 10
3
)
Untreated
1 μM NEO212
2 μM NEO212
4 μM NEO212
10 μM NEO212
Vehicle

Figure 3. 5 Effect of NEO212 on AML cell viability.
Cells were treated with increasing concentrations of NEO212 for 7 days in total. Cell number
changes were recorded by daily cell counting. (n = 2)

3.2 NEO212 overcomes MGMT-mediated drug resistances
3.2.1 Multiple cell lines express different levels of MGMT
From the MTT results above (Figure 3.2 and Figure 3.3), the IC50 differential sensitivities
for each drug, NEO212 and TMZ, vary in different cell lines. To find the reason behind this,
Western blot analysis of the level of MGMT protein was performed. MGMT is a DNA repair
protein and proven to affect TMZ sensitivity to cells (Kitange et al., 2009). For the purpose of
determining the MGMT antibody's specificity, the following controls were used. T98G are
glioblastoma cells known to be positive for MGMT expression. LN229 are glioblastoma cells
known to be negative for MGMT (Köritzer et al., 2013). LN229-M is a subline infected with a
29

construct expressing MGMT cDNA. HL60 and THP1 cells were treated for 18 hours with 15 µM
O6-benzylguanine (O6BG). O6BG is a potent small-molecule inhibitor of MGMT.
As shown in Figure 3.6, U937 and 6D10 are MGMT-negative cell lines; HL60, THP1 and
KG1 are MGMT-positive cell lines. The data revealed that those three cell lines (HL60, KG1, THP1)
with high IC50s after TMZ treatment also displayed pronounced expression levels of MGMT,
whereas the two cell lines (U937 and 6D10) with low TMZ IC50s were negative for MGMT
expression.
As shown in Figure 3.3, while it was anticipated that IC50s and MGMT levels would closely
correlate with TMZ, this relationship did not develop with NEO212. MGMT-positive HL60 and
KG1 cells were killed by NEO212 just as efficiently as MGMT-negative U937 and 6D10 cells.
NEO212 treatment consistently had a greater cytotoxic effect than TMZ on MGMT-positive THP1
cells, despite the fact that these cells were a little less sensitive to NEO212 than the other four
cell lines tested.

Figure 3. 6 Hematologic malignancy cell lines express different levels of MGMT.
A MGMT-specific antibody was used in a Western blot analysis to examine cell lysates from log-
phase-grown cells. Actin was used as the loading control.

U937
HL60
HL60 + O6BG
6D10
THP1
THP1 + O6BG
T98G
LN229
LN229-M
25
35
15
55
40
MW
(kDa)
actin
MGMT
U937
HL60
KG1
30

Based on the results of previous experiments (Figure 3.6), here the LN229 cell line was
used as a negative control, and THP1 and LN229-M cell lines were used as positive controls. As
shown in Figure 3.7, CLBL1, CLL1390 and Raji are MGMT-negative cell lines. However, according
to the results of the previous MTT assay (Figure 3.2 and Figure 3.3), the sensitivity of these three
cell lines (CLBL1, CLL1390 and Raji) to NEO212 was much higher than that of TMZ. This also
suggests that, in addition to MGMT overexpression, there are other reasons why NEO212 is not
resistant to TMZ.

Figure 3. 7 Additional hematologic malignancy cell lines express different levels of MGMT.
A MGMT-specific antibody was used in a Western blot analysis to examine cell lysates from log-
phase-grown cells. Actin was used as the loading control.

3.2.2 NEO212 treatment down-regulates MGMT protein levels.
How NEO212 and TMZ treatment affected MGMT protein levels were investigated. As
shown in Figure 3.8, MGMT protein levels were dramatically reduced after cells were treated
with NEO212, but not after cells were treated with TMZ. In THP1 cells compared to HL60, this
down-regulation was a little more pronounced and was visible as early as 16 hours after the
addition of 50 µM NEO212. In contrast, TMZ was unable to have any discernible effects on MGMT
protein levels, even at significantly higher concentrations up to 200 µM.
31

Figure 3. 8 Treatment with NEO212 reduces MGMT protein levels.
MGMT protein levels were assessed using a Western blot after THP1 and HL60 cells were exposed
to NEO212 or TMZ. A. After 24 hours, cells that had been exposed to increasing drug
concentrations were harvested. B. At 80 µM, cells were harvested at various times. C. After 24
hours, cells that had been exposed to increasing drug concentrations were harvested.

32

3.3 NEO212 induces macrophage differentiation, proliferation arrest and eventual
apoptosis
3.3.1 NEO212 increases CD11b (ITGAM) mRNA levels
RNA-seq analysis of NEO212-treated 6D10 cells was carried out to acquire some
mechanistic insight into how NEO212 might achieve its powerful anticancer effect on AML cells.
U937 cells' AraC-resistant subline is called 6D10. AraC, cytosine arabinoside, is a most used
chemotherapy for AML, and also used for lymphomas. After being exposed to NEO212 for 1, 2,
3, and 5 days, cells were contrasted with those that were left untreated or exposed to DMSO for
the same amount of time. Commercial processing of the samples was done by Novogene
(Sacramento, CA), including quality controls, library construction, and RNA sequencing on
Illumina sequencing platforms (Illumina, San Diego, CA). A sizable collection of targets that were
markedly elevated and considered to be indicators of macrophage differentiation were among
the numerous transcripts that were discovered to be impacted by NEO212. Most of these
transcripts progressively accumulated over the 5-day analysis and peaked in expression on day
5 after NEO212 was added (Figure 3.9). Five of these transcripts had fold induction levels
between 2.0 and 4.1-fold, ten had levels between 5.1 and 18.8-fold, and the final two had levels
between 131.6 and 142.0-fold. These results showed that NEO212 treatment did not just cause
a quick cytotoxic shutdown of cellular processes, but also that it initiated differentiation of these
AML cells along the macrophage pathway. Instead, even between days 3 and 5 after medication
therapy, the bulk of these transcript levels significantly increased, indicating continuing cellular
function.
33

