ARTICLE

https://doi.org/10.1038/s42003-021-02278-9 OPEN Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway

Maria Krivega1, Clara M. Stiefel1, Sahar Karbassi1, Line L. Andersen2, Narendra K. Chunduri 1, ✉ Neysan Donnelly3, Andreas Pichlmair 2,4 & Zuzana Storchová 1

Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of

1234567890():,; the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.

1 Molecular Genetics, University of Kaiserslautern, Kaiserslautern, Germany. 2 Institute of Virology, TU Munich, München, Germany. 3 Max Planck Institute of ✉ Biochemistry, Martinsried, Germany. 4 German Center for Infection Research (DZIF), Munich partner site, München, Germany. email: [email protected]. de

COMMUNICATIONS BIOLOGY | (2021) 4:831 | https://doi.org/10.1038/s42003-021-02278-9 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02278-9

hole-chromosomal aneuploidy (hereafter aneuploidy), unnecessary or dysfunctional cytoplasmic components and allows Wwhich results from chromosome segregation errors, is recycling of their constituents30. During autophagy, the phago- often detrimental to eukaryotic organisms. In humans, phore membrane carrying the lipidated form of the LC3 protein organismal aneuploidy interferes with normal development, and (LC3-II) engulfs the cytoplasmic components to be degraded, and only a low percentage of embryos with trisomies of chromosomes subsequently fuses with lysosomes to enable degradation. Under 8, 13, 18, and 21 (Warkany, Patau, Edwards, and Down syn- nutrient-rich conditions, activated mTORC1 kinase, a key drome, respectively) result in live births, although rarely1. Infants nutrient sensor, suppresses autophagosome assembly by inhibi- born with these syndromes suffer from multiple pathologies and tory phosphorylation of ULK1 and other proteins31. Starvation have a shortened life expectancy. In somatic cells, missegregating inactivates mTORC1, abrogating the inhibitory cells are often arrested in the cell cycle or die; even if they escape phosphorylations32. Other mTORC1 targets include the tran- this fate, they are outgrown by normal cells due to the pro- scription factors of the MIT/TFEB family, which regulate the liferative disadvantage associated with aneuploidy, or they may be expression of autophagy- and lysosome-specific genes33,34. In fed actively removed by immune cells2–5. At the same time, aneu- conditions, active mTORC1 phosphorylates TFEB to sequester it ploidy provides an advantage in stressful, variable environmental in the cytoplasm, whereas during starvation, when mTORC1 is conditions6,7. Aneuploidy is also a common feature in nearly 90% inactive, TFEB translocates to the nucleus and activates tran- of solid tumors. Here, the degree of aneuploidy correlates posi- scription of its targets35, thereby facilitating autophagy and tively with invasiveness, resistance to chemotherapy, immune lysosomal degradation36. Autophagy is also activated by other evasion, and poor patient prognosis8. The molecular mechanisms cellular insults, such as oxidative stress or the unfolded protein underlying the striking and variable consequences of aneuploidy response31. Recently, autophagy was shown to be activated by the remain poorly understood. innate immune response pathway in the context of genomic Recently established model mammalian somatic cell lines with instability37. While autophagy is regulated by multiple signaling defined chromosome gains have allowed in vitro studies of the effects pathways, their individual contribution to increased autophagy of aneuploidy on cellular function9–11. These studies revealed that the and lysosomal activity in cells with extra chromosomes remain gain of a single chromosome triggers a uniform and conserved enigmatic. deregulation of specific pathways that primarily affect cell prolifera- Building on our previous knowledge of the consequences of tion and genome and proteome homeostasis7,12,13.Thesecon- chromosome gain in mammalian cells and recent findings that sequences on cellular physiology are referred to as aneuploidy- the activation of both autophagy and the innate immune response associated stresses and are thought to occur largely due to the might be tightly linked, we sought to determine the cellular overexpression of proteins from the extra chromosomes, which mechanisms that contribute to the constitutive upregulation of overwhelms the cellular ability to maintain protein homeostasis autophagy and lysosomal pathways in trisomic cells. To this end, (proteostasis)7,13–16. As a result, cells with extra chromosomes we used a series of trisomic cell lines derived from RPE1 (a exhibit impaired protein folding, cytoplasmic foci of aggregated diploid, immortalized retinal pigmented epithelial cell line) and proteins, increased proteasomal activity, and sensitivity to conditions HCT116 (a diploid colorectal cancer cell line) generated by that disrupt proteostasis14,16,17. In addition, aneuploid cells suffer microcell-mediated chromosome transfer11. Strikingly, we show from genomic instability characterized by replication stress, accu- that activation of autophagy and the lysosomal pathway in con- mulation of DNA damage, and increased chromosomal stitutively trisomic cells is independent of mTORC1 status. aberrations3,18–20. Instead, the cGAS–STING pathway is constitutively active in Aneuploid model cell lines have also been shown to activate the trisomic cells and triggers type I interferon response and ISG type I interferon response, but the upstream triggers have not expression, as well as transcriptional upregulation of autophagy been identified21,22. Interestingly, DNA damage and replication and lysosomal genes. Importantly, we demonstrate that stress have recently emerged as potential triggers of the inflam- cGAS–STING-dependent activation of the inflammatory matory response in mammalian cells23–26. Here, cGAS (cyclic response and autophagy also occurs in human primary guanosine monophosphate (GMP)–adenosine monophosphate embryonic fibroblasts isolated from embryos with trisomy syn- (AMP) synthase) recognizes cytosolic DNA, which catalyzes the dromes. Taken together, our results reveal the mechanism of conversion of GTP and ATP into the second messenger cyclic autophagy activation in response to chromosome gain and extend GMP–AMP (2′3′-cGAMP)27. The latter serves as a ligand for the the link between autophagy and innate immune signaling. adapter protein stimulator of interferon (IFN) genes (STING), which dimerizes after cGAMP binding and translocates from the ER to the Golgi apparatus. Activated STING recruits and activates Results TANK-binding kinase 1 (TBK1) and I kappa B kinase (IKK), TFEB-dependent transcription and autophagy is activated in which in turn phosphorylates transcription factors IRF3 and NF- constitutive trisomies. Previously, we established isogenic series κB, respectively28. Phosphorylated IRF3 and NF-κB translocate to of human cell lines derived from HCT116 or RPE1 that carry an the nucleus, where they trigger transcription of type 1 interferons extra copy of one or more chromosomes11,38. The engineered cell and other cytokines, which subsequently elicit the expression of lines were named according to their origin (H: HCT116; R: interferon-stimulated genes (ISGs)29. Whether this signaling RPE1), the type of chromosome gain (tr: trisomic; te: tetrasomic), pathway is activated in constitutive trisomic cells has not yet been and the transferred chromosome (3, 5, 7, 8, 12, 18, or 21), fol- tested. lowed by a clone number (e.g., Htr3_11 is a trisomy of chro- Cells with extra chromosomes also upregulate autophagy and mosome 3 in HCT116, clone 11, Supplementary table 1). lysosomal genes and show increased levels of LC3-positive Proteome and transcriptome analysis revealed a general upregu- autophagosomes11,21. Moreover, murine aneuploid cells were lation of a wide range of autophagy and lysosomal factors in these selectively sensitive to treatment with chloroquine, a drug that cell lines11,21. Detailed examination of the transcriptome and impairs lysosomal function and thus blocks autophagic proteome data showed that specifically the targets of the tran- degradation17. These findings suggest that autophagic activity is scription factor TFEB, such as LC3 (MAP1LC3B), SQSTM1, constitutively increased in aneuploid cells, likely to attenuate the VPS18, and WIPI1, as well as cathepsins D, B and A, and other deleterious effects of aneuploidy-associated stresses. Autophagy is lysosome-specific proteases were upregulated in the analyzed cell a conserved, tightly regulated cellular mechanism that degrades lines (Fig. 1a, b and Supplementary Data 1). This upregulation of

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abTranscriptome Proteome c HCT116 Htr3_11 Htr8_5 RPE1 Rtr7_1 Rtr21_2 tr3_11 te5_1 BafA1 - + - + - + Hte5_4 Hte5_1 H Rtr5,12_3Rtr21_2 Htr5_6Hte5_4Htr3_11H Rtr21_2Rtr5,12_3 BafA1 - + - + - + VPS11 LC3-I LC3-I CTSD 15 LC3-II 15 ATP6V1D LC3-II 37 ATP6V1H CTSB GAPDH 37 GAPDH WIPI1 SQSTM1 CTSB MAP1LC3B MAP1LC3B +BafA1 d NS SQSTM1 CLCN7 0.0357 ATP6V0E1 32 NS CTSD CTSA 0.039 28 NS CLCN7

LAMP1 ux, 24 0.0114 UVRAG 0.0407 ATP6V1D VPS18 20 NS CTSA ATP6V1H 16 MCOLN1 12 VPS11 0.0002

LAMP1 LC3-II/LC3-I ratio Autophagy 8 CTSF VPS18 NS 4 ATG9B VPS8 1 VPS8 1 1 3 2 _2 _6 - _ _ _ ATP6V0D2 -3 -1.5 0 1.5 3 3 8_1 5 8_5 2 7_1 e5_11 tr r PE tr3 ,1 r 21 t tr tr1 tr3_11H Ht te5_4 R R 5 Rt r -2.5 -1 0 1 2.5 HCT116H H H H H tr Rt R e f Nuclei TFEB Merged 0.0001 0.0003 <0.0001 0.0004 0.0101 <0.0001 <0.0001 14 RPE1 12 <0.0001

