Spatial mitochondrial ROS microdomains drive non-genomic EGFR‑TKI resistance via S‑glutathionylation of EGFR in NSCLC

Hypothesis

In EGFR‑mutant non‑small cell lung cancer that acquires resistance to EGFR‑TKIs, confined pockets of elevated mitochondrial reactive oxygen species (ROS) in perivascular niches cause S‑glutathionylation of EGFR, preserving downstream signaling despite drug binding.

Rationale

Multi‑omics integration has shown that proteomic rewiring, not captured by genomics, underlies TKI resistance—for example, drug‑efflux proteins like glutathione S‑transferases (GSTs) are elevated without corresponding mRNA changes [1]. Spatial multi‑omics in glioma revealed locoregional metabolite enrichment that defines protective microniches [3]. Mitochondrial ROS are known to modify cysteine residues on proteins through S‑glutathionylation, a reversible post‑translational alteration that can sustain kinase activity. We propose that, in resistant NSCLC, hypoxic perivascular zones generate a localized ROS burst that specifically S‑glutathionylates EGFR at cysteine‑797, preventing TKI binding while maintaining autophosphorylation and downstream MAPK/PI3K signaling.

Testable Predictions

1. Spatial mapping of mitochondrial ROS (using MitoSOX) will colocalize with phosphorylated EGFR in resistant tumor regions but not in sensitive areas.
2. Biotin‑switch assays will detect increased S‑glutathionylation of EGFR in ROS‑high niches; this signal will diminish after mitoTEMPO treatment.
3. Disrupting mitochondrial ROS (via mitoTEMPo or genetic knockdown of SOD2) will reduce EGFR S‑glutathionylation, restore TKI binding sensitivity in vitro, and shrink xenograft tumors when combined with osimertinib.
4. Spatial proteomics will show that GSTP1 expression is elevated in the same ROS‑rich niches, linking glutathione metabolism to the modification event.

Experimental Design

• Generate EGFR‑mutant NSCLC cell lines with acquired osimertinib resistance; confirm resistance via dose‑response curves.
• Perform immunofluorescence staining for MitoSOX, p‑EGFR (Y1068), and 4‑HNE; acquire confocal z‑stacks and quantify colocalization using Pearson’s coefficient.
• Isolate ROS‑high and ROS‑low fractions by laser‑capture microdissection; run biotin‑switch followed by Western blot for EGFR.
• Treat resistant cultures with mitoTEMPo (5 µM) ± osimertinib; measure cell viability (CellTiter‑Glo), apoptosis (caspase‑3/7), and downstream signaling (p‑ERK, p‑AKT) over 72 h.
• In vivo, implant resistant xenografts in NSG mice; randomize to vehicle, mitoTEMPo, osimertinib, or combination; monitor tumor volume twice weekly and perform endpoint spatial transcript‑proteomics (slide‑seqV2) to validate niche‑specific EGFR modification.
• Statistical analysis: two‑way ANOVA with post‑hoc Tukey; significance set at p<0.05.

Falsifiability

If spatial ROS elevation does not correlate with EGFR S‑glutathionylation, or if mitoTEMPo fails to restore TKI sensitivity despite ROS reduction, the hypothesis would be refuted. Conversely, consistent niche‑specific ROS‑EGFR coupling and therapeutic reversal would support the model.