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Cytotoxic effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on canine lymphoma

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Cytotoxic effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on canine lymphoma

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CYTOTOXIC EFFECT OF NEO212, A NOVEL PERILLYL ALCOHOL-
TEMOZOLOMIDE CONJUGATE, ON CANINE LYMPHOMA

by

Nazleen Mohseni

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 2021

Copyright 2021 Nazleen Mohseni

ii
Acknowledgements
First, I would like to thank Dr. Axel Schönthal for giving me the opportunity to develop and
enhance my laboratory techniques and for allowing me to be a part of your lab. I appreciate your guidance
and patience with me during this journey and thank you for giving me plenty of opportunities to work
with canine cell lines and in-vivo projects.
I would also like to thank Dr. Stephen Swenson for answering all my questions and always finding
a way to help while still teaching me along the way. Your support and guidance is very much appreciated.
I would like to thank my thesis committee members, which includes Dr. Axel Schönthal, Dr. Joseph
R. Landolph Jr., and Dr. Stanley Tahara for their guidance during the writing of this thesis. In addition, I
would like to thank all of the Chen Glioma Laboratory Research Group. I would also like to give a special
thank you to Dr. Radu Minea, Dr. Camelia Danilov, Dr. Hee-Yeon Cho, Samantha Stack, Francesca Ferri,
and Mukund Iyer for your academic support for the past two years.
Finally, I would like to thank my friends and family for their support throughout this journey. I
would like to especially thank my parents for supporting my decision to attend USC. They taught me the
importance of education and never let me quit along the way. This accomplishment would not have been
possible without them. Thank you.

iii
Contents
Acknowledgments………..………………………………………………………………………………………………………………….…..…ii
List of Figures.…………………………………………………………………………………………………………………………….…………..v
List of Tables……………………………………………………………………………………………………………………………….…..……..vi
List of Abbreviations……………………………………………………………………………………………………………..….……………vii
Abstract………………………………………………………………………………………………………………………….………………….…viii
Chapter 1 – Introduction.…………………………………………………………………………………………………………….………….1
1.1 Canine Lymphoma……………………………………………………………………………………………………………..…1
1.1.1 Introduction………………………………………………………………………………………….……….……..1
1.1.2 Epidemiology and etiology……………………………………………………………………….…….…..…1
1.1.3 Canine tumors in comparison to human tumors…………………………………..………………..2
1.1.4 Current treatments…………………………………………………………………………………….…………3
1.2 NEO212………………………………………………………………………………………………………………………………..5
1.3 Hypothesis………………………………………………………………………………………………………………..…………7
Chapter 2- Materials and Methods…………………………………………………………………………………………………………..8
2.1 Pharmacological agents………………………………………………………………………………………………………..8
2.2 Cell lines……………………………………………………………………………………………………………….………………8
2.3 MTT assay…………………………………………………………………………………………………………………………….9
2.4 Trypan blue assay………………………………………………………………………………………………………………..10
2.5 Cell images………………………………………………………………………………………………………………………….11
2.6 In-vivo………………………………………………………………………………………………………….……………………..11
2.7 Statistical analysis……………………………………………………………………………………..………………………..12
Chapter 3- Results…………………………………………………………………………………………………..…………………………….13
3.1 NEO212 inhibits proliferation of CLBL1, 6D10 and WEHI-3 cell lines…………………………………….13
3.1.1 Effect of NEO212 on cell viability in the CLBL1 cell line……………………..…………………13
3.1.2 Images of CLBL1 cell proliferation and cell death………………………………………………….14
3.1.3 Effect of NEO212 on cell viability in the CLBL1 cell line in comparison to leukemia
cell lines; 6D10 and WEHI-3………………….………….………………………………………………………….16
3.1.4 Comparing the effects of different drugs on cell viability……….……………………………17
3.2 NEO212 causes greater cell death in comparison to its individual components…………….………20

iv
3.2.1 Cell proliferation and death measured by trypan blue assay…………………………………20
3.3 NEO212 contributes in mice survival in vivo…………………………………………………………………………25
3.3.1 Effect of NEO212 after the injection of 6D10 cells in vivo…………………………….……….25
3.3.2 Effect of NEO212 after the injection of CLBL1 cells in vivo………………………….…………27
Chapter 4- Discussion…………………………………………………………………………………………………………………………….29
Bibliography………………………………………………………………………………………………………………………………………….33

v
List of Figures
Figure 1.1: Chemical structures……………………………………………………………………………………………..……………….6
Figure 3.1: Effect of NEO212 on CLBL1 cell viability……………………………….……………………………………..….…..13
Figure 3.2: Effect of NEO212 on CLBL1, 6D10 and WEHI-3 cell lines……………………………………………..……….16
Figure 3.3: Effect of NEO212 on CLBL1 cell viability compared with its individual components………….…17
Figure 3.4: Effect of NEO212 on CLBL1 cell viability compared with AraC………………………………………..……18
Figure 3.5: Effect of NEO212 on 6D10 cell viability compared with its individual components……….….…19
Figure 3.6: Effect of NEO212 on WEHI-3 cell viability compared to TMZ…………………….…………………………20
Figure 3.7: Effect of NEO212 on CLBL1 cells as determined by Trypan blue assay…………………………………21
Figure 3.8: Effect of TMZ on CLBL1 cells as determined by Trypan blue assay……………………………………….22
Figure 3.9: Effect of POH on CLBL1 cells as determined by Trypan blue assay……………………………………….23
Figure 3.10: Effect of TMZ+POH on CLBL1 cells as determined by Trypan blue assay………..………………….24
Figure 3.11: Leukemia mice weights.…………………………………………………………………………………………………….26
Figure 3.12: Percent survival for both vehicle and NEO212 treated 6D10 mice…………...…………..…………..26
Figure 3.13: CLBL1 cells infected with GFP/luc repeatedly sorted………………………………………………………….27
Figure 3.14: Percent survival for both vehicle and NEO212 treated CLBL1 mice..………………………………….28

vi

List of Tables
Table 1.1 Annual Incidence rates (per 100,000 at risk) of cancer…………………………….……………….…………..12
Table 3.1: Images of CLBL1 cells…………………………………………………………………………………………………….....….15
Table 3.2: IC50 values for each cell line…………………………………………………………….…………………..……………..25

vii
List of Abbreviations
AraC: cytosine arabinoside
cDNA: complementary DNA sequence
CHOP: cyclophosphamide,
hydroxydaunorubicin, oncovin and
prednisone
DLBCL: diffused large B-cell lymphoma
DMSO: dimethyl sulfoxide
ER: endoplasmic reticulum
EGFP: enhanced green fluorescent
protein
FBS: fetal bovine serum
FDA: Food and Drug Administration
GFP: green fluorescent protein
HCl: hydrochloric acid
HEPES: 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid
IC50: concentration of drug that reduces
cell proliferation by 50%
MEM: minimal essential medium
MGMT: O-6-methylguanine
methyltransferase
MOPP: mustargen, vincristine,
prednisone or procarbazine
MTT: methylthiazoletetrazolium
NEO212: perillyl alcohol covalently linked to
temozolomide (TMZ-POH)
NHL: non-Hodgkin lymphoma
NEAA: non-essential amino acid
ORF: open reading frame
PBS: phosphate buffered saline
POH: perillyl alcohol
RPMI: Roswell Park Memorial Institute medium
SDS: sodium dodecyl sulfate
TMZ: temozolomide
WT: wild type

