Team:SYSU-Medicine/Description
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DESCRIPTION
Background
“Cancer is the second leading cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer.”
——WHO
Cancer and
Therapies
M1
Needless to say, there are tons of data on the importance and dangers of cancer, but it was only when we came to the hospital to practice that we really realized how prevailing and frightening this disease is. We have witnessed with our own eyes 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 moved us deeply, and led us to think deeply ——how can we actually relieve their pain? What efforts can we make as a group of medical students?
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 [2].
Compared with other oncolytic virus, the most significant advantage of M1 is that it is highly safe and can be injected intravenously. Getah virus is transmitted mainly among horses and pigs, and it has not been linked to human illness [3].
At the same time, studies have shown that M1 has a broad anti-cancer
Cancer and Therapies
Needless to say, there are tons of data on
the importance and
dangers of cancer, but it was only
when we came to the hospital to practice that we really realized how prevailing and frightening
this
disease is. 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 moved us deeply, and made us ponder -- how can we actually relieve their pain? What efforts can we make as a group of medical students?
Nowadays, 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
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. Getah virus is transmitted mainly among horses and pigs, and it has not been linked to human illness [2]. [See more in our Safety part]
At the same time, studies have shown that M1 has a broad anti-cancer spectrum. It can selectively infect and kill 13 kinds of refractory high-incidence malignant tumor cells through triple mechanism of direct oncolytic, peripheral cell killing and immune activation, but has no killing effect on normal cells[3].
To sum up, oncolytic virus M1 has the advantages of broad anti-tumor spectrum, safety, effectiveness, precision treatment, etc. It has a good potential for cancer treatment and could serve as a good foundation and platform for further synthetic biology research.
Mechanism
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【Therapies】
During the past two decades, the paradigm for cancer treatment has evolved from relatively non-specific cytotoxic agents to selective, mechanism-based therapeutics.
Cancer chemotherapies were initially identified through screens for compounds that killed rapidly dividing cells. Take actinomycin as an example, antibiotics of actinomycin series are classic drugs for tumor chemotherapy. This application is based on the ability of actinomycins to form specific stable complexes with DNA, leading to inhibition of RNA polymerase reaction and resulting in suppression of protein synthesis and cell division [4]. These drugs remain a backbone of current treatment, but are limited by a narrow therapeutic index, significant toxicities, and frequently acquired resistance.
More recently, an improved understanding of cancer pathogenesis has given rise to new treatment options, including targeted agents and cancer immunotherapy. Targeted approaches aim to inhibit molecular pathways that are critical to tumor growth and maintenance, whereas immunotherapy endeavors to stimulate a host response that effectuates long-lived tumor destruction [5].
The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumor immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients [6].
Figure 1.Fukuhara, H., Y. Ino and T. Todo, Oncolytic virus therapy: A new era of cancer treatment at dawn. Cancer Sci, 2016. 107(10): p. 1373-1379.
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【Oncolytic Virus】
Oncolytic virus (OV) immunotherapy is a therapeutic approach to cancer treatment that utilizes native or genetically modified viruses that selectively replicate within tumor cells. OVs mediate antitumor activity through two distinct mechanisms of action: selective replication within neoplastic cells, resulting in a direct lytic effect on tumor cells and induction of systemic antitumor immunity [7].
The tumor selectivity of OVs is largely conferred by tumor-specific aberrations in signaling pathways that normally sense and block viral replication [8]. It is now well established that cancer-specific aberrations in RAS, TP53, RB1, PTEN, genes encoding proteins involved in the WNT signaling pathway and other cancer-related genes can predispose cancer cells to virus infection [9]. This is due to crosstalk between oncogenic signaling and antiviral pathways, which creates a permissive environment for viral replication.
The immune response to oncolytic viruses appears to be a more important component of the antitumor effect. In nature, viruses have evolved multiple strategies to overcome immune attack, enabling OVs to promote an immune response against the tumor cells by allowing tumor antigen presentation in the context of an active viral infection.
Figure 2.Fukuhara, H., Y. Ino and T. Todo, Oncolytic virus therapy: A new era of cancer treatment at dawn. Cancer Sci, 2016. 107(10): p. 1373-1379.
Figure 3.Fukuhara, H., Y. Ino and T. Todo, Oncolytic virus therapy: A new era of cancer treatment at dawn. Cancer Sci, 2016. 107(10): p. 1373-1379.
As we gain a more complete understanding of the molecular interplay between viruses and our immune systems, it becomes increasingly possible to design therapeutic strategies that turn viruses from stealth pathogens to finely tuned ‘viroceuticals’ that initiate both viral-mediated and immune-mediated attacks against cancers [10].
