FAS Pathway Abnormalities in MF and SS

MF 和 SS 中的 FAS 通路异常

• 批准号: 8994163
• 负责人: GARY S WOOD
• 金额: --
• 依托单位: WM S. MIDDLETON MEMORIAL VETERANS HOSP
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8994163
• 关键词: Accounting; Affect; Allergic Disease; Apoptosis; Apoptotic; Autoimmune Diseases; Binding; Biopsy; Cell Death; Cell Survival; Cells; Cessation of life; Clinic; Clinical; Clinical Research; Co-Immunoprecipitations; Collection; Complex; Cutaneous; Cutaneous Lymphoma; DNA Methylation; DNA Modification Methylases; Data; Decitabine; Disease; EMSA; Epigenetic Process; Exhibits; Folic Acid Antagonists; Gene Expression; Gene Silencing; Gene Targeting; Genes; Goals; HDAC1 gene; Hand; Health; Histone Deacetylase Inhibitor; Human; Image; In Situ; In Vitro; Incidence; Inflammatory; Interferon-alpha; Leukemic Cell; Ligands; Luciferases; Malignant Neoplasms; Mediating; Mediator of activation protein; Messenger RNA; Methodology; Methods; Methotrexate; Methylation; Monitor; Mycosis Fungoides; Neoplasms; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Publishing; Regulation; Reporter; Reporting; Research; S-Adenosylmethionine; STAT3 gene; Sampling; Sezary Syndrome; Skin; Skin Cancer; Small Interfering RNA; Specimen; T cell regulation; T-Cell Lymphoma; T-Lymphocyte; TNFRSF5 gene; TNFSF6 gene; Testing; Therapeutic; Therapeutic Agents; Tumor Suppressor Genes; Ultraviolet B Radiation; Validation; Veterans; Work; analog; analytical tool; base; bench to bedside; biobank; carcinogenesis; cell growth; cell killing; clinical effect; clinical phenotype; clinical practice; cofactor; demethylation; derepression; design; epigenetic regulation; gene repression; improved; in vivo; inhibitor/antagonist; innovation; knock-down; laser capture microdissection; methotrexate analog; neoplastic; neoplastic cell; novel; novel therapeutics; p65; programs; promoter; protein expression; pyrosequencing; receptor; research study; response; transcription factor; translational approach; translational study

DESCRIPTION (provided by applicant): Skin cancer is the most common form of human neoplasia and its incidence is rising rapidly. Many cancers affecting the skin including cutaneous T cell lymphomas (CTCL) exhibit silencing of tumor suppressor genes by DNA methylation. In this study, we propose to focus on the epigenetic silencing of selected tumor suppressor genes in two types of CTCL: mycosis fungoides (MF) and the Sezary syndrome (SS). We reported that the death receptor-ligand partners, FAS and FASL, are frequently expressed only weakly in MF/SS. We observed that FAS expression is transcriptionally regulated and that there is an inverse correlation between FAS expression and promoter methylation. We found that MF/SS is generally rich in DNMT1 (the DNA methyltransferase most likely responsible for epigenetic gene silencing) and that DNMT1 knockdown reverses FAS promoter methylation. We also discovered that folate antagonists like methotrexate (MTX) can inhibit DNA methylation by depleting S- adenosylmethionine (SAM), the main methyl donor for DNMTs. Our published data involving EMSA, supershift, ChIP and luciferase reporters show that the interaction of transcription factors (e.g. NFkB p50/p65) with the FAS promoter can be decreased by methylation resulting in less FAS expression. Using laser capture microdissection, pyrosequencing and multispectral imaging, we developed a method for quantitative gene analysis of methylation and expression (Q-GAME) in cells isolated from lesional skin. Using standard skin biopsies, Q-GAME can monitor in-situ the in-vivo effects of MTX and other agents on gene methylation and expression. With these findings and analytical tools in hand, we hypothesize that analogous to the situation with FAS, DNA methylation will account at least partially for decreased expression of FASL. Furthermore, MTX acting as a demethylator will reverse this gene repression and induce MF/SS apoptosis by enhancing activation of the FAS pathway. In Aim 1, we will use FAS/FASL-low MF/SS lines (HH, SZ4) that reflect the most common MF/SS clinical phenotype to optimize the demethylating effects of 5-aza, decitabine, MTX, MTX analogs and HDAC inhibitors on FAS and FASL in MF/SS. Inhibition of MTX and its analogs by exogenous SAM will be tested to confirm our proposed mechanism of action. We will validate these in-vitro studies ex-vivo using SS leukemic cells and determine the net effects on SS cell growth and survival. In Aim 2, we will explore the mechanisms regulating tumor suppressor gene silencing in FAS/FASL-low MF/SS lines. First we will compare the effects of DNMT1 inhibition to the effects of 5-aza because our preliminary data on DNMT1 knockdown indicate that it is a key mediator of gene methylation. Then we will use ChIP and co-immunoprecipitation to identify DNMT1 cofactors (e.g. STAT3, RelA, HDAC1) that bind to FAS and FASL. Finally, we will determine the impact of inhibition of these DNMT1 cofactors and upstream DNMT1 regulators on gene methylation. These in-vitro studies will be validated ex-vivo using SS leukemic cells. Targeting gene specific cofactors might allow relatively gene specific manipulation of DNA methylation. In Aim 3, to clinically validate our in-vitro and ex-vivo findings, we will use Q- GAME to quantitatively monitor FAS and FASL gene demethylation by MTX in-situ in MF/SS patient samples and determine the impact on gene expression and clinical outcome. We will use MTX because it is a standard MF/SS therapy and we have discovered that it is an effective demethylating agent. We will also assess the clinical effect of MTX in combination with other therapies (e.g. IFNa, HDAC inhibitors, UVB) that further enhance FAS or FASL by mechanisms different than MTX. In aggregate, these aims will identify and validate novel target genes for MF/SS therapy, provide strategies for modulating and monitoring their expression in the clinical setting, and determine their impact on clinical outcome. Successful completion of these highly translational aims depends on our extensive collection of MF/SS specimens and expertise with methods tailored specifically for this proposal.

