Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy.

BACKGROUND: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. METHODS: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. RESULTS: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. CONCLUSIONS: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Immune Escape Mechanisms in Non Small Cell Lung Cancer.

Development of strong immune evasion has been traditionally associated with the late stages of solid tumor progression, since advanced cancers are more likely to have reached the third phase of the immunoediting process. However, by integrating a variety of approaches, evidence for active immune escape mechanisms has been found even in the pre-invasive lesions that later progress to the main NSCLC histotypes. Pre-invasive lesions of adenocarcinoma (LUAD) and of squamous cell carcinoma (LUSC) can show impaired antigen presentation, loss of heterozygosity at the Human Leukocyte Antigen (HLA) region, neoantigen silencing, activation of immune checkpoints, altered TH1/TH2 cytokine ratios, and immune contexture evolution. Analysis of large panels of LUAD vs. LUSC, of early stage NSCLC vs. normal lung tissue, of specific molecular subsets of NSCLC, and of distinct regions within the same tumor, indicates that all these processes of immune escape continue to evolve in the invasive stage of NSCLC, are associated with inter- and intra-tumor heterogeneity, and contribute to resistance to therapy by immune checkpoint blockade (ICB). In this review, we will discuss the most recent evidence on immune escape mechanisms developing from the precursor to invasive stage in NSCLC, and the contribution of immune evasion to resistance to ICB in lung cancer.

An actionable axis linking NFATc2 to EZH2 controls the EMT-like program of melanoma cells.

Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+ EZH2+ melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

18F-Fluorocholine PET/CT Finding of a Vertebral Hemangioma.

The uptake of F-fluorocholine (FCH), a radiopharmaceutical used to study patients with prostate cancer, follow both the phosphorylcholine and acetylcholine synthesis. FCH uptake is not specific of neoplastic cells because phospholipids are a structural constituent of the membrane of all cells. Thus, PET/CT with FCH show several areas of physiologic uptake. The skeleton concentrates only mild amounts of FCH, thus a diffuse faint uptake of the radiopharmaceutical is present at a PET/CT study. Herein we present the case of a patient in which PET/CT evidenced a sharply defined vertebral "cold" area of reduced FCH uptake corresponding to a vertebral hemangioma.

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.

Towards combinatorial targeted therapy in melanoma: from pre-clinical evidence to clinical application (review).

Over the last few years, clinical trials with BRAF and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors have shown significant clinical activity in melanoma, but only a fraction of patients respond to these therapies, and development of resistance is frequent. This has prompted a large set of preclinical studies looking at several new combinatorial approaches of pathway- or target-specific inhibitors. At least five main drug association strategies have been verified in vitro and in preclinical models. The most promising include: i) vertical targeting of either MEK or phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways, or their combined blockade; ii) association of receptor tyrosine kinases (RTKs) inhibitors with other pro-apoptotic strategies; iii) engagement of death receptors in combination with MEK-, mTOR/PI3K-, histone deacetylase (HDAC)-inhibitors, or with anti-apoptotic molecules modulators; iv) strategies aimed at blocking anti-apoptotic proteins belonging to B-cell lymphoma (Bcl-2) or inhibitors of apoptosis (IAP) families associated with MEK/BRAF/p38 inhibition; v) co-inhibition of other molecules important for survival [proteasome, HDAC and Signal transducers and activators of transcription (Stat)3] and the major pathways activated in melanoma; vi) simultaneous targeting of multiple anti-apoptotic molecules. Here we review the anti-melanoma efficacy and mechanism of action of the above-mentioned combinatorial strategies, together with the potential clinical application of the most promising studies that may eventually lead to therapeutic benefit.

NFATc2 is a potential therapeutic target in human melanoma.

The identification of intracellular signaling pathways that promote cell proliferation and resistance to cell death may lead to the development of improved treatment for advanced melanoma. Here we show that the calcineurin/nuclear factor of activated T cells c2 (NFATc2) pathway has an antiapoptotic role in melanoma cells. Expression of NFATc2 was constitutive in vitro and in vivo in human melanoma, and cyclosporin A (CsA) treatment of melanoma cells led to downmodulation of NFATc2. Inhibition of the calcineurin/NFAT pathway by CsA, or by NFATc2 silencing, led to modulation of cell cycle inhibitors and apoptosis-related proteins such as Apollon, and promoted caspase-dependent apoptosis of neoplastic cells. Calcineurin/NFATc2 targeting significantly enhanced melanoma cell death induced by antitumor agents, such as MEK- or BRAF(V600E)-specific inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand, which trigger the intrinsic or extrinsic pathway of apoptosis, respectively. These findings identify NFATc2 as a potential therapeutic target in melanoma.

Role of Apollon in human melanoma resistance to antitumor agents that activate the intrinsic or the extrinsic apoptosis pathways.

PURPOSE: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. EXPERIMENTAL DESIGN: Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents. RESULTS: Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors. CONCLUSIONS: Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways.