Abstract 5536: Short-term romidepsin treatment combined with MAPK pathway inhibition results in decreased mitochondrial hexokinase 2, increased mitochondrial Bim and apoptosis in BRAF mutant cancers
Authors: Bates Susan E. , Luchenko Victoria , Basseville Agnes , Bahr Julian , Chakraborty Arup R. , McDonald Andrew , Zimmer Alexandra , Piekarz Richard L. , Robey Robert W. 
Published: May 2015

Abstract
In ongoing efforts to increase the solid tumor efficacy of the histone deacetylase inhibitor romidepsin, we characterized romidepsin-resistant T-cell lymphoma cell lines that were found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim. This led us to examine the combination of romidepsin with MAPK pathway inhibitors in cell lines harboring the V600E BRAF mutation that leads to constitutive activation of the MAPK pathway. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF positive cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin in the presence or absence of the MAPK pathway inhibitors AZD6244 (selumetinib, 250 nM), PD0325901 (250 nM) or PLX4032 (vemurafenib, 1 µM) for 6 h, after which the cells were placed in romidepsin-free medium containing the inhibitors and incubated for an additional 42 h. Apoptosis was subsequently measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment alone (control, median annexin value 7.1%±3.8% vs. treated, median annexin value 34.7%±17.8%, p&lt;0.001). Combined treatment with romidepsin and selumetinib, PD0325901 or vemurafenib resulted in a significantly higher percent of annexin positive cells (p&lt;0.05) in 9 to 11 of the 11 BRAF mutant cell lines examined compared to treatment with romidepsin alone; expression of dephosphorylated Bim was observed. Immunoblot analysis demonstrated inhibition of the MAPK pathway at the concentrations used; apoptosis was associated with increased PARP cleavage. Using mitochondrial separation techniques, we also observed increased levels of Bim in the mitochondrial membrane fraction and reduced expression of mitochondrial hexokinase 2 in cells treated with combined romidepsin/MEK inhibitor compared to either compound alone. Our results suggest a critical role for apoptotic pathways in cell death due to romidepsin, that MAPK signaling promotes resistance via Bim degradation, and that combined treatment with romidpesin and BRAF or MEK inhibitors may be useful in the treatment of cancers harboring BRAF mutations.
Citation Format: Susan E. Bates, Victoria Luchenko, Agnes Basseville, Julian Bahr, Arup R. Chakraborty, Andrew McDonald, Alexandra Zimmer, Richard L. Piekarz, Robert W. Robey. Short-term romidepsin treatment combined with MAPK pathway inhibition results in decreased mitochondrial hexokinase 2, increased mitochondrial Bim and apoptosis in BRAF mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2014-5536