Viral mimicry acts as a tumor suppressor in colitis

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: major_revision

Abstract
Transposable elements (TEs) comprising nearly 50% of the genome are generally silenced by epigenetic mechanisms. Epigenetic anti-cancer drugs can lead to their re-expression, however, the role of TEs in tumorigenesis is unknown. Here, we demonstrate that TEs and their activation of a viral mimicry response plays an important tumor suppressive role in inflammation. We discovered that both patients and mice with colitis express TEs that lead to a viral mimicry response. Interestingly, this response inhibits stemness of cancer-initiating cells. Further activation of viral mimicry by DNA hypomethylation inhibits tumorigenesis. Conversely, knockout of the anti-viral signaling protein MAVS promotes tumorigenesis and reverses the anti-tumor effect of DNA hypomethylation, confirming a tumor suppressive role of viral mimicry. Consistent with this finding, patients with colitis-associated dysplasia show decreased expression of TEs and interferon-related genes. These findings suggest that activation of viral mimicry inhibits stemness and plays a key tumor suppressive role in inflammation.

Introduction
The vast majority of the human genome consists of noncoding intergenic DNA and intronic regions of DNA. This noncoding DNA includes repetitive sequences that consist of transposable elements (TEs), microsatellites, and DNA duplications1,2. Interestingly, TEs, including long-interspersed elements (LINEs) and small-interspersed elements (SINEs) that make up much of the repetitive DNA, are able to duplicate and move around in the genome, whereas endogenous retroviruses (ERVs) are mostly immobile3. In homeostasis, however, expression of these TEs is repressed by epigenetic mechanisms such as DNA methylation4–6 and histone modification7–12. In contrast, aberrant expression of TEs has been observed in disease states such as cancer13–15 and autoimmune diseases16–20, including the neuroinflammatory disease Aicardi–Goutières syndrome16,21 and systemic lupus erythematosus17,18,22.

Recent studies have demonstrated that anti-cancer drugs that affect DNA methylation, such as 5-AZA-2′-deoxycytidine (5-AZA), lead to re-expression of TEs that form immunostimulatory nucleic acids such as double-stranded RNAs (dsRNAs)23–26. These dsRNAs in turn activate innate immune sensors melanoma differentiation-protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) that signal through mitochondrial antiviral-signaling protein (MAVS) to induce an anti-viral type I or III interferon response and cell death23,24,27–29. This anti-viral-like immune response is known as a “viral mimicry” response. Indeed, DNA demethylating anti-cancer drugs are postulated to kill cancer cells, at least in part, through activation of “viral mimicry” and interferon signaling23,24,28,30–33.

These observations have led to speculation that re-expression of TEs during injury serves as a natural defense mechanism to prevent tumorigenesis34. It is not known, however, whether blocking the viral mimicry response makes cells prone to tumorigenesis or can reverse the anti-cancer effects of DNA demethylating drugs. Thus, we hypothesized that re-expression of TEs during injury (i.e., colitis) leads to a viral mimicry response that suppresses tumor initiation, and therefore, knockout of the viral mimicry response will promote tumorigenesis. Consistent with this hypothesis, we here provide evidence that TEs become re-expressed during inflammatory injury of the colon in both mice and humans. Moreover, we demonstrate that further upregulation of TEs by DNA hypomethylation, using 5-AZA or knockout of the DNA methyltransferase enzyme DNMT1, inhibits colonic tumorigenesis by activation of a viral mimicry response. Importantly, we show that blocking the viral mimicry response by knocking out the innate immune sensor MAVS promotes tumorigenesis, reversing the effects of 5-AZA or DNMT1 KO. Remarkably, the viral mimicry response inhibits the stemness of cancer-initiating cells in a cell-autonomous manner independent of an immune cell response. These findings suggest that viral mimicry serves as an important tumor suppressor during cancer initiation.

Results
Viral mimicry is induced in active colitis and downregulated in patients with IBD-associated dysplasia
To first examine whether colonic injury leads to re-expression of transposable elements, we examined expression of transposable elements and interferon response genes in patients with inflammatory bowel disease (IBD). We examined gene expression in the RNA-sequencing dataset GSE8368735 of surgical specimens from 61 control patients without IBD versus 77 inflamed tissues from patients with active IBD. We found that transposable elements of multiple subclasses including Alu elements (i.e., SINEs) known to engage MDA536–38 and interferon response genes such as IFNAR1/2, IRFs, ISG15, and MX1 (see Table S1 for a full list of genes) were significantly upregulated in patients with active IBD versus controls (Fig. 1a, b, Supplementary Fig. 1a). Indeed, the majority of TEs were upregulated with inflammation (p=3.21×10−21) (Supplementary Fig. 1b), and we found a positive correlation between IFN and TE expression (Supplementary Fig. 1c), consistent with the activation of a viral mimicry response. Moreover, the evolutionary age of Alus has previously been associated with their predicted dsRNA foldability37. Thus, we analyzed the Alus based on evolutionary age, but did not detect a difference in the expression levels of Alus between patients with IBD versus healthy controls when divided according to their evolutionary ages (Supplementary Fig. 1d). These data suggest that it may be specific TEs, rather than a broader class of TEs, that drive stimulation of downstream interferon signaling.

To further examine whether the viral mimicry response was altered in the setting of colitis-associated dysplasia, we analyzed a separate publicly available dataset that included RNA expression data of colonic biopsies from 41 healthy controls, 22 patients with IBD without dysplasia, and 7 patients with IBD-associated dysplasia (non-dysplastic adjacent region) (GSE23052439). Similar to the first dataset, we found expression of interferon response genes was upregulated in patients with IBD when compared to healthy controls. In patients with IBD-associated dysplasia, however, the expression of these genes was downregulated when compared to those patients without dysplasia (Fig. 1c–e). Interestingly, when comparing IBD patients with or without dysplasia, the top 10 upregulated GO term pathways in those without dysplasia were those related to interferon signaling and responses to virus (Fig. 1f). These data suggest that in the disease progression from IBD to IBD-associated dysplasia, the viral mimicry response pathways are downregulated. Indeed, transposable element expression was downregulated in patients with IBD-associated dysplasia versus patients with IBD without dysplasia (Fig. 1g). This pattern also held true for the comparison between healthy controls and patients with IBD-associated dysplasia where we similarly observed downregulation of TEs (Supplementary Fig. 1e). Furthermore, patients with sporadic colorectal cancer from the GSE230524 dataset (n=8) had decreased expression of TEs when compared to healthy controls, consistent with TE expression being reduced during tumorigenesis even in the absence of inflammatory injury (Supplementary Fig. 1f). Together, these data suggest that upon injury or inflammation, cells upregulate TEs and interferon signaling. However, with progression from inflammation to inflammation-associated dysplasia, the viral mimicry response is downregulated.

Activation of viral mimicry by DNA hypomethylation inhibits colitis-associated cancer
We next sought to validate our human observations in a mouse model of colitis. In colonic epithelial cells isolated from mice treated with dextran sodium sulfate (DSS), we found the expression of several classes of transposable elements such as LINE1, SINEs, and Mu-LV was significantly increased in colitis (Fig. 2a). Interferon response genes were similarly increased in epithelial cells from mice with colitis versus controls (Fig. 2b), consistent with the observations made in patients with IBD. To further examine the relationship between colitis and the viral mimicry response, we treated mice with different doses (0.5, 1.5, and 2.5%) of DSS (Supplementary Fig. 2a). Mice administered 2.5% DSS displayed greater body weight loss (Supplementary Fig. 2b) than those given 0.5% or 1.5% DSS, and mice administered either 1.5% or 2.5% DSS displayed decreased colon length, histological damage, and increased MPO activity when compared to controls or mice treated with 0.5% DSS (Supplementary Fig. 2c–e). Notably, mice treated with 2.5% DSS displayed the highest level of TE expression and expression of interferons or interferon response genes (Supplementary Fig. 2f, g). Taken together, these data suggest that induction of a viral mimicry response correlates with colitis severity.

