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Most RAS drug targeting strategies focus on KRAS, due to the high KRAS mutation frequency in cancer and its high expression level. Moreover, KRAS4B (hereafter KRAS) but not HRAS is a potent driver of stemness traits in cancer cells. Exactly how KRAS promotes stemness and how this activity can be targeted with drugs is not understood. Here we studied the subcellular localization of KRAS mutants to stemness mediating centriolar organelles and the effect of these mutants on the differentiation of mouse muscle C2C12 cells. This cell line contains a pool of ciliated stem/progenitor cells, which differentiate under low serum conditions, concomitant with a drop in RAS-MAPK activity. Intriguingly, expression of oncogenic KRAS retains the stem-like state, even under differentiating conditions. We hypothesized that KRAS localization at the primary cilium of C2C12 progenitor cells is propagated via the mother centriole to the mother centrosome, which is known to asymmetrically partition into the stemness retaining cell. Using confocal microscopy, we show that the trafficking chaperone PDE6D (or PDEdelta) facilitates trafficking of KRAS not only to the plasma membrane, but also to the primary cilium. Cellular BRET-interaction experiments in HEK cells demonstrate that decreasing the affinity between KRAS and PDE6D by the phosphomimetic S181D-mutation in KRAS, correlates with a reduced KRAS localization in the cilium of C2C12 cells. Likewise, treatment with Sildenafil, which increases the phosphorylation of Ser181 of KRAS, disrupts its binding to PDE6D. We then established a flow-cytometry based C2C12 cell differentiation assay to quantify accurately that mutations and pharmacological manipulations, which decrease ciliary localization of KRAS, indeed promote differentiation. These data lend a new rationale to PDE6D inhibition. By combining computational modelling and in vitro testing, we therefore developed a new, low nanomolar inhibitor, Deltaflexin-3 (structure will be disclosed), which targets the hydrophobic pocket of PDE6D. This compound is highly soluble and displays improved on-target activity as compared to previous PDE6D inhibitors, which is demonstrated by a panel of cellular BRET- and proliferation-assay data. Our results suggest that blocking of the ciliary localization of KRAS by synergistically combining PDE6D inhibitors and Sildenafil could specifically inhibit its stemness driving activity. Citation Format: Pelin Kaya, Rohan Chippalkatti, Elisabeth Schaffner-Reckinger, Bianca Parisi, Ganesh B. Manoharan, Yashar Rouzbahani, Christian Eggeling, Daniel Abankwa. Drug targeting of KRAS accumulation on the cilium inhibits its stemness driving activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3493. |