viii
Abstract
Cancer is the number one cause of death in dogs with lymphomas representing one of the most
common spontaneously occurring tumors. With no cure for canine lymphoma, many dogs relapse due to
drug resistance after treatment and require multiple therapy options. The need for more drug treatments
for cancer in the veterinary field is crucial. Even with multiple rounds of therapy, dogs diagnosed with
cancer have a poor outcome. In addition, high similarities between canine lymphomas and human non-
Hodgkin’s lymphoma allow for canines to be a reliable comparison model for human cancer research on
lymphomas.
NEO212, a covalent conjugate of perillyl alcohol and temozolomide, was tested on canine and
human cell lines to determine the effects of this novel drug on canine patients. Cell proliferation and cell
viability were measured under different concentrations of NEO212 with various cell lines to observe its
cytotoxic effects. An evaluation was also done with the individual components of NEO212, perillyl alcohol
alone and temozolomide alone. Perillyl alcohol and temozolomide were also evaluated together in a
mixture but not covalently conjugated to determine if the covalent conjugate aids in cellular uptake,
allowing a stronger cytotoxic effect at lower concentrations. We conducted methylthiazoletetrazolium
assays to determine cytotoxic effects of NEO212 on cell metabolic activity, as well as determining the
effects of NEO212 on cell viability by trypan blue assays. Cell images were captured under a light
microscope to give a visual understanding of cell viability over time. Lastly, in-vivo testing was conducted
using mice models to determine the effects of NEO212 in a biological system.
The results of our study demonstrated that NEO212 had a strong cytotoxic effect on the canine
lymphoma and human leukemia cell lines and had a greater effect at lower concentrations than TMZ
alone, POH alone, or the TMZ and POH mixture. For the canine cell line, the effects of NEO212 were
greater than temozolomide, a current treatment for dogs with relapsed lymphoma. NEO212 also
demonstrated an inhibition of cell proliferation and caused cell death at a low concentration. NEO212

ix
was shown to be an effective novel drug treatment in both the canine lymphoma cell line and the human
leukemia cell line, and a possible alternative to current treatments, especially in drug-resistant tumors.
Further studies will need to be conducted to determine its pharmacological effects and clinical potential
of NEO212.

1
Chapter 1 - Introduction
1.1 Canine Lymphoma
1.1.1 Introduction
Lymphoma is cancer that involves the monoclonal proliferation of malignant lymphocytes and can
occur in any site of the body that lymphocytes live (1). Commonly involved in the bone marrow, thymus,
lymph nodes, and spleen, lymphoma results due to excessive clonal expansion of B or T lymphocytes at
various stages of development. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in
both humans and dogs (1,2). This is due to its high spontaneity, complex pathways, rapid progression over
short periods, the aggression of its clinical course, and difficult treatment predictability (3). The canine
CLBL1 cell line is the only canine cell line that truly represents DLBCL by preserving its phenotype and
inducing tumors in a xenotransplantation setting. CLBL1 xenograft mouse models are the most reliable
way to evaluate canine B cell lymphoma for in-vivo experiments (4). A lymphoma diagnosis in dogs can be
attained by fine-needle aspirates or a lymph node biopsy. Dogs with large B-cell lymphoma have moderate
to high enlarged, non-painful peripheral lymph nodes (3). Certain dog breeds are at statistically higher risk
of developing lymphoma compared to the average risk for all dogs suggesting that there are heritable
factors involved in the disease of B cell lymphoma (1).

1.1.2 Epidemiology and etiology
Cancer is the number one cause of death in dogs. Lymphomas represent one of the most common
spontaneously occurring tumors in dogs by accounting for 90% of all hematopoietic tumors and an
average of 20-100 cases per 100,000 dogs. Lymphoma also has a higher occurrence in dogs than in humans
(5,6). Lymphoma can occur at any age but is seen to affect middle to older aged dogs with no known sex
association (6). Necropsies done on 2000 dogs showed that 23% of all dogs and 45% of dogs 10 years or

2
older had died of cancer (5). The incidence rate of canine lymphoma starts at 1.5/100,000 for dogs less
than a year old and increases to 80-85/100,000 for dogs at the age of ten (6,7). Approximately 70-80% of
lymphoma in dogs are of B-cell origin. The exact cause is unknown, but a combination of environmental
and genetic factors plays a role in the development of canine lymphoma. Medium to large breed dogs are
more likely to become afflicted by canine lymphoma, which can be related to growth factor hormone
levels. Dogs living in areas with high chemical exposure including industrial sites, waste incinerators,
radioactive sites, or polluted areas were shown to have an increased risk of developing canine lymphoma
(6,8).

1.1.3 Canine tumors in comparison to human tumors
There are many similarities between canine lymphoma and human non-Hodgkin’s lymphoma
(NHL) (6). In humans and dogs, the tumors share genetic and morphological similarities, biological
behaviors, and molecular abnormalities (1,4,9). Another factor is due to the coexistence of a common
environment amongst both groups. For example, the similar potential of exposure to carcinogens to both
the human and animal in similar environments makes the canine a good model for human cancer
research(5,8). Canine lymphoma shows a strong similarity to high-grade non-Hodgkin’s lymphoma in
humans regarding its histopathology, tumor genetics, disease progression, and response to therapy. In
canines, the remission time is much shorter compared to humans, which allows us to understand the
progression of the disease and to develop new treatments in a shorter time period. These factors signify
the importance of canines as an animal comparison model to human cancer, specifically human non-
Hodgkin’s lymphoma (5,10).
Humans and canines share many similarities in regards to tumor development. However, the
incidence rates in canines tend to be much higher per 100,000 at risk than in humans (Table 1.1).

3
Tumor location Human Dog
Mammary 108.8 198.8
Melanoma 12.6 25.0
Testes 4.7 33.9
Connective Tissue 2.5 35.8
Skin 5.2 90.4
Oral 11.6 20.4
Non-Hodgkin’s
lymphoma/leukemia
29.9 25.0
Bone 1.0 7.9

Table 1.1 Annual incidence rates (per 100,000 at risk) of cancer. The cancer rate for dogs and humans for
each tumor location of the body are quantitatively listed to compare incidence rates of each type of
tumor(7).

The abundance of certain tumor types like canine osteosarcoma and canine soft tissue sarcomas
are significantly higher than in humans, making the canine a prime animal model. Tumors in dogs typically
develop at a faster rate than in humans, allowing faster acquisition of data than in long term tumor studies
in humans (7,11). The higher incidence rate and faster development of cancer in dogs in comparison to
humans further supplements the need for new treatments in the veterinary field for canine cancer (11).
There are still very few approved chemotherapies options for dogs in the veterinary field despite the high
incidence rate of cancer and the short survival time after diagnosis.

