Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib.

Marcus J G W Ladds,Nicholas J Westwood,David P Lane,Emmet Mccormack,Ravi Bhatia,Gerald M Mcinerney,Kai Er Eng,Catherine J Drummond,Richard Svensson,Anna R Mccarthy,Ingeborg M M Van Leeuwen,Fredrik Tholander,Andrés Pastor-Fernández,Mihaela Popa,Gergana Popova,Lars Johansson,Sonia Laín

doi:10.1371/journal.pone.0195956

Abstract

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

Highlights

Tenovin-6 is a small molecule identified as an activator of p53-dependent transcription in a cell-based screen of 30,000 compounds [1]
The mechanism of autophagic flux blockage by tenovins and its therapeutic implications dose of vemurafenib and stained with propidium iodide. (C) Clonogenic assay in ARN8 melanoma cells treated as indicated for (i) 72 hours followed by staining with Giemsa to show surviving cells or (ii) 72 hours followed by four days of recovery in compound-free medium followed by staining with Giemsa stain to show cells capable of proliferating. (D) Clonogenic assay using ARN8 melanoma cells treated with (i) vemurafenib at 10 μM dose for 72 hours followed by a 72 hour treatment with vemurafenib at 10 μM and tenovin-50 at indicated doses or (ii) vemurafenib at indicated doses for 72 hours followed by 72 hours treatment with tenovin-50 at 7.5 μM
We examined the effect of a 48 h dose titration on ARN8 and Human normal dermal fibroblasts (HNDF) cells (S1 Fig panel A) and repeated the propidium iodide experiments and noted a very reproducible effect on cells (S1 Fig panel B)

Summary

Introduction

Tenovin-6 is a small molecule identified as an activator of p53-dependent transcription in a cell-based screen of 30,000 compounds [1]. It has been shown that tenovin-6 can induce apoptosis in leukaemic stem cells taken from chronic myelogenous leukaemia (CML) patient samples and achieve cure in murine CML models together with the Bcr-Abl tyrosine kinase inhibitor imatinib [4,5]. Tenovin-6 has been shown to synergise with the tyrosine-kinase inhibitor AC220 and significantly reduce acute myeloid leukaemic stem cells and lead to apoptosis of FLT3-ITD+ cells [6,7]. This remarkable anti-leukaemic stem cell effect of tenovin-6 is proposed, at least in part, to be a result of its ability to inhibit SirT1 protein deacetylase activity and subsequent activation of p53. SirT1 deacetylates lysine residues of p53 and promotes its subsequent ubiquitination and degradation [8,9]

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