To next test whether further activation of a viral mimicry response could affect inflammation-associated tumorigenesis, we examined the effects of the DNA demethylating drug 5-AZA-2′-deoxycytidine (5-AZA) on colonic tumorigenesis. 5-AZA has previously been shown to induce a viral mimicry response23,24,26,28,40. To induce colitis-associated cancer, we administered the carcinogen azoxymethane (10mg/kg i.p.×1 dose) followed by 2.5% dextran sodium sulfate (DSS) in the drinking water of C57BL6/J (wildtype) mice (Fig. 2c). Mice were then treated with six doses of 5-AZA (5mg/kg i.p. weekly) or vehicle, with the first dose given 2 days prior to DSS and the remaining five doses being given weekly 1 week post DSS (Fig. 2c). Although all mice developed tumors, mice treated with 5-AZA had significantly fewer colonic tumors than vehicle treated mice (Fig. 2d, e), with no difference in tumor size being detected (Fig. 2f). Global 5-mC levels, a measure of overall methylation, were significantly reduced in colonic epithelial cells from mice treated with two doses of 5-AZA versus vehicle (Fig. 2g). No major differences in tumor histology were observed between vehicle and 5-AZA treated mice based on H&E staining of tumors (Fig. 2h). To validate our results in another model of colitis-associated cancer, we used our previously described Dclk1CreERT2;Apcf/f;Rosa26mTmG mice41, and administered tamoxifen (6mg p.o. q2d×3 doses) followed by 5-AZA and 2.5% DSS as described above (Fig. 2i). Analogous to the AOM/DSS model, we found 5-AZA-treated mice had significantly fewer colonic tumors than vehicle treated mice (Fig. 2j, l), with significantly fewer mice having tumors than vehicle treated mice (Fig. 2k). Interestingly, in this model we also observed significantly smaller tumors in mice treated with 5-AZA versus vehicle treated mice (Fig. 2m). Importantly, we detected increased expression of transposable elements including LINEs, SINEs, and ERVs, as well as increased interferons and JAK/STAT signaling in both models of colitis-associated cancer (Fig. 2n, o, Supplementary Fig. 3a–g), consistent with DNA hypomethylation inducing a viral mimicry response. To confirm that the expression of interferons is induced by expression of TEs, we transfected HCT116 and HT29 colorectal cancer cell lines with plasmids overexpressing LINE1 or SINE B2. Expression of either LINE1 or SINE B2 induced expression of interferons and interferon response genes in both cell lines (Supplementary Fig. 4a–d), confirming that TEs induce interferon signaling. These data show that colitis injury induces a viral mimicry response and that further activation of this response by 5-AZA induced DNA hypomethylation inhibits colitis-associated tumorigenesis.

DNA hypomethylation of cancer-initiating cells activates viral mimicry to reduce stemness
To test if activation of the viral mimicry response specifically in cancer-initiating cells affects tumorigenesis, we genetically knocked out the DNA maintenance methyltransferase enzyme DNMT1 to induce a viral mimicry response as previously described40,42,43. To do this, we crossed Dclk1CreERT2;Apcf/f;Rosa26TdTomato mice to Dnmt1f/f mice. This allowed us to conditionally express a truncated form of APC and simultaneously delete DNMT1 in DCLK1+ cells upon tamoxifen administration. Mice were administered three doses of tamoxifen (6mg p.o.) followed by 2.5% DSS in the drinking water 1 week later (Fig. 3a). Fourteen weeks post DSS, mice were sacrificed for analysis. Using this model, we examined the role of the viral mimicry response specifically within the cancer-initiating cells of colitis-associated cancer. Similar to 5-AZA treated mice, significantly fewer Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice developed colonic tumors when compared to Dclk1CreERT2;Apcf/f;Rosa26TdTomato mice (Fig. 3c). Indeed, mice with DNMT1 loss in DCLK1+ cells also had significantly fewer and smaller tumors (Fig. 3b, d, e) when compared to Dclk1CreERT2;Apcf/f;Rosa26TdTomato mice that were Dnmt1+/+ or Dnmt1+/f. Importantly, DNMT1 loss did not affect DCLK1+ cell number as determined by the number of TdTomato+ cells quantified in vivo (Supplementary Fig. 5a–f) or in intestinal organoids from Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice (Supplementary Fig. 6a–d). Loss of DNMT1 in tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice was confirmed by RT-qPCR for Dnmt1 expression. Consistent with effective knockout of DNMT1, tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice had significantly lower Dnmt1 expression when compared to tumors from Dnmt1+/+ and Dnmt1+/f mice (Fig. 3f). Moreover, we confirmed that the tumors from the Dnmt1f/f mice contained only the recombined allele of Dnmt1, whereas tumors from Dnmt1+/+ mice displayed the WT allele (Fig. 3g). Notably, as previously reported44,45, DNA damage as detected by γH2AX staining was seen in 5-AZA treated mice. However, this was not the case in mice with DNMT1 loss, suggesting that the presence of DNA damage did not necessarily associate with the observed effects on tumorigenesis (Supplementary Fig. 7a–d). Importantly, global 5-mC levels showed loss of DNA methylation in tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice compared to tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato mice (Fig. 3h). To confirm activation of a viral mimicry response, tumors were isolated from Dclk1CreERT2;Apcf/f;Rosa26TdTomato and Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice, and RT-qPCR expression of TEs and interferons examined. Indeed, DNMT1 KO induced TE and interferon expression (Fig. 3i–j), confirming activation of a viral mimicry response. These data demonstrate that loss of DNMT1 in cancer-initiating DCLK1+ cells activates a viral mimicry response and inhibits colonic tumor formation.

Next, we investigated whether activation of viral mimicry inhibits tumorigenesis by blocking stemness of cancer-initiating cells. To test this, we administered three doses of tamoxifen (6mg p.o.) to Dclk1CreERT2;Apcf/f;Rosa26TdTomato and Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice followed by a low dose of DSS (0.5%) (Fig. 3k) that initiates stemness (i.e., lineage tracing from DCLK1+ cells) without promoting dysplasia46. We found significantly fewer TdTomato+ lineage-traced crypts in Dnmt1f/f mice versus Dnmt1+/+ mice (Fig. 3l–m), suggesting that loss of DNMT1 in DCLK1+ cells reduced stemness of DCLK1+ cells. To further validate these findings in vitro, we generated colonic organoids from Dclk1CreERT2;Rosa26TdTomato;Apcf/f and Dclk1CreERT2;Rosa26TdTomato;Apcf/f;Dnmt1f/f mice and treated the organoids with 4-hydroxytamoxifen to label DCLK1+ cells. These organoids were then dissociated into single cells, and the number of TdTomato+ traced organoids quantified after 7 days in culture as previously described47 (Supplementary Fig. 8a). The percentage of DCLK1+ cells that were able to give rise to lineage-traced organoids was significantly reduced upon DNMT1 loss when compared to DNMT1 wild type cells (Supplementary Fig. 8b, c).

To test whether pharmacologic DNA hypomethylation by 5-AZA can similarly inhibit DCLK1+ cell stemness, we administered 0.5% DSS to Dclk1CreERT2;Rosa26mTmG;Apcf/f mice in addition to either 5-AZA or vehicle (Fig. 3n). We observed significantly fewer GFP+ lineage-traced crypts in mice treated with 5-AZA versus vehicle (Fig. 3o, p). Moreover, in mice treated with 5-AZA, we detected a small number of single DCLK1+ cells that expressed GFP but remained quiescent and did not lineage trace crypts or tumors (Supplementary Fig. 8d, e). This is in contrast to vehicle-treated mice, where only fully lineage-traced tumors were seen. Taken together, these data suggest that DNA hypomethylation reduces the stemness of APC-mutant DCLK1+ cells.

