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）表现出通过DNA甲基化对肿瘤抑制基因的沉默。在这项研究中，我们建议专注于两种类型的CTCL中选定肿瘤抑制基因的表观遗传沉默：Fungoides（MF）和Sezary综合征（SS）。我们报告说，死亡受体配体伴侣FAS和FASL通常仅在MF/SS中弱表示。我们观察到FAS表达是在转录调控的，并且FAS表达与启动子甲基化之间存在反相关。我们发现MF/SS通常富含DNMT1（DNA甲基转移酶最有可能导致表观遗传基因沉默），并且DNMT1敲低逆转逆转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耗尽s-腺苷甲硫氨酸（SAM）（DNMTS的主要甲基供体）来抑制DNA甲基化。我们发布的涉及EMSA，Supershift，ChIP和荧光素酶报告的数据表明，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而降低，从而减少FAS的表达。使用激光捕获显微解剖，焦距测序和多光谱成像，我们开发了一种从病变皮肤分离的细胞中甲基化和表达（Q-GAME）定量基因分析的方法。使用标准的皮肤活检，Q-Game可以在原位监测MTX和其他药物对基因甲基化和表达的体内影响。 借助这些发现和分析工具，我们假设类似于FAS的情况，DNA甲基化将至少部分解释FASL的表达降低。此外，用作脱甲基的MTX将逆转该基因抑制，并通过增强FAS途径的激活来诱导MF/SS凋亡。在AIM 1中，我们将使用FAS/FASL-LOW MF/SS系（HH，SZ4）反映最常见的MF/SS临床表型，以优化5-Aza，Decitabine，Decitabine，MTX，MTX，MTX类似物和HDAC抑制剂对FAS和MF/SS中FAS的脱甲基化作用。将测试对MTX及其类似物的抑制作用，以确认我们提出的作用机理。我们将使用SS白血病细胞来验证这些体外研究，并确定对SS细胞生长和存活的净影响。在AIM 2中，我们将探索在FAS/FASL-LOW MF/SS系中调节抑制肿瘤抑制基因沉默的机制。首先，我们将DNMT1抑制作用与5-Aza的影响进行比较，因为我们对DNMT1敲低的初步数据表明它是基因甲基化的关键介体。然后，我们将使用芯片和共免疫沉淀来识别与FAS和FAS结合的DNMT1辅因子（例如STAT3，RELA，HDAC1）。最后，我们将确定抑制这些DNMT1辅因子和上游DNMT1调节剂对基因甲基化的影响。这些体外研究将使用SS白血病细胞进行验证。靶向基因特异性辅助因子可能允许对DNA甲基化的基因特异性操纵。在AIM 3中，为了在临床上验证我们的体外和前体内发现，我们将使用Q-游戏通过MTX对MF/SS患者样本进行定量监测FAS和FASL基因去甲基化，并确定对基因表达和临床结果的影响。我们将使用MTX，因为它是一种标准的MF/SS治疗，我们发现它是一种有效的脱甲基剂。我们还将评估MTX与其他疗法（例如IFNA，HDAC抑制剂，UVB）结合使用的临床作用，从而通过不同于MTX的机制进一步增强FAS或FASL。总体而言，这些目标将确定并验证用于MF/SS治疗的新型靶基因，提供调节和监测其在临床环境中表达的策略，并确定它们对临床结果的影响。这些高度转化的目标的成功完成取决于我们广泛收集的MF/SS标本和专业知识，其专门针对该建议量身定制。