Epigenome-wide association study of prostate cancer in African American men identified differentially methylated genes.

INTRODUCTION: Men with African ancestry have the highest incidence and mortality rates of prostate cancer (PCa) worldwide. METHODS: This study aimed to identify differentially methylated genes between tumor vs. adjacent normal and aggressive vs. indolent PCa in 121 African American patients. Epigenome-wide DNA methylation patterns in tumor DNA were assessed using the human Illumina Methylation EPIC V1 array. RESULTS: Around 5,139 differentially methylated CpG-sites (q < 0.01, lΔßl > 0.2) were identified when comparing normal vs. tumor, with an overall trend of hypermethylation in prostate tumors.  Multiple representative differentially methylated regions (DMRs), including immune-related genes, such as CD40, Galectin3, OX40L, and STING, were detected in prostate tumors when compared to adjacent normal tissues. Based on an epigenetic clock model, we observed that tumors' total number of stem cell divisions and the stem cell division rate were significantly higher than adjacent normal tissues. Regarding PCa aggressiveness, 2,061 differentially methylated CpG-sites (q < 0.05, lΔßl > .05) were identified when the grade group (GG)1 was compared with GG4/5. Among these 2,061 CpG sites, 155 probes were consistently significant in more than one comparison. Among these genes, several immune system genes, such as COL18A1, S100A2, ITGA4, HLA-C, and ADCYAP1, have previously been linked to tumor progression in PCa. CONCLUSION: Several differentially methylated genes involved in immune-oncologic pathways associated with disease risk or aggressiveness were identified. In addition, 261 African American-specific differentially methylated genes related to the risk of PCa were identified. These results can shedlight on potential mechanisms contributing to PCa disparities in the African American Population.

Epigenetic state determines the in vivo efficacy of STING agonist therapy.

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.

Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma.

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.

STING Signaling in Melanoma Cells Shapes Antigenicity and Can Promote Antitumor T-cell Activity.

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.

Induction of Tertiary Lymphoid Structures With Antitumor Function by a Lymph Node-Derived Stromal Cell Line.

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.

Tumor-Associated Tertiary Lymphoid Structures: Gene-Expression Profiling and Their Bioengineering.

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 various human solid tumors. We are focused on understanding the influence of TLSs on the tumor microenvironment and leveraging this understanding to both manipulate the antitumor 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.

A Cell ELISA for the quantification of MUC1 mucin (CD227) expressed by cancer cells of epithelial and neuroectodermal origin.

Quantitative analysis of MUC1, a cell membrane associated mucin, expressed by intact cells of epithelial origin previously has been limited to flow cytometry, which requires using large quantities of cells and antibodies. Here, for the first time, we report the development of a novel Cellular-based Enzyme Linked Immunosorbent Assay (Cell ELISA) to quantify the expression of MUC1 by cell lines of epithelial and neuroectodermal origin using an antibody recognizing a specific tandem repeat found in the extracellular domain of MUC1. In contrast to flow cytometry, this method requires a much lower number of cells. We report here the results obtained from two variants of this Cell ELISA in live and fixed cells. We found that the Cell ELISA in live cells was not sensitive enough to detect a difference in MUC1 levels between the normal cells and tumor cells. However, we found that Cell ELISA in fixed cells followed by whole cell staining was a dependable method of MUC1 level detection in the normal and tumor cells showing significantly higher levels of MUC1 receptor in the tumor cells when compared to the normal controls. Therefore, we conclude that the Cell ELISA in fixed cells is an efficient method for quantifying the expression of MUC1 by epithelial and neuroectodermal cancer cell lines.