Abstract C080: Lipid raft-mediated insulin signaling sustains AKT activation and contributes to osimertinib resistance in NSCLC cells

Abstract Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality. The third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), osimertinib (AZD-9291), was approved as a front-line treatment in patients with EGFR activation mutations. Although the initial response is promising, most patients develop subsequent resistance. To overcome this obstacle, we used osimertinib-resistant H1975 cells (AR cells), which were established from naïve H1975 cells harboring L858R/T790M mutations, to elucidate the underlying mechanism. We found that sustained AKT activation under osimertinib bypasses the EGFR pathway and contributes to drug resistance in AR cells. Since directly targeting AKT would cause severe side effects, we aimed to unveil the exclusive mechanism behind this lopsided AKT activation in AR cells to develop molecular-targeted strategies. AKT activation in AR cells was inhibited by osimertinib under serum-free conditions. Notably, insulin supplementation under serum starvation selectively reactivated AKT, but not ERK, in AR cells. Moreover, linsitinib, an insulin receptor (InsR) inhibitor, effectively blocked AKT activation in AR cells, indicating that AKT activation was sustained by insulin signaling in AR cells. In contrast, insulin treatment or InsR overexpression failed to rescue AKT inhibition under osimertinib in naïve cells, suggesting that another mechanism augments InsR-driven AKT activation in AR cells and contributes to resistance. The InsR-AKT axis in AR cells was blocked by the depletion of cholesterol, implying a lipid raft-dependent InsR-AKT axis in resistant cells. Sucrose gradient fractionation revealed that both EGFR and InsR were enriched in the lipid raft fractions in AR cells, whereas they were evenly distributed across all fractions in naïve cells. These findings suggest that lipid rafts act as signal transduction hubs in AR cells. Mass spectrometry identified differentially enriched proteins in the lipid rafts of AR cells. Candidate targets were functionally validated by arrayed lentiviral knockdown in AR cells, followed by an assessment of cell survival and proliferation under osimertinib. We identified flotillin-1 (FLOT1) as a key mediator of the lipid raft-associated resistance in AR cells. FLOT1 knockdown reduced both AKT activation and an IC50 of osimertinib. Co-immunoprecipitation confirmed interactions between FLOT1 and key components of insulin signaling (e.g., IRS1), and immunofluorescence showed colocalization of FLOT1 with InsR and EGFR. These results suggest that FLOT1 scaffolds insulin signaling within lipid rafts, enhancing AKT activation and resistance. In summary, we identified a previously unrecognized lipid raft-dependent InsR-AKT signaling axis that sustains osimertinib resistance in NSCLC. FLOT1 plays a pivotal role in assembling insulin signaling components within lipid rafts, enhancing aberrant AKT activation. Targeting FLOT1 re-sensitizes resistant cells to osimertinib, highlighting it as a potential molecular target to overcome EGFR-TKI resistance. Citation Format: Yen-Lin Lai, Ching-Chow Chen. Lipid raft-mediated insulin signaling sustains AKT activation and contributes to osimertinib resistance in NSCLC cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C080.