B 10 <0.0001

TFE 8 ar e 6 h ucl

N 4 <0.0001 Rtr3_1 2 1 100 0 6 1 3 1 3 80 1 _ E1 _ _ _1 _1 _2 11 3_2 8 1_ P 5 8 1 T e5_ 2 R tr tr r7 tr1 tr Rtr3 R R Rt HC Ht H Htr1 H Rtr2 60 <0.0001

40 g Nuclei LC3 LAMP2 Merged LC3+ puncta per cell 20 Nuclei LC3 LAMP2 Merged 0 1 1 HCT116 1 E _1 116 5_ 8_ 1_3 P 3_1 R tr tr8 tr1 R R HCT Hte H Htr2 i <0.0001 80 0.0088 Hte5_1 <0.0001 cell 60 ta perta c

n 40 u p RPE1 +

P2 20 LAM 0 Rtr8_1 6 1 1 1 1 _ E _1 1 8 1_3 T e5_ RP r3_1 tr8 C t tr1 tr2 Rt R H H H H

Fig. 1 Autophagy and lysosomal degradation is activated in cells with extra chromosomes. a Transcriptome and b proteome analysis of TFEB targets. The heat maps show fold changes in trisomic and tetrasomic cells compared with parental diploids. c Immunoblotting of LC3; GAPDH serves as a loading control. d Quantification of the LC3-II/LC3-I ratios upon treatment with Bafilomycin A1 (BafA1) and in control DMSO-treated cells. Each point represents one independent biological experiment, 3–10 experiments were performed in each cell line. e Localization of TFEB was assessed by immunofluorescence analysis. White arrows point to the localization of the nucleus in these cells. f Quantification of the relative TFEB nuclear localization. The difference in mean fluorescent intensity (MFI) between nuclear and cytoplasmic signals was calculated and plotted relatively to control diploid cells. At least 2000 cells for HCT116 and 1000 cells for RPE1 cell lines were analyzed in each experiment, the means of each experiment are plotted. Each point represents the mean from one independent biological experiment, 4–7 experiments were performed in each cell line. g Immunofluorescence images of LC3 and LAMP2 localization in trisomic and diploid cells. White arrows indicate double-positive puncta. h, i Quantification of LC3- and LAMP2-positive puncta per cell. Number of analyzed cells: n = 30 for LC3, n = 20 for LAMP2 in each cell line. Unpaired t-test was used for statistical analysis, unless otherwise specified. Individual measurements, mean values, p-values, and standard deviations are shown in the plots, statistical evaluation is summarized in Supplementary table 4, and source data is available in Supplementary Data 5. Scale bar 10 µm. Mann–Whitney statistical analysis was applied for data in f.

COMMUNICATIONS BIOLOGY | (2021) 4:831 | https://doi.org/10.1038/s42003-021-02278-9 | www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02278-9 autophagy and lysosome was observed in different trisomic cells, affected by other cellular stresses, such as oxidative stress or the regardless of their specific karyotype. unfolded protein response (UPR)34. Analysis of NRF2 and XBP1 Immediately after chromosome missegregation, and thus as activity, key factors in cellular antioxidant response and UPR, part of the acute response to aneuploidy, TFEB also translocates respectively, revealed no significant differences between aneu- to the nucleus and activates expression of its downstream targets. ploid and diploid cell lines (Supplementary Fig. 2f–i). However, in these settings, autophagy and lysosomal degradation We also analyzed the changes in autophagy activation in appear to be impaired39. To assess autophagic flux in constitutive aneuploid and wild-type cells after treatment with the mTORC1 trisomic cells, we determined the accumulation of lipidated LC3- inhibitor Torin 1. Efficient mTORC1 inhibition was confirmed by II, a marker of autophagosome formation, upon addition of the downregulation of p-P70S6K–T389 and p-ULK1–S575 (Supple- vacuolar H+ -ATPase inhibitor bafilomycin A1, and compared it mentary Fig. 2a, j, k). As expected, inhibition of mTORC1 with that of control cells. This analysis showed that autophagic resulted in an increased ratio of lipidated LC3-II versus flux was generally unaffected in aneuploid cells, with LC3-II nonlipidated LC3-I proteins (Supplementary Fig. 2a, l). Inhibition increasing to a similar level or more after bafilomycin treatment of mTORC1 should also increase the nuclear localization of than in the parental diploid control (Fig. 1c, d). Additionally, TFEB. As expected, nuclear TFEB was enriched in diploid cells cathepsin D levels were increased, and its activity was not after a treatment with Torin 1 to levels comparable to nuclear diminished (Supplementary Fig. 1a–c). Importantly, the LC3-II/ TFEB enrichment in trisomic cells without any treatment. The LC3-I ratio was increased in trisomic cell lines also under normal highly elevated nuclear TFEB did not increase further in trisomic culture conditions (Supplementary Fig. 1d, e). cells; a slight increase was observed only in Htr21_3, a trisomy TFEB activity is regulated by nucleocytoplasmic transport, and cell line with the lowest constitutive TFEB localization (Supple- indeed, we observed statistically significant enrichment of TFEB mentary Fig. 2m). Our results that mTORC1 inhibition does not in the nucleus in trisomic and tetrasomic cells compared with the further increase nuclear enrichment of TFEB in aneuploid cells parental control by immunofluorescence (IF) imaging (Fig. 1e, f). suggest that TFEB is nearly maximally activated in cells with extra TFE3, another factor from the MiT/TFEB family of autophagy chromosomes. Based on these results, we conclude that an and lysosomal gene regulators, was similarly enriched, but mainly mTORC1-independent mechanism is responsible for constitutive in HCT116-derived trisomic cells, likely representing physiologi- TFEB nuclear localization and increased gene expression of TFEB cal differences between HCT116 and RPE1 cell lines (Supple- targets in human trisomic cells. mentary Fig. 1f, g). These data indicate that autophagy is activated in response to chromosome gain, independently of the identity of an extra chromosome. For subsequent analysis of the The cGAS–STING–TBK1–IRF3 pathway is activated in con- mechanisms of autophagy activation, we selected five different stitutively trisomic cells. Recent evidence has suggested that trisomies, each with a different extra chromosome. autophagy can be activated via cGAS–STING signaling inde- By IF, we additionally confirmed the accumulation of LC3- pendently of mTORC137. We hypothesized that this pathway positive foci (marker of autophagosomes, Fig. 1g, h) in aneuploid might contribute to the increased TFEB activity in response to cells. Similarly, increased accumulation of lysosomes in aneuploid trisomy. This hypothesis is consistent with previous observations cells compared with diploids was observed by IF of LAMP2 that constitutive trisomy activates the type I interferon (marker of lysosomes) and by staining with LysoTracker (Fig. 1g, pathway11,21,22. Recently, endogenous DNA damage and repli- i and Supplementary Fig. 1h, i). We also used the doubly tagged cation stress, which is also a hallmark of trisomic cells3,13,19,20, mRFP–GFP–LC3 to visualize autophagic flux40. Because only the has been shown to cause accumulation of cytoplasmic DNA, mRFP signal is resistant to the acidic pH in lysosomes, this thereby contributing to activation of the type I interferon experiment allows us to estimate the ratio between autophago- pathway23,25,26,41.Wefirst analyzed the presence of dsDNA in somes (yellow signal) and autolysosomes (red signal). This the cytoplasm using a specific anti-dsDNA antibody, followed by analysis confirmed accumulation of autolysosomes in aneuploid fluorescent imaging. Indeed, trisomic cells showed an increase in cell lines (Supplementary Fig. 1j, k). Taken together, the gain of a cytoplasmic dsDNA compared with control HCT116 and chromosome causes increased nuclear localization of MiT/TFEB RPE1 cells that was similar to the increase observed in parental transcription factors and increased expression of their target diploid cells treated with the DNA damage-inducing agent ara- genes encoding regulators of autophagy and lysosomal functions; binocytosin (AraC) (Fig. 2a and Supplementary Fig. 3a). DNase I this, in turn, allows chronically aneuploid cells to maintain treatment reduced the cytoplasmic dsDNA signal, further sup- constitutively elevated autophagy. porting the specificity of the antibody (Fig. 2b and Supplementary Fig. 3b). To identify the origin of cytoplasmic dsDNA, we stained the cells with antibodies against histone H3 to label cytosolic Constitutive nuclear TFEB localization in trisomic cells is fragments of nuclear chromatin42. Antibodies against the mito- mTORC1-independent. TFEB localization is regulated by chondrial transcription factor TFAM were used to label the DNA mTORC1, and reduced activity of this kinase (e.g., during star- of mitochondrial origin43. While ~40% of cytosolic dsDNA vation) leads to nuclear accumulation of TFEB33. Analysis of the colocalized with H3 in diploid cells, this fraction increased to activity of the known upstream mTORC1 regulators AKT1 and 60–80% in cells with extra chromosomes (Fig. 2c, d and Sup- AMPK revealed no consistent p-AKT1–S473 changes and plementary Fig 3c). In contrast, almost 80% of cytosolic DNA in unchanged p-AMPK-T172 levels in aneuploid cells compared diploid cells was of mitochondrial origin, whereas this fraction with diploid parental cells (Supplementary Fig. 2a–d). To inves- was significantly reduced in aneuploid cells (Fig. 2e, f and Sup- tigate mTORC1 activity in trisomic cells, we immunoblotted the plementary Fig 3d, e). Interestingly, cytosolic chromatin is pre- cell lysates with an antibody against p-P70S6K-T389, a direct sumably removed via the autophagy pathway, as inhibition of phosphorylation target of mTORC1. We found significantly autophagosome–lysosome fusion by treatment with Bafilomycin increased phosphorylation in three out of five trisomic cell lines, A1 increased cytosolic dsDNA accumulation in trisomic cells, but confirming that the mTORC1 activity was not uniformly reduced not in diploids (Supplementary Fig. 4a, b). These observations are in trisomic cells (Supplementary Fig. 1d and Supplementary consistent with our hypothesis that DNA leakage from trisomic Fig. 2a, e). TFEB localization and autophagic flux may also be nuclei into the cytoplasm activates the cGAS–STING pathway.

