GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions

Background & Aims Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer. Autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. Approach & Results To explore the antitumor effects of GNS561, a new autophagy inhibitor, we first achieved in vitro assays using various human cancer cell lines. Having demonstrated that GNS561 displayed high liver tropism using mass spectrometry imaging, the potency of GNS561 on tumor was evaluated in vivo in two HCC models (human orthotopic patient-derived xenograft mouse model and diethylnitrosanime-induced cirrhotic immunocompetent rat model). Oral administration of GNS561 was well tolerated and decreased tumor growth in these two models. GNS561 mechanism of action was assessed in an HCC cell line, HepG2. We showed that due to its lysosomotropic properties, GNS561 could reach and inhibited its enzyme target, palmitoyl-protein thioesterase 1, resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of mTOR, lysosomal membrane permeabilization, caspase activation and cell death. Conclusions GNS561, currently tested in a global Phase 1b/2a clinical trial against primary liver cancer, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment. With an estimated 782,000 deaths in 2018, hepatocellular carcinoma (HCC) stands as the most common primary liver cancer and constitutes the fourth leading cause of cancer-related death worldwide (1). The rising incidence of HCC, the high worldwide mortality rate, and limited therapeutic options at advanced stages, make HCC a significant unmet medical need. Autophagy-related lysosomal cell death, either alone or in connection with several other cell death pathways, has been recognized as a major target for cancer therapy (2). Dysregulated autophagic-lysosomal activity and mTOR signaling were shown to allow cancer cells to become resistant to the cellular stress induced by chemotherapy and targeted therapy (3). Recently, several lysosome-specific inhibitors were shown to target palmitoyl-protein thioesterase 1 (PPT1), resulting in the modulation of protein palmitoylation and autophagy, and antitumor activity in melanoma and colon cancer models (4, 5). Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used for more than 50 years to prevent and treat malarial infections and autoimmune diseases. Based on the lysosomotropic properties and the capacity for autophagy inhibition, these molecules have been proposed as active drugs in cancer (6–9). Over 40 clinical trials have been reported to evaluate the activity of both CQ or HCQ as single agent or in combination with chemotherapy in several tumor types (6–8. However, the required drug concentrations to inhibit autophagy were not achieved in humans, leading to inconsistent results in cancer clinical trials (5, 10). This prompted research to identify novel compounds with potent inhibitory properties against autophagy for cancer therapy. We previously reported that GNS561 was efficient in intrahepatic cholangiocarcinoma (iCCA) by inhibiting late-stage autophagy (11). In this study, we investigated the mechanism of action of GNS561. We identified lysosomal PPT1 as a target of GNS561. Exposure to GNS561 induced lysosomal accumulation of unbound zinc ion (Zn2+), inhibition of PPT1 and cathepsin activity, blockage of autophagic flux and mTOR displacement. Interestingly, these effects resulted in lysosomal membrane permeabilization (LMP) and caspase activation that led to cancer cell death. This mechanism was associated with dose-dependent inhibition of cancer cell proliferation and tumor growth inhibition in two HCC in vivo models. These data establish PPT1 and lysosomes as major targets for cancer cells and led to the development of a clinical program investigating the effects of GNS561 in patients with advanced HCC. ### Competing Interest Statement SB, ER, FB, SM, MR, MN, JT, EB, JC, CD, GJ, CS, CA and PH are employees of Genoscience Pharma. SB, ER, FB, CD, CS, CA and PH are shareholders of Genoscience Pharma. SB, FB, JC and PH are co-inventors of a pending patent. The other authors declare no competing interests. * CTS : cathepsin CQ : chloroquine iCCA : intrahepatic cholangiocarcinoma DEN : diethylnitrosanime GAPDH : glyceraldehyde-3-phosphate dehydrogenase HCC : hepatocellular carcinoma HCQ : hydroxychloroquine LMP : lysosomal membrane permeabilization NtBuHA : N-tert-butylhydroxylamine PPT1 : palmitoyl-protein thioesterase 1