Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses

DNA methyltransferase inhibitors (DNMTis) upregulate immune attraction, including the interferon response, in solid tumors. We now define viral defense signaling as one mechanism for this. In epithelial ovarian cancer cells DNMTis upregulate viral defense by cytosolic sensing of double-stranded RNA (dsRNA), triggering a Type I Interferon response, upregulation of downstream interferon response genes, and increased apoptosis. Knockdown of the dsRNA sensors TLR3 and MAVS and inhibition of the interferon alpha/beta receptor blunt the DNMTi induced dsRNA response. DNMTis cause apoptosis of cancer cells, which is partially rescued by inhibiting the interferon alpha/beta receptor. We observe upregulation and demethylation of hypermethylated endogenous retroviruses (ERVs) and overexpression of individual ERVs whose sense and anti-sense transcripts may be key candidates for triggering the above signaling. Overexpression of ERVs alone is sufficient to trigger an interferon response in the absence of DNMTis. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and basal expression of the viral defense signature separates primary TCGA samples for multiple tumor types into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model. We thus define a major mechanism for how DNMTis may induce cancer cells to increase immune attraction and possibly sensitize patients to immunotherapy. Experiments determining which Aza-upregulated molecules on tumor cells are necessary for attraction and activation of host immune cells are ongoing. Citation Format: Katherine B. Chiappinelli, Pamela L. Strissel, Alexis Desrichard, Huili Li, Christine Henke, Benjamin Akman, Alexander Hein, Neal S. Rote, Leslie M. Cope, Alexandra Snyder, Vladimir Makarov, Sadna Budhu, Jedd Wolchok, Cynthia A. Zahnow, Taha Mergoub, Timothy A. Chan, Reiner Strick, Stephen B. Baylin. Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B32.

Therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, with patients exhibiting vigorous and durable responses. However, in most cancer types, fewer than half of patients respond to these immune therapies. We propose epigenetic therapy as a mechanism to sensitize these patients. DNA methyltransferase inhibitors (DNMTis) upregulate immune attraction, including the interferon response, in solid tumors. We have shown that in human epithelial ovarian cancer cells, DNMTis upregulate viral defense by cytosolic sensing of double-stranded RNA (dsRNA), triggering a Type I Interferon response and apoptosis. Demethylation and expression of bidirectionally transcribed endogenous retroviruses (ERVs) is a major component of the dsRNA that activates the response. Our work showed that treatment with the DNMTi 5-azacytidine (Aza) sensitizes mouse melanoma cells to subsequent anti-CTLA4 therapy, likely through activation of the interferon response and subsequent signaling to host immune cells. Our current work aims to verify this hypothesis. In addition, we observe that adding histone deacetylase inhibitors (HDACis) to DNMTis can augment the upregulation of specific ERVs and the resulting downstream interferon response in human cancer cell lines. Specifically, the ERV-K family as well as the Fc2 and ERV-9 families are increased by DNMTi treatment but further augmented by HDACi treatment, while HDACis alone have minimal effects on the ERVs and the downstream interferon response. We tested the hypothesis that epigenetic drugs sensitize to immune therapy by recruiting host immune cells in an immunocompetent mouse model of serous ovarian cancer. Treatment of this model with DNMTi and HDACi results in increased recruitment of (CD3+) T cells, including tumor-killing T Effector cells, to the tumor. This epigenetic therapy causes increased activation of CD8 T cells and natural killer cells, an increase in helper T cells, and a reduction in myeloid derived suppressor cells and macrophages. We observed upregulation of the immune checkpoint ligand PD-L1 on tumor cells by DNMTis and hypothesized that treatment of this mouse model with the above drug combination plus an antibody to the PD-L1 receptor (anti-PD-1) could reduce tumor burden. This combination does indeed significantly reduce tumor burden and increase survival. We thus define a major mechanism for how DNMTis and HDACis may induce cancer cells to increase attraction and activation of immune cells and sensitize patients to immunotherapy. Citation Format: Katherine B. Chiappinelli, Meredith L. Stone, Michael J. Topper, Lauren Murphy, Pamela L. Strissel, Reiner Strick, Cynthia A. Zahnow, Stephen B. Baylin. Inhibiting DNA methylation causes an interferon response in cancer cells via endogenous retroviruses and recruits immune cells to the tumor microenvironment to sensitize to immune therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4019.

