Drug resistance after long-term use of Tyrosine kinase inhibitors (TKIs) has become an obstacle for prolonging the survival time of patients with clear cell renal cell carcinoma (ccRCC). Here, genome-wide CRISPR-based screening to reveal that HDAC8 is involved in decreasing the sensitivity of ccRCC cells to sunitinib is applied. Mechanically, HDAC8 deacetylated ETS1 at the K245 site to promote the interaction between ETS1 and HIF-2α and enhance the transcriptional activity of the ETS1/HIF-2α complex. However, the antitumor effect of inhibiting HDAC8 on sensitized TKI is not very satisfactory. Subsequently, inhibition of HDAC8 increased the expression of NEK1, and up-regulated NEK1 phosphorylated ETS1 at the T241 site to promote the interaction between ETS1 and HIF-2α by impeded acetylation at ETS1-K245 site is showed. Moreover, TKI treatment increased the expression of HDAC8 by inhibiting STAT3 phosphorylation in ccRCC cells is also found. These 2 findings highlight a potential mechanism of acquired resistance to TKIs and HDAC8 inhibitors in ccRCC. Finally, HDAC8-in-PROTACs to optimize the effects of HDAC8 inhibitors through degrading HDAC8 and overcoming the resistance of ccRCC to TKIs are synthesized. Collectively, the results revealed HDAC8 as a potential therapeutic candidate for resistance to ccRCC-targeted therapies.