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Inactivating mutations in SMARCB1 confer an oncogenic dependency on EZH2 in atypical teratoid rhabdoid tumors (ATRTs), but the underlying mechanism has not been fully elucidated. We found that the sensitivity of ATRTs to EZH2 inhibition (EZH2i) is associated with the viral mimicry response. Unlike other epigenetic therapies targeting transcriptional repressors, EZH2i-induced viral mimicry is not triggered by cryptic transcription of endogenous retroelements, but rather mediated by increased expression of genes enriched for intronic inverted-repeat Alu (IR-Alu) elements. Interestingly, interferon-stimulated genes (ISGs) are highly enriched for dsRNA-forming intronic IR-Alu elements, suggesting a feedforward loop whereby these activated ISGs may reinforce dsRNA formation and viral mimicry. EZH2i also upregulates the expression of full-length LINE-1s, leading to genomic instability and cGAS/STING signaling in a process dependent on reverse transcriptase activity. Co-depletion of dsRNA sensing and cytoplasmic DNA sensing completely rescues the viral mimicry response to EZH2i in SMARCB1-deficient tumors., (© 2024. The Author(s).)

Progression to aggressive secondary acute myeloid leukaemia (sAML) poses a significant challenge in the management of myeloproliferative neoplasms (MPNs). Since the physiopathology of MPN is closely linked to the activation of interferon (IFN) signalling and that AML initiation and aggressiveness is driven by leukaemia stem cells (LSCs), we investigated these pathways in MPN to sAML progression. We found that high IFN signalling correlated with low LSC signalling in MPN and AML samples, while MPN progression and AML transformation were characterized by decreased IFN signalling and increased LSC signature. A high LSC to IFN expression ratio in MPN patients was associated with adverse clinical prognosis and higher colony forming potential. Moreover, treatment with hypomethylating agents (HMAs) activates the IFN signalling pathway in MPN cells by inducing a viral mimicry response. This response is characterized by double-stranded RNA (dsRNA) formation and MDA5/RIG-I activation. The HMA-induced IFN response leads to a reduction in LSC signature, resulting in decreased stemness. These findings reveal the frequent evasion of viral mimicry during MPN-to-sAML progression, establish the LSC-to-IFN expression ratio as a progression biomarker, and suggests that HMAs treatment can lead to haematological response in murine models by re-activating dsRNA-associated IFN signalling., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8 + T cells. In vivo HMA treatment promotes CD8 + T cell tumor infiltration and suppresses tumor growth via CD8 + T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8 + T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8 + T cell subpopulations, increasing both the number and abundance of a granzyme B high , perforin high effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response., Competing Interests: Declaration of interests D.D.D.C. received research funds from Pfizer and Nektar Therapeutics. D.D.D.C. is a co-founder and shareholder of DNAMx, Inc. C.K. is employed by and owns stock, patents, and royalties with Roche. J.S. is a permanent scientific advisory board member for and owns stock in Surface Oncology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor 1-6 . This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses 7,8 . However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands 9 . In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA 10 , which prevents activation of the MDA5 receptor 11 . We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.

Tumor progression upon treatment arises from preexisting resistant cancer cells and/or adaptation of persister cancer cells committing to an expansion phase. Here, we show that evasion from viral mimicry response allows the growth of taxane-resistant triple-negative breast cancer (TNBC). This is enabled by an epigenetic state adapted to taxane-induced metabolic stress, where DNA hypomethylation over loci enriched in transposable elements (TE) is compensated by large chromatin domains of H3K27me3 to warrant TE repression. This epigenetic state creates a vulnerability to epigenetic therapy against EZH2, the H3K27me3 methyltransferase, which alleviates TE repression in taxane-resistant TNBC, leading to double-stranded RNA production and growth inhibition through viral mimicry response. Collectively, our results illustrate how epigenetic states over TEs promote cancer progression under treatment and can inform about vulnerabilities to epigenetic therapy. SIGNIFICANCE: Drug-resistant cancer cells represent a major barrier to remission for patients with cancer. Here we show that drug-induced metabolic perturbation and epigenetic states enable evasion from the viral mimicry response induced by chemotherapy in TNBC. These epigenetic states define a vulnerability to epigenetic therapy using EZH2 inhibitors in taxane-resistant TNBC. See related commentary by Janin and Esteller, p. 1258 . This article is highlighted in the In This Issue feature, p. 1241 ., (©2020 American Association for Cancer Research.)

