Antitumor Immunity In Melanoma Research Articles

Stat3-mediated Atg7 expression enhances anti-tumor immunity in melanoma.

Epigenetic modifications to DNA and chromatin control oncogenic and tumor suppressive mechanisms in melanoma. EZH2, the catalytic component of the Polycomb repressive complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2 Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by EZH2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models we further investigated the role of pathways downstream of EZH2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of an Ezh2 Y641F epigenetic state. We found that expression of Atg7 is largely dependent on Stat3 expression and that deletion of Atg7 slows down melanoma cell growth in vivo , but not in vitro . Atg7 deletion also results in increased CD8+ T cells and reduced myelosuppressive cell infiltration in the tumor microenvironment, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.

Targeting Dectin-1 and or VISTA enhances anti-tumor immunity in melanoma but not colorectal cancer model.

Acquired resistance to immune checkpoint blockers (ICBs) is a major barrier in cancer treatment, emphasizing the need for innovative strategies. Dectin-1 (gene Clec7a) is a C-type lectin receptor best known for its ability to recognize β-glucan-rich structures in fungal cell walls. While Dectin-1 is expressed in myeloid cells and tumor cells, its significance in cancer remains the subject of controversy. Using Celc7a-/- mice and curdlan administration to stimulate Dectin-1 signaling, we explored its impact. VISTA KO mice were employed to assess VISTA's role, and bulk RNAseq analyzed curdlan effects on neutrophils. Our findings reveal myeloid cells as primary Dectin-1 expressing cells in the tumor microenvironment (TME), displaying an activated phenotype. Strong Dectin-1 co-expression/co-localization with VISTA and PD-L1 in TME myeloid cells was observed. While Dectin-1 deletion lacked protective effects, curdlan stimulation significantly curtailed B16-F10 tumor progression. RNAseq and pathway analyses supported curdlan's role in triggering a cascade of events leading to increased production of pro-inflammatory mediators, potentially resulting in the recruitment and activation of immune cells. Moreover, we identified a heterogeneous subset of Dectin-1+ effector T cells in the TME. Similar to mice, human myeloid cells are the prominent cells expressing Dectin-1 in cancer patients. Our study proposes Dectin-1 as a potential adjunctive target with ICBs, orchestrating a comprehensive engagement of innate and adaptive immune responses in melanoma. This innovative approach holds promise for overcoming acquired resistance to ICBs in cancer treatment, offering avenues for further exploration and development.

The role of tissue-resident memory T cells as mediators for response and toxicity in immunotherapy-treated melanoma-two sides of the same coin?

Tissue-resident memory T cells (TRM cells) have become an interesting subject of study for antitumor immunity in melanoma and other solid tumors. In the initial phases of antitumor immunity, they maintain an immune equilibrium and protect against challenges with tumor cells and the formation of primary melanomas. In metastatic settings, they are a prime target cell population for immune checkpoint inhibition (ICI) because they highly express inhibitory checkpoint molecules such as PD-1, CTLA-4, or LAG-3. Once melanoma patients are treated with ICI, TRM cells residing in the tumor are reactivated and expand. Tumor killing is achieved by secreting effector molecules such as IFN-γ. However, off-target effects are also observed. Immune-related adverse events, such as those affecting barrier organs like the skin, can be mediated by ICI-induced TRM cells. Therefore, a detailed understanding of this memory T-cell type is obligatory to better guide and improve immunotherapy regimens.

An iPSC-derived exosome-pulsed dendritic cell vaccine boosts antitumor immunity in melanoma

