Oncolytic virus therapy in the treatment of Melanoma TABLE OF CONTENTS1. Abstract………………………………………………………..2. Introduction……………………………………………………..3. Search Strategy…………………………………………………..4. Discussion5. Future Directions6. Appendix……………………………………………………….7. References………………………………………………………    Abstract In my opinion, oncolytic virus (OV) therapy is a new and emerging strategy for the treatment of melanoma showing superior characteristics amongst other possible treatment options. T-Vec, a specifically designed OV to target melanoma, uses a genetically engineered HSV-1 virus with characterized specificities which is injected directly at tumor site. The virus replicates in the tumor using the host’s mechanism until the tumor bursts and the immune system is induced to produce an anti-tumor response. The mechanism of action of T-Vec allows replication in only tumor cells without affecting normal cells, while producing both local and systemic immune responses provide a unique advantage for this treatment strategy. Studies comparing the overall survival rates of a few common treatment strategies for melanoma against T-Vec also show a superior efficacy of OV therapy over other treatment strategies. For future considerations, it is recommended that a combination of treatment options targeting different mechanisms should be tested to produce the optimal treatment plan to prolong survival rates in patients with melanoma.Introduction Melanoma is a cancerous condition of the skin, particularly affecting the component of skin cells known as melanocytes. Melanocytes play a role in the production of melanin and gives rise to skin pigmentation. The primary goal of melanin is to function as an ultraviolet (OV) absorbent and provide photoprotection against DNA damage caused by UV radiation.UV radiation exists in multiple forms – ultraviolet A (UVA), ultraviolet B (UVB), and ultraviolet C (UVC) – each differing in their properties and their subsequent biological effects on the skin11. UVA is highly absorbed by the skin and penetrates deep into the dermis layer whereas UVB penetrates into the epidermis layer, where melanocytes are located (Fig. 1)5. As a result of the detrimental effects of UV radiation, UVA causes indirect DNA damage by generating reactive oxygen species (ROS) which further result in single-strand breaks in DNA5. Despite the low penetrance of UVB, UV radiation from UVB is maximally and directly absorbed by DNA which also induces structural DNA damage11. As such, UV radiation poses as the primary risk factor for melanoma due to its ability to induce genetic and epigenetic changes to DNA in melanocytes11.In melanoma, the most common genetic change induced by UV radiation occurs in the B-RAF gene and protein, accounting for >60% of reported cases6. The B-RAF protein is part of the Mitogen Activated Protein Kinase (MAPK) signaling pathway which transmits extracellular signals from the cell receptor to the cell DNA via phosphorylation cascades6. The MAPK pathway comprises of protein kinases which ultimately regulate cell functions such as proliferation, gene expression, differentiation, cell survival and apoptosis9. The components of the MAPK pathway are illustrated in Figure 29. The mutation in B-RAF consists of a substation of valine (V) with glutamic acid (E) at codon 600 in exon 15, referred to as V600E9. The amino acid substitution causes conformational changes in the activation domain of the protein, such that the B-RAF protein remains constantly activated9. As a result, B-RAF constantly stimulates downstream components of the MAPK pathway independent of external stimulus and facilitates uncontrolled cell proliferation found in cancers such as melanoma (Fig. 3)9,1.Additionally, a mutation in p53 gene also plays a role in melanoma progression3. In normal cells, p53 functions as a tumor suppressor and prevents cells with damaged DNA from further replicating by inducing cell cycle arrest and cell apoptosis3. However, UV radiation is found to give rise to mutated p53 which further contributes to disease progression3,5. It should be noted that these mechanisms are not the lone pathway leading to melanoma3. Melanoma can arise from familial loss-of-function mutations in CDKN2A gene and/or MC1R gene which potentially places individuals at higher risk of developing melanoma due to greater sun sensitivity3. In most cases of melanoma, a combination of multiple genetic and epigenetic factors leads to disease.In the present day, many approaches are present in market or in the process of development which are targeted towards the treatment of melanoma, with strategies such as tumor regression, tumor growth suppression, increased anti-tumor response, and improved survival. One such strategy is the utilization of oncolytic viruses (OVs)2,4. In October 2015, talimogene laherparepvec (T-Vec) was approved by FDA as the first-ever oncolytic virus therapy, designed specifically to target melanoma2,4. The treatment strategy utilizes a genetically engineered oncolytic virus, herpes simplex virus type 1 (HSV-1), which is directly injected into melanoma tumor sites and allowed to selectively replicate using the tumor’s own mechanism2. Due to the immunosuppressed environment of the tumor, the HSV-1 virus successfully replicates and concentrates at the site of tumor until the tumor bursts, which subsequently releases tumor antigens, cytokines, and granulocyte-macrophage colony-stimulating factors (GM-CSF) into the bloodstream and lymph nodes2. As a result, the immune system becomes activated and produces tumor antigen-specific T cells which recognize, target and destroy the melanoma tumor cells2. Overall, OVs induce a local response by directly causing tumor cell lysis, as well as produces a systemic anti-tumor immune response2.The specificity of T-Vec to target only the tumor cells while ensuring the normal cells remain unaffected is aided by the genetic engineering modifications in the HSV-1 OVs2,4. Specifically, mutations (deletions) were engineered in the infectious cell proteins (ICP) 35.4 and 472. Normally, ICP35.4 interacts with PP1? to dephosphorylate eIF2? and allow viral replication in host cells (Fig. 4)4. However, a mutated ICP34.5 is non-functional and prevents de-phosphorylation of eIF2?, which ensures that viral translation is shut off and prevents replication in normal cells4. Since tumor sites have impaired anti-viral mechanisms, viral replication can proceed4. As such, genetic engineering of ICP34.5 protects normal cells from HSV-1 while providing tumor selectivity4. Similarly, a deletion mutation in ICP47 is also induced in HSV-1 and replaced with a gene insertion encoding GM-CSF2,4. Normal ICP47 blocks antigen presentation to the immune response as a means of increasing virus proliferation4. However, a deleted ICP47 removes this mechanism whereas the GM-CSF gene increases immune stimulation to enhance the anti-tumor immune response4. Therefore, genetic engineering of HCV-1 enhances the efficacy of oncolytic virus therapy in order to destroy melanoma tumors and induce a systemic immune response4. The aim of this opinion paper is to present OV as an emerging treatment for melanoma and discuss the beneficial advantages of the therapy which make it superior to other treatment strategies. Search Strategy The University of Windsor – Leddy library website were accessed. Link to “Journal Articles and Research Tools” was used and the sub-category of “Chemistry and Biochemistry” was selected.The following journal articles were used:National Centre for Biotechnology Information (NCBI)PubMedGoogle ScholarScience DirectThe following keywords were used:MelanomaMalignant melanomaTalimogene laherparepvecT-VecOncolytic virus therapyMAPK pathwayB-RAF functionUltraviolet radiationDiscussionThe strategy of OVs provide a promising area of treatment in cancerous diseases. When considering T-Vec as a treatment for melanoma, the strategy provides many advantages and superior properties than other treatment options available in the market. The biggest advantage provided by T-Vec is the ability to target selectively for tumor cells facilitated by the genetic modifications in HSV-1 virus in order to avoid viral replication in normal cells2. The selectivity allows for a concentration of viruses to replicate at the intended site and obtain the desired effect of rupturing the tumor cells, while ensuring minimal side effects of viral invasion are exposed to the patient2. In comparison to other strategies (i.e. drugs), OV treatment avoids non-specific interactions in other pathways and prevents undesired side effects. Additionally, the T-Vec treatment strategy also benefits from the immune response generated as a result of viral invasion2,4. Due to the injection of HSV-1 at the tumor site, the rupture of tumor cells induces a local response, as well as a systemic response via the release of cytokines, GM-CSF and tumor specific antigens2,4. The combined amplified immune response is beneficial and helps to achieve the selective tumor cell-killing capacity of the virus4. Lastly, during the approval process of T-Vec, clinical trials Phase I-III proved the therapy to have a safe pharmacokinetic profile while establishing high efficacy levels and safe tolerability in patients10.While many treatment strategies are available for melanoma with biological targets derived from various pathways, a comparison between the following treatments was conducted to determine their relative success rates: monoclonal antibody targeting CTLA-4 to activate immune system (Ipilimumab), B-RAF inhibitors (Vemurafenib), and T-Vec12. A summary of the trials specifications and patient characteristics are listed in Figure 512. Since trials were conducted separately and compiled for comparison purposes, results were adjusted based on patient and disease characteristics for overall survival (OS) rates (in months) for Ipilimumab and Vemurafenib12. The OS values for all treatments before and after adjustment are listed in Figure 612. The OS curves for Ipilimumab versus Vemurafenib versus T-Vec treatments are illustrated in Figure 712. The OS curves for Ipilimumab only versus T-Vec treatments are illustrated in Figure 812. The OS curves for Vemurafenib only versus T-Vec treatments are illustrated in Figure 912. Based on the analysis of all results, it is evident that OS was higher with the T-Vec treatment strategy compared to the other treatments12. As such, data from the comparison study show that using using T-Vec for melanoma may be superior than other treatment strategies, however, a compiled study comparing treatments to each other is required to conclusively determine