Abstract 5440: Study of the mechanism of action and pharmacodynamic response of JNJ-26481585, a potent histone deacetylase inhibitor in the setting of melanoma

Abstract

Abstract Background: JNJ-26481585 is a potent histone deacetylase (HDAC) inhibitor. It is undergoing evaluation in phase I trials in solid tumours. The drug has shown plasma concentrations consistent with activity in preclinical models and target inhibition in surrogate and tumour tissue. Purpose: To determine the mechanism of action of JNJ-26481585 and to study factors determining sensitivity to the drug in-vitro in the setting of melanoma cell lines. Experimental Procedures: Array comparative genomic hybridization (array CGH) was carried out to study gene copy number. The growth inhibitory (GI50) concentrations of JNJ-26481585 and 17-allylamino, 17-demethoxygeldanamyin (17-AAG) were determined by sulforhodamine (SRB) assays. A panel of melanoma cell lines was exposed to GI50 and 5 X GI50 for 24hrs and gene expression profiling was performed using gene expression microarrays. Changes in protein expression were studied by western blot analysis. Results: Cells exposed to GI50 and 5X GI50 of 17-AAG for 24hrs showed a molecular signature of HSP90 inhibition including C-RAF depletion and HSP70 induction while this was not the case in equitoxic concentrations of JNJ-26481585. In addition, JNJ-26481585 did not reproducibly induce acetylation of tubulin which is a surrogate for cytoplasmic acetylation of proteins such as HSP90. JNJ-26481585 however induced robust acetylation of histone H3 suggestive of its mechanism of action predominantly as a class I HDAC inhibitor. The micropthalmia associated transcription factor (MITF) locus was amplified in the SKMEL-28 cells. The GI50 concentrations of JNJ-26481585 in the cell lines SKMEL-28, WM266.4, A2058, SKMEL-2 and SKMEL-5 were 5.4 (SD 0.86) nM, 20 (SD 0.58) nM, 28.1 (SD 0.7) nM, 30.4 (SD 3.6) nM, 33.4 (SD 3.6) nM respectively. The cell line panel was exposed to GI50 and 5 X GI50 of JNJ-26481585 for 24hrs. Protein levels of MITF was depleted in addition to MITF m-RNA levels being significantly reduced; p=0.002 (Welch T test with Benjamini and Hochberg correction for multiple testing; FDR 5%) at the time points studied, suggesting down regulation of MITF gene expression as a possible mechanism of action of JNJ-26481585. All the cell lines studied underwent apoptosis as evidenced by demonstration of cleaved PARP on western blotting. Conclusions: In this model, the mechanism of action of JNJ-26481585 is not predominantly due to HSP90 inhibition caused by acetylation of HSP90 in melanoma cells. Melanoma cell lines which have an amplification of MITF are sensitive to the HDAC inhibitor JNJ-26481585. Further exploration of MITF protein to evaluate pharmacodynamic response of JNJ-26481585 is warranted. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5440.