Induced pluripotent stem cells (iPSCs) express a broad spectrum of tumor-associated antigens and exert prophylactic effects on various tumors. However, some problems remain, such as potential tumorigenicity, challenges in transport to the lymph nodes and spleen, and limited antitumor effects. Thus, designing a safe and effective iPSC-based tumor vaccine is necessary. We prepared iPSC-derived exosomes and incubated them with DCs (dendritic cells) for pulsing to explore their antitumor effects in murine melanoma models. The antitumor immune response induced by the DC vaccine pulsed with iPSC exosomes (DC+ EXO) was assessed invitro and invivo. After DC+ EXO vaccination, extracted spleen Tcells effectively killed a variety of tumor cells (melanoma, lung cancer, breast cancer, and colorectal cancer) invitro. In addition, DC+ EXO vaccination significantly inhibited melanoma growth and lung metastasis in mouse models. Furthermore, DC+ EXO vaccination induced long-term Tcell responses and prevented melanoma rechallenge. Finally, biocompatibility studies showed that the DC vaccine did not significantly alter the viability of normal cells and mouse viscera. Hence, our research may provide a prospective strategy of a safe and effective iPSC-based tumor vaccine for clinical use.

Abstract 4072: Class I HLA-independent lysis of immunotherapy-resistant melanoma by CD8 T cells

Abstract Cancer immunotherapy with immune checkpoint blockade (ICB) has transformed the treatment of melanoma, although intrinsic or acquired resistance develops in nearly half of patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity in melanoma and other cancers, and single-cell RNA-sequencing has identified T cell states associated with improved clinical response to ICB, as well as adoptive T cell therapy (ACT). Despite these advances, strategies to identify and analyze tumor-reactive TILs in ICB-resistant patients remain limited. Here, we demonstrate that TILs from ICB-resistant melanoma patients can recognize and eliminate autologous tumor cells independent of class I HLA-TCR interactions. TILs eliminated matched melanoma cells in a time and dose-dependent fashion associated with secretion of effector cytokines. Strikingly, the deletion of B2M (resulting in loss of class I HLA surface expression) did not alter the activity of these TILs. Immunophenotyping studies confirmed that TILs are largely (>95%) effector memory (Tem) CD8 T cells (CD45RA-CD45RO+CCR7-) and give rise to terminal effector cells after co-culture with matched melanoma cells. Further, the elimination of melanoma cells by TILs required intact JAK1/2 signaling, although interferon-gamma (IFNγ) was neither necessary nor sufficient for tumor cell elimination. Together, these findings demonstrate that expanded TILs from ICB-resistant melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism. Citation Format: Hongyan Xie, Aiping Jiang, Anne Jenney, Yi Sun, Tatyana Sharova, Moshe Sade-Feldman, Or-yam Revach, Angelina Cicerchia, Martin Q. Rasmussen, Nir Hacohen, Robert T. Manguso, Russell W. Jenkins. Class I HLA-independent lysis of immunotherapy-resistant melanoma by CD8 T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4072.

Abstract 1380: Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance

Abstract While immunotherapies have been revolutionary for the treatment of many cancers including melanoma, most patients do not respond, or develop resistance to these therapies. Several epigenetic modulators have been identified as potential targets for augmenting anti-tumor immunity, however the full breadth of their role is not well-understood. To identify modulators of anti-tumor immunity in melanoma, we performed a whole-genome CRISPR screen using the YUMMER-G model and identified members of the HUSH complex and its associated methyltransferase, Setdb1, as drop-out hits. To validate these findings, we used CRISPR-Cas9 to knockout Setdb1 individually and observed that Setdb1-/- tumors are cleared by their host mice in a CD8+ T-cell dependent manner. To determine the source of immunogenicity in Setdb1-/- cells, we performed RNA-sequencing, cytokine profiling, and evaluated antigen presentation by flow cytometry. These assays revealed increased MHC-I levels, elevated secretion of Ifn-, and induction of an interferon-stimulated gene (ISG) expression program, consistent with the observed immunogenicity. We further determined that treating Setdb1-/- cells with IFNAR-blocking antibodies restores MHC-I levels to wild-type levels, suggestive of a tumor-intrinsic interferon loop. We hypothesized that type-I interferon signaling is generated through sensing of expressed endogenous retroviral elements (ERVs), and consistently observe upregulation of ERVs in Setdb1-/- cells. Previous studies have described the potential for ERV-derived antigens to act as substrates for anti-tumor activity by CD8+ T cells. We identified T cells in the tumor microenvironment of Setdb1-/- tumors that bind tetramer carrying the P15E peptide derived from ERV envelope. Together, these findings suggest that tumors lacking expression of Setdb1-/- upregulate ERV transcripts, driving expression of type-I interferons which generate an inflamed tumor microenvironment and potentiate efficient tumor clearance of Setdb1-/- tumors. Overall, this study emphasizes a critical role for regulators of ERV expression and type-I interferon expression, and suggests their potential as therapeutic targets for augmenting anti-cancer immune responses. Citation Format: Meaghan K. McGeary, William Damsky, Drew Daniels, Goran Micevic, Eric Song, Hua Jane Lou, Clotilde Calderwood, Sateja Paradkar, Akiko Iwasaki, David Calderwood, Benjamin Turk, Marcus Bosenberg. Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1380.

