Abstract 1683: Autophagy inhibition for cancer therapy.
Authors: Balaburski Gregor , Leu Julia I-Ju , Beeharry Neil , Hayik Seth , Andrake Mark D. , Zhang Gao , Herlyn Meenhard , Villanueva Jessie , Dunbrack Roland L. , Yen Tim , George Donna L. , Murphy Maureen E. 
Published: August 2014

Abstract
In tumor but not normal cells, the stress-inducible heat shock protein 70 (HSP70) is abundantly expressed and becomes incorporated into lysosome membranes, where it serves to stabilize and protect lysosome function. Inhibition of HSP70 thus results in defective lysosome function, along with impaired autophagy. We previously reported that the HSP70 inhibitor phenylethynesulfonamide (PES) is a potent and effective autophagy inhibitor (1). PES shows marked cytotoxicity to tumor cells, but minimal effects in normal, non-transformed cells. We previously reported that PES binds to both HSP70 and to the constitutively-expressed family member HSC70; these are critical co-chaperones for HSP90, and we previously reported that treatment of tumor cells with PES leads to decreased function of HSP90 client proteins like HER2, AKT and CDK4 (2). We have recently performed a preliminary structure-activity relationship for PES. These studies revealed a novel PES analogue that we call PES-Cl, which shows 10-fold decreased IC50 for tumor cells, minimal cytotoxicity to normal cells, and a greatly enhanced ability to inhibit autophagy. In vitro we show that PES-Cl is cytotoxic to melanoma cells, including those with both intrinsic and acquired resistance to BRAF inhibitors, but shows minimal cytotoxicity to primary melanocytes. In a pre-clinical model for B-cell lymphoma (Eu-myc transgenic mouse), we show that PES-Cl demonstrates significant ability to extend the life of mice (p=0.000006), with no evidence for liver pathology or toxicity. We report that PES binds to the substrate-binding domain of HSP70, and requires the C-terminal helical ‘lid’ of this protein (amino acids 573-616) in order to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in HSP70, and we identify point mutants that fail to interact with this compound. Our cell cycle analyses of PES-Cl-treated cells show that this compound induces G2/M arrest. Interestingly, we also show that this HSP70 inhibitor impairs the activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in cell-free extracts. PES-Cl is thus a promising new HSP70 inhibitor that binds to the C-terminal ‘lid’ of HSP70, and multiple mechanisms of action: inhibition of autophagy, inhibition of HSP90 function, and inhibition of the Anaphase Promoting Complex/Cyclosome (APC/C).
1. Leu et al, Molecular Cell, 6(1):15-27, 2009
2. Leu et al, Molecular Cancer Research, 9(7):936-47, 2011
Citation Format: Gregor Balaburski, Julia I-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn, Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, Maureen E. Murphy. Autophagy inhibition for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2013-1683