Targeting endolysosomal trafficking to overcome paclitaxel resistance in triple-negative breast cancer

Background: Taxane therapy is the mainstay for triple-negative breast cancer (TNBC) treatment yet acquired paclitaxel resistance occurs in >90% of metastatic cases, leaving patients without approved second-line therapies and a dismal 5-year survival of <30%. Resistant TNBC cells survive by upregulating two complementary endolysosomal pathways: macropinocytosis to scavenge nutrients and protective autophagy to sequester/degrade paclitaxel. Both processes critically depend on the lipid kinase PIKfyve and its product PI(3,5)P₂. We have developed low-nanomolar PIKfyve inhibitors that exploit the reliance of resistant TNBC cells on endolysosomal trafficking pathways, by substantially enhancing macropinocytic paclitaxel uptake while blocking autophagic flux, converting these adaptive survival mechanisms into selective lethality in paclitaxel-resistant TNBC. Methods: The PIKfyve inhibitors were rationally designed, synthesized, and evaluated for kinase inhibition using the ADP-Glo assay. Paclitaxel-resistant TNBC cells (SUM159PT/PAC200; PTX IC₅₀ >1000 nM) were treated with inhibitors ± paclitaxel. Macropinocytosis was quantified by 70 kDa FITC-dextran uptake (flow cytometry/confocal) ± EIPA (25 µM). Autophagic flux was assessed by LC3-II/p62 immunoblotting. Viability was determined by 72 h SRB & MTT assays; synergy calculated using Chou-Talalay combination index (CompuSyn). Paclitaxel sensitization and apoptosis induction by PIKfyve inhibitors were also evaluated in organoids derived from SUM159PT/PAC200 cells. Experiments were performed in triplicate; statistical significance by one-way ANOVA followed by Dunnett's post hoc test. Results: Lead PIKfyve inhibitors (PSG-06 and PSG-09) potently induced macropinocytosis (>3-fold increase in dextran uptake at 10 µM) while completely blocking autophagic flux (marked p62 accumulation). This dual effect dramatically enhanced intracellular paclitaxel accumulation and restored sensitivity, resulting in combination indices as low as 0.64, >50% reduction in viability, cell cycle arrest, and apoptosis at clinically relevant paclitaxel concentrations (5-20 nM). The macropinocytosis inhibitor EIPA completely abolished resensitization, confirming an on-target mechanism. Notably, PSG-induced effects were reversible, providing a transient window for paclitaxel accumulation and cytotoxicity, potentially minimizing long-term toxicity associated with sustained macropinocytosis and autophagy inhibition. Furthermore, PSG-6 combined with paclitaxel induced dose-dependent apoptosis in paclitaxel-resistant SUM159PT/PAC200 3D organoids, as confirmed by Western blot detection of cleaved PARP. Conclusions: Low-nanomolar PIKfyve inhibitors exploit the paradoxical ability of PIKfyve blockade to simultaneously drive macropinocytic paclitaxel uptake and inhibit protective autophagy, creating a potent synthetic-lethal combination in otherwise resistant TNBC cells. This single-agent, dual-hit strategy bypasses P-glycoprotein efflux, prevents autophagic sequestration and degradation of paclitaxel, and provides a clear translational path for taxane-refractory TNBC and other macropinocytosis-addicted tumors.