Transcription Of Endogenous Retroviruses Research Articles

Transcription of endogenous retroviruses in senescent cells contributes to the accumulation of double-stranded RNAs that trigger an anti-viral response that reinforces senescence

An important epigenetic switch marks the onset and maintenance of senescence. This allows transcription of the genetic programs that arrest the cell cycle and alter the microenvironment. Transcription of endogenous retroviruses (ERVs) is also a consequence of this epigenetic switch. In this manuscript, we have identified a group of ERVs that are epigenetically silenced in proliferating cells but are upregulated during replicative senescence or during various forms of oncogene-induced senescence, by RAS and Akt, or after HDAC4 depletion. In a HDAC4 model of senescence, removal of the repressive histone mark H3K27me3 is the plausible mechanism that allows the transcription of intergenic ERVs during senescence. We have shown that ERVs contribute to the accumulation of dsRNAs in senescence, which can initiate the antiviral response via the IFIH1-MAVS signaling pathway and thus contribute to the maintenance of senescence. This pathway, and MAVS in particular, plays an active role in shaping the microenvironment and maintaining growth arrest, two essential features of the senescence program.

Leukemic Cell-Intrinsic Innate Nucleic Acid Receptor Signaling Governs Efficacy of Hypomethylating Agents and Immunosurveillance in AML

Introduction: Hypomethylating agents (HMA), such as Azaciditine (AZA), are a long-established treatment option for acute myeloid leukemia (AML). Besides epigenetic reprogramming of malignant cell clones, HMAs are recognized to facilitate antineoplastic immunity. In vitro assessments in solid tumor models suggest that a major mechanism underlying AZA-triggered immune responses may be the activation of innate nucleic acid sensing pathways. As such, the RNA- and DNA-sensing receptor systems, RIG-I/MDA-5 and cGAS/STING - and subsequent type I interferon (IFN-I) signaling seem to be triggered by the transcriptional upregulation of otherwise DNA-hypermethylated and thus silenced endogenous retroviruses (ERVs). In AML, it is unknown whether similar processes are involved and/or by which molecular mechanisms, HMAs modulate anti-leukemic immunity against AML blasts, and how these processes are affected during acquired HMA resistance. Methods: Murine (c1498 and Wehi-3B) and human (MOLM-13) AML cell lines were exposed to AZA in vitro and analyzed for expression of ERVs. Nucleic acid receptor sensing pathways and IFN-I genes are not usually DNA methylated by AZA at promoter regions. However, the re-expression of endogenous nucleic acids or ERVs could trigger their activation. Protein and transcriptional expression of major components of nucleic acid receptor sensing pathways and IFN-I genes were, therefore, analysed. To understand the importance of AML cell-intrinsic nucleic acid receptor signaling in HMA-induced immunosurveillance, syngeneic c1498 and Wehi-3B AML models were employed. Mice received 2 cycles of 2.5mg/kg AZA (6 days on, 5 days off, 6 days on) intraperitoneally to mimic the principal treatment course used in patients. To address the contribution of the nucleic acid-sensing pathways to AZA treatment efficacy in vitroand in vivo, AML cells with acquired AZA resistance (AZA-R) or gene engineered AML cells deficient in RNA receptors (RIG-I/MDA-5), or the RNA- and DNA-sensing adapter proteins (MAVS and STING) were employed. Results: A targeted ERV screen revealed upregulation of various ERV transcripts in c1498 and Wehi-3B cells and was similarly observed in MOLM-13 AML cells following AZA exposure in vitro. Increased RNA transcript and protein expression of RIG-I/MAVS and (to a lesser degree) STING was observed, resulting in activation of downstream constituents including higher phosphorylation of the transcription factors IRF3/7 and NF-kB, IFN-I, and other ISGs. This was abrogated in RIG-I and MAVS, but not STING, deficient murine and human AML cells. Interestingly, expression of RIG-I/MDA-5, STING and all other downstream constituents, but not MAVS, were diminished in AZA-R AML cells. In mice harboring MAVS deficient AML (c1498), HMA treatment failed to show anti-leukemic activity resulting in rapid disease progress and death of recipient animals. Loss of STING in leukemic cells also impacted AZA treatment efficacy, although to a much less pronounced degree. These findings were similarly reproduced with Wehi-3B AML-engrafted mice. Following antibody-mediated depletion of T cells but not NK cells, mice rapidly succumbed to AML disease and demonstrated no anti-leukemic effect of AZA treatment. CyTOF and flow cytometric analysis also revealed increased bone marrow and spleen infiltration as well as activation of CD4 +/CD8 + T cells, NK cells, and MHC-II + APCs/Myeloid cells in AZA treated mice bearing wildtype AML, compared to untreated. This was not observed in AZA-treated mice bearing MAVS-deficient or AZA-R AML. Conclusion: Our findings indicate that nucleic acid receptor signaling (specifically RIG-I/MAVS) within leukemic cells is imperative for AZA efficacy and immunosurveillance in AML. Whether these RNA-sensing receptors are triggered by ERVs or alternate (endogenous) RNAs remains to be determined. Elucidating the molecular mechanisms of how nucleic acid sensors within leukemic blasts shape treatment efficacy of AZA may guide development of systematic immunotherapeutic strategies for AZA-R AML by targeted combinatorial approaches.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

