scholarly journals Epigenetic reprogramming of tumor cell–intrinsic STING function sculpts antigenicity and T cell recognition of melanoma

2021 ◽  
Vol 118 (15) ◽  
pp. e2013598118
Author(s):  
Rana Falahat ◽  
Anders Berglund ◽  
Ryan M. Putney ◽  
Patricio Perez-Villarroel ◽  
Shota Aoyama ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cell ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Immune Escape ◽  
Type I ◽  
Cell Recognition ◽  
Melanoma Tumor ◽  
T Cell Recognition ◽  
Melanoma Cell Lines

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.

scholarly journals 760 In vivo demethylation-mediated reversal of tumor cell-intrinsic cGAS silencing improves the efficacy of STING agonist therapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A795-A795
Author(s):  
Rana Falahat ◽  
Patricio Perez-Villarroel ◽  
Anders Berglund ◽  
Shari Pilon-Thomas ◽  
Glen Barber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Fold Increase ◽  
Cell Recognition ◽  
Agonist Therapy ◽  
T Cell Recognition ◽  
Tumor Bearing ◽  
Melanoma Cell Lines

BackgroundWhile STING-activating agents have shown limited efficacy in early phase clinical trials, multiple lines of evidence suggest the importance of the so far unappreciated tumor cell-intrinsic STING function in antitumor immune responses. Accordingly, we have shown that although there is a widespread impairment of STING signaling among human melanomas, its restoration through epigenetic reprogramming can augment antigenicity and T cell recognition of melanoma cells.1 2 In this study, we determined if rescue of tumor cell-intrinsic STING signaling using a DNA methyltransferase inhibitor can improve the therapeutic efficacy of a STING agonist in mouse models of melanoma.MethodsWe subjected three distinct murine melanoma cell lines (B16-F10, B16-ISG and Yumm1.7) to treatment with 5-aza-2'-deoxycytidine (5AZADC) and evaluated their activation of STING following stimulation with the STING agonist ADU-S100. Using a B16-F10 subcutaneous model, we assessed the effect of 5AZADC treatment on the efficacy of intratumorally administered ADU-S100 in STINGgt/gt mice. Additionally, we performed mechanistic studies using T-cell depletion experiments as well as phenotypic and gene expression profiling.ResultsWe observed reconstitution of cGAS in all three 5AZADC-pretreated cell lines as well as up to a 46-fold increase in induction of IFN-beta (p < 0.001) and a 4.5-fold increase in MHC class I surface expression (p < 0.01) compared to untreated controls following stimulation with ADU-S100. In B16-F10 tumor-bearing mice, while treatment with a combination of 5AZADC plus ADU-S100 resulted in a marked increase in Ifnb1 transcripts within tumors (p < 0.001), it significantly delayed tumor growth compared to treatment with ADU-S100 alone (p = 0.0244 on day 22). Antibody-mediated depletion studies in mice receiving the combination therapy further indicated that this antitumor activity depends on the generation of functional tumor antigen-specific CD8+ T cells (p = 0.0111 on day 22); however, tumor growth remained unaltered by the depletion of CD4+ T cells.ConclusionsWe show that reversal of methylation silencing of cGAS 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 is capable of inducing 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, depending on tumor cell-intrinsic STING signaling status, its pharmacologic restoration should be considered for improving therapeutic efficacy of STING agonists in future clinical studies.AcknowledgementsFunding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mulé JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunol Res 2019;7(11):1837–48.Falahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell–intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS 2021;118(15).

Effector T Cell Analysis of Melanoma Tumor-infiltrating Lymphocyte Cultures Using HLA-ABC Semimatched Melanoma Cell Lines

2008 ◽  
Vol 31 (7) ◽  
pp. 633-643 ◽  
Author(s):  
Björn Carlsson ◽  
Arian Sadeghi ◽  
Mats Bengtsson ◽  
Gunnar Wagenius ◽  
Thomas H. Tötterman
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Cell Analysis ◽  
Melanoma Tumor ◽  
Effector T Cell ◽  
Tumor Infiltrating Lymphocyte ◽  
Lymphocyte Cultures ◽  
Melanoma Cell Lines

scholarly journals Synergy, Additivity, and Antagonism between Cisplatin and Selected Coumarins in Human Melanoma Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 537
Author(s):  
Paula Wróblewska-Łuczka ◽  
Aneta Grabarska ◽  
Magdalena Florek-Łuszczki ◽  
Zbigniew Plewa ◽  
Jarogniew J. Łuszczki
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Type I ◽  
Antiproliferative Effects ◽  
Isobolographic Analysis ◽  
Natural Substances ◽  
Additive Interactions ◽  
Melanoma Cell Lines