Figure 3. 9 Treatment with NEO212 induces the expression of markers for macrophage
differentiation.
30 µM NEO212 was used to treat AraC-resistant 6D10 cells. The cells were harvested after 1, 2,
3, and 5 days and then processed for RNA-seq analysis. Cells in comparison cell cultures were
either left untreated or received a 5-day DMSO vehicle treatment. After 5 days of drug treatment,
the right column (fold induction) indicates the fold increase of the relevant transcript in
comparison to untreated cells. Green on the heatmap represents pre-treatment levels, while
yellow denotes an improvement. Graph prepared by Dr. Axel Schönthal.

The most common marker for macrophage differentiation, CD11b (also known as ITGAM:
integrin subunit alpha M), increased by 5.1-fold in the aforementioned transcriptomic analysis
(Figure 3.9). This effect was verified in other AML cell lines. In addition to 6D10 cells, NEO212
was applied to U937, HL60, and THP1 cells for 3 or 5 days. Following drug treatment, RNA was
extracted, and all samples underwent RT-qPCR. This showed that CD11b transcript induction was
always significantly increased (p <0.01). (Figure 3.10). As a positive control, the phorbol ester
TPA, which is a recognized trigger for AML cells to differentiate into macrophages, was employed.
fold
induction
34

Additionally, TPA increased CD11b expression (Figure 3.10E). The results of treating the cells with
equimolar concentrations of TMZ combined with POH (Figure 3.10E), however, did not result in
elevated CD11b expression, demonstrating that the effects of the conjugated product cannot be
replicated by the combination of the individual components that make up NEO212.

Figure 3. 10 Treatment with NEO212 elevates the levels of CD11b mRNA.
AML cell lines that had received drug treatment were collected, and RT-qPCR was used to
measure CD11b transcript levels. Control cells only got the vehicle (vh) (DMSO). A. U937 cells
were treated with 10 µM NEO212 for the indicated times. B. 6D10 cells were treated with 10 µM
NEO212. C. THP1 cells were treated with 50 µM NEO212. D. HL60 cells were treated with 10 µM
NEO212. E. THP1 cells were treated for 4 days with TMZ in combination with equimolar
concentrations of POH. As a positive control, these were also treated with 10 nM TPA for 2 days.
Asterisks: * = p < 0.05, ** = p < 0.01, *** = p < 0.001; n.s. = not significant.

A
CD1 1b
CD1 1b
CD1 1b
0
5
10
15
20
U937-CD11b
Relative Levels
Vehicle
3d NEO212
5d NEO212
U937 vh. NEO212
days 3 3 5
n.s.
**
**
B
CD1 1b
CD1 1b
CD1 1b
0
2
4
6
8
Relative Levels
6D10-CD11b
Vehicle
3d NEO212
5d NEO212
*
***
***
6D10 vh. NEO212
days 5 3 5
CD1 1b
CD1 1b
CD1 1b
0
2
4
6
Relative Levels
THP1-CD11b
Vehicle
3d NEO212
5d NEO212
THP1 vh. NEO212
days 3 3 5
n.s.
***
***
C
CD1 1b
CD1 1b
CD1 1b
0
5
10
15
U937-CD11b
Relative Levels
Vehicle
3d 75 µM NEO212
3d 100 µM NEO212
*
**
***
HL60 vh. NEO212
µM 0 75 100
D
E
n.s.
n.s.
***
CD11b CD11b CD11b CD11b
0
2
4
6
Relative Levels
THP1 vh. TMZ + POH TPA
µM 0 80 100 0.01
Figure 6
35

3.3.2 NEO212 increases CDKN1A (p21) mRNA levels
In addition to the role that the macrophage differentiation pathway plays in the
anticancer impact of NEO212, the ability of NEO212 for proliferation arrest and apoptosis was
also investigated.
CDKN1A, also called p21, is a tumor suppressor gene which encodes a potent cyclin-
dependent kinase (CDK) inhibitor 1A (Al & Gali, 2019). By adhering to and preventing the activity
of cyclin-cyclin-dependent kinase 2 (Cdk2) or cyclin-cyclin-dependent kinase 4 (Cdk4) complexes,
the encoded protein acts as a regulator of cell cycle progression in the G1 stage. The expression
of the CDKN1A gene is tightly regulated by the tumor suppressor protein p53, which causes the
p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. Overall,
CDKN1A overproduction results in G1 arrest (Harper et al., 1995).
6D10 and U937 cells were treated with NEO212 for 3 or 5 days at 30 µM. HL60 was
treated with NEO212 for 3 days at 75 and 100 µM. After drug treatment, RNA was extracted, and
all samples underwent RT-qPCR. As observed in Figure 3.11 below, 3-day treatment with NEO212
increased the expression of CDKN1A in comparison to both the untreated and vehicle group.
36

Figure 3. 11 Treatment with NEO212 elevates the levels of CDKN1A mRNA.
AML cell lines that had received drug treatment were collected, and RT-qPCR was used to
measure CDKN1A transcript levels. Control cells only got the vehicle (vh) (DMSO). HL60 cells were
treated with 75 µM and 100 µM NEO212 for 3 days. U937 cells were treated with 30 µM NEO212
for 3 and 5 days separately. 6D10 cells were treated with 30 µM NEO212 for 3 and 5 days
separately. (n = 2)

