Robert Silverman, Ph.D.
The Mal and Lea Bank Chair
Lerner Research Institute,
9500 Euclid Avenue, Cleveland, Ohio 44195
Phone: (216) 445-9650
Fax: (216) 445-6269
Our studies probe fundamental immune responses that profoundly impact cancer and viral infections. Interferons (IFNs) are critically important cytokines that control antiviral innate immunity. One the ways IFNs work is through the 2’,5’-oligoadenylate (2-5A) synthetase (OAS)-RNase L system, the focus of our studies. Viruses produce double-stranded (ds) RNA that activates IFN-inducible OAS enzymes to produce 2-5A. The 2-5A then binds to the ubiquitous, but latent, RNase L, causing its dimerization and activation. Once activated, RNase L cleaves viral and cellular RNA. Virus replication is inhibited and cell death occurs by apoptosis. The pro-apoptotic activity of OAS-RNase L has long been thought to contribute to the control of cancer cell proliferation by IFNs. Previously we purified and cloned RNase L and established its antiviral activities in genetically deficient mice. We reported that RNase L activates the NLPR3 inflammasome, contributing to viral mediated inflammation. How precisely viruses evade OAS-RNase L by degrading 2-5A is one current area of study. We are studying viral and host enzymes that degrade 2-5A and can prevent activation of RNase L.
Recently we showed that even uninfected cells sometimes produce dsRNA that triggers the OAS-RNase L pathway. These studies have implications for controlling cancer cell proliferation. We found that dsRNA transcribed from repetitive regions of the genome can activate OAS enzymes. Drugs that cause epigenetic modification of DNA, such as 5-azacytidine (AZA), are used clinically to treat myelodysplastic syndromes and acute myeloid leukemia. In addition, AZA is being investigated for use against a range of different types of solid tumors, including lung cancer. Treatment with AZA causes demethylation of DNA, thus increasing RNA synthesis, including the synthesis of dsRNA, which is otherwise produced in virus-infected cells. We determined that cell death in response to AZA requires the OAS-RNase L pathway. These results identify a drug target for enhancing the anticancer activity and reducing the toxicity of AZA and related drugs. In addition, OAS1 expression correlates with AZA sensitivity in tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. Finally, we reported that deficiencies in the RNA editing enzyme ADAR1 (adenosine deaminase acting on dsRNA 1) that edits and destabilizes dsRNA, activates OAS-RNase L resulting in apoptotic cell death.
In other words ...
In other words…. Dr. Silverman has investigated the role interferons play in protecting the body from viruses and cancer for the past 40 years. Interferons are proteins made and released by host cells in response to the presence of pathogens. Dr. Silverman is particularly interested in how the enzyme system OAS-RNase L, which protects higher vertebrates from both RNA and DNA viruses, contributes to this process. As often happens, however, viruses have adapted and evolved to inhibit OAS-RNase L activity. He continues on in his decades-long quest to better understand how exactly RNase L works on a cellular and molecular level. This knowledge may help researchers prevent harmful viruses from evading the protective effects of RNase L, and therefore reduce viral infections and their often serious negative health effects. Recent studies implicate the OAS-RNase L pathway in the anti-tumor effects of the DNA demethylating drug AZA, providing a more comprehensive understanding about how AZA and dsRNA prevent the proliferation of cancer cells. It also offers important insights about how a host antiviral protein can, under some conditions, function against cancer cells. Engineering and training the body’s cells to use “self” to fight cancer is a growing area of interest among researchers.
The intersection between innate immunity and tumor suppression is the focus of the Silverman Lab. Our studies probe fundamental molecular and cellular processesthat impact microbial infections and cancer. We seek a better understanding of how the mammalian cell resists viral infections and how the virus antagonizes the host response to infection. We are also investigating the role of the RNase L gene in hereditary prostate cancer. In addition, we are developing a combination therapy for late-stage metastatic cancer by combining an oncolytic virus with a drug.
The interferon (IFN) system is a frontline host defense against invading viral pathogens. The 2',5'-oligoadenylate (2-5A)synthetase (OAS)-RNase L pathway is among the most potent IFN-induced antiviral effectors, blocking viral infections by several mechanisms, including directly cleaving viral single-stranded RNA genomes, depleting viral and host mRNA available for translation, and enhancing type I IFN induction. Type I IFNs bind to the cell surface receptor IFNAR initiating JAK-STAT signaling to the OAS genes, which results in elevated levels of OAS proteins. When activated by viral double-stranded (ds)RNA, certain OAS isoforms use ATP to synthesize 5'-triphosphorylated 2-5A. Trimer and longer species of 2-5A bind with high specificity and affinity to the inactive monomeric RNase L, causing it to dimerize and become active.
