Piperine Targets the FANCL/UBE2T Complex to Inhibit the FA Pathway and Sensitize Bladder Cancer to Cisplatin.

Platinum-based chemotherapy remains a mainstay for bladder cancer treatment, yet resistance often arises through activation of the Fanconi anemia (FA) DNA repair pathway. The monoubiquitination of the FANCI-FANCD2 (ID2) complex by FANCL and UBE2T is a critical step in repairing cisplatin-induced interstrand crosslinks (ICLs). Identifying small molecules that block this process may improve the therapeutic efficacy of cisplatin. We investigated the effects of piperine, a natural alkaloid from black pepper, on FA pathway activation in bladder cancer cells. A combination of immunoblotting, immunofluorescence, co-immunoprecipitation, qPCR-blocking assays, dot blot analyses, in vitro ubiquitination/discharge assays, biolayer interferometry (BLI), and differential scanning fluorimetry (DSF) were employed to characterize the molecular mechanism. Xenograft models were used to evaluate in vivo efficacy. Piperine pretreatment markedly suppressed cisplatin-induced monoubiquitination of FANCI and FANCD2 and reduced FANCD2 foci formation in T24, 5637, and RT4 cells. Co-immunoprecipitation confirmed diminished recruitment of downstream nucleases and repair factors (FANCP, FANCQ, PCNA). qPCR-blocking assays showed delayed ICL repair, while dot blot analyses revealed that intrastrand cisplatin adduct removal was unaffected, indicating selective inhibition of ICL repair. Piperine did not alter mRNA or protein expression of FANCL, UBE2T, USP1, or UAF1, nor did it enhance deubiquitinase activity. Instead, in vitro assays demonstrated that piperine blocked FANCL-mediated ubiquitin transfer from UBE2T∼Ub to the ID2 complex, without impairing E2 charging or FANCL-UBE2T binding. BLI confirmed unaltered binding affinity, whereas DSF revealed a significant ΔTm shift for UBE2T, consistent with allosteric modulation. In xenografts, combined cisplatin and piperine treatment significantly reduced tumor growth and attenuated FANCI/FANCD2 monoubiquitination. Our findings uncover piperine as a natural compound that allosterically inhibits UBE2T activity within the FA pathway, thereby impairing ID2 monoubiquitination and enhancing cisplatin sensitivity in bladder cancer. This study highlights the therapeutic potential of piperine and provides a rationale for targeting the FA repair axis to overcome platinum resistance.