3.3.3 NEO212 upregulated γ-H2AX and cl. caspase7 protein level

In addition to NEO212 promoting the cancer cell macrophage differentiation and
proliferation arrest, established markers of apoptosis were also investigated, such as p-H2AX and
cl. caspase7 by Western blot analysis.
Phosphorylated histone variant H2AX identifies DNA damage and the site of DNA repair.
Double stranded breaks (DSBs), which can result from endogenous or external DNA damage, are
always followed by the phosphorylation of the histone H2AX. The H2A protein family, which
makes up the histone octamer in nucleosomes, includes the variant H2AX. It is phosphorylated
in the PI3K pathway by protein kinases such ataxia telangiectasia mutated (ATM) and ATM-Rad3-
related (ATR). γ -H2AX, a freshly phosphorylated protein, is the starting point for attracting and
37

locating DNA repair proteins. DSBs can be brought on by cytotoxic chemicals or ionizing radiation,
for example, and as a result, γ-H2AX foci are swiftly formed (Kuo & Yang, 2008). These foci serve
as a biomarker for damage because they accurately depict DSBs. By using flow cytometry to
identify and visualize γ-H2AX, DNA damage, associated DNA damage proteins, and DNA repair
can all be evaluated. Other uses for γ -H2AX include the detection of genomic damage brought
on by cytotoxic chemical agents, as well as environmental and physical damage, particularly in
the context of cancer therapy and treatment.
Caspases, a family of proteolytic enzymes, are known as the central regulators of
apoptosis. Caspase-7 contains a catalytic cysteine residue in its active site that helps to cleave
various substrates such as PARP. Caspase 7 functions downstream of the mitochondrial-initiated
apoptosis including producing ROS, which promotes cell separation during apoptosis (Julien &
Wells, 2017). The phosphorylation of caspase 7 inhibits its activity and reduces cellular apoptosis
(Eron et al., 2017).
The THP1 cell line was treated with increasing concentrations of 40, 60, and 80 µM
NEO212 and TMZ for 52 hours. The expression level of cl. caspase 7 increased after 52-hour
treatment with NEO212 at 80 µM compared to the untreated group and TMZ treated groups.
The CLL1390 cell line was treated with increasing concentrations of NEO212 at 40 and 70 µM and
TMZ at 80 and 150 µM for 72 hours. The expression levels of both γ-H2AX and cl. caspase 7
increased after 72-hour treatment with NEO212 at 70 µM compared to the untreated group.
Conversely, the expression level of both γ-H2AX and cl. Caspase 7 of TMZ treated groups was not
upregulated compared to untreated or vehicle groups even when the concentration of TMZ (80
and 150 µM) was higher than NEO212 (40 and 70 µM). The HL60 cell line was treated with
38

increasing concentrations at 10, 30, 50, 60, and 75 µM of NEO212 for 96 hours. Compared to the
untreated or vehicle group, the expression level of cl. caspase 7 increased after the treatment
with NEO212 at 10 µM or higher. The CLBL1 cell line was treated with increasing time (0, 1, 2, 3,
and 4 days) of NEO212 at 6 µM. The expression levels of both γ-H2AX and cl. caspase 7 were
increased over time with NEO212 treatment. In conclusion, the protein expression levels of γ-
H2AX and cl. Caspase 7 increased after NEO212 treatment. However, TMZ didn’t have this effect,
even when the concentration of TMZ was as high as 150 µM. Additionally, this agrees with the
outcomes of the MTT assay. These two cell lines (HL60 and CLBL1) showed such an impact at
relatively low therapeutic doses of NEO212, 10 µM or lower.

Figure 3. 12 Treatment with NEO212 induces γ-H2AX and cl. caspase 7 protein levels.
γ-H2AX and cl. caspase 7 protein levels were assessed using a Western blot after THP1, CLL1390,
HL60, and CLBL1 cells were exposed to NEO212 or TMZ. A. After 52 hours, THP1 cells that had
39

been exposed to increasing NEO212 and TMZ concentrations were harvested. B. After 3 days,
CLL1390 cells that had been exposed to increasing NEO212 and TMZ concentrations were
harvested. C. After 4 days, HL60 cells that had been exposed to increasing NEO212 concentrations
were harvested. D. At 6 µM, CLBL1 cells were harvested at various times.

3.4 NEO212 contributes to mice survival in vivo
Then, we went on to in vivo research. We have previously demonstrated that NEO212,
after being implanted into mice, exhibited therapeutic effectiveness against MGMT-negative
blood cancer cells. We used this model in our mouse studies because the above in vitro findings
showed that NEO212 also had strong cytotoxic effects against MGMT-positive blood cancer cells.
We used TMZ-resistant MGMT-positive HL60 human cells, AraC- resistant MGMT-positive 6D10
human cells, MGMT-negative Raji human cells, and TMZ-resistant CLBL1 canine cells
Immune-compromised mice were injected with HL60 and 6D10 cell lines, then given
NEO212 or vehicle as the placebo. NEO212 was administered in cycles, with one cycle involving
a once-daily oral gavage for five days straight. Following this cycle, a treatment break lasting a
few days to a week was followed by another. There were no more treatments after that.

Figure 3. 13 Survival rates for HL60 mice treated with NEO212 versus control (vehicle).
40

The group treated with NEO212 had 7 mice, compared to the vehicle group's 6 mice. Experiment
and graph prepared by Dr. Steve Swenson. ***p-value<0.001.

