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Adding to the toolbox: Receptor tyrosine kinases as potential targets in the treatment of hepatitis C
International Hepatology
Adding to the toolbox: Receptor tyrosine kinases as potential targets in the treatment of hepatitis C Nikolaus Jilg, Raymond T. Chung⇑ Gastrointestinal Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA
COMMENTARY ON: EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M, Gorke S, Royer C, Fischer B, Zahid MN, Lavillette D, Fresquet J, Cosset FL, Rothenberg SM, Pietschmann T, Patel AH, Pessaux P, Doffoël M, Raffelsberger W, Poch O, McKeating JA, Brino L, Baumert TF. Nat Med. 2011 May;17(5):589–95. Copyright (2011). Abstract reprinted with permission from MacMillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/21516087 Abstract: Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection. Ó 2011 European Association for the study of the Liver. Published by Elsevier B.V. All rights reserved. The longtime standard therapy for chronic hepatitis C consisting of peginterferon and ribavirin is fraught with numerous absolute and relative contraindications, limited efficacy, and an unfavorable side effect profile. In view of the global burden of the disease, novel therapeutic strategies are therefore urgently needed. Direct antiviral agents (DAA) against several of the viral proteins have been developed. Eagerly awaited by both patients and their doctors, the first two of these drugs (the NS3/4A protease inhibitors
Keywords: HCV; HCV entry; Entry factors; EGFR; Host factors; Receptor tyrosine kinases. Received 1 June 2011; received in revised form 4 June 2011; accepted 6 June 2011 ⇑ Corresponding author. Tel.: +1 617 724 7562; fax: +1 617 643 0446. E-mail address: [email protected] (R.T. Chung).
telaprevir and boceprevir) have recently been approved by the US FDA [1,2]. While representing a significant leap forward in the treatment of hepatitis C, this new class of drugs will likely not be the final solution for all patients and there remains a significant need for additional drugs with different and novel antiviral modes of action. Viral components and proteins represent the most obvious target of antiviral treatment, but direct-acting antiviral therapies in general are limited by their selection of resistant viral variants. Viral life cycles depend on a multitude of host factors, that, not least, contribute to species specificity and are responsible for many of the interindividual differences seen in the susceptibility to and the course of viral diseases [3,4]. Therefore, efforts have recently been directed toward identifying these host factors and blocking their access to viruses and their ability to exploit them. Cyclophilin A, for instance, is a chaperone that is essential for HCV replication and can be specifically inhibited by alisporivir, an oral agent demonstrating promise in ongoing hepatitis C clinical trials [5]. In recent years, novel tools such as high-throughput assays have been developed to comprehensively screen for the relevant host factors in viral diseases [6,7]. In the May issue of Nature Medicine, Lupberger et al. provide conclusive evidence for a crucial role of receptor tyrosine kinases (RTKs) in HCV infection [8]. Furthermore, they open the possibility of clinical application by robustly demonstrating the impact of RTK inhibitors on HCV infection. RTKs play important roles in basic cellular functions such as cell proliferation, differentiation and cell morphology. Sorafenib, an inhibitor of several protein kinases, is an approved drug in the treatment of renal cell carcinoma and hepatocellular carcinoma (HCC) and has recently been shown to be effective against HCV infection in vitro [9]. The authors screened an siRNA library against 691 human kinases and associated proteins in cell culture, and identified 58 host kinases (11.2% of the human kinome) that impair HCV entry. Of these hits, they choose the RTKs epidermal growth factor receptor (EGFR) and the ephrin A2 receptor (EphA2) for further validation. Both RTKs play important roles in basic physiologic functions such as cell proliferation, differentiation, and cell morphology. Both have been shown to be involved in oncogenesis, and several inhibitors of EGFR and EphA2 are currently approved or under investigation for the treatment of malignant diseases, including non-small cell lung cancer and CML. Erlotinib, one of the small molecule inhibitors of EGFR, is being evaluated in clinical trials for HCC [10].