Antiviral Mechanisms of 2-5A Dependent RNase L:
RNase L is a principal mediator of the innate antiviral response and is thus critically important for human health. Previously, we purified RNaseL(JBC, 1988, PMID: 3366783), cloned RNase L (Cell, 1993, PMID: 7680958), and knocked out the RNase L gene in mice (EMBO J, 1997, PMID: 9351818). We showed that RNA cleavage products of self-RNA produced by RNase L function as PAMPs in amplifying innate immune signaling (Nature, 2007, PMID: 17653195). In a continuation of these studies, we are investigating how RNase L amplifies production of antiviral cytokines and how it eliminates virus particles through a process known as autophagy (Journal of Virology, 2012, PMID: 22875977). Our long-term objectives are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles, spread and pathogenesis. The knowledge to be gained from the proposed studies could contribute to improved treatments for viral infections and cancer.
Control of Viral Pathogenesis by Regulation of 2-5A Levels:
(a collaborative project with Susan Weiss, University of Pennsylvania)
The ability of viruses to evade the IFN antiviral response plays an important role in viral tropism and disease pathogenesis. Relevant to this project, the murine coronavirus ns2 protein has 2’,5’-phosphodiesterase (PDE) activity that antagonizes the IFN regulated OAS-RNase L antiviral pathway by eliminating 2-5A thereby promoting hepatitis; several other viruses and host cells have proteins with similar activities (Cell Host & Microbe, 2012, PMID: 22704621). Recently, with the teams of Susan Weiss and John Patton (NIAID, NIH), we reported group A rotavirus, an important cause of acute gastroenteritis in children worldwide, encodes a similar 2’,5’-PDE (PNAS, 2013, PMID: 23878220). The discovery and use of inhibitors of viral and cellular PDEs to enhance RNase L activity selectively in virus-infected cells potentially provides a novel mode of regulation of the antiviral response, with broad implication for the control of viral infections.
Role of RNase L in Prostate Cancer:
Prostate cancer is the leading cause of non-cutaneous malignancies and the second leading cause of cancer-related deaths among American men. Hereditary prostate cancer (HPC), which accounts for 43% of early onset cases and about 9% of all cases, is due to germline mutations in HPC genes. In 2002, we co-authored with the group of Jeffrey Trent and others a study a report that the HPC1 locus maps to the RNase L gene (Nature Genetics, 2002, PMID: 11799394). We are currently investigating the potential role of RNase L as a suppressor of prostate cancer by focusing on basic cellular processes regulated by RNase L including autophagy and apoptosis.
Combination OncolyticVirotherapy for Late-Stage Cancer:
The use of lytic viruses to preferentially infect and eliminate cancer cells while sparing normal cells is a promising experimental therapeutic approach for treating cancer. However, the efficacy of oncolyticvirotherapy is often limited by two innate immunity pathways, the protein kinase PKR and the 2’,5’-OAS/RNase L systems, which are widely present in many but not all tumor cell types. We reported that the anti-cancer drug, sunitinib, an inhibitor of VEGF-R and PDGF-R, has off-target effects against both PKR and RNase L(JBC, 2012, PMID: 21636578). Recently, we showed that combining sunitinib treatments with infection by an oncolytic virus, vesicular stomatitis virus, led to the elimination of prostate, breast and kidney malignant tumors in mice (Molecular Therapy, 2013, PMID: 23732991). In contrast, either virus or sunitinib alone slowed tumor progression but did not eliminate tumors. Results indicate that transient inhibition of innate immunity with sunitinib enhances oncolyticvirotherapy allowing the recovery of tumor-bearing animals. We are currently extending these findings to other oncolytic viruses.
Selected from 249 publications:
Silverman, R.H. and Atherly, A.G. The search for ppGpp and other unusual highly phosphorylated nucleotides in eukaryotes. Microbiological Reviews 43, 27-41, 1979.
Silverman, R.H., Cayley, P.J., Knight, M., Gilbert, C.S., and Kerr, I.M. Control of the ppp(A2'p)nA system in HeLa cells: effects of interferon and virus infection. Eur. J. Biochem. 124, 131-138, 1982.
Silverman, R.H., Watling, D., Balkwill, F.R., Trowsdale, J., and Kerr, I.M. The ppp(A2'p)nA and protein kinase systems in wild type and interferon-resistant Daudi cells. Eur. J. Biochem. 126, 333-341, 1982.