Genome‐wide identification of endogenous retrovirus elements and their active transcription in mink genome

AbstractMammalian endogenous retroviruses (ERVs) are ancient retroviruses that have been integrated into genomes. ERVs were believed to be inactive until the discovery of ERV transcription in the mouse genome. However, the transcription level and function of ERV elements in mammalian genomes are not well understood. In this study, we performed the first genome‐wide scanning of ERV loci in the American mink (Neogale vison) genome (NeoERV) followed by transcriptomic analysis to detect actively transcribed NeoERV elements. A total of 365,791 NeoERV loci were identified, and161,205 (44%) of these loci were found to be actively transcribed based on transcriptomic data from three types of tissues (amygdala, trachea and lung). More than one third of the actively transcribed NeoERV loci were tissue‐specific. Furthermore, some of the active loci were associated with host gene transcription, and the level of NeoERV transcription was positively correlated with that of host genes, specifically when active loci were located in overlapped gene regions. An in‐depth analysis of the envelope protein coding env gene showed that, in general, its transcription level was higher than that of NeoERVs, which is believed to be associated with host immunity.

18P A translational project of the WSG-ADAPT-TN trial demonstrates immunomodulatory and anti-viral defense gene networks predicting pathological complete response (pCR) and survival after de-escalated neoadjuvant chemotherapy (NACT) in early triple-negative breast cancer (eTNBC)

NACT is the standard treatment for eTNBC, yet, molecular phenotypes driving chemotherapy sensitivity are poorly understood. Whole transcriptome profiling of baseline tumor biopsies from the neoadjuvant WSG-ADAPT-TN phase II trial (NCT01815242) was performed to identify gene networks predictive and prognostic for pCR and survival. Patients with cT1c-cT4c, cN+, centrally confirmed eTNBC were randomized to 12 weeks of nab-paclitaxel+gemcitabine (Arm A; n=182) or nab-paclitaxel+carboplatin (Arm B; n=154). The primary endpoint was pCR (ypT0/is, ypN0), secondary endpoints included survival and translational research. AmpliSeq RNA sequencing allowing simultaneous analysis of the expression of >20,000 genes was performed in 137 patients (Arm A/B: n=72/65). Differentially expressed genes were then evaluated in training (n=67) and validation (n=68) sets and two polygenic scores (PS) with a high diagnostic performance for prediction of pCR (PS:pCR) and invasive disease-free survival (PS:iDFS) were found. 49/135 (36.3%) patients had pCR; 30 iDFS events occurred during 60-month median follow-up. Genes and networks associated with immune recruitment, endogenous retroviral transcription, and viral defense were strongly associated with pCR and iDFS. PS:pCR achieved a high diagnostic accuracy (ROC AUC: 83%). In multivariate analysis (validation set) adjusted for clinical factors, pCR was associated with PS:pCR (OR 7.24; 95%CI 2.05, 25.58; p=0.002). Addition of PS:pCR to clinical factors increased ROC AUC for pCR from 0.707 to 0.887. iDFS was associated with PS:iDFS (HR 2.06; 95%CI 1.01, 4.20; p=0.046). Polygenic scores based on immunomodulatory and anti-viral defense gene networks may have utility in predicting pCR and survival, and could represent an opportunity for selecting patients for de-escalated NACT in eTNBC. This transcriptome network analysis also identifies potential new targets for personalized medicine approaches for patients without response to chemotherapy.

