Novel Hydroxamic Acid-Derived HDAC Inhibitor LBH589 Potently Activates Intrinsic and Extrinsic Apoptotic Pathways, and Induces Tubulin Hyperacetylation in Multiple Myeloma.

Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematological malignancies. LBH589 is a novel hydroxamic acid derivative HDAC inhibitor that inhibits myeloma cell growth at a median inhibitory concentration of less than 10 nM at 48 hours in multiple myeloma (MM) cell lines resistant to conventional therapies, as well as MM patient cells, as determined by MTS assay and tritiated-thymidine uptake. Cell cycle analysis revealed G1 arrest and a significant increase in the sub-G1 populations in these cells. MM.1S myeloma cell proliferation was also inhibited when co-cultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Pro-apoptotic effects were confirmed by Western blot. Significant cleavage of caspase 8, caspase 9, caspase 3 and PARP were observed within 24h with 20nM of LBH589 in MM.1S cells, indicating involvement of both the intrinsic and extrinsic apoptotic pathways in LBH589-mediated apoptosis. There was associated up-regulation of p21 and down-regulation of c-myc. Synergistic activity was observed in combination with proteasome inhibitor bortezomib. LBH589-induced HDAC inhibition resulted in histone hyperacetylation at low nanomolar concentrations. Acetylation of alpha-tubulin is a post-translational modification that consists of the addition of an acetyl group to lysine 40, which is reversed by HDAC6, or tubulin deacetylase (TDAC). Tubulin acetylation plays an important role in the differentiation of microtubule structure and function. Furthermore, TDAC constitutively binds both polyubiquitinated misfolded proteins and dynein, recruiting misfolded protein to dynein motors for transport to aggresomes along microtubules, which serves an important defense mechanism against apoptosis in MM cells. Importantly, dose-dependent inhibition of TDAC in MM.1S cells was observed at low nanomolar concentrations of LBH589. These findings provide the rationale for clinical studies with LBH589, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM.