Oncogenic Transformation Drives DNA Methylation Loss and Transcriptional Activation at Transposable Element Loci.

Transposable elements (TE) are typically silenced by DNA methylation and repressive histone modifications in differentiated healthy human tissues. However, TE expression increases in a wide range of cancers and is correlated with global hypomethylation of cancer genomes. We assessed expression and DNA methylation of TEs in fibroblast cells that were serially transduced with hTERT, SV40, and HRASR24C to immortalize and then transform them, modeling the different steps of the tumorigenesis process. RNA sequencing and whole-genome bisulfite sequencing were performed at each stage of transformation. TE expression significantly increased as cells progressed through transformation, with the largest increase in expression after the final stage of transformation, consistent with data from human tumors. The upregulated TEs were dominated by endogenous retroviruses [long terminal repeats (LTR)]. Most differentially methylated regions (DMR) in all stages were hypomethylated, with the greatest hypomethylation in the final stage of transformation. A majority of the DMRs overlapped TEs from the RepeatMasker database, indicating that TEs are preferentially demethylated. Many hypomethylated TEs displayed a concordant increase in expression. Demethylation began during immortalization and continued into transformation, while upregulation of TE transcription occurred in transformation. Numerous LTR elements upregulated in the model were also identified in The Cancer Genome Atlas datasets of breast, colon, and prostate cancer. Overall, these findings indicate that TEs, specifically endogenous retroviruses, are demethylated and transcribed during transformation. SIGNIFICANCE: Analysis of epigenetic and transcriptional changes in a transformation model reveals that transposable element expression and methylation are dysregulated during oncogenic transformation.

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

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 Trp53-/- 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 Trp53-/- 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-ß 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.

Epigenetic Therapies in Ovarian Cancer Alter Repetitive Element Expression in a TP53-Dependent Manner.

Epithelial ovarian carcinomas are particularly deadly due to intratumoral heterogeneity, resistance to standard-of-care therapies, and poor response to alternative treatments such as immunotherapy. Targeting the ovarian carcinoma epigenome with DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) increases immune signaling and recruits CD8+ T cells and natural killer cells to fight ovarian carcinoma in murine models. This increased immune activity is caused by increased transcription of repetitive elements (RE) that form double-stranded RNA (dsRNA) and trigger an IFN response. To understand which REs are affected by epigenetic therapies in ovarian carcinoma, we assessed the effect of DNMTi and HDACi on ovarian carcinoma cell lines and patient samples. Subfamily-level (TEtranscripts) and individual locus-level (Telescope) analysis of REs showed that DNMTi treatment upregulated more REs than HDACi treatment. Upregulated REs were predominantly LTR and SINE subfamilies, and SINEs exhibited the greatest loss of DNA methylation upon DNMTi treatment. Cell lines with TP53 mutations exhibited significantly fewer upregulated REs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the TP53-mutant cell line had significantly higher baseline expression of REs but upregulated fewer upon epigenetic treatment. In addition, p53 activation increased expression of REs in wild-type but not mutant cell lines. These data give a comprehensive, genome-wide picture of RE chromatin and transcription-related changes in ovarian carcinoma after epigenetic treatment and implicate p53 in RE transcriptional regulation. SIGNIFICANCE: This study identifies the repetitive element targets of epigenetic therapies in ovarian carcinoma and indicates a role for p53 in this process.

MMP-2 is a novel histone H3 N-terminal protease necessary for myogenic gene activation.

BACKGROUND: Selective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown. RESULTS: In this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation. CONCLUSIONS: This study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.

Mitochondrial Enzymes of the Urea Cycle Cluster at the Inner Mitochondrial Membrane.

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein-"variable segment," which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.