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The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2024 Apr;389(1):51–60. doi: 10.1124/jpet.123.002021

Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo

Daniel P Radin 1, Sophie Shifman 1, Ian R Outhwaite 1, Aryan Sharma 1, Robert Bases 1, Markus A Seeliger 1, Stella E Tsirka 1,✉
PMCID: PMC10949164  PMID: 38296645

Abstract

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment.

SIGNIFICANCE STATEMENT

We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

Introduction

Gliomas are tumors that may arise from multiple cell types in the central nervous system. Glioblastoma (GBM), a stage IV glioma, is the most common and aggressive primary brain tumor diagnosed in adults. GBM carries a poor prognosis despite treatment with surgery, temozolomide (TMZ) chemotherapy, radiation, and the newly added tumor treating fields (TTF) therapy (Stupp et al., 2017). Median survival is still only 16 to 20 months (Stupp et al., 2017). When GBM recurs, there exists no effective treatment modality to significantly extend patient survival.

The nonsurgical treatments used in the management of GBM all involve targeting rapidly dividing cells and the eventual induction of DNA-damage-dependent cell death. To that end, cellular mechanisms that confer resistance to TMZ by augmenting DNA repair may be induced and rapidly confer resistance to ionizing radiation and TTF therapy. Of the multiple pathways initiated by DNA damage in glioma cells, principle among them is the induction of cytoprotective autophagy (Kanzawa et al., 2004; Ito et al., 2005; Lomonaco et al., 2009; Lin et al., 2012; Knizhnik et al., 2013; Zou et al., 2014; Hori et al., 2015; Lee et al., 2015; Ye et al., 2016; Shteingauz et al., 2018). Autophagy has not only been implicated in resistance to TMZ, radiation, and TTF but also supports tumor cell survival in areas of hypoxia (Hu et al., 2012; Jawhari et al., 2016; Abdul Rahim et al., 2017) and dampens antitumor immune responses (Liang et al., 2012; DeVorkin et al., 2019; Valdor et al., 2019; Sharma et al., 2020; Kim et al., 2021). The latter may partially explain the lack of efficacy of programmed cell death protein 1 inhibitors in GBM patients. Therefore, inhibiting autophagy in GBM may offer an effective method of managing this intractable neoplasia.

Lucanthone, a known antischistosome agent, has been since described as an inhibitor of topoisomerase I and II (Bases and Mendez, 1997; Dassonneville and Bailly, 1999) during replication and transcription, as well as of AP endonuclease (Mendez et al., 2002; Luo and Kelley, 2004; Naidu et al., 2011), a key enzyme in base excision repair. We and others have shown that lucanthone acts as an inhibitor of autophagy (Carew et al., 2011; Radin et al., 2022) and is efficacious in the treatment of some types of solid tumors when paired with ionizing radiation. Lucanthone crosses the blood-brain barrier (Bases and Mendez, 1997) and induces regression of breast cancer metastases (Del Rowe et al., 1999). It is eliminated via the liver and kidney. Patients tolerate a total dose of 10 mg/kg per day (which achieves serum levels of ∼3–4 μg/ml or 8–12 μM) well, with no signs of nausea or other side effects (Del Rowe et al., 1999). Our published data revealed that lucanthone is effective against mouse and human GBM lines in culture and in vivo and preferentially targets glioma stem-like cells (GSC), normalizes tumor vasculature, reduces tumor hypoxia, and increases cytotoxic T cell infiltration into developing tumors (Radin et al., 2022). Lucanthone is structurally similar to chloroquine/hydroxychloroquine and may target palmitoyl protein thioesterase 1 (PPT1), whose pro-tumorigenic function has been evidenced in several types of peripheral tumors (Amaravadi and Winkler, 2012; McAfee et al., 2012; Rebecca et al., 2017, 2019; Nicastri et al., 2018; Sharma et al., 2020). Indeed, newer generation PPT1 inhibitors exhibit oncolytic activity against tumors arising from cells that are resistant to radiation and immunotherapy (Rebecca et al., 2017), suggesting that PPT1 inhibition may be useful for the treatment of cancer after recurrence.

In the current study we investigate in detail lucanthone’s mechanism of action in GBM and show that it is able to slow the growth of tumors derived from cells resistant to high concentrations of TMZ by inhibiting autophagy and reducing the number of Olig2+ GSC. We also find that lucanthone abates tumor microtube (TM) network formation in GSC. Importantly, we provide evidence that lucanthone may act to inhibit PPT1 in patient-derived glioma cell lines. Our data further support the need to investigate clinical applications of lucanthone in TMZ-resistant GBΜ.

