Flim-FRET Imaging of Plitidepsin-eEF1A2 Complexes in Live Cancer Cells using the Phasor Approach

Eukaryotic Elongation Factor 1A2 (eEF1A2) is an isoform of the alpha subunit of eEF1A complex. Differently from the A1 isoform, the expression of eEF1A2 is restricted to brain, heart and skeletal muscle. eEF1A2 is overexpressed in tumors, including multiple myeloma (MM), prostate, pancreas, and ovarian, and has also an oncogenic behavior favoring tumor cell proliferation while inhibiting apoptosis. Thus, eEF1A2 is an interesting target for cancer treatment. Plitidepsin (Aplidin®, APL) is an antitumor agent, originally isolated from the marine tunicate Aplidium albicans, which is being tested in MM patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone. HeLa-WT and HeLa-APLR (resistant to Aplidin®) cell lines have been previously described. HeLa-APLR present lower eEF1A2 levels than HeLa-WT cells. Herein we reveal the interaction of Plitidepsin with eEF1A2 using a fluorescent Aplidin® derivative, APL-DMAC as a FRET donor. We stably transfected HeLa-WT and HeLA-APLR cells with plasmids encoding GFP-tagged eEF1A2 (FRET acceptor), and selected clones expressing physiological levels of the fusion proteins. We applied the FLIM-phasor approach [Redford and Clegg 2005; Digman et al. 2008], to localize and quantify APL-DMAC molecular species and to measure FRET efficiencies, DMAC - GFP, thus identifying different eEF1A2-APL complexes [Losada et. al. 2016].