Team:SYSU-Medicine/Inspiration
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INSPIRATION
Nowadays, there are tons of data on the importance and dangers of cancer. We have witnessed the suffering of our patients, the incessant coughing of patients with lung cancer, the excruciating appearance of patients with stomach cancer, and the huge abdomen filled with ascites of patients with liver cancer. All this inspired us to look for a treatment that combines the strengths of the current approaches.
Previous studies have shown that targeted and immune-based therapies have already transformed the standard-of care for several malignancies. Oncolytic virus, as an attractive therapeutic combination of the advantage of tumor-specific cell lysis together with immune stimulation, are emerging as important agents in cancer treatment.
M1 is a strain of Getah-like alphavirus that was isolated from culicine mosquitoes collected on Hainan Island of China which possesses selective and potent antitumor activity through intravenous administration [1]. Compared with other oncolytic virus, the most significant advantage of M1 is that it is highly safe and can be injected intravenously. In addition, M1 virus has high targeting ability and broad anticancer spectrum, however, it does not have a significant oncolytic effect on all subtypes of tumor cells. Thus we further study the mechanism of M1, it is generally believed that the limited application of M1 virus is mainly attributed to pathways involved by ZAP, RHOQ, IRE1α and MXRA8 in insensitive tumor cells, lead to the impairment of invasion and replication of the virus [2]. However, since the underlying genesis and development mechanism of tumor itself is still far from being thoroughly studied, it is more reasonable to find a solution so that the anti-cancer spectrum of M1 can no longer be restricted by the limitations of the pathways in tumor cells.
Figure1：The key molecular mechanisms of oncolytic virus M1 targeting tumor cells include zinc finger antiviral protein ZAP, RHO family protein TC10, and UPR signaling molecule IRE1α.
One of the most remarkable features of tumors is their limitless replicative potential which was acquired during tumor progression and was essential for the development of their malignant growth state [3]. The ability of unlimited proliferation is reflected in the hyperactive transcription process at the cellular level. Therefore, we want to find a means to interfere with tumor cells at the transcriptional level and then enhance the infection rate and lethality of M1.
Surprisingly, many chemotherapy drugs work through this mechanism. Actinomycin D, as a typical representative among actinomycins, has been widely used in clinical practice since 1954 as an anticancer drug for treating many tumors. The two main mechanisms of its anti-tumor ability are intercalation to DNA and the stabilization of cleavable complexes of topoisomerases I and II with DNA, which interferes with cell transcription, but does not affect alphavirus replication or cytopathic vacuoles formation [4].
Figure 2: Targeting sites and binding modes of selected type IIA topoisomerase inhibitors and regulatory proteins. Delgado, J.L., et al., Topoisomerases as anticancer targets. Biochem J, 2018. 475(2): p. 373-398.
As early as 1963, a study suggested that actinomycin D could enhance the growth of Chikungunya virus, which is also an alphavirus, within tumor cells through some cellular mechanism [5]. Also, the yield of SFV could be increased at least 50% in the cells pretreated with actinomycin D [4]. More studies have also confirmed that actinomycin D promotes the replication of alphavirus in tumor cells significantly, for it can help to suppress cellular functions which inhibit production of infectious virus particles. Those possible mechanism may include interferon and cyclopentenone prostaglandin A1 (PGA1) [5-7].
These chemotherapies, which act on the transcription process, could effectively increase the infection rate of M1 against a variety of tumor cells, however, the broad-spectrum effect also causes serious side effects. All this suggests that there might be a feasible way to expand the anti-cancer profile of M1 while reducing the side effects of chemotherapy drugs by combining the therapies together, fostering their strengths and circumventing the weaknesses.
Therefore, we further designed the system to combine M1 virus with chemotherapy drugs for cancer treatment. We engineered M1 with enzyme, and after enzyme was delivered to the cancer cell by M1 and expressed, the enzyme can activate subsequently administered prodrugs of chemicals (such as actinomycin D, CB1954) to potent drugs.
Initial Ideas
Prior to our initial survey, we had many different ideas for potential projects:
| Purpose | Benefits | Limitations | |
| Directed evolution | We constructed the M1 with low fidelity RNA polymerase to increase the probability of mutation caused by adaptation, to expand the anti-cancer spectrum of M1 virus by continuous passage. | Overcome the limitations of the complex regulation mechanism of virus-host interaction. | Mutations are unpredictable; Uncertainty. |
| Engineer M1 with ligands | We inserted ligands of receptors that are highly expressed in some tumors, such as EGF, HER2, etc., which aims to enhance the affinity between M1 and insensitive tumor cells. | Enhance the affinity between M1 and insensitive tumor cells. | The insertion of ligands into the M1 genome may affect the replication of M1. |
| M1-directed enzyme system | We used M1 as a viral vector to deliver the gene of enzyme, after the enzyme is expressed, the ability to specifically activate subsequently administered and otherwise inert prodrugs to potent drugs, which allows for a specific therapy with limited systemic side effects. | Increase the lethality to tumor cells. It can significantly increase the targeting of chemotherapy drugs and reduce its side effects. | The prodrug and M1 virus need to be administered separately. |
| 3D culture | Better simulation of the vivo environment, to improve the affinity of M1 to insensitive cells and enhance its anti- tumor effect. | Reduce the affinity of normal cells and improve the safety of M1; Enhance the oncolysis of M1. | The lack of a standard approach. |
| M1 armed with Cytokines | Encode cytokine such as IL-2、 IL-4 in the M1 vector to induce T cell responses of sufficient magnitude | Enhance adaptive immune responses. | Causes premature elimination of M1; It is limited by the rate of infection. |
| Combination with PD-1 or PD-L1 antibody | Using OVs to heat up ‘cold’ tumors for immune checkpoint blockade, such as PD L1 or PD1, and then increase the antitumor effect. | In a number of other preclinical studies, ICIs have been shown to increase the efficacy of OV therapy involving a variety of viral platforms. | Hard to regulate the concentration of drug administration; This brings substantially increased toxicity to patients. |
Final Project Decision
Though interviewed the professor Yan, and taking into account the innovation and feasibility of each project, we subsequently decide to try the following three projects: 1) Directed evolution; 2) Engineer M1 with ligands; 3) M1-directed enzyme system. However, after we did the pre-experiments of the first two projects, the results can not satisfy our expectation. Thus we decided to the third project to expand the anti-cancer spectrum of M1 virus. We designed to engineer M1 to specifically encode and express enzymes that can convert prodrugs into active therapeutic metabolites and enhance bystander effects of chemicals. As the result have shown, we proved that this project was feasible.
REFERENCE
[1] Hu J, Cai XF, Yan G. Alphavirus M1 induces apoptosis of malignant glioma cells via downregulation and nucleolar translocation of p21WAF1/CIP1 protein. Cell Cycle. 2009;8(20):3328–3339.
[2] K. Li, C. Hu, F. Xinget al. Deficiency of the IRE1α-Autophagy Axis Enhances the Antitumor Effects of the Oncolytic Virus M1. J Virol，2018;92（6）
[3] Grimley, P.M., et al., Specific membranous structures associated with the replication of group A arboviruses. J Virol, 1972. 10(3): p. 492-503.
[4] Douglas Hanahan, Robert A Weinberg, The Hallmarks of Cancer, Cell, Volume 100, Issue 1, 2000, Pages 57-70, ISSN 0092-8674.
[5] GIFFORD, G.E. and E. HELLER, EFFECT OF ACTINOMYCIN D ON INTERFERON PRODUCTION BY 'ACTIVE' AND 'INACTIVE' CHIKUNGUNYA VIRUS IN CHICK CELLS. Nature, 1963. 200: p. 50-1.
[6] Carter, M.F., et al., Effects of actinomycin D and ultraviolet and ionizing radiation on Pichinde virus. J Virol, 1973. 12(1): p. 33-8.
[7] Burlandy, F.M. and M.A. Rebello, Inhibition of Mayaro virus replication by prostaglandin A(1) in Vero cells. Intervirology, 2001. 44(6): p. 344-9.