Matthew David

 Introduction: Retroviruses have become endogenized into germline cells throughout mammalian evolution, with amplification resulting in the genomic content of these endogenous retroviruses (ERVs) to ~8% in humans.¹ Epigenetic repression of ERVs and subsequent accumulation of mutations allow host silencing,² but open chromatin enrichment of ERVs in embryonic stem cells and cancer cells suggests roles in regulatory function.³ With the exception of the evolutionarily young HERV-K family, ERVs have shown distinct down-regulation when tested in cancer cells, similar to the hypermethylation-mediated suppression of tumor suppressor genes.^4,5 This suggests a possible mechanism for epigenetic immuno-oncology therapy, and previous attempts to correlate successful epigenetic therapies to non-ERV mechanisms have been unsuccessful.⁵ Methods:⁵ HCT116 colorectal cancer cells were treated with a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-AZA-CdR). Gene expression microarray and consensus clustering were used to classify response to demethylation of interferon-responsive genes, and DNA methylation BeadChips were used to establish correlation between expression and DNA methylation. An ingenuity pathway analysis was performed to identify canonical pathway association, and type-I interferon signals were assessed by qPCR and ELISA. The upstream pathway for cytosolic pattern recognition receptors RIG1 and MDA5 was assessed by knockdown with small hairpin RNAs to detect late onset 5-AZA-CdR responsive genes, and similar knockdown of the downstream signaling protein MAVS was performed. Primary colorectal cancer cells from a surgical resection biobank were treated with 5-AZA-CdR in an in vitro assay for sphere initiation, and MAVS knockdown was performed. Finally, direct agonist stimulation of the RIG1/MDA5/MAVS pathway was assessed by transfection of colorectal cancer cells with dsRNAs. Results:⁵ Colorectal cancer cells showed sustained activity of late-onset interferon responsive genes when treated with 5-AZA-CdR, not correlated with canonical INF-a or INF-b pathways. This was demonstrated to be dependent on the intracellular viral nucleotide receptor pathway RIG1/MDA5/MAVS, a known type-III interferon signal pathway. Treatment with 5-AZA-CdR resulted in a 10-fold decrease in frequency of sphere-initiating cells, and transfection of dsRNA showed bidirectional retroviral transcription to be a specific pathway agonist. Conclusion: These results demonstrate a mechanism by which intracellular immune signaling of cancer cells may be enhanced through endogenous retroviral transcription, utilizing a known epigenetic therapy 5-AZA-CdR. While prior correlations with tumor suppressor and interferon response genes had unreliably predicted DNA demethylation treatment response, this cytosolic viral nucleotide recognition pathway reliably predicts the type III interferon cascade responsible for both prior mechanism proposals, and cancer cell models correspondingly demonstrate reduced proliferative capacity.⁵

1. Gonzalez-Cao, M., Iduma, P., Karachaliou, N., Santarpia, M., Blanco, J., & Rosell, R. (2016). Human endogenous retroviruses and cancer. Cancer biology & medicine, 13(4), 483–488. doi:10.20892/j.issn.2095-3941.2016.0080
2. Ohtani, H., Liu, M., Zhou, W., Liang, G., & Jones, P. A. (2018). Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses. Genome research, 28(8), 1147–1157. doi:10.1101/gr.234229.118
3. Jacques PÉ, Jeyakani J, Bourque G (2013) The Majority of Primate-Specific Regulatory Sequences Are Derived from Transposable Elements. PLOS Genetics 9(5): e1003504. https://doi.org/10.1371/journal.pgen.1003504
4. Tongyoo, P., Avihingsanon, Y., Prom-On, S., Mutirangura, A., Mhuantong, W., & Hirankarn, N. (2017). EnHERV: Enrichment analysis of specific human endogenous retrovirus patterns and their neighboring genes. PloS one, 12(5), e0177119. doi:10.1371/journal.pone.0177119
5. David Roulois, Helen Loo Yau, Rajat Singhania, Yadong Wang, Arnavaz Danesh, Shu Yi Shen, Han Han, Gangning Liang,Peter A. Jones, Trevor J. Pugh, Catherine O’Brien, Daniel D. De Carvalho. DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts. Cell. Volume 162, Issue 5, 27 August 2015, Pages 961-973. https://doi.org/10.1016/j.cell.2015.07.056