Combination Therapy with the Histone Deacetylase Inhibitor Vorinostat Plus the Novel Aurora Kinase A Inhibitor MK-5108 Leads to Enhanced Lymphoma Cell Death Due to Acetylation of p53 and Repression of c-Myc, hTERT, and miRNA Levels.

Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few options after salvage therapy and transplant, and new agents are thus needed. MK-5108 is a novel aurora kinase inhibitor (AKI) with specificity against aurora kinase A, that produces G2/M phase cell cycle arrest. We show that addition of vorinostat, a histone and protein deacetylase inhibitor, to AKI treatment results in reactivation of proapoptotic genes and enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination, using cell growth, apoptosis, and flow cytometry assays, followed by molecular studies.

MK-5108 alone at 0.1 – 3 mM results in significant growth inhibition and apoptosis in multiple cell lines representing Hodgkin, Burkitt, and Non-Hodgkin lymphoma types, interestingly,DHL-4 and DHL-6 cells were more sensitive to this agent than to the pan-AKI MK-0457. Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by 50% or more in all lines tested. The combination of 1.5 mM vorinostat and 100 nM MK-5108 results in over 85% apoptosis of multiple lymphoma lines tested at 72 hours. Cell cycle analyses by FACS of MK-5108 treated cells show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat the G2/M peak decreases and there is a significant increase in the apoptotic sub-G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination revealed that vorinostat treatment leads to alteration in pro-apoptosis, growth arrest, and DNA damage response genes. Myc mRNA and protein levels are reduced by vorinostat, and repression of microRNAs (miRNAs) in the Myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, occurs with vorinostat and TSA. Prosurvival genes such as bcl-X_L and hTERT are downregulated five-fold by vorinostat treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. Vorinostat treatment leads to enhanced acetylation of p53, with a corresponding increase in the p53 target genes p21 and Noxa. To analyze the role of Myc inhibition in the sensitization by vorinostat of lymphoma cells to MK-5108, siRNA-mediated knock-down of Myc expression in L540 cells was performed. The siRNA-Myc transfected L540 cells showed enhanced sensitivity to MK-5108 as compared to control siRNA-null cells, as well as decreased hTERT levels, confirming the role of Myc inhibition by vorinostat as an integral part of the sensitization of lymphoma cells to MK-5108.

The HDACi vorinostat leads to both transcriptional and post-transcriptional changes that create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the aurora kinase A inhibitor MK-5108. These preclinical data support clinical trials of MK-5108 plus vorinostat in patients with relapsed or refractory lymphomas. [We acknowledge Merck Inc for providing Vorinostat, MK-0457, MK-5108, and research support.]

Kretzner:Merck: Research Funding. Yen:Merck: Research Funding. Kirschbaum:Merck: Research Funding, Speakers Bureau.