- Email: [email protected]
cGAS Micro-Manages Genotoxic Stress
Immunity
Previews cGAS Micro-Manages Genotoxic Stress Mona Motwani1 and Katherine A. Fitzgerald1,*
1Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2017.09.020
Maintenance of genome integrity is essential to prevent cancer. Genotoxic stress drives damaged DNA out of the nucleus by forming micronuclei. Two studies in Nature reveal how the cytosolic DNA sensor cGAS gains access to the cargo within micronuclei to drive type I IFN responses. Genotoxic stress leads to inflammatory responses, which include induction of type I interferons (IFNs), a family of cytokines best known for their role in anti-viral defense. Type I IFNs exhibit antitumor effects by inhibiting the proliferation of cancer cells and promoting apoptosis. Exactly how damaged DNA from the nucleus is coupled to the induction of type I IFNs is poorly understood. Two studies published in Nature (Mackenzie et al., 2017), (Harding et al., 2017) reveal that cGAS, a cytosolic DNA sensor, samples the contents of micronuclei in cells exposed to genotoxic stress. These studies reveal how material normally compartmentalized within the nucleus can be accessible to sensors in the cytosol, findings that have important implications for cancer and autoimmunity. The innate immune system is exquisitely sensitive to dsDNA. Microbial DNA that accumulates in the cytosol during infection is recognized by cGAS, a nucleotidyl transferase enzyme that generates a second-messenger cGAMP (Chen et al., 2016). cGAMP in turn binds and activates STING leading to activation of IRF3 and transcription of type I interferons. cGAS also recognizes self-DNA contributing to autoimmunity. Genetic studies in humans and mice have revealed how mutations in nucleases such as Dnase II and Dnase III (also called Trex1) lead to DNA accrual in the cytosol, which activate the cGAS-STING pathway. Cellular sources of immune stimulatory self-DNA include endogenous retroelements, mitochondrial DNA, and DNA from phagolysosomal compartments containing apoptotic corpses. Growing evidence also supports the idea that the nucleus is a source of selfDNA with immune stimulatory potential. Studies in non-phagocytic cells lacking
Dnase II reveal that damaged DNA is extruded from the nucleus in small budding structures, where it accumulates in the cytosol ligating the STING pathway (Lan et al., 2014). Prior to these findings, several studies have found that type I IFNs are induced in cells exposed to DNA damage agents (Brzostek-Racine et al., 2011; Yu et al., 2015). Other studies have suggesting that small DNA fragments leak from sites of DNA damage through the interphase nuclear envelope into the cytosol to engage cGAS-STING pathway. Most recently two studies defined the cGAS-STING pathway as a crucial regulator of cellular senescence €ck et al., 2017). (Yang et al., 2017; Glu All of these studies clearly indicate that nuclear material can engage the cGASSTING pathway. The two Nature studies advance on these findings and identify micronuclei as the source of immune stimulatory DNA. Micronuclei are secondary nuclei that contain chromosomes or chromosomal fragments that fail to segregate during mitosis or cell division. Micronuclei formation occurs due to loss of tumor suppressor genes such as p53, or in response to genotoxic insults such as DNA damage or irradiation (Crasta et al., 2012). Both studies also found that micronuclei become unstable when the cell enters the cell cycle and as a result their cargo can be detected by cGAS (Figure 1). The first of these studies focused on RnaseH2, an enzyme involved in DNA replication and repair. Prior work has linked mutations in RnaseH2 to Aicardi– Goutie`res syndrome (AGS), a type I IFN dependent autoimmune disease. Genetic studies in mice have identified the cGASSTING pathway as a driver of type I IFNs in cells lacking RnaseH2 (Pokatayev et al., 2016). Exactly how defects in RnaseH2, which compromise genomic integ-
