Expression of HDAC11 may be involved in myeloid cell biology.
Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and ...malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL‐10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage‐restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.
The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase ...inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure–activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.
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.
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.
APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone ...deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation--but no changes in STAT3 acetylation--as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.
BackgroundDespite its common epigenetic suppression in multiple cancers, STING signaling has emerged as a major pathway for augmenting tumor cell antigenicity and initiation of T cell responses.1 2 ...Another aspect of intact activation of STING signaling in tumor cells is downstream induction of T cell-homing chemokines including CXCL10 and CCL5. These chemokines are also among our earlier reported 12-chemokine (12-CK) gene expression signature (GES) predicting the presence of tumor-localized tertiary lymphoid structures (TLSs), which are increasingly shown to correlate with improved survival in certain solid tumor types.3 4 Based on these findings, we hypothesized that epigenetic silencing of STING signaling genes through promoter hypermethylation would be inversely associated with the presence of TLSs.MethodsWe assessed the correlation between the expression of STING signaling genes and the chemokines present in the 12-CK GES across melanomas and urothelial bladder carcinomas using cBioPortal datasets. To extend these studies beyond these tumor types, we performed correlative and survival analyses using the TCGA PanCancer Atlas. Additionally, we determined the correlation between the promoter methylation levels of STING signaling genes and the 12-CK GES score. We also evaluated STING expression in TLS+ and TLS- melanoma samples in situ by immunohistochemistry (IHC).ResultsWe identified a distinct correlation between STING-expressing tumors and each of the twelve chemokines among melanoma and urothelial bladder carcinoma samples. In particular, STING expression was positively correlated with secondary lymphoid organ-associated chemokines, CCL19 (p=0.0077), CCL21 (p=0.0046), and CXCL13 (p=0.0034) in urothelial bladder carcinomas. The presence of TLSs in STING-expressing melanomas was further confirmed by IHC. Using TCGA PanCancer datasets, we observed a strong correlation between the expression of cGAS (Pearson’s r=0.46) and STING (Pearson’s r=0.37) with the 12-CK GES score. In contrast, the methylation levels of cGAS and STING were inversely correlated with the 12-CK GES score (Pearson’s r=-0.37 and -0.41, respectively). Similarly, hypermethylation of STING was correlated with inferior disease-specific survival (DSS) (p<0.0001) in lung adenocarcinomas. Survival analysis on the TCGA skin cutaneous melanoma (SKCM) dataset also indicated significant DSS advantage in 12-CK GES scoreHigh cGASHighpatients (p<0.0001).ConclusionsWe provide evidence that epigenetic state of cGAS and STING cannot only shape tumor antigenicity but is also associated with the 12-CK GES and the presence of TLSs. Considering the well-established prognostic value of TLSs, these findings argue that targeting epigenetic suppression of STING signaling should be considered as a strategy to guide effective immunotherapy-based interventions.AcknowledgementsThis work was supported by the Moffitt Cancer Center Tissue Core and Analytic Microscopy Core Facilities, all comprehensive cancer center facilities designated by the National Cancer Institute (P30 CA076292). This work was funded by: NCI-NIH (1R01 CA148995, 1R01 CA184845, P30 CA076292, P50 CA168536), CJG Fund, Chris Sullivan Fund, V Foundation, Melanoma Research Foundation, and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat 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).Falahat R, Berglund A, Perez-Villarroel P, Putney RM, Hamaidi I, Kim S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic state determines the in vivo efficacy of STING agonist therapy. Nature Communications. 2023;14(1):1573.Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, Schell MJ, Sondak VK, Weber JS, Mulé JJ. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy?. Scientific reports. 2012;2(1):765.Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science. 2022;375(6576):eabf9419.
BackgroundIt is incompletely understood which populations of tumor-infiltrating lymphocytes (TIL) respond to checkpoint blockade (CB) and when. Recent studies in murine MC-38 colon carcinoma ...demonstrate CD4+ T cells are among the most prominent responders,1 but these studies were undertaken late in tumor growth, weeks after CB blockade was initiated. Here, we profile how the landscape of CB-responding TIL change between early and late MC-38 tumor growth, and uncover a novel switch that occurs between natural killer T (NKT) and conventional CD4/CD8 T cell responses.MethodsWe treated C57BL/6 mice bearing subcutaneous MC-38 tumors with anti-PD-1 and/or anti-CTLA-4 antibodies, and analyzed TIL 11 or 21 days later using a 23-paramter flow cytometry panel that includes three markers of effector function: TNF-alpha, IFN-gamma, and CD107a. We then investigated major populations, including NKT TIL, in ex vivo cytotoxicity assays and in vivo tumor growth studies using CD1d overexpressin MC-38 cells.ResultsOur analysis identified 37 TIL populations in MC-38 tumors, representing CD4+ or CD8+ T cells, natural killer (NK), and NKT cells. The distribution and effector function among TIL shift dramatically between early and late MC-38 growth. At 11 days, the immune response is dominated by TNF-alpha-producing NKT, which represent 53.5 ± 3.7% of all TIL. These are accompanied by modest frequencies of CD4+ and CD8+ TIL, producing low levels of IFN-gamma. After 21 days, NKT populations are reduced to 15.2 ± 1.5%, giving way to increased NK, CD4+, and CD8+ TIL, with increased IFN-gamma production. CB hastens this switch, markedly reducing NKT to less than 20% of all TIL, downregulating TNF-alpha production across NKT and CD4+ T cell subpopulations, increasing CD4+ and CD8+ TIL frequencies, and significantly up-regulating IFN-gamma production at 11 days. CD107a expression patterns suggest degranulation is most associated with NK and NKT TIL (figure 1). NKT displayed no CD1d-restricted cytotoxicity against MC-38 ex vivo. However, CD1d overexpression on MC-38 significantly delayed tumor growth in vivo, suggesting early NKT activity may indirectly suppress tumor progression, but by what precise mechanism(s) is currently unknown.Abstract 226 Figure 1t-SNE analysis of effector TIL populations identifies distinct, IFN-gamma and TNF-alpha-producing cells at early (day 11) and late (day 21) time points of subcutaneous MC38 growth. (a) Combined pseudocolored density plot of t-SNE parameters of viable, non-aggregated, CD45.2+, CD3+ and/or NK1.1+ cells from all time points and treatment conditions. (b) MFI values of clustering parameters from identified TIL populations were used in a hierarchical clustering analysis. Major clustering groups were then broadly identified as: TC, cytotoxic T cells; TH, helper T cells; gamma delta-like, gamma delta T cells or T cells clustering with gamma delta T cells; NK, natural killer cells; or O, other TIL. (c) Expression of effector molecules CD107a (top), IFN-gamma (middle), and TNF-alpha (bottom) among each identified TIL population. The extent of background signal for each effector molecule is denoted by a red-dashed FMO line. (d) A heat map of effector molecule MFIs overlaid onto the t-SNE analysis. (e) Analyses of TNF-alpha expression for P5 day 11. Included is the population location (upper left), TNF-alpha expression versus side-scatter (upper right), P5 frequency with check point blockade (lower left), and TNF-alpha MFI with check point blockade (lower right) (f) Analyses of IFN-gamma expression for P32 day 11. Included is the population location (upper left), IFN-gamma expression versus side-scatter (upper right), P32 frequency with check point blockade (lower left), and percent IFN-gamma with check point blockade (lower right).ConclusionsDespite evidence of an indirect benefit of early NKT activity, CB hastens a switch from TNF-alpha-driven NKT involvement toward a IFN-gamma-driven CD4+ and CD8+ T cell response in subcutaneous MC-38 tumors. These results corroborate earlier findings that CD4+ TIL are a major CB-responding population, and introduce a NKT/T cell switch that may be a key feature of the CB response in certain tumors.Ethics ApprovalAnimal experiments in this study were performed according to protocols approved by the University of South Florida’s institutional animal care and use committee (IACUC) committee, number R IS00005710.ReferenceWei S, Levine J, Coghill A, Zhao Y, Anang N, Andrews M, Sharma P, Wang J, Wargo J, Pe’er D, Allison J. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell 2017 Sep 7; 170(6): 1120–1133.e17.
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
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.
Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, ...we investigate the therapeutic, antimelanoma potential of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma. Utilizing murine and human melanoma cell lines, we analyzed the effects of LBH589 on proliferation and survival. In addition, we analyzed the expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589-treated melanoma to activate antigen-specific T cells. Finally, we assessed the in-vivo effects of LBH589 in a mouse melanoma model. Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of major histocompatibility complex and costimulatory molecules on melanoma cells leading to an increased ability to activate antigen-specific T cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost. Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.
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).
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