Protein acetylation, especially histone acetylation, is the subject of both research and clinical investigation. At least four small-molecule histone deacetylase inhibitors are currently in clinical trials for the treatment of cancer. These and other inhibitors also affect microtubule acetylation. A multidimensional, chemical genetic screen of 7,392 small molecules was used to discover ''tubacin,'' which inhibits ␣-tubulin deacetylation in mammalian cells. Tubacin does not affect the level of histone acetylation, gene-expression patterns, or cell-cycle progression. We provide evidence that class II histone deacetylase 6 (HDAC6) is the intracellular target of tubacin. Only one of the two catalytic domains of HDAC6 possesses tubulin deacetylase activity, and only this domain is bound by tubacin. Tubacin treatment did not affect the stability of microtubules but did decrease cell motility. HDAC6 overexpression disrupted the localization of p58, a protein that mediates binding of Golgi elements to microtubules. Our results highlight the role of ␣-tubulin acetylation in mediating the localization of microtubuleassociated proteins. They also suggest that small molecules that selectively inhibit HDAC6-mediated ␣-tubulin deacetylation, a first example of which is tubacin, might have therapeutic applications as antimetastatic and antiangiogenic agents.

We have shown that the proteasome inhibitor bortezomib (formerly known as PS-341) triggers significant antitumor activity in multiple myeloma (MM) in both preclinical models and patients with relapsed refractory disease. Recent studies have shown that unfolded and misfolded ubiquitinated proteins are degraded not only by proteasomes, but also by aggresomes, dependent on histone deacetylase 6 (HDAC6) activity. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins followed by significant cell stress and cytotoxicity in MM cells. To prove this hypothesis, we used bortezomib and tubacin to inhibit the proteasome and HDAC6, respectively. Tubacin specifically triggers acetylation of ␣-tubulin as a result of HDAC6 inhibition in a dose-and time-dependent fashion. It induces cytotoxicity in MM cells at 72 h with an IC 50 of 5-20 M, which is mediated by caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity by c-Jun NH 2-terminal kinase͞caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow. Finally, adherence of MM cells to bone marrow stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of tubacin and bortezomib triggers toxicity even in adherent MM cells. Our studies therefore demonstrate that tubacin combined with bortezomib mediates significant anti-MM activity, providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.histone deacetylase