The DAC Inhibitor LBH589 Is Highly Effective in Both “Therapy”-Sensitive and -Resistant Lymphomas and Enhances the Anti-Tumor Activity of Chemotherapy Agents, Monoclonal Antibodies, and Bortezomib

Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Histones are one of the target proteins, which is why DACs (Class I) are sometimes referred to as histone deacetylases (HDACs). DACs also target non-histone proteins, which include transcription factors, a-tubulin, and HSP90 (Class II DACs). LBH589 is a novel and potent DAC Inhibitor that inhibits both class I and II of DAC activity, inducing cell death in tumor cells undergoing pre-clinical and clinical testing. Other investigators had demonstrated that DACs can down-regulate Bcl-2 expression in lymphoma cells (Duan, Heckman, Mol Cell Biol.2005; 25(5):1608-19), and potentially overcome chemotherapy resistance in B-cell lymphomas. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphomas we studied the effects of LBH589 at various doses (2nM to 20nM) and schedules of administration (before, after or concurrent administration) in the anti-tumor activity of chemotherapy agents and monoclonal antibodies in a panel of rituximab-sensitive (RSCL) (Raji and RL cells) and rituximab-resistant cell lines (Raji 2R, Raji 4RH and RL-4RH) (RRCL). In addition, we utilized lymphoma cells isolated from patients with treatment-naive or refractory/relapsed B-cell lymphomas. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells lines were exposed to the following chemotherapy agents or monoclonal antibodies: CDDP (1 to 100mM), Doxorubicin (4 to 16mM), Vincristine (1 to 5mM), Bortezomib (1 to 10nM) or Rituximab (10mg/ml), A20 (10mg/ml), or Trastuzumab (Isotype, 10mg) alone or in combination with LBH589. In dose-sequence studies the treatment with LBH589 preceded or followed in vitro exposure to the chemotherapy agent or the monoclonal antibody by 24 hrs. Changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 15 minute intervals for 24 and 48 hrs. Patient-derived tumor cells (10 samples up to date) were exposed to either LBH589, bortezomib or both at different doses. Changes in ATP content were determined using the cell titer glow assay. LBH859 was active as a single agent against rituximab-sensitive or resistant cells tested as well as in tumor cells derived from lymphoma patients. Significant anti-tumor activity was more pronounced after 48 hrs of drug exposure even at 2nM doses of LBH859. In addition, significant synergistic activity was observed by combining LBH589 and chemotherapy agents (CDDP, doxorubicin or Vincristine), bortezomib or the two mAbs targeting CD20 studied (rituximab and veltuzumab). The sequence of administration impacted the responses observed with bortezomib and mAbs, but not the chemotherapy agents evaluated. Pre-incubation of NHL cells with LBH589 prior to bortezomib or mAb therapy leads to the maximum optimization of each agent than another sequence of administration. In summary, our data suggests that LBH589 is active against various RSCL, RRCL and patient-derived tumor cells and potentiate the anti-tumor effects of chemotherapy agents, mAbs targeting CD20, and bortezomib. Findings suggest that LBH589 added to systemic anti-CD20 and/or chemotherapy could result in a novel and potent treatment strategy against B-cell lymphomas.