Novel role of melanin-carbonyls in progression of NRAS mutant 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)具有相当于一个紫外线光子的三重态能量。MC产生了大约50%的 总环丁烷嘧啶二聚体(CPDS)，紫外线特异的DNA加合物，在完全没有紫外线的情况下。这个 MECH期间和之后合谋的化学事件，特别是MC的作用仍不清楚 黑色素瘤。我们建议鉴定和表征黑色素-羰基-DNA(MCD)和黑色素- 黑色素瘤进展过程中的羰基蛋白加合物及其对当前治疗药物的耐药性 接近了。其理论基础是，一氧化氮合酶大多是过度活跃的，并与黑色素瘤的不良预后有关 而色素沉着在黑色素瘤中的作用仍存在争议。我们假设高一氧化氮合酶诱导 MECH，导致慢性MC产生，从而失调黑素细胞生理，促进黑色素瘤 通过“MCD和MCP加合物”或其他未知机制进展和治疗耐药。这个 假说与黑色素瘤中的三个独特的手臂有关。首先是色素沉着。黑色素通过以下途径促进黑色素瘤 激活缺氧诱导因子-1α。相反，黑色素的物理存在通过弹性调节来抑制转移， 但仍需接受严格的考验。第二个是机械加工，它被认为是黑色素瘤的中心轴。 然而，生物学从未在实验中进行过测试。第三是黑色素瘤中过度活跃的一氧化氮合酶 病情严重，预后不良，且仅与黑色素有关。将这三个分支与 通过MCD和MCP加合物的作用机制，我们将在黑色素瘤中识别一个引导的正环 通过一氧化氮合酶、机械和MC在以下两个目标：在目标1，我们假设由于过度活跃的一氧化氮合酶， 慢性MC生成导致MCD和MCP加合物。使用点击化学标记细胞黑色素- 黑色素单体的类似物(CCA)(已制定的方案)，我们将通过化学或UV和 用高效液相色谱-串联质谱仪纯化和鉴定CCA标记的基因组和蛋白质组分 增殖性黑色素瘤信号转导中的CPD热点与富含赖氨酸和半胱氨酸的蛋白 由于-NH2和-SH基团是AIM 2中羰基加成的主要靶点，我们假设MCD和 MCP加合物促进黑色素瘤进展和治疗抵抗。我们使患者产生过敏反应 黑色素瘤细胞通过清除羰基和抑制一氧化氮合酶进行靶向治疗。分析范围： 黑素细胞、发育不良痣和黑色素瘤，以及自建的有色性和非有色性黑色素瘤患者 衍生的黑色素瘤细胞将确定这种敏化背后的可能机制，在我们的初步研究中 观察到，与BRAF或NRAS等驱动突变无关。AIM 1的综合分析 和2将确定MC介导的、对黑色素瘤进展和治疗耐药性的非经典调节 可能被用作对抗黑色素瘤的一种新的脆弱性。

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.