Arjun Kanjarpane

5
IR-3/09GT
5.3.18
Data Analysis: Report

Topic:
The researcher hopes to discover the inter-relational bonds between his three controls and
more about them in general. Each of my controls, autophagy/mitophagy, tumor translational
mediation and drug-modulated immunosuppression by means of either a T-cell blockade or other
depressant, releases on specific bodily processes which are reliant on other bodily processes.
Observing these tactics facilitates further research and the complicated procedures needed to
ensure that each system can work together. The overall goal, or the end goal, however is to
ultimately reduce antiviral responses which oncolytic viruses face from the immune system.

Method​:
For this data collection, the researcher plans to do a literature review of 3 articles
surrounding his three topics. The researcher also hopes to use the knowledge provided by those
articles and surrounding background knowledge to produce possible results. To execute the type
of data collection usually required for this topic (experimental) would require a full lab, potent
viruses and many laboratory techniques and skills which he either does not have access to, or
have not learned.

Articles Used:
For each of the controls, the researcher hypotheses that 2-3 articles with great detail
would be sufficient, but more could be added later. The first two controls, cellular autophagy and
immunosuppression are both accounted for, with 3 vetted, published articles. Translational affect
is a more novel idea and thus the researcher has less of a knowledge base.
 Articles:

Mitophagy/ Autophagy:
1. Ding, Wen-Xing, and Xiao-Ming Yin. “Mitophagy: Mechanisms, Pathophysiological

Roles, and Analysis.” ​Biological Chemistry​, vol. 393, no. 7, July 2012, pp. 547–64,

doi:​10.1515/hsz-2012-0119​.

2. Meng, Gang, et al. “Mitophagy Promotes Replication of Oncolytic Newcastle Disease

Virus by Blocking Intrinsic Apoptosis in Lung Cancer Cells.” ​Oncotarget​, vol. 5, no. 15,

July 2014, pp. 6365–74, doi:​10.18632/oncotarget.2219​.

3. Xia, Mao, Patrick Gonzalez, et al. “Mitophagy Enhances Oncolytic Measles Virus

Replication by Mitigating DDX58/RIG-I-Like Receptor Signaling.” ​Journal of Virology​,

vol. 88, no. 9, May 2014, pp. 5152–64, doi:​10.1128/JVI.03851-13​.

4. Xia, Mao, Gang Meng, et al. “Mitophagy Switches Cell Death from Apoptosis to

Necrosis in NSCLC Cells Treated with Oncolytic Measles Virus.” ​Oncotarget​, vol. 5,

no. 11, May 2014, pp. 3907–18,

Immunosuppression:
1. Rajani, Karishma R., and Richard G. Vile. “Harnessing the Power of

Onco-Immunotherapy with Checkpoint Inhibitors.” ​Viruses​, vol. 7, no. 11, Nov. 2015,

pp. 5889–901, doi:​10.3390/v7112914​.

2. Thomas, Maria A., et al. “Immunosuppression Enhances Oncolytic Adenovirus

Replication and Antitumor Efficacy in the Syrian Hamster Model.” ​Molecular Therapy :
 The Journal of the American Society of Gene Therapy​, vol. 16, no. 10, Oct. 2008, pp.

1665–73, doi:​10.1038/mt.2008.162​.

3. Engeland, Christine E., et al. “CTLA-4 and PD-L1 Checkpoint Blockade Enhances

Oncolytic Measles Virus Therapy.” ​Molecular Therapy​, vol. 22, no. 11, Nov. 2014, pp.

1949–59, doi:​10.1038/mt.2014.160​.

Translation:

1. Jacobson, Blake A., et al. “Cap-Dependent Translational Control of Oncolytic Measles

Virus Infection in Malignant Mesothelioma.” ​Oncotarget​, vol. 8, no. 38, June 2017, pp.

63096–109, doi:​10.18632/oncotarget.18656​.

2. Pelletier, Jerry, et al. “TARGETING THE EIF4F TRANSLATION INITIATION

COMPLEX: A CRITICAL NEXUS FOR CANCER DEVELOPMENT.” ​Cancer

Research​, vol. 75, no. 2, Jan. 2015, pp. 250–63, doi:​10.1158/0008-5472.CAN-14-2789​.

Intended Audience:​ The intended audience for this research is either the scientific community
specific to oncology and the frontiers in immunotherapies or researchers in this field.

Distribution Plan:​ The distribution plan is either through the researcher’s advisor and her broad
network of contacts or submission to a scholarly journal such as ​nature.

Result:​ The distribution plan is in the works: the paper is being constructed and will soon be
integrated into the primary background paper.
 Part 2: Data
Autophagy

Mitophagy Enhances Oncolytic Measles Virus Replication by Mitigating DDX58/RIG-I-Like

Receptor Signaling.
Translation

“Translational Control of Oncolytic Measles Virus Infection in Malignant Mesothelioma.”