The effectiveness of OVs has been demonstrated in many preclinical studies and recently in humans, with US Food and Drug Administration approval of the oncolytic herpesvirus Talimogene laherparepvec (T-Vec) in advanced melanoma, a breakthrough for the field [11]. Moreover, some of the cancer-targeted multimechanistic oncolytic viruses have been proven to be well-tolerated in clinical trials, with patients exhibiting only mild flu-like symptoms, offering great potential for increasing efficacy while eliminating the side effects [12]. Thus, the OV approach to cancer therapy is becoming more interesting for scientists, clinicians, and the public.
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【M1】
As mentioned above, M1 virus was first isolated from culicine mosquitoes in Baoting Li and Miao autonomous county, Hainan island in 1964. Phylogenetic analysis showed that M-1 grouped with GETV first and then grouped together with SAGV. The genome of M1 is single-plus RNA, with a total length of 11,696 nt, encoding 4 non-structural proteins (NS1, NS2, NS3 and RdRP) and 5 structural proteins (E1, E2, E3, C and 6K) [13].
Compared with the oncolytic virus (including modified poxvirus, herpes virus, measles virus, Newcastle disease virus, etc.) currently in clinical trials and the approved H101 and T-Vec viruses, the most significant advantage of M1 is that it is highly safe and can be injected intravenously. Getah virus, which is highly similar to M1 virus, mainly infects horses and pigs, causing mild symptoms such as fever, rash, edema of hind limbs and enlarged lymph nodes, and generally recovered within 1 week, presenting a benign course [14]. Although Getah virus is widely distributed in southeast Asia, Australia, Japan, Mongolia, Russia and China, no human diseases caused by Getah virus have been reported so far. Domestic and foreign epidemiological studies have shown that Getah virus antibody can be detected in normal people, which explains the advantages of a virus with lower risk of disease from another perspective [15].
Figure 4.Taguchi, S., et al., Current status of clinical trials assessing oncolytic virus therapy for urological cancers. International Journal of Urology, 2017. 24(5): p. 342-351.
At the same time, studies have shown that oncolytic virus M1 has a broad anti-cancer spectrum. It can selectively infect and kill 13 kinds of refractory high-incidence malignant tumor cells through triple mechanism of direct oncolytic, peripheral cell killing and immune activation, but has no killing effect on normal cells. Studies have shown [16-18] that 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. These three proteins play an antiviral role in the intracellular life history of M1: ZAP binds and degrades viral RNA; TC10 inhibits virus transport; IRE1α removes viral proteins by activating autophagy. Their low expression in tumors allows M1 virus to selectively replicate in large numbers of tumor cells and play a role in its oncolytic effect.
Figure 5. Y. Lin, H. Zhang, J. Lianget al. Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers. Proceedings of the National Academy of Sciences of the United States of America，2014;111（42）：E4504-E4512.
Overall animal experiments showed that M1 injected by caudal vein and intratumoral injection could significantly accumulate in tumor tissues and inhibit tumor growth, while normal organs were not affected. In addition to cell and animal experiments, the oncolytic effect of oncolytic virus M1 ex vivo was further confirmed [16]
Despite all those advantages of M1, however, results from normal and tumor cell models, immunodeficient and immunohealthy tumor-bearing mice models, and ex vivo clinical tumor culture models have revealed its drawbacks. The sensitivity rate of the cell model was 48% (32/67 tumor cells) and that of the ex vivo model was 36% (17/47 tumor samples) [3]. Obviously, the existing oncolytic virus M1 still has the necessary and development space to enhance the anti-tumor spectrum and anti-cancer activity.
Figure 6. Sensitivity of tumor cells and in vitro living tissue to oncolytic virus M1. A) of the 67 tumor cells, 32 were sensitive to M1, with A sensitivity rate of 48%. B) among the 47 clinical tumor specimens, 17 were sensitive to M1, with a sensitivity rate of 36%. Sensitive cells or cases are shown in red. [3]
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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 [18]. Research shows that using inhibitors to reduce those protein levels can to some extent improve the curative effect of oncolytic viruses in several tumors, which may be a clue to further research.
However, since the underlying genesis and development mechanism of tumor itself is still far from being thoroughly studied, it is unrealistic to increase M1 infection rate by studying the pathway of tumor cells one by one. 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. In other words, could we find a pathway more upstream than ZAP, TC10 and IRE1α, that is common to all tumor cells, to be utilized by M1?
Figure 7. Diagram of the growth signaling circuitry of the mammalian cell, which may serve as potential anti-tumor targets [20].
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 [19]. 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 [20].
Figure 8. Sites for inhibition of nucleic acid synthesis by antibiotics and drugs. Waring, M. J. (1981). DNA Modification and Cancer. Annual Review of Biochemistry, 50(1), 159–192.