Epigenetics Regulates Antitumor Immunity in Melanoma

Melanoma is the most malignant skin cancer, which originates from epidermal melanocytes, with increasing worldwide incidence. The escape of immune surveillance is a hallmark of the tumor, which is manifested by the imbalance between the enhanced immune evasion of tumor cells and the impaired antitumor capacity of infiltrating immune cells. According to this notion, the invigoration of the exhausted immune cells by immune checkpoint blockades has gained encouraging outcomes in eliminating tumor cells and significantly prolonged the survival of patients, particularly in melanoma. Epigenetics is a pivotal non-genomic modulatory paradigm referring to heritable changes in gene expression without altering genome sequence, including DNA methylation, histone modification, non-coding RNAs, and m6A RNA methylation. Accumulating evidence has demonstrated how the dysregulation of epigenetics regulates multiple biological behaviors of tumor cells and contributes to carcinogenesis and tumor progression in melanoma. Nevertheless, the linkage between epigenetics and antitumor immunity, as well as its implication in melanoma immunotherapy, remains elusive. In this review, we first introduce the epidemiology, clinical characteristics, and therapeutic innovations of melanoma. Then, the tumor microenvironment and the functions of different types of infiltrating immune cells are discussed, with an emphasis on their involvement in antitumor immunity in melanoma. Subsequently, we systemically summarize the linkage between epigenetics and antitumor immunity in melanoma, from the perspective of distinct paradigms of epigenetics. Ultimately, the progression of the clinical trials regarding epigenetics-based melanoma immunotherapy is introduced.

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects.

SUMMARYDevelopmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

EZH2 Inhibitor Enhances the STING Agonist‒Induced Antitumor Immunity in Melanoma

STING agonists are a new class of drugs for cancer immunotherapy that activate both innate and adaptive antitumor immunity. Recently, multiple clinical trials of STING agonists have been conducted in hematological malignancies and solid tumors. However, STING is commonly suppressed in melanoma through mechanisms that remain unclear. We found that STING expression was epigenetically suppressed by H3K27me3 in melanoma, and EZH2 inhibitor could induce an H3K27 shift from trimethylation to acetylation, resulting in increased expression of STING. Furthermore, a combination of STING agonist and EZH2 inhibitor upregulated major histocompatibility complex class I expression and chemokine production. Whole-transcriptome analysis showed that IFN-1‒related genes were significantly upregulated in the combination treatment group. In addition, the combination treatment synergistically reduced tumor growth and increased CD8+ T-cell infiltration in a poorly immunogenic melanoma mouse model B16-F10. These results showed, to our knowledge, a previously unreported mechanism underlying the epigenetic regulation of STING expression in melanoma; a combination of STING agonists and EZH2 inhibitors can boost the antitumor immune response and would be apromising treatment option for patients with melanoma who are refractory to current immunotherapies.