The roles of epidermal Blimp-1 in atopic dermatitis

Abstract Keratinocytes are the main cell type constructing the epidermis of animals, which can further differentiate into corneocytes and express multiple innate immune receptors. B lymphocyte-induced maturation protein-1 (Blimp-1) plays a crucial rule in directing cornification of keratinocytes. We previously reported that keratinocyte-specific deletion of Prdm1, the gene encoding Blimp-1, induced spontaneous inflammation in the skin of adult mice. We further found that deletion of Prdm1in keratinocytes in Balb/c genetic background caused hyper-production of type 2 cytokines and thymic stromal lymphopoietin (TSLP) in the skin, and staphylococci colonization, which are characteristics of atopic dermatitis (AD). Since K5-CreER+Prdm1f/f(CKO) mice in Balb/c background exhibited AD-like symptoms, we wondered whether they are more susceptible to the disease progression in a murine AD model induced by MC903, a vitamin D3 analog enhancing TSLP production by keratinocytes. We found that CKO mice showed more immune cell infiltration in ear skin, developed more severe epidermal hyperplasia, and induced more TSLP secretion in the skin following topical application of MC903. Recent studies showed that staphylococci colonization activated the transcription of endogenous retroviruses (ERVs) in murine skin, while excess ERV expression led to skin inflammation. As staphylococci colonized on our CKO mouse skin, we hypothesized that enhanced ERV expression may also link to skin inflammation of CKO mice. Indeed, both in vivoand in vitrodeletion of Prdm1in keratinocytes resulted in elevated levels of ERV transcripts. We are studying the contribution of elevated ERV transcripts in Blimp-1-reduction-mediated AD-like skin inflammation.

Activation of endogenous retrovirus triggers microglial immuno-inflammation and contributes to negative emotional behaviors in mice with chronic stress

BackgroundThe “missing” link of complex and multifaceted interplay among endogenous retroviruses (ERVs) transcription, chronic immuno-inflammation, and the development of psychiatric disorders is still far from being completely clarified. The present study was aimed to investigate the mechanism of protective role of inhibiting ERVs on reversing microglial immuno-inflammation in basolateral amygdala (BLA) in chronic stress-induced negative emotional behaviors in mice.MethodsMale C57BL/6 mice were exposed to chronic unpredictable mild stress (CUMS) for 6 w. Negative emotional behaviors were comprehensively investigated to identify the susceptible mice. Microglial morphology, ERVs transcription, intrinsic nucleic acids sensing response, and immuno-inflammation in BLA were assessed.ResultsMice with chronic stress were presented as obviously depressive- and anxiety-like behaviors, and accompanied with significant microglial morphological activation, murine ERVs genes MuERV-L, MusD, and IAP transcription, cGAS–IFI16–STING pathway activation, NF-κB signaling pathway priming, as well as NLRP3 inflammasome activation in BLA. Antiretroviral therapy, pharmacological inhibition of reverse transcriptases, as well as knocking-down the ERVs transcriptional regulation gene p53 significantly inhibited microglial ERVs transcription and immuno-inflammation in BLA, as well as improved the chronic stress-induced negative emotional behaviors.ConclusionsOur results provided an innovative therapeutic approach that targeting ERVs-associated microglial immuno-inflammation may be beneficial to the patients with psychotic disorders.

Susceptibility and resilience to maternal immune activation are associated with differential expression of endogenous retroviral elements

Endogenous retroviruses (ERVs) are ancestorial retroviral elements that were integrated into the mammalian genome through germline infections and insertions during evolution. While increased ERV expression has been repeatedly implicated in psychiatric and neurodevelopmental disorders, recent evidence suggests that aberrant endogenous retroviral activity may contribute to biologically defined subgroups of psychotic disorders with persisting immunological dysfunctions. Here, we explored whether ERV expression is altered in a mouse model of maternal immune activation (MIA), a transdiagnostic environmental risk factor of psychiatric and neurodevelopmental disorders. MIA was induced by maternal administration of poly(I:C) on gestation day 12 in C57BL/6N mice. Murine ERV transcripts were quantified in the placentae and fetal brains shortly after poly(I:C)-induced MIA, as well as in adult offspring that were stratified according to their behavioral profiles. We found that MIA increased and reduced levels of class II ERVs and syncytins, respectively, in placentae and fetal brain tissue. We also revealed abnormal ERV expression in MIA-exposed offspring depending on whether they displayed overt behavioral anomalies or not. Taken together, our findings provide a proof of concept that an inflammatory stimulus, even when initiated in prenatal life, has the potential of altering ERV expression across fetal to adult stages of development. Moreover, our data highlight that susceptibility and resilience to MIA are associated with differential ERV expression, suggesting that early-life exposure to inflammatory factors may play a role in determining disease susceptibility by inducing persistent alterations in the expression of endogenous retroviral elements.

