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Despite the outstanding clinical results of immune checkpoint blockade (ICB) in melanoma and other cancers, clinical trials in breast cancer have reported low responses to these therapies. Current efforts are now focused on improving the treatment efficacy of ICB in breast cancer using new combination designs such as molecularly targeted agents, including histone deacetylase inhibitors (HDACi). These epigenetic drugs have been widely described as potent cytotoxic agents for cancer cells. In this work, we report new noncanonical regulatory properties of ultra-selective HDAC6i over the expression and function of epithelial–mesenchymal transition pathways and the invasiveness potential of breast cancer. These unexplored roles position HDAC6i as attractive options to potentiate ongoing immunotherapeutic approaches. These new functional activities of HDAC6i involved regulation of the E-cadherin/STAT3 axis. Pretreatment of tumors with HDAC6i induced critical changes in the tumor microenvironment, resulting in improved effectiveness of ICB and preventing dissemination of cancer cells to secondary niches. Our results demonstrate for the first time that HDAC6i can both improve ICB antitumor immune responses and diminish the invasiveness of breast cancer with minimal cytotoxic effects, thus departing from the cytotoxicity-centric paradigm previously assigned to HDACi.Significance:Ultraselective HDAC6 inhibitors can reduce tumor growth and invasiveness of breast cancer by noncanonical mechanisms unrelated to the previously cytotoxic properties attributed to HDAC inhibitors.

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Wound Assay vehicle

Introduction: Pan-histone deacetylase (HDAC) inhibitors have been used as anti-cancer agents due to their cytotoxicity. However, studies from our group have reported that intervention of class-specific HDAC inhibitors can differentially affect immune-related pathways in macrophages and influence their pro- or anti-inflammatory phenotype and function. For example, specific HDAC6 inhibitors demonstrated promising anti-tumor effects by suppressing tumor-promoting M2 macrophages in the tumor microenvironment (TME). In contrast, inhibition of HDAC11 promoted the anti-inflammatory phenotype of macrophages. Besides the divergent roles and distinct signaling pathways, a comprehensive study on the effect of HDCA6 and HDAC11 inhibition on macrophage phenotype and the implications on TME and inflammation is warranted. Objective: To determine the effect of HDAC6 and HDAC11 inhibition on the phenotype and function of macrophages in the context of the TME. Methods: Bone marrow-derived macrophages (BMDMs) isolated from C57BL/6 mice were pre-treated with HDAC6 (NexturastatA) or HDAC11 (FT895) inhibitors prior to polarizing them to M1 and M2 phenotypes. Similarly, BMDMs were isolated from total knockouts (KO) of HDAC6 and HDAC11 mice. The changes in gene expression were identified by RNA-seq analysis. Furthermore, single-cell secretome analysis on Isoplexis platform was performed from human and mice BMDMs on a panel of cytokines and chemokines. Results: Transcriptomic analysis revealed that HDAC6 inhibition in M1 polarized BMDMs sustained inflammatory gene signature while suppressing the tumor-promoting and anti-inflammatory genes in M2 macrophages. This supported our previous reports where HDAC6i increased M1/M2 ratio in murine melanoma models. In contrast, HDAC11 inhibition in M2 macrophages upregulated classical M2 markers, whereas M1-associated gene signature was minimally affected. These results were recapitulated in HDAC6KO and HDAC11KO macrophages, suggesting a high specificity of the inhibitors. Further validation at protein level by single-cell secretome analysis indicated that HDAC11 inhibition in M2 polarized BMDMs increased secretion of growth factors such as Egf and Pdgf and of anti-inflammatory cytokines such as Il10 and Il13. On the contrary, HDAC6 inhibition upregulated inflammatory cytokines such as Ifng and Tnf and T-cell recruiting chemokine Cxcl10 in the M1 phenotype. Conclusions:Transcriptomics and single-cell secretome analyses of macrophages indicate that HDAC6 and HDAC11 affects macrophage function in diametrically opposite directions. Therefore, our study underscores the importance of class-selective inhibition of HDACs over pan-HDAC inhibitors and the potential use of selective HDAC inhibitors as a therapeutic option to control macrophage phenotype in cancer and other conditions. Citation Format: Manasa Suresh, Hawa Coulibaly, Xintang Li, David Quiceno-Torres, Nithya Gajendran, Karen Tan, Matias Hepp, Karthik Musunuri, Satish Noonepalle, Alejandro Villagra. Inhibition of HDAC6 and HDAC11 has opposite effects on inflammation and the modulation of the functional phenotype of macrophages in the tumor microenvironment [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 2880.