Abstract 1893: The dynamics of T cell clonal repertoire under targeted therapy vs. immunotherapy in melanoma

Abstract Introduction. The current standard of care for metastatic melanoma patients utilizes either MAPK pathway inhibitors (MAPKi)-based targeted therapies or immune checkpoint inhibitors (ICI)-based immunotherapies. Almost every melanoma patient treated with MAPKi will relapse in a median of one year, whereas a small subset of ICI-treated melanoma patients experienced prolonged tumor control. Multiple reports have shown that both MAPKi and ICI treatment will induce increased T cell infiltration into the tumor. However, the T-cell receptor (TCR) clonotype dynamics across different therapies (MAPKi vs. ICI) and/or clinical response (responding vs. resistant) is still unknown. This study aims to compare the TCR repertoire dynamics and their associated tumor microenvironment (TME) phenotypes in melanoma pre- vs. post-ICI or MAPKi to see if there are unique patterns associated with better tumor control. Methods. Publicly available bulk RNA-seq data of 345 melanoma samples pre- and post- treatment were downloaded, including two MAPKi (n=116 tumors) and two ICI data sets (n = 229 tumors). All samples were classified into three groups: pre-treatment (PT), on-treatment responding (OT-R), and on-treatment non-responding (OT-NR). T cell clonotype and gene expression were quantified from the RNA-seq data using trust4 and HISAT2/htseq-count, respectively. Although the transcripts of T cells are mixed with tumor cells in the bulk RNA-seq data, the TCR clonotype abundance showed significant correlation with T cell marker expression, offering a measure of robustness in the identification of TCR from bulk tumor derived RNA-seq data. Results. Both MAPKi and ICI therapies significantly increase the TCR count, abundance, diversity, and clonality in OT-R samples compared to PT and OT-NR samples. Intriguingly, we observed significantly higher clonal replacement of PT samples' T cell clonotypes with novel, OT-specific clonotypes under both therapies. Of note, in the tumor subset that responded to the ICI, the increase of these novel TCR were significantly associated with the enrichment of naïve/memory CD4 and CD8 T cell signatures. Conclusions. In melanoma, intratumoral TCR dynamics patterns in response to MAPKi and ICI are mostly similar, except for the upregulation of naïve/memory T cell signatures in the subset of ICI-responder tumors. These insights may eventually lead to better markers for therapy-induced, productive anti-tumor immunity in melanoma. Citation Format: Lizhong Ding, Katie M. Campbell, Egmidio Medina, Kevin Chen, Willy Hugo. The dynamics of T cell clonal repertoire under targeted therapy vs. immunotherapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1893.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2) are essential to maintain tissue homeostasis. In cancer, ILC2 can harbor both pro- and anti-tumorigenic functions but we know very little about their underlying mechanisms, nor whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2 are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) which coordinate the recruitment and activation of eosinophils to enhance anti-tumor responses. Tumor-infiltrating ILC2 expressed programmed cell death protein-1 (PD-1), which limited their intratumoral accumulation, proliferation and anti-tumor effector functions. This inhibition could be overcome in vivo by combining IL-33-driven ILC2 activation with PD-1 blockade to significantly increase anti-tumor responses. Together, our results identified ILC2 as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for anti-tumor immunotherapies.

A20 regulates the therapeutic effect of anti-PD-1 immunotherapy in melanoma

BackgroundThe therapeutic effect of immune checkpoint blockers, especially the neutralizing antibodies of programmed cell death (PD-1) and its ligand programmed death ligand 1 (PD-L1), has been well verified in melanoma....

Abstract IA09: Tumor cell oxidative metabolism as a barrier to antitumor immunity in melanoma