Abstract 6301: DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML

Abstract DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p<0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p<0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.

Abstract 5593: DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity

Abstract DNA methyltransferase inhibitors (DNMTis) modulate genome-wide methylation patterns, previously linked by our group to downregulation of DNA double strand break repair (DSBR) that sensitizes non-small cell lung cancer (NSCLC) to poly(ADP-ribose) polymerase inhibitors (PARPis). DNMTi hypomethylation of endogenous retroviral (ERV) elements produces cytosolic aggregation of double-stranded RNA (dsRNA) that activates antiviral interferon (IFN) and inflammasome signaling. Accordingly, DNMTi treatment of NSCLC cell lines (A549, H460) increases expression of 1) multiple ERV transcripts; 2) dsRNA sensors RIG-I and MDA5; 3) downstream mitochondrial antiviral signaling protein (MAVS); 4) MAVS-activated antiviral kinases (TBK1, IKKα/β) and transcriptional regulators (IRF3/7, NFκB); and 5) IFNβ and IFN-stimulated genes (ISGs). A second mechanism of cellular antiviral signaling is STING (stimulator of interferon genes) activation triggered by cytosolic dsDNA. Following DNMTi treatment, we observe mitochondrial dysfunction including impairment of oxidative phosphorylation, reactive oxygen species generation, and reduced mitochondrial membrane potential. This dysfunction is associated with increased mitochondrial DNA (mtDNA) damage and accumulation of cytosolic mtDNA, leading to increased STING expression and IFN activation that can be abrogated by siSTING or IKK inhibition. Treatment with PARPi, which activates STING via cytosolic accumulation of damaged nuclear DNA, further enhances DNMTi-induced IFN signaling. We previously linked IFN activation to DSBR downregulation in breast and ovarian cancer, and now present TCGA analysis in NSCLC indicating a similar negative correlation, corroborated by reduced DSBR in NSCLC cell lines after IFNβ treatment. Computational interrogation indicates multiple DSBR promoters contain IFN-stimulated response elements (ISREs), including potential negative regulators, that may account for these findings. Notably, we find that precise patterns of immune activation may be TP53 dependent: NSCLC cell lines with missense mutations (H23, H441, H1115), as observed in ~30% patients, exhibit increased NFκB inflammasome signaling and decreased IFN activation compared to wildtype (A549, H460). TP53 mutation is associated with significantly reduced baseline STING expression, potentially affording a more robust DNMTi treatment response; accordingly, TP53-mutant NSCLC cells exhibit significantly greater sensitivity to DNMTi/PARPi. Our new data expands on our published work to further understanding of novel mechanisms of 1) inflammasome induction and 2) mitochondrial dysfunction through which DNMTis activate cellular antiviral signaling and downregulate DSBR to induce PARPi sensitivity in NSCLC. We also identify TP53 as a possible biomarker for future personalization of DNMTi therapy. Citation Format: Rachel Abbotts, Lora Stojanovic, Michael J. Topper, Brian M. Polster, Stephen B. Baylin, Feyruz V. Rassool. DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5593.

TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment.

Telomerase plays a pivotal role in tumorigenesis by both telomere-dependent and telomere-independent activities, although the underlying mechanisms are not completely understood. Using single-sample gene set enrichment analysis (ssGSEA) across 9,264 tumour samples, we observe that expression of telomerase reverse transcriptase (TERT) is closely associated with immunosuppressive signatures. We demonstrate that TERT can activate a subclass of endogenous retroviruses (ERVs) independent of its telomerase activity to form double-stranded RNAs (dsRNAs), which are sensed by the RIG-1/MDA5-MAVS signalling pathway and trigger interferon signalling in cancer cells. Furthermore, we show that TERT-induced ERV/interferon signalling stimulates the expression of chemokines, including CXCL10, which induces the infiltration of suppressive T-cell populations with increased percentage of CD4+ and FOXP3+ cells. These data reveal an unanticipated role for telomerase as a transcriptional activator of ERVs and provide strong evidence that TERT-mediated ERV/interferon signalling contributes to immune suppression in tumours.

