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. Significance Although cancer immunotherapies have shown potential in inducing durable tumor regression in patients with metastatic melanoma, their efficacy remains highly variable and patient specific. Here, we present one strategy to overcome resistance to T cell–based immunotherapies by pharmacologically modulating the STING pathway, a relatively common suppressed DNA-sensing pathway in human melanomas. We show that rescue of this pathway in human melanoma cells using a clinically available DNA methylation inhibitor can augment their antigenicity and recognition by tumor-infiltrating lymphocytes. These findings have important clinical implications for the development of optimal therapeutic protocols that consider the status of tumor-intrinsic STING activity and provide a strategy to design clinical interventions using T cell–based immunotherapies with appropriate patient selection in melanoma.

STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells are known to be involved in this process, insight into the participation of tumor cell–intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell–intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.

While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8⁺ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.

Tertiary lymphoid structures (TLSs) associate with better prognosis in certain cancer types, but their underlying formation and immunological benefit remain to be determined. We established a mouse model of TLSs to study their contribution to antitumor immunity. Because the stroma in lymph nodes (sLN) participates in architectural support, lymphogenesis, and lymphocyte recruitment, we hypothesized that TLSs can be created by sLN. We selected a sLN line with fibroblast morphology that expressed sLN surface markers and lymphoid chemokines. The subcutaneous injection of the sLN line successfully induced TLSs that attracted infiltration of host immune cell subsets. Injection of MC38 tumor lysate-pulsed dendritic cells activated TLS-residing lymphocytes to demonstrate specific cytotoxicity. The presence of TLSs suppressed MC38 tumor growth in vivo by improving antitumor activity of tumor-infiltrating lymphocytes with downregulated immune checkpoint proteins (PD-1 and Tim-3). Future engineering of sLN lines may allow for further enhancements of TLS functions and immune cell compositions.

Tertiary lymphoid structures (TLSs) have been identified in the parenchyma and/or in the peripheral margins of human solid tumors. Uncovering the functional nature of these structures is the subject of much intensive investigation. Studies have shown a direct correlation of the presence of human tumor-localized TLS and better patient outcome (e.g., increase in overall survival) in certain solid tumor histologies, but not all. We had identified a tumor-derived immune gene expression signature, encoding 12 distinct chemokines, which could reliably identify the presence of TLSs, of different degrees, in a variety of human solid tumors. We are focused on understanding the influence of TLSs on the tumor microenvironment and leveraging this understanding to both manipulate the anti-tumor immune response and potentially enhance immunotherapy applications. Moreover, as not all human solid tumors show the presence of these lymphoid structures, we are embarking on bioengineering approaches to design and build ‘designer’ TLSs to address, and potentially overcome, an unmet medical need in cancer patients whose tumors lack such lymphoid structures.