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

“无功能”lyso-PAF（一种无活性的血小板激活因子）在 Nras 突变黑色素瘤中的重要性

• 批准号: 10362359
• 负责人: Jing Chen
• 金额: $ 40.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-23 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10362359
• 关键词: 3-hydroxy-3-methylglutaryl-coenzyme A; Acetates; Acetoacetates; Address; Attenuated; BRAF gene; Binding; Biochemistry; Biological; Biological Process; Bypass; Cell Line; Cell Proliferation; Cells; Chondroitin; Chondroitin Sulfate A; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Drug Screening; Enzymes; Exhibits; Human; In Vitro; Inflammation; Ketone Bodies; Knock-out; Leukocytes; Libraries; Lyase; MAP Kinase Gene; MAP2K1 gene; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Melanoma Cell; Messenger RNA; Metabolic; Metabolism; Molecular; Mutation; Oncogenes; PAK2 kinase; PTEN gene; Pathway interactions; Phospholipase; Phospholipid Degradation Pathway; Phospholipids; Phosphorylation; Phosphotransferases; Physiological; Platelet Activating Factor; Platelet Activating Factor Degradation; Production; Proto-Oncogene Proteins c-akt; Research; Role; Screening Result; Series; Signal Transduction; Structure; Succimer; T-Lymphocyte; The Cancer Genome Atlas; Vascular Permeabilities; Warburg Effect; cancer cell; casein kinase II; design; dietary supplements; extracellular; in vivo; inhibitor; ketogentic; knock-down; lyso-PAF; macrophage; mast cell; melanoma; mutant; patient derived xenograft model; prostate cancer cell; recruit; small hairpin RNA; small molecule; therapeutic target; tumor; tumor growth

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.

项目概要/摘要 尽管许多人类癌症具有相似的代谢改变，包括瓦尔堡效应，但它仍然存在 我们还不清楚肿瘤的发展是否需要癌基因特异性的代谢改变。 磷脂酶 A2G7 (PLA2G7) 作为黑色素瘤细胞中 Nras Q61K/R 突变体的“合成致死”伴侣， 对于表达突变体的黑色素瘤细胞的细胞增殖和肿瘤生长潜力具有选择性重要 Nras，但不在表达 BRAF V600E（又名血小板激活因子乙酰水解酶）的细胞中。 (PAF-AH)) 是一种由巨噬细胞、T 细胞和肥大细胞等白细胞产生的分泌酶， 催化磷脂血小板激活因子 (PAF) 的降解并产生无生物活性的物质 磷脂产物 Lyso-PAF，从机制上阻断 PAF 诱导的炎症和血管通透性。 我们发现 PLA2G7 的细胞内信号传导功能令人惊讶，敲低 PLA2G7 会导致细胞内信号传导功能下降。 细胞中 Raf-1 的 S338 磷酸化对于 Raf-1 激活至关重要，因此对于突变体至关重要 Nras 依赖性 MAPK 激活，但对于绕过 Raf-1 的 BRAF V600E 激活 MAPK 来说是可有可无的。 这解释了 PLA2G7 仅在表达突变型 Nras 的细胞中具有选择性重要性。 PAF 的生物非活性形式被认为“无功能”，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，可能在催化作用中 裂，通过稳定 ATP 结合导致 PAK2 激酶活性增强。 “无功能”Lyso-PAF 具有细胞内信号传导作用，对突变型 Nras 具有选择性重要作用 通过促进 Raf-1 的 PAK2 依赖性 S338 磷酸化来进行转化，PLA2G7 代表 我们已经鉴定并选择性地治疗表达突变 Nras 的黑色素瘤细胞的替代治疗靶点。 验证了化合物 Succimer 作为选择性且有效的 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力，该突变在表达 BRAF V600E 的细胞中被“绕过”，使用 (2) 探索分子结构 Lyso-PAF 通过直接结合 PAK2 催化裂隙对 PAK2-Raf-1 轴做出贡献的机制 从而稳定 ATP 结合 (3) 评估 PLA2G7 作为选择性的替代靶标。 减弱表达突变型 Nras 的黑色素瘤细胞的体外增殖和肿瘤生长潜力，以及 分别使用我们新鉴定的 PLA2G7 体内黑色素瘤患者来源的异种移植 (PDX) 模型 抑制剂、琥珀酸，并阐明进一步结构激活的潜在结构机制 关系（SAR）研究。