Signaling importance of ‘functionless’ lyso-PAF, an inactive form of platelet activating factor, in melanoma with mutant Nras

项目摘要/摘要 尽管许多人类癌症都有相似的代谢变化，包括华宝效应，但它仍然 目前尚不清楚肿瘤发生是否需要癌基因特异性代谢改变。我们确认了 磷脂酶A2G7(PLA2G7)作为NRAS Q61K/R突变体在黑色素瘤细胞中的合成致死伙伴 对表达突变的黑色素瘤细胞的细胞增殖和肿瘤生长潜能具有选择性的重要作用 NRAS，但表达BRAF V600E的细胞不表达。PLA2G7(又名血小板活化因子乙酰水解酶 (PAF-AH)是一种由包括巨噬细胞、T细胞和肥大细胞在内的白细胞产生的分泌酶，它 催化磷脂血小板活化因子(PAF)的降解和生物活性物质的产生 磷脂产物lyso-PAF，阻断PAF引起的炎症和血管通透性。从机械上讲， 我们发现PLA2G7具有惊人的细胞内信号功能。PLA2G7基因敲除导致细胞数量减少 S338 Raf-1在细胞中的磷酸化，这对Raf-1的激活至关重要，因此对突变体是必不可少的 依赖于NRAS的MAPK激活，但对于绕过Raf-1的BRAF V600E激活MAPK是必不可少的。 这解释了PLA2G7只在表达NRAS的突变细胞中具有选择性重要性。此外，Lyso-PAF，a 生物活性失活的PAF被认为是无功能的，可能与p21激活有关 依赖于激酶2(PAK2)的S338通过直接与PAK2结合而使Raf-1磷酸化，可能是在催化 裂解，通过稳定ATP结合而导致PAK2激酶活性增强。因此，我们假设 “无功能”的lyso-PAF对突变型NRAS具有选择性重要的细胞内和信号转导作用 通过促进PAK2依赖的S338磷酸化Raf-1进行转化，而PLA2G7代表一种 选择性治疗表达突变NRAS的黑色素瘤细胞的替代治疗靶点。我们已经确定并 验证了一种化合物Succmer作为一种选择性和有效的PLA2G7抑制剂。提出了三个具体目标： (1)确定PLA2G7-lyso-PAF轴在增殖和肿瘤生长中的选择性重要性 表达突变NRAS的黑色素瘤细胞的潜能，该突变NRAS在表达BRAF V600E的细胞中被绕过 不同的人类黑色素瘤细胞系和“等基因”细胞系对。(2)探索分子和结构 Lyso-PAF通过直接结合PAK2催化裂解参与PAK2-Raf-1轴的机制 从而稳定三磷酸腺苷结合。(3)评价PLA2G7作为选择性靶点 体外抑制表达突变型NRAS的黑色素瘤细胞的增殖和肿瘤生长能力 使用我们新发现的PLA2G7分别在体内建立患者来源的异种移植(PDX)黑色素瘤模型 抑制剂，蔗糖分子，并阐明进一步结构活化的潜在结构机制 关系(SAR)研究。

Project Summary/Abstract Although many human cancers share similar metabolic alterations, including the Warburg effect, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. We identified phospholipase A2G7 (PLA2G7) as a “synthetic lethal” partner of Nras Q61K/R mutants in melanoma cells, which is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant Nras, but not in cells expressing BRAF V600E. PLA2G7 (a.k.a. platelet-activating factor acetylhydrolase (PAF-AH)) is a secreted enzyme produced by leukocytes including macrophages, T cells, and mast cells, which catalyzes the degradation of phospholipid platelet activating factor (PAF) and production of a biologically inactive phospholipid product Lyso-PAF, blocking PAF-induced inflammation and vascular permeability. Mechanistically, we found a surprising intracellular signaling function of PLA2G7. Knockdown of PLA2G7 results in decreased S338 phosphorylation of Raf-1 in cells, which is crucial for Raf-1 activation and consequently essential for mutant Nras-dependent MAPK activation, but dispensable for MAPK activation by BRAF V600E, which bypasses Raf-1. This explains the selective importance of PLA2G7 only in mutant Nras-expressing cells. Moreover, Lyso-PAF, a biological inactive form of PAF that has been suggested to be “functionless”, may contribute to p21-activated kinase 2 (PAK2)-dependent S338 phosphorylation of Raf-1, through direct binding to PAK2, likely in the catalytic cleft, leading to enhanced PAK2 kinase activity by stabilizing ATP binding. Thus, we hypothesize that “functionless” Lyso-PAF has an intracellular and signaling role that is selectively important for mutant Nras transformation by contributing to PAK2-dependent S338 phosphorylation of Raf-1, and PLA2G7 represents an alternative therapeutic target to selectively treat melanoma cells expressing mutant Nras. We have identified and validated a compound Succimer as a selective and potent PLA2G7 inhibitor. Three specific aims are proposed: (1) To determine the selective importance of the PLA2G7-Lyso-PAF axis in the proliferative and tumor growth potential of melanoma cells expressing mutant Nras, which is “bypassed” in cells expressing BRAF V600E, using diverse human melanoma cell lines and “isogenic” cell line pairs. (2) To explore the molecular and structural mechanisms by which Lyso-PAF contributes to PAK2-Raf-1 axis through directly binding to PAK2 catalytic cleft and consequently stabilizing ATP binding. (3) To evaluate PLA2G7 as an alternative target to selectively attenuate proliferative and tumor growth potential of mutant Nras-expressing melanoma cells in vitro, and in patient-derived xenograft (PDX) models of melanoma in vivo, respectively, using our newly identified PLA2G7 inhibitor, Succimer, and elucidate the underlying structural mechanism for further structure-activation relationship (SAR) studies.