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 [21]. Also, the yield of SFV could be increased at least 50% in the cells pretreated with actinomycin D [20]. 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) [21-23].
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.
Problem Definition
【Shortages of Current Cancer Therapies】
【limitations of chemotherapy drugs】
【Low Sensitivity of M1】
【Lack of Effective Regulation】
Shortages of Current Cancer Therapies
Current cancer treatments are either limited in their effectiveness or have large side effects.
limitations of chemotherapy drugs
Chemotherapy drugs remain a backbone of current treatment, but are limited by a narrow therapeutic
index, significant toxicities, and frequently acquired resistance.
Low Sensitivity of M1
Oncolytic virus M1 may be a viable solution for its high targeting ability and broad anticancer
spectrum, however, it does not have a significant oncolytic effect on all subtypes of tumor cells.
Lack of Effective Regulation
Although M1 virus is safe to normal cells and not pathogenic to humans, as an RNA virus with a high
mutation rate, further application of M1 virus requires it to be more controllable if its potential
as a tool of synthetic biology is to be fully realized.
Approach
Enzyme Prodrug System
Riboswitch
Enzyme Prodrug System
Our project is to engineer M1 to specifically encode and express enzymes that can convert
prodrugs
into active therapeutic metabolites, and enhance bystander effects.
Riboswitch
We also utilize riboswitch to enhance the dynamic regulation of M1. It comprises a ribozyme actuator
and RNA aptamer sensor and is engineered into the genome and subgenome 5’UTR.
Future Perspectives
Ideal M1-Prodrug Enzyme System
Drug Screening
Ideal M1-Prodrug Enzyme System
As described in the experimental results, we first verified the significant effect of prodrug enzyme
system CB1954/NTR and M1 combination on the anti-tumor activity. Furthermore, we verified that the
chemotherapy drug actinomycin D can in turn promote M1 infection on a variety of tumor cells.
Therefore, if we can combine the advantages of the two, this would be an ideal M1 prodrug enzyme
system. To this end, we designed the prodrug structure of actinomycin D (see design for details) and
screened a variety of drugs with similar mechanism of action to actinomycin D. We plan to carry out
further experimental verification soon.
Drug Screening
In our project, we proved that the efficacy of M1-directed nitroreductase system in combination with
CB1954, which made it possible to use M1 vector to combine with other chemotherapy drugs. Previous
studies have shown that there are many side effects of chemotherapy, such as myelosuppression,
gastrointestinal reactions, liver function impairment etc. So it is necessary to improve the target
of these drugs. Therefore, we plan to screen for more effective chemotherapy drugs, such as
Etoposide, Daunorubicin etc., to combine with M1-directed enzyme prodrug system.
Directed Evolution
Clinical Application
Directed Evolution
In the experiment, we observed that the expression level of nitroreductase in the cell itself would
affect the transformation of prodrug, and at the same time, actinomycin D played a role in promoting
M1 virus infection. It is reasonable to speculate that this feature can be used to exert selective
pressure on the evolution of M1-prodrug enzyme system, by administrating drugs to tumor cells with
low expression level of nitroreductase for targeted evolutionary screening of M1. In our previous
study, a laboratory member has already successfully obtained virus strains with directed evolution
using similar strategies.
Clinical Application
Virus-directed enzyme prodrug therapy is one of the major strategy of the targeted delivery of
cytotoxic compounds to tumor tissue, increasing cytotoxicity of bioreductive agents [24]. The vitro
experiment of the prodrug system has demonstrated the effectiveness of the combined therapy. The
5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) is a mature anticancer prodrug that is efficiently
reduced by nitroreductase, requiring reduction to the 2-and 4-hydroxylamines to be cytotoxic CB1954.
M1 virus has good target and safety, which can achieve the targeting of CB1954. With the merit of
increasing effective killing of tumor cells and being less damage to the surrounding normal tissues,
this combined therapy is possible to be applied to medical and clinical treatment in the future.
Although M1-directed nitroreductase therapy have shown effective bystander effect of CB1954 in
vitro, the study to prove this system in vivo has not been done. Therefore, more researches are
needed to improve this therapy.
REFERENCE
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[14] Li, Y.Y., et al., From discovery to spread: The evolution and phylogeny of Getah virus. Infect Genet Evol, 2017. 55: p. 48-55.
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[18] 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）
[19] Grimley, P.M., et al., Specific membranous structures associated with the replication of group A arboviruses. J Virol, 1972. 10(3): p. 492-503.
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[22] 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.
[23] 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.
[24] Szewczuk, M., et al., Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives. Tumour Biol, 2017. 39(7): p. 1010428317713675.