Here we characterize the effects of p53 status on the immune microenvironment of tumors. Specifically, we focus on the immune response induced by treatment with epigenetic modulators like 5-azacytidine (Aza), a DNA methyltransferase inhibitor (DNMTi), and whether this differs in tumors with and without functional p53. We and others have previously shown that cancer cells elicit an immune response when treated with epigenetic modulators such as DNMTis. These drugs inhibit methylation-induced gene silencing and cause re-expression of repetitive elements. Furthermore, they trigger interferon signaling in tumors by upregulation of double-stranded RNA (dsRNA) including DNA methylated endogenous retroviruses (ERVs), remains of exogenous retroviruses that integrated into the germline millions of years ago. The tumor suppressor protein p53, which is mutated in over half of all cancers, acts as a transcriptional repressor of ERVs. Little is known about the interaction between ERVs and p53 in a pathophysiologic model such as cancer. We hypothesize that p53 mutations influence the epigenetic regulation and ERV-induced immune signaling in cancer cells treated with Aza. We injected mouse ovarian cancer cells with p53 wild-type or null status into a syngeneic animal model for ovarian cancer and treated these mice with vehicle or Aza, an FDA-approved epigenetic modulator for myelodysplastic syndrome. We report that after 15 weeks, mice injected with p53 null cells present with a significantly higher tumor burden reflected in occurrence and volume of abdominal ascites fluid, weight gain over time, and shorter survival compared to mice injected with p53 wild-type cells. Further, Aza treatment compared to mock has a significant positive effect on tumor burden and survival but exclusively in mice with p53 null cells. No significant differences between treatments in the groups containing p53 wild-type cells were observed, indicating that the presence of p53 in these tumor cells likely abrogates the immunologic antitumor effects of Aza. This makes sense as p53 is a major transcriptional regulator of repetitive elements such as ERVs, which are upregulated after Aza treatment in this tumor model. We are currently characterizing differences in immune regulation of these treatment groups, in particular, infiltrating lymphocytes and Type I interferon signaling, which is crucial for the antitumor effect of Aza. We have studied how functional or lack of p53 contributes to the epigenetic regulation of ERVs and their potential to sensitize patients to immune therapy. We are now creating isogenic ovarian cancer cells containing wild-type or null as well as p53 point mutations, which occur in the majority of cancers. In doing so, we will gain novel information about the effects of mutant p53 on the ERV-induced immune response in tumor cells. Considering the vast amount of cancers harboring p53 mutations, assessing the p53 mutational status in cancers could become a useful biomarker for epigenetic therapies activating ERVs. Citation Format: Elisa Arthofer, Noor Diab, Katherine Bakshian Chiappinelli. p53 regulation of repetitive elements and the interferon response in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B20.

Highlights from Recent Cancer Literature

BackgroundImmune checkpoint inhibition (ICI) has been established as an essential treatment for lung and other cancers. Preclinical studies revealed that DNA-demethylating agents induced type I interferon (IFN-I) response and primed...

Activation of T Cells Specific to Endogenous Retroviral Peptides: Possible Association with Clinical Response to Azacitidine in Myeloid Malignancies

Oral Vitamin C Supplementation to Azacitidine in Patients with Myeloid Cancer: Normalization of Plasma Vitamin C Induces Epigenetic Changes

While therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, less than 10% of ovarian cancer (OC) patients have responded. Previous work from our group showed that DNA methyltransferase inhibitors (DNMTis) activate the interferon response in tumors through upregulation of repetitive elements (REs) including endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline several million years ago. DNMTis increase ERVs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill the tumors. Importantly, nearly all high-grade serous OCs harbor a mutation in the TP53 gene, encoding the tumor suppressor protein P53. p53 may transcriptionally repress ERVs to ensure genome stability; 30% of all p53 binding sites are in ERV-derived sequences. We treated seven human cancer cell lines, including four OC lines (3 TP53 wild-type, 4 TP53 mutant) with DNMTi and analyzed repetitive element expression (RNA-Seq), open chromatin (ATAC-Seq), and DNA methylation (MeDIP + MRE). Interestingly, RE upregulation was significantly higher in the P53 mutant cell lines. This was true for the SINE, LINE, and ERV groups of repetitive elements. The DNA methylation of these elements was not significantly different between TP53 wild-type and mutant cell lines, but the open chromatin peaks (ATAC-Seq) were significantly increased in the TP53 mutant cell lines. We thus hypothesize that wild-type TP53 may partner with DNA methylation to transcriptionally repress RE transcription. Treating TP53 wild-type cells with Nutlin-3A, which activates P53, represses transcription of some ERVs. When we overexpressed R175H or R273H mutant P53 constructs in the SKOV3 P53-null cell line and treated with DNMTi, we observed much higher upregulation of ERVs after DNMTi treatment than when we overexpressed the control wild type P53 vector. We predicted that higher ERVs would induce a higher interferon response and perhaps change the microenvironment in P53 mutant tumors. We used the ID8 MOSE mouse ovarian cancer model modified by the MacNeish laboratory to compare p53+/+ and p53-/- tumor response to DNMTi treatment. Characterization of the tumor microenvironment indicated a significantly higher frequency of lymphocytes, Treg cells, MDSCs, and myeloid cells in the p53-/- model in comparison to the p53 wild-type model. In current work, we are creating isogenic cell lines of human and mouse wild-type, mutant, and null p53 and comparing the response to DNMTi in vitro and in vivo. We will then determine whether P53 binding directly regulates repetitive elements transcriptionally, and more broadly, whether mutant P53 cells are more sensitive to DNMTi upregulation of repetitive elements and induction of an immune response. Results of this work will be particularly relevant for high-grade serous OC, which is driven by mutant P53 and is generally unresponsive to immunotherapies. Citation Format: Stephanie Gomez, James I. McDonald, Elisa Arthofer, Noor Diab, Aneil Srivastava, Paul Austin, Katherine B. Chiappinelli. The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B58.

The Effect of TET Mutations on DNA Methyltransferase Cytotoxicity and ERV Induction in B and T Cell Malignancies

Epithelial ovarian cancer is a deadly disease due to late detection and lack of targeted therapies. Novel therapies for ovarian cancer are clearly needed and epigenetic agents have emerged as promising new therapies for ovarian cancer and other solid tumors. Clinical success observed for patients with lung cancer suggests that the actions of the DNA methyltransferase inhibitor 5-azacytidine (AZA) may stimulate the immune response and target immune cells to clear tumors. Ovarian cancer is a good candidate for immune therapy, as infiltrating lymphocytes predict longer time to recurrence. Therapies that upregulate the immune system have been shown to be effective in mouse and human ovarian cancers. To determine how epigenetic therapy affects ovarian cancer cells, we utilized genome-wide methylation and expression profiling on 17 ovarian cancer cell lines treated with low-dose AZA. We discovered upregulation of immunomodulatory pathways including viral defense, type I interferon signaling, antigen processing and presentation, and immune evasion via Gene Set Enrichment Analysis. In addition, we observed strong upregulation of cancer testis antigens and molecules that attract and activate natural killer cells. Endogenous retroviruses were also increased; increased transcription of these elements caused by reversal of DNA methylation may trigger the viral defense/ interferon response. This was a specific, not an off-target, effect of AZA, as a colon cancer cell line (DKO) genetically haploinsufficient for DNMT1 and lacking DNMT3b showed a similar upregulation of immune genes. Treatment with the chemotherapeutic agent carboplatin did not upregulate the same immune response genes. The cell lines that had the highest upregulation of immune genes after AZA treatment were often the best responders to AZA when grown as xenografts in NOD/SCID mice. We hypothesize that AZA activates the immune response in cancer cells, resulting in tumor cell killing. Validation of immune target genes showed that AZA treatment activates the interferon response, including transcription of interferon beta. Media transferred from AZA-treated cells to naïve cells was sufficient to cause an interferon response in the target cells, as evidenced by increased levels of interferon-stimulated genes. FACS staining confirmed the upregulation of antigen-presenting MHC Class I molecules on the cell surface of AZA-treated ovarian cancer cells. Adding the histone deacetylase inhibitor MS275 (entinostat) to AZA treatment increased AZA-induced expression of interferon-stimulated genes IRF7 and IFI27. This combination treatment also caused an increase in the activating chromatin mark H3Ac at the IRF7 promoter. AZA increased transcript levels and cell surface expression of the PD-L1 molecule, ligand for PD-1 on lymphocytes and responsible for evasion of the host immune system by tumors, in ovarian cancer cell lines. We predict that AZA plus anti-PD-L1 (or PD-1) treatment, which has shown success in non-small cell lung cancer, might be effective for ovarian cancers. Future work will involve combination treatment of AZA and anti-PD-1 in immune competent ovarian cancer mouse models. The immune pathways upregulated by AZA were used to query hundreds of primary ovarian cancer samples from the Cancer Genome Atlas project (TCGA). Ovarian tumors classified clearly into “high” and “low” interferon gene expression subsets. The “high” interferon group was associated with higher levels of antigen presentation as well as an expression signature associated with better prognosis. The “low” interferon group indicates a group of solid tumors that could be targeted by AZA to upregulate levels of immunomodulatory pathways, and thus apoptosis or targeting by host immune cells. These preliminary results point to an “immune priming” role for the DNA methyltransferase inhibitor 5-azacytidine and could lead to clinical trials with combined AZA and immune therapies in epithelial ovarian cancer. This abstract is also presented as Poster B79. Citation Format: Katherine B. Chiappinelli, Ray-Whay Chiu Yen, Huili Li, Michael Topper, Cynthia A. Zahnow, Stephen B. Baylin. Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR12.