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a b

Nuclei dsDNA Merged Nuclei dsDNA Merged

RPE1 RPE1 +DNaseI

Rtr3_1 Rtr3_1 +DNaseI

c d <0.0001 100 <0.0001

Nuclei H3 dsDNA Merged 3 80 ion H t d Nuclei H3 dsDNA Merged a z

li 60 an a

HCT116 A

DN 40 ds cta coloc of of

n 20 u % p Htr18_1 0 1 _1 _1 8_1 T116 RPE1 tr3 tr8 tr1 tr21_3 R R HC Hte5_H H

e f 0.0006 0.0034 0.0002 Nuclei TFAM dsDNA Merged 100 <0.0001 Nuclei TFAM dsDNA Merged <0.0001 80 HCT116 FAM FAM 60

40 colocalization a t Htr18_1 f c 20 un p % of dsDNA and T 0 E1 _1 8_1 P 8 R tr CT116te5_1tr1 tr21_3 Rtr3_1R H H H H

Fig. 2 Increased presence of cytoplasmic dsDNA in cells with extra chromosomes. a Examples of immunofluorescent images of nuclear and cytoplasmic dsDNA. White arrows point to the dsDNA staining in the cytoplasm. b Staining of cytoplasmic dsDNA in cells treated with DNase I. c Immunofluorescence images of dsDNA colocalization with H3 (white arrows). d Quantification of the percentages of dsDNA puncta copositive for H3. e Immunofluorescence of dsDNA with TFAM colocalization (white arrows). Yellow arrows indicate dsDNA puncta negative for TFAM. f Quantification of the percentages of puncta positive for both dsDNA and TFAM. Individual measurements, mean values, p-values, and standard deviations are shown in the plots, statistical evaluation is summarized in Supplementary table 4, and source data is available in Supplementary Data 5. At least 20 cells were analyzed for the puncta quantification for each cell line. Unpaired t-test was used for statistical analysis. Scale bar 10 µm.

Activation of the cGAS–STING pathway is manifested by treated with AraC (Fig. 3i, j), or after transformation with plasmid phosphorylation and relocalization of pathway members. Indeed, in DNA (Supplementary Fig. 5e, f). In contrast, NF-κBtargetswerenot trisomic cells, we observed increased levels of the downstream kinase consistently upregulated, as shown by transcriptome analysis and TBK1 and p-TBK1–S172, as well as of STING and p-STING–S366 confirmed by qRT-PCR of the canonical NF-kB target IL6 (Fig. 3h (Fig. 3a–e). Consistent with this observation, IF of STING revealed and Supplementary Data 3). Activation of the NF-kB pathway is also increased accumulation and clustering of the STING signal in the accompanied by reduced level of its inhibitor IkBα,butitsabundance cytoplasm of trisomic cells (Fig. 3f, g). The cGAS–STING–TBK1 axis was not reduced in trisomic cell lines (Supplementary Fig. 5c, d). triggers expression of the interferon type I factors either via the IRF3 These results indicate that the type I interferon response is triggered or NF-kB transcriptional factors; transcriptome analysis suggested via IRF3 activation upon chromosome gain. that specifically targets of IRF3 are overexpressed in trisomic cells Nuclear localization of STAT1, a transcription factor contribut- (Fig. 3h and Supplementary Data 2). The increased expression of ing to ISG expression44, was also increased in aneuploid cells IRF3 targets and ISGs in aneuploid cells was confirmed by qRT-PCR (Supplementary Fig. 6a, b). Induction of ISG expression in trisomic (Supplementary Fig. 5a, b). Activation of IRF3 by chromosome gain cell lines was lower compared with induction upon treatment with was confirmed by its increased nuclear localization in aneuploid cells, the synthetic immunostimulant poly-IC or with purified inter- which was comparable to IRF3 nuclear accumulation in diploid cells feron, but comparable to levels observed in parental diploid cells

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a 0.0329 0.0409 bc0.0190 0.0283 4 0.0066 15

T116 te5_1 r18_1 E1 r3_1 172 10 H t S HC Ht Htr21_3 RP R Rtr8_1 3 NS 0.0004 100 0.0027 0.0295 p-TBK1-S172 K1 levels 6 0.0005 TBK1-

75 B 2 - TBK1 100 4 75 -actin 50 1 2

37 Relative T 50 Relative p 0 p-STING-S366 0 37 3 1 1 6 1 1 1 16 _ _ E _1 1 PE1 8_ 11 5 8_1 1_3 r8 50 e5_118_1 R tr T te 1 2 RP STING CT r tr21_ Rtr3 R Rtr3_Rt 37 H Ht Ht H HC H Htr Htr -actin 50 0.0024 37 d 0.0026 e 0.0303 0.0286 2.5 0.0004 3.5 f NS 3.0 Nuclei STING Merged 2.0 NS 2.5 0.0286 1.5 2.0 HCT116 eSTINGlevels 1.0 1.5 v 1.0 0.5

Relati 0.5 0.0 0.0 Relative p-STING-S366 levels 6 1 1 Hte5_1 _1 E _ 6 1 1 3 1 1 1 5 8_1 3 1 _ E _ _ 1 21_3 RP 1 8 1_ P 3 8 T 1 R tr r Hte Rtr Rtr8_1 C r2 R Rt HCT11 Htr Htr H Hte5_Htr Ht

g 0.0097 RPE1 0.0128 30 <0.0001 25 h te5_4 tr3_11tr5_6 Rtr3_1 20 H Hte5_1H H Rtr5,12_3Rtr21_2 Proteome GFPT2 * Direct target 15 IFIT2 genes 0.0344 IRF3 10 NR3C1 0.0039 IFIT1 NF-kB i Nuclei IRF3 Merged 5 IFIT3 Cytoplasmic STING intensity 1 REL 0 AHNAK HCT116 1 1 1 1 ISG15 _ _1 3 _ 5 3_ 8 T116 e 18 21_ RPE r PLCG2 C r r Rt Rtr H Ht Ht Ht B4GALT1 NFKB2 HCT116 TNFAIP2 +AraC SDC4 TNIP1 j 0.0305 NFKB1 <0.0001 <0.0001 2.0 -5 -3 -1 0 1 3 5 Hte5_1 1.8 1.6 1.4

Nuclear IRF31.2 RPE1 1.0 0.8

6 1 3 1 1 1 1 C _ _1 C _ _ ra 5 8 1_ E- ra 8 Rtr8_1 T1 A e P A C + R + tr3 tr Ht tr1 tr2 1 R R H 16 H H 1 RPE- HCT

Fig. 3 The cGAS–STING–TBK1–IRF3 pathway is constitutively active in trisomic cells. a Representative image of immunoblotting of TBK1, p-TBK1–S172, STING, and p-STING–S366. β-actin serves as a loading control. Quantification of TBK1 (b), p-TBK1–S172 (c), STING (d), and p-STING–S366 (e) by signal intensity of the protein bands on the gel. The signal was normalized to parental controls. Each point represents the mean from one independent biological experiment, 4–8 experiments were performed in each cell line. f Immunofluorescence images of STING localization. White arrows indicate increased clustering in trisomic cells. g Quantification of cytoplasmic STING fluorescence intensity. Delta MFI from the total cell and the nucleus was calculated for the cytoplasmic signal. At least 200 cells were analyzed in each independent biological experiment, means of each experiment are shown, and 5–12 experiments were performed in each cell line. h Proteome analysis of IRF3 and NF-kB targets. The heat map shows fold changes in trisomic and tetrasomic cells compared with parental diploid cells. *GFPT2 is located on chromosome 5, which may explain its increased abundance in cell lines with trisomy 5. i Examples of immunofluorescent images of IRF3 localization. White arrows point to the nuclei. Yellow arrow indicates perinuclear space occupied by IRF3 protein.jQuantification of the relative intensity of the nuclear IRF3 signal. Delta MFI data, characterizing IRF3 abundance in the nucleus, were quantified as a difference between nuclear and cytoplasmic MFI for each individual cell. Delta MFI data were plotted after normalization to the parental diploid controls. Diploid cells treated with DNA-damaging agent AraC were used as a positive control. At least 1000 cells were analyzed per each cell line, means of each experiment are shown, and 4–17 independent experiments for each cell line were performed. Unpaired t-test was used for statistical analysis of all the data; in e Mann–Whitney test was applied. Individual measurements, mean values, p-values, and standard deviations are shown in the plots. Statistical evaluation is summarized in Supplementary table 4, source data is available in Supplementary Data 5. Scale bar 10 µm.

