Despite significant strides in cancer research, current treatments for a lethal form of brain cancer — glioblastoma — remain palliative and ultimately futile. However, recent experimental research utilizing a unique brand of viruses demonstrates the real possibility of developing a successful treatment option for patients of glioblastoma or other forms of cancer.    

           The public was taken by storm during 2015 when news broke out that Zika virus had reached the Americas. Despite the World Health Organization declaring Zika virus as an international public health emergency by February 2016,[1] many were already aware of the disease’s nature. While adult symptoms of the disease tend to be asymptomatic, in utero damage can occur if the infected person is pregnant. Zika’s most severe consequence is microcephaly (as shown in Figure 1), an occasionally fatal condition in which the baby is born with an abnormally small head and brain, causing cognitive deficits[2]. However, you may be surprised that despite the potentially severe consequences of infection, Zika is being explored — along with many other viruses  — as a means of treating cancer. These viruses, studied for their cancer-treating properties, are referred to as oncolytic viruses.

Figure 1: Microcephaly http://labblog.uofmhealth.org/sites/lab/files/2016-08/UMH_L_microcephaly%401x.jpg
Figure 2: Necrosis vs. Apoptosis
http://medicinembbs.blogspot.com/2011/03/programmed-cell-death-apoptosis.html

A discussion of oncolytic viruses (the prefix being the Latin onco, which means tumor) requires some knowledge of the dual mechanism by which they accomplish tumor extinction via an immune response: a) their ability to kill individual tumor cells and b) initiate systemic immunity. To begin with, viruses normally rely on a form of cell death – apoptosis – via an intracellular pathway devoted to controlled cell death. The purpose is to reduce the probability of releasing viral progeny; however, for an oncolytic virus to be successful, it must spread to trigger an immune response. Therefore, it can be inferred that the limitation of apoptosis during tumor infection would be beneficial.[3] The consequence of denying apoptosis is forcing the cell to default to necrosis as its form of cell death, such that the cellular membrane lyses and disintegrates, releasing its contents as part of the immunogenic response (see Figure 2). This is exactly what we see in virus-infected tumor cells; rejection of apoptosis provides additional time for virus proliferation. But, what is it that differentiates the method of cell death between neoplastic (tumorous) and normal infected tissues?

Research from the turn of the last century implicated a protein as the cause of the shift towards necrosis in neoplastic tissue. A culture of cells exposed to reovirus (a family of nonpathogenic viruses used in pathogenesis research) proved resistant to infection until the activation of the Ras protein, a catalytic protein utilized by the cell during signal transduction. Upon its activation, phosphorylation of another enzyme, Protein-Kinase R (PKR), was prevented. This eliminated its function as an inhibitor of RNA translation (shown in Figure 3), allowing for reovirus to access the cellular machinery for translation.[4] Later research identified PKR as a detector of viral infection, and current models now postulate that normal antiviral machinery, including PKR, may be abnormal in neoplastic tissue, preventing the initiation of apoptosis and extending viral proliferation.

Avoiding the apoptotic pathway is not the only obstacle scientists have had to confront to promote treatment efficiency. After necrosis, antigens referred to as PAMP’s (pathogen associated molecular patterns) are released and detected by cells responsible for activating the immune response: macrophages and dendritic cells. However, in neoplastic tissue, abnormal PKR activity indirectly affects a signaling pathway responsible for inhibiting virus replication via release of interferon (IFN) protein (Figure 4, steps 1 to 2). Genetic engineers have successfully been able to circumvent this issue by taking advantage of the another way to activate the immune response, By altering the genetic makeup of oncolytic viruses to permit cytokine production via insertion of the GM-CSF gene, macrophages and dendritic cells can be activated independent of the former mechanism (steps 2 to 3). Release of synthesized GM-CSF promotes the presentation of tumor-associated antigens (e.g. PAMPs)  by dendritic cells in order to induce an immune response via CT8+ T cells (step 4). Concurrently, GM-CSF encourages the maturation of additional dendritic cells, while IFN can directly activate innate “natural killer” cells (step 4). The consequence of both enhanced antigen presentation and activation of innate cells is are that systemic anti-tumor immunity can be achieved. This allows for elimination of neoplastic tissue unaffected by the virus.[5]