Identification of an endogenous retroviral signature to predict anti-PD1 response in advanced clear cell renal cell carcinoma: an integrated analysis of three clinical trials.

Endogenous retrovirus (ERV) elements are genomic footprints of ancestral retroviral infections within the human genome. Previous studies have demonstrated that dysregulated ERV transcription level is associated with immune cell infiltration in cancers, but the association between ERV expression and programmed cell death protein 1 (PD-1) blockade response is currently unraveled for solid cancers, such as advanced clear cell renal cell carcinoma (ccRCC). ERV mRNA profiles were obtained from three clinical trials of ccRCC where the patients were treated with anti-PD-1 (CM-009, CM-010, CM-025, and TCGA-KIRC data). Patients treated with nivolumab were divided into training and test cohort, while the TCGA-KIRC cohort was used as an external validation. Univariate Cox regression analysis and least absolute shrinkage and selection operator regression were used to establish the signature. Immune cell infiltration analysis and gene set enrichment analysis were performed to explore potential biological mechanisms. An ERV signature was established based on nine ERV expression patterns. In the training cohort, the median overall survival in the low- and high-risk group was 45.2 and 19.6 months [hazard ratio (HR) = 0.49, 0.32-0.75, p < 0.001], respectively. The results were confirmed in the test (HR = 0.41, 0.20-0.83, p = 0.013), and in the TCGA-KIRC cohort (HR = 0.55, 0.34-0.90, p = 0.017). Moreover, in the CM-025 cohort, the low-risk group that received nivolumab had a more favorable survival compared with those that received the mTOR inhibitor everolimus, while no significant differences were observed in the high-risk group. CD8+ T cells were enriched in the low-risk group, while immune suppressive pathways were suppressed. The newly identified ERV signature is not only a prognostic, but also a predictive biomarker for advanced ccRCC patients who received anti-PD-1 therapy, which can guide personalized treatment in cancer patients in the future.

Influence of retroelements on oncogenes and tumor suppressors in carcinogenesis: A review

Analysis of scientific literature data has revealed several ways in which retrotransposons, when activated, are involved in carcinogenesis. First, retroelements can encode oncogenic proteins. For example, the Np9 protein is translated from HERV-K endogenous retrovirus transcript. Second, retroelements are used as alternative promoters of protooncogenes. Accordingly, their activation contributes to the enhanced expression of oncogenes (e.g. CSF1R, IRF5, MET, RAB3IP, CHRM3). Third, retroelements are located in the introns of some genes, and upon their activation, they form chimeric transcripts, such as LTR2-FABP7, LTR-ALK, LTR-ERBB4, LINE1-MET, which have pronounced oncogenic activity. Fourth, retroelements are transposed into tumor suppressor genes (e.g. APC, NF1, MSH2, PTEN, RB1, TSC2, STK11, VHL) and inactivate them, which is associated with the presence of hot spots of insertional mutagenesis in them. As a result, the growth of tumors and the survival of their cells are stimulated. It is important to note that protein products of tumor suppressor genes, such as TP53, RB1, VHL, BRCA1, ATM, are characterized by the ability to inhibit the activity of retroelements. Accordingly, when even one oncosuppressive gene is inactivated, a kind of "vicious circle" can be triggered when the control of expression of retroelements is weakened. The latter, in turn, inactivate other tumor suppressors containing hot spots of insertional mutagenesis. This stimulates new pathways of carcinogenesis and the production of oncogenes associated with transposons. Thus, it is possible to explain in a new way the mechanisms of tumor formation in hereditary tumor syndromes. This is due to the fact that the weakening of the function of an oncosuppressor in a germinal heterozygous mutation may be sufficient to trigger a vicious circle involving retroelements, oncogenes and other oncosuppressors. Similar mechanisms are likely for sporadic malignant tumors. However, the initiating event in them can be the direct activation of transposons under the influence of stressors, chemical and physical carcinogens. In addition to the events described, activation of retroelements causes genomic instability, which contributes to complex genomic rearrangements often observed in malignant tumors. MicroRNAs and long noncoding RNAs, the sources of which are retroelements, also play an important role in the evolution of tumors. Their study is promising for the development of targeted therapy for neoplasms.