(1) Cisplatin (CDDP) is used in melanoma chemotherapy, but it has many side effects. Hence, the search for natural substances that can reduce the dose of CDDP, and CDDP-related toxicity, is highly desired. Coumarins have many biological properties, including anticancer and antiproliferative effects. (2) An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on two human melanoma cell lines (FM55P and FM55M2) examined the antitumor properties of CDDP and five naturally occurring coumarins (osthole, xanthotoxin, xanthotoxol, isopimpinellin, and imperatorin). The antiproliferative effects produced by combinations of CDDP with the coumarins were assessed using type I isobolographic analysis. (3) The most potent anticancer properties of coumarins were presented by osthole and xanthotoxol. These compounds were characterized by the lowest median inhibitory concentration (IC50) values relative to the FM55P and FM55M2 melanoma cells. Isobolographic analysis showed that for both melanoma cell lines, the combination of CDDP and osthole exerted synergistic and additive interactions, while the combination of CDDP and xanthotoxol exerted additive interactions. Combinations of CDDP with xanthotoxin, isopimpinellin, and imperatorin showed antagonistic and additive interactions in two melanoma cell lines. (4) The combination of CDDP and osthole was characterized by the most desirable synergistic interaction. Isobolographic analysis allows the selection of potential candidates for cancer drugs among natural substances.

282 EFFECT OF THE HBV PRES2 MUTANT ON T CELL RECOGNITION OF HEPATOCYTE TUMOR CELL LINES

2011 ◽  
Vol 54 ◽  
pp. S115
Author(s):  
A. Gehring ◽  
J. Jo ◽  
Z.Z. Ho ◽  
S. Konduru ◽  
A. Bertoletti
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Cell Recognition ◽  
Tumor Cell Lines ◽  
T Cell Recognition

scholarly journals Conformation-dependent recognition of a protein by T cells requires presentation without processing

1989 ◽  
Vol 259 (3) ◽  
pp. 731-735 ◽  
Author(s):  
M Z Atassi ◽  
G S Bixler ◽  
T Yokoi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Protein Antigen ◽  
Mouse Strains ◽  
Immune Recognition ◽  
Cell Recognition ◽  
T Cell Recognition ◽  
T Cell Lines

Presentation of a protein antigen to T cells is believed to follow its intracellular breakdown by the antigen-presenting cell, with the fragments constituting the trigger of immune recognition. It should then be expected that T-cell recognition of protein antigens in vitro will be independent of protein conformation. Three T-cell lines were made by passage in vitro with native lysozyme of T cells from two mouse strains (B10.BR and DBA/1) that had been primed with the same protein. These cell lines responded well to native lysozyme and very poorly to unfolded (S-sulphopropyl) lysozyme. The response of the T-cell lines to the antigen was major histocompatibility complex (MHC)-restricted. A line from B10.BR was selected for further studies. This line responded to the three surface-simulation synthetic sites of lysozyme (representing the discontinuous antigenic, i.e. antibody binding, sites) and analogues that were extended to a uniform size by a nonsense sequence. T-cell clones prepared from this line were specific to native lysozyme and did not respond to the unfolded derivative. Furthermore, several of these clones showed specificity to a given surface-simulation synthetic site. The exquisite dependency of the recognition by the clones on the conformation of the protein antigen and their ability to recognize the surface-simulation synthetic sites indicate that the native (unprocessed) protein was the trigger of MHC-restricted T-cell recognition.

DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines

Tumor Biology ◽  
2016 ◽  
Vol 37 (8) ◽  
pp. 11209-11218 ◽  
Author(s):  
Ya-Ping Chen ◽  
Xiao-Yang Hou ◽  
Chun-Sheng Yang ◽  
Xiao-Xiao Jiang ◽  
Ming Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Malignant Melanoma ◽  
Histone Acetylation ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Malignant Melanoma Cell ◽  
Melanoma Cell Lines

scholarly journals NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

2009 ◽  
Vol 119 (5) ◽  
pp. 1251-1263 ◽  
Author(s):  
Tadepally Lakshmikanth ◽  
Shannon Burke ◽  
Talib Hassan Ali ◽  
Silvia Kimpfler ◽  
Francesco Ursini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Nk Cell ◽  
Cell Recognition ◽  
Mouse Melanoma ◽  
Mouse Melanoma Cell ◽  
Human And Mouse ◽  
Melanoma Cell Lines

scholarly journals EBV Latent Membrane Proteins (LMPs) 1 and 2 as Immunotherapeutic Targets: LMP-Specific CD4+Cytotoxic T Cell Recognition of EBV-Transformed B Cell Lines

2008 ◽  
Vol 180 (3) ◽  
pp. 1643-1654 ◽  
Author(s):  
Tracey A. Haigh ◽  
Xiaorong Lin ◽  
Hui Jia ◽  
Edwin P. Hui ◽  
Anthony T. C. Chan ◽  
...  
Keyword(s):  
T Cell ◽  
Membrane Proteins ◽  
B Cell ◽  
Cell Lines ◽  
Cytotoxic T Cell ◽  
Cell Recognition ◽  
T Cell Recognition

scholarly journals 598 Reversal of epigenetic silencing of cGAS and STING in melanoma enhances the activity of tumor infiltrating lymphocytes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A633-A633
Author(s):  
Rana Falahat ◽  
James Mulé ◽  
Anders Berglund ◽  
Patricio Perez- Villarroel ◽  
Ryan Putney ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Promoter Hypermethylation ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Infiltrating Lymphocytes ◽  
Melanoma Cell Lines

BackgroundIt is becoming more evident that STING activity in tumor cells can have a functional role in mediating antitumor immune responses. We have recently shown that activation of STING signaling in human melanoma cell lines enhances their antigenicity and susceptibility to lysis by human melanoma tumor infiltrating lymphocytes (TIL) through the augmentation of MHC class I molecules.1 However, the frequent impairment of this pathway through loss of cGAS and/or STING expression in melanoma cell lines limits their antigen presentation and subsequently their sensitivity to cytotoxic T cell mediated killing. In this study, we asked if this suppression is, in part, epigenetically regulated and if it is indeed a driver of melanoma resistance to T cell-based immunotherapies.MethodsTo determine the role of DNA methylation in melanoma STING and cGAS silencing, we performed genome-wide DNA methylation profiling across a panel of 16 human melanoma cell lines. We subjected melanoma cell lines that indicated STING and/or cGAS promoter hypermethylation to treatment with 5-aza-2’-deoxycytidine (5AZADC) and evaluated their protein expression by immunoblot. We next assessed phosphorylation of IRF3 and induction of IFN-β and CXCL10 in 5AZADC-treated melanoma cells following their stimulation with dsDNA or 2’3’-cGAMP. We also co-cultured 5AZADC-pretreated melanoma cell lines with their HLA-matched human melanoma TIL in the presence or absence of dsDNA or 2’3’-cGAMP and assessed TIL production of IFN-γ.ResultsUsing whole genome methylation profiling, we identified a distinct correlation between promoter hypermethylation and loss of STING and cGAS expression in human melanoma cell lines. Reconstitution of STING and cGAS expression through DNA demethylation reinstated functional STING signaling in at least half of the examined cell lines as indicated by STING-dependent phosphorylation of IRF3, induction of CXCL10 (~300 pg/ml, P < 0.0001) and IFN-β (~900 pg/ml, P < 0.0001) and upregulation of MHC class I. We also observed up to a 8-fold increase in TIL production of IFN-γ in co-culture studies using 5AZADC-pretreated melanoma cells compared to untreated controls in the presence of dsDNA or 2’3’-cGAMP (~2,000 pg/ml, P < 0.001).ConclusionsWe provide evidence that methylation silencing of cGAS and STING is not only a notable mechanism of STING signaling dysfunction in melanoma, but also plays a role in tumor antigen presentation and recognition by TIL. Collectively, these observations argue that targeting epigenetic loss of STING signaling in melanomas should be considered as a strategy to improve the efficacy of clinical interventions using T cell-based immunotherapies.AcknowledgementsFunding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferenceFalahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mulé JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunology Research 2019; 7(11):1837–48.

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