BackgroundNovel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response.MethodsHuman OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8Trp53-/-mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8Trp53-/-cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.ResultsWe show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.ConclusionIn summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

Cancer, traditionally viewed as a disease driven by genomic alterations, is now perceived as an accumulation of genetic mutations as well as global epigenetic changes to the chromatin that regulate gene expression (1, 2). Genetic alterations to either tumor suppressors or oncogenes can result in dramatic gene expression changes leading to cancer; however, changes in the epigenome are rather subtle. Despite similar genomic sequences in all the cell types, the epigenome can vary considerably, resulting in distinct gene expression patterns and, therefore, distinct cellular functions. Epigenetic modifications to chromatin include DNA methylation, histone modifications, nucleosome positioning, and non-coding RNAs that can regulate access of DNA to transcription factors and other cis-regulatory elements, thereby affecting gene expression (3). It is now recognized that genetic and epigenetic components complement each other to drive tumor initiation and progression (4). Recent technical advances in high throughput sequencing have improved understanding of the epigenomic landscape at a higher resolution. Massive datasets and databases, including the encyclopedia of DNA elements (ENCODE) (5), The Cancer Genome Atlas (TCGA) ((https://www.cancer.gov/tcga), the NIH Roadmap Epigenomics Mapping Consortium (6), the Epigenome browser (7), have enhanced our ability to understand the interplay between cancer cells, tumor microenvironment (TME) and immune cells. Therefore, a new classification of molecular epigenetic modifications is needed to differentiate “cancer intrinsic” and “cancer extrinsic” mechanisms influencing anti-tumor immune responses. The schematic shows a graphical representation of various intrinsic and extrinsic factors affecting the cancer cells and thereby regulate the TME ( Figure 1 ). The International Human Epigenome Consortium (IHEC) provides high-resolution reference epigenomes of major primary human cell types (8). Based on the data from these projects, genetic and epigenetic crosstalk in cells is evident, and it has led to the identification of novel biomarkers and the development of novel therapeutic strategies. The articles in this Research Topic on Genetic and Epigenetic Control of Immune Responses address both cell-intrinsic and cell-extrinsic mechanisms controlling the immune response to tumors. Open in a separate window Figure 1 Tumor intrinsic and extrinsic factors regulating immune responses in the tumor microenvironment. Cancer cell intrinsic factors such as genomic mutations, chromatin modifiers and non-coding RNA regulate tumor initiation, propagation as well as immunogenicity. Epigenetic modifications such as DNA methylation, histone acetylation and methylation regulate gene expression. Non-coding RNA including long non-coding RNA, microRNA, circular non-coding RNA regulate gene transcription as well as mRNA stability. Other mechanisms intrinsic to cancer cells include expression of immunosuppressive cytokines to facilitate escape from anti-tumor immunity, expression of immunosuppressive molecules such as PD-L1 and PD-L2, suppression of antigen processing and presentation machinery and tumor associated antigens. Cancer cell extrinsic factors include tumor infiltrated immune cells, fibroblasts, stromal cells and endothelial cells. Extrinsic factors also include secretory factors such as cytokines, chemokines, metabolites, growth factors and immune checkpoint molecules. Tumor associated antigens presented by antigen presenting cells such as macrophages, dendritic cells activate CD8 T-cells for effective anti-tumor immunity. However, immune checkpoint molecules expressed by cancer cells regulate the inflammatory status of the tumor tamping down the inflammation. Use of epigenetic modifiers such as DNMT inhibitors, HDAC inhibitors, BET inhibitors etc. can alter these reversible modifications to enhance anti-tumor immunity. Figure created with BioRender.com.

