SUMMARY
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. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two HCC in vivo models. We showed that GNS561, which is an effective lysosomotropic agent, can reach and inhibit 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. Accordingly, 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.
INTRODUCTION
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 (Bray et al. 2018). 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 (Aits and Jaattela 2013). 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 (Klempner et al. 2013). Recently, several lysosome-specific inhibitors were shown to target palmitoyl-protein thioesterase 1 (PPT1), resulting in the modulation of protein palmitoylation and antitumor activity in melanoma and colon cancer models (Rebecca et al. 2017, Rebecca et al. 2019). PPT1 palmitoylates proteins, enabling their degradation and intracellular trafficking of membrane-bound proteins. This process was shown to play a central role in the control of cellular autophagy. PPT1 was reported to be highly expressed in several cancer cell lines as well as in advanced stage cancers in patients (Rebecca et al. 2019).
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 (Dolgin 2019, Pérez-Hernández et al. 2019) and have been extensively investigated in recent years (Kimura et al. 2013, Manic et al. 2014, Zhang et al. 2015, Shi et al. 2017, Verbaanderd et al. 2017, Xu et al. 2018). 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 (Manic et al. 2014, Shi et al. 2017, Verbaanderd et al. 2017). However, the required drug concentrations to inhibit autophagy were not achieved in humans, leading to inconsistent results in cancer clinical trials (Pascolo 2016, Rebecca et al. 2017, Plantone and Koudriavtseva 2018). 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 and inducing a dose-dependent build-up of enlarged lysosomes (Brun et al. 2019). In this study, we investigated the lysosomotropism of GNS561 and then the disruption of related lysosomal functions such as autophagy and lysosomal enzymatic activity. We also identified lysosomal PPT1 as a target GNS561. Exposure to GNS561 was shown to induce lysosomal unbound zinc ion (Zn2+) accumulation, 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 several 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.
RESULTS
GNS561 displays activity against human cancer cell lines and patient-derived cells
The effects of GNS561 on cell viability were investigated in a panel of human cancer cell lines, including HCC, iCCA and colon, renal cell, breast, prostate, lung, and ovarian carcinoma as well as acute myeloid leukemia, glioblastoma, and melanoma. As shown in Table 1, GNS561 showed potent antitumor activity ranging from 0.22 ± 0.06 μM for the most sensitive cell line (LN-18, a glioblastoma cell line) to 7.27 ± 1.71 μM for the least sensitive cell line (NIH:OVCAR3, an ovarian cancer cell line). GNS561 was at least 10-fold more effective than HCQ in cultured cancer cells. GNS561 also displayed activity in primary HCC patient-derived cells and was on average 3-fold more potent than sorafenib, a reference drug in HCC treatment (mean IC50 3.37 ± 2.40 μM for GNS561 vs 10.43 ± 4.09 μM for sorafenib).
GNS561 has antitumor properties in HCC in vivo models
The whole-body tissue distribution of GNS561 was investigated in rats after repeated oral administration of GNS561 at a dose of 40 mg/kg/day for 28 days. Seven hours after the last administration, the GNS561 level was measured by mass spectrometry imaging in the liver, lung, stomach, brain, eye, salivary gland, kidney, heart, fat, muscle, testis, and skin (Figure 1). GNS561 mainly accumulated in the liver, stomach and lung as shown by the calculated organ/blood ratio (Figure 1B). Lower concentrations of GNS561 were also detected in eyes, skin, brain and testis, indicating that GNS561 crosses the blood/brain barrier and the blood/testis barrier to a limited extent (brain to blood and testis to blood ratios were 0.21 and 0.40, respectively).
Based on the high concentrations of GNS561 in the liver and potent in vitro activity against HCC cells, the effects of GNS561 were investigated in vivo using two liver cancer models, including the human HCC orthotopic patient-derived LI0752 xenograft mouse model and the diethylnitrosanime (DEN)-induced immunocompetent rat HCC model.