Journal of Hepatology 2012 vol. 56 j 282–284
JOURNAL OF HEPATOLOGY Lupberger et al. confirmed the relevance of the RTKs EGFR and Eph2A for HCV entry by markedly downregulating infection with specific protein kinase inhibitors of EGFR (erlotinib, gefitinib, lapatinib) and EphA2 (dasatinib) in cell lines and primary human hepatocytes. Entry of murine leukemia virus and measles virus, however, was not impaired by the same treatment. The inhibitors do not compromise viral replication in an entry-independent replicon model system, suggesting specificity for the entry process. In addition, the authors show that HCV entry is markedly enhanced by the two EGFR-ligands, EGF and TGF-a. These effects are reversible by treatment with erlotinib or an EGFR antibody [8]. Furthermore, the authors sought to fine-map the mechanism by which EGFR and EphA2 influence HCV entry: silencing the RTKs has no effect on expression levels of the known HCV entry factors SR-B1, CD81, CLDN1, and OCLN. The group elegantly demonstrates that EGFR and EphA2 are not required for initial viral
attachment, but promote the formation of CD81–CLDN1 coreceptor complexes, a prerequisite to HCV entry and are involved in viral glycoprotein-dependent membrane fusion (Fig. 1). Very interestingly, Lupberger et al. are able to verify erlotinib’s inhibitory effect in an in vivo model of hepatitis C: chimeric urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice repopulated with human hepatocytes and infected with serum-derived HCV [11]. After discontinuation of erlotinib, viral loads in the treatment group reached similar levels as in placebo-treated animals. In line with the presented results, the authors further hypothesize that EGFR-ligand binding may activate downstream signaling that is required for HCV entry and that EGFR and EphA2 may be part of the same entry regulatory pathway. Of note, a recent case report describes the rapid clearance of serum HCV RNA in a patient undergoing treatment with erlotinib for recurrent HCC following liver transplantation [12].
Antibodies against host factors (e.g. EGFR, CD81)
Antibodies against viral envelope proteins
Viral binding
RTK ligands (e.g. EGF, Ephrin-A1)
Clathrin mediated endocytosis Dimerization
TIR
Binding TIR TIR
P
GAG
SR-B1 LDLR
CLDN1 OCLN
P TIR Interaction
RTK
TIR
RTK activation
RTKI (e.g. Erlotinib, Dasatinib)
CD81
Downregulation of host factors (RNAi)
+ +
Fig. 1. HCV entry in the hepatocyte – relevant host factors and possible antiviral strategies. HCV binding to the cell membrane is facilitated by glycosaminoglycans (GAG) and the low-density lipoprotein receptor (LDLR). The surface proteins scavenger receptor class B1 (SR-B1), CD81, claudin-1 (CLDN1), and occludin (OCLN) are known to be crucial for viral binding and clathrin-mediated endocytosis. Receptor tyrosine kinases (RTK), such as epidermal growth factor receptor (EGFR) and ephrin receptor A2, have been now identified as additional host factors that promote at least two steps during the viral entry process: the association of CD81 and CLDN1, as well as membrane fusion during clathrin mediated endocytosis. They are activated by binding their specific ligands, which leads to dimerization of receptor molecules and phosphorylation of tyrosine residues that are part of the intracellular domains (P, phosphate residue; Tyr, tyrosine residue). Increasing knowledge about these events during viral infection lays the foundation for attractive inhibition strategies: antibodies against cell surface proteins such as EGFR, CD81, and CLDN1 appear to be promising candidates for novel therapies. The kinase domain of RTKs such as EGFR can be specifically targeted by small molecule RTK inhibitors (RTKI, e.g., erlotinib). In addition, viral infection is substantially compromised in knock down experiments with siRNAs directed against these entry factors.