 Immunosuppression

Immunosuppression Enhances Oncolytic Adenovirus Replication and Antitumor Efficacy in the

Syrian Hamster Model.
 Part 3: Analysis
Oncolytic Virotherapy (OV) is a cutting-edge research breakthrough within the domain
of cancer immunotherapeutics. It’s genesis was driven by the intensity of researching innovative
treatments against one of the most devastating diseases in the 21st Century-cancer. Most
oncolytic viruses feature a deletion of the E1B gene, which causes apoptosis in infected cells.
This deletion allows for greater virus replication and greater expression of the E1A protein,
which stabilizes the tumor suppressor, p53 gene. The deletion of p53 not only allows for
advanced tumor suppressive abilities, but combined with the E1B deletion, promotes the lack of
replication in healthy, non cancerous tissues. As more oncolytic viruses come into the forefront
of cancer research and treatment, their adverse characteristics must be addressed to increase
efficacy. The primary therapeutic challenge facing OV and other immunotherapeutics are the
human body’s natural reaction to these treatments, specifically, antiviral responses. Three
specific modalities to optimize OV efficacy are autophagy, immunosuppression and translatory
modulation. Each of these modalities have individually shown promise by increasing viral titer
and oncolytic rates in the tumor microenvironment. However, to maximize therapeutic efficacy,
the researcher seeks to use all three modalities together. In order to work together and produce a
result which is not antagonistic, the method and location in and by which the modality functions,
must be independent.
“Immunosuppression Enhances Oncolytic Adenovirus Replication and Antitumor
Efficacy in the Syrian Hamster Model”, presents the advantage that immunosuppressant drugs
can have on the overall therapeutic efficacy of an oncolytic virus, as measured with tumor
volume. The effect of cyclophosphamide (CP) on the oncolytic efficacy of oncolytic
adenoviruses VX-007 and AD5 were measured with the immunosuppressive and alone (without)
on hamster adenocarcinoma models. The experiment was measured over the course of five
weeks post-administration of CP and oncolytic adenoviruses against adenocarcinomas. The
independent variable of the study observed the activity of the tumor alone (vehicle), CP alone,
the viruses alone, and the viruses with CP in a tumor microenvironment. The gross tumor
volume (µl), with the vehicle alone at the end of 40 days measured volumes upward of 5,200μl
but did not see any signs of decreased volume. The CP alone did not have a statistically
significant effect on the growth of the tumor and is evidenced by the similar pattern it takes when
growing over the course of 40 days. The overall volume decreased very slightly, about 50μl
compared to the vehicle alone, but this is still statistically insignificant. The infusion of the
oncolytic viruses VRX-OO7 (an adenovirus with an overexpression of the Ad death protein) and
AD5 (an un-attenuated adenovirus serotype) into the environment without CP yielded
satisfactory results with both viruses reaching 2000μl from a previous 8,000μl 40 days post
infection (from a starting 1000μl at day 0). This was significant oncolysis as compared to the
vehicle, but growth was further reduced after CP was added, with tumor volume reaching below
1000μl 40 days post infection with the wild Ad5 virus being slightly more efficacious as it
reached 970μl compared to VX-007, which reached 1,000μl.. With the vehicle alone, with a
7,000μl volume, the oncolytic viruses brought the overall tumor volume lower than the starting
point, and lower than the viruses alone, proving the modality as beneficial to overall therapeutic
efficacy. Specifically, the first two weeks saw both immunosuppressant and immunocompetent
environments display no noticeable differences but researchers report that immunocompetent
hamsters began to display tumor regrowth, which is represented on the first graph by the incline
in tumor volume around day 14 and 15. The overall conclusion represents the ability for
immunocompetent environments to host viruses for longer periods of time leading to longer
oncolysis and in turn, a prolonged period of cancer remission.
While secondary factors such as drug-modulated immunosuppression aid in the process
of prolonging viral life and oncolytic, viruses also inherent unique abilities to defend against the
body’s defenses. The following study used the Edmonston strain of the Measles Virus measured
the effect of autophagy and autophagic flux rates when autophagic stimulants and depressants
were used. This rate was measured by the frequency of autophagosome formation. Lung cancer
cells in two variants, the AF49 and H1299 cells were observed with and without MV-Edmonston
infection. The autophagosome moved towards cells with the EGFP-MAP1LC3B transgene. The
result number of vesicles increased, evidenced by the GFP being embedded into the transgene,
displaying that the overall lysis of the lung cancer cells. Cells containing EGFP-MAP1LC3B
were subject to MV-EDM infection, at 0.5 viral titer and cultured for 4h. Following this
culturation, cells were stained for MV H protein, to highlight the protein. After 6 hours, increases
in viral titer nor a increase in MV H protein were present and EGFP-MAP1LC3B levels and
