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

项目总结/摘要 尽管许多人类癌症都有类似的代谢改变，包括瓦尔堡效应，但它仍然存在。 目前尚不清楚肿瘤发生是否需要癌基因特异性代谢改变。我们确定 磷脂酶A2G7（PLA2G7）作为黑色素瘤细胞中Nras Q61 K/R突变体的"合成致死"伴侣， 对于表达突变体黑色素瘤细胞的细胞增殖和肿瘤生长潜力是选择性重要的 Nras，但不表达BRAF V600 E的细胞。PLA2G7（a.k.a.血小板活化因子乙酰水解酶 （PAF-AH））是一种由白细胞（包括巨噬细胞、T细胞和肥大细胞）产生的分泌酶， 催化磷脂血小板活化因子（PAF）的降解和生物学上无活性的 磷脂产物Lyso-PAF，阻断PAF诱导的炎症和血管通透性。机械地说， 我们发现了PLA2G7令人惊讶的细胞内信号传导功能。PLA2G7的敲低导致降低的 细胞中Raf-1的S338磷酸化，这对于Raf-1的激活至关重要，因此对于突变体Raf-1的表达至关重要。 Nras依赖的MAPK激活，但BRAF V600 E绕过Raf-1激活MAPK。 这解释了PLA2G7仅在突变型Nras表达细胞中的选择重要性。此外，Lyso-PAF a PAF的生物学非活性形式已被认为是"无功能的"，可能有助于p21激活 Raf-1的激酶2（PAK2）依赖性S338磷酸化，通过直接结合PAK2，可能在催化 裂缝，导致增强PAK2激酶活性稳定ATP结合。因此，我们假设， "无功能" Lyso-PAF具有细胞内和信号传导作用，其对于突变型Nras选择性地重要。 通过促进Raf-1的PAK2依赖性S338磷酸化转化，PLA2G7代表了一种新的转化途径。 选择性治疗表达突变型Nras的黑色素瘤细胞的替代治疗靶点。我们已经确定， 验证了化合物琥珀酰亚胺作为选择性和有效的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 表达突变型Nras的黑素瘤细胞的潜力，其在表达BRAF V600 E的细胞中被"绕过"，使用 不同的人黑色素瘤细胞系和"同基因"细胞系对。(2)为了探索分子和结构 Lyso-PAF通过直接结合PAK2催化裂缝而促进PAK2-Raf-1轴的机制 从而稳定ATP结合。(3)为了评估PLA2G7作为选择性地治疗的替代靶标， 在体外减弱表达突变型Nras的黑色素瘤细胞的增殖和肿瘤生长潜力， 分别使用我们新鉴定的PLA2G7， 抑制剂，琥珀酸，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.