Journal of Hepatology 2012 vol. 56 j 282–284
283
International Hepatology In conclusion, Lupberger et al. identify receptor tyrosine kinases as important host factors for HCV entry and show feasibility of their specific inhibition as a novel way to counteract HCV infection. These findings suggest a potentially promising host-centered approach to treatment of HCV, and may find particular utility among those patients who have proven refractory to direct acting antiviral agents. Their already defined role in treating human cancer may further enhance their attractiveness in a group of patients already at high risk for HCC. Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Hutchison JG, Manns MP, Muir AJ, Terrault NA, Jacobson IM, Afdhal NH, et al. Telaprevir for previously treated chronic HCV infection. New Engl J Med 2010;362:1292–1303. [2] Poordad F, McCone Jr J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. New Engl J Med 2011;364:1195–1206.
284
[3] Schwegmann A, Brombacher F. Host-directed drug targeting of factors hijacked by pathogens. Sci Signal 2008;1:re8. [4] Tan SL, Ganji G, Paeper B, Proll S, Katze MG. Systems biology and the host response to viral infection. Nat Biotechnol 2007;25:1383–1389. [5] Flisiak R, Feinman SV, Jablkowski M, Horban A, Kryczka W, Pawlowska M, et al. The cyclophilin inhibitor Debio 025 combined with PEG IFNalpha2a significantly reduces viral load in treatment-naive hepatitis C patients. Hepatology 2009;49:1460–1468. [6] Tai AW, Benita Y, Peng LF, Kim SS, Sakamoto N, Xavier RJ, et al. A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Cell Host Microbe 2009;5:298–307. [7] Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 2008;319:921–926. [8] Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat Med 2011;17:589–595. [9] Himmelsbach K, Sauter D, Baumert TF, Ludwig L, Blum HE, Hildt E. New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication. Gut 2009;58:1644–1653. [10] Pircher A, Medinger M, Drevs J. Liver cancer: targeted future options. World J Hepatol 2011;3:38–44. [11] Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:927–933. [12] Bardou-Jacquet E, Lorho R, Guyader D. Kinase inhibitors in the treatment of chronic hepatitis C virus. Gut 2011;60:879–880.
Journal of Hepatology 2012 vol. 56 j 282–284
Adding to the toolbox: Receptor tyrosine kinases as potential targets in the treatment of hepatitis C Nikolaus Jilg, Raymond T. Chung⇑ Gastrointestinal Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA
COMMENTARY ON: EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M, Gorke S, Royer C, Fischer B, Zahid MN, Lavillette D, Fresquet J, Cosset FL, Rothenberg SM, Pietschmann T, Patel AH, Pessaux P, Doffoël M, Raffelsberger W, Poch O, McKeating JA, Brino L, Baumert TF. Nat Med. 2011 May;17(5):589–95. Copyright (2011). Abstract reprinted with permission from MacMillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/21516087 Abstract: Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection. Ó 2011 European Association for the study of the Liver. Published by Elsevier B.V. All rights reserved. The longtime standard therapy for chronic hepatitis C consisting of peginterferon and ribavirin is fraught with numerous absolute and relative contraindications, limited efficacy, and an unfavorable side effect profile. In view of the global burden of the disease, novel therapeutic strategies are therefore urgently needed. Direct antiviral agents (DAA) against several of the viral proteins have been developed. Eagerly awaited by both patients and their doctors, the first two of these drugs (the NS3/4A protease inhibitors
Keywords: HCV; HCV entry; Entry factors; EGFR; Host factors; Receptor tyrosine kinases. Received 1 June 2011; received in revised form 4 June 2011; accepted 6 June 2011 ⇑ Corresponding author. Tel.: +1 617 724 7562; fax: +1 617 643 0446. E-mail address: [email protected] (R.T. Chung).