Lessons Learned Entinostat at the selected dose levels in combination with a standard dose of enzalutamide showed a promising safety profile in this small phase I study Background Entinostat inhibits prostate cancer (PCa) growth and suppresses Treg cell function in vitro and in vivo. Methods This was a phase I study to explore the safety and preliminary efficacy of entinostat (3 and 5 mg orally per week) in combination with enzalutamide in castration resistant PCa (CRPC). The study was carried out in an open-label two-cohort design. Patients who had developed disease progression on or were eligible for enzalutamide were enrolled in the study. The safety profile of the combination therapy, Prostate specific antigen (PSA) levels, the pharmacokinetics of enzalutamide after entinostat administration, peripheral T-cell subtype (including Treg quantitation), and mononuclear cell (PBMC) histone H3 acetylation were analyzed. Results Six patients with metastatic CRPC were enrolled. There was no noticeable increment of fatigue related to entinostat. Toxicities possibly or probably related to entinostat or the combination therapy included grade 3 anemia 1/6 (17%), grade 2 white blood cell (WBC) decrease 1/6 (17%), and other self-limiting grade 1 adverse events (AEs). Median duration of treatment with entinostat was 18 weeks. Entinostat did not affect the steady plasma concentration of enzalutamide. Increased PBMC histone H3 acetylation was observed in blood samples. No evident T-cell subtype changes were detected, including in Treg quantitation. Conclusion Entinostat 5 mg weekly in combination with enzalutamide showed an acceptable safety profile in this small phase I study. A planned phase II part of the trial was terminated because of sponsor withdrawal.

Irregular inflammatory responses are a major contributor to tissue dysfunction and inefficient repair. Skin has proven to be a powerful model to study mechanisms that regulate inflammation. In particular, skin wound healing is dependent on a rapid, robust immune response and subsequent dampening of inflammatory signaling. While injury-induced inflammation has historically been attributed to keratinocytes and immune cells, a vast body of evidence supports the ability of non-immune cells to coordinate inflammation in numerous tissues and diseases. In this review, we concentrate on the active participation of tissue-resident adipocytes and fibroblasts in pro-inflammatory signaling after injury, and how altered cellular communication from these cells can contribute to irregular inflammation associated with aberrant wound healing. Furthering our understanding of how tissue-resident mesenchymal cells contribute to inflammation will likely reveal new targets that can be manipulated to regulate inflammation and repair.

Selective inhibition of histone deacetylase 6 (HDAC6) is being recognized as a therapeutic approach for cancers. In this study, we designed a new HDAC6 inhibitor, named Suprastat, using in silico simulations. X-ray crystallography and molecular dynamics simulations provide strong evidence to support the notion that the aminomethyl and hydroxyl groups in the capping group of Suprastat establish significant hydrogen bond interactions, either direct or water-mediated, with residues D460, N530, and S531, which play a vital role in regulating the deacetylase function of the enzyme and which are absent in other isoforms. In vitro characterization of Suprastat demonstrates subnanomolar HDAC6 inhibitory potency and a hundred- to a thousand-fold HDAC6 selectivity over the other HDAC isoforms. In vivo studies reveal that a combination of Suprastat and anti-PD1 immunotherapy enhances antitumor immune response, mediated by a decrease of protumoral M2 macrophages and increased infiltration of antitumor CD8+ effector and memory T-cells.

