Dauricine Overcomes Osimertinib Resistance in Lung Cancer by Inducing Ferroptosis via Stabilizing SAT1

Abstract

Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) widely used to treat advanced nonsmall cell lung cancer (NSCLC) with EGFR mutations. However, resistance to osimertinib frequently develops, limiting its long-term effectiveness. In this study, we used osimertinib-resistant lung cancer cell lines and lung cancer patient-derived organoids to demonstrate the potential of dauricine, a bioactive compound derived from menispermum dauricum, to overcome osimertinib resistance in lung cancer cells. Mechanistic studies reveal that dauricine, when combined with osimertinib, efficiently induces ferroptosis in resistant lung cancer cells. Notably, RNA interference and pharmacological inhibition assays identify SAT1, a key enzyme involved in polyamine metabolism and oxidative stress regulation, as a critical mediator of the synergistic effects observed with the dauricine-osimertinib combination therapy. Furthermore, we show that dauricine can directly interact with and stabilize SAT1 to enhance its activity. In vivo, when administered with osimertinib, dauricine significantly suppresses tumor growth in osimertinib-resistant lung cancer models. These findings provide novel insights into the role of SAT1 in overcoming osimertinib resistance and suggest that combining dauricine with osimertinib could be a promising therapeutic strategy to improve the efficacy of EGFR-TKI therapy in resistant NSCLC.

Keywords: EGFR‐TKIs resistance; NSCLC; dauricine; ferroptosis; osimertinib; spermidine/spermine N1‐acetyltransferase 1.

FIGURE 1

Establishment of osimertinib‐resistant lung cancer cell lines and lung cancer patient organoids. (A) Evaluating IC₅₀ values for osimertinib in PC9, PC9‐OR, H1975 and H1975‐OR; (B) Western blot analysis of p‐EGFR, p‐ERK, total EGFR, and ERK in PC9, PC9‐OR, H1975 and H1975‐OR cells treated with osimertinib for 48 h; (C) Representative images of osimertinib‐sensitive and resistant lung cancer organoid; (D) Determination of osimertinib IC₅₀ values for resistant and their parental organoids.

FIGURE 2

Screening of TCM monomers and identification of dauricine to improve the sensitivity of osimertinib. (A) Screening of 302 TCM monomers to overcome osimertinb resistance in PC9‐OR and H1975‐OR cells; (B) Evaluating IC₅₀ values of dauricine in PC9‐OR and H1975‐OR cells; (C) Dose–response curves showing cell viability of PC9, H1975 and PC9‐OR, H1975‐OR with or without 10 μM DAC treatment; (D) SynergyFinder analysis of the synergy between dauricine and osimertinib in PC9‐OR and H1975‐OR cells; (E) Colony formation assay of PC9‐OR and H1975‐OR cells treated with osimertinib, dauricine, or a combination of both, along with the quantification of the results; (F) Determination of osimertinib IC₅₀ values for resistant and parental organoids treated with or without dauricine, along with representative images of osimertinib‐resistant lung cancer organoids treated with osimertinib, dauricine, or their combination. Data were expressed as means SD (*p < 0.05;**p < 0.01;***p < 0.001;****p < 0.0001).

FIGURE 3

Combined dauricine and osimertinib triggers ferroptosis in osimertinib‐resistant lung cancer cells. (A) GSEA of ferroptosis‐related genes in PC9‐OR cells with or without dauricine and osimertinib treatment; (B) Cell death assay in PC9‐OR cells co‐treated with dauricine, osimertinib, and specific cell death inhibitors; (C) Immunoblot analysis of ferroptosis proteins in PC9‐OR cells treated with osimertinib, dauricine, or their combination; (D) Immunoblot analysis of ferroptosis proteins in dauricine and osimertinib‐treated PC9‐OR cells with or without DFO pretreatment; (E) FACS analysis of ROS and lipid peroxidation levels in PC9‐OR cells treated with osimertinib, dauricine, or their combination; (F) FACS analysis of ROS and lipid peroxidation in PC9‐OR cells treated with dauricine, osimertinib, or both, with or without DFO pretreatment. Data were expressed as means SD (*p < 0.05; **p < 0.01;***p < 0.001; ****p < 0.0001).