Figure 3: How viral infection is influenced by Ras & HSV-1 Gene ICP34.5, adapted from https://www.researchgate.net/figure/227681656_fig3_Figure-3-Mechanisms-of-tumor-cell-selectivity-of-reovirus-and-HSV1716-During-the-normal
Figure 4: An MRI of a glioblastoma patient
http://www.aboutcancer.com/mri_gbm.htm

Oncolytic viruses provide many convincing arguments for clinical implementation. They act as in situ vaccines modified with immunomodulatory transgenes which can be used in conjunction with other therapies. Over time, the volume of virus dosage increases due to prolonged viral proliferation, a sharp contrast to classical drug pharmacokinetics. Viral proliferation (upon intratumoral administration) also remains confined to neoplastic tissue, with minimal systemic toxicity. Moreover, genetic engineering can reduce pathogenicity and increase safety by altering viral tropism (its effect on cellular pathways). Lastly, there’s a large list of proposed viral candidates other than Zika virus such as: measles virus, poliovirus, poxvirus, adenovirus, and importantly, herpes simplex virus (HSV-1).[6]

The significance of HSV-1 is because a modified version of it (T-VEC) was recently approved in the United States by the FDA for regulatory use in the treatment of advanced melanoma. This attenuated version of HSV-1 contains significant deletions and inclusions. The GM-CSF gene mentioned beforehand makes the cut, while the ICP34.5 and ICP47 genes were deemed too dangerous to include (see Figure 3). Deletion of the latter set of genes not only was found to eliminate the neuropathic nature of HSV-1, but also the promotion of antigen presentation. However, despite positive results in a phase III clinical trial, the treatment has not been adopted as quickly as anticipated by the oncology community due to biosafety concerns and the emergence of other melanoma-specific therapies.[7]

The potential for Zika virus to be used in the treatment of glioblastoma (a form of brain cancer pictured in Figure 5) is perhaps a more exciting prospect than that of T-VEC. While stage 1A/B melanoma (its most diagnosed form) has a ten year survival rate no less than 86%,[8] only about 2% of patients with glioblastoma survive three years, with a median survival rate of less than one year.[9] After surgical removal, glioblastoma often returns near the area of resection, and its localization in the brain makes the cancer difficult to treat. So, why is Zika virus the prime candidate to treat this lethal form of cancer?

An important link with regards to neurodevelopment can be made between microcephaly and the nature of glioblastoma. The tumor is the result of the accumulation of glioblasts (which are partially differentiated cells ideally destined to form glial cells) as well as the stem cells that produce them (stem cells are undifferentiated and can potentially become anything). With the knowledge that microcephaly in newly-born infants is due to virally-compromised neurodevelopment, it can be inferred that Zika’s preferential target in the fetus is that of these partially differentiated cells (glioblasts) and of undifferentiated, progenitor neurons (the neural stem cells). Zika’s documented propensity to attack neural stem cells makes it an oncolytic agent of interest in targeting glioblastoma stem cells (GSCs).[10] Researchers found that Zika virus preferentially infected and killed GSCs, while differentiated (regular) glioma cells remained resilient to viral infection, as did normal neural tissue. While this may seem fine, a common property of glioma cells is an inability to commit to differentiation. One study found that few GSCs were found to differentiate in response to growth factors (BMPs),  while even those cells that did often re-entered the cell-cycle. Whether Zika infection can affect these re-undifferentiated cells remains to be seen due to their preserved DNA methylation patterns. However, current researchers believe the lack of commitment to be the product of excessive SOX2 expression; it can be inferred that SOX2 expression is therefore a marker for GSC activity.[11]  

With this in mind, researchers documented the potency of Zika virus as an oncolytic virus by monitoring concentrations of SOX2, a transcription factor which positively regulates the expression of additional proteins responsible for maintaining pluripotency (which translates to the lack of commitment observed). Researchers found in a GSCs culture that the effects of Zika virus on cell number were associated with the reductions in SOX2 expression. This remained consistent across human glioblastoma organoid models (a simplified, three-dimensional “organ” grown to mimic cellular heterogeneity) as well mouse models. Notably, the survival time of tumor-bearing mice infected with Zika virus proved to be greater than those left untreated. Additionally, localization of caspase-3 (an enzyme which ultimately triggers the apoptotic pathway) two weeks post-treatment confirmed that infection indirectly induced programmed cell death in differentiated cells, though Zika virus itself did not infect those differentiated tumor cells and trigger necrosis.[12]  

Figure 4: An overview of the immunological response to an oncolytic virus, adapted from http://cancerimmunolres.aacrjournals.org/content/canimm/2/4/295/F1.large.jpg

The jury may still be out as to how Zika virus is able to preferentially target GCSs. However, these in vitro human models and in vivo mouse models show promise towards developing a novel therapy. Given the lack of glioblastoma treatments, a clinical trial may be in the works. Perhaps in the near future, oncolytic viruses will finally make their deserved entrance into the world of clinical oncology.

 

References:

[1] WHO Director-General summarizes the outcome of the Emergency Committee regarding clusters of microcephaly and Gullian-Barré syndrome. (2016). http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-microencephaly/en

[2] Zika Virus. (2017, August 28). Retrieved September 17, 2017, from https://www.cdc.gov/zika/about/overview.html

[3] Thomson, B. J. (2008). Viruses and apoptosis. International Journal of Experimental Pathology, 82(2), 65-76. doi:10.1111/j.1365-2613.2011.iep195.x

[4] Strong, J.E., Coffey, M.C., Tang, D., Sabinin, P., & Lee, P.W. (1998). The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus. The EMBO Journal, 17(12), 3351-3351. doi:10.1093/emboj/17.12.3351

[5] Kaufman, H.L., Kohlhapp, F.J., & Zloza, A. (2015). Oncolytic viruses: a new class of immunotherapy drugs. Nature, 14, 642-662. doi:10.1038/nrd4663

[6] Chiocca, A.E., & Rabkin, S.D. (2014). Oncolytic Viruses and Their Application to Cancer Immunotherapy. Cancer Immunology Research, 2(7), 699-699. doi:10.1158/2326-6066. CIR-14-0015

[7] Rehman, H., Silk, A., Kane, M., & Kaufman, H. (2016). Into the clinic: Talimogene laherparepvec (T-VEC), a first-in-class intratumoral oncolytic viral therapy. Journal for ImmunoTherapy of Cancer, 4(53).

[8] Survival Rates for Melanoma Skin Cancer, by Stage. (2016, May 20). Retrieved September 23,, 2017, from https://www.cancer.org/cancer/melanoma-skin-cancer/detection-diagnosis-staging/survival-rates-for-melanoma-skin-cancer-by-stage.html

[9] Walid, M. (2008). Prognostic Factors for Long-Term Survival after Glioblastoma. The Permanente Journal, 12(4). doi:10.7812/tpp/08-027

[10] Hughes, B.W., Addanki, K.C., Sriskanda, A.N., Mclean, E., & Bagasra, O. (2016). Infectivity of Immature Neurons to Zika Virus: A Link to Congenital Zika Syndrome. EBioMedicine, 10, 65-70. doi:10.1016/j.ebiom.2016.06.026

[11] Carén, H., Stricker, S., Bulstrode, H., Gagrica, S., Johnstone, E., Bartlett, T., …Pollard, S. (2015). Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest. Stem Cell Reports, 5(5), 829-842. doi:10.1016/j.stemcr.2015.09.014

[12] Zhu, Z., Gorman, M., McKenzie, L., Chai, J., Hubert, C., Pager, B., …Chheda, M (2017). Zika virus has oncolytic activity against glioblastoma stem cells. The Journal of Experimental Medicine, 214(9). doi:10.1084/jem.20171093

 

 

Posted by Sebastian Mendez

Sebastian is a pre-medical student pursuing a Neuroscience major and Psychology minor at JHU. He is a member of HMR’s marketing team. He is a research assistant at the Bakker Memory Laboratory, assisting in the identification of non-invasive in vivo biomarkers for Alzheimer’s. In addition, Sebastian volunteers for Thread, providing socio-academic support to select public school students. He is also a violinist with the Hopkins Concert Orchestra.

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