For 17c a DC50 value of 14 nM was determined in MM.1S cells. Overall, our data confirms that 17c is a highly potent, selective, and non-toxic HDAC6 degrader. Thus, this promising tool compound was selected for a more comprehensive evaluation. HDAC6 degradation is dependent on the cyclic imide degron of 17c. To investigate, if the degradation of HDAC6 is mediated by the fluorothalidomide cyclic imide degron of 17c, a methylated, non-degrading control, NC-17c (Figure ), was synthesized (see Scheme S1, Supporting Information) and evaluated head-to-head with 17c. As depicted in Figure , 17c showed potent degradation of HDAC6, but not of HDAC1. NC-17c did not show any degradation of HDAC1 or HDAC6. These results confirm the necessity of the free imide group for HDAC6 degradation, which is essential for binding and recruiting CRBN. Additionally, the hyperacetylation of the HDAC6 substrate α-tubulin was determined by immunoblot analysis (Figure ). Notably, hyperacetylation of the substrate was not found in NC-17c-treated cells. NC-17c was determined to inhibit HDAC6 at 0.389 µM, which is similar to the inhibitory activity of 17c (0.295 µM, Table ). Only by degradation of HDAC6 through 17c, we found significantly hyperacetylated α-tubulin. This effect was more pronounced at 5 µM than at 0.5 µM, which is possibly due to a combination of HDAC6 degradation and inhibition of residual protein. HDAC6 degradation is mediated by the ubiquitin-proteasome system. Proteasomal rescue experiments were performed to confirm that HDAC6 degradation by 17c is mediated via the ubiquitinproteasome pathway. For this purpose, MM1.S cells were pretreated for 30 minutes with 10 M of the binding competitors tubastatin A, pomalidomide or the NEDD8-activating enzyme inhibitor MLN4924 before 17c was added. As illustrated in Figure , HDAC6 levels were almost completely rescued when the cells were pretreated with these compounds. The rescue experiments confirmed that 17c induced HDAC6 degradation is neddylation dependent and requires the interaction with both CRBN and HDAC6.