Abstract 3037: RSK4 inhibition promotes pseudo-hypoxic metabolic responses in lung adenocarcinoma

Background: RSK4 is a member of the p90 Ribosomal S6 kinase family that promotes metastasis and chemoresistance in non-small cell lung cancer and its inhibition improves therapeutic outcome in in vivo and ex vivo models. Here, we show that RSK4 inhibition activates HIF-1alpha and also impacts glucose and glutamine metabolism in lung adenocarcinoma cells. Methods: RSK4 was downregulated in lung adenocarcinoma cells by RNA interference or CRISPR/Cas9 technology. Glycolytic rate assay and mitochondrial stress test were performed using the Seahorse XFe96 analyzer. ATP levels were determined by the CellTiter Glo assay. Intracellular metabolites were profiled by untargeted mass spectrometry and U-13C glutamine labelling. 3D spheroids viability was assessed by CellTiter Glo 3D assay with or without glycolysis inhibition using 2-deoxy-D-glucose (2DG) or glutaminase inhibition using 6-Diazo-5-oxo-L-norleucine (DON). Protein expression was determined by Western blotting. Results: RSK4 downregulation enhanced glycolysis, oxidative phosphorylation, and ATP production in A549 cells. The RSK4-silenced cells showed increased lactate production and glutamine consumption via reductive carboxylation. This sensitized RSK4-silenced cells to 2DG and DON treatments. Mechanistically, RSK4 knockdown upregulates HIF-1alpha in normoxia through decreased activation of PHD2, an enzymes that targets HIF-1alpha for degradation. Stabilization of HIF-1alpha resulted in increased transcription of HK2, the rate limiting enzyme of glycolysis, and GLS1, which hydrolyzes glutamine. Conclusion: RSK4 knockdown promotes a pseudo-hypoxic phenotype in lung adenocarcinoma cells characterized by increased glycolysis and enhanced glutamine utilization via reductive carboxylation. Targeting these changes potentiates the effects of RSK4 inhibition and may represent new therapeutic opportunities for this tumor type. Citation Format: Lucksamon Thamlikitkul, Peng Chai, Cristina Balcells Nadal, Rajat Roy, Xiao-Ming Sun, Marion MacFarlane, Hector Keun, Michael Seckl, Olivier Pardo. RSK4 inhibition promotes pseudo-hypoxic metabolic responses in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3037.