5-AZA induces DNA hypomethylation of transposable elements in colonic epithelial cells
Given that 5-AZA inhibited tumor formation, we next sought to confirm that 5-AZA led to transposable element expression by DNA hypomethylation of these elements during cancer initiation. WT mice were treated with 5-AZA or vehicle in the presence or absence of 2.5% DSS induced colitis. Two weeks following DSS, colonic tissues were collected (Fig. 4a). Notably, 5-AZA-treated mice had significantly less weight loss during the acute period of DSS, but had no difference in colon length (Supplementary Fig. 9a, b) or histology (Supplementary Fig. 9c) when compared to vehicle-treated mice (day 19 post DSS initiation). To examine DNA methylation, we isolated DNA from epithelial cells dissociated from the colon of mice in each of the four groups and determined methylation status using the Infinium Mouse Methylation BeadChip Array from Illumina, covering more than 285,000 CpGs. As predicted, we found that 5-AZA significantly reduced global DNA methylation levels as indicated by lower mean β-values (Fig. 4b) in colonic epithelial cells treated with 5-AZA. When comparing control and 5-AZA treated mice we found that 54.5% of all CpG s analyzed were significantly differentially methylated (FDR < 0.01, effect size > 0.1) (Fig. 4c) with 154,488 CpGs being hypomethylated and one CpG being hypermethylated (CpG: uk10321) (Fig. 4d). When comparing DSS and 5-AZA + DSS treated mice we observed 43.7% of all CpGs analyzed were significantly differentially methylated (Fig. 4c) with hypomethylation of more than 120,000 CpGs, and only one CpG (CpG: cg47977693) being hypermethylated (FDR < 0.01, effect size > 0.1) (Fig. 4e). Similarly, we detected regional DNA hypomethylation with 5-AZA treatment both in the presence or absence of colitis (Fig. 4f). Importantly, we detected DNA hypomethylation of repeat elements as a genomic region (Fig. 4f). We next checked whether 5-AZA treatment leads to DNA hypomethylation of different classes of TEs within colonic epithelial cells. We found that all elements, including SINEs, LINEs, satellite repeats, and endogenous retroviruses (ERVs), were hypomethylated following 5-AZA treatment versus vehicle and DSS-treated mice (Fig. 4g). When looking at specific CpGs within each class of transposable elements, we found 18 CpGs in satellite repeats, 9 CpGs in LTRs, 43 CpGs in LINEs, 51 CpGs in ERV1s, 310 CpGs in ERV2s, 30 CpGs in ERV3s, and 16 CpGs in retroelements that were significantly hypomethylated between either control and 5-AZA-treated mice, or DSS and 5-AZA + DSS treated mice (Fig. 4h, Supplementary Fig. 10a, b). When comparing the CpGs that were hypomethylated between control and 5-AZA, and between DSS and 5-AZA + DSS treated mice, we found 127 CpGs (26.4%) common to both comparisons. In contrast, 223 CpGs (46.4%) were specifically hypomethylated between control and 5-AZA-treated mice, while 131 CpGs (27.2%) were specifically hypomethylated only between DSS and 5-AZA + DSS-treated mice (Supplementary Fig. 10c). This suggests that 5-AZA alters the expression of many of the same transposable elements regardless of the inflammation present. To exclude the possibility that interferon response genes, including Mavs, were upregulated due to hypomethylation of the promoter region of these genes, we looked at methylation of CpGs located in CpG islands, shelves, or shores of these genes. We found that these were not different between groups (Supplementary Fig. 10d, e), suggesting that activation of interferon response genes was indeed a result of TE upregulation. These data suggest that 5-AZA induces a viral mimicry response by hypomethylating DNA of TEs in colonic epithelial cells.

Knockout of the viral mimicry response promotes tumorigenesis and reverses the effect of DNA hypomethylation
Next, to examine whether the main antitumor effect of DNA hypomethylation is mediated via activation of a viral mimicry response, we compared colonic tumorigenesis in WT or Mavs-tm1Zjc (MAVS-KO) mice treated with 5-AZA or vehicle in the AOM/DSS colitis-associated cancer model (Fig. 5a). Given that MAVS-KO mice are more susceptible to DSS colitis48,49, a lower dose of DSS (1.5%) was used to induced colitis in these mice. Interestingly, MAVS-KO mice had reduced survival irrespective of treatment when compared to WT mice (Fig. 5c). Furthermore, the percentage of mice with tumors was significantly higher in MAVS-KO mice versus 5-AZA-treated WT mice (Fig. 5d). MAVS-KO mice also had increased tumors when compared to WT mice, regardless of treatment (5-AZA versus vehicle) (Fig. 5b, e). We confirmed that MAVS-KO mice did not have any Mavs gene expression as detected by RT-qPCR (Fig. 5f). As predicted, expression of transposable elements was significantly increased upon 5-AZA treatment in all groups (Fig. 5g), whereas increased gene expression of interferons was only seen in 5-AZA-treated WT mice and not MAVS-KO mice (Fig. 5h). We next validated these observations in a second model of colitis-associated cancer. Here, we crossed our Dclk1CreERT2;Rosa26TdTomato;Apcf/f;Dnmt1f/f mice to MAVS-KO mice and induced tumor formation (Fig. 5i). We found that the percentage of mice with tumors was significantly lower with DNMT1 loss compared to controls (Fig. 5j). Knockout of MAVS in the setting of DNMT1 loss resulted in 100% of mice developing tumors, and reversal of the anti-tumor effects of DNMT1 loss (Fig. 5j). Similar to 5-AZA treatment, we detected significantly fewer tumors with DNMT1 loss compared to controls, whereas knockout of MAVS reverted tumor number back to control levels (Fig. 5k). These data clearly suggest that the main antitumor effect of DNMT1 loss is activation of the viral mimicry pathway. No difference in tumor size was seen between groups (Fig. 5l). Importantly, tumors collected from controls, Dnmt1f/f mice and Dnmt1f/f;Mavs-tm1Zjc mice revealed that tumors with DNMT1 loss had lower Dnmt1 expression compared to controls (Fig. 5m). Tumors with MAVS KO had undetectable expression of Mavs compared to both control and Dnmt1f/f tumors (Fig. 5m). Together, these data show that loss of the viral sensing protein MAVS reverses the anti-tumor effect of DNA hypomethylation, consistent with activation of a viral mimicry response being essential to the anti-tumor effect of DNA hypomethylation.

Viral mimicry suppresses stemness of cancer-initiating cells
To next investigate whether the effects of viral mimicry on stemness are cell autonomous versus dependent on extracellular signaling, we used an in vitro epithelial-derived organoid culture model. Colonic organoids were established from Dclk1CreERT2;Rosa26TdTomato mice wildtype for the Apc gene or crossed to Apcf/f, Apcf/f;Dnmt1f/f, Apcf/f;Mavs-tm1Zjc, or Apcf/f;Dnmt1f/f;Mavs-tm1Zjc mice. Following 4-hydroxytamoxifen treatment for 48h, organoids were dissociated into single cells, and TdTomato+ cells counted. Seven days post dissociation, fully traced TdTomato+ organoids were counted (Fig. 6a). Importantly, while DCLK1+ cells wildtype for the Apc allele did not form organoids, 5–10% of DCLK1+ cells that were Apcf/f showed organoid-forming ability (Fig. 6b). As shown previously (Supplementary Fig. 8b, c), DNMT1 loss significantly reduced the number of TdTomato+ cells able to give rise to organoids (Fig. 6b, c). Remarkably, knockout of MAVS promoted stemness of DCLK1+ cells as measured by increased lineage tracing capacity of TdTomato+ cells as well as increased organoid size even in the setting of DNMT1 loss (Fig. 6b–d). Similarly, 5-AZA significantly reduced the lineage tracing capacity of DCLK1+ Tomato labeled cells, whereas MAVS-KO reversed this effect and increased organoid size (Fig. 6e–h). These findings suggest that the viral mimicry response inhibits the stemness of APC-mutant DCLK1+ cells in a cell-autonomous manner (Fig. 7).