Purpose: To evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically 'cold' solid tumors to immune checkpoint inhibitor durvalumab., Experimental Design: PD-L1/PD-1 inhibitor naïve patients with advanced microsatellite stable colorectal cancer; platinum resistant ovarian cancer; and estrogen receptor positive, HER2 negative breast cancer were enrolled in this single-institution, investigator-initiated trial. Two 28 day regimens, regimen A (CC-486 300 mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500 mg intravenous day 15) and regimen B (CC-486 100 mg QD days 1-21 (cycle 1 and beyond), vitamin C 500 mg once a day continuously and durvalumab 1500 mg intravenous day 15) were investigated. Patients underwent paired tumor biopsies and serial peripheral blood mononuclear cells (PBMCs) collection for immune-profiling, transcriptomic and epigenomic analyzes., Results: A total of 28 patients were enrolled, 19 patients treated on regimen A and 9 on regimen B. The combination of CC-486 and durvalumab was tolerable. Regimen B, with a lower dose of CC-486 extended over a longer treatment course, showed less grade 3/4 adverse effects. Global LINE-1 methylation assessment of serial PBMCs and genome-wide DNA methylation profile in paired tumor biopsies demonstrated minimal changes in global methylation in both regimens. The lack of robust tumor DNA demethylation was accompanied by an absence of the expected 'viral mimicry' inflammatory response, and consequently, no clinical responses were observed. The disease control rate was 7.1%. The median progression-free survival was 1.9 months (95% CI 1.5 to 2.3) and median overall survival was 5 months (95% CI 4.5 to 10)., Conclusions: The evaluated treatment schedules of CC-486 in combination with durvalumab did not demonstrate robust pharmacodynamic or clinical activity in selected immunologically cold solid tumors. Lessons learned from this biomarker-rich study should inform continued drug development efforts using these agents., Trial Registration Number: NCT02811497., Competing Interests: Competing interests: PLB: Consultant for: Bristol-Myers Squibb (compensated), Sanofi (compensated), Pfizer (compensated). Grant/Research support from (Clinical Trials for aarinstitution): Bristol-Myers Squibb, Sanofi, AstraZeneca, Genentech/Roche, Servier, GlaxoSmithKline, Novartis, SignalChem, PTC Therapeutics, Nektar, Merck, Seattle Genetics, Mersana, Immunomedics, Lilly. AAR: Honoraria: Boehringer Ingelheim. Consultant for: Lilly (compensated), Merck (compensated), Boehringer Ingelheim (compensated). Grant/Research support from (Clinical Trials): CASI Pharmaceuticals, Boehringer Ingelheim, Lilly, Novartis, Deciphera, Karyopharm Therapeutics, Pfizer, Genentech/Roche, Boston Biomedical, Bristol-Myers Squibb, MedImmune, Amgen, GlaxoSmithKline, Blueprint Medicines, Merck, Abbvie, Adaptimmune. ARH: Advisory/Consulting/Research for Genentech/Roche, Merck, GSK, Bristol-Myers Squibb, Novartis, Boston Biomedical, Boehringer-Ingelheim, AstraZeneca, Medimmune. AS: Consultant for (Advisory Board): Merck (compensated), Bristol-Myers Squibb (compensated), Novartis (compensated), Oncorus (compensated), Janssen (compensated). Grant/Research support from (Clinical Trials): Novartis, Bristol-Myers Squibb, Symphogen AstraZeneca/Medimmune, Merck, Bayer, Surface Oncology, Northern Biologics, Janssen Oncology/Johnson & Johnson, Roche, Regeneron, Alkermes, Array Biopharma. DC: Consulting/Advisory: Agendia, AstraZeneca, GSK, Merck, Novartis, Pfizer, Puma, Roche, Dynamo Therapeutics. Grant/Research support from (Clinical Trials): GlaxoSmithKline, Merck, Pfizer. Intellectual Property: Biomarkers for TTK inhibitors (assigned to institution). MOB: Consulting for: Bristol-Myers Squibb, Novartis, Merck, GlaxoSmithKline, EMD Serono, Sanofi, Immunocore. Grant/Research support from (Clinical Trials): Merck, Takara Bio. AMO: Consultant for: AstraZeneca, MERCK, Clovis Oncology, TESARO (all non-compensated). SL: Consultant for: Merck (compensated), AstraZeneca (compensated), GlaxoSmithKline (compensated), Roche (compensated). PO: Consulatnt for: Providence (compensated), Symphogen (compensated), Tessa (compensated), Research support from: EMD Serono. LS: Consultant for: Merck (compensated), Pfizer (compensated), Celgene (compensated), AstraZeneca/Medimmune (compensated), Morphosys (compensated), Roche (compensated), GeneSeeq (compensated), Loxo (compensated), Oncorus (compensated), Symphogen (compensated), Seattle Genetics (compensated), GSK (compensated), Voronoi (compensated), Treadwell Therapeutics (compensated), Arvinas (compensated), Tessa (compensated), Navire (compensated). Grant/Research support from (Clinical Trials for institution): Novartis, Bristol-Myers Squibb, Pfizer, Boerhinger-Ingelheim, GlaxoSmithKline, Roche/Genentech, Karyopharm, AstraZeneca/Medimmune, Merck, Celgene, Astellas, Bayer, Abbvie, Amgen, Symphogen, Intensity Therapeutics, Mirati, Shattucks, Avid. Stockholder in: Agios (spouse). DDC: Research support from Pfizer and Nektar therapeutics., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