616 Immunity 47, October 17, 2017 ª 2017 Elsevier Inc.
rity result in cGAS activation has remained unclear and have been difficult to explain because RNasH2 is nuclear localized and cGAS resides in the cytosol. Mackenzie et al. found that micronuclei formed in cells lacking Rnaseh2b and that cGAS was recruited there. In an independent study, Harding et al. observed similar findings in cells exposed to gamma radiation and other DNA-damaging agents. Moreover, micronuclei formed in cells due to mechanical stress or those formed following the addition of nocodazole, a compound which induces lagging chromosomes to be encapsulated in micronuclei also recruited cGAS. Exactly how the contents of micronuclei, which are surrounded by nuclear membranes, become accessible to cGAS was an obvious question arising from these studies. Both groups found that micronuclei membranes ruptured, allowing cGAS access to the cargo inside. Both of the studies highlight the importance of the cell cycle in controlling cGAS-dependent recognition of micronuclei. Micronuclei formation alone is insufficient to trigger cGAS activation. Cells must also progress through the cell cycle to destabilize micronuclei membranes and expose their inner contents. As such, pharmacological inhibition of mitosis prevented cGAS activation and IFN induction. Forced rupturing of micronuclear membranes using mechanical force could bypass the requirement for entry into mitosis. These observations indicate that progression of cells into the cell cycle facilitates micronuclear membrane disintegration liberating the stored cargo DNA. A second question arising from these studies concerned the nature of the DNA within these micronuclei and whether cGAS could recognize chromatin. Harding et al. found that cGAS
Immunity
Previews AIM2 can be activated upon DNA release from the nucleus when nuclear envelope integrity is compromised by Nelfinavir treatment (Di Micco et al., 2016). The expectation therefore would be that pyroptotic cell death would follow if AIM2 detected DNA from micronuclei. It remains to be determined therefore whether like cGAS, AIM2 also micromanages genotoxic stress. A final area not addressed in these studies relates to the fate of the micronuclei and their DNA cargo and whether autophagy regulates their clearance. Future studies addressing these questions might address these mechanisms and bring our understanding of genome instability and immune surveillance to a new level. Figure 1. cGAS Detects DNA from Leaky Micronuclei (A) Under homeostatic conditions, a cell counteracts DNA damage by cell-cycle arrest or by repairing damaged DNA. Failure to repair the DNA results in cellular apoptosis. A healthy cell then undergoes regulated cell division resulting in two daughter cells with no micronuclei or immune activation. (B) Cells that accrue genomic instability due to the loss of tumor suppressor genes such as p53 or due to external genomic insults such as gamma irradiation override cell-cycle arrest and cellular apoptosis. Division of such cells results in daughter cells with micronuclei. Micronuclei rupture their nuclear membranes as they progress through cell cycle and contain damaged DNA. Due to compromised membrane integrity, the immune DNA sensor cGAS accesses the DNA of these micronuclei. DNA binding results in production of second messenger cGAMP that binds and activates STING resulting in the induction of type I interferons. The fate of these micronuclei through autophagy and the role of other DNA sensors such as AIM2 in these processes remains unclear.
transiently bound chromatin during mitosis and this event dissipated when the cell divided. Mackenzie et al. found that circular DNA, fragmented plasmid DNA or even DNA packed in nucleosomes could activate cGAS. These observations are somewhat surprising since structural work had suggested that cGAS was unlikely to bind chromatin since the active cGAS dimer formed with each monomer binding a dsDNA molecule. Moreover, it remains unclear what happens during cell division when cGAS transiently binds chromatin. Does this lead to activation of cGAS or are there regulatory hurdles that prevent downstream signaling? Further work will be needed to better understand these mechanisms. The ability of self-DNA recognition to drive anti-tumor immunity is well established and forms the basis for STINGdirected cancer therapeutics. Using a mouse model of radiation-induced DNA damage, Harding et al. found that cGAS activation boosted the anti-tumor activity of radiation combined with checkpoint