6 COMMUNICATIONS BIOLOGY | (2021) 4:831 | https://doi.org/10.1038/s42003-021-02278-9 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02278-9 ARTICLE treated with the DNA damage-inducing agent AraC or with the I protein ratio was increased in parental diploids upon interferon-stimulatory DNA ISD (Supplementary Figs. 5a, b, g and Amlexanox treatment, it remained unchanged or was reduced 6c–e). Finally, accumulation of the cGAS product cGAMP was in trisomic cells (Supplementary Fig. 7d, e). Accordingly, nuclear modestly increased in trisomic cells compared with diploid localization of TFEB was not affected by inhibition of TBK1 parental cell lines (Supplementary Fig. 6f). Thus, constitutively activity. In fact, nuclear TFEB enrichment further increased after trisomic cells activate a modest, but persistent inflammatory TBK1 inhibition in trisomic cells, but not in diploid cells response via the cGAS–STING–TBK1–IRF3 axis. (Supplementary Fig. 7f, g). In conclusion, cGAS and STING are TBK1 kinase is an upstream regulator of the transcription crucial for both innate immune response and transcriptional factor IRF329. After treatment with Amlexanox, a selective activation of autophagy in aneuploid cells, whereas down- inhibitor of TBK1 kinase activity, we observed reduced regulation of TBK1 activity in trisomic cells impairs only the phosphorylation of TBK1 and reduced nuclear localization of innate immune response. IRF3 in all trisomic cell lines (Fig. 4a–d). Additionally, the expression of OAS3, IFIT1,orIFIT3 was decreased in the trisomic cell lines after TBK1 inhibition (Supplementary Fig. 7a–c). Thus, Primary cells from trisomy syndromes activate IFN type I active TBK1 is required for the IRF3-dependent gene expression response and autophagy via the cGAS–STING signaling path- in trisomic cells. way. We next asked whether similar pathways were activated in To determine whether the expression of IRF3 targets and ISGs primary cells from individuals or embryos with trisomy syn- in trisomic cells is mediated by the cGAS–STING pathway, we dromes. Interestingly, cells from individuals with Down syn- depleted cGAS with siRNA. The depletion efficiency determined drome express a specificinflammatory signature that has been by WB was approximately 50% (Fig. 4e, f). Importantly, this attributed to chromosome 21 gene composition, although many modest cGAS depletion diminished the expression of its targets of the upregulated genes are not located on this chromosome47. IFIT1 or IFIT3 in constitutive trisomic/tetrasomic cells, and in We hypothesized that this pattern reflects more a general diploid cells treated with AraC. In contrast, the expression response to the presence of an extra chromosome. This hypoth- remained largely unchanged in diploid cells upon cGAS depletion esis is supported by the fact that the same transcriptional (Fig. 4g). We conclude that the gain of a single chromosome inflammatory signature of Down syndrome patients is also found causes genotoxic stress that induces type I interferon response via in our model trisomies, regardless of the identity of the additional the cGAS–STING–TBK1–IRF3 pathway. chromosome (Supplementary Fig. 8a and Supplementary Data 4). To determine whether the constitutive cGAS–STING-dependent activation of TFEB and IRF3 is general to naturally occurring The cGAS–STING signaling pathway activates autophagy and trisomies, we compared six primary fibroblast cultures from TFEB-dependent transcription in trisomic cells. We hypothe- embryonic material trisomic for chromosomes 8, 15, 18, or 21 sized that activation of the cGAS–STING pathway is also with diploid primary fibroblasts. By IF, we detected a significant responsible for autophagy activation in trisomic cells. To test this increase in cytoplasmic dsDNA of nuclear origin in all trisomic idea, we depleted cGAS using siRNA and evaluated the markers primary embryonic cells compared with the diploid control of autophagy in these cells. For these experiments, we used the (Fig. 7a, b and Supplementary Fig. 8b–g). By WB, we observed same conditions that were sufficient to reduce the innate immune increased levels of STING, p-STING, and p-TBK1–S172, as well response (Fig. 4e–g). Strikingly, nuclear accumulation of TFEB as increased STING clustering in the cytoplasm in trisomic cells was reduced to 30–50% in trisomic cell lines upon cGAS deple- (Fig. 7c–i). Accordingly, nuclear localization of IRF3 was sig- tion, but not in diploids (Fig. 5a). We also detected a modestly nificantly enhanced in all trisomic primary cells compared with reduced expression of the TFEB target genes LC3, SQSTM1, and the diploid control (Fig. 7j, k and Supplementary Fig. 8h). Finally, LAMP2 in trisomic cells, but not in diploid controls (Fig. 5b). by mass spectrometry, we observed an increased cGAMP pro- Because we achieved only a 50% reduction of the cGAS levels by duction in Tr.21 primary fibroblasts (Supplementary Fig. 8i). This siRNA, we decided to eliminate cGAS and STING using CRISPR/ suggests that accumulation of cytoplasmic dsDNA in primary CAS9 double nickase. By IF, we observed that loss of cGAS and trisomic fibroblasts contributes to the chronic innate immune STING significantly reduced the accumulation of nuclear TFEB in response, similar to what we observed in model trisomic cell lines. trisomic cells, but not in diploid cells (Fig. 5c–f). Loss of cGAS Finally, we asked whether autophagy was increased in these also reduced nuclear IRF3 levels, with diploid cells less affected cells. Indeed, the primary fibroblasts showed significantly increased than trisomic cells (Fig. 5g, h). We additionally used our pre- levels of cytoplasmic LC3 puncta, lysosomes, as well as accumula- viously established system of CRISPRi in trisomic cells45. For this, tion of nuclear TFEB (Fig. 8a–f and Supplementary Fig. 9a). An we expressed a dCAS9–KRAB CRISPRi vector in diploid control increased LC3-II/LC3-I protein ratio was observed in all trisomic as well as in a cell line trisomic for chromosome 5 (Htr5_6). We primary fibroblasts (Supplementary Fig. 9b, c). This was then transduced the cell lines with two different guide RNAs accompanied by increased relative autophagic flux, as documented (gRNAs) targeting STING. As shown in the WB, both gRNAs by an increased LC3-II/LC3-I protein ratio after treatment with strongly reduced STING levels, whereas the control gRNA Bafilomycin 1A (Supplementary Fig. 9d, e). Importantly, the showed no effect (Fig. 6a, b). Importantly, IF imaging revealed a increased autophagic flux was independent of mTORC1 activity, as significant reduction in nuclear accumulation of TFEB and IRF3 most trisomic cells showed increased p-P70S6K–T389 phosphor- after STING depletion in trisomic cells, but not in diploid control ylation and no significant changes in p-ULK1–S757 levels cells (Fig. 6c–f). Reduced STING also caused a decrease in (Supplementary Fig. 9b, f, g). To confirm that the cGAS–STING cytoplasmic LC3 and LAMP2 signal, but only in trisomic cells pathway contributes to autophagy activation in primary trisomic (Fig. 6g–j). This confirms our hypothesis that cGAS–STING fibroblasts, we again employed the CRISRP/CAS9-mediated signaling is required for autophagy activation in response to knockout of STING and cGAS. Indeed, loss of these two factors chromosome gain. significantly reduced the accumulation of nuclear TFEB in trisomic Previously, the cGAS–STING pathway was reported to primary fibroblasts, similarly as observed in model trisomic cell activate autophagy via mTOR–TBK146. To determine whether lines (Fig. 8g–j). Taken together, our data suggest that chromo- nuclear TFEB localization in trisomic cells depends on TBK1, we some gain induces interferon type I response and autophagy via used Amlexanox to inhibit TBK1 activity. While the LC3-II/LC3- the cGAS–STING–IRF3 signaling pathway.

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a b 2.0

HCT116 RPE1 +AraC HCT116 Hte5_1 Htr18_1Htr21_3 +AraC RPE1 Rtr3_1 Rtr8_1 ls 1.8 0.0297 0.0159 ve

Amlx - + - + - + - + - + - + - + - + - + e 0.0079 0.0152 0.0357 l 1.6

p-TBK1-S172 75 100 1.4 0.0286 0.0152 -actinin 100 0.0159 0.0112 1-S172 1.2 1.0 -T BK p

c 0.8 Nuclei IRF3 Merged Nuclei IRF3 Merged ve

ati 0.6 el

R 0.4 HCT116 RPE1 0.2 0.0 Amlx - + - + - + - + - + - + - + - + - +

HCT116 RPE1 T116raC e5_1 RPE1 RPE1raC tr8_1 CT116 A t A Rtr3_1 R +AraC +AraC H HC + H Htr18_1Htr21_3 + d 4.0 0.0143

HCT116 RPE1 3.5 +AraC +AraC 0.0008 +Amlx 0.0036 +Amlx 3.0 <0.0001 3 F 2.5 <0.0001 IR r a 2.0 Htr21_3 Rtr3_1 <0.0001

ucle <0.0001 <0.0001

N 1.5 NS

1.0

Htr21_3 0.5 +Amlx Rtr3_1 +Amlx 0.0 Amlx - + - + - + - + - + - + - + - + - +

T116 RPE1 RPE1raC tr3_1 tr8_1 C AraC te5_1 tr18_1 R R e HCT116 Hte5_1 Htr18_1 Htr21_3 RPE1 Rtr3_1 Rtr8_1 HCT116H + H H Htr21_3 +A CTRL siRNA + + + + + + + cGAS siRNA + + + + + + + 75 cGAS IFIT1 IFIT3 50 g  1.6 0.0281 -actinin 100 NS 0.0200 0.0195 1.4 0.0366 NS f 0.0345 0.0500