telaprevir and boceprevir) have recently been approved by the US FDA [1,2]. While representing a significant leap forward in the treatment of hepatitis C, this new class of drugs will likely not be the final solution for all patients and there remains a significant need for additional drugs with different and novel antiviral modes of action. Viral components and proteins represent the most obvious target of antiviral treatment, but direct-acting antiviral therapies in general are limited by their selection of resistant viral variants. Viral life cycles depend on a multitude of host factors, that, not least, contribute to species specificity and are responsible for many of the interindividual differences seen in the susceptibility to and the course of viral diseases [3,4]. Therefore, efforts have recently been directed toward identifying these host factors and blocking their access to viruses and their ability to exploit them. Cyclophilin A, for instance, is a chaperone that is essential for HCV replication and can be specifically inhibited by alisporivir, an oral agent demonstrating promise in ongoing hepatitis C clinical trials [5]. In recent years, novel tools such as high-throughput assays have been developed to comprehensively screen for the relevant host factors in viral diseases [6,7]. In the May issue of Nature Medicine, Lupberger et al. provide conclusive evidence for a crucial role of receptor tyrosine kinases (RTKs) in HCV infection [8]. Furthermore, they open the possibility of clinical application by robustly demonstrating the impact of RTK inhibitors on HCV infection. RTKs play important roles in basic cellular functions such as cell proliferation, differentiation and cell morphology. Sorafenib, an inhibitor of several protein kinases, is an approved drug in the treatment of renal cell carcinoma and hepatocellular carcinoma (HCC) and has recently been shown to be effective against HCV infection in vitro [9]. The authors screened an siRNA library against 691 human kinases and associated proteins in cell culture, and identified 58 host kinases (11.2% of the human kinome) that impair HCV entry. Of these hits, they choose the RTKs epidermal growth factor receptor (EGFR) and the ephrin A2 receptor (EphA2) for further validation. Both RTKs play important roles in basic physiologic functions such as cell proliferation, differentiation, and cell morphology. Both have been shown to be involved in oncogenesis, and several inhibitors of EGFR and EphA2 are currently approved or under investigation for the treatment of malignant diseases, including non-small cell lung cancer and CML. Erlotinib, one of the small molecule inhibitors of EGFR, is being evaluated in clinical trials for HCC [10].
Journal of Hepatology 2012 vol. 56 j 282–284
JOURNAL OF HEPATOLOGY Lupberger et al. confirmed the relevance of the RTKs EGFR and Eph2A for HCV entry by markedly downregulating infection with specific protein kinase inhibitors of EGFR (erlotinib, gefitinib, lapatinib) and EphA2 (dasatinib) in cell lines and primary human hepatocytes. Entry of murine leukemia virus and measles virus, however, was not impaired by the same treatment. The inhibitors do not compromise viral replication in an entry-independent replicon model system, suggesting specificity for the entry process. In addition, the authors show that HCV entry is markedly enhanced by the two EGFR-ligands, EGF and TGF-a. These effects are reversible by treatment with erlotinib or an EGFR antibody [8]. Furthermore, the authors sought to fine-map the mechanism by which EGFR and EphA2 influence HCV entry: silencing the RTKs has no effect on expression levels of the known HCV entry factors SR-B1, CD81, CLDN1, and OCLN. The group elegantly demonstrates that EGFR and EphA2 are not required for initial viral
attachment, but promote the formation of CD81–CLDN1 coreceptor complexes, a prerequisite to HCV entry and are involved in viral glycoprotein-dependent membrane fusion (Fig. 1). Very interestingly, Lupberger et al. are able to verify erlotinib’s inhibitory effect in an in vivo model of hepatitis C: chimeric urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice repopulated with human hepatocytes and infected with serum-derived HCV [11]. After discontinuation of erlotinib, viral loads in the treatment group reached similar levels as in placebo-treated animals. In line with the presented results, the authors further hypothesize that EGFR-ligand binding may activate downstream signaling that is required for HCV entry and that EGFR and EphA2 may be part of the same entry regulatory pathway. Of note, a recent case report describes the rapid clearance of serum HCV RNA in a patient undergoing treatment with erlotinib for recurrent HCC following liver transplantation [12].