Silverman, R.H., Skehel, J.J., James, T.C., Wreschner, D.H., and Kerr, I.M. Ribosomal RNA cleavage as an index of ppp(A2'p)nA activity in interferon-treated encephalomyocarditis virus-infected cells. J. Virol. 46, 1051-1055, 1983.
Silverman, R.H., Jung, D.D., Nolan-Sorden, N.L., Dieffenbach, C.W., Kedar, V.P., and SenGupta, D. Purification and analysis of murine 2-5A-dependent RNase. J. Biol. Chem. 263, 7336-7341, 1988.
Zhou, A., Hassel, B.A., and Silverman, R.H. Expression cloning of 2-5A-dependent RNase-a uniquely regulated mediator of interferon action. Cell, 72, 753-765, 1993.
Hassel, B.A., Zhou, A., Sotomayor, C., Maran, A., and Silverman, R.H. A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J., 12, 3297-3304, 1993.
Zhou, A., Paranjape, J., Brown, T.L., Nie, H., Naik, S., Dong, B., Chang, A., Trapp, B. Fairchild, R., Colmenares, C., and Silverman, R.H. Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate dependent RNase L. EMBO J. 16, 6355-6363,1997.
Der, S., Zhou, A., Williams, B.R.G., and Silverman, R.H.Identification of Genes Differentially Regulated by IFN-α β or γ or Using Oligonucleotide Arrays.Proc. Natl. Acad. Sci. U.S.A 95,15623-15628, 1998.
Zhou, A., Paranjape, J.M., Der, S.D., and Williams, B.R.G. and Silverman, R.H. Novel Innate Mechanisms of Interferon Action are Revealed in Triply Deficient Mice. Virology, 258, 435-440, 1999.
Carpten, J., N., et al. Germline Mutations in the Ribonuclease L (RNase L) Gene in Hereditary Prostate Cancer 1 (HPC1) -Linked Families. Nature Genetics, 30, 181-184, 2002.
Casey, G., et al.RNASEL R462Q variant is implicated in 13% of prostate cancer cases. Nature Genetics, 32, 581-583, 2002. Silverman, R.H. Implications for RNase L in Prostate Cancer Biology.Biochemistry, 42, 1805-1812, 2003.
Xiang, Y., Wang,Z., Murakami, J., Plummer, S., Klein, E.A., Carpten, J., Trent, J., Isaacs W., Casey, G., and Silverman, R. H. Effects of RNase L mutations associated with prostate cancer on apoptosis induced by 2',5'-oligoadenylates. Cancer Res. 63: 6795-6801, 2003.
Malathi, K., Paranjape, J.M., Bulanova, E., Shim, M., Guenther-Johnson, J.M., Faber, P.W., Eling, T.E., Williams, B.R.G., and Silverman, R.H. A novel transcriptional signaling pathway in the interferon system mediated by 2'-5'-oligoadenylate activation of RNase L. Proc. Natl. Acad. Sci. U.S.A., 102, 14533-14538, 2005.
Malathi, K., Dong, B., Gale, M., and Silverman, R.H. Small Self RNA Generated by RNase L Amplifies Antiviral Innate Immunity. Nature, 448: 816-819, 2007.
Malathi, K., Saito, T., Crochet, N., Barton, D.J.,Gale, M.and Silverman, R.H. RNase L Releases a Small RNA from HCV RNA that Refolds into a Potent PAMP. RNA, 16: 2108-2119, 2010.
Jha, B.K., Polyakova, I., Kessler, P., Dong, B., Dickerman, B., Sen, G.C., and Silverman, R.H. Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity. J. Biol. Chem.,286: 26319-26326. 2011.
Zhao, L., Jha, B., Wu, A., Elliott, R., Ziebuhr, J., Gorbalenya, A.E., Silverman, R.H., Weiss, S.R. 2012. Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology. Cell Host and Microbe, 11:607-616, 2012.
Chakrabarti, A., Ghosh, P.K., Banerjee, S., Gaughan, C., and Silverman, R.H. RNase L triggers autophagy in response to viral infections. J. Virol. 86: 11311-21, 2012.
Jha, B.K., Dong, B., Nguyen, C.T., Polyakova, I. and Silverman, R.H. Suppression of antiviral innate immunity by sunitinib enhances oncolytic virotherapy. Molecular Therapy, 21:1749-57, 2013.