Figure 3. 14 Survival rates for 6D10 mice treated with NEO212 versus control (vehicle).
The group treated with NEO212 had 4 mice, compared to the vehicle group's 5 mice. Experiment
and graph prepared by Dr. Steve Swenson. ***p-value<0.001.

Immune-compromised mice were injected with Raji or CLBL1 cell lines, then given
NEO212 or TMZ or vehicle as the placebo. NEO212 was administered in cycles, with one cycle
involving a once-daily oral gavage for five days straight. Following this cycle, a treatment break
lasting a few days to a week was followed by another. There were no more treatments after that.
41

Figure 3. 15 Survival rates for HL60 mice treated with NEO212, TMZ and control (vehicle).
The groups treated with NEO212 and TMZ both had 7 mice, compared to the vehicle group's 6
mice. Experiment and graph prepared by Dr. Steve Swenson. **p-value<0.01.

Figure 3. 16 Survival rates for HL60 mice treated with NEO212, TMZ and control (vehicle).
The groups treated with NEO212 had 12 mice. The groups treated with TMZ had 7 mice compared
to the vehicle group's 11 mice. Experiment and graph prepared by Dr. Steve Swenson. ****p-
value<0.0001.
42

Chapter 4 – Discussion
NEO212 is a novel hybrid molecule produced by conjugation of POH and TMZ. Five
different AML cell lines (U937, 6D10, THP1, HL60, and KG1), including chemoresistant cells
(6D10) to AraC and three chemoresistant cells (HL60, KG1, THP1) to TMZ, were inhibited from
proliferating by NEO212. The expression of MGMT is highly expressed in the three TMZ-resistant
cell lines. The growth inhibition was accompanied by the appearance of a series of markers of
macrophage differential, proliferation arrest and apoptosis. In two in vivo mouse models with
chemoresistant cells (HL60 and 6D10), all treated animals survived for 300 days after receiving a
few brief cycles of oral NEO212. In addition to its effect on AML cell lines, NEO212 blocked the
proliferation of two different TMZ-resistant lymphoma cell lines (Raji and CLBL1) in vitro. In these
two mouse models, a few short cycles of oral administration of NEO212 resulted in a substantial
increase in survival time with some of the treated animals surviving to 300 days resulting in a
dramatic increase in survival rate in treated animals.
TMZ is a well-known anticancer medication that works by methylating the O6 position of
guanine. The fatal kind of methylation that occurs in the O6 position of guanine is thought to be
what causes TMZ's cytotoxicity. MGMT repairs DNA by removing the methyl group that the
alkylating agent places at the O6-guanine position and moving the group to itself. This also causes
the enzyme to be degraded via the ubiquitin/proteasomal system. Due to the stochastic nature
of this process, it can be used to assess the degree of DNA methylation at the O6-guanine
position. It's interesting to note that TMZ was unable to cause this downregulation of MGMT
(Figure 3.8) at concentrations comparable to or even double those of NEO212. On the contrary,
NEO212 treatment was discovered to reduce the amount of intracellular MGMT proteins,
43

indicating that the medication is very effective at preventing DNA methylation by O6-guanine.
This difference may be explained by the fact that NEO212 enters the cell more frequently than
TMZ and accumulates more methyl-O6-guanine molecules, placing a heavier burden on MGMT-
mediated repair in NEO212-treated cells. The earlier studies, which produced the following
results, provide evidence in favor of this model. NEO212 degrades to its constituents TMZ and
POH after being absorbed by cells (Cho et al., 2020). It's interesting to note that intracellular TMZ
concentrations are higher after NEO212 treatment than after TMZ treatment, which is consistent
with more thorough O6-guanine DNA methylation.
In the case of MGMT-negative cells, the effect of TMZ was comparable to that of NEO212
in some cell lines (U937 and 6D10) (Figure 3.6), which appears to refute the theory that NEO212's
greater efficacy may be attributable to its superior cell entry. We speculate that this might be
the result of an unusual cell type-specific effect, though we are currently unable to offer a
satisfactory explanation. The cytotoxicity IC50 of NEO212 was consistently found to be
significantly lower than that of TMZ when we previously compared the cytotoxicity of the two
drugs in a variety of MGMT-negative cell lines from various tumor types. As other MGMT-
negative cell lines used (Raji, CLBL1 and CLL390) also proved this point once again (Figure 3.2 and
Figure 3.3). In these three MGMT-negative cell lines, the cytotoxic IC50 of NEO212 was
significantly lower than the IC50 of TMZ. The U937 cell line and its AraC-resistant 6D10 subline
(Schönthal et al., 2021) are a notable exception in this regard and requires further investigation.
NEO212's cytotoxic potential can be explained by its ability to alkylate O6-guanine, but in
addition to this mechanism, NEO212 also exhibits some ability to induce macrophage
differentiation, trigger proliferative arrest, and promote apoptosis. First, NEO212 has the ability
44