Despite the outstanding clinical results of immune checkpoint blockade (ICB) in melanoma and other cancers, clinical trials in breast cancer have reported low responses to these therapies. Current efforts are now focused on improving the treatment efficacy of ICB in breast cancer using new combination designs such as molecularly targeted agents, including histone deacetylase inhibitors (HDACi). These epigenetic drugs have been widely described as potent cytotoxic agents for cancer cells. In this work, we report new noncanonical regulatory properties of ultra-selective HDAC6i over the expression and function of epithelial–mesenchymal transition pathways and the invasiveness potential of breast cancer. These unexplored roles position HDAC6i as attractive options to potentiate ongoing immunotherapeutic approaches. These new functional activities of HDAC6i involved regulation of the E-cadherin/STAT3 axis. Pretreatment of tumors with HDAC6i induced critical changes in the tumor microenvironment, resulting in improved effectiveness of ICB and preventing dissemination of cancer cells to secondary niches. Our results demonstrate for the first time that HDAC6i can both improve ICB antitumor immune responses and diminish the invasiveness of breast cancer with minimal cytotoxic effects, thus departing from the cytotoxicity-centric paradigm previously assigned to HDACi.Significance:Ultraselective HDAC6 inhibitors can reduce tumor growth and invasiveness of breast cancer by noncanonical mechanisms unrelated to the previously cytotoxic properties attributed to HDAC inhibitors.

Cancer cells are metabolically rewired to activate glycolysis, which results in the generation and excretion of lactate as a byproduct. Although lactate was previously thought to be a waste product of anaerobic respiration, increasing evidence has revealed that distinct cell populations within tumors use lactate as a source of energy through TCA cycle. This metabolic symbiosis between distinct cell populations, hypoxic lactate consumers, and oxidative glycolytic lactate exporters within the tumor is regulated by differential expression of lactate transporters, mainly MCT1 and MCT4. Therefore, dual targeting of MCT1 and MCT4 is a novel approach to treat cancer. We have developed a compound, NGY-B, which binds and inhibits both MCT1 and MCT4 transporters. This compound has shown to significantly increase intracellular lactate levels in melanoma models and thereby decreasing in vitro cell proliferation. When cultured in the media with exogenous lactate to mimic the tumor microenvironment, NGY-B greatly reduced lactate uptake by cancer cells. Mechanistically, NGY-B deactivated glycolytic pathway by reducing hexokinase 2 (HK2), phosphoglycerate dehydrogenase (PHGDH), and pyruvate dehydrogenase kinase 1 (PDK1) expression. Additionally, NGY-B treatment strongly decreased STAT3 and mTOR signaling pathways. Furthermore, the in vivo therapeutic efficacy of NGY-B was validated in xenograft and syngeneic mouse models of melanoma. While a significant reduction in tumor size was observed in A375 xenograft model treated with NGY-B, a robust tumor growth reduction was evident in SM1 immunocompetent syngeneic melanoma mouse model, suggesting the involvement of immune cells for higher response in this model. Supporting this premise, we also found that NGY-B treated cells showed a decrease in PD-L1, B7-H3, and B7-H4 expression. Overall, our results indicate that NGY-B inhibits melanoma by targeting metabolic symbiosis and activating antitumor immune response mechanisms. Citation Format: Sambad Sharma, Gregory Goreczny, Satish Kumar Noonepalle, Erica Palmer, Maria Garcia-Hernandez, Daliya Banerjee, Jaime Escobedo, Alejandro Villagra, Vincent Sandanayaka. A novel treatment approach for melanoma by dually targeting MCT1 and MCT4 lactate transporters [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 1268.

The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including non-canonical functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the HDAC6i Nexturastat A as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot”, and potentially augment immune check-point blockade therapies. This combination modality demonstrated to significantly reduce tumor growth in syngeneic melanoma tumor models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed profound changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of individual components of the tumor microenvironment suggested that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor-associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies.

