Abstract 3306: Inhibition of ribosomal protein S6 (rpS6) phosphorylation and cell proliferation by a novel chloroquine analog in lung cancer cells

Autophagy is an evolutionarily conserved self-defense mechanism that allows tumor cells to overcome stress during oncogenesis and after treatment with chemotherapy. It is the basis of clinical trials using the autophagy inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ). While searching for novel agents targeting autophagy, we discovered a compound, named EAD1, that is structurally related to CQ but is up to 16-fold more potent an inhibitor of autophagy and of lung cancer cell proliferation than CQ. Interestingly, one of the cell lines whose proliferation was inhibited by EAD1 were H1650 NSCLC cells. This was surprising as H1650 cells have a biallelic deletion within the Atg7 locus. Atg7 is a critical component of the canonical autophagy pathway and its loss renders cells autophagy-deficient. This observation was confirmed in H460 Atg7-knockout cells. That these cells had the same sensitivity to EAD1 (and HCQ) as did cells with an intact autophagy pathway suggests that inhibition of autophagy was not the mechanism by which those drugs inhibit cell proliferation. We therefore conducted exploratory experiments to identify other possible sites of action of EAD1. The only molecular change we observed with EAD1 was the selective inhibition of ribosomal protein S6 (rpS6) phosphorylation, which correlated with its inhibition of cell proliferation. rpS6 is a component of the 40S ribosome subunit, and is only one of two ribosomal proteins known to be phosphorylated in a regulated manner. The decrease in rpS6 phosphorylation also occurred in the Atg7-deficient cells, indicating it was not a consequence of autophagy inhibition. rpS6 regulates cell size, survival, and migration. Consistent with these reports, we found that EAD1 caused a significant decrease in the size of the H460 cells (when controlled for cell cycle distribution), and this decrease was not seen with HCQ. rpS6 is phosphorylated and activated by two known families of kinases, p70 S6K and p90 RSK, that are downstream targets of the mTORC1 and the RAS/MEK/ERK pathways, respectively. However, we have not seen any changes in the expression or phosphorylation of the members of these pathways, leading us to hypothesize that EAD1 is either having a direct effect on rpS6, or that it is blocking the kinase activity of an already activated p70S6K or p90RSK. Further studies are needed to determine the role of rpS6 in cancer cells, and to assess if it is a viable target for future drug development. Citation Format: Juan Sironi, Evelyn Aranda, Lars Nordstroem, Roman Perez Soler, Edward L. Schwartz. Inhibition of ribosomal protein S6 (rpS6) phosphorylation and cell proliferation by a novel chloroquine analog in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2017-3306