to induce macrophage differentiation. It is common knowledge that the typical inducer that
promotes macrophage differentiation in AML cells is the phorbol ester TPA. TPA can induce
macrophage markers like the integrin CD11b and proliferation arrest (Otte et al., 2011;
Prudovsky et al., 2002). In this TPA-induced differentiation state, cells gradually enter apoptosis
and perish after a few days. As a result, many similarities between TPA-treated cells and those
treated with NEO212 were found, including growth arrest, a notable rise in a group of
macrophage differentiation markers, and a slow entry into apoptosis. Second, in addition to
inducing macrophage differentiation, NEO212 also exhibits an ability to increase intracellular
CDKN1A protein expression by causing cell cycle arrest. CDKN1A is a tumor suppressor that
causes growth inhibition of cancer cells. This growth inhibition is triggered by binding two
different domains. One is the PCNA domain and the other is the CDK-cyclin inhibitory domain
(Rousseau et al., 1999). However, CDKN1A behaves as an oncogene in some cellular
environments, which complicates the direct theory of CDKN1A as a tumor suppressor. the
rationale for CDKN1A as an oncogene is that it has an inhibitory effect on apoptosis. Therefore,
to further investigate the mechanism behind the upregulation of CDKN1A protein expression by
NEO212, upstream or downstream factors of CDKN1A in cells after NEO212 treatment can be
selectively investigated in the future. This will further confirm whether NEO212 does cause
reproductive arrest in cancer cells (Abbas & Dutta, 2009). Third, in addition to inducing
macrophage differentiation and causing cell cycle arrest, NEO212 also has the potential to
directly cause apoptosis. For example, protein expressions of apoptosis markers (γ-H2AX and cl.
caspase 7) were upregulated in cancer cells after NEO212 treatment.
45

It is noted that prior research has also demonstrated that the organosulfur compound
DMSO treatment can cause the differentiation process to occur. However, according to reports,
DMSO is effective only at concentrations above 1% (Galvao et al., 2014; Hoyberghs et al., 2021).
It is essential to rule out in the experiments that NEO212-induced differentiation is not caused
by the DMSO carrier because the majority of our medications use DMSO as a solvent. While we
did observe that DMSO has a growth-inhibiting effect, it was not visible at concentrations of 0.4%
and lower. Instead, it became very effective at 1.5% after beginning to show effects at around
0.8%. (Figure 3.4). We were able to rule out the possibility that the low concentrations of DMSO
used in our experiments had detectable biological activity because the concentration of DMSO
was kept below 0.2% and it was used as a comparator for untreated cells in all experiments.
The striking result in the study was the finding that NEO212 could significantly cure mice
carrying drug-resistant AML cells. None of the animals treated with NEO212 died from the
disease; instead, they continued to grow until a predetermined endpoint of 300 days (Figures
3.13 and 3.14), which is comparable to 33 years in humans (Dutta & Sengupta, 2016). As a result,
NEO212 application could be clinically advantageous for AML patients. The therapeutic effect of
NEO212 was weaker in mice carrying drug-resistant lymphoma cells compared to mice carrying
drug-resistant AML cells (Figure 3.15 and Figure 3.16). However, the effect of NEO212 was still
significantly better than that of TMZ. These findings imply that NEO212 is a therapeutic agent for
hematologic malignancies that needs to be taken into consideration for development.

46

Bibliography
1. Abbas, T., & Dutta, A. (2009). p21 in cancer: intricate networks and multiple activities. Nat
Rev Cancer, 9(6), 400-414. https://doi.org/10.1038/nrc2657
2. Al, B., & Gali, M. (2019). The Role of the Cyclin Dependent Kinase Inhibitor p21cip1/waf1
in Targeting Cancer: Molecular Mechanisms and Novel Therapeutics. Cancers, 11(10),
1475. https://doi.org/10.3390/cancers11101475
3. Alard, E., Butnariu, A. B., Grillo, M., Kirkham, C., Zinovkin, D. A., Newnham, L., Macciochi,
J., & Pranjol, M. Z. I. (2020). Advances in Anti-Cancer Immunotherapy: Car-T Cell,
Checkpoint Inhibitors, Dendritic Cell Vaccines, and Oncolytic Viruses, and Emerging
Cellular and Molecular Targets. Cancers (Basel), 12(7).
https://doi.org/10.3390/cancers12071826
4. Auberger, P., Tamburini-Bonnefoy, J., & Puissant, A. (2020). Drug Resistance in
Hematological Malignancies. Int J Mol Sci, 21(17). https://doi.org/10.3390/ijms21176091
5. Chen, T. C., Fonseca, C. O., & Schönthal, A. H. (2015). Preclinical development and clinical
use of perillyl alcohol for chemoprevention and cancer therapy. Am J Cancer Res, 5(5),
1580-1593. https://www.ncbi.nlm.nih.gov/pubmed/26175929
6. Cho, H. Y., Swenson, S., Thein, T. Z., Wang, W., Wijeratne, N. R., Marin-Ramos, N. I., Katz,
J. E., Hofman, F. M., Schönthal, A. H., & Chen, T. C. (2020). Pharmacokinetic properties of
the temozolomide perillyl alcohol conjugate (NEO212) in mice. Neurooncol Adv, 2(1),
vdaa160. https://doi.org/10.1093/noajnl/vdaa160
7. Cho, H. Y., Wang, W., Jhaveri, N., Lee, D. J., Sharma, N., Dubeau, L., Schönthal, A. H.,
Hofman, F. M., & Chen, T. C. (2014). NEO212, temozolomide conjugated to perillyl
alcohol, is a novel drug for effective treatment of a broad range of temozolomide-
resistant gliomas. Mol Cancer Ther, 13(8), 2004-2017. https://doi.org/10.1158/1535-
7163.MCT-13-0964
8. Crowell, P. L. (1999). Prevention and therapy of cancer by dietary monoterpenes. J Nutr,
129(3), 775S-778S. https://doi.org/10.1093/jn/129.3.775S
9. Dutta, S., & Sengupta, P. (2016). Men and mice: Relating their ages. Life Sci, 152, 244-248.
https://doi.org/10.1016/j.lfs.2015.10.025
10. Eron, S. J., Raghupathi, K., & Hardy, J. A. (2017). Dual Site Phosphorylation of Caspase-7
by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms. Structure, 25(1), 27-39.
https://doi.org/10.1016/j.str.2016.11.001
47