Zhao, L., Birdwell, D. Wu, A., Elliott, R., Rose, K., Phillips, J., Li, Y., Grinspan, J., Silverman, R., and Weiss, S. Cell type specific activation of the OAS-RNase L pathway by a murine coronavirus. J. Virol., 87:8408-18, 2013. [Featured article in "JVI Spotlight"]
Sorgeloos, F., Jha, B.K., Silverman, R.H., and Michiels, T.Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L. PLoS Pathogens, 9(6):e1003474, 2013.
Zhang, R., Jha, B.K., Ogden, K., Dong, B., Zhao, L., Elliot, R., Patton, J.T., Silverman, R.H.* and Weiss, S.R.* Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc. Natl. Acad. Sci., 110:13114-9, 2013. [*Co-corresponding authors].
Zhou, Y., Kang, M.J., Silverman, R.H., Lee, C.G., and Elias, J.A. Role of RNase L in viral pathogen-associated molecular pattern/influenza virus and cigarette smoke-induced remodeling. J. Immunol., 191:2637-46, 2013.
Zhang, A., Dong, B., Doucet, A.J., Moldovan, J.B., Moran, J.V., and Silverman, R.H. RNase L restricts the mobility of engineered retrotransposons in cultured human cells. Nucleic Acids Research, 42: 3803-20, 2013.
Huang, H., Zeqiraj, E., Dong, B., Jha, B.K., Duffy, N., Orlicky, S., Thevakumaran, M., Pillon, M.C., Ceccarelli, D.F., Wan, L., Juang, Y.C., Mao, D.Y.L., Gaughan, C., Brinton, M.A., Perelygin, A.A., Kourinov, I., Guarne, A., Silverman, R.H.*, Sicheri,F.* Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity. Molecular Cell 53(2):221-34, 2014. [*Co-corresponding authors] [highlighted as an Editor’s Select commentary in Cell, 2014, “The Fantastic RNase L”]
Silverman, R.H. and Weiss, S.R. Viral phosphodiesterases that antagonize double strand RNA signaling to RNase L by degrading 2-5A. J. Interferon & Cytokine Res., 34(6): 455-463, 2014., 2014.
Cooper, D.A., Jha, B.K., Silverman, R.H., Hesselberth, J.R., and Barton, D.J. Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. Nucleic Acids Res., 42:5202-16, 2014.
Banerjee, S., Chakrabarti, A., Jha, B.K., Weiss, S.R., and Silverman, R.H. Cell type-specific effects of RNase L on viral induction of IFN-. mBio, 5(2): e00856-14, 2014.
Gusho, E., Zhang, R., Jha, B.K., Thornbrough, J.M., Dong, B., Gaughan, C., Elliott, R., Weiss, S.R. and Silverman, R.H. Murine AKAP7 has a 2’,5’-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene. mBio,34:455-63, 2014.
Franchi, L., Eigenbrod, T., Munoz-Planillo, Ozkurede, U., Kim, Y.G., Chakarbarti, A., Gale, M., Silverman, R.H., Colonna, M., Akira, S., Nunez, G. Cytosolic double-stranded RNA activates the NLRP3 inflammasome via MAVS-induced membrane permeabilization and K+ efflux. J. Immunol. 193, 4214-4222. 2014.
Cooper, D., Banerjee, S., Chakrabarti, A., Garcia-Sastre, A., Hesselberth, J., Silverman, R.H., Barton, D.J. Ribonuclease L targets distinct sites in influenza A virus RNAs. J. Virol. 89, 2764-2776, 2015.
Chakrabarti, A., Banerjee, S., Franchi, L., Loo, Y.M., Gale, M., Nunez, G., Silverman, R.H. RNase L activates the NLRP3 inflammasome during viral infections. Cell Host & Microbe, 17: 466-477, 2015 [On the Cover].
Silverman, R.H. Caps off to poxviruses. Cell Host & Microbe, 17: 2878-2879, 2015.
Ogden, K.M., Hu, L., Jha, B.K., Sankaran, B., Weiss, S.R., Silverman, R.H., Patton, J.T., and Prasad, B.V.V. Structural basis for 2¢-5¢-oligoadenylate binding and enzyme activity of a viral RNase L antagonist. J. Virol., 89: 6633-6645, 2015.
Banerjee, S., Li, G., Li, Y., Gaughan, C., Baskar, D., Parker, Y., Lindner, D.J., Weiss, S.R., and Silverman, R.H. RNase L is a negative regulator of cell migration. Oncotarget, 6: 44360-44372, 2015.
Birdwell, L.D., Zalinger, Z.B., Li, Y., Wright, P.W., Elliott, R., Rose, K.M., Silverman, R.H., Weiss,. S.R. Activation of RNase L by murine coronavirus in myeloid cells is dependent on basal Oas gene expression and independent of virus-induced interferon. J. Virology, 90:3160-72, 2016.
