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)： 皮肤癌是人类最常见的肿瘤类型，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)，通过DNA甲基化表现出肿瘤抑制基因的沉默。在这项研究中，我们建议集中在两种类型的CTCL中选择的肿瘤抑制基因的表观遗传沉默：真菌病(MF)和Sezary综合征(SS)。我们报道了死亡受体-配体配对Fas和FASL在MF/SS中只有微弱的表达。我们观察到Fas的表达受转录调控，并且Fas的表达与启动子甲基化之间存在负相关。我们发现，MF/SS普遍富含DNMT1(最可能导致表观基因沉默的DNA甲基转移酶)，并且DNMT1基因敲除逆转了Fas启动子的甲基化。我们还发现，叶酸拮抗剂如甲氨蝶呤(MTX)可以通过耗尽DNMT的主要甲基供体S-腺苷蛋氨酸(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技术，我们建立了一种从皮肤细胞分离的甲基化和表达的定量基因分析方法(Q-GAME)。使用标准的皮肤活检，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，我们假设类似于Fas的情况，DNA甲基化将至少部分解释FASL表达下降的原因。此外，MTX作为去甲基剂将逆转这种基因抑制，并通过促进Fas途径的激活而诱导MF/SS细胞凋亡。在目标1中，我们将利用反映最常见的MF/SS临床表型的Fas/FASL-low MF/SS株(HH、SZ4)来优化5-氮杂、地西他滨、MTX、MTX类似物和HDAC抑制剂对MF/SS中Fas和FASL的去甲基化作用。我们将测试外源SAM对MTX及其类似物的抑制作用，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。目的2探讨Fas/FASL-low MF/SS细胞中抑癌基因沉默的调控机制。首先，我们将比较DNMT1抑制和5-aza的影响，因为我们对DNMT1基因敲除的初步数据表明，它是基因甲基化的关键媒介。然后，我们将使用芯片和免疫共沉淀来鉴定与Fas和FASL结合的DNMT1辅助因子(如STAT3、RELA、HDAC1)。最后，我们将确定这些DNMT1辅因子和上游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。靶向基因特异性辅因子可能允许相对基因特异性的DNA甲基化操作。在目的3中，为了在临床上验证我们的体外和体外研究结果，我们将使用Q-GAME定量监测MTX原位检测MF/SS患者样本中Fas和FASL基因去甲基化，并确定其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疗法，我们已经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(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。总而言之，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它们的表达的策略，并确定它们对临床结果的影响。成功完成这些高度翻译的目标依赖于我们广泛收集的MF/SS样本和专门为本提案量身定做的方法的专业知识。