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

Abstract

ABSTRACT 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.