In the HCC patient-derived LI0752 xenograft BALB/c nude mouse model, tumor volume and weight were reduced by 37.1% and 34.4%, respectively, in mice treated with GNS561 at 50 mg/kg compared to the control (Figure S1A and B). Consistently, GNS561 treatment induced a decrease in serum AFP levels in a dose-dependent manner and was significantly different from the control at days 21 and 28 after treatment (Figure S1C-G).
Since HCC often develops in cirrhotic livers in humans, we further characterized the antitumor effects of GNS561 in a DEN-induced cirrhotic rat model of HCC. Rats with already developed HCC were either treated with sorafenib at 10 mg/kg, GNS561 at 15 mg/kg, or the combination of both drugs (Figure S2). In this model, tumor progression was significantly reduced by sorafenib (33.0%) and GNS561 (33.0%) compared to an untreated control group, and the greatest decrease in tumor progression was observed by the combination (68%) that displayed an additive effect (Figure 2A). Magnetic resonance imaging analyses further showed a significant increase in the mean tumor size of 9.97 ± 0.97 mm in control rats compared to 6.45 ± 0.35 mm with sorafenib, 5.48 ± 1.00 mm in GNS561 and 3.83 ± 0.52 mm in the combination group (Figure 2B). Following liver resection, the macroscopic counting of tumor nodules revealed significantly lower numbers in all treated groups compared to the control group (Figure 2C). Immunohistochemical analyses of liver tumors showed a significantly lower Cyclin D1-positive nuclear staining in the tumors of rats treated with GNS561 or by the combination of GNS561 with sorafenib compared to the control group (Figure 2D). GNS561 and combination treatments also significantly reduced Ki67 staining compared to the control group (Figure 2E). The effects on Cyclin D1 and Ki67 were primarily related to GNS561 exposure, as sorafenib alone showed no statistically significant differences in Cyclin D1 or Ki67 staining compared to the control group. Our results further showed that GNS561 and the combination treatment did not interfere with lipid or glucose metabolism or kidney function but slightly affected some liver functions (Table S1).
GNS561 activates the caspase-dependent apoptosis pathway
We further wanted to characterize the antitumor effect of GNS561 and to determine whether GNS561 could trigger apoptotic cell death. To this end, annexin V/propidium iodide (PI) analysis was performed by flow cytometry after 48 h of GNS561 exposure in HepG2 cells. Early (Annexin V+/PI-staining) and late (Annexin V+/PI+ staining) apoptosis increased in a dose-dependent manner after GNS561 exposure (Figure 3A). The induction of apoptosis was confirmed by immunodetection of poly-ADP-ribose polymerase cleavage in GNS561-treated cells (Figure 3B). We further examined whether GNS561-induced apoptosis was related to caspase activation. After 6 h of exposure, GNS561 had no effect on caspase 8 and caspase 3/7 activity in HepG2 cells (Figure 3C). In contrast, activation of caspase 8 and caspase 3/7 was observed after 24 h of treatment with GNS561, and this effect was sustained at 30 h. A decrease in cell viability was concomitant with caspase activation (Figure 3C). The induction of caspase activation was confirmed by flow cytometry (Figure 3D) and by detection of cleavage of caspase 3 using immunoblot analysis (Figure 3E). Moreover, to confirm that GNS561-induced cell death is caspase-dependent apoptosis, pretreatment (1 h) with the cell-permeable pan-caspase inhibitor Z-VAD-FMK (5 μM) was performed. Cell viability was restored in the presence of Z-VAD-FMK (Figure 3F), further confirming that GNS561 induced a caspase-dependent apoptotic cell death.