1.4 ssion 1.2 0.0498 e 0.0256 0.0181 0.0055 r 0.0179 0.0074 0.0022 xp 1.0 1.2 0.0066 0.8

1.0 tive e a 0.6 rel

0.8 s T 0.4 0.6 IFI

lative cGAS levels levels cGAS lative 0.2 e

R 0.4 0.0 0.2 CTRL siRNA + + + + + + + + + cGAS siRNA + + + + + + + + + 0.0 CTRL siRNA + + + + + + + T116 raC te5_1 RPE1 RPE1raC tr3_1 cGAS siRNA + + + + + + + H tr21_3 R Rtr8_1 HC HCT116+A Htr18_1 H +A

T116 te5_1 RPE1 tr8_1 tr21_3 Rtr3_1 R HC H Htr18_1 H

Fig. 4 Activation of the innate immune response in constitutive trisomic cells depends on cGAS and TBK1. a Representative images of immunoblotting of p-TBK1–S172 inhibition upon treatment with Amlexanox (Amlx) (100 µM in HCT116 and 50 µM in RPE1 cell lines, 4 h of incubation). b Quantification of the TBK1 phosphorylation after treatment with Amlx. The data were normalized to the corresponding DMSO controls. In total, 3–6 independent experiments were performed in each cell line. c Examples of immunofluorescence images of IRF3 localization after treatment with Amlx. White arrows point to the nuclei and yellow indicates the perinuclear space. Scale bar 10 µm. d Quantification of the nuclear IRF3 (delta MFI) after Amlexanox treatment. At least 1500 cells in HCT116 and 500 cells for RPE1-derived cell lines were analyzed in each independent experiment; values normalized to the corresponding control are plotted, 6–17 experiments were performed in each cell line. Unpaired t-test was used for statistical analysis. e Representative immunoblot of cGAS upon treatment with siRNA. f Quantification of the cGAS protein levels upon depletion with cGAS siRNA, 3–9 independent experiments were performed. g qRT-PCR data of IRF3 target gene expression changes upon cGAS siRNA. IFIT1 and IFIT3 were measured relative to the endogenous control RPL27 and SPIKE control. All samples were normalized to the corresponding control siRNA. In total, 3–6 independent experiments were performed in each cell line. Mann–Whitney test was applied for statistical analysis, unless otherwise specified. Individual measurements, mean values, p- values, and standard deviations are shown in the plots, statistical evaluation is summarized in Supplementary table 4, and source data is available in Supplementary Data 5.

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a b LC3 SQSTM1 LAMP2 5.0 0.0286 NS 1.3 NS 4.5 NS 0.0195 0.0195 NS 0.0498 1.2 0.0358 0.0286 4.0 0.0286 0.0080 NS 0.0043 0.0139 0.0357 1.1 0.0238 NS

3.5 0.0028 ession r

3.0 0.0086 xp 1.0 0.0003 e 2.5 0.0286 0.0286 0.9 NS

Nuclear TFEB 2.0 0.8

1.5 rget gene 0.7 -ta 1.0 0.6 FEB 0.5 T 0.5 0.0 0.4 CTRL siRNA + + + + + + + + + CTRL siRNA + + + + + + + + + + + + + + + cGAS siRNA + + + + + + + + + cGAS siRNA + + + + + + + + + + + + + + +

aC RPE1aC RPE1 r tr3_1 tr8_1 te5_1 RPE1tr3_1tr8_1 RPE1 tr3_1 te5_1 Hte5_1 R R R R tr21_3 R Rtr8_1 CT116H tr21_3 HCT116HCT116+Ar Htr18_1 Htr21_3 +A HCT116H Htr21_3 HCT116Hte5_1H H H c d f cGAS-CRISPR-GFP cGAS TFEB Nuclei Merged 1.50 2.5 NS 0.0305 1.25 0.0004 2.0 NS y

Rtr3_1 it 1.00

ns 1.5 0.75 -GFP cells nte i

1.0 B 0.50 TFEB intensity FE

STING-CRISPR-GFP STING TFEB Nuclei Merged T 0.5 e 0.25 in CRISPR in CRISPR-GFP cells 0.00 0.0 CTRL STING-KO CTRL STING-KO Rtr8_1 CTRL cGAS-KO CTRL cGAS-KO Diploid Trisomic Diploid Trisomic h 1.50 0.0009 g cGAS-CRISPR-GFP IRF3 TFEB Nuclei Merged 1.25 0.0571 1.00 Rtr8_1 0.75

F3 intensity intensity F3 0.50 R I

in CRISPR-GFP cells 0.25 0.00 CTRL cGAS-KO CTRL cGAS-KO Diploid Trisomic

Fig. 5 The activation of autophagy in constitutive trisomic cells depends on cGAS. a Quantification of TFEB nuclear localization upon cGAS siRNA treatment. The nuclear TFEB was quantified from delta MFI similarly as in Fig. 1f and plotted relative to CTRL siRNA for every cell line. In total, 3–14 independent experiments were performed in each cell line. b qRT-PCR data of selected TFEB target genes after cGAS depletion with siRNA. Relative LC3, SQSTM1, and LAMP2 levels were calculated using endogenous controls RPL27 and SPIKE. The data were plotted after normalization to the corresponding control siRNA sample for each cell line. In total, 3–6 independent experiments were performed for each cell line. c, e, g Examples of immunofluorescence images of TFEB localization upon cGAS or STING knockout with CRISPR/Cas9 nickase. Yellow arrow marks the cells efficiently transfected with the CRISPR–CAS9–GFP RNP that lack either nuclear TFEB (c, e) or IRF3 (g). White arrow marks the untransfected cells with normal nuclear expression of cGAS, IRF3, and TFEB. Scale bar 10 µm. d, f, h Quantification of the MFI of TFEB (d, f) and IRF3 (h) signals in cGAS or STING knockout cells transfected with CRISPR–CAS9–GFP. The data are presented in comparison with cells transfected with control CRISPR–CAS9–GFP. Both HCT116 and RPE1 diploid and all aneuploid cell lines were grouped together. About 4–11 measurements were performed in each sample group. Mann–Whitney test was applied for the statistical analysis of the graphs a and b, unpaired t-test was used for the graphs d, f, h. Individual measurements, mean values, p value, and standard deviations are shown in the plots, statistical evaluation is summarized in Supplementary table 4; source data is available in Supplementary Data 5.

Discussion interferon response—are intimately linked in aneuploid cells. We Model constitutive trisomic cell lines have provided useful demonstrate that increased DNA damage leads to accumulation insights into the consequences of abnormal chromosome copy of cytoplasmic dsDNA in trisomic cells that, in turn, activates the numbers13. A gain of a single chromosome in somatic mamma- cGAS–STING-mediated innate immune response. This further lian cells triggers a conserved gene expression pattern that reflects activates the TBK1/IRF3-dependent expression of interferon- the proteotoxic and genotoxic stress in these cells. Correspond- stimulated genes, as well as the TFEB-dependent expression of ingly, trisomic cells suffer from impaired protein folding and lysosomal and autophagosomal factors (Fig. 8k). increased proteasomal and autophagic activity, as well as from an Previous data revealed an increased expression of autophagoso- accumulation of DNA damage and impaired replication. Human mal and lysosomal factors and increased levels of autophagosomes trisomic cells also show increased expression of several factors and lysosomes accompanied by a reliance on phagolysosome that are involved in interferon type I signaling21,22. Activation of activity in somatic trisomic mammalian cells11,17. The presence of both the inflammatory response and autophagy has been pre- extra chromosomes leads to the production of excessive proteins viously recognized in trisomic cells, but the upstream triggers that may overwhelm the capacity of lysosomal degradation, as was have never been identified. Here, we show that these three phe- observed in cells acutely missegregating chromosomes39.Wepro- notypes—increased DNA damage, autophagy activation, and pose that cells with extra chromosomes adapt to the altered protein

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ab2.0 0.0286

ls 0.0286 0.0265 e 1.5 HCT116 Htr5_6 RL D1 D2 CTRL K KD2 CT KD1 K 1.0 STING 37 ive STING lev STING ive -actinin 100 t 0.5 Rela 0.0 L TR D2 D2 C KD1 K CTRL KD1 K HCT116 Htr5_6

c <0.0001 CTRL-CRISPR STING-CRISPR d 3.0 Nuclei TFEB Merged Nuclei TFEB Merged 2.5 NS 2.0 HCT116 1.5 1.0 Nuclear TFEB Nuclear 0.5 Htr5_6 0.0 L L R D1 D2 TR D1 D2 CT K K C K K HCT116 Htr5_6

0.0044 0.0121 e Nuclei IRF3 Merged Nuclei IRF3 Merged f 5

4 HCT116 NS 3

2 Nuclear IRF3 Htr5_6 1

0 L R RL T D2 D1 C KD1 K CT K KD2 HCT116 Htr5_6 gh Nuclei LC3 Merged Nuclei LC3 Merged 80 <0.0001

60 HCT116 NS ae per cell t c 40

Htr5_6 20

LC3 positive pun 0 L TR TRL D1 C KD1 KD2 C K KD2 HCT116 Htr5_6 i j Nuclei LAMP2 Merged Nuclei LAMP2 Merged 80 <0.0001

HCT116 60 NS

40

Htr5_6 positive punctae per cell 20 2

LAMP 0 L R RL T D2 T D1 D2 C KD1 K C K K HCT116 Htr5_6 homeostasis and increased requirement for autophagy by con- activated as a part of the ER stress-induced unfolded protein stitutive nuclear localization of TFEB, which augments the response48,49. While these pathways may contribute to TFEB acti- expression of the required autophagy and lysosomal factors. Strik- vation in individual trisomic cell lines, our data suggest that none of ingly, the constitutive nuclear localization of TFEB is independent them serves as a universal trigger of nuclear TFEB localization and of mTOR in trisomic cells. Several mTORC1-independent transcriptional activation of autophagy in our model system as well mechanisms of TFEB activation were recently described, includ- as in primary embryonic fibroblasts. Instead, we propose that ing through the activation of AKT kinase or via PERK, which is nuclear TFEB localization depends on the cGAS–STING pathway.