Methods
Animals
Animal studies were approved by the Western University Animal Care Committee according to guidelines established by the Canadian Council on Animal Care. Mice were housed in the London Regional Cancer Program vivarium. Mice were exposed to a 12-h light-dark cycle and housed with controlled temperature (19–22°C) and humidity (40–60%), with water and regular chow ad libitum. Equal numbers of male and female mice were used for each experiment. All mice were inbred on a C57BL/6J background. For practical reasons, the strains were maintained as homozygous when possible. Mouse lines used include C57Bl6/J (Jackson Labs), Dclk1CreERT2 (Westphalen and Asfaha41), Rosa26mTmG (Jackson Labs), Rosa26TdTomato (Jackson Labs), Dnmt1f/f (Mutant Mouse Resource and Research Centers), Mavs-tm1Zjc (kindly gifted by Dr. Daniel De Carvalho from University of Toronto), and Apcf/f (National Cancer Institute).

Dclk1CreERT2;Apcf/f mice were crossed to Dnmt1f/f mice to generate inducible knockout mice. For induction of Cre-mediated recombination, mice of 6–10 weeks of age received three doses of 6mg tamoxifen (MP Biomedicals) dissolved in corn oil every 2 days by oral gavage. For AOM administration, mice of 6–8 weeks of age received a single i.p. injection of 10mg/kg AOM (Sigma-Aldrich). Mice were exposed to 0.5–2.5% dextran sodium sulfate (DSS) (Gojira Fine Biomedicals) in the drinking water for 5 days. During DSS and the 5 days following DSS, mice were scored daily in regards to weight loss, stool consistency, movement, and survival. Following the acute period of DSS, body weights were measured once a week until experimental or humane endpoint. 5-AZA-2′-deoxycytidine (Sigma-Aldrich) was dissolved in PBS and given at a dose of 5mg/kg by i.p. injection.

Tissue preparation, immunofluorescence, and microscopy
Tissue was harvested from euthanized mice and fixed in 4% paraformaldehyde (PFA) for 6–8 h at 4°. The tissue was cryopreserved in 30% sucrose at 4°, embedded in optimal cutting temperature (OCT) compound (Fisher Healthcare), and frozen on dry ice. Sections were cut at 6μm. For paraffin embedding, tissue was harvested, fixed in 10% formalin (Fisher Chemical) for 24h, and processed, followed by embedding in paraffin. Sections were cut at 5μm.

For hematoxylin and eosin staining, sections were deparaffinized and rehydrated, followed by staining with CAT hematoxylin (BioCare Medical) and Eosin-Y (Fisher Chemical). Slides were mounted with Permount Mounting Medium (Fisher Scientific) for viewing. Fluorescence and brightfield images were acquired using EVOS FL Auto.

Plasmid preparation
Plasmid E. coli strains of pB2-neo-TET (Plasmid #51287) and pBS-L1PA1-CH-mneo (Plasmid #51288) were purchased from Addgene. Plasmid preparation was carried out with Presto Mini Plasmid Kit (Geneaid Biotech). Briefly, an individual bacterial clone was picked from an E. coli LB agar plate and cultured at 37°C overnight with shaking in TB medium with ampicillin (100μg/ml). Bacteria were then pelleted by centrifugation at 8000×g for 1 min. The pellet was next resuspended and lysed. Neutralization buffer was added to the lysate and centrifuged at 16,000 xg for 5 min. The supernatant was then transferred to a binding column and centrifuged at 16,000 xg for 30s. The column was washed, and the plasmid eluted.

Transfection of cell lines
HT29 (ATCC, HTB-38) and HCT116 (ATCC, CCL-247) colorectal cancer cells were seeded at 150,000 cells per well in a 6-well plate in DMEM media (Multicell) supplemented with 1× Glutamax (Gibco) and Pen Strep (Gibco). Transfection of cells was performed 24h after seeding using jetPRIME transfection reagents (PolyPlus) according to the manufacturer’s instructions. Cells were collected 48h after transfection for downstream analysis.

Small intestinal and colonic mouse organoid culturing
Organoid culturing was performed as previously described93. Briefly, the scraped mouse small intestine (SI) or colon was harvested and incubated in 2.5mM (SI) or 10mM (colon) EDTA for 60min. Following mechanical disruption of tissues, isolated crypts were plated in Matrigel (Corning), and cultured in Advanced DMEM/F12 (Gibco) supplemented with Primocin (InvivoGen), 10mM HEPES (Gibco), 1x Glutamax (Gibco), 1xB27 (Gibco), 1mM N-acetyl-L-cysteine (Sigma-Aldrich), and 1x N-2 (Gibco) as well as 50ng/mL EGF (PeproTech), 100ng/mL Noggin (PeproTech), and 1μg/mL R-spondin 1 (ENR). 50% Wnt3a-conditioned media (made in house) was used for colonic cultures. 2–3 days post-culture, 2μM 4-hydroxytamoxifen was added to the culture media for 48h for Cre-mediated recombination. For 5-AZA treatment, 1μM 5-AZA was added to the media for 24h.

For single-cell dissociation, TrypLE Express was added to each well following 4-hydroxytamoxifen treatment and incubated until organoids were dissociated into single cells. The cells were replated into new Matrigel, and the ROCK inhibitor Y-27632 (Selleck Chemicals LLC) added to the media to prevent cell death. The number of TdTomato+ cells was counted on day 1, and the number of TdTomato+ organoids was counted on day 7 post single cell dissociation. Notably, when we quantified traced organoids, these were additionally confirmed to be consistent with stem cell labeling as they could be passaged a minimum of five times, except APC-WT TdTomato+ cells and TdTomato+ cells treated with 5-AZA or with DNMT1 loss that could not be maintained post passaging.

Organoid size was measured using ImageJ94.

RNA isolation and RT-qPCR
RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. Lithium chloride purification of the RNA was performed for all tissues, as DSS is known to inhibit RT-qPCR amplification of mRNA95. The RNA was reverse transcribed using iScript RT Supermix (Biorad). RT-qPCR was conducted using SYBR Green (Applied Biosystems). Reactions were run on a QuantStudio5 thermocycler. Gene expression levels were quantified using the ddCT method and normalized to the CT value of GAPDH. Primers are listed in Table S2. For tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice, RT-qPCR was only performed on tumors confirmed to have recombination of Dnmt1.

Small intestinal and colonic epithelial cell isolation and FACS
In preparation for hybridization to the Illumina Mouse Methylation BeadChip array, colonic tissue was harvested and incubated in 10mM EDTA in PBS for 1h. After incubation, the tissue pieces were transferred to 10% Fetal Bovine Serum (FBS) (Multicell) in PBS and mechanically disrupted. The solution was transferred through a 100μm strainer and centrifuged at 300×g for 5 min to pellet colonic crypts.

For FACS, SI, and colonic tissues were harvested and washed in PBS. SI and colonic tissues were incubated in 2.5mM and 3mM EDTA, respectively, for 1h. Tissues were then mechanically disrupted in 10% FBS in PBS and passed through a 100μm strainer. Samples were then centrifuged at 300×g for 5 min. The pellet was then resuspended and incubated in TrypLE Express (Gibco) for 30 min until dissociation into single cells. The cells were then filtered through a 40μm strainer, centrifuged at 300×g for 5 min, and resuspended in 2% FBS in PBS for sorting. TdTomato-expressing cells were sorted on a FACS SH800 (SONY) into 2% FBS in PBS. Viable single cells were gated based on the FSC-W versus FSC-A profile.

DNA isolation and genotyping
DNA isolation was performed using the Invitrogen DNA isolation kit. Briefly, tissues were digested in the Digestion Buffer provided by the kit, followed by the addition of the Lysis/Binding buffer. Lysate was added to a PureLink Spin Column, washed, and eluted. For the Infinium Mouse Methylation BeadChip Array, the DNA was extracted from isolated colonic epithelial cells. For confirmation of Dnmt1 recombination, DNA was isolated from tumors from Dclk1CreERT2;Apcf/f;Rosa26TdTomato and Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice. Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers listed in Table S3 were used for confirmation of Dnmt1 recombination, whereas 2X Taq Frogga mix (FroggaBio) was used for all other genotyping.