GNS561 is a lysosomotropic agent
The intracellular localization of GNS561 in HepG2 cells was visualized using GNS561D, the photoactivable analog of GNS561 containing a diazide moiety (Figure 4A). GNS561D showed a punctuate fluorescent signal that colocalized with the intracellular vesicle-like structure stained by LAMP1 (Figure 4B), demonstrating that GN561 accumulated in lysosomes and is a lysosomotropic agent. Pretreatment with NH4Cl, a weak base that rapidly increases lysosomal pH, was further used to validate the lysosomotropic character of GNS561. As shown in Figure 4B, NH4Cl pretreatment strongly prevented lysosomal accumulation of GNS561D. Then, we investigated whether GNS561 lysosomotropism was related to induced cell death. For this purpose, HepG2 cells were pretreated for 2 h with NH4Cl and then treated with GNS561 for 24 h. Although a concentration of 20 mM NH4Cl alone slightly decreased viability (Figure 4C), it significantly attenuated the larger decrease in viability induced by GNS561. These results were confirmed by pretreatment with bafilomycin A1 (Baf A1), an inhibitor of the vacuolar H+-ATPase (Figure S3). Therefore, disrupting GNS561 lysosomal localization protected against GNS561-mediated cell death. These results suggested that GNS561 antitumor activity in HepG2 cells is caused by its lysosomotropism.
GNS561 modulates lysosomal functions
The GNS561 lysosomotropism-dependent cell death prompted us to examine GNS561 capacity to modulate lysosomal characteristics and functions.
Following continuous exposure to GNS561, staining of LysoTracker, which is a reagent allowing the identification of the lysosomal compartment, increased in HepG2 cells (Figure 4D), suggesting that GNS561 prompted a dose-dependent build-up of enlarged lysosomes. We therefore examined the enzymatic activity of three prominent lysosomal proteinases, two cysteine cathepsins B (CTSB) and L (CTSL), and aspartic cathepsin D (CTSD). After 6 and 24 h of treatment, GNS561 significantly impaired, in a dose-dependent manner, the enzymatic activity of cathepsins (Figure 4E). However, this decreased activity did not relate to a direct GNS561-dependent inhibition of cathepsin activities (Figure S4). Based on the literature, depressed proteolytic activity of cathepsins may result from an increased Zn2+ lysosomal concentration and/or altered maturation of cathepsin precursors. Indeed, it has been described that Zn2+ may downregulate the proteolytic activity of CSTB and CTSL (Lockwood 2010, Lockwood 2013, Lockwood 2019). We investigated whether GNS561 modified unbound Zn2+ localization in HepG2 cells. As shown in Figure 4D, GNS561 induced a strong accumulation of unbound Zn2+ in lysosomes, as evidenced by colocalization of the fluorescent signals of Fluozin and LysoTracker in the merged images. This increase in lysosomal unbound Zn2+ could explain the decreased proteolytic activity of CTSL and CTSB. Cathepsins are synthesized as inactive zymogens, which are converted to their mature active forms by other proteases or by autocatalytic processing (Turk et al. 2012). As depicted in Figure 4F, GNS561 did not impact CTSB maturation, while it impaired the maturation of both CTSL and CSTD (increase of precursor forms) and decreased their catalytic activity accordingly.
As GNS561 induced lysosomal dysfunction, the effect of GNS561 on the autophagic process was investigated. Herein, we showed that the GNS561-induced accumulation of light chain 3 phosphatidylethanolamine conjugate was not enhanced in the presence of BafA1 (Figure S5), suggesting that GNS561 blocked autophagic flux.
PPT1 is a target of GNS561
Since PPT1 is critical for lysosomal function and is described to be the molecular target of chloroquine derivatives (Rebecca et al. 2017, Rebecca et al. 2019), we investigated whether PPT1 could be a molecular target of GNS561. First, the binding of GNS561 to recombinant PPT1 was analyzed in vitro by nano differential scanning fluorimetry using HCQ as a positive control (Rebecca et al. 2019). In the presence of GNS561 and HCQ, we observed a significant dose-dependent decrease in PPT1 melting temperature (Figure 5A). Additionally, inhibition of PPT1 enzymatic activity was observed in HepG2 cells treated with GNS561 (Figure 5B). Moreover, the chemical mimetic N-tert-butylhydroxylamine (NtBuHA) attenuated autophagy inhibition associated with GNS561 (Figure 5C), indicating that inhibition of PPT1 function by GNS561 induced the observed anti-autophagy effect.
