PTBP1-Mediated Alternative Splicing of DNAJB6 Promotes Everolimus Resistance in Clear Cell Renal Cell Carcinoma via EIF4B/PKIB/AKT/mTOR Positive Feedback Loop.

BACKGROUND: Everolimus (EVE) is a key treatment for advanced clear cell renal cell carcinoma (ccRCC), mainly targeting AKT/mTOR signaling. However, developed resistance remains a major challenge for EVE treatment. Aberrant alternative splicing, particularly regulated by PTBP1, may represent a critical mechanism enabling the activation of these resistance-associated pathways. METHODS: Single-cell transcriptome sequencing is performed to reveal the association between PTBP1 expression, EVE resistance and AKT/mTOR pathway activity. Patient-derived specimens, animal models, and in vitro assays are used to evaluate the role of PTBP1 in EVE resistance. Full-length transcriptome sequencing and crosslinking immunoprecipitation (CLIP) sequencing are employed to identify PTBP1-regulated splicing targets. Co-immunoprecipitation, proteomic analyses, and molecular docking further elucidate the mechanism by which DNAJB6b re-activated AKT/mTOR signaling. RESULTS: PTBP1 is upregulated in EVE-resistant tumors and its expression positively correlates with AKT/mTOR signaling activation. PTBP1 promotes the expression of the DNAJB6b isoform through alternative splicing. EIF4B is identified as a direct binding partner of DNAJB6b, forming a complex that enhances oncogenic translation and activates a PKIB-dependent AKT/mTOR signaling cascade. CONCLUSION: This study demonstrates that PTBP1 drives EVE resistance in ccRCC by upregulating DNAJB6b, which recruits EIF4B to initiate a PKIB/AKT/mTOR positive feedback loop, sustaining oncogenic translation and therapeutic evasion.