Western blot
Whole colonic tissue from mice treated with two doses of 5mg/kg 5-AZA or vehicle was homogenized in 200μL RIPA buffer (containing 0.2mM PMSF, 0.5mM NaF, 0.1mM Na3VO4, 1x protease/phosphatase inhibitor cocktail). Protein was quantified using a Bradford assay (BioRad). 20μg protein lysates were loaded on an 8% SDS-PAGE gel and transferred to nitrocellulose membrane (BioRad) using a wet electroblotting system (BioRad). The membrane was incubated using the manufacturer protocols for p-STAT1 Ab (#9167, Cell Signaling Technologies, 1:1000), STAT1 Ab (#9172, Cell Signaling Technologies, 1:1000), β-Actin Ab (#4970, Cell Signaling Technologies, 1:1000), and Anti-rabbit IgG-HRP-linked Ab (#7074, Cell Signaling Technologies, 1:2000). The membrane was rinsed briefly in enhanced chemiluminescence substrate (BioRad) and imaged using the BioRad ChemiDoc MP Image System. Images were analyzed with ImageJ (version 1.53).

Myeloperoxidase (MPO) measurement
MPO measurements were performed on distal colonic tissues as previously described96. Briefly, colonic tissues were homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer according to tissue weight. Homogenate was added in triplicate to a 96-well plate. O-dianisidine solution was prepared by combining 16.7mg o-dianisidine dihydrochloride, 90mL dH2O, 10mL 50mM potassium phosphate buffer, and 50μL 1.2% H2O2. Next, 200μL O-dianisidine solution was added to each well containing tissue homogenate. Absorbance was measured at 450nm, and MPO calculated as units of MPO/mg tissue.

Global DNA methylation
Global DNA methylation was measured on DNA isolated from colonic epithelial cells or tumors from either Dclk1CreERT2;Apcf/f;Rosa26TdTomato and Dclk1CreERT2;Apcf/f;Rosa26TdTomato;Dnmt1f/f mice using the MethylFlash Global DNA methylation (5-mC) ELISA Easy Kit (EpigenTek) according to the manufacturer’s instructions. Briefly, the binding solution and DNA were added to a 96-well plate. Wells were then washed, and 5-mC Detection Complex solution added to each well followed by Developer Solution. Stop Solution was then added, and the plate read at 450nm. Percentage methylated DNA was calculated based on a standard curve.

Infinium mouse methylation beadchip array
DNA was isolated from colonic epithelial cells isolated from mice treated with vehicle (controls), DSS, 5-AZA, or 5-AZA + DSS (n=3 in each group) and run on the Infinium Mouse Methylation BeadChip Array (GSE283386). Preprocessing of IDAT files was done using the R package SeSaMe (version 1.20)97 and included calculation of detection p values (probes with a detection p value of <0.05 were retained), background subtraction, strain interference, and dye bias correction. Differentially methylated CpGs were identified using the DML function, which models β values using a linear model. Sample group was used as the predictor. Sex-specific CpGs in colonic tissues were removed from the analysis to account for unequal distribution of sexes between the four groups analyzed (Ctrl, DSS, 5-AZA, and 5-AZA+DSS). This was done by identifying differentially methylated CpGs in colonic tissues between male (n=2) and female (n=3) mice using samples available in the dataset GSE18441098. Only colonic samples in this dataset were used for analysis. IDAT files were preprocessed as described above, and the DML function was used to identify differentially methylated CpGs in GSE184410 samples with sex as the predictor. CpGs were considered significant for a sex effect if the false discovery rate (FDR) was less than 0.05 and the effect size was greater than 0.1. 1223 CpGs were identified as being sex-specific and removed from the analysis. Following these quality control steps, differentially methylated CpGs were defined as those with FDR less than 0.01 and an effect size greater than 0.1, unless otherwise stated. Heatmap were generated using the pheatmap package (version 1.0.12) and volcano plots using ggplot2 (version 3.5.1).

Human RNA-sequencing data retrieval and processing
Two publicly available datasets containing RNA-sequencing was analyzed for this study. Shaw et al.39 contained RNA-sequencing of colonic biopsies from 41 healthy controls, 22 patients with IBD without dysplasia, and 7 patients with IBD-associated dysplasia (biopsy of non-dysplastic region adjacent to dysplasia). Peters et al.35 contained RNA-sequencing of surgical specimens from 61 control patients that had colon resection due to sporadic CRC and inflamed tissues from 77 patients with active IBD. Using the SRA toolkit (version 3.1.0), fastq files obtained and processed by Shaw et al.39 and Peters et al.35 were retrieved. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to identify sequencing artifacts that were then removed using bbduk (http://jgi.doe.gov/data-and-tools/bb-tools/). The resulting reads were aligned to the hg38 human genome (GRCh38.p14, GENCODE) with HISAT299 (version 2.2.1), excluding multi-mapping reads and incorporating splice junctions as previously described36. These splice junctions were specified through a custom GTF file merging GRCh38.p14 transcripts (GENCODE) and RepeatMasker repeat element loci from the UCSC genome browser. Samtools100 (version 1.18) indexed the resulting alignments, which were later passed to htseq-count101 to quantify custom GTF gene expression (version 0.11.1). Final datasets from Shaw et al. and Peters et al. were compiled into separate matrices for further analysis in R (version 4.3.2).

Downstream gene expression analysis
DESeq2102 (version 1.42.0) was chosen for differential gene expression analysis across the specified groups, for which the default parameters were used. When comparing IBD patients to those with IBD and dysplasia in data from Shaw et al., genes identified as upregulated and significant (Benjamini-Hochberg adjusted p value < 0.05) were passed into enrichGO (clusterProfiler103, version 4.10.0) for gene ontology (GO) enrichment analysis. The ten most significantly enriched biological processes (BP) GO terms were selected from these results and visualized with ggplot2 (version 3.4.4).

To identify patterns in interferon genes, the count matrix was adjusted for sequencing depth (using the DESeq2 counts function), normalized relative to the average expression in the non-inflamed control samples, and then log2 transformed. The resulting data was visualized using ComplexHeatmap104 (version 2.18.0). Additionally, the differential expression of repetitive elements, identified through RepeatMasker, was visualized using an EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano, version 1.20.0) plot, emphasizing the most significantly altered elements.

Similarly, DESeq2 was used to compare control and IBD samples from the data in Peters et al. Repetitive elements with significant differences in expression were visualized using ComplexHeatmap (version 2.18.0). A two-sided binomial test, performed under the null hypothesis (alpha=0.05), demonstrated that the number of repetitive elements upregulated in IBD samples (174) compared to control samples (36) was statistically significant (p=3.21×10−21).

