Hypoxia-driven HIF-1alpha/YAP-AXL signaling drives adaptive resistance to TKIs in NSCLC

Background: Intratumoral hypoxia is a hallmark of solid cancers and promotes invasion, metastatic progression, and drug resistance. However, the mechanisms by which hypoxia contributes to resistance against tyrosine kinase inhibitors (TKIs) in driver-mutant non-small cell lung cancer (NSCLC) remain insufficiently defined. To address this gap, we systematically examined how hypoxic conditions reprogram signaling networks that undermine TKI responses. Methods: EGFR-mutant PC-9 and HCC4011 and ALK-rearranged H2228 cells were cultured under normoxia (21% O₂) or hypoxia (1% O₂) and treated with osimertinib or brigatinib. Drug response and signaling were assessed by MTT assays, immunoblotting, and phospho-RTK arrays. Bulk RNA-seq (PC-9; normoxia vs hypoxia, 72 h) supported pathway-level analyses. Mechanistic perturbations included siRNA knockdown, co-immunoprecipitation, nuclear/cytoplasmic fractionation, and immunofluorescence. For spatial analysis of human tumors, multiplex immunofluorescence (HIF-1alpha, AXL, alpha-SMA, DAPI) was performed on FFPE EGFR-mutant lung adenocarcinoma specimens and quantified with whole-slide single-cell segmentation and tumor/cancer-associated fibroblast (CAF) classification; CytoMAP nearest-neighbor and 50-µm neighborhood metrics assessed CAF proximity to hypoxic tumor cells. Results: Hypoxia reduced sensitivity to osimertinib in PC-9 and HCC4011 and to brigatinib in H2228. RNA-seq in PC-9 highlighted enrichment of hypoxia and PI3K/AKT pathways, indicating RTK network rewiring under hypoxia. Integrative phospho-RTK profiling and downstream validation pinpointed AXL as the dominant hypoxia-responsive node. AXL knockdown restored TKI growth inhibition and the suppression of downstream AKT signaling under hypoxia, and the AXL inhibitor ONO-7475 phenocopied these effects. Mechanistically, hypoxia decreased LATS1/YAP phosphorylation, promoted nuclear YAP, and enhanced HIF-1alpha-YAP interaction; silencing HIF-1alpha or YAP inhibited hypoxia-induced AXL expression. In patient specimens, HIF-1alpha-high tumor cells showed higher AXL intensity and closer proximity to alpha-SMA⁺ CAFs within 50-µm neighborhoods, linking hypoxia, AXL upregulation, and CAF-enriched niches. Conclusions: Hypoxia induces adaptive resistance to TKIs via HIF-1alpha/YAP-AXL signaling, linking oxygen stress to RTK reprogramming. Spatial analyses of patient tumors reveal CAF-proximal hypoxic niches with elevated AXL, providing tissue context for this mechanism. These findings support AXL inhibition as a rational combination partner to TKIs to suppress hypoxia-driven adaptive resistance in NSCLC, and motivate future studies to define the clinical conditions under which targeting this axis improves treatment durability.