11. Friedman, H. S., Kerby, T., & Calvert, H. (2000). Temozolomide and treatment of malignant
glioma. Clin Cancer Res, 6(7), 2585-2597.
https://www.ncbi.nlm.nih.gov/pubmed/10914698
12. Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., & Cordeiro, M. F. (2014).
Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J, 28(3), 1317-1330.
https://doi.org/10.1096/fj.13-235440
13. Garza-Morales, R., Gonzalez-Ramos, R., Chiba, A., Montes De Oca-Luna, R., McNally, L.,
McMasters, K., & Gomez-Gutierrez, J. (2018). Temozolomide Enhances Triple-Negative
Breast Cancer Virotherapy In Vitro. Cancers, 10(5), 144.
https://doi.org/10.3390/cancers10050144
14. Harper, J. W., Elledge, S. J., Keyomarsi, K., Dynlacht, B., Tsai, L. H., Zhang, P., Dobrowolski,
S., Bai, C., Connell-Crowley, L., Swindell, E., & et al. (1995). Inhibition of cyclin-dependent
kinases by p21. Mol Biol Cell, 6(4), 387-400. https://doi.org/10.1091/mbc.6.4.387
15. Hoyberghs, J., Bars, C., Ayuso, M., Van Ginneken, C., Foubert, K., & Van Cruchten, S.
(2021). DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo
Developmental Toxicity Assay. Front Toxicol, 3, 804033.
https://doi.org/10.3389/ftox.2021.804033
16. Inno, A., Fanetti, G., Di Bartolomeo, M., Gori, S., Maggi, C., Cirillo, M., Iacovelli, R., Nichetti,
F., Martinetti, A., de Braud, F., Bossi, I., & Pietrantonio, F. (2014). Role of MGMT as
biomarker in colorectal cancer. World J Clin Cases, 2(12), 835-839.
https://doi.org/10.12998/wjcc.v2.i12.835
17. Islami, F., Ward, E. M., Sung, H., Cronin, K. A., Tangka, F. K. L., Sherman, R. L., Zhao, J.,
Anderson, R. N., Henley, S. J., Yabroff, K. R., Jemal, A., & Benard, V. B. (2021). Annual
Report to the Nation on the Status of Cancer, Part 1: National Cancer Statistics. J Natl
Cancer Inst, 113(12), 1648-1669. https://doi.org/10.1093/jnci/djab131
18. Julien, O., & Wells, J. A. (2017). Caspases and their substrates. Cell Death Differ, 24(8),
1380-1389. https://doi.org/10.1038/cdd.2017.44
19. Kim, Y. J., & Wilson, D. M., 3rd. (2012). Overview of base excision repair biochemistry.
Curr Mol Pharmacol, 5(1), 3-13. https://doi.org/10.2174/1874467211205010003
20. Kitange, G. J., Carlson, B. L., Schroeder, M. A., Grogan, P. T., Lamont, J. D., Decker, P. A.,
Wu, W., James, C. D., & Sarkaria, J. N. (2009). Induction of MGMT expression is associated
with temozolomide resistance in glioblastoma xenografts. Neuro Oncol, 11(3), 281-291.
https://doi.org/10.1215/15228517-2008-090
21. Köritzer, J., Boxhammer, V., Schäfer, A., Shimizu, T., Klämpfl, T. G., Li, Y.-F., Welz, C.,
Schwenk-Zieger, S., Morfill, G. E., Zimmermann, J. L., & Schlegel, J. (2013). Restoration of
48

Sensitivity in Chemo — Resistant Glioma Cells by Cold Atmospheric Plasma. PLoS ONE,
8(5), e64498. https://doi.org/10.1371/journal.pone.0064498
22. Koumarianou, A., Antoniou, S., Kanakis, G., Economopoulos, N., Rontogianni, D.,
Ntavatzikos, A., Tsavaris, N., Pectasides, D., Dimitriadis, G., & Kaltsas, G. (2012).
Combination treatment with metronomic temozolomide, bevacizumab and long-acting
octreotide for malignant neuroendocrine tumours. Endocrine-Related Cancer, 19(1), L1-
L4. https://doi.org/10.1530/erc-11-0287
23. Kuo, L. J., & Yang, L. X. (2008). Gamma-H2AX - a novel biomarker for DNA double-strand
breaks. In Vivo, 22(3), 305-309. https://www.ncbi.nlm.nih.gov/pubmed/18610740
24. Li, J., Koczor, C. A., Saville, K. M., Hayat, F., Beiser, A., McClellan, S., Migaud, M. E., & Sobol,
R. W. (2022). Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD+
Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase. Cancers, 14(15), 3572.
https://doi.org/10.3390/cancers14153572
25. Marin-Ramos, N. I., Perez-Hernandez, M., Tam, A., Swenson, S. D., Cho, H. Y., Thein, T. Z.,
Hofman, F. M., & Chen, T. C. (2019). Inhibition of motility by NEO100 through the calpain-
1/RhoA pathway. J Neurosurg, 1-12. https://doi.org/10.3171/2019.5.JNS19798
26. Mason, W. (2009). Temozolomide: The evidence for its therapeutic efficacy in malignant
astrocytomas. Core Evidence, 93. https://doi.org/10.2147/ce.s6010
27. McCabe, M. T., Brandes, J. C., & Vertino, P. M. (2009). Cancer DNA Methylation: Molecular
Mechanisms and Clinical Implications. Clinical Cancer Research, 15(12), 3927-3937.
https://doi.org/10.1158/1078-0432.ccr-08-2784
28. Morgan-Meadows, S., Dubey, S., Gould, M., Tutsch, K., Marnocha, R., Arzoomanin, R.,
Alberti, D., Binger, K., Feierabend, C., Volkman, J., Ellingen, S., Black, S., Pomplun, M.,
Wilding, G., & Bailey, H. (2003). Phase I trial of perillyl alcohol administered four times
daily continuously. Cancer Chemotherapy and Pharmacology, 52(5), 361-366.
https://doi.org/10.1007/s00280-003-0684-y
29. Muvarak, N. E., Chowdhury, K., Xia, L., Robert, C., Choi, E. Y., Cai, Y., Bellani, M., Zou, Y.,
Singh, Z. N., Duong, V. H., Rutherford, T., Nagaria, P., Bentzen, S. M., Seidman, M. M.,
Baer, M. R., Lapidus, R. G., Baylin, S. B., & Rassool, F. V. (2016). Enhancing the Cytotoxic
Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for
Cancer. Cancer Cell, 30(4), 637-650. https://doi.org/10.1016/j.ccell.2016.09.002
30. Ortiz, R., Perazzoli, G., Cabeza, L., Jimenez-Luna, C., Luque, R., Prados, J., & Melguizo, C.
(2021). Temozolomide: An Updated Overview of Resistance Mechanisms,
Nanotechnology Advances and Clinical Applications. Curr Neuropharmacol, 19(4), 513-
537. https://doi.org/10.2174/1570159X18666200626204005
49

