Combination Therapy with Histone Deacetylase Inhibitor Vorinostat Plus Aurora Kinase Inhibitor MK-0457 Leads to Enhanced Lymphoma Cell Killing with Stabilization of p53 and Repression of C-Myc, hTERT, and Myc-Responsive miRNAs

Background: Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few therapeutic options after salvage therapy and transplant, and new agents are thus needed. MK-0457 is a novel aurora kinase inhibitor (AKI) which leads to polyploidy and G2 arrest by disregulating mitosis. We demonstrate that the addition of vorinostat, a broad spectrum HDACi, leads to the reactivation of proapoptotic and DNA damage response elements leading to enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination using MTS, Annexin V, and flow cytometry studies. Results: Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by at least 50% in all lines tested. For example, 1.5 mM vorinostat results in averages of 50% growth reduction and 30% apoptotic cells at 48 hours in the L540 HL cell line. MK-0457 alone at 100 nM – 500 nM results in up to a 50% reduction in cell growth and 20% apoptosis in the tested cell lines. However, the combination of 1.5 mM vorinostat and 100 nM MK-0457 results in 50 – 70% apoptosis of the L540 and KM-H2 Hodgkin cell lines at 48 hours. Cell cycle analyses by FACS show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat there is a significant increase in the sub G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination suggest an epigenetic as well as a protein acetylation mechanism for this activity. Prosurvival genes such as bcl-X_L and hTERT are downregulated five-fold by combination drug treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. The p53 tumor suppressor is stabilized both by increased acetylation in response to vorinostat as well as by reduced inhibitory phosphorylation at Ser315 by MK-0457-inhibited aurora kinase, leading to a corresponding increase in the p53 target genes p21 and Noxa. Protein and mRNA levels of c-myc are reduced . Repression of microRNAs (miRNAs) in the myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, is demonstrated with vorinostat and TSA. Conclusions: The HDACi vorinostat leads to both transcriptional and post-transcriptional changes which create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the AKI, MK-0457. These preclinical data support a clinical trial of MK-0457 plus vorinostat in patients with relapsed or refractory Hodgkin lymphoma. [We acknowledge Merck Inc for providing Vorinostat, MK-0457, and research support.]<