1.1.4 Current Treatments
Combination chemotherapies are a successful treatment option for dogs with lymphoma, but
with current protocols the goal is to ease the severity of the patient’s pain rather than curing the patient

4
(9,12). The most effective therapies for canine lymphomas include doxorubicin, cyclophosphamide,
alkaloids, L-asparaginase, and corticosteroids (7). The combination of vincristine, cyclophosphamide,
doxorubicin, and prednisone is a common chemotherapy for dogs known as CHOP (cyclophosphamide,
hydroxydaunorubicin [doxorubicin], oncovin [a trade name of vincristine], and prednisone) and is
considered the gold standard for canine tumor treatment (9,13,14). Other protocols include a non-
doxorubicin protocol called COP. This protocol is typically well tolerated, less expensive, and easier to
administer but less effective than the CHOP protocol (13). Tanovea, under the generic name
Rabacfosadine, has been conditionally approved by the US Food and Drug Administration (FDA) for the
treatment of a variety of canine lymphomas, making it the first anti-tumor drug ever approved by the US
FDA for the treatment of lymphoma in dogs. This is a good option for CHOP-relapsed cancer since clinicians
must begin to utilize other options once the drugs included in the CHOP based protocol fail to actively
work against the lymphoma when the patient has relapsed (15).
Most veterinarians use a concoction of drugs in alternating combinations to result in an 86-91%
response rate. The initial response rate to chemotherapy is high and survival time is about 200-300 days
but ultimately drug resistance develops (7,9). Due to drug resistance, a dog’s health will regress which is
why alternative treatment methods are needed to combat the adverse health effects of drug resistance
(6). Drug resistance is the major factor limiting successful treatment of canine lymphoma. Drug resistance
can occur due to many different factors, one being if the tumor cell has increased DNA repair, it can repair
drug-induced DNA damage via 06-methyl-guanine-DNA-methyltransderase (MGMT). Cells with high levels
of MGMT can develop drug resistance and become difficult to treat with temozolomide (TMZ), a current
secondary treatment option for canine lymphoma (16). In previous studies, canine lymphoma cell lines
have been shown to have a high mRNA expression of DNA repair genes, leading to resistance to alkylating
agents like TMZ (17). Other reasons for drug resistance include decreased drug uptake, increased drug
efflux, increased resistance to apoptosis, changes in metabolism and changes in drug target. For this

5
reason, veterinarians tend to use a discontinuous chemotherapy protocol as opposed to a continual
chemotherapy approach. For a discontinuous chemotherapy approach, the chemotherapy is discontinued
for patients at the end of treatment but once there is a sign of recurrence of the lymphoma, the original
chemotherapy is reintroduced. Studies have shown that patients in a discontinuous chemotherapy
approach are more likely to achieve a second remission when they relapse than patients who used a
continuous or maintenance chemotherapy (13).
A very small number of dogs are cured with the current treatments used in practice today. With
drug resistance, every time a new drug is needed, it becomes difficult to find one that the cancer will
respond to, which further emphasizes why new drug therapies need to become available for canine
patients. Drug options like L-asparaginase, MOPP (mustargen/vincristine/prednisone/procarbazine),
tanovea, mitoxantrone, dacarbazine, temozolomide, and prednisone are used as a subsequent method to
the CHOP based protocol, once the dog has developed resistance and has relapsed (18). Options range in
pricing per treatment and vary case by case. With current therapies, the average survival time for dogs
with B-cell lymphoma is about 12 months. With no therapy, a dog with high-grade lymphoma has an
estimated survival of about 4-6 weeks (7). Breed type can have an effect on disease progression and
recovery after treatment as well (1).

1.2 NEO212
NEO212 is a conjugate of temozolomide (TMZ) and perillyl alcohol (POH) (Figure 1.1). NEO212 has
been shown to have therapeutic activity in a variety of preclinical cancer models (19). NEO212 has been
tested on TMZ-resistant glioma cell lines and was shown to be more cytotoxic than TMZ to the TMZ-
resistant cells and not toxic to non-malignant cells. NEO212 works similarly to TMZ by functioning as a
DNA alkylating agent but NEO212 induces endoplasmic reticulum (ER) stress and inhibits autophagy (20).

6

Figure 1.1 Chemical structures. Shown are (A) perillyl alcohol and (B) temozolomide; (C) NEO212, a perillyl
alcohol and temozolamide covalently conjugated via a carbamate bridge (21).

TMZ is an alkylating agent that is the current chemotherapeutic standard of care for patients with
malignant gliomas (22,23). TMZ methylates the O6-position of guanine (mO6G) which is responsible for
triggering cell death via double stranded DNA breaks. MO6G can be repaired by the DNA repair enzyme
06-methyl-guanine-DNA-methyltransferase (MGMT). If a cell has high levels of MGMT, after TMZ
methylation occurs, the methyl group is removed to prevent cell death by DNA damage. Thus, cells with
high levels of MGMT are highly resistant to TMZ therapy (19,23).
POH, a naturally occurring monoterpene and a metabolite of limonene, is found in essential oils
and is a natural constituent of lavender, peppermint, cherries, sage, citrus fruit, spearmint, cranberries,
and lemongrass (24,25). At high doses, POH has cytotoxic effects on a variety of tumor cells, including
those resistant to the effects of TMZ (20).

7
1.3 Hypothesis
There is a lack of chemotherapeutic drugs in veterinary medicine and more need to be
developed to combat drug resistance in canines (6). TMZ is currently used for the treatment of human
and canine cancer but is shown to be needed at high concentrations, which can lead to the development
of drug resistance. Covalently conjugating TMZ to POH can increase the effects of TMZ by including the
cytotoxic properties of POH, resulting in a greater effect at lower concentrations (19). Based on previous
studies with NEO212, we hypothesize that NEO212 will have a greater cytotoxic effect on the canine cell
lines than TMZ alone, POH alone, or the non-covalently conjugated mixture of TMZ+POH. If this
hypothesis is found to be true, then NEO212 could be a novel treatment against canine lymphoma and
possibly an improvement over current treatments used today. Due to drug resistance being a major
problem in veterinary oncology, novel treatments can positively impact the current issue being faced in
regards to relapse in dogs. Also, with the similarities between canine lymphoma and human non-
Hodgkin’s lymphoma, if NEO212 is found to be effective in canine models, NEO212 could be a novel drug
treatment in humans as well. Further studies will need to be conducted, including clinical trials in
canines, to determine the full effects of NEO212.

8
Chapter 2 - Material and methods
2.1. Pharmacological agents
NEO212 was provided by NeOnc Technologies (Los Angeles, CA) and was dissolved in dimethyl
sulfoxide (DMSO) at 100 mM. TMZ was obtained from the pharmacy at the University of Southern
California (USC) or from Sigma Aldrich (St. Louis, MO), and dissolved in DMSO (Santa Cruz
Biotechnology, Dallas, TX) to a concentration of 50 mM. POH was purchased from SigmaAldrich and
diluted with DMSO at 100 mM. Stock solutions were stored in -20°C. Phosphate buffered saline (PBS)
and Roswell Park Memorial Institute (RPMI) medium were 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).

2.2. Cell lines
The CLBL1, 6D10 and WEHI-3 cell lines were used in this study. CLBL1 cells are a B-cell lymphoma
cell line derived from a dog with confirmed stage IV diffuse large cell lymphoma. The cell line was
established in the laboratory of Drs. Barbara C. Rütgen and Armin Saalmüller at the University of
Veterinary Medicine in Vienna, Austria and was provided to USC by Barbara Rose in Dr. Douglas H.
Thamm’s laboratory from the Flint Animal Cancer Center at Colorado State University in Fort Collins,
Colorado. The 6D10 cells are an AraC-resistant pro-monocytic myeloid human leukemia cell that was
established in Dr. David A. Largaespada’s laboratory at the University of Minnesota in Minneapolis,
Minnesota. WEHI-3 cells are a BALB/c mouse myelomonocytic leukemia provided by Alan L. Epstein, M.D.,
Ph. D. in the Department of Pathology in the Keck School of Medicine at the University of Southern
California. The cells were grown in 500 mL RPMI medium with L-glutamine, 10% fetal bovine serum
(FBS)/fetalclone I, 1% non-essential amino acids, 1% sodium pyruvate (100 mM), 1% penicillin-

9
streptomycin (10,000 U/ml of penicillin and 10,000 g/ml of streptomycin) and 0.5% 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) (1M). The CLBL1 cell line was grown in complete minimal essential
medium (MEM) which contains a basal medium with the addition of 20 mL MEM vitamin solution, 10 mL
L-glutamine 200 mM, 10 mL sodium pyruvate solution 100 mM, 10 mL non-essential amino acid (NEAA)
solution, 10 mL of antibiotic/antimycotic solution and 10% FBS. The CLBL1 and 6D10 cell lines were stably
expressing the luciferase and green fluorescent protein reporters (GFP). The labeling of the 6D10 and
CLBL1 cells was performed by Dr. Radu Minea by infecting the parental cells with a lentiviral construct in
which the cDNA sequence of firefly luciferase was cloned behind an EF1α promoter while the cDNA for
enhanced green fluorescent protein (EGFP) was cloned as a separate ORF behind a CMV promoter. The
prepackaged lentiviral particles, for the lentiviral construct, were purchased from VectorBuilder (Chicago,
IL). Both parental cells were incubated for up to 24 hours with the packaged lentiviral particles in the
presence of cationic polybrene (10 μg/ml). The cells were then spun, resuspended in complete RPMI
medium, and cultured for an additional 48 hours before being sorted based on their GFP signal in a BD
SORP FACSAria IIu instrument (BD Biosciences). The sorted GFP positive fractions of both cell lines were
then returned to culture, expanded, and subjected to two additional rounds of sorting for further
enrichment in the GFP signal. Finally, these homogenous GFP populations were checked for
bioluminescence (i.e., functional firefly luciferase) in the presence of D-luciferin in a Varioskan Lux
(Thermo Fisher Scientific) microplate reader. All cells were kept in a humidified incubator at 37°C and 5%
CO 2 atmosphere.

2.3. MTT assay
The Methylthiazoletetrazolium (MTT) assay was performed to determine the metabolic activity
of the cells. The assay determines the number of viable cells by the capability to reduce the tetrazolium
dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to insoluble formazan, which can

10
be measured by an absorbance reader due to its purple color (26,27). Cells were seeded at a volume of
50 µl per well at 3.0-6.0x10
5
cells/ml into a 96-well plate. An additional 50 µl was added to each of the
wells which contained various concentrations of the drug or vehicle. The cells were then incubated for
different lengths of time from 72 hours to 120 hours. After incubation, the cells were followed by the
addition of 12 µl thiazolyl-blue tetrazolium (i.e. MTT; Sigma Aldrich) from a stock solution of 5 mg/ml in
PBS. The cells were returned to the cell culture incubator for 4 hours to allow the cells to reduce the
tetrazolium dye to formazan by their NADPH-dependent cellular oxidoreductase. The reaction was then
stopped by the addition of 100 µl of solubilization solution made by adding 0.1 M hydrochloric acid (HCL)
to 10% sodium dodecyl sulfate (SDS). The 96-well plate was left in the cell culture incubator overnight and
the optical density was measured the next day in a Varioskan Lux Reader by Thermo Scientific (Waltham,
MA) at 560 nm. Background values were used as optical density controls, which contained medium
without cells, MTT dye, and solubility solution, and were subtracted from all the values measured. Each
experiment was repeated independently several times and each treatment was set up in either
quadruplets or octuplets.

2.4. Trypan blue assay
This assay determines live versus dead cells due to the dye being impermeable to live cells and
permeable to dead cells. The dead cells then become a blue color, and the live cells are colorless under a
microscope. CLBL1 cells were seeded at a volume of 2 ml per well at 5.0x10
4
-1.0x10
5
cells/ml into a 6-
well plate. Fresh medium was given to the cells at the 72 hour time point. Cell proliferation was assessed
by counting cells at the 0, 24, 48, 72, and 96 hour time points. Each well was exposed to different
concentrations of drug. Aliquots of cells were removed from each well, mixed with Trypan blue (Sigma
Aldrich, St, Louis, MO), and counted in a hemocytometer. Cells stained blue were considered dead, while
the unstained cells were counted as alive.

11
2.5. Cell Images
CLB1 cells were seeded at a volume of 2 mL per well at 1.0x10
5
cells/ml into a 6 well plate. Fresh
medium was given to the cells at the 72 hour time point. Images were taken with a Canon digital camera
under a light microscope at 72, 96, and 168 hour time points. Each well was exposed to different
concentrations of drug.

2.6. In-Vivo
For tumorigenicity, an intraperitoneal (IP) injection of 50,000 6D10-luc cells in 50 µL per SCID nude
mouse was conducted. Drug treatment began 11 days after tumor cell implantation. Oral gavage of 25
mg/kg of NEO212 was given to 5 mice, and the other 5 mice were given the vehicle of DMSO. The mice
treated with NEO212 were labeled red while the mice given DMSO were labeled blue, to distinguish the
treated and vehicle groups from one another. Treatment was for 5 consecutive days followed by a 2 week
treatment break. On day 25, a second cycle of NEO212 was given to the treatment group while DMSO was
given to the vehicle group by oral gavage. Treatment was for 5 consecutive days, followed by a 2 week
treatment break. At day 48, a third cycle of NEO212 was given to the treatment group.
An IP injection of 50,000 CLBL1-luc cells in 50 µL per SCID nude mouse was conducted. Treatment
began 5 days after tumor cell implantation. An oral gavage of 25 mg/kg of NEO212 was given to 5 mice
and the other 5 mice were given the vehicle of DMSO. The mice treated with NEO212 were labeled red
while the mice given DMSO were labeled blue to distinguish the treated and vehicle groups from one
another. Treatment was for 5 consecutive days followed by a 2 week treatment break. At day 23, a second
cycle of NEO212 was given to the treatment group while DMSO was given to the vehicle group by oral
gavage. Treatment was for 5 consecutive days.

13
2.7. Statistical analysis
All data were analyzed using Excel. T-tests were applied to calculate the statistical significance of
the differences between values. A probability value (p)<0.05 was considered statistically significant.

13
Chapter 3 - Results
3.1. NEO212 inhibits proliferation of CLBL1, 6D10, and WEHI-3 cell lines
To determine the cytotoxic effect of NEO212 an assay that measures cell metabolic activity,
known as an MTT assay, was performed. NADPH-dependent cellular oxidoreductase enzymes reflect the
number of viable cells remaining in each well (25). Cell images were also taken to visually determine the
proliferation and inhibition of the CLBL1 cells by NEO212.

3.1.1 Effect of NEO212 on cell viability in the CLBL1 cell line
First, CLBL1 cells were tested by adding different concentrations of NEO212 in separate wells and
determining the cytotoxic activity of NEO212.

Figure 3.1 Effect of NEO212 on CLBL1 cell viability. CLBL1 cells were treated with increasing
concentrations of NEO212 for 72 hours by an MTT assay. Percent survival was calculated relative to the
untreated cells set at 100%. Data points are mean ±SE with each data point in quadruplets. P-values
comparing treated vs untreated cells *** p-value< 0.001 ****p-value<0.0001

Cell survival decreased after the addition of NEO212 and this was noticed at low concentrations
72 hours post drug administration. The p-values showed to be statistically significant from 3 µM. NEO212
inhibited cell proliferation resulting in the IC50, which is the concentration of drug that reduces cell
proliferation by 50%, to be between 3 and 10 µM. This demonstrates the cytotoxic effects of NEO212 on
CLBL1 cell viability even at low concentrations.
0
20
40
60
80
100
120
Untreated Vehicle 3 10 20 30 100
% Survival
NEO212 [µM]
***
****
***
*
****
**
**
**** ****

14
3.1.2 Images of CLBL1 cell proliferation and cell death
Different concentrations of NEO212 were added to CLBL1 cells to visually determine the effects
of the novel drug on the cell line. This allows a visual comparison of the CLBL1 cells with different
concentrations of NEO212 over time. Based on the MTT assays, the concentrations 3 µM, 10 µM, 30 µM,
and 100 µM were used and images were taken at 72, 120, and 168 hours.

15
Day 3 (72 h) Day 5 (120 h) Day 7 (168 h)

1

0 µM
3 µM
10 µM
30 µM
100 µM
DMSO

16
Table 3.1: Images of CLBL1 cells. Cells were treated with different concentrations of NEO212. Images
were taken at 72, 120, and 168 hours. Fresh medium was given to the cells at 72 hours.

Cell proliferation is noticeable at low concentrations of 0 µM and 3 µM. As concentration and time
increased, cell death began to occur, and this was seen by 168 hours with 30 µM of NEO212. By 100 µM
cell death was noticeable as early as 72 hours.

3.1.3 Effect of NEO212 on cell viability in the CLBL1 cell line in comparison to leukemia cell lines;
6D10 and WEHI-3
NEO212 was tested on 6D10 and WEHI-3 cell lines at different concentrations to determine the
cytotoxicity in comparison to the CLBL1 cell line.

Figure 3.2 Effect of NEO212 on CLBL1, 6D10 and WEHI-3 cell lines. Cells were treated with increasing
concentrations of NEO212. The treatment was for 72 hours for CLBL1 and 6D10 cell lines and 96 hours for
the WEHI-3 cells measured by an MTT assay. Percent survival was calculated relative to the untreated
cells set at 100%. Data points are mean ±SE with each data point in quadruplets.

Cell viability decreased as the concentration of NEO212 increased in all three cell lines. Despite
being both leukemia cell lines, the WEHI-3 cells were less sensitive to the drug in comparison to the 6D10
cells. The IC50 for the WEHI-3 cells ranged from 30-35 µM, while both the CLBL1 cells and 6D10 cells had
an IC50 of approximately 6 µM. The WEHI-3 cell line had a higher IC50 in comparison to the CLBL1 cells
0
20
40
60
80
100
120
0 20 40 60 80 100
% Survival
NEO212 [µM]
CLBL1
6D10
WEHI-3

17
while the 6D10 cell line had a similar IC50 to the CLBL1 cell line. NEO212 demonstrated high cytotoxic
activity in the CLBL1 and 6D10 cell lines at low concentrations.

3.1.4 Comparing the effect of different drugs on cell viability
NEO212 has shown high cytotoxic activity in regard to the CLBL1 cell line, demonstrating a
reduction in cell survival. Now, we wanted to determine whether NEO212, a covalently bonded conjugate
of TMZ and POH, is more effective than TMZ alone, POH alone, and TMZ and POH together without being
covalently conjugated.

Figure 3.3 Effect of NEO212 on CLBL1 cell viability compared with its individual components. An MTT
assay was performed to treat CLBL1 cells with increasing concentrations of NEO212, TMZ alone, POH
alone, and a mixture of POH and TMZ. The treatment was for 72 hours and percent survival was calculated
relative to the untreated cells set at 100%. Data points are mean ±SE with each data point in quadruplets
or octuplets.

The MTT assay demonstrated that NEO212 decreased cell viability and survival more effectively
than TMZ alone, POH alone, or a mixture of TMZ and POH (TMZ+POH). The IC50 of NEO212 ranged from
6 to 10 µM while the IC50 of TMZ+POH was approximately 60 µM. This demonstrated that NEO212, a
covalent conjugation of TMZ and POH, has increased effectiveness in comparison to a mixture of TMZ and
POH. TMZ alone and POH alone had an effect on the cell line with an IC50 of 45 µM and 175 µM
0
20
40
60
80
100
120
0 50 100 150 200 250 300
% Survival
Drug [µM]
TMZ
TMZ+POH
POH
NEO212

18
respectively, but both required a higher concentration than NEO212 demonstrating the efficiency of
NEO212 on cell viability in comparison to its individual components.
NEO212 demonstrated promising results in comparison to its individual parts. We now wanted
to compare NEO212 to AraC (cytosine arabinoside), a current leukemia treatment, on the CLBL1 cell line.

Figure 3.4 Effect of NEO212 on CLBL1 cell viability compared with AraC. An MTT assay was performed to
treat CLBL1 cells with increasing concentrations of NEO212 and AraC. The treatment was for 72 hours and
percent survival was calculated relative to the untreated cells set at 100%. Data points are mean ±SE with
each data point in quadruplets.

Though AraC was more effective, especially at low concentrations, NEO212 still had a strong
effect on decreasing cell survival and can show promising results in future in-vivo experiments. AraC is
used to treat myeloid leukemia and non-Hodgkin’s lymphoma in humans. Patients can develop drug
resistance to AraC resulting in AraC resistant cells which include the 6D10 cell line, an AraC-resistant pro-
monocytic myeloid human leukemia. By comparing AraC and NEO212 with the CLBL1 cell line, a good
canine cell model for human non-Hodgkin’s lymphoma, we can show the potential effectiveness of
NEO212 in both canines and humans. The CLBL1 and 6D10 cell lines have similar effects to NEO212,
resulting in NEO212 potentially being an effective alternative drug to AraC resistant cells.
0
20
40
60
80
100
120
0 10 20 30 40 50
% Survival
Drug [µM]
AraC
NEO212

19
Next, we investigated if the difference in the effect of NEO212 observed between its individual
components in CLBL1 cells was also present in 6D10 cells due to the cells having a similar response to
NEO212.

Figure 3.5 Effect of NEO212 on 6D10 cell viability compared with its individual components. An MTT
assay was performed to treat 6D10 cells with increasing concentrations of NEO212, TMZ alone, POH
alone, and a mixture of POH and TMZ. The treatment was for 72 hours and percent survival was
calculated relative to the untreated cells set at 100%. Data points are mean ±SE with each data point in
quadruplets.

NEO212 was very effective in both the CLBL1 and 6D10 cells, with an IC50 being approximately 6
µM but the 6D10 cells were just as sensitive to TMZ and the combination of TMZ+POH with an IC50 of 6
µM as well. TMZ and TMZ+POH were able to inhibit the growth of the 6D10 cells similar to NEO212,
whereas in the CLBL1 cells, TMZ and TMZ+POH were effective at higher concentrations. The IC50 of the
6D10 cells with NEO212, TMZ, and TMZ+POH were all at 6 µM, but at 100 µM NEO212 was more toxic
and resulted in lower cell survival than TMZ and TMZ+POH. The IC50 was 300 µM TMZ and 300 µM
TMZ+POH when the cells reached zero percent survival while the same result was achieved with NEO212
at 100 µM. There was a slight effect of POH on the 6D10 cells but was less effective than POH on the
CLBL1 cell line.

0
20
40
60
80
100
120
0 50 100 150 200 250 300
% Survival
Drug [µM]
TMZ
POH
NEO212
TMZ+POH

20
Next, we investigated NEO212 in comparison to TMZ with the WEHI-3 cell line since the WEHI-3
cells were less sensitive to NEO212 compared to the CLBL1 and 6D10 cell lines.