Statistical analysis
Statistical analyses were performed using two-tailed Student’s t-tests when comparing two groups or standard One-way ANOVA analysis with Tukey’s multiple comparison test when comparing 3 or more groups. For survival analysis, a Log-rank test was performed. Error bars denote the mean ± SEM. Unless otherwise specified, a P value of less than 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "While the study does show an interesting case of viral mimicry associated with colitis, a very interesting topic, there are a couple of concerns which prevent greater enthusiasm.\nIn particular, what the authors term viral mimicry is a co-expression of transposable elements and other repeats with interferon and in the presence of demethylating agents. Its unclear whether the agents expressed are capable of engaging the pattern recognition receptors of the innate immune system. The agents most capable of forming dsRNAs are inverted repeats, which as Alus in humans and a subset of SINE elements in mice, or CpG rich satellite repeats such as HSATII. The authors should provide some more detail as to whether the specific repeats they see upregulated are likely to form innate agonists such as in here:\nhttps://www.biorxiv.org/content/10.1101/2021.11.04.467016v2\nLikewise the authors would need to show the interferon is induced by there repeats. In and of itself it is unsurprising that demethylating agents lead to repeat upregulations – elements such as active LINE-1 are known to be CpG rich. But there needs to be a clearer demonstration of causality over correlation.\nFor what its worth repeat upregulation was found to be associated with HCMV induced colitis here:\nhttps://pubmed.ncbi.nlm.nih.gov/30626867/\nSo in summary to move forward the authors would have to show the repeats upregulated likely engage PRRs and that such repeats directly lead to IFN upregulation to establish causality.", "reviewer_2": "The manuscript by Larsen et al establishes a link that epigenetic changes (DNA demethylation) results in the ectopic expression of transposable elements (TEs) which otherwise would be remain silenced. They further propose that such ectopic TE expression induces a cell autonomous viral mimicry response through IFN signaling which inhibits cancer cell stemness and tumorigenesis.\nUsing pharmacological and genetic in vivo mouse models as well as in vitro organoid cultures the authors show that experimentally demethylating DNA and thus TE elements by epigenetic drugs or genetic models increases TE expression and reduces tumor formation in mouse models of colitis associated cancer.\nThe results in this manuscript are timely and most claims well supported by control experiments to exclude confounding effects. Moreover, the authors show, using multiple models, both genetical and pharmacological, a convincing link between DNA methylation, transposable elements and colonic tumor burden. However, it is the opinion of this reviewer that the link between TE expression and viral mimicry response, mediated by IFN is not sufficiently backed by data. The authors are requested to strengthen this claim.\nMajor points:\n• The general working hypothesis of the manuscript is unclear. The authors show that inflammation /colitis increases transposable elements (TE) expression, while at the same time the manuscript shows that TE expression serves as a tumor suppressive mechanism. However, inflammation (and thus TE expression) is strongly associated with tumor initiation and the authors themselves leverage this fact in all their models. (see also minor comment (6). The authors are therefore asked to clearly state their hypothesis and discuss why tumors, despite having abundant TE expression escape suppression via viral mimicry response.\n• While the sequencing data linking demethylation to increased TE expression is convincing, the direct link between increased TE expression and cell autonomous viral mimicry response (IFN) need to be strengthened, in this context in depth interrogation of JAK-STAT pathway activation in 5-AZA treated mice or the genetic models described (DNMT K.O.) is needed.\nMinor points:\n1. The original dataset GSE83687 contains only 134 samples, yet the authors specify the dataset contains samples of 61 control patients and 77 patients with IBD (total 138) please explain or correct.\n2. Sup. Fig1B: it would be more informative to plot relative expression levels (as is done in the rest of the manuscript) instead of absolute counts, as sequencing depth might be different among samples\n3. The original dataset GSE230524 contains in total 110 patients with IBD and 56 healthy controls why were not all samples included?\n4. Line 87-88 the statement ‘leading to tumorigenesis’ is too strong especially since this connection has not yet been made this early in the manuscript, please remove\n5. Fig. 2D, J are insufficiently clear, as tumors are quite large, a larger format image or a swiss role preparation would more clearly show the number of tumors.\n6. The authors argument that\n1) tumorigenesis requires the need of overcoming viral mimicry response (discussion 300-302) and\n2) the observation of TE downregulation in human dysplasia IBD\nis not in line with data presented in Sup Fig. 2 C, D which shows sustained upregulation of both TE and IFN gene expression within the tumor tissue itself (upon 5-AZA treatment or without). The authors are asked to discuss these discrepancies or adjust the hypotheses stated in the discussion (line 300-302) and human results (line 86-88).\n7. Line 189: remove ‘to’ in “we next to sought ...”\n8. Line 208-215: The largest demethylating effects of 5-AZA treatment is found in regions which directly or indirectly modulate gene expression (such as TSS, enhancer and genebody regions). To exclude that the observed effects on IFN response are not directly related to opening of these genomic regions, the authors are asked to show methylation status of enhancer and promotor regions of genes involved in IFN expression.\n9. Line 223-228: The statement that inflammation (DSS treatment) alters many of the same TEs as 5-AZA treatment is not in line with data presented in Fig. 4B, which shows overall high methylation levels in DSS treatment alone.\n10. Fig. 5 G, the authors are asked to comment on the fact that MAVS-KO mice have an increased sensitivity to 5-AZA in respect to TE expression. In this context TE expression in MAVS-KO mice not exposed to DSS but treated with/without 5-AZA would be informative.\n11. Fig 5H: despite the complete lack of MAVS expression, as shown in Fig 5F, IFN response is only very slightly reduced in vehicle treated MAVS-KO animals exposed to DSS as compared to vehicle MAVS-WT mice exposed to DSS. Suggestive that the IFN response in response to DSS (as shown in Fig 2 B) is still very much activated.\n12. Line 305: please correct the statement that normally quiescent Dclk1+ cells can transition upon knockout of MAVS. Data presented n in Fig 6. only shows that in the absence of ARC, Dclk1+ cells can transition. The effect of MAVS-KO in the presence of wt ARC was not assessed, but would be informative.\n13. Rework all images of immunofluorescence to include labels of different channels/colours shown", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This manuscript reported that activation of transposable elements elicits a viral mimicry response to foster a tumor suppressive function in inflammation. This viral mimicry, induced by DNA hypomethylation, inhibits tumorigenesis by regulating the stemness of cancer-initiating cells. This study is interesting and convincing, however, there are several fundamental questions need to be addressed or thoroughly discussed, such as, how to reconcile the results reported in this study with the inflammation-induced tumorigenesis model? What is the relationship between inflammation and TEs activation? How to distinguish the tumor-promoting and tumor-suppressive functions of TEs? Specific questions or comments are as follow:\n1. The authors discovered patients and mice with colitis express TEs that lead to a viral mimicry response, inducing TEs by DNA hypomethylation in inflammation inhibits tumorigenesis. However, many studies have also found that TEs are activated in patients with colorectal cancer and promotes cancer progression (DOI: 10.1158/2159-8290.CD-21-1117,DOI: 10.1038/s41467-022-31197-4). Does TEs play different roles in the process of tumorigenesis and tumor progression? Or does TEs exhibit an inhibitory effect specifically on patients with colitis-associated cancer?\n2. Related to above, using a mouse model with AOM/DSS administration, the levels of TEs in the colonic tissue of C57Bl6/J mice treated with different concentrations of DSS and the vehicle, as well as in the tumor tissues, should be detected.\n3. How can we explain the phenomenon that patients who experienced IBD are more prone to developing colorectal cancer? When comparing patients with active IBD without TEs activation to those with TE activation, does the latter group exhibit a lower incidence of colorectal cancer?\n4. In Fig. 1 and Fig. S1, although the majority of TEs are upregulated, the expression of some TEs is downregulated in patients with active IBD. What accounts for the selective activation of a subset of TEs? And how do the expression of these TEs in another dataset, GSE230524?\n5. Compared with control patients, is there an enrichment of genes associated with viral or interferon signaling pathways in patients with active IBD from GSE230524 as determined by GO term pathways analysis?