描述（由申请人提供）： 皮肤癌是人类肿瘤最常见的形式，其发病率正在迅速上升。许多影响皮肤的癌症，包括皮肤 T 细胞淋巴瘤 (CTCL)，都表现出 DNA 甲基化导致的抑癌基因沉默。在这项研究中，我们建议重点关注两种类型 CTCL 中选定抑癌基因的表观遗传沉默：蕈样肉芽肿 (MF) 和塞扎里综合征 (SS)。我们报道了死亡受体配体伙伴 FAS 和 FASL 在 MF/SS 中经常仅微弱表达。我们观察到 FAS 表达受到转录调控，并且 FAS 表达与启动子甲基化之间存在负相关。我们发现 MF/SS 通常富含 DNMT1（最有可能导致表观遗传基因沉默的 DNA 甲基转移酶），并且 DNMT1 敲低可逆转 FAS 启动子甲基化。我们还发现，甲氨蝶呤 (MTX) 等叶酸拮抗剂可以通过消耗 DNMT 的主要甲基供体 S-腺苷甲硫氨酸 (SAM) 来抑制 DNA 甲基化。我们发表的涉及 EMSA、supershift、ChIP 和荧光素酶报告基因的数据表明，转录因子（例如 NFkB p50/p65）与 FAS 启动子的相互作用可以通过甲基化来减少，从而导致 FAS 表达减少。利用激光捕获显微切割、焦磷酸测序和多光谱成像，我们开发了一种对从病变皮肤分离的细胞中甲基化和表达进行定量基因分析（Q-GAME）的方法。使用标准皮肤活检，Q-GAME 可以原位监测 MTX 和其他药物对基因甲基化和表达的体内影响。 有了这些发现和分析工具，我们假设与 FAS 的情况类似，DNA 甲基化至少部分解释了 FASL 表达的降低。此外，MTX 作为去甲基剂将逆转这种基因抑制，并通过增强 FAS 途径的激活来诱导 MF/SS 凋亡。在目标 1 中，我们将使用反映最常见 MF/SS 临床表型的 FAS/FASL 低 MF/SS 系（HH、SZ4）来优化 5-aza、地西他滨、MTX、MTX 类似物和 HDAC 抑制剂的去甲基化作用关于 MF/SS 中的 FAS 和 FASL。将测试外源 SAM 对 MTX 及其类似物的抑制，以证实我们提出的作用机制。我们将使用 SS 白血病细胞在体外验证这些体外研究，并确定对 SS 细胞生长和存活的净影响。在目标 2 中，我们将探索 FAS/FASL-low MF/SS 系中抑癌基因沉默的调控机制。首先，我们将比较 DNMT1 抑制与 5-aza 的影响，因为我们关于 DNMT1 敲除的初步数据表明它是基因甲基化的关键介质。然后我们将使用 ChIP 和免疫共沉淀来鉴定与 FAS 和 FASL 结合的 DNMT1 辅助因子（例如 STAT3、RelA、HDAC1）。最后，我们将确定这些 DNMT1 辅助因子和上游 DNMT1 调节因子的抑制对基因甲基化的影响。这些体外研究将使用 SS 白血病细胞进行体外验证。靶向基因特异性辅助因子可能允许对 DNA 甲基化进行相对基因特异性的操作。在目标 3 中，为了临床验证我们的体外和离体研究结果，我们将使用 Q-GAME 定量监测 MF/SS 患者样本中 MTX 引起的 FAS 和 FASL 基因去甲基化，并确定对基因表达和临床结果。我们将使用 MTX，因为它是标准的 MF/SS 疗法，而且我们发现它是一种有效的去甲基化剂。我们还将评估 MTX 与其他疗法（例如 IFNa、HDAC 抑制剂、UVB）联合使用的临床效果，这些疗法通过与 MTX 不同的机制进一步增强 FAS 或 FASL。总的来说，这些目标将识别和验证 MF/SS 治疗的新靶基因，提供在临床环境中调节和监测其表达的策略，并确定它们对临床结果的影响。这些高度转化性目标的成功完成取决于我们广泛收集的 MF/SS 标本和专门为此提案量身定制的方法的专业知识。