Figure 3.6 Effect of NEO212 on WEHI-3 cell viability compared to TMZ. An MTT assay was performed to
treat WEHI-3 cells with increasing concentrations of NEO212 and TMZ. Treatment was for 96 hours for
NEO212 and 120 hours for TMZ and percent survival was calculated relative to the untreated cells set at
100%. Data points are mean ±SE with each data point in quadruplets.

Though in comparison to the 6D10 and CLBL1 cell lines NEO212 was not as effective in the
WEHI-3 cells, NEO212 demonstrated higher cytotoxicity compared to TMZ alone. The IC50 for WEHI-3
cells with NEO212 ranged from 30-35 µM while with TMZ it was 70 µM, almost double the
concentration. NEO212 had a greater cytotoxic effect on WEHI-3 cell viability than TMZ alone,
demonstrating that the covalent conjugation of POH to TMZ had an effect on reducing cell viability.
3.2. NEO212 causes greater cell death in comparison to its individual components
3.2.1 Cell proliferation and death measured by trypan blue assay
MTT assays measure cell metabolic activity to determine cell viability and the cell’s ability to
proliferate. However, to determine cell survival and cell death caused by NEO212, a trypan blue assay was
performed. The assay measures the number of live cells and determines cell survival and death under
specific drug concentrations over time.
0
20
40
60
80
100
120
0 50 100 150 200 250 300
% Survival
Drug [µM]
TMZ
NEO212

21
Different concentrations of NEO212 were administered to the CLBL1 cells to determine cell
survival, death and proliferation. Cell counting was performed daily starting at zero hours and every 24
hours until reaching 96 hours.

Figure 3.7 Effect of NEO212 on CLBL1 cells as determined by Trypan blue assay. CLBL1 cells were treated
with different concentrations of NEO212. Fresh medium was given at 72 hours. Cell density was based on
the number of live cells. Vehicle used was DMSO. P-values are comparing treated vs untreated cells. *p-
value<0.05; **p-value<0.01.

At 72 hours the 3 and 10 µM concentrations of NEO212 cell numbers peaked, which indicated
that cell proliferation was halted and the CLBL1 cells were proliferating at a slower rate or no longer
proliferating due to the effects of NEO212. At 96 hours, 3 µM NEO212 slowed down cell proliferation
while 10 µM NEO212 decreased in cell numbers indicating cell death. A statistically significant drug
response was observed at 3 µM. At higher concentrations, there was a decrease starting at 24 hours and
reaching zero cells as early as 72 hours for 100 µM and after 96 hours for 30 µM.
To compare results to the MTT assays performed, the trypan blue assay was conducted with TMZ
alone, POH alone, and TMZ+POH mixture.
0
100
200
300
400
500
600
0hrs 24hrs 48hrs 72hrs 96hrs
Cell Density (cell/mlx10
3
)
DMSO 0µM 3µM 10µM 30µM 100µM
**
*

22

Figure 3.8 Effect of TMZ on CLBL1 cells as determined by Trypan blue assay. CLBL1 cells were treated with
different concentrations of TMZ. Fresh medium was given at 72 hours. Cell density was based on the
number of live cells. Vehicle used was DMSO. P-values are comparing treated vs untreated cells. *p-
value<0.05; **p-value<0.01.

Similar to the MTT assay, NEO212 in comparison to TMZ had a greater effect on cell proliferation.
At 10 µM TMZ, the CLBL1 cells were not inhibited, even at 96 hours, indicating that proliferation was
continuing. With 10 µM NEO212, by 72 hours cell survival plateaued and a decrease in cell density
occurred by 96 hours. With TMZ at 30 µM an effect on cell survival was observed at 96 hours; TMZ at 100
µM and 200 µM began to have an effect on the CLBL1 cells at 48 hours, later than the effect of 100 µM
NEO212. A statistically significant drug response was observed at 30 µM for TMZ while for NEO212 this
was noticed at 3 µM. This demonstrates that NEO212 has a greater effect on cell proliferation than TMZ
at lower concentrations.

0
50
100
150
200
250
300
350
400
0hrs 24hrs 48hrs 72hrs 96hrs
Cell Density (cell/mlx10
3
)
DMSO 0µM 10µM 30µM 100µM 200µM
*
**
**

23

Figure 3.9 Effect of POH on CLBL1 cells as determined by Trypan blue assay. CLBL1 cells were treated with
different concentrations of POH. Fresh medium was given at 72 hours. Cell density was based on the
number of live cells. P-values are comparing treated vs untreated cells. *p-value<0.05; **p-value<0.01.

At 24 hours, the 100 µM POH cell numbers reached a plateau with no to very little cell
proliferation or cell death. This is a higher concentration than the plateau reached with TMZ and
NEO212 on the CLBL1 cell line. By 24 hours, higher doses of POH caused complete cell death. This
coincided with the MTT graphs (Fig. 3.3) indicating that the IC50 for POH was roughly around 175 µM
since at 100 µM there was very little cell death or cell proliferation while at 200 µM we saw complete
cell death by 48 hours. A statistically significant drug response was observed at 50 µM, a much higher
concentration than what was noticed with both TMZ and NEO212.

0
50
100
150
200
250
300
350
400
450
0hrs 24hrs 48hrs 72hrs 96hrs
Cell Density (cell/mlx10
3
)
0µM 50µM 100µM 200µM 300µM 500µM
*
**
**

24

Figure 3.10 Effect of TMZ+POH on CLBL1 cells as determined by Trypan blue assay. CLBL1 cells were
treated with different concentrations of a mixture of TMZ+POH. Fresh medium was given at 72 hours.
Cell density was based on the number of live cells. Vehicle used was DMSO. P-values are comparing
treated vs untreated cells. **p-value<0.01 ***p-value<0.001

The mixture of TMZ+POH had a similar effect to TMZ alone but the mixture was slightly less
effective than TMZ alone. A statistically significant drug response was noticed at 30 µM, similar to TMZ.
At 100 and 200 µM, cell density decreased by 24 hours indicating cell death and no to very little cell
proliferation was occurring. By 96 hours, 30 µM drug mixture showed a plateau on cell number while for
30 µM TMZ alone a decrease in cell number was apparent by 96 hours. 10 µM drug for both cases acted
similarly by demonstrating an increase in cell density but at a slower rate than 0 µM and vehicle. An
effect caused by TMZ+POH was noticed at 72 hours for 10 µM but acted similarly to untreated cells
before the 72 hour time mark. In comparison to NEO212, TMZ+POH had less of an effect on inhibiting
cell proliferation thus more cells were alive at lower concentrations of TMZ+POH than NEO212,
underscoring the importance of the covalent conjugation of TMZ and POH in increasing drug
effectiveness.
The table below shows the different concentrations of each drug that will reduce cell proliferation
by 50% in each cell line discussed.
0.0
50.0
100.0
150.0
200.0
250.0
300.0
350.0
400.0
450.0
0hrs 24hrs 48hrs 72hrs 96hrs
Cell Density (cell/mlx10
3
)
DMSO 0µM 10µM 30µM 100µM 200µM
**
***
***

25

Cell Line IC50 NEO212
[µM]
IC50 TMZ
[µM]
IC50
TMZ+POH
[µM]
IC50 POH
[µM]
IC50 AraC
[µM]
CLBL1 6-10 45 60 175 0.3
6D10 6 6 6 300

WEHI-3 30-35 70

Table 3.2 IC50 values for each cell line.

3.3. NEO212 contributes to mice survival in vivo
3.3.1 Effect of NEO212 after the injection of 6D10 cells in vivo
IP implantation of the 6D10-luc cells were conducted on 10 SCID nude mice. Five of the ten mice
were given NEO212 and the other 5 were given a vehicle, both by oral gavage. Mice were weighed
periodically on day 4, day 10, day 17, day 27, and day 40. Mouse 1 of the vehicle group was found dead 4
days post implantation and was not included in the figure.

26

Figure 3.11 Leukemia mice weights. Vehicle and treated mice weighed in grams. Graph and weights as
indicated were obtained by Dr. Steve Swenson

The weights of the mice increased over time, but an extreme increase of weights in the vehicle
mice was noticed. Soon after the spike in weight, vehicle 2, 4, and 5 mice were found dead. All treated
mice had a consistent slow increase in body weight and all survived past day 125.

Figure 3.12 Percent survival for both vehicle and NEO212 treated 6D10 mice. NEO212 treated group had
5 mice while the vehicle group consisted of 4 mice. Graph prepared by Dr. Steve Swenson. **p-value<0.01

0
5
10
15
20
25
30
Treated Red
1
Treated Red
2
Treated Red
3
Treated Red
4
Treated Red
5
Vehicle Blue
2
Vehicle Blue
3
Vehicle Blue
4
Vehicle Blue
5
Weight in grams
Day 4 Day 10 Day 17 Day 27 Day 40

27
Mice treated with NEO212 had a 100% survival rate 125 days post tumor implantation, but
percent survival decreased to 80% by 130 day post tumor implantation. All vehicle-treated mice did not
survive past 35 days post 6D10 tumor implantation. This demonstrated the effectiveness of NEO212
against 6D10 cells in vivo. The mice were treated with 3 rounds of 5 day treatments of NEO212 with 2
week breaks in between and left untreated afterwards.

3.3.2 Effect of NEO212 after the injection of CLBL1 cells in vivo
An IP injection of the CLBL1-luc cells was conducted on 10 SCID nude mice. CLBL1-luc cells were
infected with GFP/luc lenti and repeatedly sorted as shown below. This was similarly done to the 6D10
cells as well.

Figure 3.13 CLBL1 cells infected with GFP/luc repeatedly sorted. The cells were infected and sorted by
Dr. Radu Minea.

The cells were sorted based on their GFP expression. The first graph (left) includes the wild type
(WT) cells, indicated in blue, prior to sorting. The cells were then sorted and cells with GFP positive-
fractions are located in the P3 section of the graph in green. The sorted GFP positive fractions of both cell
lines were then returned to culture, expanded, and subjected to two additional rounds of sorting for
further enrichment of GFP signal. Finally, these homogenous GFP populations were checked for
bioluminescence.

28

Figure 3.14 Percent survival for both vehicle and NEO212 treated CLBL1 mice. Both groups had 5 mice
each. Graph prepared by Dr. Steve Swenson. **p-value<0.01

Mice treated with NEO212 have a 40% survival rate as of 55 days post tumor implantation, and
all the vehicle treated mice did not survive 4 weeks post tumor implantation. This demonstrated the
effectiveness of NEO212 against CLBL1 cells in vivo compared to the vehicle treated mice.

32
Chapter 4 – Discussion

In the veterinary oncology field, there are not many treatment options for dogs with lymphoma
that allow for a long and sustainable life after treatment. There are a variety of available treatment
options but a majority of patients will relapse and develop drug resistance to what is currently
accessible. Most patients with current treatments, even with multiple therapies, have an average
survival time of one year.
NEO212 has shown encouraging results in previous studies involving the treatment of tumors in
humans as well as in TMZ resistant tumors. Therefore, NEO212 was explored as a potential treatment
option for canine lymphoma. TMZ is a current drug used in the veterinary field for lymphoma in relapsed
patients, but it is not used as a primary option. The effectiveness of TMZ in canine patients is low. Only
30% of canines achieve remission, and the average patient only responds to one treatment cycle (28).
NEO212, a covalent conjugation of TMZ and POH, was considered as an alternative option due to the
limited number of effective current treatments and the idea that the covalent conjugation of TMZ to
POH will increase the effects of TMZ by including the cytotoxic properties of POH and increasing drug
uptake into the cell. Canine lymphoma cell lines and human leukemia cell lines were used to
demonstrate the effects of NEO212 due to the similarities between canines and humans in cancer
biology. Canine lymphoma best represents human non-Hodgkin’s lymphoma, and results from this study
can be used for future studies in human lymphoma and leukemia treatment with canine models.
The results of the study showed the cytotoxic effects of NEO212 on the CLBL1 cell line at low
concentrations. The low concentrations seen are good indicators of the cytotoxic effects of NEO212,
with little risk of developing side effects. MTT assays revealed that at concentrations of 6 to 10 µM,
NEO212 demonstrated cytotoxic effects on the CLBL1 cell line by reducing cell survival by 50% (Fig. 3.1,

32
Fig. 3.2). This low concentration was seen for the 6D10 cells as well. NEO212 also had a greater cytotoxic
effect on the CLBL1 and 6D10 cells than on the WEHI-3 cell line (Fig. 3.2).
Next, it was observed that the CLBL1 cells treated with NEO212 showed a better cytotoxic
response and greater cell death than cells treated with TMZ alone, POH alone, and TMZ+POH not
covalently conjugated (Fig. 3.3). Covalently conjugating TMZ to POH increased the effects of TMZ by
including the cytotoxic properties of POH, resulting in a greater effect at lower concentrations. We
wanted to determine whether the covalent conjugation of the two drugs was necessary to result in
greater cytotoxic effects or if mixing the two drugs was sufficient. The results showed a reduction of cell
survival by 50% for NEO212 at 6-10 µM and TMZ+POH at 60 µM (Fig. 3.3). NEO212 had a greater
cytotoxic effect than TMZ+POH mixture, indicating that the covalent conjugation is necessary to increase
cytotoxicity in the CLBL1 cell line. NEO212 was also shown to have a significant effect on the CLBL1 cell
line in comparison to TMZ alone, which establishes NEO212 as a potential novel drug treatment for
canine lymphoma. For the 6D10 cell line, NEO212 was effective by reducing cell proliferation by 50% at a
low concentration of 6 µM, but TMZ alone and TMZ+POH also have similar effects (Fig. 3.5). NEO212
reached 0% cell survival in the 6D10 cells at a concentration of 100 µM while both TMZ and TMZ+POH
did not reach 0% cell survival until 300 µM indicating the efficiency of NEO212 at causing cell death in
comparison to TMZ and TMZ+POH. NEO212, even though at a higher concentration than the other two
cell lines, was more effective than TMZ on the WEHI-3 cells (Fig. 3.6). These findings demonstrate how
the covalent conjugation of TMZ and POH can increase the effectiveness, making NEO212 an effective
drug choice. TMZ is a drug currently used in the veterinary field as a secondary option to drug-resistant
relapsed tumors. With the results demonstrating that NEO212 has greater effects, in regards to
cytotoxicity and cell death, on canine lymphoma at a lower concentration than TMZ, this data can be
vital in future canine lymphoma treatments.