blockade. The ability of cGAS to detect micronuclei represents an important step in mobilizing cell intrinsic anti-tumor response. Reports demonstrating that expression of cGAS and STING is lost in various cancers further support the importance of this response. To sum up these recent studies reveal that compromised genome stability either from RnaseH2b disruption, genotoxic stress following DNA damage or irradiation is subjected to a cell intrinsic immune surveillance pathway controlled by cGAS. The ability of cGAS to sense genotoxic stress has direct relevance in cancer and also sheds new light on the potential for nuclear material to engage the cGAS pathway in autoimmunity. Although these studies provide these new insights, they also raise important questions. cGAS is not the sole DNA sensor in the cytosol. Absent in melanoma (AIM2), a cytosolic sensor that forms a caspase-1 activating inflammasome, should in principle also recognize DNA liberated from micronuclei. Indeed,
REFERENCES Brzostek-Racine, S., Gordon, C., Van Scoy, S., and Reich, N.C. (2011). J. Immunol. 187, 5336–5345. Chen, Q., Sun, L., and Chen, Z.J. (2016). Nat. Immunol. 17, 1142–1149. Crasta, K., Ganem, N.J., Dagher, R., Lantermann, A.B., Ivanova, E.V., Pan, Y., Nezi, L., Protopopov, A., Chowdhury, D., and Pellman, D. (2012). Nature 482, 53–58. Di Micco, A., Frera, G., Lugrin, J., Jamilloux, Y., Hsu, E.T., Tardivel, A., De Gassart, A., Zaffalon, L., Bujisic, B., Siegert, S., et al. (2016). Proc. Natl. Acad. Sci. USA 113, E4671–E4680. €ck, S., Guey, B., Gulen, M.F., Wolter, K., Kang, Glu T.W., Schmacke, N.A., Bridgeman, A., Rehwinkel, J., Zender, L., and Ablasser, A. (2017). Nat. Cell Biol. 19, 1061–1070. Harding, S.M., Benci, J.L., Irianto, J., Discher, D.E., Minn, A.J., and Greenberg, R.A. (2017). Nature 548, 466–470. Lan, Y.Y., London˜o, D., Bouley, R., Rooney, M.S., and Hacohen, N. (2014). Cell Rep. 9, 180–192. Mackenzie, K.J., Carroll, P., Martin, C.A., Murina, O., Fluteau, A., Simpson, D.J., Olova, N., Sutcliffe, H., Rainger, J.K., Leitch, A., et al. (2017). Nature 548, 461–465. Pokatayev, V., Hasin, N., Chon, H., Cerritelli, S.M., Sakhuja, K., Ward, J.M., Morris, H.D., Yan, N., and Crouch, R.J. (2016). J. Exp. Med. 213, 329–336. Yang, H., Wang, H., Ren, J., Chen, Q., and Chen, Z.J. (2017). Proc. Natl. Acad. Sci. USA 114, E4612–E4620. Yu, Q., Katlinskaya, Y.V., Carbone, C.J., Zhao, B., Katlinski, K.V., Zheng, H., Guha, M., Li, N., Chen, Q., Yang, T., et al. (2015). Cell Rep. 11, 785–797.
Immunity 47, October 17, 2017 617
Previews cGAS Micro-Manages Genotoxic Stress Mona Motwani1 and Katherine A. Fitzgerald1,*
1Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2017.09.020
Maintenance of genome integrity is essential to prevent cancer. Genotoxic stress drives damaged DNA out of the nucleus by forming micronuclei. Two studies in Nature reveal how the cytosolic DNA sensor cGAS gains access to the cargo within micronuclei to drive type I IFN responses. Genotoxic stress leads to inflammatory responses, which include induction of type I interferons (IFNs), a family of cytokines best known for their role in anti-viral defense. Type I IFNs exhibit antitumor effects by inhibiting the proliferation of cancer cells and promoting apoptosis. Exactly how damaged DNA from the nucleus is coupled to the induction of type I IFNs is poorly understood. Two studies published in Nature (Mackenzie et al., 2017), (Harding et al., 2017) reveal that cGAS, a cytosolic DNA sensor, samples the contents of micronuclei in cells exposed to genotoxic stress. These studies reveal how material normally compartmentalized within the nucleus can be accessible to sensors in the cytosol, findings that have important implications for cancer and autoimmunity. The innate immune system is exquisitely sensitive to dsDNA. Microbial DNA that accumulates in the cytosol during infection is recognized by cGAS, a nucleotidyl transferase enzyme that generates a second-messenger cGAMP (Chen et al., 2016). cGAMP in turn binds and activates STING leading to activation of IRF3 and transcription of type I interferons. cGAS also recognizes self-DNA contributing to autoimmunity. Genetic studies in humans and mice have revealed how mutations in nucleases such as Dnase II and Dnase III (also called Trex1) lead to DNA accrual in the cytosol, which activate the cGAS-STING pathway. Cellular sources of immune stimulatory self-DNA include endogenous retroelements, mitochondrial DNA, and DNA from phagolysosomal compartments containing apoptotic corpses. Growing evidence also supports the idea that the nucleus is a source of selfDNA with immune stimulatory potential. Studies in non-phagocytic cells lacking