31. Ortiz, R., Perazzoli, G., Cabeza, L., Jiménez-Luna, C., Luque, R., Prados, J., & Melguizo, C.
(2021). Temozolomide: An Updated Overview of Resistance Mechanisms,
Nanotechnology Advances and Clinical Applications. Current Neuropharmacology, 19(4),
513-537. https://doi.org/10.2174/1570159x18666200626204005
32. Otte, A., Mandel, K., Reinstrom, G., & Hass, R. (2011). Abolished adherence alters
signaling pathways in phorbol ester-induced human U937 cells. Cell Commun Signal, 9,
20. https://doi.org/10.1186/1478-811X-9-20
33. Pawlowska, E., Szczepanska, J., Szatkowska, M., & Blasiak, J. (2018). An Interplay between
Senescence, Apoptosis and Autophagy in Glioblastoma Multiforme-Role in Pathogenesis
and Therapeutic Perspective. Int J Mol Sci, 19(3). https://doi.org/10.3390/ijms19030889
34. Pegg, A. E. (2011). Multifaceted roles of alkyltransferase and related proteins in DNA
repair, DNA damage, resistance to chemotherapy, and research tools. Chem Res Toxicol,
24(5), 618-639. https://doi.org/10.1021/tx200031q
35. Pietanza, M. C., Waqar, S. N., Krug, L. M., Dowlati, A., Hann, C. L., Chiappori, A.,
Owonikoko, T. K., Woo, K. M., Cardnell, R. J., Fujimoto, J., Long, L., Diao, L., Wang, J.,
Bensman, Y., Hurtado, B., de Groot, P., Sulman, E. P., Wistuba, II, Chen, A., . . . Byers, L. A.
(2018). Randomized, Double-Blind, Phase II Study of Temozolomide in Combination With
Either Veliparib or Placebo in Patients With Relapsed-Sensitive or Refractory Small-Cell
Lung Cancer. J Clin Oncol, 36(23), 2386-2394. https://doi.org/10.1200/JCO.2018.77.7672
36. Prudovsky, I., Popov, K., Akimov, S., Serov, S., Zelenin, A., Meinhardt, G., Baier, P., Sohn,
C., & Hass, R. (2002). Antisense CD11b integrin inhibits the development of a
differentiated monocyte/macrophage phenotype in human leukemia cells. Eur J Cell Biol,
81(1), 36-42. https://doi.org/10.1078/0171-9335-00219
37. Regan, D., Garcia, K., & Thamm, D. (2018). Clinical, Pathological, and Ethical
Considerations for the Conduct of Clinical Trials in Dogs with Naturally Occurring Cancer:
A Comparative Approach to Accelerate Translational Drug Development. ILAR J, 59(1), 99-
110. https://doi.org/10.1093/ilar/ily019
38. Rousseau, D., Cannella, D., Boulaire, J., Fitzgerald, P., Fotedar, A., & Fotedar, R. (1999).
Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct
mechanisms and is regulated by ubiquitin-proteasome pathway. Oncogene, 18(30), 4313-
4325. https://doi.org/10.1038/sj.onc.1202686
39. Schönthal, A. H., Swenson, S., Bonney, P. A., Wagle, N., Simmon, V. F., Mathew, A. J.,
Hurth, K. M., & Chen, T. C. (2022). Detection of perillyl alcohol and its metabolite perillic
acid in postsurgical glioblastoma tissue after intranasal administration of NEO100:
illustrative case. J Neurosurg Case Lessons, 4(8). https://doi.org/10.3171/CASE22215
50

