Abstract 4110: Downregulation of telomerase activity in response to epoxomicin in MM cells.

Background Proteasome inhibitors are effective drugs against multiple myeloma (MM). The mechanism of action of these pleiotropic agents has not been yet fully elucidated. The importance of telomerase activity (TA) in MM has been repeatedly demonstrated. Previously we showed that the first generation proteasome inhibitor bortezomib (B) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance. In the current study we evaluated the anti telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG132 (MG) in order to clarify whether telomerase inhibition represents a class effect. Methods MM cell lines, ARP-1, CAG, RPMI 8226 and U266 were exposed to EP or MG for 24-48 hours. Viability was assessed by the WST-1 and Trypan Blue exclusion assays, TA by the TRAP assay, hTERT expression by real time PCR, transcription factors (TF) binding by the ChIP assay and post-translational modifications were evaluated by IP and Western blot. Results EP and MG differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and hTERT (the gene encoding for telomerase) expression was faster in CAG than ARP-1 cells. EP was more potent than MG and therefore further mechanistic studies were performed on this compound. The inhibition of TA was mainly transcriptionally regulated and the phosphorylation of the enzyme was not changed. The binding of three positive regulator transcription factors (TF): SP1, c-Myc and NFκB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular concentrations. In ARP-1 cells the SP1 and c-MYC binding and content were similarly affected by EP while NFκB was not affected. Interestingly the transcription factor WT-1 (Wilms’ tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged. Conclusions Our results demonstrate the effects of EP and MG on TA in MM. These results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors. The inhibitory effect on TA is exerted on several regulatory levels, transcriptional and post translational. SP1, C-Myc and NFkB were involved in mediating these effects. A novel finding of this study is the role of WT-1. WT-1 appears as a negative regulator of hTERT expression. The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in MM. Citation Format: Orit Uziel, Naama Shalem, Einat Beery, Jardena Nordenberg, Mary Bakhanashvili, Anna Gutkin, Meir Lahav. Downregulation of telomerase activity in response to epoxomicin in MM cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4110. doi:10.1158/1538-7445.AM2013-4110