To determine whether inhibition of PPT1 function was responsible for the antitumoral activity of GNS561, HepG2 cells were treated with GNS561 with or without NtBuHA pretreatment. As shown in Figure 5D, NtBuHA partially prevented the antitumor activity of GNS561, as evidenced by the increased viability of cells pretreated with NtBuHA. The same rescue effect of NtBuHA pretreatment was observed for HCQ used as a positive control (Figure S7). After demonstrating that NtBuHA had no impact on GNS561 lysosomal localization (Figure S6), we validated that the impact of NtBuHA pretreatment was due to its PPT1 mimetism, suggesting that inhibition of PPT1 function by GNS561 was partially liable for its antitumoral activity.
The results of Rebecca et al. suggested that PPT1 inhibition could result in mTOR inhibition through the displacement of mTOR from the lysosomal membrane (Rebecca et al. 2017, Rebecca et al. 2019). Thus, we investigated the localization of mTOR after GNS561 treatment using immunofluorescence microscopy. HCQ and EAD1 were used as positive controls (Sironi et al. 2019). As shown in Figure 5E, GNS561 treatment, as well HCQ and EAD1 treatments, significantly impaired mTOR localization to the lysosomal surface. Therefore, GNS561-induced PPT1 inhibition resulted in displacement of mTOR from the lysosomal membrane and consequently likely inhibited the mTOR signaling pathway.
GNS561 induces LMP and cathepsin-dependent cell death
To characterize GNS561-induced changes in lysosomes, we analyzed LMP. To this end, we took advantage of the steady endocytic capacity of cells to load fluorescent dextran into lysosomes and the translocation of lysosomal localized dextran into the cytosol after LMP-inducing insult. Fluorescent dextran in healthy cells appears in dense punctate structures representing intact lysosomes, whereas after LMP, a diffuse staining pattern throughout the cytoplasm is seen. After GNS561 treatment, such diffuse dextran staining was observed (Figure 6A), suggesting an induction of LMP. As seen in Figure 6B, the loss of membrane integrity, which is the hallmark of LMP, was observed by transmission electron microscopy of HepG2 cells treated with 3 μM GNS561 for 24 h. To confirm this effect, cathepsin localization was studied after GNS561 treatment. After 48 h of treatment, GNS561 decreased cathepsin staining (Figure 6C), indicating that cathepsins were released into the cytosol, thus validating LMP.
As cathepsin release into the cytosol after LMP may trigger cytosolic cellular death signaling (Oberle et al. 2010), we evaluated the role of cathepsins in GNS56-induced cell death. To this end, HepG2 cells were pretreated with an inhibitor of CTSD, pepstatin A, or an inhibitor of CTSB, CA-074-Me. Under these conditions, cell viability was partially rescued (Figure 6D and E), suggesting that the GNS561-induced apoptotic pathway is at least partially cathepsin-dependent.
DISCUSSION
Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal functions. Lysosomes are acidic and catabolic organelles found in nucleated human cells that are responsible for the disposal and recycling of used and damaged macromolecules and organelles, as well as the assimilation of extracellular materials incorporated into the cell by endocytosis, autophagy, and phagocytosis. Increased autophagic flux and changes in lysosomal compartments in cancer cells have been shown to promote invasion, proliferation, tumor growth, angiogenesis, and drug resistance. Consistently, lysosomal changes are expected to sensitize cells to lysosome-targeting anticancer drugs (Kallunki et al. 2013). Many steps in the autophagy pathway represent potentially druggable targets and several clinical trials have aimed to inhibit autophagy by inhibiting lysosomal functions using CQ and HCQ. Unfortunately, CQ and HCQ failed to demonstrate consistent antitumor effects possibly due to subeffective anticancer concentrations in humans, even with high doses. Drug screening led us to identify GNS561 as a lead compound that displays lysosomotropism and significantly higher antiproliferative effects in human cancer cells compared to HCQ.
