Neuropilin-2b facilitates resistance to tyrosine kinase inhibitors in non-small cell lung cancer

Objective Innate and acquired resistance is the principle factor limiting the efficacy of tyrosine kinase inhibitors (TKIs) in lung cancer. We have observed a dramatic upregulation of the cell surface co-receptor neuropilin-2b (NRP2b) in lung cancers clinically treated with TKIs correlating with acquired resistance. We hypothesize that NRP2b plays a functional role in acquired TKI resistance. Methods NSCLC proliferation and survival were determined during chronic TKI exposure in the presence or absence of NRP2b knock down. Interactions of NRP2a and NRP2b isoforms with PTEN and GSK3β were assessed by immunoprecipitation. NRP2a and NRP2b mutants deleted for their cytoplasmic domains were utilized to identify regions responsible for NRP2b-GSK3β interaction. Since GSK3β is known to phosphorylate and degrade PTEN, phospho-PTEN and total PTEN levels were assessed following transfection of NRP2a and NRP2b wild type and mutant constructs. Results NSCLC cells chronically treated with gefitinib or osimertinib developed drug resistance and exhibited logarithmic growth in the presence of EGFR TKIs. However, NRP2b knockdown cells remained sensitive to gefitinib. Similarly, NRP2b knockdown suppressed and NRP2a knockdown enhanced cellular migration. Acquired drug resistance and cell migration correlated with NRP2b-dependent AKT activation with the intermediate step of GSK3β-dependent PTEN degradation. A specific binding site for GSK3β on the cytoplasmic domain of NRP2b was identified with truncated protein constructs and computer modeling. Conclusions NRP2b facilitates NSCLC resistance to TKIs and this biological effect relates to AKT activation. NRP2b-GSK3β interactions appear to be essential for PTEN degradation and AKT activation in lung cancer cells. Disruption of the NRP2b-GSK3β interaction may represent a novel treatment strategy to preserve sensitivity to TKIs in NSCLC.