Abstract 3515: Down the rabbit hole with structural effects of FK866 in primary CLL B cells

Abstract

Abstract FK866 (Daporinad or APO866) is a specific inhibitor of NAMPT (nicotinamide phosphoribosyltransferase), a rate limiting enzyme involved in NAD generation. It is a prospective cancer treatment utilized in several clinical trials and has generated much interest. It has been previously demonstrated that FK866 (10nM) induces cell death in CLL patient derived B-cells (CLL-B cells) by early depletion of NAD (1 day after treatment) followed by a subsequent decrease in cellular ATP levels (2 days after treatment). Upon further study we observed that CLL-B cells have structural changes after treatment with FK866 for either 6 or 18-20 hours via electron microscopy. There is a presence of bound structures as well as nuclear invaginations. These structures are only observed in cells that were treated with FK866 and not with any other drugs used (fludarabine, bendamustine, chlorambucil) nor with the DMSO or untreated controls. We stained f-actin (a cytoskeletal protein) with FITC labelled phalloidin in CLL-B cells with and without FK866 treatment and then viewed with confocal microscopy to exclude the chance that these structures were artifacts. Changes in the f-actin were only observed in the FK866 treated cells. Phalloidin staining was performed at 6 and 18 hours. We observed holes in the f-actin framework that passed through the entire cell and in some instances, went through the nuclei too. A comparison of the phalloidin staining was performed in non-CLL based cell lines that were known to be sensitive to treatment with FK866 though structural differences had not been observed at the same time points (6 and 18-20 hours). Two Ewing's sarcoma cell lines and two small cell lung cancer cell lines were used. All four cell lines showed no changes in the f-actin. Labeled dextran beads were observed via confocal microscopy to be taken up more readily by the FK866 treated CLL -B cells as compared to the vehicle control. This would imply that the treated cells are actively removing nutrients from the environment. A lysosome stain, LysoTracker, was employed to determine if there was any involvement of lysosomes in this phenomenon. No change was observed between the untreated, vehicle control and the FK866 treated CLL patient derived B-cells. We were curious about the mitochondrial dynamics in the FK866 treated cells so they were stained with MitoTracker. We were able to detect mitochondria in the cytoplasm of the cells in all treatments but the FK866 treated cells displayed a more distinct punctate pattern. Interestingly, we also observed the mitochondria were appearing at or near the holes in the cytoskeleton. As the FK866 treated cells are deprived of their NAD, the CLL cells may be undergoing macropinocytosis, an mTOR mediated phenomenon used by cells to extract nutrients from the environment. We have shown that FK866 causes an increase in 4EBP1 phosphorylation compared to controls. In conclusion, FK866 may mediate macropinocytosis in CLL cells in an mTOR dependent manner. Citation Format: Cheryl Peltier, Eileen McMillan-Ward, Elizabeth Henson, Nabanita Chatterje, Donna Hewitt, Danielle Desautels, Matthieu Bourrier, Cornelia Mutz, Iris Gehrke, Eric Bouchard, Heinrich Kovar, Shantanu Banerji, Spencer Gibson, Sara Israels, Versha Banerji. Down the rabbit hole with structural effects of FK866 in primary CLL B cells [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 3515.