We previously reported that GNS561 yielded antiproliferative activity in iCCA, inhibited late-stage autophagy, and induced a dose-dependent enlargement of lysosomes (Brun et al. 2019). Based on these preliminary results, we further investigated the cellular mechanisms by which GNS561 may lead to lysosomal changes and death in cancer cells. In this study, we confirmed that GNS561 antitumor properties are strongly dependent on its lysosomotropic properties. In accordance with the hypothesis proposed in our previous study (Brun et al. 2019), we showed here that GNS561 induced a dose-dependent increase in the number of enlarged lysosomes, LMP leading to cytosolic cathepsin release, caspase activation, and apoptotic cell death. These observations confirm prior reports that highlight the capability of lysosomotropic agents to cause lysosomal stress and lysosomal enlargement (Wang et al. 2018). Moreover, studies demonstrated that lysosomal unbound Zn2+ buildup led to lysosomal swelling, LMP, release of lysosomal enzymes, and cell death (Hwang et al. 2008, Chung et al. 2009, Hwang et al. 2010, Yu et al. 2010). Further investigations are needed to identify the upstream signals that initiate LMP in GNS561-treated cells.
PPT1, an enzyme involved in the removal of thioester-linked fatty acyl groups in proteins and thus subsequently enabling the degradation and intracellular trafficking of membrane-bound proteins, plays a central role in the control of cellular autophagy. PPT1 is highly expressed in several cancer cell lines as well as in advanced stage cancers in patients (Rebecca et al. 2019). Recent data have shown that lysosome-specific inhibitors targeting PPT1 can modulate protein palmitoylation and display antitumor activity in melanoma and colon cancer models (Rebecca et al. 2017). Our data showed that PPT1 acts as a molecular target of GNS561. GNS561 bound to PPT1 and inhibited its activity in cells. Cells treated with the chemical mimetic NtBuHA were partially resistant to GNS561-mediated cytotoxicity and attenuated GNS561-associated autophagic flux inhibition, suggesting that inhibition of the thioesterase activity of PPT1 is essential for the anti-autophagic and antitumoral effects of GNS561.
In our study, we observed that GNS561 modified the intracellular distribution and localization of mTOR. This is in accordance with previous studies showing that inhibition of PPT1 may displace the mTOR protein from the lysosomal membrane as a result of the inhibition of vATPase/Ragulator/Rag GTPase interactions (Sancak et al. 2010, Korolchuk et al. 2011, Carroll et al. 2016, Rebecca et al. 2017, Rabanal-Ruiz and Korolchuk 2018, Rebecca et al. 2019, Sironi et al. 2019). It was also described that lysosomal mTORClocalization brings it in close vicinity to its main regulator, Rheb, and that as a result, the mTOR/Rheb interaction can activate mTOR kinase activity leading to the phosphorylation of downstream effectors (Carroll et al. 2016). Consistently, we hypothesized that GNS561-induced PPT1 inhibition led to mTOR signaling pathway inhibition.
