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Background: Synthetic alkyl-phospholipid analogs (APLs), such as Edelfosine and Perifosine, are a class of anti-tumor agents that target cell membranes to induce growth arrest and apoptosis. In addition, APLs enhance the cytotoxic effect of radiation in preclinical models making these compounds attractive candidates as radiosensitizers. We previously found that different mechanisms determine ALP accumulation and consequent apoptotic toxicity in lymphoma versus carcinoma cells. Yet, despite decades of research, it is still unclear how ALPs are transported across the plasma membrane and how they affect, either positively or negatively, key intracellular signaling pathways to alter tumor cell behavior. Here we examine (i) the possible role of lipid flippases in mediating transbilayer transport of ALP-like lysophospholipids (notably lysoPC and lysoPE), and (ii) how Edelfosine affects gene expression in human breast cancer cells. Methods: The uptake of fluorescent lysophospholipids (TopFluor-LPC and -LPE) was monitored over time in wild-type HAP1 and CDC50A -/- cells using single-cell live-imaging. For RNA-seq analyses, MDA-MB-231 and Hs5788T triple-negative breast cancer (TNBC) cells were used. Results: Ad (i). Knockout of the flippase subunit CDC50a slows and attenuates the uptake of fluorescent lysoLPC and lyoPE. This suggests that CDC50a mediates, at least in large part, the active uptake of cytotoxic ALPs, similarly to natural lysophophoslipids. Ad (ii). Whole genome RNA-seq analysis reveals that Edelfosine rapidly induces a unique gene expression program in breast cancer cells. When compared to bioactive lysoPA (LPA), Edelfosine upregulated about 25 genes after 4 hrs, whereas LPA upregulated about 300 genes under the same conditions. Strikingly, the Edelfosine- and LPA-induced early genes showed significant overlap, including transcription factors (FOS, EGR1, EGR3), CSF2 and the EGFR/HER2 inhibitor ERRFI1 (MIG6). Increased MIG6 expression by Edelfosine was confirmed by Western blot. Basal expression of MIG6 was high in MDA-MB-231 cells, but undetectable in Hs5788T TNBC cells. The latter cells were more sensitive than MDA-MB-231 cells to Edelfosine-induced apoptosis. Conclusions: We conclude that Edelfosine “hijacks” LPA receptor signaling pathways at early time points. Edelfosine-induced upregulation of the anti-proliferative gene MIG6, a negative regulator of the EGFR family, is of particular interest. Since MIG6 is induced by the G-actin-MAL transcriptional pathway, these results suggest that Edelfosine affects tumor cell behavior in part by influencing F-actin polymerization, a new scenario that warrants further studies. Citation Format: Elisabetta Argenzio, Wouter H. Moolenaar, Marcel Verheij. New insights into the mode of action of cytotoxic alkyl-phospholipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5454.