Targeting GRB2 with Polyphyllin H overcomes PIKFYVE inhibitor resistance in bladder cancer by blocking...

Targeting PIKFYVE has emerged as a promising anticancer strategy; however, its efficacy is often limited by adaptive resistance. This study aimed to investigate the efficacy and resistance mechanisms of PIKFYVE inhibitors in bladder cancer, and discover a phytochemical capable of overcoming the adaptive resistance, thereby establishing a potent and safe combination therapy.

The anti-proliferative effects of PIKFYVE inhibitors and phytochemical polyphyllin H (PPH) were evaluated in a panel of cancer cell lines. Apoptosis was assessed by flow cytometry. Molecular mechanisms were investigated using integrated transcriptomic and proteomic analyses, complemented by functional validation using gene knockdown and pharmacological inhibition. The interaction between GRB2 and PPH was characterized by surface plasmon resonance, cellular thermal shift assays, and molecular dynamics simulations. Gene expression, correlation, and survival data were analyzed using public databases. Therapeutic synergy between PPH and PIKFYVE inhibitors was assessed both in vitro and in vivo.

PIKFYVE inhibitors suppressed bladder cancer growth and overcame cisplatin resistance via autophagy blockade, but concurrently triggered a growth factor receptor binding protein 2 (GRB2)-dependent EGFR-Akt-SREBP lipogenic axis as a compensatory adaptive resistance mechanism. We identified PPH as a novel GRB2 inhibitor that binds specifically to the c-SH3/SH2 interface via a steric bottleneck involving key residues (Phe62, Ile65, Phe182). PPH suppressed the Akt-SREBP1-SCD1 cascade, inhibiting lipogenesis and tumor proliferation even under lipid-rich conditions. PPH disrupted this resistance mechanism, leading to strong synergistic antitumor activity without toxicity.

Our findings reveal a GRB2-mediated adaptive resistance pathway centered on lipid reprogramming and establish PPH as a clinically translatable GRB2 inhibitor that synergizes with PIKFYVE-targeted therapy.