Inhibition of HDACs and Aminopeptidases Is Highly Synergistic in Myeloma Cells Resulting in Cell Death Via the Upregulation of BIRC3, a Key Mediator of NF-KappaB Signalling.

Abstract 607

Myeloma cells rely on a number of mechanisms to maintain their survival, including the silencing of genes that would normally check uncontrolled proliferation and lead to apoptosis. Epigenetic alterations such as histone deacetylation contribute significantly to the pathogenesis of both solid and haematological malignancies and are associated with the silencing of tumour suppressor genes. Pan-histone deacetylase inhibitors (HDACs), such as SAHA, panobinostat, depsipeptide, and numerous others have been published to have anti-myeloma activity. Here we report the effects of a novel pan HDAC inhibitor, CHR-3996, on multiple myeloma cells. Results: CHR-3996 was potently cytotoxic against a panel of myeloma cell lines (H929, KMS11, LP-1, MM1s, RPMI-8226, and U266) with a GI50 ranging from 9 to 65 nM and was equally as potent in primary patient myeloma cells. The loss of cell viability was associated with an increase in apoptotic cells; EndonucleaseG and Noxa were both up-regulated and caspase 9 was cleaved. Furthermore a decrease in apoptosis was demonstrated in the presence of a pan-caspase inhibitor indicating cell death is largely dependent on caspase-mediated apoptosis. There was no effect on bone marrow stromal (BMS) cell viability but there was an observed decrease in the IL-6 and VEGF the BMS cells secrete, both of which promote the growth and survival of myeloma cells in the bone marrow microenvironment. CHR-3996 caused an increase in acetylated H3K9 but there was minimal change in the levels of ubiquitinated proteins in the cell or acetylated alpha-tubulin, indicating low activity against HDAC6 or the proteasome (also demonstrated by an assay to specifically measure proteasome function). In addition to induction of apoptosis, cell cycle analysis showed an increased proportion of cells in G0/G1 indicating cell cycle arrest. In-keeping with data from treating myeloma cells with SAHA and panobinostat, an increase in cell cycle inhibitor p21 was observed. Gene expression profiling (GEP) identified changes in cell cycle regulators, indications of increased cell stress (elevated CHOP, ATF3, and TAO kinase 3), repression of Wnt (up-regulation of NLK, GSK3beta) and mTOR (decreased 4E-BP1) signalling, and changes in key pro- and anti-apoptotic proteins (for example SMAD3, BCL2, BIM, BID, and BIRC5). When used in combination studies CHR-3996 was highly synergistic in vitro with tosedostat, an aminopeptidase inhibitor, which we have previously shown to have anti-myeloma activity via the induction of the amino acid deprivation response and autophagy. One of the largest changes the GEP analysis identified was BIRC3 (CIAP2), an inhibitor of NF-kappaB signalling, which increased 23.5 fold with CHR-3996 as a single agent and over 100-fold when added to H929 cells in combination with tosedostat. Additionally both CHR-3996 and todestat independently up-regulated expression of members of the IkappaB family. Altered expression and nuclear localisation of canonical and non-canonical NF-kappaB family members were observed by immuno-fluorescence and immunoblotting, suggesting targeting of NF-kappaB signalling as a reason for the high degree of synergy between these two compounds. Early data suggest oral CHR-3996 is effective in a subcutaneous in vivo myeloma model and, reflecting the in vitro data, there is a degree of synergy when administered with tosedostat. Conclusions: The novel compound CHR-3996 is a potent HDAC inhibitor that leads to increased H3K9 acetylation but has no detectable activity against HDAC6 or proteasome activity in myeloma. It induces apoptosis of myeloma cells without affecting BMS cell viability. CHR-3996 shows a very high degree of synergy with an aminopeptidase inhibitor, tosedostat, potentially through targeting the NF-kappaB pathway. It has exciting therapeutic potential either as a mono-therapy or in combination with other agents.