APOBEC enzymes are strong mutagens in breast cancer. High APOBEC3B (A3B) expression in tumors is associated with poor prognosis, but clinical significance of the other APOBEC3s (A3A, C-H) is unclear. We investigated the associations of A3A and C-H with survival and tumor immune activity in 1091 primary breast cancer tumors of The Cancer Genome Atlas project. Tumor cytolytic activity, T cell receptor (TCR) diversity, and immune cell fractions were estimated from RNA sequencing data. Patients were divided into 3 equal groups by gene expression to compare high and low expressors. Cox, Spearman and t tests were used for survival, correlation and comparison analyses, respectively. Hallmark gene-sets were used for enrichment analysis. RNA editing was determined from whole exome and RNA sequencing data for 5,208 sites of 3,630 gene transcripts that are known to undergo APOBEC3-mediated RNA editing. Examination of 55 breast cancer cell-lines of Cancer Cell Line Encyclopedia showed that A3B and A3C represented 91% of A3 gene expression. Examination of Illumina Human Body Map showed that all APOBEC3s are expressed in leukocytes at a 4-450x higher level compared to breast. In patients, expression of A3B but not other APOBEC3s was higher in tumors compared to normal tissue (4.5x). A3C-H expression levels correlated positively with both leukocyte and lymphocyte fractions in tumor (r = 0.29-0.70 and 0.20-0.50, resp.). Expression of genes related to immune function like interferon response and complement activation was enriched in high A3C-H expressors, which also had significantly more CD4 and CD8 T cells, and TCR diversity (2.3-4.0x, 2.1-5.4x and 1.3-2.1x, resp.). Concordantly, for each of A3C-H, expression correlated with tumor immune cytolytic activity (r = 0.31-0.79), which was increased 3.1-7.9x in high expressors. A3B or A3A gene expression levels had no effect on overall survival (OS), but higher expression for each of A3C-H was significantly associated with improved OS (HR = 0.45-0.66). To obtain insights on any causative mechanism for the prognostic value of A3C-H expression, we examined APOBEC3-mediated C>U RNA editing in the tumors. RNA editing at a mean level of 6.3% (SD = 4.3%) was observed among tumors for 767 genes. Editing was not associated with tumor subtype or pathologic stage but tumors with high editing had 1.4-11x higher gene expression of A3A and C-H (P <0.05 for all). TCR diversity and immune cytolytic activity in tumors correlated with RNA editing (P <0.001 for both), and increased editing was associated with improved disease-free interval (P <0.05). Even though APOBEC3C-H are DNA mutators, their high expression in tumor is associated with a strong immune response and improved survival in breast cancer. This is also observed for APOBEC3-mediated RNA editing, suggesting that RNA editing of specific genes by APOBEC3s may be promoting immune activity against cancer. Note: This abstract was not presented at the meeting. Citation Format: Mariko Asaoka, Santosh K. Patnaik, Eriko Katsuta, Takashi Ishikawa, Kazuaki Takabe. APOBEC3C-H gene expression and APOBEC3-mediated RNA editing in breast cancer tumors are associated with immune response and improved outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3416.