HDACs, originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins that are involved in diverse cellular processes unrelated to the chromatin environment. This includes deacetylation of nonhistone targets involved in multiple signaling pathways. In this regard, a considerable number of reports have analyzed the role of nonspecific inhibition of HDACs through pan-HDACi in cancer as well as processes of immune regulation. However, with pan-HDACi there is a lack of understanding about the exact contribution of inhibition of each individual HDAC, which makes the rational design of improved drug candidates extremely difficult. Additionally, current approaches using nonselective HDACi in the clinic have critical limitations, including pan-HDACi which elicit poor activity in solid tumors and cardiac toxicity, class I HDACi which activate multiple apoptotic pathways, limiting its use for longer periods of time, and class I-HDAC6i that evidenced a number of adverse effects in initial clinical trials. Therefore, there is a growing interest in the identification of more selective HDACi, and the subsequent development of accurate functional tests to identify the effectiveness and selectivity of these inhibitors. In this chapter, we are describing some selected methodologies to identify the individual activities of HDACs. In addition, we present specific methods to identify enzymatic and nonenzymatic molecular targets of HDACs.

In addition to their canonical role in transcriptional regulation, Histone deacetylases (HDACs) regulate other cellular functions by maintaining a balance of lysine acetylation on key cellular proteins. HDAC inhibitors are under development for use in treating cancers, neurological, and immunological diseases. A barrier to the development of HDAC inhibitors has been the toxicity associated with non-selective pan-HDAC inhibitors, which simultaneously inhibit multiple HDAC isoforms. Isoform selective HDAC inhibitors offer disease targeting with an improved toxicity profile. HDAC6 presents an important target due to its role in immune regulatory processes involved in antitumor immune responses. We have shown that selective inhibition of HDAC6 regulates macrophages to recognize, phagocyte, and present antigens to the adaptive immune system. Treatment with our selective HDAC6 inhibitor, SP-2-225, was shown to enhance the production of cancer-associated antigens and macrophage antigen cross-presentation to T cells. Through a non-cytotoxic mechanism, SP-2-225 significantly reduced the tumor volume in a syngeneic SM1 melanoma model. Analysis of the tumors from treated animals showed a significant shift in the M1/M2 ratio of the infiltrating macrophages as compared to control, indicating that SP-2-225 caused a pro-inflammatory, antitumor shift in the macrophage equilibrium surrounding the tumor. This was identified as the putative mechanism through adoptive cell therapy where macrophages from naive animals were harvested and treated ex-vivo with SP-2-225. Reimplantation of these SP-2-225 ex-vivo treated macrophages resulted in reduced tumor volumes when compared to both vehicle and untreated macrophage animal cohorts. We propose that this selective inhibitor of HDAC6 offers potential as a stand-alone cancer therapy and in combination with other immunotherapies and radiation treatments. Citation Format: Scott Grindrod, Satish Noonepalle, Nima Aghdam, Alfredo Velena, Maria Gracia-Hernandez, Christian Zevallos-Delgado, Mira Jung, Anatoly Dritschilo, Alejandro Villagra. Immune-mediated tumor growth inhibition by selective HDAC6 inhibitor SP-2-225 [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO023.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including non-canonical functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the HDAC6i Nexturastat A as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot”, and potentially augment immune check-point blockade therapies. This combination modality demonstrated to significantly reduce tumor growth in syngeneic melanoma tumor models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed profound changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of individual components of the tumor microenvironment suggested that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor-associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

The generation of reactive oxygen species (ROS) plays an important role for the maintenance of cellular processes and functions in the body. However, the excessive generation of oxygen radicals under pathological conditions such as acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) leads to increased endothelial permeability. Within this hallmark of ALI and ARDS, vascular microvessels lose their junctional integrity and show increased myosin contractions that promote the migration of polymorphonuclear leukocytes (PMNs) and the transition of solutes and fluids in the alveolar lumen. These processes all have a redox component, and this chapter focuses on the role played by ROS during the development of ALI/ARDS. We discuss the origins of ROS within the cell, cellular defense mechanisms against oxidative damage, the role of ROS in the development of endothelial permeability, and potential therapies targeted at oxidative stress.