\n6. Is there an overlap between the TEs that are upregulated in patients with active IBD compared to control patients and the TEs that are downregulated in patients with IBD-associated dysplasia compared to patients with IBD without dysplasia? And why?\n7. The selection criteria for interferon response gene expression in Fig. 1 is unclear. Were these genes identified based on established databases? Why do the genes shown in Fig. 1A and Fig. 1C differ? In addition, why is there an inconsistency between the TEs and interferon response genes detected by the authors in Figures 2, 3, and 5?\n8. The authors have shown that DSS can induce the expression of TEs. Then, why is it necessary to use 5-AZA to further activate TEs? Does 5-AZA inhibit tumorigenesis by promoting inflammation to activate TEs? Is there a difference in the degree of inflammation between C57Bl6/J mice treated with DSS and those treated with 5–AZA in Fig. 2?\n9. The authors utilized multiple mouse models to demonstrate that DNA hypomethylation activates viral mimicry and suppresses colitis-associated cancer. However, the rationale for administering 5-AZA before DSS treatment remains unclear.\n10. The authors found the level of IFNα increases in the colonic tissues of Dclk1CreERT2;Apcf/f mice treated with 5-AZA (Fig. 2B), while it decreases in the tumor tissues of Dnmt1f/f mice (Fig. 3H), What could be the underlying reason for this divergent phenomenon?\n11. the authors induced colitis in C57Bl/6J mice by treating them with 2.5% DSS in Fig. 2. Dclk1CreERT2;TdTomato;Apcfl/fl;Dnmt1+/+ mice were treated with 0.5% DSS in Fig. 3, and 1.5% DSS was used to in MAVS KO mice in Fig. 5. Why the DSS concentrations vary across mouse models? Any explanation?\n12. The authors found global 5-mC levels were significantly reduced in colonic epithelial cells from mice treated with 5-AZA versus vehicle, which supports 5AZA suppresses DNA methylation. To further confirm this hypothesis, the percentage 5-mC (5-mC/total DNA) in colonic epithelial cells from Dnmt1+/+ and Dnmt1f/f mice treated with tamoxifen should also be examined.\n13. In Fig. 2A, it is indicated that DSS activates the expression of TEs. However, in Fig. 4B, it is found that DSS has no effect on DNA methylation. So, how does DSS influence the expression of TEs?\n14. Although 5-AZA caused DNA hypomethylation of repeat elements, the methylation in other regions is also regulated by 5-AZA (Fig. 4F). Therefore, the suppression of tumorigenesis by 5-AZA cannot be attributed to the activation of TE.\n15. The authors found that treatment with 5AZA reduced AOM-DSS induced tumorigenesis of wild-type mice in Fig. 2. However, in Fig. 5, the authors discovered that there was no difference in the survival of wild-type mice with or without 5AZA treatment. Why?\n16. MAVS gene expression is increased upon 5-AZA treatment in WT mice (Fig 5F). Does the methylation level of the MAVS promoter decrease with 5-AZA treatment?\n17. In Fig. 3 and Fig. 5, the mRNA of DNMT1 can still be detected in the tumor tissues of DNMT1fl/fl mice. Why is this the case?\n18. The authors generated colonic organoids from Dclk1CreERT2;Rosa26TdTomato;Apcf/f and Dclk1CreERT2;Rosa26TdTomato;Apcf/f;Dnmt1f/f mice and treated the organoids with 4-hydroxytamoxifen to label DCLK1+ cells (Fig. 6). Why not treat mice directly with tamoxifen to label the DCLK1+ cells in vivo and subsequently dissociate the colonic DCLK1+ cells for organoid culture?\n19. The authors observed that the number of lineage traced crypts per colonic section is lower in Dclk1CreERT2;Rosa26mTmG;Apcf/f mice treated with 5-AZA compared to those treated with the vehicle in Fig. 6. Could this phenomenon be attributed to the inhibitory effect of 5-AZA on the proliferation of DCLK1+ cells? Please add the number and size of DCLK1+ cells per organoids over time treated with 5-AZA and vehicle.\n20. Can other drugs such as HDAC inhibitors that activate TEs also suppress the colitis-associated cancer?\n21. The authors found that DNA hypomethylation triggers a viral mimicry response to inhibit stemness of cancer-initiating cells. Is it possible there is an increase in the stemness of intestinal stem cells in IBD patients with dysplasia, accompanied by an increase in DNA methylation level?\n22. Please keep the text size within the figure are maintained as uniformly as possible.\n23. Gene names need to be italicized to differentiate them from protein names."}, "author_response": "Point-by-point response to reviewers\nWe thank the reviewers for the reviews of our manuscript entitled “Viral mimicry acts as a tumor suppressor in inflammation”. We appreciate the opportunity to address their concerns.\nReviewer 1:\nWhile the study does show an interesting case of viral mimicry associated with colitis, a very interesting topic, there are a couple of concerns which prevent greater enthusiasm.\nIn particular, what the authors term viral mimicry is a co-expression of transposable elements and other repeats with interferon and in the presence of demethylating agents. Its unclear whether the agents expressed are capable of engaging the pattern recognition receptors of the innate immune system. The agents most capable of forming dsRNAs are inverted repeats, which as Alus in humans and a subset of SINE elements in mice, or CpG rich satellite repeats such as HSATII. The authors should provide some more detail as to whether the specific repeats they see upregulated are likely to form innate agonists such as in here: https://www.biorxiv.org/content/10.1101/2021.11.04.467016v2\nWe thank the reviewer for this point and agree with the reviewer’s comment. Thus, to demonstrate that the TEs we observe expressed in colitis are capable of activating the innate immune system, we have now divided the human and mouse TE expression data according to their respective subclasses (i.e. ERVs, LINEs, SINEs etc.). As the reviewer points out “...the agents most capable of forming dsRNAs are inverted repeats, which are Alus in humans and a subset of SINE elements in mice, or CpG rich satellite repeats such as HSATII”. Thus, we now present the human IBD data in a new heatmap that divides the subclasses of TEs (see heatmap below) and bar graph in manuscript Figure 2a for the mouse data. Indeed, our data highlights the fact that we do see upregulation of elements known to form dsRNA such as AluYg6 and AluY in patients with IBD (see Figure 1 below) and SINE elements in mice with colitis (see manuscript Figure 2a). Additionally, it would be ideal to determine whether dsRNAs form in patients with IBD and what elements they are derived from, however, due to the limitations of current technology this remains challenging to do as antibodies used to capture dsRNA are designed for in vitro use (as outlined by the manufacturer (https://www.exalpha.com/products/scicons-anti-dsrna-antibody-j2/10010200)).\nFigure 1 – Heatmap of the expression of different classes of TEs in patients with IBD compared to controls\nLikewise, the authors would need to show the interferon is induced by these repeats. In and of itself it is unsurprising that demethylating agents lead to repeat upregulations – elements such as active LINE-1 are known to be CpG rich. But there needs to be a clearer demonstration of causality over correlation.\nWe thank the reviewer for this point. To address this comment, we provide new data in which we transfected HCT116 and HT29 colorectal cancer cells with a plasmid overexpressing LINE1 or a plasmid overexpressing SINE B2. The cells were then collected 48 hours after transfection. We first confirmed that both elements are overexpressed (see Figure 2 below) and found that cells that express either LINE1 or SINE B2 have increased expression of interferons and interferon response genes when compared to control transfected cells. These data clearly show that expression of both these elements that we see upregulated in humans and mice with colitis can directly induce interferon signaling (See Figure 2 below). This data has now been included in the manuscript in Supplementary Fig. 4.\nFurthermore, we provide evidence that MAVS-KO can reverse the antitumor effect of both 5-AZA and DNMT1 loss (see Figures 5 and 6 in manuscript). This observation suggests that activation of the MDA5/MAVS pathway by TEs occurs following DNA hypomethylation. Moreover, in Figure 5H we also show MAVS-KO results in loss of expression of interferon response genes and JAK/STAT signaling genes even in the setting of 5-AZA treatment. These data confirm that increased interferon signaling observed with 5-AZA treatment is due to activation of MDA5/MAVS signaling.\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have clarified their hypothesis and have addressed all my previous concerns and the manuscript in its current form has been greatly improved.\nMy sole recommendation would be to add a graphical abstract summarisingg the cascade of events leading to VM and prevention of tumorigenesis including the interventions the authors use (eg. MAVS k.o. , Dnmt1 k.o.)", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have adequately addressed my major concerns. However, the potential tumor promoting activity of certain transposable elements has not been discussed to declare the limitations of the current study, and several relevant literatures are left uncited (e.g. Nat Commun, 2022).\nNonetheless, the manuscript is significantly improved, and I think with minor modification it is ready for publication.\n***Additional notes: I was also asked to comment on the previous report provided by Reviewer #1, who is unavailable for this round of review. Here is my feedback on authors' responses to Reviewer #1's previous comments.