Materials and Methods

Glioma Cell Lines.

Glioma cell lines were described previously (Radin et al., 2022). Briefly, luciferase+ GL261 cells (GLUC2) were a kind gift from Dr. Michael Lim’s laboratory. They are derived from a chemically induced astrocytoma in C57BL/6 mice (Ausman et al., 1970). Luciferase+ KR158 cells were obtained from the laboratories of Drs. Tyler Jacks and Behnam Badie and are derived from genetically engineered Nf1/Tp53 mutants (Reilly et al., 2000). Two primary patient-derived glioma cell lines were also used in these studies: GBM43 and GBM9, which carry Nf1 and Tp53 mutations and Kras and Tp53 mutations, respectively. The lines were obtained from the Mayo Clinic Brain Tumor Patient-Derived Xenograft National Resource. Cells were maintained in DMEM, 10% serum, 1% antibiotic, and 1% sodium pyruvate and incubated at 37°C with 5% CO2. To enrich for glioma stem-like cells (GSC), serum was reduced stepwise over a week as described previously (Yi et al., 2013). GSC were cultured in serum-free DMEM medium containing F12 supplement along with pyruvate, antibiotics, N2 supplement, epidermal growth factor, fibroblast growth factor, and heparin (Yi et al., 2013).

Phalloidin Texas-Red Stain.

Phalloidin Texas Red Stain was performed according to manufacturer’s instructions. Briefly, GBM9 and GBM43 GSC were plated onto glass coverslips that had been precoated with a 5% Matrigel solution for two hours and allowed to adhere overnight. Cultures were treated with medium or 3 µM lucanthone for 24 hours after which the medium was aspirated and cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes. Plates were then washed 3x with 0.3% TX-100 in PBS and wells were blocked with 3% normal goat/donkey serum/0.3% TX-100 in PBS for 1 hour. Cultures were then stained with Phalloidin Texas Red diluted 1:40 in PBS according to manufacturer’s instructions for an hour. Cells were then washed 3x with PBS, counterstained with DAPI and imaged under confocal microscopy.

Animals.

C57/Bl6 (WT) mice were bred under maximum isolation on a 12:12 hour light:dark cycle with food ad libitum.

Murine Glioma Model.

Gliomas established in immunocompetent mice were established in 3- to 4-month-old male and female mice as described previously (Miyauchi et al., 2016, 2018; Caponegro et al., 2021). Parental and TMZ-resistant GLUC2 GSC were dissociated with accutase and counted. Mice were anesthetized with 20 mg/kg avertin; a midline incision was made in the scalp, the skin was retracted, and a small burr hole was drilled in the skull at the following stereotactic coordinates from bregma: –1mm anteroposterior and +2 mediolateral. 1x105 GLUC2 GSC resuspended in PBS were injected over a period of 4 minutes at a depth of 3 mm. At the end of the injection, the needle was kept in the injection site for a further 3 minutes. After needle removal, the incision was sutured and mice were placed on a heating pad until they fully recovered from anesthesia. During the disease course if mice were found to have lost more than 15% of their initial body weight, they were euthanized. All animal procedures were approved by the Stony Brook University Institutional Animal Care and Use Committee.

Lucanthone Treatment In Vivo.

Lucanthone was supplied by Dr. Robert Bases (Albert Einstein College of Medicine). Lucanthone was solubilized in 10% DMSO, 40% 2-Hydroxypropyl-β-cyclodextrin in PBS. After confirming the presence of gliomas on day 7 [using in vivo imaging system (IVIS) bioluminescence visualization], mice bearing GLUC2 GSC were randomly divided into control and experimental groups and treated with either saline or 50mg/kg lucanthone i.p. daily (days 7 to 20). On day 21, tumors were visualized by bioluminescent imaging, as previously described. For studies utilizing tumors derived from TMZ-resistant GLUC2 spheroids, mice were treated starting on day 7 through day 26 and then imaged and euthanized on day 27. Eleven control mice (five male, six female) were used in the experiments. For the lucanthone-treated mice, nine were used (four male, five female).

RNA Extraction from Tumors.