3O Identification of an endogenous retroviral signature to predict PD-1 blockade response in advanced clear cell renal cell carcinoma: Integrated analysis of three clinical trials

Endogenous retrovirus (ERV) elements represent genomic footprints of ancestral retroviral infections within the human genome. Previous studies have demonstrated that ERV transcription has been associated with increased immunogenicity in cancers. This study focused on the identification of an ERV signature for predict PD-1 blockade response in advanced clear cell renal cell carcinoma (ccRCC).

Abstract 1960: Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors

Abstract Cytoplasmic nucleic acid sensors for double-stranded (ds) RNA (RIG-I/MDA5) and DNA (cGAS-STING) are pattern recognition receptors (PRRs) key to intracellular anti-viral responses. Recent research has highlighted roles for PRR agonists, including oncolytic virotherapy agents, in anti-tumor immunotherapy. Reovirus type 3 Dearing (Rt3D) is an oncolytic dsRNA virus with limited single-agent activity in clinical studies, but potential for use in combination regimens. We sought synergistic drug-virotherapy combinations using an unbiased screening approach that highlighted the CDK4/6 inhibitor, palbociclib, as a leading hit. We found that, when combined with Rt3D, palbociclib augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling. Combined Rt3D-palbociclib treatment potently increased interferon signaling and endogenous retroviral transcripts. Knockdown (siRNA) studies indicated key UPR proteins and the RNA sensor, RIG-I, were essential to the phenotype observed. Mechanistically independent experiments, using canonical RIG-I agonists and the ER stress inducer (thapsigargin), confirmed cross-talk between RNA sensing and ER stress pathways that augment cancer cell death and interferon production. Combined Rt3D-palbociclib increased innate immune activation and effector function. Our findings demonstrate that UPR signaling and innate immune RNA sensor crosstalk can be exploited to enhance anti-cancer efficacy with pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, as well as broadening the therapeutic remit of CDK4/6 inhibitors to include their role as ER stress sensitizers. Citation Format: Victoria Roulstone, Joan Kyula, Richard Elliot, Christopher J. Lord, Nik Matthews, Vicki Jennings, Harriet Whittock, David Mansfield, Jyoti Choudhary, James Wright, Lu Yu, Alan Melcher, Richard Vile, Matt Coffey, Martin McLaughlin, Kevin Harrington. Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1960.

Preferential In Vitro and In Vivo Induction of Endogenous Retrovirus Transcription from a Monoallelic Chromosome 7q in Decitabine (DAC) Treated AML Blasts