40. Schönthal, A. H., Swenson, S., Minea, R. O., Kim, H. N., Cho, H., Mohseni, N., Kim, Y. M., &
Chen, T. C. (2021). Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-
Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid
Leukemia. Cancers (Basel), 13(14). https://doi.org/10.3390/cancers13143385
41. Shinsato, Y., Furukawa, T., Yunoue, S., Yonezawa, H., Minami, K., Nishizawa, Y., Ikeda, R.,
Kawahara, K., Yamamoto, M., Hirano, H., Tokimura, H., & Arita, K. (2013). Reduction of
MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of
glioblastoma. Oncotarget, 4(12), 2261-2270. https://doi.org/10.18632/oncotarget.1302
42. Song, T., Li, H., Tian, Z., Xu, C., Liu, J., & Guo, Y. (2015). Disruption of NF-kappaB signaling
by fluoxetine attenuates MGMT expression in glioma cells. Onco Targets Ther, 8, 2199-
2208. https://doi.org/10.2147/OTT.S85948
43. Song, X., Liu, L., Chang, M., Geng, X., Wang, X., Wang, W., Chen, T. C., Xie, L., & Song, X.
(2019). NEO212 induces mitochondrial apoptosis and impairs autophagy flux in ovarian
cancer. J Exp Clin Cancer Res, 38(1), 239. https://doi.org/10.1186/s13046-019-1249-1
44. Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J.,
Belanger, K., Brandes, A. A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R. C., Ludwin,
S. K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J. G., Eisenhauer, E., Mirimanoff, R.
O., . . . National Cancer Institute of Canada Clinical Trials, G. (2005). Radiotherapy plus
concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 352(10), 987-
996. https://doi.org/10.1056/NEJMoa043330
45. Syro, L. V., Rotondo, F., Camargo, M., Ortiz, L. D., Serna, C. A., & Kovacs, K. (2018).
Temozolomide and Pituitary Tumors: Current Understanding, Unresolved Issues, and
Future Directions. Front Endocrinol (Lausanne), 9, 318.
https://doi.org/10.3389/fendo.2018.00318
46. Thakur, A., Faujdar, C., Sharma, R., Sharma, S., Malik, B., Nepali, K., & Liou, J. P. (2022).
Glioblastoma: Current Status, Emerging Targets, and Recent Advances. J Med Chem,
65(13), 8596-8685. https://doi.org/10.1021/acs.jmedchem.1c01946
47. Wang, C.-C., Lin, S.-Y., Lai, Y.-H., Liu, Y.-J., Hsu, Y.-L., & Chen, J. J. W. (2012). Dimethyl
Sulfoxide Promotes the Multiple Functions of the Tumor Suppressor HLJ1 through
Activator Protein-1 Activation in NSCLC Cells. PLoS ONE, 7(4), e33772.
https://doi.org/10.1371/journal.pone.0033772
48. Zandvliet, M. (2016). Canine lymphoma: a review. Vet Q, 36(2), 76-104.
https://doi.org/10.1080/01652176.2016.1152633
49. Zhang, J., Stevens, M. F., & Bradshaw, T. D. (2012). Temozolomide: mechanisms of action,
repair and resistance. Curr Mol Pharmacol, 5(1), 102-114.
https://doi.org/10.2174/1874467211205010102

Asset Metadata

Creator Yang, Zhuoyue (author)

Core Title Development of a temozolomide-perillyl alcohol conjugate, NEO212, for the treatment of hematologic malignancies

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2023-05

Publication Date 01/26/2023

Defense Date 01/17/2023

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

Tag hematologic malignancy,NEO212,OAI-PMH Harvest,temozolomide

Format theses (aat)

Language English

Advisor Schonthal, Axel (committee chair), Swenson, Steve (committee member), Tahara, Stanley (committee member)

Creator Email zhuoyuey@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC112719088

Unique identifier UC112719088

Identifier etd-YangZhuoyu-11440.pdf (filename)

Legacy Identifier etd-YangZhuoyu-11440

Document Type Thesis

Format theses (aat)

Rights Yang, Zhuoyue

Internet Media Type application/pdf

Type texts

Source 20230126-usctheses-batch-1004 (batch), 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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract Hematologic malignancy continues to claim the lives of many patients. The alkylating drug temozolomide (TMZ) has been studied in the past to treat AML and has been found to be only partially effective; however, tumor cells that contain the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) confer profound therapeutic resistance to TMZ. The novel anticancer compound NEO212 that we are creating has TMZ covalently linked to perillyl alcohol (POH). In numerous preclinical cancer models, NEO212 has demonstrated strong therapeutic activity. We examined its effects on human hematologic malignancy cell lines in the present study and discovered that it exerts cytotoxic effects even on MGMT-positive cells that are extremely resistant to TMZ. In addition, NEO212 significantly increased the expression of numerous markers associated with macrophages, such as CD11b/ITGAM, as well as markers associated with growth inhibition, such as CDKN1A/p21, and apoptosis, such as γ-H2AX and cl. caspase 7. When cells were treated with equimolar mixtures of either TMZ or POH, the anticancer effects of NEO212 could not be duplicated. Two 5-day cycles of 25 mg/kg NEO212 resulted in a significant cure in a mouse model implanted with TMZ-resistant, MGMT-positive AML and lymphoma cells. In the AML model, mice survived for more than 300 days without exhibiting any symptoms. In the lymphoma model, the number and time of survival of NEO212 treated mice is much greater than that of the non-administered group. The results indicate that NEO212 affects hematologic malignancy cells in a variety of ways, including through differentiation, proliferative arrest, and ultimately cell death. Also, the superior cytotoxic effects of NEO212 appear to involve a downregulation of MGMT protein levels. NEO212 was well tolerated in vivo even at doses significantly higher than those required for treatment, indicating a broad therapeutic window. These findings imply that NEO212 is a therapeutic agent for hematologic malignancies that needs to be taken into consideration for development.