To prepare RNA from the glioma tissue, tumor-bearing mice were deeply anesthetized, euthanized, and perfused with 30 ml ice-cold PBS. Tumors were microdissected from brains and mechanically dissociated in Trizol (Caponegro et al., 2021). To obtain cDNA, one microgram of RNA was reverse transcribed on a Veriti thermocycler using the High Capacity cDNA Reverse Transcription Kit. Amplification was performed on a StepOnePlus real-time polymerase chain reaction machine using a SYBR green kit (Applied Biosystems).

Immunohistochemistry.

Mice were anesthetized with 20 mg/kg avertin and transcardially perfused with 30ml PBS followed by 30ml 4% paraformaldehyde in PBS. Brains were removed and post-fixed in 4% paraformaldehyde in PBS overnight. They were dehydrated for 48 hours in 30% w/v sucrose in PBS. Brains were then embedded in optimal cutting temperature compound (Tissue-Tek) and 20 μm coronal sections throughout the tumor were taken on a Leica cryostat (Nusslock, Germany) and collected on Superfrost plus microscope slides. For immunohistochemical analysis, slides were brought to room temperature, washed 3x with 0.3% TX-100 in PBS, and then blocked with 1% bovine serum albumin/0.3% TX-100 in PBS for 1 hour. Slides were incubated overnight with appropriate primary antibodies [Ki67, Olig2, P62, Cathepsin D, lysosomal associated membrane protein 1 (LAMP1)]. The primary antibody was removed and slides were washed 3x 0.3% TX-100 in PBS and incubated with appropriate secondary antibodies for 1 hour. Slides were washed 3x with PBS, and counterstained with DAPI. Immunoreactivity was visualized by confocal imaging using the Leica SP8-x system, with white light and argon lasers.

Western Blot.

Immunoblotting was done as described previously (Radin et al., 2022). Briefly, cells were lysed in 50mM Tris-HCl (pH 7.4) with 1% Nonidet P-40, 0.25% sodium deoxycholate, 150mM NaCl, 1% SDS, and 1mM sodium orthovanadate. Proteins were denatured by boiling and treatment with β-mercaptoethanol. Proteins were run on SDS-page gels and transferred onto PVDF membranes (Immobilon; Millipore). Membranes were washed with Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked in a 5% bovine serum albumin in TBST solution for 1 hour. Membranes were then incubated with LC3 (1:1000, CST 2775S), PPT1 (1:1000, Thermo Fisher PA5-102896), and β-Actin (1:2000; Sigma-Aldrich) primary antibodies overnight at 4°C. Membranes were rinsed in TBST, probed with associated horseradish peroxidase-conjugated secondary antibodies, and exposed to SuperSignal West Pico PLUS Chemiluminescent Substrate for 1 minute (Thermo Fisher Scientific), and subsequently developed using the iBright Imaging System.

Molecular Docking.

Receptors were prepared for docking using PDBs 1eh5 (Bellizzi et al., 2000) and 3gro (Dobrovetsky et al., 2009). Residues in bovine PPT1 (PDB 1eh5) were mutated to corresponding human residues. Unmodeled residues were built in 3gro. Selenomethionines in 3gro were replaced with methionines. Nonprotein atoms were removed and hydrogen atoms were added to both structures. Charges were added with AMBER ff14sb (Maier et al., 2015) and structures were minimized. Charges were added to lucanthone using AMBER ff14sb. Lucanthone was docked using a standard DOCK6 flexible docking protocol (Mukherjee et al., 2010) against both receptors. Importantly, DOCK6 binding spheres were generated across each receptor to explore a variety of potential binding sites. Poses were ranked by DOCK6 gridscore, and the highest-scoring pose was selected. Figure images were generated in UCSF Chimera (Pettersen et al., 2004).

Statistical Analysis.

Data comparing two population means with a normal distribution were analyzed using Student’s t test. Data with nonnormal distributions were analyzed using a Mann–Whitney test. Differences in cumulative distributions were assessed with the Kolomogorov–Smirnov test. Tumor cell circularity was calculated using the equation Circularity = 4*π*[area/(perimeter2)]. ANOVA and Tukey test was used to analyze Western blot data. The alpha value was set at 0.05 prior to starting experiments. Power analysis was used to determine the appropriate number of animals used in each experiment. Viability experiments were replicated with the two tumor lines. Statistical analysis was performed using Graphpad Prism (Graphpad Software Inc., La Jolla, CA).

Results

Lucanthone Exhibits Increased Potency over TMZ in Perturbing GSC Growth, Proliferation, and Sphere Formation.