Background: Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in developing areas and is among the top 10 deadliest cancers with a dismal 5-year survival rate of 10~20%. ESCC molecular pathogenesis has not been well characterized, while chemoradiotherapy remains the routine treatment option besides surgery, highlighting the need for a thorough understanding of ESCC and new treatment options. Tumor protein p63 (TP63), encoding p63, plays fundamental roles in stratified epithelial homeostasis. In the esophagus, ΔNp63 is the predominant isoform and is required for normal epithelial development. ESCC retains high ΔNp63 expression level, but its contribution to ESCC development is not fully understood. Results: We found ΔNp63 maintains ESCC development, depletion of which results in great attenuation and regression of cell line-derived xenograft growth in mice. Transcriptomic profiling showed type-I interferon (IFN-I) signaling-related pathways are the top pathways enriched in ΔNp63-depleted cells. This was further verified by quantitative PCR confirming up-regulations of interferon-stimulated genes upon ΔNp63 depletion in cell lines, and by transcriptomic profiling on our latest panel of naïve ESCC patient-derived organoids (PDOs) showing TP63 expression level negatively associated with IFN-I signaling-related pathways. Elevated endogenous retrotransposon-encoded RNA expression induces cancer cell IFN-I signaling through mediating tumor-suppressive viral mimicry response, an anti-viral state triggered by endogenous stimuli (1). We found cancer cell ΔNp63 depletion results in increased retrotransposon expression triggering dsRNA sensing and downstream signaling activation; Interferon regulatory factor 1 plays a critical role in mediating viral mimicry response downstream of ΔNp63. We further showed cells with lower ΔNp63 level and higher IFN-I signaling activity (ΔNp63-depleted cell lines and ΔNp63lo PDOs) display stronger responses to Decitabine, an anti-cancer drug and viral mimicry booster (1), as compared to control cell lines or ΔNp63hi PDOs, respectively. Conclusion: We identified a novel function of ΔNp63 in repressing cancer retrotransposon expression and explored the therapeutic potential of enhancing viral mimicry response, which may guide future ΔNp63/viral mimicry response-targeted therapy. Acknowledgement: We acknowledge the Research Grants Council (TRS T12-701/17-R to MLL) and the Food and Health Bureau (HMRF 06171566 to VZY) of Hong Kong SAR for funding supports. We acknowledge DSMZ for the KYSE cell lines. We acknowledge the HKUMed-CPOS for providing imaging facilities. Reference: 1.Chiappinelli KB, et al. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Cell 2015 Citation Format: Valen Zhuoyou Yu, Shan Shan So, Bryan Chee-Chad Lung, Carissa Wing-Yan Wong, Ian Yu-Hong Wong, Claudia Lai-Yin Wong, Desmond Kwan-Kit Chan, Fion Siu-Yin Chan, Betty Tsz-Ting Law, Ka-On Lam, Anthony Wing-Ip Lo, Josephine Mum-Yee Ko, Wei Dai, Alfred King-Yin Lam, Dora Lai-Wan Kwong, Simon Law, Maria Li Lung. p63 constrains cancer cell transposable element expressions and viral mimicry response to sustain esophageal cancer development and indicates therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2611.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses

NSD2 expression remodels DNA methylation, creating a targetable epigenetic dependency in t(4;14) multiple myeloma