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the immunological modulation of the HDAC6i Nexturastat A in combination with anti-PD-1 checkpoint blockade therapy and the use of this HDAC6i as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot” in order to more specifically augment immune check-point blockade therapies. This combination of Nexturastat A and anti-PD-1 therapy demonstrated a significant reduction of tumor growth in syngeneic melanoma tumor animal models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed that the pre-treatment with selective HDAC6i induced major changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of the effect of HDAC6i on individual immune components suggest that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies. Note: This abstract was not presented at the meeting. Citation Format: Tessa J. Knox, Eva Sahakian, Debarati Banik, Melissa Hadley, Erica Palmer, Jennifer Kim, Satish Noonepalle, John Powers, Maria Gracia-Hernandez, Vasco Oliveira, Fengdong Cheng, Jie Chen, Cyril Barinka, Javier Pinilla-Ibarz, Norman Lee, Alan Kozikowski, Alejandro Villagra. Priming the tumor microenvironment with epigenetic modifiers to overcome resistance to immune checkpoint inhibitors [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 LB-074.

Solid tumors often have altered metabolism that depend on aerobic glycolysis for energy and macromolecules for cell survival thereby generating lactate as a metabolic byproduct. Lactate is transported across the cell membrane into tumor microenvironment (TME) using monocarboxylate transporters, MCT1 and MCT4. MCTs facilitate proton linked transport of monocarboxylate molecules such as lactate, pyruvate, and ketone bodies across the plasma membrane. Lactate released by glycolytic cells into the TME results in a metabolic symbiosis when other tumor cells utilize lactate as an energy source that undergo oxidative metabolism which is referred to as “lactate shuttle”. Moreover, lactate induces inhibitory pathways on immune cells, especially on cytotoxic CD8+ T-cells resulting in local immunosuppression in the TME. Additionally, acidosis of TME through lactate results in local inflammation and angiogenesis by activation of VEGFR signaling. The net result of an increased lactate in TME is that it generates a conducive milieu for tumor growth and metastasis. Although studies show that MCT inhibitors mitigate the effects of lactate and promote immune function, its effect on immune cells in the context of tumor infiltration is yet to be explored. In this study, we used a MCT inhibitor NGY-A to determine the effect of suppressing lactate levels in the TME and restoring the anti-tumor immunity. In our preliminary studies to determine the cytotoxicity, NGY-A showed significant cancer cell killing in multiple cell lines in vitro. To determine the in vivo effect of NGY-A, syngeneic mouse model of SM1 melanoma cells harboring BRAF mutation in C57BL/6 mice was studied. When treated with NGY-A (10 mg/kg, i.p injection) (n=15), a significant decrease in tumor volumes was observed compared to controls (n=15). Gene expression analysis of total RNA isolated from SM1 tumors by quantitative RT-PCR indicated that expression of immunosuppressive molecules such as Cd274 (PD-L1), Cd276 (B7-H3) and Lgals9 (Galactin-9) was down regulated in NGY-A (n=5) treated tumors compared to the control tumors (n=5). An increase in IFN-γ (IFNG) gene expression in NGY-A treated samples indicated an increase in CD8+ T-cell tumor infiltration. Future studies would include analysis of phenotype of tumor infiltrating immune cells and cytokine profiles after treatment with NGY-A in combination with immune checkpoint blockade inhibitors which could potentially be an effective strategy to treat melanoma. Citation Format: Satish Kumar Reddy Noonepalle, Jennifer Kim, Melissa Hadley, Erica Palmer, Debarati Banik, Tessa Knox, Sophiya Ephrame, Prathima Vembu, Summer Rudish, Sara Moufarrij, Jaime Escobedo, Vincent Sandanayaka, Alejandro Villagra. Modulation of lactate in the tumor microenvironment with lactate transporter inhibitor in a melanoma syngeneic mouse model [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 5126.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.

