Novel role of melanin-carbonyls in progression of NRAS mutant melanoma

黑色素羰基在 NRAS 突变黑色素瘤进展中的新作用

• 批准号: 10648486
• 负责人: Sanjay Premi
• 金额: $ 46.34万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-22 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648486
• 关键词: Affinity Chromatography; BRAF gene; Biology; Carboxylic Acids; Cells; Chemicals; Chemistry; Chromatin; Chronic; DNA; DNA Adduction; DNA Adducts; Darkness; Data; Dysplastic Nevus; Elasticity; Environmental Risk Factor; Enzymes; Event; Fibroblasts; Generations; Genetic Transcription; Genome; Genomics; Goals; High Pressure Liquid Chromatography; Homeostasis; Hour; Human; Hyperactivity; Indoles; Label; Lysine; MAP Kinase Gene; Mediating; Melanins; Melanoma Cell; Mutation; Neoplasm Metastasis; Nitric Oxide; Nitric Oxide Synthase; Pathogenesis; Pathway interactions; Patients; Periodicity; Photochemistry; Photons; Physiological; Physiology; Pigmentation physiologic function; Pigments; Probability; Production; Prognosis; Proteins; Proteomics; Protocols documentation; Pyrimidine Dimers; Regulation; Resistance; Role; Side; Signal Transduction; Skin; Skin Cancer; Source; Sulfhydryl Compounds; Testing; Text; Therapeutic; Transcriptional Regulation; Triplet Multiple Birth; UV Radiation Exposure; Ultraviolet Rays; adduct; albino mouse; analog; arm; carcinogenesis; carcinogenicity; cell type; comparative; cysteine rich protein; driver mutation; glycogen synthase kinase 3 beta; keratinocyte; melanocyte; melanoma; monomer; mutant; novel; novel therapeutics; p38 Mitogen Activated Protein Kinase; response; skin cancer prevention; targeted treatment; therapy resistant; triplet state

We discovered melanin-chemiexcitation (MeCh) where Nitric oxide synthase (NOS) oxidizes melanin into Melanin-carbonyls (MCs) that have triplet energy equivalent to a UV photon. The MCs generate ~50% of the total cyclobutane pyrimidine dimers (CPDs), UV specific DNA adducts, in complete absence of UV. The chemical events that conspire during and after MeCh, and specifically the role of MCs remain unknown in melanoma. We propose to identify and characterize the role of melanin-carbonyl-DNA (MCD) and melanin- carbonyl-protein (MCP) adducts in melanoma progression and resistance against current therapeutic approaches. The rationale is that NOS is mostly hyperactive, and associated with poor prognosis of melanoma patients, and the role of pigmentation is still controversial in melanoma. We hypothesize that high NOS induces MeCh, leading to chronic MC production which dysregulates melanocytic physiology, and promotes melanoma progression and therapy resistance through “MCD and MCP adducts” or other unknown mechanisms. The hypothesis relates to three unique arms in melanoma. First is pigmentation. Melanin promotes melanoma by activating HIF-1α. Contrarily, melanin’s physical existence inhibits metastasis through elasticity modulations, but remains to be tested stringently. Second is MeCh which is proposed to be a central axis in melanoma biology though never tested experimentally. Third is the NOS enzyme, hyperactive in melanoma, correlates strongly with poor prognosis, and exclusively associated with melanin. Integrating these three arms with the mechanisms operated by MCD and MCP adducts, we will identify a positive loop in melanoma that is steered by NOS, MeCh, and MCs in the following two aims: In aim 1, we hypothesize that owing to hyperactive NOS, the chronic MC production leads MCD and MCP adducts. Labeling the cellular melanin using click-chemistry- analogs (CCA) of melanin-monomers (protocols established), we will induce MeCh chemically or with UV and purify and characterize CCA labeled genomic and proteomic fraction using HPLC-MS/MS. Focus will be on CPD hotspots in the genome and Lysine and Cysteine rich proteins from proliferative melanoma signaling since -NH2 and -SH groups are prime targets for carbonyl adduction In aim 2, we hypothesize that MCD and MCP adducts promote melanoma progression and therapy resistance. We sensitized patient derived melanoma cells to targeted therapies by scavenging carbonyls and inhibiting NOS. Analyses across melanocytes, dysplastic nevi, and melanoma, and the self-established pigmented and non-pigmented, patient derived melanoma cells will identify probable mechanisms behind this sensitization which in our preliminary observations, was independent of the driver mutations like BRAF or NRAS. An integrated analysis of Aim 1 and 2 will identify MC-mediated, non-classical regulation of melanoma progression and therapy resistance that could be used as a novel vulnerability against melanoma.

我们发现了黑色素化学激发（MeCh），其中一氧化氮合酶（NOS）将黑色素氧化成 黑色素-羰基（MC）具有相当于UV光子的三重态能量。MC产生约50%的 总环丁烷嘧啶二聚体（CPD），UV特异性DNA加合物，完全不存在UV。的 在MeCh期间和之后密谋的化学事件，特别是MC的作用仍然未知， 黑素瘤我们建议确定和表征黑色素羰基DNA（MCD）和黑色素- 羰基蛋白（MCP）加合物在黑色素瘤进展和对当前治疗药物的耐药性中的作用 接近。其基本原理是NOS大多是过度活跃的，并且与黑色素瘤的预后不良相关 患者，色素沉着的作用仍然是有争议的黑色素瘤。我们假设高NOS诱导 MeCh，导致慢性MC产生，其失调黑素细胞生理学，并促进黑色素瘤 通过“MCD和MCP加合物”或其他未知机制导致疾病进展和治疗抗性。的 假设涉及黑素瘤中的三个独特臂。首先是色素沉着。黑色素促进黑色素瘤， 激活HIF-1α。首先，黑色素的物理存在通过弹性调节抑制转移， 但仍有待严格检验。其次是MeCh，它被认为是黑素瘤的中心轴 生物学虽然从未经过实验测试。第三是NOS酶，在黑色素瘤中过度活跃，与 预后差，且仅与黑色素相关。将这三个臂与 通过MCD和MCP加合物操作的机制，我们将确定黑色素瘤中的一个正循环， 通过NOS、MeCh和MC在以下两个目的中：在目的1中，我们假设由于NOS过度活跃， 慢性MC产生导致MCD和MCP加合物。用点击化学标记细胞黑色素 黑色素单体的类似物（CCA）（建立的方案），我们将化学诱导MeCh或用UV诱导， 使用HPLC-MS/MS纯化和表征CCA标记的基因组和蛋白质组部分。 基因组中的CPD热点以及来自增殖性黑素瘤信号传导的富含赖氨酸和半胱氨酸的蛋白质 由于-NH 2和-SH基团是羰基加合的主要目标。在目标2中，我们假设MCD和 MCP加合物促进黑色素瘤进展和治疗抗性。我们从病人身上 黑色素瘤细胞的靶向治疗，通过清除羰基和抑制NOS。分析 黑素细胞、发育不良痣和黑色素瘤，以及自身确定的色素沉着和非色素沉着患者 衍生的黑色素瘤细胞将确定这种致敏背后的可能机制，在我们的初步研究中， 观察到，这与BRAF或NRAS等驱动突变无关。对目标1的综合分析 和2将鉴定MC介导的黑色素瘤进展和治疗抗性的非经典调节， 可以作为一种新的针对黑色素瘤的弱点。