The production of cytokines in response to DNA damage events may be an important host defense response to help prevent the escape of pre-cancerous cells. The innate immune pathways involved in these events are known to be regulated by cellular molecules such as STING (stimulator of interferon genes), which controls type I interferon and pro-inflammatory cytokine production in response to the presence of microbial DNA or cytosolic DNA that has escaped from the nucleus. STING signaling has been shown to be defective in a variety of cancers, such as colon cancer and melanoma, actions which may enable damaged cells to escape the immunosurveillance system. Here, we report through examination of databases that STING signaling may be commonly suppressed in a greater variety of tumors due to loss-of-function mutation or epigenetic silencing of the STING/cGAS promoter regions. In comparison, RNA activated innate immune pathways controlled by RIG-I/MDA5 were significantly less affected. Examination of reported missense STING variants confirmed that many exhibited a loss of function phenotype and could not activate cytokine production following exposure to cytosolic DNA or DNA-damage events. Our data implies that the STING signaling pathway may be recurrently suppressed by a number of mechanisms in a considerable variety of malignant disease and be a requirement for cellular transformation.

Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell–based immunotherapies. Evidence suggests that activation of stimulator of interferon genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell–based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of cGAS and STING genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell–based immunotherapies.

Background Epigenome-wide association studies are emerging in the field of cancer epidemiology with the rapid development of large-scale methylation array platforms. Until recently, these methods were only valid for DNA from fresh frozen (FF) tissues. Novel techniques for repairing DNA from formalin-fixed paraffin-embedded (FFPE) have emerged; however, a direct comparison of FFPE DNA repair methods prior to analysis on genome-wide methylation array to matched FF tissues has not been conducted. Methods We conducted a systematic performance comparison of two DNA repair methods (REPLI-g Ligase vs. Infinium HD Restore Kit) on FFPE-DNA compared to matched FF tissues on the Infinium 450K array. A threshold of discordant methylation between FF-FFPE pairs was set at Δβ>0.3. The correlations of β-values from FF-FFPE pairs were compared across methods and experimental conditions. Results The Illumina Restore kit outperformed the REPLI-g ligation method with respect to reproducibility of replicates(R2>0.970), highly correlated β-values between FF-FFPE(R2>0.888), and fewest discordant loci between FF-FFPE(≤0.61%). The performance of the Restore kit was validated in an independent set of 121 FFPE tissues. Conclusions The Restore kit outperformed RELPI-g ligation in restoring FFPE-derived DNA prior to analysis on the Infinium 450K methylation array. Our findings provide critical guidance that may significantly enhance the breadth of diseases that can be studied by methylomic profiling. Impact Epigenomic studies using FFPE tissues should now be considered among cancers that have not been fully characterized from an epigenomic standpoint. These findings promote novel epigenome-wide studies focused on cancer etiology, identification of novel biomarkers, and developing targeted therapies.

and HUYA Biosciences. A provisional patent application titled "Methylation modulates the tumor immune synapse" (application serial no. 62/889,981) has been filed.

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