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mTOR inhibitors including everolimus and temsirolimus have been approved by US FDA for treatment of clear cell renal cell carcinoma (ccRCC) in clinic. However, resistance to these drugs has been inevitable and the underlying mechanism remains poorly understood. Histone modifier ZMYND8 paradoxically acts as a transcription coactivator or corepressor. Here we show that SPOP, a CULLIN3-RING E3 ubiquitin ligase (CRL) substrate-binding protein that is often overexpressed in ccRCC in patients, promotes K63-linked polyubiquitination of ZMYND8 at lysine 398, which inhibits ZMYND8 to form phase separation compartments and drives the formation of ZMYND8-ZHX2 transactivation complex, resulting in aberrant NEK7 kinase gene transcription, alternative activation of p70S6K, and mTOR inhibitor-resistant cell growth. Inhibition of either SPOP or NEK7 increases ccRCC cell sensitivity to mTOR inhibitor. Treatment with a NEK7 proteolysis-targeting chimera (PROTAC) effectively inhibits aberrant p70S6K activation and overcame everolimus resistance in ccRCC cells in vitro and in mice. Our findings uncover a function switch of ZMYND8 driven by overexpressed SPOP as a key mechanism that causes mTOR inhibitor resistance and nominate NEK7 as a potential target of thwarting mTOR inhibitor resistance in ccRCC.