Abstract
BRAF inhibitors (BRAFi) show remarkable benefit in melanoma patients with BRAFV600E mutations, but drug resistance develops rapidly. Combined or sequential therapy is promising for overcoming resistance. HDAC3 has been shown to deacetylate membrane-based GM3, thus activating Akt-mTOR signaling. HDAC3 inhibition prevents BRAFi resistance, but the mechanism is unclear. To evaluate the impact of HDAC3 expression on BRAFi resistance, HDAC3 was stably overexpressed in five BRAFV600E melanoma lines. BRAFi (1 mM vemurafenib) reduced mean Bcl-2 expression by 5.2-fold and increased caspase-3 11.3-fold, as shown by immunoblotting and cas-3 activity ELISA. In addition, BRAFi suppressed cell proliferation (by WST) by 5.6-fold and increased cell death (56.3+3.5% vs. 4.2+0.5%, by live/dead cell flow cytometry) at day 5 post BRAFi treatment (all p<0.001). Overexpression of HDAC3 promoted BRAFi resistance (BRAF Res), partially reversing these changes (all p<0.05). A greater ratio of cytoplasmic: nuclear HDAC3 localization was seen in metastatic > primary melanoma > normal melanocytes, suggesting an important role for HDAC3 translocation in promoting metastasis. Here, we found the combination of BRAFi and KPT-185 (nuclear export inhibitor analog) to significantly inhibit cell survival and proliferation in BRAF Res and HDAC3-overexpressing cells, almost to the level of naïve BRAF mutant cells treated with only BRAFi, suggesting increased BRAFi sensitivity. In addition, overexpression of chromosome region maintenance 1 (CRM1), a nuclear export receptor for HDAC3 transport, also increased BRAF Res, but not when cells were treated with HDAC3 inhibitors (MS-275, YC-5-164, -165 and -169). These data further emphasize the central role of HDAC3 in modulating the BRAFi response and suggest that inhibiting both nuclear export and BRAF simultaneously could maintain the melanoma drug response.
Published Version
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