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Fig. 6 The activation of autophagy in constitutive trisomic cells depends on STING. a Immunoblotting of STING protein upon knockdown using the STING–CRISPR–CAS9–KRAB system (two different gRNAs were used, KD1 and KD2). α-actinin was used as a loading control. b Quantification of three independent knockdown experiments from a). Four experiments were performed in each cell line. Mann–Whitney test was applied. c Immunofluorescence images of TFEB in STING–CRISPR–CAS9–KRAB and CTRL–CRSIPR–CAS9–KRAB-transfected cells. White arrows point to nuclei negative for TFEB and yellow arrows point to the nucleus positive for TFEB. d Quantification of nuclear TFEB intensities upon STING knockdown. e IRF3 immunofluorescence images in STING-KD cells. White arrows indicate the absence of IRF3 in the nucleus, while yellow arrows indicate IRF3-positive nuclei. In total, 400 cells were analyzed in each cell line, 11–16 independent experiments were performed. f Quantification of the IRF3 nuclear intensity in STING-KD cells normalized to the control cells. In total, 400 cells were analyzed in each cell line, means of individual experiments are shown, and 5–14 experiments were performed. g LC3 immunofluorescent images with STING-KD. White arrows point to LC3-positive puncta. h Quantification of the number of LC3-positive puncta per cell with STING-KD. i Immunofluorescence images of LAMP2 in STING–CRISPR–CAS9–KRAB KD cells. White arrows point to LAMP2-positive puncta. j Quantification of LAMP2-positive puncta per cells in STING-KD cells. In each experiment, 30–60 cells were analyzed for LC3 and LAMP2 puncta quantification. For all graphs, unpaired two-tailed t-test was used for statistical analysis, unless otherwise specified; statistical evaluation is summarized in Supplementary table 4, source data is available in Supplementary Data 5. Individual measurements, mean values, and standard deviations are shown on the plots. Scale bar 10 µm.

Can trisomy per se activate the cGAS–STING pathway? Here, trisomic cells. Moreover, while we analyzed several different tri- we demonstrate that trisomic cells contain increased levels of somic cell lines, it remains to be determined whether this phe- cytoplasmic dsDNA, a well-known activator of the cGAS–STING notype is universal, or whether it can be influenced by the identity pathway. of the extra chromosome. Work from several laboratories recently revealed that increased Recently, an evolutionary conserved role of cGAS–STING DNA damage can lead to elevated levels of cytoplasmic dsDNA, signaling in autophagy activation was proposed37. Here, we which is recognized by the cytosolic receptor cGAS23,25,41. Gain demonstrate that the nuclear localization of TFEB in model tri- of a chromosome was shown to cause genotoxic stress due to somic cells at least partly depends on cGAS activity. abnormal replication3,19,20, and we propose that this may be the cGAS–STING can activate TFEB via TBK1 and mTORC1, as has reason for the accumulation of dsDNA in the cytosol of trisomic been proposed using chronic immune activation in a Trex1−/− cells. The DNA may also originate from damaged mitochondria mouse model60, but also via unknown mechanisms indepen- that are often found in trisomic cells50; however, our data suggest dently of TBK137, suggesting that the involvement of TBK1 in rather a nuclear origin of the cytosolic DNA. Micronuclei arising autophagy regulation depends on the context. We show that from missegregation of chromosomes were previously reported to inhibition of TBK1 did not influence autophagy in trisomic cells, activate the cGAS–STING pathway and, subsequently, the NF- nor did it reduce the nuclear localization of TFEB. The exact κB-mediated transcription51–53; however, the used model tri- mechanism of TFEB regulation upon cGAS–STING pathway somic cell lines do not missegregate chromosomes at a sig- activation in trisomic cells will be the subject of future studies. nificantly higher rate than the parental diploids20. Thus, We made the initial observations in our model human cell lines chromosomally unstable cells that continuously missegregate engineered to carry one or two extra copies of individual chro- chromosomes, thereby producing micronuclei, may activate mosomes, but, notably, the observed phenotypes also hold true cGAS–STING via a different mechanism than constitutive triso- for primary embryonic fibroblasts from trisomic embryos. By mies with low rates of chromosomal instability. testing four different trisomies from six embryos of different sex It should be noted that the abundance of the cGAS protein and and origin, we demonstrate that these cells accumulate cyto- the cGAS–STING activity varies in different cell lines. Indeed, plasmic dsDNA and activate the innate immune response as well there is some discrepancy regarding the detection of cGAS as autophagy in an mTORC1-independent manner. Individuals expression in HCT116 and RPE1 cell lines, where some labora- with Down syndrome are often predisposed to autoimmune tories have found no cGAS protein54, while others did (e.g., disorders, such as insulin-dependent diabetes mellitus, celiac refs. 45,55–57). We show that there is a functional cGAS–STING disease, and others61. Recently, it was proposed that DS indivi- pathway in HCT116 and RPE1 cell lines that becomes chronically duals exhibit dysregulated interferon signaling due to the chr. 21- activated in constitutively aneuploid cells. This finding is also specific overexpression of immune factors. Yet, transcriptional supported by data we obtained in primary fibroblasts from tri- analysis of differentially regulated genes in cells from patients somic embryos. with trisomy 21 showed that the top 13 upregulated factors are Importantly, cytoplasmic dsDNA that accumulates in trisomic IFN-related factors, which are mostly not encoded on chromo- cells activates the cGAS–STING pathway, as documented by some 2147,62. We present here evidence that the upregulated increased cGAMP production, followed by increased TBK1 interferon response in trisomic cells is independent of the identity kinase activity. Our data demonstrate that TBK1, via IRF3 and of the extra chromosome, as we observed the same response in STAT1, is responsible for the elevated expression of type I cells trisomic for chromosomes 8, 15, 18, or 21. We propose that interferon response and interferon-stimulated genes in trisomic the upregulation instead arises as a direct consequence of con- cells. The observed activation of the innate immune response in stitutive innate immune pathway activation in trisomic cells that multiple different trisomic cells was consistent, significant, and leads to low-grade inflammation. independent of the identity of the extra chromosome, although Our data, together with previously published findings, suggest markedly lower than upon interferon stimulation or poly-IC the following model (Fig. 8k). Cells with any additional chro- transfection. This is consistent with previous observations of a mosome suffer from a chronically imbalanced proteome that moderate increase in ISG expression in response to genotoxic causes well- documented aneuploidy-associated stresses7,13. This conditions52,58,59. Collectively, our data show that a gain of even a negatively impacts DNA replication and repair, which leads to single chromosome induces modest, but chronic activation of the accumulation of cytoplasmic dsDNA and subsequent activation innate immune pathway accompanied by ISG expression. In of the cGAS–STING pathway. We propose that the cGAS–STING future experiments, it should be determined whether and how the signaling not only activates the type I interferon response, but expression of ISGs affects the survival and proliferation of also executes a transcriptional program to upregulate autophagy

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ab Nuclei dsDNA Merged <0.0001 c 2.5 0.0013 0.0014 0.0026 Nuclei STING Merged inset 2.0 0.0004 Diploid <0.0001 Diploid

asmic dsDNA asmic 1.5 l

Cytop 1.0 Tr.8 Tr.18_A

B 15 _B _ Tr.8 r. _A 8 1 T 8 1 r.1 r. .21_A .2 Diploid T T Tr Tr d efg 0.0286 NS 0.0003 Diploid NS 0.0286 8 Tr.8 Tr.18_A Tr.18_B Tr.21-A 0.0007 100 3.0 4 7 0.0008 p-TBK1-S172 75 s 0.0126 2.5 vel 6 100 e TBK1 75 3 5 2.0 -actin 50 37 4 levels BK1 1.5 2 50 T

p-STING-S366 TBK1-S172 l 3 37 - p tive

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0.0006 h i 0.0238 jk <0.0001 0.0206 0.0043 3.5 0.0003 <0.0001 <0.0001 0.0022 Nuclei IRF3 Merged 0.0009 5 8 3.0 0.0149 s l <0.0001 ve 7 2.5 4 els

6 le 6 Diploid 6 2.0 3 5 G-S3 1.5 N 4 I T Nuclear IRF3

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l 1 e

R d 8 5 B i r. A _A 0 0 lo T r.1 8_ p T 1 18_B 21_ i r. r. r.21 r. 8 B d A B D T T T T . A _ _ Tr loi Tr.8 1_A 8_ 18 18 2 .1 r. r. r.18_ r. Diploid Tr T Tr.21_A Dip T T T

