Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that is considered curable; however, even with rituximab-based chemoimmunotherapy 40% relapse and die of disease. New agents with novel mechanisms of action are needed for this disease. We have demonstrated that treatment of DLBCL cells with the histone deacetylase (HDAC) inhibitor LBH589 as a single agent induced growth inhibition at nano-molar doses in association with inhibition of constitutively activated Akt in a time and dose dependent manner. The aim of the present study is to explore the mechanism by which LBH causes Akt deactivation in DLBCL. Mechanistically, this Akt dephosphorylation might be mediated through the deactivation of upstream kinases or the activation of a downstream phosphatase. To discern the role of transcriptional activation by LBH, we assessed the expression levels of a series of signaling proteins related to the regulation of Akt signaling pathways in LBH treated DLBCL cell line DHL-6. Proteins investigated included the p85 regulatory subunits of PI3K, PDK-1, Akt, PP1, and PP2A. We have shown that LBH exposure did not alter the expression level of any of these signaling proteins, excluding the involvement of transcriptional activation in altering the status of Akt phosphorylation. Consequently, we investigated a potential link between LBH-mediated Akt down-regulation and protein phosphatase activation by examining the effect of the PP1 inhibitor calyculin A on phospho-Akt levels in LBH-treated DHL-6 cells. Calyculin A completely abrogated the effect of LBH on phospho-Akt, suggesting the involvement of PP1 in LBH-facilitated Akt dephosphorylation. Next we investigated whether LBH causes PP1-mediated Akt dephosphorylation through HDAC inhibition. Since LBH treatment did not lead to increased PP1 levels, we hypothesized that the HDAC inhibitor might cause disruption of HDAC-PP1complexes, which would free PP1 to interact with Akt. LBH treatment produced disruption of the HDAC-PP1 complex accompanied by a dose dependent increase in PP1-Akt associations. We also found that DLBCL lines expressed both class I and class II HDACs and LBH treatment significantly inhibited HDAC3 and HDAC4 expression, while had only moderate effects on other HDACs. Further, we used HDAC3 and HDAC4 specific siRNAs to selectively knock down the expression of HDACs 3 and 4 and found that repressed expression of HDAC3 but not HDAC4 led to decreased Akt phosphorylation. In summary, the above findings suggest that LBH causes PP1-mediated Akt dephosphorylation by sequestering HDAC 3. This novel HDAC-PP1-Akt mechanism will allow the design of more effective strategies to optimize the use of these agents in DLBCL as single-agents or in combination with other drugs.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-208.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009