Antibodies against host factors (e.g. EGFR, CD81)
Antibodies against viral envelope proteins
Viral binding
RTK ligands (e.g. EGF, Ephrin-A1)
Clathrin mediated endocytosis Dimerization
TIR
Binding TIR TIR
P
GAG
SR-B1 LDLR
CLDN1 OCLN
P TIR Interaction
RTK
TIR
RTK activation
RTKI (e.g. Erlotinib, Dasatinib)
CD81
Downregulation of host factors (RNAi)
+ +
Fig. 1. HCV entry in the hepatocyte – relevant host factors and possible antiviral strategies. HCV binding to the cell membrane is facilitated by glycosaminoglycans (GAG) and the low-density lipoprotein receptor (LDLR). The surface proteins scavenger receptor class B1 (SR-B1), CD81, claudin-1 (CLDN1), and occludin (OCLN) are known to be crucial for viral binding and clathrin-mediated endocytosis. Receptor tyrosine kinases (RTK), such as epidermal growth factor receptor (EGFR) and ephrin receptor A2, have been now identified as additional host factors that promote at least two steps during the viral entry process: the association of CD81 and CLDN1, as well as membrane fusion during clathrin mediated endocytosis. They are activated by binding their specific ligands, which leads to dimerization of receptor molecules and phosphorylation of tyrosine residues that are part of the intracellular domains (P, phosphate residue; Tyr, tyrosine residue). Increasing knowledge about these events during viral infection lays the foundation for attractive inhibition strategies: antibodies against cell surface proteins such as EGFR, CD81, and CLDN1 appear to be promising candidates for novel therapies. The kinase domain of RTKs such as EGFR can be specifically targeted by small molecule RTK inhibitors (RTKI, e.g., erlotinib). In addition, viral infection is substantially compromised in knock down experiments with siRNAs directed against these entry factors.
Journal of Hepatology 2012 vol. 56 j 282–284
283
International Hepatology In conclusion, Lupberger et al. identify receptor tyrosine kinases as important host factors for HCV entry and show feasibility of their specific inhibition as a novel way to counteract HCV infection. These findings suggest a potentially promising host-centered approach to treatment of HCV, and may find particular utility among those patients who have proven refractory to direct acting antiviral agents. Their already defined role in treating human cancer may further enhance their attractiveness in a group of patients already at high risk for HCC. Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Hutchison JG, Manns MP, Muir AJ, Terrault NA, Jacobson IM, Afdhal NH, et al. Telaprevir for previously treated chronic HCV infection. New Engl J Med 2010;362:1292–1303. [2] Poordad F, McCone Jr J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. New Engl J Med 2011;364:1195–1206.
284
[3] Schwegmann A, Brombacher F. Host-directed drug targeting of factors hijacked by pathogens. Sci Signal 2008;1:re8. [4] Tan SL, Ganji G, Paeper B, Proll S, Katze MG. Systems biology and the host response to viral infection. Nat Biotechnol 2007;25:1383–1389. [5] Flisiak R, Feinman SV, Jablkowski M, Horban A, Kryczka W, Pawlowska M, et al. The cyclophilin inhibitor Debio 025 combined with PEG IFNalpha2a significantly reduces viral load in treatment-naive hepatitis C patients. Hepatology 2009;49:1460–1468. [6] Tai AW, Benita Y, Peng LF, Kim SS, Sakamoto N, Xavier RJ, et al. A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Cell Host Microbe 2009;5:298–307. [7] Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 2008;319:921–926. [8] Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat Med 2011;17:589–595. [9] Himmelsbach K, Sauter D, Baumert TF, Ludwig L, Blum HE, Hildt E. New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication. Gut 2009;58:1644–1653. [10] Pircher A, Medinger M, Drevs J. Liver cancer: targeted future options. World J Hepatol 2011;3:38–44. [11] Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:927–933. [12] Bardou-Jacquet E, Lorho R, Guyader D. Kinase inhibitors in the treatment of chronic hepatitis C virus. Gut 2011;60:879–880.
Journal of Hepatology 2012 vol. 56 j 282–284