success rates without bias12.Despite the numerous benefits offered by OV therapy, it must be noted that the treatment option contains some drawbacks which can pose as limitations for the therapy. The main drawback for viral therapy is the efficacy of the vector to induce the desired effect in the body8. The vector must be resistant to rejection by the host and induce viral replication after administration8. However, numerous changes introduced to the virus genome via genetic engineering allowed to overcome vector rejection and improve vector properties, such as mutations of ICP34.5 and ICP472. Another major drawback of using viruses as a treatment strategy is the ability of the host to develop resistant mechanisms towards the virus and recognize the virus as a foreign invader, leading to rejection7. Although this possibility is not yet observed, the chances of developing resistance mechanisms exist due to the nature of viruses7. Given that T-Vec is a new therapy approach which received FDA approval in October 2015, the long-term pharmacokinetic profile and side effects remain unexamined7. As such, it can be concluded that the current data available for OV therapy renders it as a beneficial treatment approach with certain drawbacks resolved via genetic engineering. Future Directions Currently, T-Vec has shown high efficacy and success in the treatment of melanoma. However, due to the complexity of cancer pathway and severity of cancer across patients, the treatment course consisting of only one treatment strategy (i.e. T-Vex) may not be sufficient to give the optimal results7. As such, future directions to optimize treatment strategies may need to investigate the combination of multiple treatment strategies, such as B-RAF inhibitors, immune checkpoint blockades, immunostimulatory treatments and/or anti-tumor response treatments7. The combination of multiple treatments will provide the benefits of enhancing the immune response towards tumor directed cell death while inhibiting further tumor growth7. As such, I believe it is important to investigate the best combination of treatments in future studies to optimize endpoints such as overall survival, tumor regression, tumor growth suppression, and increased anti-tumor response in melanoma patients.                                  Appendix Figure 1: Comparison of penetration depth of UVA and UVB5. Figure 2: Components of the MAPK pathway9.  Figure 3: Comparison of normal versus. abnormal MAPK pathway (CITE).  Figure 4: Role of ICP34.5 in presence of virus4.   Figure 5: Descriptions of trials used for comparison study of various melanoma treatment strategies12. Figure 6: Overall survival values (in months) for all treatments before and after adjustment12.       Figure 7: Overall survival curves for Ipilimumab versus Vemurafenib versus T-Vec treatments12.  Figure 8: Overall survival curves for Ipilimumab versus T-Vec treatments12. Figure 9: Overall survival curves for Vemurafenib versus T-Vec treatments12. ReferencesAscierto, P. A., Kirkwood, J. M., Grob, J.-J., Simeone, E., Grimaldi, A. M., Maio, M., … Mozzillo, N. (2012). The role of BRAF V600 mutation in melanoma. Journal of Translational Medicine, 10, 85. http://doi.org/10.1186/1479-5876-10-85Bayan, C. A. Y., Lopez, A. T., Gartrell, R. D., Komatsubara, K. M., Bogardus, M., Rao, N., … & Pradhan, J. S. (2018). The Role of Oncolytic Viruses in the Treatment of Melanoma. Current oncology reports, 20(10), 80.Box, N. F., Vukmer, T. O., & Terzian, T. (2014). Targeting p53 in melanoma. Pigment Cell & Melanoma Research, 27(1), 8–10. http://doi.org/10.1111/pcmr.12180Braidwood, L., Graham, S. V., Graham, A., & Conner, J. (2013). Oncolytic herpes viruses, chemotherapeutics, and other cancer drugs. Oncolytic virotherapy, 2, 57.Brenner, M., & Hearing, V. J. (2008). The Protective Role of Melanin Against UV Damage in Human Skin. Photochemistry and Photobiology, 84(3), 539–549. http://doi.org/10.1111/j.1751-1097.2007.00226.xCandido, S., Rapisarda, V., Marconi, A., Malaponte, G., Bevelacqua, V., Gangemi, P. … Libra, M. (2014). Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure. Oncology Reports, 31, 1079-1082. https://doi.org/10.3892/or.2014.2977Lawler, S. E., Speranza, M. C., Cho, C. F., & Chiocca, E. A. (2017). Oncolytic viruses in cancer treatment: a review. JAMA oncology, 3(6), 841-849.Lundstrom, K. (2018). New frontiers in oncolytic viruses: optimizing and selecting for virus strains with improved efficacy. Biologics: Targets & Therapy, 12, 43–60. http://doi.org/10.2147/BTT.S140114McCain, J. (2013). The MAPK (ERK) Pathway: Investigational Combinations for the Treatment Of BRAF-Mutated Metastatic Melanoma. Pharmacy and Therapeutics, 38(2), 96–108.Pol, J., Kroemer, G., & Galluzzi, L. (2016). First oncolytic virus approved for melanoma immunotherapy. Oncoimmunology, 5(1), e1115641. http://doi.org/10.1080/2162402X.2015.1115641Riker, A. I., Zea, N., & Trinh, T. (2010). The Epidemiology, Prevention, and Detection of Melanoma. The Ochsner Journal, 10(2), 56–65.Quinn, C., Ma, Q., Kudlac, A., Palmer, S., Barber, B., & Zhao, Z. (2016). Indirect Treatment Comparison of Talimogene Laherparepvec Compared with Ipilimumab and Vemurafenib for the Treatment of Patients with Metastatic Melanoma. Advances in Therapy, 33, 643–657. http://doi.org/10.1007/s12325-016-0313-x