\nI think the authors have adequately addressed the concerns raised by Reviewer #1 by performing a series of additional experiments and analyses. After inclusion of those mentioned references, the manuscript in my opinion is ready for publication.", "reviewer_5": "In this revised manuscript by Larsen et al, the authors have addressed multiple aspects of the concerns raised in relation to the first version. In this paper, the authors demonstrate that colitis leads to activation of transposable elements which act as viral mimetics and then are reduced as the inflammation progresses to dysplasia. The central hypothesis is an interesting one and is further supported by revisions to the manuscript. The major concern regarded the mechanistic link between TEs and viral mimicry mediated by MAVS. In particular, more information regarding the type of TEs induced in human and in mice in colitis and the demonstration that the induced TEs are the cause of the interferon response. The authors addressed these concerns adequately, providing detailed analysis of TEs subclasses. We acknowledge it is technically very difficult to directly link one particular TE as a causative agent, the experiments addressing this in vitro by overexpressing 2 specific TEs was strong. There are a few specific points that would be useful to address to make the study stronger.\n1. The concern that the correlation between TE and repeat expression with interferon activation needs further analysis was addressed through sub-setting TEs based on families, but these families can contain a large number of individual repeats and whether any of these are capable of forming dsRNA is not clear and not fully addressed by the revised data. For instance, identifying the expression of those that are old and have degenerated compared to those which are young and mobile would be of interest.\n2. The new analysis shows that there are many LINES and LTR transposons that are highly expressed in some patients (Supplemental figure 1) however the aggregated analysis shows downregulation of some LTRs. What accounts for this inconsistency?\n3. The new data showing expression of TEs subclasses by patients indicates that some samples have high expression of many subclasses, while others have unique patterns. If the patient samples are segregated based on TE expression – i.e. the universal high expressing samples vs. the low expressing samples or unique patterns – does that alter the correlation with expression of immune genes or, visa versa, are the patients with the most robust immune response those that have the highest TE expression? In particular, does Alu, SINE or HSATII expression correlate the most strongly with immune activation?\n4. The hypothesis that MAVS is detecting dsRNA downstream of TE actiation predicts that MAVS deficient cells should still have the same TE expression, and presumably the same amount of dsRNA. Is that true?\n5. One overlooked point from the initial submission is that DNMT1 depletion and 5-Aza are well established DNA damaging agents. This has not been examined or mentioned and should be investigated and added as an important caveat to these approaches.", "reviewer_6": "The authors have sufficiently addressed comments from the original submission. I have two main concerns on this paper which hopefully can be resolved with an addition to the discussion section. The role of ERVs in cancer are multifaceted: on one hand ERVs can cause insertional mutagenesis, deactivate tumor suppressor genes or act as cis-promoters for proto-oncogenes. Additionally, certain ERVs can form proteins that can induce cancer formation i.e. HERV-K. The authors of the manuscript mainly focus on dsRNA elements, rather than any protein coding ERVs or LINE-1 that can play a role in tumor formation. Therefore the balance between these two should be discussed briefly.\nThe last concern is that the title is too broad. The title of the article should reflect the disease which is being studied: colitis and should reflect that."}, "author_response": "Point-by-point response to reviewers\nWe thank the reviewers for the reviews of our manuscript entitled “Viral mimicry acts as a tumor suppressor in inflammation”. We greatly appreciate the opportunity to address their concerns.\nReviewer #2:\nThe authors have clarified their hypothesis and have addressed all my previous concerns and the manuscript in its current form has been greatly improved.\nWe thank the reviewer for these comments and kindly appreciate the positive feedback on the previous revisions.\nMy sole recommendation would be to add a graphical abstract summarising the cascade of events leading to VM and prevention of tumorigenesis including the interventions the authors use (eg. MAVS k.o. , Dnmt1 k.o.)\nWe agree with this suggestion and have added a graphical summary (see Figure 7) as recommended.\nReviewer #4:\nThe authors have adequately addressed my major concerns. However, the potential tumor promoting activity of certain transposable elements has not been discussed to declare the limitations of the current study, and several relevant literatures are left uncited (e.g. Nat Commun, 2022).\nNonetheless, the manuscript is significantly improved, and I think with minor modification it is ready for publication.\nWe thank the reviewer for these comments and the positive feedback regarding the previous revisions.\nWe have now added a paragraph to the discussion highlighting the tumor-promoting vs inhibiting functions of TEs (please see lines 363-371) including the Nature communications paper from 2022 that was highlighted by the reviewer.\n***Additional notes: I was also asked to comment on the previous report provided by Reviewer #1, who is unavailable for this round of review. Here is my feedback on authors' responses to Reviewer #1's previous comments.\nI think the authors have adequately addressed the concerns raised by Reviewer #1 by performing a series of additional experiments and analyses. After inclusion of those mentioned references, the manuscript in my opinion is ready for publication.\nOnce again, we thank the reviewer for acknowledging the additional changes we made to address Reviewer #1’s comments. We have included the aforementioned references as recommended by the reviewer.\nReviewer #5:\nIn this revised manuscript by Larsen et al, the authors have addressed multiple aspects of the concerns raised in relation to the first version. In this paper, the authors demonstrate that colitis leads to activation of transposable elements which act as viral mimetics and then are reduced as the inflammation progresses to dysplasia. The central hypothesis is an interesting one and is further supported by revisions to the manuscript.\nWe thank the reviewer for these comments and for acknowledging the novelty of our work.\n[...]"}, {"round": 3, "reviewer_comments": {"reviewer_2": "I have already supported publication of this nice paper in the previous round, and I still support it.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed all my concerns. I have no additional comments.", "reviewer_5": "This is an interesting study. While the work that went into the revision is appreciated and the responses to the previous comments are helpful, it is curious that none of them were included in the revised manuscript. All of those comments were intended to improve the manuscript, and I believe that all of the data in the response to comments should be included in the manuscript.", "reviewer_6": "The authors have addressed all my concerns."}, "author_response": "Point-by-point response to reviewers\nWe thank the reviewers for the reviews of our manuscript entitled “Viral mimicry acts as a tumor suppressor in colitis”.\nReviewer #2:\nI have already supported publication of this nice paper in the previous round, and I still support it.\nWe thank the reviewer for their positive feedback on the manuscript.\nReviewer #4:\nThe authors have addressed all my concerns. I have no additional comments.\nWe thank the reviewer for their time and review of the manuscript.\nReviewer #5:\nThis is an interesting study. While the work that went into the revision is appreciated and the responses to the previous comments are helpful, it is curious that none of them were included in the revised manuscript. All of those comments were intended to improve the manuscript, and I believe that all of the data in the response to comments should be included in the manuscript.\nWe thank the reviewer for these comments. We agree that much of our new data from the recent revisions would be of interest to the broader readership, and therefore, have now incorporated the majority of new data included. Specifically, we included the data looking at the expression of SINEs based on evolutionary age in patients with IBD vs controls in supplementary figure 1d. The same data, however, comparing patients with IBD and no dysplasia to patients with IBD-associated dysplasia was not included as this was similar to the analysis comparing controls to patients with IBD and did not reveal a difference in expression.\nAdditionally, we added the correlation between IFN-related gene expression and TE expression in controls and patients with IBD in supplementary figure 1c. In our analysis, we also compared different subclasses of TEs, however, did not detect a correlation that was common to the different subclasses. Therefore, we combined all the TEs together to look for a correlation and displayed our analysis in this manner as we felt this would be more informative to the reader.\nWe also added the y-H2AX staining for 5-AZA treated mice and DNMT1 tumors in supplementary figure 7.\nReviewer #6:\nThe authors have addressed all my concerns.\nWe thank the reviewer for their time."}]

Decision letter