During cancer initiation and progression, the somatic epigenome is broadly reprogrammed. This reprogramming can be a consequence of several processes, including altered transcriptional profiles and mutations. In addition, immune cells infiltrating the tumor microenvironment display a reprogrammed epigenome. For instance, tumor infiltrating T cells frequently exhibit an exhausted phenotype characterized by aberrant DNA methylation. Moreover, these aberrant epigenomes of cancer cells and infiltrating immune cells may represent a cancer vulnerability. Accumulating evidence supports the potential of using epigenetic therapy to not only reactivate silenced genes in cancer cells, but to also increase antitumor immunogenicity, by reactivation of endogenous retroviruses, to increase tumor immune-infiltration, and to reinvigorate T cell exhaustion. These findings highlight the potential synergies between epigenetic therapies and immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nucleic acid sensing is a central mechanism for innate immune defense against foreign molecules that culminates with an activation of interferon signaling pathways. This involves detection of molecular patterns associated with extracellular or intracellular pathogens by specialized receptors within the cell. In addition to foreign molecules, cells also sense endogenous molecules. One specific arm of nucleic acid sensors detects dsRNA structures. In this chapter, we discuss principles of dsRNA recognition and downstream activation of signaling pathways important in the process of antiviral responses. We also discuss various mechanisms by which endogenous dsRNA can form in a cell, in particular, through epigenetic regulation. Finally, we provide approaches for measuring and quantifying dsRNA accumulation and downstream activation in human colorectal cancer cells., (© 2019 Elsevier Inc. All rights reserved.)

The use of liquid biopsies for cancer detection and management is rapidly gaining prominence 1 . Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations 2-5 . By contrast, large-scale epigenetic alterations-which are tissue- and cancer-type specific-are not similarly constrained 6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns.