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. 2010 Apr;95(4):613-21.
doi: 10.3324/haematol.2009.013870. Epub 2010 Feb 4.

Vorinostat interferes with the signaling transduction pathway of T-cell receptor and synergizes with phosphoinositide-3 kinase inhibitors in cutaneous T-cell lymphoma

Magdalena B Wozniak  1 , Raquel Villuendas, James R Bischoff, Carmen Blanco Aparicio, Juan F Martínez Leal, Paloma de La Cueva, Ma Elena Rodriguez, Beatriz Herreros, Daniel Martin-Perez, Maria I Longo, Marta Herrera, Miguel A Piris, Pablo L Ortiz-Romero
Affiliations

Vorinostat interferes with the signaling transduction pathway of T-cell receptor and synergizes with phosphoinositide-3 kinase inhibitors in cutaneous T-cell lymphoma

Magdalena B Wozniak et al. Haematologica. 2010 Apr.

Abstract

Background: Vorinostat (suberoylanilide hydroxamic acid, SAHA), an inhibitor of class I and II histone deacetylases, has been approved for the treatment of cutaneous T-cell lymphoma. In spite of emerging information on the effect of vorinostat in many types of cancer, little is yet known about this drug's mechanism of action, which is essential for its proper use in combination therapy. We investigated alterations in gene expression profile over time in cutaneous T-cell lymphoma cells treated with vorinostat. Subsequently, we evaluated inhibitors of PI3K, PIM and HSP90 as potential combination agents in the treatment of cutaneous T-cell lymphoma.

Design and methods: The genes significantly up- or down-regulated by vorinostat over different time periods (2-fold change, false discovery rate corrected P value<0.05) were selected using the short-time series expression miner. Cell viability was assessed in vitro in cutaneous T-cell lymphoma cells through measuring intracellular ATP content. Drug interactions were analyzed by the combination index method with CalcuSyn software.

Results: The functional analysis suggests that vorinostat modifies signaling of T-cell receptor, MAPK, and JAK-STAT pathways. The phosphorylation studies of ZAP70 (Tyr319, Tyr493) and its downstream target AKT (Ser473) revealed that vorinostat inhibits phosphorylation of these kinases. With regards to effects on cutaneous T-cell lymphoma cells, combining vorinostat with PI3K inhibitors resulted in synergy while cytotoxic antagonism was observed when vorinostat was combined with HSP90 inhibitor.

Conclusions: These results demonstrate the potential targets of vorinostat, underlining the importance of T-cell receptor signaling inhibition following vorinostat treatment. Additionally, we showed that combination therapies involving histone deacetylase inhibitors and inhibitors of PI3K are potentially efficacious for the treatment of cutaneous T-cell lymphoma.

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Figures

Figure 1.
Figure 1.
Gene expression profiles of vorinostat (SAHA)-regulated genes. (A) Numbers of genes up- and down-regulated at each time point after treatment with vorinostat. Number of genes with significant profile that were differentially expressed by at least 2-fold from time 0 h to at least one later time point in response to vorinostat are shown. (B) Short time series expression miner (STEM) analysis flowchart. The figure represents selected up- and down-regulated profiles and significant genes within selected profiles are visualized for each cell line. Genes selected as differentially expressed were clustered using CLUSTER and visualized by TreeView. Genes were hierarchically clustered based on standard correlation coefficients. (C) Functional clustering of vorinostat-regulated T-cell receptor associated genes. The red asterisks indicate genes chosen for quantitative RT-PCR validation.
Figure 2.
Figure 2.
Effect of vorinostat (SAHA) on TCR-related genes. HuT78 and Myla cells were incubated with 5 μM (+) or 25 μM (++) of vorinostat for 0.5, 1, 6 and 24 h. Whole cell lysates were examined by western blotting for phospho-ZAP70 (Tyr319 and Tyr493) (A) and phospho-AKT (Ser473) (B). Total ZAP-70 and AKT were used as controls, respectively. Tubulin was used as a loading control of total proteins.
Figure 3.
Figure 3.
Combination studies of vorinostat with PI3K, PIM and HSP90 inhibitors in cutaneous T-cell lymphoma (CTCL) cells. (A) Effect of vorinostat on the targets of the inhibitors used for combination studies: PI3K inhibitors (GSK-3β - left panel), PIM inhibitor (PIM1 and PIM2 – middle panel) and HSP90 inhibitor (17-AAG – right panel) in five CTCL cell lines. The HH, HuT78, MJ, MYLA, and SeAx cells were treated with 5 μM of vorinostat over 24 h. The gene expression values were normalized with respect to time 0. The selected genes were visualized using CLUSTER and TreeView. (B) Synergistic interactions between vorinostat and PI3K inhibitors (LY294002 and ETP-45658). (C) Additive interactions for concomitant and sequential co-administration of vorinostat with PIM inhibitor (ETP-39010). (D) Antagonistic interactions between vorinostat and HSP90 inhibitor (17-AAG). (B, C, D) Synergistic, additive, or antagonistic effects of the drug combinations were determined by calculating the combination index value (CI) by isobologram analysis with CalcuSyn software. A CI = 1 indicates synergism, CI ± 1 indicates additivity and CI > 1 indicates antagonism.
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