As previously observed in iCCA (Brun et al. 2019), we showed here that GNS561 induced a significant decrease in the enzymatic activity of cathepsins. This decreased activity is unlikely due to a direct inhibition of CTSL, CTSB and CTSD by GNS561 but rather could be the consequence of both impairment of CTSL and CTSD maturation and lysosomal unbound Zn2+ accumulation. As cathepsin activity is optimal in acidic pH (Gieselmann et al. 1985, Turk et al. 1999), we could also speculate that GNS561 may negatively influence the proteolytic activity of cathepsins by inducing an increase in lysosomal pH via PPT1 inhibition. In fact, other authors have shown that PPT1 deficiency in Cln1-/- mice disrupted the delivery of the v-ATPase subunit V0a1 to the lysosomal membrane, leading to a dysregulation of lysosomal acidification (Bagh et al. 2016). The authors suggested that S-palmitoylation by PPT1 may play a critical role in the trafficking of the V0a1 subunit of v-ATPase to the lysosomal membrane and in lysosomal pH regulation.
Based on prior studies, GNS561 was neither a zinc ionophore nor a zinc chelator (data not shown), unlike CQ (Xue et al. 2014). However, our hypothesis that GNS561-induced PPT1 inhibition could lead to lysosomal deacidification could also explain the observed lysosomal unbound Zn2+ accumulation after GNS561 treatment. In fact, as lysosomal pH is mainly regulated by cation/anion movement across the lysosomal membrane, it was suggested that a proton motive force was required to mediate unbound Zn2+ efflux (Lockwood 2013, Bin et al. 2019).
In summary, GNS561-induced PPT1 inhibition may lead to two main mechanisms inducing cancer cell death. One is related to lysosomal deacidification, which induces lysosomal unbound Zn2+ accumulation, a decrease in the enzymatic activity of cathepsins, inhibition of autophagic flux, lysosomal swelling, LMP, cathepsin release, and caspase-dependent apoptosis. The other is linked to prevention of the interaction between v-ATPase and the Ragulator complex, blockage of mTOR lysosomal recruitment, impairment of mTOR–Rheb interaction and finally the inhibition of mTOR signaling pathway. Thus, by targeting PPT1, GNS561 acts as a regulator of autophagy and mTOR, two major processes that drive cancer aggressiveness. Finally, as lysosomes and autophagy are associated with adaptive mechanisms of resistance to mTOR inhibition (Xie et al. 2013), GNS561 can disable mTOR function and downregulate adaptive mechanisms of resistance.
An extensive preclinical program has been conducted to evaluate the antitumor activity, pharmacological properties and toxicology of GNS561. Our data showed that GNS561 displays antiproliferative effects in several human cancer cells (cell lines and primary patient-derived cells) and that GNS561 was more potent than HCQ. Analysis of the whole-body tissue distribution of GNS561 in rats after repeated oral dosing of GNS561 showed that GNS561 was mainly concentrated in the liver, stomach and lung. The data are consistent with the basic lipophilic nature of GNS561 and with studies showing that basic lipophilic drugs show high lysosomal tropism and high uptake in lysosome-profuse tissues, such as the liver and the lung (Daniel and Wójcikowski 1997). As GNS561 had a high liver tropism, the effect of GNS561 on tumor growth in vivo was evaluated using two liver cancer models: one orthotopic human liver cancer xenograft mouse model (with an HCC patient-derived cell line, LI0752) and one DEN-induced cirrhotic rat model with HCC. These studies showed that GNS561 administered by oral gavage was well tolerated up to the doses of 50 mg/kg/day for 6 days in mice and up to 15 mg/kg/day for 6 weeks in rats and induced significant antitumor growth activity that was either comparable to or higher than sorafenib. In addition, ina DEN-induced cirrhotic rat model with HCC, the combination of GNS561 with sorafenib exerted an additive effect in controlling tumor progression and cell proliferation. Furthermore, instead of that observed with CQ and HCQ (Harder et al. 2018), the distribution of GNS561 into the central nervous system was limited. Inactivating PPT1 mutations have long been known to induce infantile neuronal cerebral lipofuscinosis and induce retinopathy during childhood (Metelitsina et al. 2016). Germline PPT1 mutations were shown to selectively affect the central nervous system, with no effects in other tissues. Prior clinical experience using CQ and HCQ showed that retinopathy was one of the major toxicities in patients (Marmor et al. 2011). Authors have suggested that novel PPT1 inhibitors may take advantage of not crossing the blood-brain barrier to avoid retinal toxicity (Rebecca et al. 2019). Interestingly, our data shown that the disposition of GNS561 displays limited penetration into the brain in rats, consistent with the lack of neurological and retinal toxicity observed in the current Phase 1b/2a clinical trial of GNS561 (ClinicalTrials.gov).