Abstract The treatment of melanoma has been fundamentally changed by immunotherapies like blockade of coinhibitory “checkpoint” molecules like CTLA-4 and PD-1. However, despite these remarkable successes, even within T cell-inflamed, high-mutational-burden tumors, response rates remain low, likely due to additional immune inhibitory mechanisms present in the tumor microenvironment. It is now generally appreciated that the tumor microenvironment is characterized by a distinct metabolic landscape, having low oxygen tension, depleted levels of essential nutrients, and buildup of toxic byproducts. We sought to determine whether this metabolic landscape was variable from patient to patient and how this landscape affected antitumor immunity and thus response to immunotherapy. Using extracellular metabolic flux analysis, we profiled the metabolic derangement of patient-derived cell lines as well as direct ex vivo patient tumor samples, revealing striking heterogeneity not only in the degree of metabolic derangement but also in the type of metabolism a tumor cell was dependent on. Analysis of the tumor infiltrate revealed that a tumor’s preference for oxidative metabolism was associated with immune dysfunction, suggesting that the ability to generate hypoxic regions in vivo is a key aspect of immune resistance. We confirmed this finding in vivo using a novel model of melanoma in which individual metabolic pathways can be selectively targeted in a single-cell derived line: loss of oxidative metabolism in tumor cells sensitizes animals to PD-1 blockade immunotherapy, while loss of glycolytic metabolism has no appreciable effect on antitumor immunity. Analysis of melanoma patient biopsy samples prior to initiation of immunotherapy revealed that increased tumor oxidative metabolism and intratumoral hypoxia was associated with poorer responses to checkpoint blockade. Our data highlight the use of tumor microenvironment hypoxia as a potential predictor of immunotherapy resistance and shed light on the use of modulators of tumor cell oxidation as a means to improve responses to checkpoint blockade. Citation Format: Greg M. Delgoffe. Tumor cell oxidative metabolism as a barrier to antitumor immunity in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA09.

Abstract 5695: Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies

Abstract Growth and remodeling of tumor-associated lymphatics, termed lymphangiogenesis, has been known to increase metastasis rate in cancer patients. However, a recent study from our group has shown that tumor-associated lymphatics can improve anti-tumor immunity in melanoma upon treatment with immunotherapies. The role of lymphangiogenesis in shaping anti-tumor immune responses in breast cancer, however, remains unexplored. Furthermore, recently approved checkpoint inhibitor immunotherapies have had limited success in breast cancer patients. A better understanding of the role of tumor-associated lymphatics in shaping anti-tumor immune responses could lead to more informed decisions when treating breast cancer patients with immunotherapies. In this study, we investigated the role of induced tumor lymphangiogenesis in modulating responsiveness to different immunotherapies in a murine triple-negative mammary carcinoma model. Spontaneously metastatic 4T1 tumor cells were transduced to over-express the pro-lymphangiogenic growth factor VEGF-C (VC) to induce tumor lymphangiogensis in vivo. Flow cytometry and immunohistochemistry analyses confirmed that VEGF-C over-expressing tumors have an increase in tumor-associated lymphatics than control ones. Higher infiltrations of conv CD4 T cells and macrophages were observed in lymphangiogenic 4T1 tumors. Lymphangiogenic tumors treated with aPD-1 and aCTLA-4 checkpoint inhibitors, or an agonist of the Stimulator of Interferon Genes (STING) pathway, showed improved responsiveness to the therapy compared to the control tumors, as indicated by a reduced tumor growth rate. Moreover, positive correlations between VEGF-C expression and CD4 T cell and macrophage gene expressions were observed in primary human triple-negative breast tumor samples from The Cancer Genome Atlas (TCGA). In conclusion, our study shows that increase in tumor-associated lymphatics in triple-negative breast cancer enhances responsiveness to immunotherapies. Citation Format: Peyman Hosseinchi, Aslan Mansurov, Léa Maillat, Lambert Potin, Gavin Fujimori, Jeffrey A. Hubbell, Melody A. Swartz. Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5695.

Expression of cancer cell-associated Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) regulates protective anti-tumor immunity in melanoma

