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

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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.