Multiple myeloma (MM) is a deadly plasma cell cancer that resides in the bone marrow (BM). Numerous studies have demonstrated the involvement of the BM microenvironment supporting tumor growth, angiogenesis, bone disease and drug resistance. Reciprocal interactions between the different components of the BM microenvironment and the MM cells are necessary to regulate migration, differentiation, proliferation and survival of the malignant plasma cells. In this review we focus on the interactions and molecular mechanisms by which the BM microenvironment exert these effects. Better understanding of these interactions and the study of the epigenetic changes that tumor cells undergo are necessary in order to improve current treatments and for the discovery of new therapies that may eventually lead to a potential cure.

Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.

Introduction Histone Deacetylases Classification Histone-dependent mode of action Non-histone targets Cell cycle-related histone and non-histone substrates of histone deacetylases Retinoblastoma Protein 53 transcription factor Cyclin-dependent kinase inhibitor p21Cip1 Proliferating cell nuclear antigen Inhibition of histone deacetylases in liver cells Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes Pathophysiological condition Liver malignancy - hepatocellular carcinoma Liver fibrosis Histone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotype The role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascades Conclusions The transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction. In general, though not exclusively, histone acetylation is associated with a positive regulation of transcription, whereas histone deacetylation is correlated with transcriptional silencing. The elucidation of unequivocal links between aberrant action of HDACs and tumorigenesis lies at the base of key scientific importance of these enzymes. In particular, the potential benefit of HDAC inhibition has been confirmed in various tumour cell lines, demonstrating antiproliferative, differentiating and pro-apoptotic effects. Consequently, the dynamic quest for HDAC inhibitors (HDIs) as a new class of anticancer drugs was set off, resulting in a number of compounds that are currently evaluated in clinical trials. Ironically, the knowledge with respect to the expression pattern and function of individual HDAC isoenzymes remains largely elusive. In the present review, we provide an update of the current knowledge on the involvement of HDACs in the regulation of fundamental cellular processes in the liver, being the main site for drug metabolism within the body. Focus lies on the involvement of HDACs in the regulation of growth of normal and transformed hepatocytes and the transdifferentiation process of stellate cells. Furthermore, extrapolation of our present knowledge on HDAC functionality towards innovative treatment of malignant and non-malignant, hyperproliferative and inflammatory disorders is discussed.

Multiple myeloma (MM) is a B-cell malignancy, which often remains incurable because of the development of drug resistance governed by the bone marrow (BM) microenvironment. Novel treatment strategies are therefore urgently needed. In this study, we evaluated the anti-MM activity of JNJ-26481585, a novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor, using the syngeneic murine 5TMM model of MM. In vitro, JNJ-26481585 induced caspase cascade activation and upregulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 toward cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM-bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity that can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

Novel drugs such as bortezomib and high dose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow micro-environment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC) inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells. Here we summarize the molecular actions of HDACi as a single agent or in combination with other drugs in different in vitro and in vivo myeloma models and in (pre)clinical trials.

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

The transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction. In general, though not exclusively, histone acetylation is associated with a positive regulation of transcription, whereas histone deacetylation is correlated with transcriptional silencing. The elucidation of unequivocal links between aberrant action of HDACs and tumorigenesis lies at the base of key scientific importance of these enzymes. In particular, the potential benefit of HDAC inhibition has been confirmed in various tumour cell lines, demonstrating antiproliferative, differentiating and pro-apoptotic effects. Consequently, the dynamic quest for HDAC inhibitors (HDIs) as a new class of anticancer drugs was set off, resulting in a number of compounds that are currently evaluated in clinical trials. Ironically, the knowledge with respect to the expression pattern and function of individual HDAC isoenzymes remains largely elusive. In the present review, we provide an update of the current knowledge on the involvement of HDACs in the regulation of fundamental cellular processes in the liver, being the main site for drug metabolism within the body. Focus lies on the involvement of HDACs in the regulation of growth of normal and transformed hepatocytes and the transdifferentiation process of stellate cells. Furthermore, extrapolation of our present knowledge on HDAC functionality towards innovative treatment of malignant and non-malignant, hyperproliferative and inflammatory disorders is discussed.