Tumor infiltrating lymphocytes (TILs) play a critical role in regulating the immunomodulatory properties of triple negative breast cancer (TNBC). However, the specific adaptations that TNBC tumors undergo when challenged by lymphocyte infiltration remain unclear. In order to address this gap in knowledge, we conducted an immuno-phenotype comparison using mRNA sequencing between the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were co-cultured with activated human T-cells. Although the cytokine-induced immune signature of the two cell lines were similar, MDA-MD-231 cells were able to transcribe the tryptophan catabolizing enzyme IDO1 at a significantly higher level than MCF7 cells. Stimulation with IFNg was able to differentially induce IDO protein expression and enzymatic activity in ER- cell lines compared to ER+ cell lines, though no differences were observed in upstream JAK/STAT1 signaling or IDO1 mRNA stability between the two cell lines. Further experiments showed that treatment with the demethylating agent 5-aza-deoxycytidine was able to reverse suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation serves as a potential determinant in IDO1 induction. Analysis of TCGA and other previously published breast cancer datasets revealed subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal-like subtypes exhibiting lower promoter methylation and higher mRNA expression than ER+/luminal subtypes. Bisulfite pyrosequencing validated the subtype-specific association of decreased promoter methylation with increased IDO1 expression in breast cancer cell lines and an independent cohort of primary breast tumors. In addition, decreased IDO1 promoter methylation and elevated IDO1 expression in basal-like breast tumors was found to be associated with increased levels of kynurenine, the metabolic product of IDO1, as well as higher numbers of CD8+ TILs. Furthermore, high kynurenine levels in breast tumors were associated with worse patient survival. Taken together, these findings suggest that subtype-specific IDO1 promoter methylation regulates the ability of breast tumors to escape from antitumor immune responses driven by CD8+ TILs and could be used as a predictive biomarker for IDO inhibitor-based immunotherapy. Citation Format: Gu F, Noonepalle SK, Lee E-J, Choi J-H, Shull AY, Pei L, Sreekumar A, Ambs S, Shi H. Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-08.

An intrinsic claudin-low subtype within the TNBC was recently identified based on low/absent expression of luminal differentiation markers, enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Interleukin-6 (IL6), a potent pleiotropic cytokine, is induced in response to acute and chronic inflammation. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Suppressor of cytokine signaling 3 (SOCS3) mediated Stat3/NF-κB pathway. SOCS3 is a critical negative regulator of IL6-mediated pathways. We have showed that constitutive activation of inflammatory loop in transformed cells but not in their untransformed counterparts depends on proteolytic degradation of SOCS3. Several studies indicate that activation of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) represents a key pathway suppressing anti-tumor immunity. Moreover a recent study in dendritic cells found that SOCS3 binds IDO via phosphotyrosine-containing peptides and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. IDO is shown to be constitutively expressed by many tumors. We analyzed a panel of breast cancer cell lines and found an inverse correlation between SOCS3 and IDO1 protein levels. Interestingly IDO protein and transcript levels were significantly increased in TNBC compared to luminal cell lines, thus confirming that IL6-JAK-STAT inflammatory loop is responsible for IDO regulation. Enforced expression of stable form of SOCS3, lacking the PEST and SOCS box domain in TNBC cell lines resulted in decreased expression of IDO. Therapeutic targeting of the IDO-IL6-SOCS3 inflammatory loop would revert the immune suppression mediated by IDO in this breast cancer subtype. These studies would provide a strong rationale for development of inflammatory pathway targeted agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Maria Ouzounova, EunMi Lee, Ena Novakovic, Satish Kumar Noonepalle, Raziye Piranlioglu, Huidong Shi, Max Wicha, Hasan Korkaya. SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1984. doi:10.1158/1538-7445.AM2015-1984

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060