Epigenetic Reprogramming Driven By Metabolic Alterations As A Mechanism Of EGFR – Tyrosine Kinase Inhibitor Resistance In Human Lung Adenocarcinoma

Abstract

Osimertinib, an EGFR tyrosine kinase inhibitor (TKI), is approved for the treatment of lung adenocarcinoma (LUAD) patients with activating EGFR mutations. However, acquired resistance to osimertinib is common. Resistance results from genomic alterations, but also through perturbations in enzyme activities, and epigenetic modifications. We aimed to identify pathways that mediate drug resistance and target them by identifying potent targeted therapies. Using quantitative proteomics and transcriptomics, we revealed altered metabolic pathways such as oxidative phosphorylation, citric acid cycle, and glutamate pathway in LUAD. Osimertinib‐resistant tumors were dependent on alpha‐ketoglutarate (aKG) pool derived from glutamine but not dependent on citrate, aspartate or fumarate for their survival. Moreover, fumarate, a competitive inhibitor of histone demethylases (HDMs) which use aKG as a cofactor, inhibited the viability of resistant cells. We validated these findings in our clinical trial (NCT02759835). Using RNAseq, we also show that in LUAD patients, oxidative phosphorylation, citric acid cycle, and glutamate pathway genes, including glutaminase (GLS) was significantly over expressed upon resistance when compared to their baseline (pre‐treatment) tumors. Moreover, the osimertinib resistant patient‐derived xenograft tumors were resensitized to osimertinib therapy when treated in combination with telaglenastat, a GLS inhibitor. Given that aKG is essential for HDMs, we investigated the activity of HDMs. Compared to baseline, upon development of resistance patients’ tumors and resistant cells exhibited several over expressed HDMs and decreased histone methylation (H3K27me3), which was restored upon treatment with telaglenastat or transient knock down of GLS, suggesting that reduced intracellular aKG due to the inhibition of GLS could have reduced the activity of HDMs. Osimertinib‐resistant xenograft tumors were susceptible to both HDM inhibitor (GSK J4), and to depletion of KDM6B, an aKG‐dependent demethylase. GSK J4 treatment and depletion of KDM6B increased histone methylation in resistant tumors and decreased their viability, thereby potentially suggesting its role in the maintenance of resistant tumors. We further investigated the role of transcription factors in regulating GLS expression. We found that JUN and RELA were over expressed and activated in resistant tumors. Transient depletion of JUN and RELA reduced GLS expression leading to increased histone methylation and reduced viability of resistant cells. Together, these results suggest that the increased glutaminase expression and activity increase the reliance of cells on aKG dependent HDMs, resulting in the transcriptional reprogramming that may be driving the osimertinib resistance that can be potentially targeted by telaglenastat and GSK‐J4 to overcome resistance.Support or Funding InformationIntramural Research Program, National Institutes of Health. Major Opportunity Grant on Metabolism, Center for Cancer Research, National Cancer Institute.