EZH2-AR non-canonical axis activation in TKI-resistant RCC: Differential effects of lenvatinib and cabozantinib

Tyrosine kinase inhibitors (TKIs) are critical to the treatment of renal cell carcinoma (RCC). However, recurrent and metastatic RCC frequently develop resistance to TKIs, limiting therapeutic options. Understanding the molecular mechanisms underlying this resistance is crucial for improving patient outcomes. Our previous studies have identified EZH2 and the androgen receptor (AR) as key regulators of acquired resistance to the TKI . Inhibiting EZH2 or AR restores sunitinib sensitivity in vitro and in vivo, highlighting these molecules as therapeutic targets potentially shared across TKIs .To investigate the interplay between EZH2 and AR, we compared sunitinib-resistant 786-0 cells (786-0 RS ) with drug-naive 786-0 cells overexpressing AR (786-0 AR ). Chromatin immunoprecipitation sequencing revealed that AR and EZH2 co-occupy genomic loci exclusively in resistant cells. Sunitinib treatment induced nuclear translocation of AR in 786-0 AR , with AR binding at EZH2-occupied sites. Mass spectrometry further showed high phosphorylation of AR at S81 and S213, markers of ligand-independent activation, in 786-0 RS , which was absent in 786-0 AR .786-0 RS cells undergo EZH2-dependent reprogramming of the tyrosine kinome via a likely non-canonical mechanism. Co-expression of a phosphomimetic EZH2 mutant (S21D) with AR promoted sunitinib resistance, whereas wild-type or phosphonull (S21A) EZH2 did not. Expanded phosphoproteomic analyses revealed extensive rewiring of serine/threonine signaling networks in sunitinib resistant cells, including enrichment of EGFR-driven MAPK and AKT pathways. Interestingly, preliminary data indicate that cabozantinib does not induce AR and EZH2 upregulation like other TKIs such as lenvatinib. Moreover, cabozantinib appears to prevent compensatory pathway activation triggered by lenvantinib, suggesting a distinct resistance profile. Consistently, AR expression is observed in RCC lines resistant to sunitinib, dovitinib, and lenvatinib, but not with cabozantinib treatment. Ongoing phosphoproteomic analysis will dissect the difference across the TKIsdrugs. Overall, our findings suggest that EZH2-dependent kinase reprogramming drives AR phosphorylation and activation, contributing to drug resistance to selective TKIs. Cabozantinibunique ability to bypass AR/EZH2 upregulation underscores the need to explore alternative resistance pathways.