Dnase II reveal that damaged DNA is extruded from the nucleus in small budding structures, where it accumulates in the cytosol ligating the STING pathway (Lan et al., 2014). Prior to these findings, several studies have found that type I IFNs are induced in cells exposed to DNA damage agents (Brzostek-Racine et al., 2011; Yu et al., 2015). Other studies have suggesting that small DNA fragments leak from sites of DNA damage through the interphase nuclear envelope into the cytosol to engage cGAS-STING pathway. Most recently two studies defined the cGAS-STING pathway as a crucial regulator of cellular senescence €ck et al., 2017). (Yang et al., 2017; Glu All of these studies clearly indicate that nuclear material can engage the cGASSTING pathway. The two Nature studies advance on these findings and identify micronuclei as the source of immune stimulatory DNA. Micronuclei are secondary nuclei that contain chromosomes or chromosomal fragments that fail to segregate during mitosis or cell division. Micronuclei formation occurs due to loss of tumor suppressor genes such as p53, or in response to genotoxic insults such as DNA damage or irradiation (Crasta et al., 2012). Both studies also found that micronuclei become unstable when the cell enters the cell cycle and as a result their cargo can be detected by cGAS (Figure 1). The first of these studies focused on RnaseH2, an enzyme involved in DNA replication and repair. Prior work has linked mutations in RnaseH2 to Aicardi– Goutie`res syndrome (AGS), a type I IFN dependent autoimmune disease. Genetic studies in mice have identified the cGASSTING pathway as a driver of type I IFNs in cells lacking RnaseH2 (Pokatayev et al., 2016). Exactly how defects in RnaseH2, which compromise genomic integ-
616 Immunity 47, October 17, 2017 ª 2017 Elsevier Inc.
rity result in cGAS activation has remained unclear and have been difficult to explain because RNasH2 is nuclear localized and cGAS resides in the cytosol. Mackenzie et al. found that micronuclei formed in cells lacking Rnaseh2b and that cGAS was recruited there. In an independent study, Harding et al. observed similar findings in cells exposed to gamma radiation and other DNA-damaging agents. Moreover, micronuclei formed in cells due to mechanical stress or those formed following the addition of nocodazole, a compound which induces lagging chromosomes to be encapsulated in micronuclei also recruited cGAS. Exactly how the contents of micronuclei, which are surrounded by nuclear membranes, become accessible to cGAS was an obvious question arising from these studies. Both groups found that micronuclei membranes ruptured, allowing cGAS access to the cargo inside. Both of the studies highlight the importance of the cell cycle in controlling cGAS-dependent recognition of micronuclei. Micronuclei formation alone is insufficient to trigger cGAS activation. Cells must also progress through the cell cycle to destabilize micronuclei membranes and expose their inner contents. As such, pharmacological inhibition of mitosis prevented cGAS activation and IFN induction. Forced rupturing of micronuclear membranes using mechanical force could bypass the requirement for entry into mitosis. These observations indicate that progression of cells into the cell cycle facilitates micronuclear membrane disintegration liberating the stored cargo DNA. A second question arising from these studies concerned the nature of the DNA within these micronuclei and whether cGAS could recognize chromatin. Harding et al. found that cGAS
Immunity
Previews AIM2 can be activated upon DNA release from the nucleus when nuclear envelope integrity is compromised by Nelfinavir treatment (Di Micco et al., 2016). The expectation therefore would be that pyroptotic cell death would follow if AIM2 detected DNA from micronuclei. It remains to be determined therefore whether like cGAS, AIM2 also micromanages genotoxic stress. A final area not addressed in these studies relates to the fate of the micronuclei and their DNA cargo and whether autophagy regulates their clearance. Future studies addressing these questions might address these mechanisms and bring our understanding of genome instability and immune surveillance to a new level. Figure 1. cGAS Detects DNA from Leaky Micronuclei (A) Under homeostatic conditions, a cell counteracts DNA damage by cell-cycle arrest or by repairing damaged DNA. Failure to repair the DNA results in cellular apoptosis. A healthy cell then undergoes regulated cell division resulting in two daughter cells with no micronuclei or immune activation. (B) Cells that accrue genomic instability due to the loss of tumor suppressor genes such as p53 or due to external genomic insults such as gamma irradiation override cell-cycle arrest and cellular apoptosis. Division of such cells results in daughter cells with micronuclei. Micronuclei rupture their nuclear membranes as they progress through cell cycle and contain damaged DNA. Due to compromised membrane integrity, the immune DNA sensor cGAS accesses the DNA of these micronuclei. DNA binding results in production of second messenger cGAMP that binds and activates STING resulting in the induction of type I interferons. The fate of these micronuclei through autophagy and the role of other DNA sensors such as AIM2 in these processes remains unclear.
