Lipid addiction via Fatty Acid Synthase activates PI3K signaling in B cell non-Hodgkin’s Lymphoma (bNHL) [Affymetrix]. Homo sapiens

Dysregulated lipid metabolism is an oncogenic mechanism that leads to increased de novo fatty acid biosynthesis via elevated expression of fatty acid synthase (FASN) in cancer. FASN catalyzes biosynthesis of (C16) palmitic acid by using Acetyl CoA, malonyl CoA and NADPH as substrates. Palmitic acid and its derivatives participate as signaling lipids andinvolved in the regulation of receptor functions associated with many biological processes. and furtherThis includes contribute formation of cell structures, and all these processes which are essential for both normal and malignant cell growth and proliferation. In this study, we investigated the biological impact of FASN inhibition in B cell Non Hodgkin Lymphoma (bNHL) cells using FASN inhibitors Cerulenin and other novel agents (TVB3166, TVB3567). Inhibition of FASN resulted in a dose dependent increase in cell death and apoptosis in the bNHL and primary tumor cells. Transcriptomic and comprehensive systems biology analysis of cerulenin, TVB3166 or TVB3567 treated bNHL cells revealed conserved activation of (TNF) immune and PI3K signaling, and inhibition of cell cycle pathways as prominent responses to FASN inhibition. Cell fractionation and western blot analysis of cCerulenin treated cells showed translocation of TNF receptor adaptor proteins (TRAF2 and RIP), docking of AKT and PI3K/p110 subunits to cell membrane and activation of PI3K/AKT pathways by phosphorylation in SUDHL2 and SUDHL4 bNHL cells, while these mechanisms were constitutively active in cerulenin treated SUDHL10 bNHL cells. Disruption of lipid raft formation by methyl β cyclodextrin inhibited PI3K signaling and downregulated FASN expression in SUDHL10 cells. Subsequently, we observed that combination of FASN (cerulenin) and PI3K (Buparlisib) inhibitors resulted in synergistic cell death in bNHL cell lines and increased apoptosis accompanied with down-regulation of FASN expression in bNHL cell lines and primary cells, implicating the importance of PI3K in the regulation of lipid metabolism. Further mass spectrometric evaluation of cerulenin and Buparlisib treated DLBCL cells fed with C14C glucose, synergistically decreased the biosynthesis of palmitic acid and its derivatives compared to single agent treatments. Our conclusions from these investigations suggest that simultaneous blocking of PI3K and FASN is likely to exert stronger inhibition of lipid metabolism and enhance cell death in lymphoma. Overall design: SUDHL10 and Raji cells were treated with Cerulenin for 48 hours, RNA was isolated using RNeasy Minikit (Qiagen), following instructions recommended supplied by the manufacturer. Purity and the yield of isolated RNA was determined using Bioanalyzer. These experiments were performed in biological triplicates. Microarray experiment was performed on Affy Human Gene Chip 2.0, at UMASS Medical School Genomic Core facility. Data for lines were background adjusted and quantile normalized using RMAExpress. Statistically relevant genes were determined by applying LIMMA with a FDR < 0.05 that resulted in 1092 significant genes for the Raji experimental group and 1600 significant genes for the SUDHL10 experimental group.