quantities remained constant. 24 hours after virus infusion, MV H protein levels rose
astronomically along with a subliminal decrease in EGFP-MAP1LC3B levels. The 9th square
merged these factors and provided evidence to a strong viral titer decrease. This contributed to
the overall understanding that autophagy and the late stage, autophagic flux is induced in greater
quantities after viral infection, and this is specific to the Measles Virus-EDM. Autophagic
stimulants, in this study were added via extra tumoral infusion via injection. In the future, these
types of injections are subject to mutation and will complicate the tumor microenvironment. To
operate more seamlessly, MV-EDM should feature RNA coding for inherent autophagic
stimulants as part of the viral genome. This process now allows the virus to replicate and while
translating, the virus automatically makes autophagic stimulants. However, autophagy may be of
more benefit when stimulated even further through translation.
The process of taking mRNA and feeding information to the ribosomes to conduct
protein synthesis is known as translation. Virus infusion and control relies heavily on translatory
measures, as by itself, viruses are not a stable platform to induce protein synthesis needed for
viral survival and transmission. Measuring the effect of transitional control in a tumor of viral
control measures the rate at which proteins are produced within the tumor, and inferentially the
virus. This rate, is increased, increases both the rate of tumor multiplication and virus multiple.
To measure the effect of translational control on virus survivability and overall efficacy, a
translational inhibitor called eIF4E targeted antisense oligonucleotide (ASO) was used to treat
H514, H2461, H2596 mesothelioma cancer cells with MV-CEA, a measles virus which over
expresses the carcinoembryonic antigen. A mismatch control ASO was used for control
purposes. 48 hours later, cell viability in H256 cells decreased from 1.1 to 0.5, more than 200%
reduction. Levels of CEA were present in all cells inside the tumor environment, however cells
infected by MV-CEA and not mmAS, 4EASO displayed 300ng/ml of CEA compared to the
other two CEA acquiring methods in H513 cells, while the CEA levels in H2373 cells was closer
to 200 in MV-CEA cells. These results display that the mismatch control item had an inhibitory
effect upon the H513 cell line. 75% of cells tested displayed unviability while 25% displayed
continued viability with continued use of the MV-CEA virus as well as 3EASO. Transitional
stimulants such as IGF-I, after 24 hours in use with MV-CEA drastically lowered CEA levels,
reaching a staggering 60 ng/ml of CEA in cells.
The beneficial effects of three oncolytic viral antiviral mitigatory methods, autophagy,
immunosuppression and translation were analyzed in this literature review. The purpose was to
study the efficacy of the three modalities and analyze the possibility that when these are used
concurrently, the result is a clinically significant increase in Onco-Virotherapeutic efficacy. Each
review article outlined the specific oncolytic virus being used, modality specifics, and modal
effect on treatment efficacy. Cellular autophagy operates based on stimulants which are protein
related. This direct infusion can occur via direct fusion as well as gene editing prospects (Xia et
al). Recoding the genetic information in the virus can yield a higher output of autophagic
stimulants. While autophagy and translation handle the immune system from the inside,
immunosuppression weakens the immune system from the outside, allowing body-foreign agents
to be safely introduced in vivo. Immunosuppression is largely drug-dependent with
cyclophosphamide (CP) used in the reviewed study. CP is a prodrug, meaning it requires
metabolic activation in the liver, which produces 4-hydroxycyclophosphamide. This process also
does not rely on further external factors that are required for activation. The two modalities of
autophagy and immunosuppression worked flawlessly and optimized therapeutic efficacy
independent of each other. Both modalities operate on external cells which are unaffected by
virus infection. The third modality of translational control presents a slight problem in that the
very process of translation which requires genetic information from the nucleus to become
mRNA and travel to the ribosome. The interactions between genetic changes used to produce
autophagic stimulants may coincide with genetic information transferred to ribosomes due to the
translational stimulants.
 Works Cited
Thomas, Maria A., et al. “Immunosuppression Enhances Oncolytic Adenovirus Replication and
Antitumor Efficacy in the Syrian Hamster Model.” ​Molecular Therapy : The Journal of
the American Society of Gene Therapy​, vol. 16, no. 10, Oct. 2008, pp. 1665–73,
doi:​10.1038/mt.2008.162​.

Xia, Mao, Patrick Gonzalez, et al. “Mitophagy Enhances Oncolytic Measles Virus Replication
by Mitigating DDX58/RIG-I-Like Receptor Signaling.” ​Journal of Virology​, vol. 88, no.
9, May 2014, pp. 5152–64, doi:​10.1128/JVI.03851-13​.

Jacobson, Blake A., et al. “Cap-Dependent Translational Control of Oncolytic Measles Virus
Infection in Malignant Mesothelioma.” ​Oncotarget​, vol. 8, no. 38, June 2017, pp.
63096–109, doi:​10.18632/oncotarget.18656​.