Inhibiting DNA methylation and RNA editing upregulates immunogenic RNA to transform the tumor microenvironment and prolong survival in ovarian cancer

Stephanie Gomez, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Olivia L. Cox, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Reddick R. Walker, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Uzma Rentia, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Melissa Hadley, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Elisa Arthofer, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Noor Diab, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Erin E. Grundy, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Tomas Kanholm, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
James I. McDonald, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Julie Kobyra, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Erica Palmer, Department of Biochemistry, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Satish Noonepalle, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA.
Alejandro Villagra, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA.
David Leitenberg, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Catherine M. Bollard, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Yogen Saunthararajah, Department of Hematology and Medical Oncology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.
Katherine B. Chiappinelli, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Cancer Center, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA kchiapp1@gwu.edu.

Document Type

Journal Article

Publication Date

11-1-2022

Journal

Journal for immunotherapy of cancer

Volume

10

Issue

11

DOI

10.1136/jitc-2022-004974

Keywords

adjuvants, pharmaceutic; genital neoplasms, female; interferon inducers; lymphocytes, tumor-infiltrating; tumor microenvironment

Abstract

BACKGROUND: Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS: Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days. RESULTS: We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION: In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

Department

Pediatrics

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