Abstract 4542: Targeting KRAS4b plasma membrane localization in cells

Abstract

Abstract All RAS isoforms, including the oncogenic variants, must localize to the plasma membrane (PM) to initiate downstream signaling. H-RAS, N-RAS and the KRAS splice variant KRAS4a all transit to the PM through the Golgi, and rely on cycles of reversible palmitoylation on one or two cysteines in the hyper variable region (HVR) to regulate exchange on and off the PM. In contrast, the KRAS4b HVR contains a series of positively charged lysine residues, which target KRAS4b to regions of the PM rich in negatively charged phospholipids. Though the details have not been fully worked out, evidence suggests that KRAS4b trafficking to and from the PM is mediated by chaperones such as PDE6δ and smgGDS. Since its path to the PM is distinct, and since membrane localization is a requirement for downstream signaling, perturbing the spatial organization of KRAS4b may uncover new molecular targets for KRAS-dependent cancers. To this end, we are developing a high content assay and image analysis pipeline to screen for molecules that disrupt the PM localization of KRAS4b. The assay is based on a doxycycline-inducible GFP-KRASG12V expressing HeLa cell line, and uses confocal microcopy to automatically image cells at sub-micron resolution in multi-well glass bottom plates. To specifically counterstain the PM compartment and nucleus, we use fluorescently labelled Concanavalin A (ConA) and Hoechst (a DNA specific dye) respectively. An image analysis pipeline was developed to segment and quantitate the levels of GFP-KRAS-G12V in the PM compartment. The pipeline includes a supervised machine learning algorithm to convert the grey-scale intensity values of the PM channel (ConA) to the probability that the ConA signal belongs to the PM. These probability values are subsequently used to segment the cell boundary and PM using a graph-cut method and a thresholding technique. We use the nuclear channel to eliminate any segmented boundaries that do not belong to a cell. Calculated Z’ factors using the mean values per well of membrane localized GFP-KRAS-G12V in doxycycline treated and untreated cells show acceptable values (0.7) for high content screening (HCS). Furthermore, as a secondary assay to evaluate hits from the primary HCS, we are developing fluorescence correlation spectroscopy (FCS) techniques to measure the temporal dynamics of GFP-KRAS-G12V membrane localization in live cells. In preliminary FCS experiments, we tested farnesyl thiosalicylic acid (FTS), a molecule reported to dissociate RAS molecules from the PM. We observed increased lateral diffusion (2.38±0.55 μm2/s) in drug treated cells (15-30 mins after treatment) compared to untreated cells (0.85±0.12 μm2/s) consistent with values previously reported using fluorescence recovery after photobleaching measurements (Niv H. et al. J. Biol. Chem. 1999; 274:1606-1613). Taken together, we demonstrate that our screening platform can be used to identify small molecule disruptors of oncogenic KRAS4b spatial and temporal localization in the PM of cells. Citation Format: Alla Brafman, Prabhakar Gudla, Kaustav Nandy, John Columbus, De Chen, Karen Worthy, Stephen Lockett, Thomas Turbyville. Targeting KRAS4b plasma membrane localization in cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4542. doi:10.1158/1538-7445.AM2015-4542