32
The difference in the effectiveness of NEO212 compared to TMZ could be due to the presence of
MGMT in the CLBL1 cell line. Studies have shown that TMZ resistance can be caused by high levels of
DNA repair genes including MGMT (17). TMZ methylates the O6-position of guanine (mO6G) which is
responsible for triggering cell death via double stranded DNA breaks. MO6G can be repaired by the DNA
repair enzyme O6-methyl-guanine-DNA-methyltransderase (MGMT). If a cell has high levels of MGMT,
after TMZ methylation, MGMT removes the methyl group and prevents cell death. The presence of
MGMT in cell lines can increase drug resistance which makes NEO212 a good option for TMZ resistant
tumor cells. Further research will need to be conducted to determine the levels of MGMT or other DNA
repair mechanisms in the CLBL1 cell line leading to a potential cause of drug resistance in canines.
Next, NEO212 had greater cell death on the CLBL1 cells than its individual components based on
trypan blue assays. Comparing the effects of each individual component of NEO212 to the effects of the
novel drug solidifies that the covalent conjugation of TMZ and POH increases the effectiveness. While
MTT assays consider the metabolic activity of the cells after administering NEO212, the trypan blue
assay looks at overall cell survival and death to determine the inhibition of cell growth. This was time
and dose dependent and the results indicated that NEO212 was more effective in CLBL1 cell death at
lower concentrations and at earlier time points than TMZ alone, POH alone, and TMZ+POH (Fig. 3.7, Fig.
3.8, Fig. 3.9, Fig. 3.10). Images of the CLBL1 cells give a visual understanding of how time and dose is
important for the inhibition of cell growth by NEO212 (Table 3.1)
Finally, using mice models, we IP injected 6D10 and CLBL1 to determine the effects of NEO212 in
vivo. In the 6D10 mouse model, the vehicle mice were at 0% survival by day 35 while the mice treated
with NEO212 are at 80% survival 130 days post tumor implantation (Table 3.12). This shows promising
results for human leukemia patients with AraC resistant tumors. This also demonstrates the
effectiveness of NEO212 in drug resistant cells, which is important in canine cancer research due to the
high incidence rate of drug resistance. In the CLBL1 mouse model, the vehicle-treated mice reached 0%

32
survival before day 30 compared to the NEO212 mice which were at 100% survival (Table 3.14). This
shows promising results regarding NEO212 as a potential treatment option for canines. Canines with no
treatment for their lymphoma typically survive 4-6 weeks, which is shown with the vehicle-treated mice
not surviving past the 4 week time point (7). 40% of the mice are currently still alive 55 days post tumor
implantation (Table 3.14). TMZ, a current drug used in canine lymphoma treatment, exhibits a 30%
chance of remission with the patient only responding to one treatment (28). My results demonstrate
that NEO212 can be used as a potential treatment option for canine lymphoma, since the mice treated
with NEO212 have undergone multiple rounds of treatment and are still responsive to the drug.
The results shown are promising for future studies with canine tumor cell lines but more studies
will need to be conducted to ultimately reach drug approval. Determining the safety, efficacy, and
effectiveness of the drug and an optimal dosing schedule are important prior to clinical trials in the
target animal. The maximum tolerated dose of NEO212 has been previously determined in canines to be
50 mg/kg with repeated doses. While mice models are good in vivo models for canine lymphoma,
moving into a canine clinical trial is the next step.
Working with dogs in a clinical or research setting comes with ethical considerations due to
canines being companion animals and since clinical trials in canines will require patients with lymphoma,
owners will be volunteering their pet to test the novel drug. Though results look promising in both in
vitro and in vivo experiments, more research will need to be conducted prior to testing on volunteer
patients to ensure the best outcome. Other research options include testing NEO212 in vivo and in vitro
on a variety of different canine and human tumor cell lines to determine if NEO212 is not only effective
for canine lymphoma but other canine tumors. Western blot analysis of drug resistance mechanisms,
which include probing for DNA repair proteins, apoptotic markers and ER stress markers. Measuring cell
death and cell viability by FACS analysis to further determine the effects of NEO212 on canine
lymphoma. Also, the pharmacodynamics and pharmacokinetics of NEO212 needs to be better

32
understood when transitioning from canine to human trials (9). Lastly, comparing NEO212 in vivo and in
vitro to other current canine lymphoma drug treatments can give a better understanding of NEO212 as a
potential drug option. More experiments will need to be conducted to determine NEO212’s potential in
the veterinary field.

33
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Abstract (if available)

Abstract Cancer is the number one cause of death in dogs with lymphomas representing one of the most common spontaneously occurring tumors. With no cure for canine lymphoma, many dogs relapse due to drug resistance after treatment and require multiple therapy options. The need for more drug treatments for cancer in the veterinary field is crucial. Even with multiple rounds of therapy, dogs diagnosed with cancer have a poor outcome. In addition, high similarities between canine lymphomas and human non-Hodgkin’s lymphoma allow for canines to be a reliable comparison model for human cancer research on lymphomas. ❧ NEO212, a covalent conjugate of perillyl alcohol and temozolomide, was tested on canine and human cell lines to determine the effects of this novel drug on canine patients. Cell proliferation and cell viability were measured under different concentrations of NEO212 with various cell lines to observe its cytotoxic effects. An evaluation was also done with the individual components of NEO212, perillyl alcohol alone and temozolomide alone. Perillyl alcohol and temozolomide were also evaluated together in a mixture but not covalently conjugated to determine if the covalent conjugate aids in cellular uptake, allowing a stronger cytotoxic effect at lower concentrations. We conducted methylthiazoletetrazolium assays to determine cytotoxic effects of NEO212 on cell metabolic activity, as well as determining the effects of NEO212 on cell viability by trypan blue assays. Cell images were captured under a light microscope to give a visual understanding of cell viability over time. Lastly, in-vivo testing was conducted using mice models to determine the effects of NEO212 in a biological system. ❧ The results of our study demonstrated that NEO212 had a strong cytotoxic effect on the canine lymphoma and human leukemia cell lines and had a greater effect at lower concentrations than TMZ alone, POH alone, or the TMZ and POH mixture. For the canine cell line, the effects of NEO212 were greater than temozolomide, a current treatment for dogs with relapsed lymphoma. NEO212 also demonstrated an inhibition of cell proliferation and caused cell death at a low concentration. NEO212 was shown to be an effective novel drug treatment in both the canine lymphoma cell line and the human leukemia cell line, and a possible alternative to current treatments, especially in drug-resistant tumors. Further studies will need to be conducted to determine its pharmacological effects and clinical potential of NEO212.

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Asset Metadata

Creator Mohseni, Nazleen (author)

Core Title Cytotoxic effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on canine lymphoma

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 03/29/2021

Defense Date 03/04/2021

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

Tag 6D10,B-cell lymphoma,canine large B-cell lymphoma,canine lymphoma,canine lymphoma treatment,CLBL1,comparative oncology,cytotoxicity,human leukemia,NEO212,NHL,non-Hodgkin lymphoma,OAI-PMH Harvest,perillyl alcohol,temozolomide,veterinary medicine,veterinary oncology

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Schönthal, Axel H. (committee chair), Landolph, Joseph R., Jr. (committee member), Tahara, Stanley (committee member)

Creator Email nazleenmohseni@gmail.com,nmohseni@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-433669

Unique identifier UC11667457

Identifier etd-MohseniNaz-9367.pdf (filename),usctheses-c89-433669 (legacy record id)

Legacy Identifier etd-MohseniNaz-9367.pdf

Dmrecord 433669

Document Type Thesis

Rights Mohseni, Nazleen

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...

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