Fig. 7 Innate immune response is activated in primary fibroblasts with trisomy. a Examples of immunofluorescence images of nuclear and cytoplasmic dsDNA. White arrows point to the dsDNA-positive signal outside of the nucleus. b Quantification of the relative intensity of cytoplasmic dsDNA signal. At least 700 cells were analyzed in each experiment, 6–9 experiments were performed in each cell line, and means of individual experiments are plotted. Delta MFI data for the cytoplasmic dsDNA were calculated as the difference between the signals from the total cellular area and the nuclear area; plotted are data normalized to the diploid controls. c Immunofluorescence images of STING. White arrows indicate the STING-positive clusters. d Quantification of the cytoplasmic STING intensities, calculated similarly as in b. In total, 100 cells were analyzed in each experiment, means of each experiment are plotted, and 8–12 experiments were performed for each cell line. e Immunoblot showing total and phosphorylated levels of TBK1 and STING proteins and its relative quantification. f–i In total, 3–7 experiments were performed in each cell line. j Localization of IRF3 in primary fibroblasts. White arrows point to the nuclei with a strong nuclear IRF3 signal in trisomic cells. k Quantification of the relative IRF3 nuclear localization. At least 1400 cells were analyzed in 5–10 independent experiments, means of each experiment are plotted. Delta MFI data for the nuclear IRF3 were calculated and normalized to the diploid controls. Unpaired two-tailed t-test was used for statistical analysis; f, g, i were analyzed with Mann–Whitney test. Individual measurements, mean and p- values, and standard deviations are shown in the plots; statistical evaluation is summarized in Supplementary table 4, source data is available in Supplementary Data 5. Scale bar 10 µm. and lysosomal biogenesis independently of mTORC1 signaling, (University of Manchester, UK). HCT116 H2B-GFP was generated by lipofection thus connecting genetic instability of trisomic cells to autophagy (FugeneHD, Roche) of HCT116 (ATCC No. CCL-247) with pBOS–H2B–GFP (BD ’ 63 activation. Our findings provide a rationale for why aneuploid Pharmingen) according to the manufacturer s protocols . Trisomic and tetrasomic cell lines were generated by microcell-mediated chromosome transfer as previously cells are recognized by the immune system and removed from described11. The cell line Hte5_1 was kindly provided by Minoru Koi, Baylor tissues2,3. These cellular changes occur independently of the University Medical Centre, Dallas, TX, USA. All cell lines were maintained at 37 °C identity of the extra chromosome and occur also in primary with 5% CO2 atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) con- embryonic fibroblasts with trisomy syndromes. Importantly, the taining 10% fetal bovine serum (FBS), 100 U penicillin, and 100 U streptomycin. All cell lines tested negative for mycoplasma contamination. The human primary observed activation of innate immunity by chromosome gain embryonic fibroblasts were purchased from the cell repository of Coriell Institute provides a new insight into possible causes of chronic low-grade for Medical Research, NJ 08103, USA. Cells were propagated in similar culture inflammation that is frequently observed in cancer and in trisomy conditions for up to three passages and DMEM was supplemented with 15% of syndromes. FBS. All experiments were performed at subconfluent conditions. The full list of cells used in the study is presented in Supplementary table 1.

Methods Cells used in the study and culture conditions. RPE1 hTERT (referred to as siRNA, ISD, and DNA transfection. The siRNA transfection was performed RPE1) and RPE1 hTERT H2B-GFP were a kind gift from Stephen Taylor according to the manufacturer’s protocol. In total, 2 × 105 cells per well were seeded

12 COMMUNICATIONS BIOLOGY | (2021) 4:831 | https://doi.org/10.1038/s42003-021-02278-9 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02278-9 ARTICLE

a c e LC3 Nuclei Merged Nuclei Lysotracker Merged Nuclei TFEB Merged

Diploid Diploid Diploid

Tr.18_A Tr.21_A Tr.18_A

<0.0001 p<0.001 0.0023 bdp<0.001 f 4.0 120 80 <0.0001 70 3.5 100 NS 60 3.0 80 50 FEB 2.5 T 60 40 2.0

cta per cell 30 40 n 1.5

20 Nuclear pu

20 ysotracker positive 1.0

L 10 0 0 0.5 LC3 positive puncta per cell 0.0 8 d B A r. B A i _A T _A 8_ _ lo Tr.8 8 8_ 1_ Tr.15 .1 21 .21_B .1 1 2 8 5 B r.18 r r. r r. id r. 1 _A _ _A _B Diploid T T T Tr Dip T Tr. T o T r. 8 1 1 pl T 1 2 i r.18 r. . r.2 D T T Tr T

g cGAS-CRISPR-GFP cGAS TFEB Nuclei Merged j h 0.002 0.0136 Tr.18_A 5 3.0 NS 2.5 4 NS 2.0 3 1.5 i STING-CRISPR-GFP STING TFEB Nuclei Merged 2 1.0 Nuclear TFEB Nuclear TFEB 1 0.5 in CRISPR-GFP cells Tr.18_A in CRISPR-GFP cells 0 0.0 CTRL cGAS-KO CTRL cGAS-KO CTRL STING-KO CTRL STING-KO Diploid Trisomic Diploid Trisomic

k mTORC1 ULK1 AMPK1 TBK1 STING ? calcineurin autophagosome cGAMP TFEB TFEB-p IRF5 TBK1-p IRF7 cGAS ATG3, ATG7 LC3, P62 fusion IRF3 IRF3-p with lysosome cytoplasmic DNA gene expression IRF3-p DNA damage interferon  nucleus TFEB 2N+1 lysosome & autophagy

IFIT1, IFIT3, OAS3 STAT-p ISG15, MX1 IFN type I response

in DMEM media with supplements in a six-well dish and incubated overnight; and plasmid DNA were transfected with a similar strategy and collected after 4 and media was replaced with DMEM without antibiotics 4 h ahead of the transfection. 6 h for qPCR analysis. First, 20 pmol siRNA (cGAS siRNA sc-95512, CTRL siRNA sc-36869, Santa Cruz) was diluted in 100 µL of transfection medium (SC-36868, Santa Cruz) and 6 µL of transfection reagent (SC-29528, Santa Cruz) were diluted to 100 µL with trans- Drug treatment. For Amlexanox (4857, Tocris) experiments, 2.5 × 105 RPE1 cells fection medium. Then, siRNA and transfection reagent solution were gently mixed or 1 × 106 HCT116 cells were seeded into 6-cm dishes and incubated overnight. and incubated at room temperature for 30 min. Next, the entire 200-µL siRNA/ The next day, 100 µM or 50 µM concentrations were used for RPE1 and HCT116 transfection solution was added, dropwise, on top of cells. Cells were then left to cell lines, respectively. For western blot, cells were incubated with Amlexanox for 4 incubate for 16 h. The next day, the media was exchanged for fully supplemented h, which was sufficient to decrease the levels of p-TBK1–S172. For qRT-PCR media, and the cells were incubated for 24 h before collection for RT-qPCR, experiments, cells were incubated overnight. For the positive control, AraC (C6645, Western blot, or immunofluorescence. Double- stranded ISD DNA oligo (10 μg) Sigma) was added at 50 µM to the control cells and incubated overnight. To inhibit

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Fig. 8 Autophagy is activated in primary fibroblasts with trisomy. a Immunofluorescence images of LC3 in trisomic fibroblasts. White arrows indicate LC3- positive puncta. b Quantification of the numbers of LC3-positive puncta per cell. In total, 6–25 cells were analyzed in each experiment. c Representative images of cells labeled with lysotracker. d Quantification of the number of lysotracker-positive puncta per cell. In total, 8–25 cells were analyzed in each experiment. e Representative images of TFEB localization in primary fibroblasts. White arrows indicate nuclei positive for the TFEB protein. f Quantification of the relative nuclear TFEB signal. Delta MFI data were calculated similarly as in Fig. 1f and plotted after normalization to diploid control cells. At least 1400 cells were analyzed in 5–9 independent experiments, means of each experiment are plotted. g, i Representative images of TFEB localization in trisomic fibroblasts transfected with GFP-positive cGAS–CRISPR–CAS9 RNP (g)orwithSTING–CRISPR–CAS9 RNP (i). Transfected cells (GFP positive) are marked with yellow arrows, untransfected cells with white arrows. h, j Quantification of nuclear TFEB intensity inhibited upon cGAS (g) and STING (i)CRISPR–CAS9-KO. Data were normalized to the control transfected with CTRL–CRISPR–CAS9 RNP. In total, 5–12 cells were analyzed in each group. Unpaired t-test was used for statistical analysis. Individual measurements, mean and p-values, and standard deviations are shown on the plots, statistical evaluation is summarized in Supplementary table 4, and source data is available in Supplementary Data 5. Scale bar 10 µm in all images. k Schematics of cGAS–STING involvement in TFEB-mediated activation of autophagy in trisomic cells. DNA damage due to genotoxic stress in trisomic cells leads to accumulation of dsDNA in the cytosol, where it is bound by the cytosolic receptor cGAS. Activated cGAS produces cGAMP -signaling molecule that induces STING clustering and TBK1 activation and, subsequently, IRF3-dependent transcription of its target genes. Additionally, activated STING triggers translocation of TFEB to the nucleus in an mTOR- independent manner. In this context, TFEB induces expression of lysosomal and autophagy factors, which in turn promotes autophagy and lysosomal degradation.