transiently bound chromatin during mitosis and this event dissipated when the cell divided. Mackenzie et al. found that circular DNA, fragmented plasmid DNA or even DNA packed in nucleosomes could activate cGAS. These observations are somewhat surprising since structural work had suggested that cGAS was unlikely to bind chromatin since the active cGAS dimer formed with each monomer binding a dsDNA molecule. Moreover, it remains unclear what happens during cell division when cGAS transiently binds chromatin. Does this lead to activation of cGAS or are there regulatory hurdles that prevent downstream signaling? Further work will be needed to better understand these mechanisms. The ability of self-DNA recognition to drive anti-tumor immunity is well established and forms the basis for STINGdirected cancer therapeutics. Using a mouse model of radiation-induced DNA damage, Harding et al. found that cGAS activation boosted the anti-tumor activity of radiation combined with checkpoint
blockade. The ability of cGAS to detect micronuclei represents an important step in mobilizing cell intrinsic anti-tumor response. Reports demonstrating that expression of cGAS and STING is lost in various cancers further support the importance of this response. To sum up these recent studies reveal that compromised genome stability either from RnaseH2b disruption, genotoxic stress following DNA damage or irradiation is subjected to a cell intrinsic immune surveillance pathway controlled by cGAS. The ability of cGAS to sense genotoxic stress has direct relevance in cancer and also sheds new light on the potential for nuclear material to engage the cGAS pathway in autoimmunity. Although these studies provide these new insights, they also raise important questions. cGAS is not the sole DNA sensor in the cytosol. Absent in melanoma (AIM2), a cytosolic sensor that forms a caspase-1 activating inflammasome, should in principle also recognize DNA liberated from micronuclei. Indeed,
REFERENCES Brzostek-Racine, S., Gordon, C., Van Scoy, S., and Reich, N.C. (2011). J. Immunol. 187, 5336–5345. Chen, Q., Sun, L., and Chen, Z.J. (2016). Nat. Immunol. 17, 1142–1149. Crasta, K., Ganem, N.J., Dagher, R., Lantermann, A.B., Ivanova, E.V., Pan, Y., Nezi, L., Protopopov, A., Chowdhury, D., and Pellman, D. (2012). Nature 482, 53–58. Di Micco, A., Frera, G., Lugrin, J., Jamilloux, Y., Hsu, E.T., Tardivel, A., De Gassart, A., Zaffalon, L., Bujisic, B., Siegert, S., et al. (2016). Proc. Natl. Acad. Sci. USA 113, E4671–E4680. €ck, S., Guey, B., Gulen, M.F., Wolter, K., Kang, Glu T.W., Schmacke, N.A., Bridgeman, A., Rehwinkel, J., Zender, L., and Ablasser, A. (2017). Nat. Cell Biol. 19, 1061–1070. Harding, S.M., Benci, J.L., Irianto, J., Discher, D.E., Minn, A.J., and Greenberg, R.A. (2017). Nature 548, 466–470. Lan, Y.Y., London˜o, D., Bouley, R., Rooney, M.S., and Hacohen, N. (2014). Cell Rep. 9, 180–192. Mackenzie, K.J., Carroll, P., Martin, C.A., Murina, O., Fluteau, A., Simpson, D.J., Olova, N., Sutcliffe, H., Rainger, J.K., Leitch, A., et al. (2017). Nature 548, 461–465. Pokatayev, V., Hasin, N., Chon, H., Cerritelli, S.M., Sakhuja, K., Ward, J.M., Morris, H.D., Yan, N., and Crouch, R.J. (2016). J. Exp. Med. 213, 329–336. Yang, H., Wang, H., Ren, J., Chen, Q., and Chen, Z.J. (2017). Proc. Natl. Acad. Sci. USA 114, E4612–E4620. Yu, Q., Katlinskaya, Y.V., Carbone, C.J., Zhao, B., Katlinski, K.V., Zheng, H., Guha, M., Li, N., Chen, Q., Yang, T., et al. (2015). Cell Rep. 11, 785–797.
Immunity 47, October 17, 2017 617