Abstract Activation of moderated endoplasmic reticulum (ER) stress in cancer cells promotes cellular adaptation and survival in response to the adverse conditions of the tumor microenvironment (TME). However, how modulation of ER stress in cancer cells culminates in the development of an immunosuppressive TME remains poorly understood and therapeutically unexploited. Upon disruption of ER homeostasis, mediator proteins IRE1a, ATF6 and PERK induce activation of the unfolded protein response (UPR). Although maladaptive priming of UPR mediators in immune cells deactivates their capacity for anti-tumor immunity, the immunosuppressive potential of UPR mediators in cancer cells, in particular PERK, remains unknown. Thus, in this study, we hypothesized that activation of PERK in tumor cells shapes the formation of the immunosuppressive TME and impairs the development of anti-tumor immunity. Elimination of PERK in cancer cells impaired survival upon chemically-induced ER stress and promoted caspase-independent cell death. Interestingly, in vivo ablation of PERK in melanoma impaired tumor growth, which surprisingly occurred in a T cell-dependent manner. Additionally we observed that ablation of PERK shifted the TME from an immunosuppressive to immunostimulatory environment, enhanced accumulation of effector T cells and directed reprogramming of myeloid derived suppressor cells (MDSCs) into immunostimulatory cells. Additional experiments indicated that ablation of PERK induced expression of mediators associated with immunogenic cell death. Collectively, our findings demonstrate the immunoregulatory role of PERK in tumor cells and highlight the therapeutic potential of targeting UPR mediators and tumor adaptive processes.

LB1122 Myeloid targeting in combination with PD1 inhibition boosts anti-tumor immunity in melanoma

LB1122 Myeloid targeting in combination with PD1 inhibition boosts anti-tumor immunity in melanoma

Abstract 5190: EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation

Abstract Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remain key obstacles. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Additionally, EZH2 has been shown to interact with DNA methyltransferases (DNMTs). Aberrant EZH2 in cancer results in the silencing of hundreds of tumor suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq, microarrays and DNA methylation sequencing, we identified a subset of EZH2 target genes that are silenced by both histone and DNA methylation in melanoma. Interestingly many of these genes play a role in the anti-tumor immune response, including RASSF5 and ITGB2 that facilitate lymphocyte infiltration into tumors and correlate with better survival outcomes in melanoma patients when their expression is high. We show that such genes can be significantly up-regulated by combined treatment with inhibitors of both EZH2 and DNMT in melanoma cells in vitro. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the anti-tumor immune response. The progression of several preclinical, small molecule inhibitors of EZH2 and DNMTs remains promising. Citation Format: Jessamy Tiffen, Dilini Gunatilake, Stuart Gallagher, Peter Hersey. EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5190.

Abstract 497: Characterization of TIGIT expression using MultiOmyxTM hyperplexed immunofluorescence assay in solid tumors

Abstract TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a recently identified immune-receptor that is expressed on T cells, natural killer (NK) cells and NKT cells. TIGIT has emerged as an important coinhibitory receptor. TIGIT expression on CD8+ tumor infiltrating lymphocytes (TILs) has been shown to be upregulated in solid cancers such as melanoma, colon cancer, and NSCLC and have been associated with a dysfunctional phenotype in TILs. TIGIT can also be activated on a subset of Regulatory T cells (Tregs) and may be critical in driving CD8+ T cell dysfunction. A second mechanism in which TIGIT inhibits immunosurveillance is through both competition and direct inhibition of CD226, impairing its ability to activate immunosurveillance. TIGIT has also been reported to inhibit T cell responses indirectly by triggering CD155 expression in dendritic cells (DCs), thereby preventing DC maturation. These findings render the TIGIT pathway as an attractive candidate for cancer immunotherapy In this study, we seek to use MultiOmyx hyperplexed immunofluorescence (IF) assay to exploit this new pathway and characterize the TIGIT expression in a total of 20 melanoma and NSCLC samples. The cancer FFPE slides will be stained with a 13-marker panel including TIGIT, CD226, CD155, CD3, CD4, CD8, FOXP3, CD56, CD45, CD11b, CD11c, PD1, LAG3, TIM3 and PanCK. This panel will enable the detection of TIGIT, CD226 and CD115 expression in the Melanoma and NSCLC samples. The TIGIT expression will be further characterized on different TILs including CD4+ helper T cells, CD8+ cytotoxic T cells, Tregs and NK cells. Using the MultiOmyx proprietary algorithm, we can quantify different subtypes of TIGIT expressing cells and measure the distance of different TIGIT expressing cells to the tumor. Increased expression of TIGIT and PD1 has been demonstrated in NSCLC and melanoma. Moreover, TIGIT+ Tregs have been reported to upregulate TIM-3 expression in mice tumor models. In this study, we will also evaluate the expression of TIGIT in conjunction with other coinhibitory receptors such as PD1, LAG3 and TIM3. The percentage of TILs that co-express TIGIT/PD1, TIGIT/LAG3 and TIGIT/TIM3 will be quantified and analyzed. Leveraging TIGIT in combination with other immune therapy may achieve more robust clinical outcomes. There are currently multiple phase I clinical trials using TIGIT monoclonal antibody (for instance, BMS-986207 by Bristol-Myers Squibb and MTIG7192A by Genetech) in combination with anti PD1/PDL1 antibodies in solid tumors. Our data can help provide more insight into how TIGIT modulate antitumor immunity in melanoma and NSCLC tumors. And the findings in this study can also be used to understand the synergistic effects between TIGIT and other coinhibitory receptors and help identify the additional opportunity for combination immunotherapy using checkpoint inhibitors. Citation Format: Qingyan Au, Arezoo Hanifi, Erinn Parnell, Judy Kuo, Eric Leones, Flora Sahafi, Kathy Pham, RaghavKrishna Padmanabhan, Nicholas Hoe, Josette William. Characterization of TIGIT expression using MultiOmyxTM hyperplexed immunofluorescence assay in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 497.

