Modulation of Signaling Cascades by Inhibitors of Histone Deacetylase and Akt

In multiple myeloma (MM), the bone marrow (BM) microenvironment plays a crucial role in promoting tumor cell proliferation, survival, migration, and drug resistance. It is composed of different types of cellular components, including hematopoietic stem cells, progenitor and precursor cells, immune cells, erythrocytes, BM stromal cells (BMSCs), BM endothelial cells, as well as osteoclasts and osteoblasts. These cells not only physically interact with MM cells, but also secrete growth and/or antiapoptotic factors, including interleukin (IL)-6, insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), stromal cell–derived factor (SDF) 1 , and B-cell activating factor (BAFF). These factors trigger several proliferative/antiapoptotic signaling cascades in MM cells: phosphatidylinositide-3 kinase (PI3K)/Akt; Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-related kinase (ERK); Janus kinase (JAK) 2/signal transducers and activators of transcription (STAT)-3; and nuclear factor (NF)– B.1 Therefore, cytokines, their receptors, and their downstream protein kinases and transcription factors represent potential therapeutic targets in MM. Histone deacetylases (HDACs) are divided into 4 classes: class I, HDAC 1, 2, 3, and 8; class II, HDAC 4, 5, 6, 7, 9, and 10; class III, silent information regulator genes (SIR)1, 2, 3, 4, 5, 6, and 7; and in class IV, HDAC 11. Importantly, class I and IV HDACs are constitutively localized in the nucleus, whereas class II HDACs can shuttle between the nucleus and the cytoplasm interacting with 14-3-3 protein. HDAC inhibitors have shown promise as novel antitumor agents for many malignancies, including structurally diverse HDAC inhibitors purified from natural sources or synthetically developed. Hypoacetylation of histones is associated with condensed chromatin, resulting in repression of gene transcription, whereas acetylated histones are associated with open chromatin structure and activation of transcription; therefore, HDAC inhibitors trigger transcription. However, inhibition of HDAC activity ultimately triggers growth arrest and/or apoptosis of tumor cells. Although possible mechanisms of HDAC inhibitor antitumor activities have been extensively studied, their growth inhibitory effects in MM cells have not yet been fully characterized. Importantly, recent studies have shown that acetylation of proteins other than histones may be altered by HDACs and their inhibitors. HDAC6 has an essential role in recruitment of ubiquitinated proteins for transport to aggresomes, ultimately leading to lysosomal protein degradation.2 We have shown that combined inhibition of proteasomes with bortezomib and of aggresomes with HDAC6-specific inhibitor tubacin3 triggers synergistic cytotoxicity in MM cell lines and MM patient tumor cells in vitro.4 We further examined whether inhibition of HDAC6 modulates acetylation of proteins. Interestingly, HDAC6 was constitutively associated with heat-shock protein (Hsp) 90, which was enhanced by tubacin treatment. Consistent with previous studies showing that HDAC6 modulates Hsp90 acetylation,5 tubacin enhanced acetylation of Hsp90 in MM cells. Since Akt is a client protein of Hsp90, we next examined whether acetylation of Hsp90 via inhibition of HDAC6 could modulate the activity of Akt. Tubacin enhanced phosphorylation of Akt; conversely, Hsp90 inhibitor 17-AAG inhibited its effect. Interestingly, enhanced interaction of Hsp90 with Akt was blocked by tubacin. Our results therefore suggest that HDAC6 negatively regulates phosphorylation of Akt. LBH589 (Novartis Pharmaceuticals) is a hydroxamic acid analog that blocks classes I and II HDAC and has significant antiMM activities.6 Importantly, LBH589 synergistically augments MM cell cytotoxicity induced by bortezomib; an international multicenter clinical trial evaluating LBH589 with bortezomib in MM is ongoing. We found that LBH589 significantly inhibited phosphorylation of JAK2 and its downstream molecule STAT3, without inhibiting Akt or ERK phosphorylation. IL-6 and BMSC coculture markedly augmented JAK2/STAT3 phosphorylation; however, LBH589 completely abrogated this effect. It has also been shown that acetylation of p65 (RelA) is crucial for its transcriptional Signaling Pathways in Myeloma