Sui, B., Huang, J., Jha, B.K., Yin, P., Zhou, M., Fu, Z.F., Silverman, R.H., Weiss, S.R., Peng, G., and Zhao, L. Crystal structure of the mouse hepatitis virus ns2 phosphodiesterase domain that antagonizes RNase L activation. J. gen. Virol., 97:880-886, 2016.
Li, Y., Banerjee, S., Wang, Y., Goldstein, S.A., Dong, B., Gaughan, C. *Silverman, R.H., and *Weiss, S.R. Activation of RNase L is dependent on OAS3 expression during infection with diverse viruses. Proc. Natl. Acad. Sci., 113: 2241-6, 2016. [*Co-corresponding authors]
Thornbrough, J.M., Jha, B.K., Yount, B., Goldstein, S.A., Li, Y., Elliott, R., Sims, A.C., Baric, R.S., Silverman, R.H., and Weiss, S.R. Middle east respiratory syndrome coronavirus NS4b protein inhibits host ribonuclease L activation. mBio, 7(2). pii: e00258-16, 2016.
Goldstein, S.A., Thornbrough, J.M., Zhang, R., Jha, B.K., Li, Y., Elliott, R., Quiroz-Figueroa, Chen, A.I., Silverman, R.H., and Weiss, S.R. Lineage A Betacoronavirus NS2 proteins and homologous Torovirus Berne pp1a- carboxyterminal domain are phosphodiesterases that antagonize activation of RNase L. J. Virol., Feb 14;91(5). pii: e02201-16. doi: 10.1128/JVI.02201-16, 2017.
Kindler, E., Gil-Cruz, C., Spanier, J., Li, Y., Wilhelm, J., Rabouw, H., Zust, R., Hwang, M. V’kovski, P., Stalder, H. Marti, S., Habjan, M., Cervantes-Barragan, L., Elliot, R., Karl, M., Gaughan, C., van Kuppeveld, F., Silverman, R.H., Keller, M., Ludewig, B., Bergmann, C., Ziebuhr, Weiss, S.R., Kalinke, U., Thiel, V. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathogens Feb 3;13(2):e1006195. doi: 10.1371/journal.ppat.1006195, 2017.
Li, Y., Banerjee, S., Goldstein, S., Dong, B., Gaughan, C., Rath, S., Donovan, J., Korennykh, A., Silverman, R.H.*, Weiss, S.R.* Ribonuclease L mediates the cell-lethal phenotype of the double-stranded RNA editing enzyme ADAR1 in a human cell line. eLife, Elife. Mar 31;6. pii: e25687. doi: 10.7554/eLife.25687, 2017. [*Co-corresponding authors].
Drappier, M., Jha, B.K., Stone, S., Elliott, R., Zhang, R., Vertommen, D., Weiss, S.R., Silverman, R.H., Michiels, T. A novel mechanism of RNase L inhibition: Theiler’s virus L* protein prevents 2-5A from binding to RNase L. PLoS Pathogens, 14(4):e1006989, 2018.
Tang, W., Wallace T.A., Yi, M., Magi-Galluzzi, C., Dorsey, T.H., Onabajo, O.O., Obajemu, A., Jordan, S.V., Loffredo, C.A., Stephens, R.M., Silverman, R.H., Stark, G.R., Klein, E.A., Prokunina-Olsson, L., Ambs, S. IFNL4-ΔG allele is associated with an interferon signature in tumors and survival of African-American men with prostate cancer. Clin Cancer Res. 24: 5471-5481, 2018.
Banerjee, S., Gusho, E., Gaughan, Dong, B., Gub, X., Holvey-Bates, E., Talukdar, M., Li, Y., Weiss, S.R., Sicheri, F., Saunthararajah, Y., Stark, G.R., Silverman, R.H. The OAS-RNase L innate immune pathway mediates the cytotoxicity of a DNA demethylating drug. Proc. Natl. Acad. Sci., 116:5071-5076, 2019.
A new paper published in Proceedings of the National Academy of Sciences shows for the first time how 5-azacytidine (AZA), a drug commonly prescribed to treat myelodysplastic syndromes and acute myeloid leukemia, causes cancer cell death and identifies several therapeutic targets that may help to amplify the drug’s already powerful disease-fighting effects.
With a recent five-year, $2 million grant renewal from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), Robert Silverman, PhD, staff, Department of Cancer Biology, Lerner Research Institute, has now received 36 years of NIH funding for the same project—a rare and impressive accomplishment.