FIGURE 4

Dauricine stabilizes SAT1 in osimertinib‐resistant lung cancer cells. (A) Volcano plot of differential gene expression in dauricine and osimertinib‐treated PC9‐OR cells vs. control; (B) Validation of different expressed ferroptosis‐related genes by qPCR in PC9‐OR; (C) Cell viability assay of PC9‐OR cells with dauricine and osimertinib treatment after knocking down SAT1, ACSL1, LC3B, PRNP, HMOX1, and PCBP1; (D) The diagram showing the role and relationship of ACSL1 and SAT1 in ferroptosis. Image created with BioRender.com; (E) CETSA analysis of SAT1 expression in PC9‐OR cells at different temperature gradients with or without dauricine treatment; (F) Immunoblot analysis of SAT1 expression in PC9‐OR cells treated with different concentrations of dauricine at 45°C; (G) CETSA analysis of ACSL1 expression in PC9‐OR cells at different temperature gradients with or without dauricine treatment; (H) Immunoblot analysis of ACSL1 expression in PC9‐OR cells treated with different concentrations of dauricine at 45°C; (I) DARTS assay of SAT1 and ACSL1 expression in PC9‐OR cells treated with pronase and different concentrations of dauricine; (J) The docking assays between SAT1 and Dauricine using Autodock; (K) Western blot analysis of SAT1 in PC9 or PC9‐OR cells treated with osimertinib; (L) Western blot analysis of SAT1 in PC9‐OR cells treated with osimertinib and different concentrations of dauricine; (M) Immunoblot analysis of SAT1 expression in cycloheximide treated PC9‐OR cells with or without dauricine treatment; (N) The degradation curve of SAT1 in cycloheximide treated PC9‐OR cells with or without dauricine treatment. Data were expressed as means SD (*p < 0.05; **p < 0.01;***p < 0.001; ****p < 0.0001).

FIGURE 5

Dauricine overcomes osimertinib resistances via SAT1. (A) Cell viability assay and IC50 evaluation of dauricine and osimertinib in sh‐NC and sh‐SAT1 PC9‐OR cells; (B) Immunoblot analysis of GPX4 and TFRC in dauricine and osimertinib treated sh‐NC or sh‐SAT1 PC9‐OR cells; (C) FACs analysis of ROS levels and lipid peroxidation levels in dauricine and osimertinib treated sh‐NC or sh‐SAT1 PC9‐OR cells; (D) Cell viability assay of PC9‐OR cells treated with dauricine and osimertinib with or without pentamidine pretreatment (1 μM, 24 h), along with the evaluation of IC₅₀ values of dauricine and osimertinib in PC9‐OR cells pretreated with or without pentamidine (1 μM, 24 h); (E) Overexpression of SAT1 in PC9‐OR cells, with cell viability assay and evaluation of IC₅₀ values of dauricine and osimertinib in control and SAT1‐OE PC9‐OR cells. Data were expressed as means SD (*p < 0.05; **p < 0.01;***p < 0.001; ****p < 0.0001).

FIGURE 6

Dauricine can effectively enhance the sensitivity of lung cancer‐resistant cells to osimertinib in vivo. (A) Schematic diagram of drug administration for PC9‐OR xenograft mice, mice were treated with vehicle (Control), osimertinib (OSI), dauricine (DAC), or osimertinib combined with dauricine (DAC + OSI); n = 5 per group. Image created with BioRender.com; (B) Subcutaneous tumor growth curve of Control, OSI, DAC or DAC + OSI treated mice; (C) Representative images of dissected tumors from Control, OSI, DAC or DAC + OSI mice; (D) The quantification of tumor volume in (C); (E) The body weight curve of Control, OSI, DAC or DAC + OSI treated mice; (F) Quantification of MDA in tumor tissues from Control, OSI, DAC or DAC + OSI treated mice; (G) Representative images of Ki67 staining from tumor tissues isolated from Control, OSI, DAC or DAC + OSI treated mice; (H) Quantification of Ki67 staining in (G); (I) Representative H&E staining results from tumor tissues of Control, OSI, DAC or DAC + OSI treated mice; (J)Western blot analysis of SAT1 in tumor tissue lysates from Control, OSI, DAC or DAC + OSI treated mice. Data were expressed as means SEM (*p < 0.05; **p < 0.01;***p < 0.001; ****p < 0.0001).