575 GZ17-6.02 promotes autophagy and cell death in actinic keratoses via ATM-dependent mTOR inhibition

Abstract

GZ17-6.02 (602) is a novel investigational compound composed of curcumin, harmine, and isovanillin undergoing phase I clinical trials in oncology for solid tumors and lymphoma (NCT03775525). The goal of the present study is to determine the efficacy of 602 in killing actinic keratoses (AK) and to elucidate the molecular mechanisms underlying these effects. In a trypan assay, low concentrations of 602 killed AK cells to a greater degree than 5-fluorouracil (p<0.05). Alterations in cell signaling caused by 602 in AK cells were evaluated via fluorescence microscopy. 602 activated ATM, AMPK, and ULK1, resulting in inactivation of AKT, mTORC1 and mTORC2, reduced expression of K-RAS, N-RAS, HSP90, HSP70, and GRP78, reduced phosphorulation of ERBB 1/2/3/4, and increased phosphorylation of PERK and eIF2α. Mechanistic roles for individual proteins during the killing process were investigated via knockdown gene expression of suspected modulators of cell death. Knock down of Beclin1, ATM, AMPK, ULK1, or eIF2α suppressed the lethality of 602. Expression of constitutively active mTOR reduced its lethality. In control cells expressing LC3-GFP-RFP, 602 enhanced the formation of GFP+ vesicles after 4h. After 8h, the numbers of GFP+ vesicles declined, while RFP+ vesicle levels increased. Activated mTOR suppresed GFP+ autophagosome formation after 4h and 8h and no increase in RFP+ autolysosomes was observed. In summary, 602 induces autophagy, increases endoplasmic reticulum stress signaling, reducing expression of K-RAS and N-RAS, and inactivating AKT and mTOR. Expression of activated mTOR reduced killing, autophagosome formation and abolished autophagic flux. Our data argue that 602 causes cell death in AK cells via a targeted signaling mechanism and indicate that 602 may represent a novel therapeutic agent in the treatment of actinic keratoses.