Background: Hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/7q-, often embedded in a complex, monosomal karyotype (Lübbert et al., Haematologica 2012). Integrative methylome/transcriptome studies have provided evidence for aberrant hypermethylation/silencing on monoallelic gene loci, including tumor suppressor genes (TSGs, Kotini et al., Nat. Biotechnol. 2015). Recently, an alternative mechanism of HMA action was described (Roulois et al., Chiappinelli et. al, Cell 2015): induction of silenced endogenous retroviral (ERV) dsRNA transcription, resulting in activation of an interferon-mediated, antitumor immune response (“viral mimicry”). To unravel in vivo HMA activity in different (cyto)genetic backgrounds, we hypothesized that in AML haploinsufficient for 7q, transcriptional repression of monoallelic genes in this region may be preferentially reversed by HMA treatment.Materials and Methods: For identification of genes induced by HMAs, two AML cell lines were selected, ELF-153 (ELF) and UCSD-AML1 (AML1): copy number variations were analyzed by Genome-Wide Human SNP Array 6.0 (Affymetrix), and revealed no monosomies but loss of 7p; in AML1 loss of one entire chromosome 7 was the only numerical aberration. Cells were treated with DAC at equitoxic and -effective concentrations for 4 days, harvested on day 5. Total RNA was depleted of ribosomal RNA (Ribo-Zero rRNA Removal Kit, Illumina) and cDNA libraries (strand-specific, 50 bp) were sequenced with ≥33 million paired-end reads, read quality checked with FastQC (≥95% >Q30). Alignment to the reference genome hg19 was performed with TopHat2, read-counting with HTseq, differential expression testing with DESeq2 (protein-coding transcripts only). Selected target genes were validated by RT-qPCR. Peripheral blood mononuclear cells (PBMC) were collected from 9 AML pts (5 pts with 7q-, 4 cytogenetically normal [CN] pts) treated with DAC (i.v. 20 mg/m2 for 5 days) within the DECIDER trial (NCT00867672). Leukemic blasts before treatment and at day 8 were isolated using automatic magnetic sorting of cells labeled with anti-human CD34 and CD117 MACS microbeads.Results: DAC treatment of AML1 and ELF cells resulted in massive transcriptome changes in both cell lines, with 1,704 genes induced in AML1 (total of 3,583) and 1,206 in ELF (total of 1,867; adjusted FDR <0.01). Comparing DAC-induced expression changes only of genes on 7q, we identified 43 genes significantly differentially expressed only in AML1 cells (up: 15), 24 only in ELF (up: 21), and 19 in both (up: 12). The 15 genes on 7q that were selectively upregulated in AML1 included 3 with TSG features: zyxin (ZYX; up 6.0-fold), homeodomain interacting protein kinase 2 (HIPK2; up 3.8-fold), and high mobility group box transcription factor 1 (HBP1, up 3.1-fold). However, the most heavily upregulated transcripts in AML1 were the endogenous retrovirus 3 group member 1 (ERV3-1 ; up 86.4-fold in AML1 vs 14.3-fold in ELF) and the neighboring zinc finger 117 (ZNF117; up 119.4-fold in AML1vs. 6.5-fold in ELF), both transcribed from the ERV3-1 promoter. ERV3-1 induction was accompanied by induction of the interferon type I response genes retinoic acid inducible gene I (RIG-I) and interferon regulatory factor 7 (IRF7) in both cell lines. Therefore, we next interrogated induction of these genes by DAC in vivo, utilizing serially sorted, matched primary AML blasts (purified before and 3 days after DAC administration). Stronger induction of ERV3-1 and RIG-I mRNA was seen in the 7q- pts compared to the CN pts: By qRT-PCR, median ERV3-1 and RIG-I expression was induced 1.5- and 1.4-fold, respectively, in the pts with 7q- (range: 1.1-2.1 and 1.0-3.2), 1.1- and 0.7-fold, respectively, in the CN pts (range: 0.8-1.7 and 0.3-0.9).Conclusions: We successfully developed an unbiased RNA-seq approach of AML cell lines either mono- or bi-allelic for 7q, demonstrating that DAC treatment preferential upregulates several monoallelic TSGs, and massively activates the ERV3-1 gene (implicated in “viral mimicry”). Induction of ERV3-1 and the dsRNA sensor RIG-I was validated in vivo, and preferentially seen in 7q- AML purified primary blasts. Thus, under clinically established treatment conditions, both the ERV and an interferon response gene can be activated by DAC, supporting a combination with immunotherapy. DisclosuresLübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

E3 ubiquitin ligase HECTD2 mediates melanoma progression and immune evasion

The ubiquitin-proteasome system maintains protein homoeostasis, underpins the cell cycle, and is dysregulated in cancer. However, the role of individual E3 ubiquitin ligases, which mediate the final step in ubiquitin-mediated proteolysis, remains incompletely understood. Identified through screening for cancer-specific endogenous retroviral transcripts, we show that the little-studied E3 ubiquitin ligase HECTD2 exerts dominant control of tumour progression in melanoma. HECTD2 cell autonomously drives the proliferation of human and murine melanoma cells by accelerating the cell cycle. HECTD2 additionally regulates cancer cell production of immune mediators, initiating multiple immune suppressive pathways, which include the cyclooxygenase 2 (COX2) pathway. Accordingly, higher HECTD2 expression is associated with weaker anti-tumour immunity and unfavourable outcome of PD-1 blockade in human melanoma and counteracts immunity against a model tumour antigen in murine melanoma. This central, multifaceted role of HECTD2 in cancer cell-autonomous proliferation and in immune evasion may provide a single target for a multipronged therapy of melanoma.

Functions and Regulation of Endogenous Retrovirus Elements during Zygotic Genome Activation: Implications for Improving Somatic Cell Nuclear Transfer Efficiency.

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.

Nanopore direct RNA sequencing detects DUX4-activated repeats and isoforms in human muscle cells.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor, which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent long terminal repeat (LTR)-retrotransposons and pericentromeric satellite repeats (Human Satellite II). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs, producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes and discovered 61 differentially expressed repeat loci, which are near DUX4–ChIP peaks. We also identified 247 gene–ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu elements are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short-read complementary DNA sequencing.