mTOR complex activity, Torin 1 (4247, Tocris) was applied overnight at a con- protocol. Information regarding the used antibodies can be found in Supplemen- centration of 2 µM. To measure the autophagic flux, the lysosome inhibitor Bafi- tary table 2. lomycin A1 (1334, Tocris) was used for 4 h at 100 nM. Autophagy activity assay. HCT116 and RPE1 diploid and aneuploid cells were fl CRISPR/CAS9 cGAS and STING depletion. cGAS and STING Double Nickase seeded 1 day before transfection to achieve 40% con uency at the transfection day. Plasmid (sc-403354-NIC, sc-403148-NIC, Santa Cruz Biotechnology) was used to The ptfLC3 plasmid (mRFG-GFP-LC3, Addgene) was transfected using Lipo- fectamine 2000 according to the manufacturer’s protocol. Two days after trans- remove the target protein. Control Double Nickase Plasmid was used in parallel in fi the same experimental conditions (sc-437281, Santa Cruz Biotechnology). The fection, cells were xed with ice-cold methanol for 10 min and SlowFade Gold transfection procedure was performed according to the manufacturer’s protocol Antifade reagent with DAPI. mRFP-positive (red puncta) autolysosomes and GFP/ mRFP-positive (yellow puncta) autophagosomes were visualized with ×60 objective available online, using UltraCruz Transfection Reagent (sc-395739, Santa Cruz fl Biotechnology), Plasmid Transfection Medium (sc-108062, Santa Cruz Bio- and counted to estimate the autophagy ux. technology). The transfected cells were visualized with GFP encoded by the same plasmids. Microscopy. Spinning-disk confocal laser microscopy was performed using a fully An alternative approach to depleted STING was to use HCT116 and Htr5_6 cell automated Zeiss inverted microscope (AxioObserver Z1) equipped with a MS-2000 lines that stably express dCAS9–KRAB. Using viral transduction, we introduced stage (Applied Scientific Instrumentation, Eugene, OR), the CSU-X1 spinning-disk two variable guide RNAs for successful STING knockdown, in a similar strategy as confocal head (Yokogawa), and LaserStack Launch with selectable laser lines we described before45. (Intelligent Imaging Innovations, Denver, CO). Image acquisition was performed using a CoolSnap HQ camera (Roper Scientific) and a 20x-air, 40x-air, or 63x-oil objective (Plan Neofluar × 40/0.75, Plan Neofluar ×20/0.75) under the control of Immunoblotting. To prepare the whole-cell lysate, cells were lysed with RIPA the SlideBook 6 × 64 program (SlideBook Software, Intelligent Imaging Innova- buffer and sonicated. After spinning down, the supernatant was used for protein tions, Denver, CO, USA). concentration measurements with the Bradford protein assay. To prepare samples for SDS-PAGE, the whole-cell lysate was diluted to 1 µg/µL with Lämmli solution and water, and the samples were boiled for 5 min at 95 °C. Gels were loaded with Image analysis. CellProfiler, a cell image analysis software (https://cellprofiler.org/), 10 µg of sample and run for 15 min at 100 V, followed by 210 V for 35 min. was used to quantify microscopy images. A mask of the nucleus of each cell using Precision Plus 50 Protein All Blue Standard was used as a marker. The proteins nuclear staining was taken and applied to an image of specific staining to quantify were transferred onto a nitrocellulose blotting membrane via semidry transfer the nuclear signal. To quantify the cytoplasmic signal intensity, the mask area was using Bjerrum Schafer–Nielson transfer buffer and the Trans-Blot® Turbo™ extended around the nucleus, without counting the nucleus. The difference in values (BioRad Laboratories, Hercules, USA). Next, the membranes were blocked for 30 for nuclear and cytoplasmic signals of specific staining was used to determine min with 5% milk solution, and primary antibodies were added and followed by increased or decreased presence of proteins in the nucleus. To collect the full signal incubation at 4 °C overnight. The next day, secondary antibodies were added fol- from the cytoplasm, the cell mask was used and the nuclear signal was subtracted as lowed by incubation for 1 h at room temperature. To visualize the signal on the described above. Three independent biological replicates were performed for each membranes, a horseradish peroxidase solution (ECL) was used and imaged with experiment, at least 300 cells were scored in each experiment. In the CRISPR/Cas9 the Azure c500 system (Azure Biosystems, Dublin, USA). ImageJ software was then experiment with CRISPR–GFP double-nickase experiment, all imaged cells were used to quantify each protein band intensity, which was then normalized to its scored. respective loading control. Protein-loading amount was controlled for either using α Ponceau staining or -actinin intensity. Information regarding the used antibodies Quantitative real-time PCR. To isolate RNA, Qiagen instructions for RNeasy can be found in Supplementary table 2. All immunoblotting experiments were Mini Kit and On-column Dnase Digestion (Qiagen) were followed. cDNA was performed in at least three biological replicates. made with the iScriptTM Advanced cDNA Synthesis Kit (1725037, BioRad) using 2 µg of RNA. SPIKE (RS25SI, TATAA Biocenter AB) was used as an exogenous reference to control for technical variability. RPL27 primers were used as an Immunofluorescence. HCT116 and RPE1 cells and their aneuploid derivatives 4 3 endogenous reference to control for RNA quantity and quality in each sample. were seeded to a 96-well plate (HCT116: 10 cells per well, RPE1: 10 cells per well) qPCR was performed in a 96-well plate, where each sample was loaded in triplicate, and incubated at 37 °C and 5% CO2 on the day before experiments. For cell ’ fi according to the SYBR Master Mix manufacturer s instructions. The results were xation, a 3% formaldehyde solution was used for 15 min at room temperature. examined with the BioRad CFX Manager. Primer sequences are in Supplementary Next, cells were permeabilized with 0.1% Triton for 20 min at room temperature. table 3. Then, cells were preblocked with 3% bovine serum for 30 min at room tempera- ture. Primary antibodies were diluted in blocking solution (Supplementary table 2) and incubated overnight at 4 °C. The next day, cells were washed and secondary Transcriptome and proteome analysis. Genome-wide proteome and tran- antibodies were added in a concentration of 1 mg per ml followed by incubation for scriptome expression profiling of HCT116- and RPE1-derived aneuploid cell lines 1 h in the dark at room temperature. SYTOX® green (Invitrogen) or DAPI was previously performed11,21. As previously described, bioinformatics analysis of (Invitrogen) were used to localize the nucleus. For cytoplasmic staining, the HCS the proteomic and transcriptomic data was performed using Perseus (1.6.2.3) as Cell Mask (H327 12 Component, 701618, Invitrogen) was applied. The cells were part of the MaxQuant Software Package. For comparison of each aneuploid cell line covered with SlowFade Gold Antifade (Invitrogen). To localize mitochondria, with the corresponding control cell line, gene expression fold change ratios were MitoTrackerTM Red CMXRos (M7512, Invitrogen) was used (100 nM for 45-h calculated. We used the same datasets to analyze autophagy- and immune incubation at 37 °C). 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45. Vasudevan, A. et al. Single-chromosomal gains can function as metastasis Acknowledgements suppressors and promoters in colon cancer. Dev. Cell 52, 413–428.e416 (2020). We thank Yvonne Kraus and Johanna Buchheit for their help with the experiments. The 46. Bodur, C. et al. The IKK-related kinase TBK1 activates mTORC1 directly in work was supported by the Rheinland-Palatine Research Initiative BioComp 3.0 and by response to growth factors and innate immune agonists. EMBO J. 37,19–38 the German Research Foundation STO 918/5 and 918/7 to ZS; by an ERC consolidator (2018). grant (ERC-CoG ProDAP, 817798), the German Research Foundation (PI 1084/5, 47. Sullivan, K. D. et al. Trisomy 21 consistently activates the interferon response. TRR179/TP11, TRR237/A07), and the German Federal Ministry of Education and Elife 5, e16220 (2016). Research (COVINET) to AP. 48. Palmieri, M. et al. mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases. Nat. Commun. 8, 14338 (2017). Author contributions 49. Hsu, C. L. et al. MAP4K3 mediates amino acid-dependent regulation of MK performed most experiments, supervised CMS and SK, and analyzed the data, CMS, autophagy via phosphorylation of TFEB. Nat. Commun. 9, 942 (2018). LLA, and NKC performed experiments related to the inflammatory response, SK per- 50. Yim, A. et al. mitoXplorer, a visual data mining platform to systematically formed experiments related to TBK1 function, ND performed experiments related to analyze and visualize mitochondrial expression dynamics and mutations. autophagy activation, AP and ZS obtained funding and supervised the project, and ZS – Nucleic Acids Res. 48, 605 632 (2020). initiated the project and wrote the paper; all authors commented on the paper. 51. Bakhoum, S. F. et al. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature 553, 467–472 (2018). 52. Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome Funding instability to innate immunity. Nature 548, 461–465 (2017). Open Access funding enabled and organized by Projekt DEAL. 53. Harding, S. M. et al. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466–470 (2017). 54. Chen, J. et al. Cell cycle checkpoints cooperate to suppress DNA- and RNA- associated molecular pattern recognition and anti-tumor immune responses. Competing interests Cell Rep. 32, 108080 (2020). The authors declare no competing interests. 55. Basit, A. et al. The cGAS/STING/TBK1/IRF3 innate immunity pathway maintains chromosomal stability through regulation of p21 levels. Exp. Mol. Med. 52, 643–657 (2020). Additional information 56. Cao, Y. et al. 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