The HDAC inhibitor AR42 interacts with pazopanib to kill trametinib/dabrafenib-resistant melanoma cells in vitro and in vivo

// Laurence Booth 1 , Jane L. Roberts 1 , Cindy Sander 3 , John Lee 4 , John M. Kirkwood 3 , Andrew Poklepovic 2 , Paul Dent 1 1 Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298-0035, USA 2 Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298-0035, USA 3 University of Pittsburgh Cancer Institute Melanoma and Skin Cancer Program, Hillman Cancer Research Pavilion Laboratory L1.32c, Pittsburgh PA 15232, USA 4 Chan Soon-Shiong Institute of Molecular Medicine, Culver City, CA 90232, USA Correspondence to: Paul Dent, email: paul.dent@vcuhealth.org Keywords: autophagy, chaperone, death receptor, ER stress Received: December 20, 2016 Accepted: January 19, 2017 Published: January 27, 2017 ABSTRACT Studies focused on the killing of activated B-RAF melanoma cells by the histone deacetylase (HDAC) inhibitor AR42. Compared to other tumor cell lines, PDX melanoma isolates were significantly more sensitive to AR42-induced killing. AR42 and the multi-kinase inhibitor pazopanib interacted to activate: an eIF2α–Beclin1 pathway causing autophagosome formation; an eIF2α–DR4/DR5/CD95 pathway; and an eIF2α-dependent reduction in the expression of c-FLIP-s, MCL-1 and BCL-XL. AR42 did not alter basal chaperone activity but increased the ability of pazopanib to inhibit HSP90, HSP70 and GRP78. AR42 and pazopanib caused HSP90/HSP70 dissociation from RAF-1 and B-RAF that resulted in reduced ‘RAF’ expression. The drug combination activated a DNA-damage-ATM-AMPK pathway that was associated with: NFκB activation; reduced mTOR S2448 and ULK-1 S757 phosphorylation; and increased ULK-1 S317 and ATG13 S318 phosphorylation. Knock down of PERK, eIF2α, Beclin1, ATG5 or AMPKα, or expression of IκB S32A S36A, ca-mTOR or TRX, reduced cell killing. AR42, via lysosomal degradation, reduced the protein expression of HDACs 2/5/6/10/11. In vivo , a 3-day exposure of dabrafenib/trametinib resistant melanoma cells to the AR42 pazopanib combination reduced tumor growth and enhanced survival from ~25 to ~40 days. Tumor cells that had adapted through therapy exhibited elevated HGF expression and the c-MET inhibitor crizotinib enhanced AR42 pazopanib lethality in this evolved drug-resistant population.