Abstract 2997: Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells: Regulatory pathways and clinical implications
Authors: Uziel Orit , Weiss Hana , Wolach Ofir , Nordenberg Jardena , Beery Einat , Bulvik Shlomo , Kanfer Gil , Cohen Olga , Ram Ron , Bakhanashvili Mary , Nativ-Magen Hila , Lahav Meir 
Published: June 2012

Abstract
Background: The proteasome inhibitor bortezomib (B) is an effective drug in multiple myeloma (MM). However, its exact mode of action is not yet fully understood. The importance of telomerase activity (TA) in the biology and prognosis MM is well established, but its response to B has not been assessed yet. In light of common signaling pathways connecting telomerase regulation and B known mechanisms of action, we surmised that the drug may affect the activity of telomerase in MM cells.
Methods: Two MM cell lines, ARP-1 and CAG, were exposed to B for 24-72 hours. Viability was assessed by the WST-1 assay, TA by the TRAP assay, hTERT expression by real time PCR, transcription factors binding by the ChIP (Chromatin Immuno Precipitation) assay, post-translational modifications were evaluated by Western blot. Ex vivo studies included the isolation of mononuclear cells from bone marrow aspirates of MM patients scheduled to treatment with B.
Results: B downregulated TA in all MM cell lines. However, the kinetics of this downregulation differed between the two lines. Whereas in ARP-1 cells TA decreased 24 hours after B treatment, in CAG cells the effect was observed only after 48 hours. These finding imply different regulatory mechanisms between these lines. In both lines B transcriptionally downregulated TA by inhibiting hTERT expression. ChIP analysis showed that SP-1 but not C-Myc or NFκB transcription factors mediate this transcriptional downregulation of telomerase. However, the two lines differed in phosphorylation of PKCα, which phosphorylates telomerase. Whereas in ARP-1 cells the phosphorylated form of PKCα was downregulated in response to B, the drug did not affect PKCα phosphorylation in CAG cells. These findings explain the different kinetics of telomerase downregulation and are in keeping with previous data showing different PKCα response between these two cell lines. We also show that telomerase phosphorylation decreased in response to B. These in vitro results were supported by ex vivo data from cells isolated from MM patients. Notably, the response of TA to B correlated with the clinical response of the patients to the drug. More over, this difference was dependent on the levels of p-PKCα in response to B.
Conclusions: Taken together, our results shed light on the effects and mechanism of B on TA in MM. The differential response of the cells to B may depend on the response of pPKCα pathway to the drug. Telomerase inhibition by B may vary in subsets of MM patients in relation to their pPKCα dependency. A correlation between the effect of B on TA and resistance to B in MM was found. As such, the ex vivo assessment of TA in response to treatment may serve as a prognostic feature and add to the therapeutic armamentarium by the addition of telomerase inhibitors in a defined subsets of patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2997. doi:10.1158/1538-7445.AM2011-2997