AIM: To evaluate the screening of histone deacetylase inhibitors (HDACi) based on cellular potency using the murine multiple myeloma 5T33MM model. METHODS: The cellular potencies of 10 structurally related compounds of the natural HDACi Trichostatin (TSA) were screened by measuring the DNA synthesis in primary murine 5T33MMvv cells. The anti-tumor activity of the three most potent analogues was further confirmed in the 5T33MMvt cell line using proliferation, viability and active caspase-3 assays. The data of this 5T33MM model were compared with the HDAC IC 50 of these compounds which were preliminarily determined in normal hepatocyte extract. RESULTS: The TSA analogues with a tetranoic spacer (compounds 7-10) had no significant effect on the DNA synthesis of 5T33MM cells, whereas the three most potent analogues (compounds 1, 3 and 4), having a pentanoic spacer, significantly reduced the DNA synthesis and the viability. We further observed a significant increase of active caspase-3 in the 5T33MM cells when treated with compound 3 or 4, reflecting their apoptosis-inducing capability. When comparing with the HDAC-inhibitory activities of these compounds in normal hepatocyte extract, a similar result was obtained to suggest that compound 4 had the most potent anti-cancer activity. CONCLUSION: Screening the cellular potency of TSA analogues using the 5T33MM model is a reliable and efficient system to select the most potent analogue early in the development of HDACi and proves the value of this model for evaluation of HDACi.

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug-target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Insulin-like growth factor-1 (IGF-1) is an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Bcl-2 like 11 (Bim) is a pro-apoptotic protein belonging to the BH3-only group of Bcl-2 family members. Three major forms of Bim are known: BimEL, BimL and BimS. In leukemia, beta1 integrin mediated adhesion has been shown to reduce Bim levels by enhanced proteasome activation. We used the 5T33MM syngeneic, immunocompetent murine model for MM. Both the exclusively in vivo growing 5T33MM vv cells and the stroma independent in vitro growing 5T33MM vt cells were used. The present study showed that Bim levels were reduced in MM cells after IGF-1 treatment. IGF1-mediated regulation of Bim expression in 5T33MM vv cells was demonstrated to be a result of reduced gene expression on the one hand and increased proteasomal-mediated degradation of Bim protein levels on the other hand. Reduced expression was related to activation of the Akt pathway and inactivation of the transcription factor FoxO3a. Increased degradation of Bim was related to activation of the mitogen-activated protein kinase pathway, as IGF-1 treatment resulted in phosphorylation of ERK1/2. Reducing Bim levels by transducing 5T33MMvt cells with a lentiviral vector with a short hairpin RNA cassette inhibited serum starved and the clinical relevant deacetylase inhibitor panobinostat (LBH589, Novartis) induced cell death. Together our data indicate that disrupting IGF-1-mediated regulation of Bim degradation may increase the efficacy of drugs. In the past few years, methylation of DNA and acetylation of histones have been proven to be important regulators of gene expression. Moreover, acetylation of non-histone proteins has emerged as a dynamic posttranslational modification regulating numerous cellular processes. Co-treatment of 5T33MM vv and 5T33MM vt cells for 24h with the demethylation agent 5-Aza-2′deoxycytidine and the deacetylase inhibitor panobinostat resulted in a strong induction of Bim expression at protein level. In the future, we will investigate the contribution of IGF-1 to the epigenetic silencing of Bim by i) comparing the Bim promoter methylation pattern of both human IGF-1 responsive cell lines and murine 5T33MM vv cells treated or not with IGF-1 using bisulfite PCR sequencing and ii) using the epigenetic modulating agents 5-Aza-2′deoxycytidine and panobinostat.

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells

In multiple myeloma (MM) monoclonal plasma cells accumulate in the bone marrow (BM) where they interact with neighboring cells to receive supportive signals. Since BM adipocytes are known to participate in normal and malignant hematopoiesis, we presumed interactions between BM adipocytes and myeloma cells. Using an adipocytic cell line and primary isolated BM adipocytes, we performed functional assays indicating that adipocytes support MM cell development by affecting proliferation, apoptosis, cell migration and adhesion. We subsequently analyzed the secretion of different cytokines by BM adipocytes and analyzed the contribution of a specific adipokine, leptin, to these processes. We confirmed the expression of leptin by adipocytes and leptin receptor by myeloma cells. Immunostaining showed strong presence of leptin receptor on MM cells in 13 of 28 patients tested. This expression was not correlated to disease stage. On cell proliferation, leptin acted as a moderate growth factor. We conclude that, next to the earlier described interactions within the BM, adipocytes influence MM cell behavior by affecting proliferation, apoptosis and migration and that part of these effects may be contributed to leptin.