In brief, our findings strengthen the importance of PPT1 and lysosomes as cancer targets. Recently, it was shown that PPT1 inhibition by CQ derivatives or genetic Ppt1 inhibition increases the antitumor activity of anti-PD-1 antibody in melanoma by M2 to M1 phenotype switching in macrophages and a reduction in myeloid-derived suppressor cells in the tumor (Sharma et al. 2020). As such, GNS561 represents a promising new candidate for drug development in HCC either alone or in combination with other drugs, such as anti-PD-1 antibody.
METHODS
Details of the materials and methods are provided in the Supplementary Methods.
Cell culture
All cell lines were cultured in the presence of 5% CO2 and 95% air in a humidified incubator and were maintained in medium containing 1% penicillin-streptomycin (Dutscher, #P06-07100) and 10% fetal bovine serum (HyClone, #SV30160.03C), except NIH:OVCAR3 and KG-1 cell lines, which were cultured in medium supplemented with 20% fetal bovine serum.
Animal models
The animals were checked daily for clinical signs, effects of tumor growth and any other abnormal effects. For experiments involving the mouse model (performed in CrownBio facilities), the protocol and any amendment(s) or procedures involving the care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of CrownBio prior to experimentation, and during the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care. For the rat model, all animals received humane care in accordance with the Guidelines on the Humane Treatment of Laboratory Animals (Directive 2010/63/EU), and experiments were approved by the animal Ethics Committee: GIN Ethics Committee No.004.
Statistical analysis
Statistical analyses were performed using Prism 8.4.3 software (GraphPad Software Inc., CA, USA). For datasets with normal distribution, multiple comparisons were performed using one-way ANOVA with Dunnett’s post hoc analysis. The parametric Student t-test was used to compare two groups of data with normal distribution. Data are presented as the mean values ± standard error mean (SEM) unless stated otherwise. Statistical significance was defined as a p-value < 0.05 and has been indicated by an asterisk in all figures.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online.
AUTHOR CONTRIBUTIONS
Conceptualization, S.B, E.R, F.B and P.H.; Methodology, S.B., F.B., T.D, P.H; Validation, S.B., Z.M.J, G.L, J.S, T.S, T.D; Formal Analysis, S.B., Z.M.J, M.N, J.T, A.H, G.L, R.L, T.S, M.G.P, J.P.B; Investigation, Z.M.J, S.M., M.N, J.T, A.H, L.V, E.B, R.L, M.G.P, G.R; Resources, J.C, C.A; Data Curation, C.D, G.J; Visualization, S.B, Z.M.J; Supervision, S.B, F.B, P.H; Project Administration, S.B; Writing – original draft preparation, S.B.; Writing – review and editing, S.B., E.R, F.B., Z.M.J, M.R, G.L, C.S, and P.H.; Funding Acquisition, P.H.
DECLARATION OF INTERESTS
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.
ACKNOWLEDGMENTS
The authors are very grateful to Dr. Sebastian Müller and Dr. Raphaël Rodriguez from Curie Institute for mechanistic analysis, Pr. Thierry Levade and Dr. Nathalie Andrieu from CRCT for the PPT1 enzymatic assay, Keerthi Kurma and Seyedeh Tayebeh Ahmad Pour for the Institute for Advanced Biosciences for technical support during animal experiments and Dr. François Autelitano, Dr. Marie Guillemot and Philippe Fabre for Zn2+ localization analysis.
Footnotes
↵15 Lead contact