Multiple antigen-engineered DC vaccines with or without IFN\u03b1 to promote antitumor immunity in melanoma

BackgroundCancer vaccines are designed to promote systemic antitumor immunity and tumor eradication. Cancer vaccination may be more efficacious in combination with additional interventions that may build on or amplify their effects.MethodsBased on our previous clinical and in vitro studies, we designed an antigen-engineered DC vaccine trial to promote a polyclonal CD8+ and CD4+ T cell response against three shared melanoma antigens. The 35 vaccine recipients were then randomized to receive one month of high-dose IFNα or observation.ResultsThe resulting clinical outcomes were 2 partial responses, 8 stable disease and 14 progressive disease among patients with measurable disease using RECIST 1.1, and, of 11 surgically treated patients with no evidence of disease (NED), 4 remain NED at a median follow-up of 3 years. The majority of vaccinated patients showed an increase in vaccine antigen-specific CD8+ and CD4+ T cell responses. The addition of IFNα did not appear to improve immune or clinical responses in this trial. Examination of the DC vaccine profiles showed that IL-12p70 secretion did not correlate with immune or clinical responses. In depth immune biomarker studies support the importance of circulating Treg and MDSC for development of antigen-specific T cell responses, and of circulating CD8+ and CD4+ T cell subsets in clinical responses.ConclusionsDC vaccines are a safe and reliable platform for promoting antitumor immunity. This combination with one month of high dose IFNα did not improve outcomes. Immune biomarker analysis in the blood identified several predictive and prognostic biomarkers for further analysis, including MDSC.Trial registrationNCT01622933.

Abstract 1384: The epigenetic modifier EZH2 represses antitumor immunity in melanoma

Abstract Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remains a key obstacle. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Aberrant EZH2 in cancer results in the silencing of hundreds of tumor-suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq and microarrays to reveal downstream EZH2 target genes, we identified many tumor-suppressor genes including some that play a role in the antitumor immune response. Interestingly, xenografts in immune-deficient NOD SCID mice showed no significant inhibition in tumor growth when treated with an EZH2 inhibitor. However, growth inhibition was achieved when the same experiment was performed in nude mice, which retain some mediators of innate immunity. The effect of EZH2 overexpression in melanoma on immune-mediated rejection was studied in an inducible in vivo model. Subcutaneous B16 tumors expressing model antigen were initially rejected but relapsed in the majority of immunodeficient mice transferred with CD4 T cells. Long-term (120-day) survival was reduced from 40% to 22% if the tumors also overexpressed EZH2. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the antitumor immune response. The progression of several preclinical, small-molecule inhibitors of EZH2 remains promising. Citation Format: Jessamy Tiffen, Elena Shklovskaya, Stuart Gallagher, Dilini Gunatilake, Peter Hersey. The epigenetic modifier EZH2 represses antitumor immunity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1384.