TMZ is the Food and Drug Administration-approved chemotherapeutic agent for GBM shown to selectively kill non-stem glioma cells. The nontargeted GSCs subsequently drive tumor recurrence (Chen et al., 2012). Increasing concentrations of TMZ were used to treat the GSC lines and cell survival was quantified. TMZ exhibited an IC50 of > 200 μM in each cell line and an IC90 of > 750 μM (Fig. 1A). Given that TMZ accumulates in brain tumors at a concentration less than 20 μM (Portnow et al., 2009), these results reemphasize that TMZ only poorly targets GSC.

Fig. 1.

Fig. 1.

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Lucanthone exhibits increased potency over TMZ in perturbing GSC growth, proliferation, and sphere formation. GBM43 and GBM GSC were treated with TMZ (A) or lucanthone (B) for 5 days, after which an MTT assay was performed. Bars are mean +/− S.E.M. N = 3–4 independent experiments ANOVA P < 0.01 for each drug treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Dunnett’s multiple comparison test to control-treated cells. (C) Calcein-AM stained cells after treatment with lucanthone for 5 days, illustrating reductions in cell number, sphere formation, and sphere size. (D) Limiting dilution assay. GBM43 and GBM9 were plated in 96-well plates at low densities (100, 200, 400 cells per well) and treated with indicated concentrations of lucanthone for 2 weeks, after which sphere formation was assessed and quantified. Line indicates median sphere formation. N = 6–7 independent experiments ANOVA P < 0.0001 for each cell line. ****P < 0.0001, Dunnett’s multiple comparison test compared with control-treated cultures.

We previously showed that lucanthone at 10 μM was effective in reducing the growth of patient-derived GSC (Radin et al., 2022). To gain a better understanding of the effective dose-response range of lucanthone against GSC, cultures were treated with up to 3 μM lucanthone. Lucanthone exhibited an IC50 of ∼1.5 μM for each cell line with an IC90 of ∼3 μM (Fig. 1B).

We also assessed the ability of tumor cells to form spheres upon lucanthone treatment. GSC were dissociated with accutase and plated in 96-well plates overnight. GSC were then treated with 0, 1, 2, or 3 μM lucanthone for 5 days, after which survival was assessed. In both cell lines concentrations of 2 and 3 μM lucanthone strongly reduced GSC proliferation and sphere formation by 75% and 90%, respectively (Fig. 1C). In a limiting dilution assay a small number of cells (100, 200, 400) were treated with 400 or 800 nM lucanthone (Mulkearns-Hubert et al., 2019). Cultures were allowed to grow for 2 weeks and then were scored for sphere formation using light microscopy. Four hundred nM lucanthone markedly reduced GBM9 and GBM43 sphere formation ability by more than 60%. Eight hundred nM treatment nearly abolished self-renewal in these cultures (Fig. 1D). As lucanthone can penetrate the blood-brain barrier and was shown to accumulate in the serum of patients at 8 to 12 µM (Del Rowe et al., 1999), these data highlight lucanthone’s ability to limit GSC self-renewal at clinically relevant concentrations.

Lucanthone Reduces Tumor Microtube Formation in GSC.

As reported in other types of tumors (Osswald et al., 2015; Weil et al., 2017; Venkataramani et al., 2019; Horne et al., 2021; Xie et al., 2021; Joseph et al., 2022), GSC cells in our cultures seemed to form connections with other cells, through structures that resemble tumor microtubes. Cultures treated with lucanthone for 5 days had fewer connections. To interrogate this possibility quantitatively, GSCs were plated in Matrigel-covered glass cover slips and treated with vehicle or 3 µM lucanthone for 24 hours, after which cultures were stained with Texas Red Phalloidin to visualize actin-based networks. In GBM43 GSC, control-treated cultures exhibited extensive TM formation, with some TMs extending beyond 100 µm (Fig. 2A). Twenty-four-hour lucanthone treatment resulted in a pronounced decrease in the number of these connections between GBM43 GSC as well as a modest decrease in mean TM length (Fig. 2, A and B). The morphology of dissociated GBM43 GSC treated with lucanthone correlated with an increase in cell circularity (Fig. 2, A and B). GBM9 GSC were also interconnected at baseline (Fig. 2, C and D), and lucanthone treatment reduced the number of TMs per cell and the length of TMs (Figs. 2C, 3D). Similarly, lucanthone-treated GBM9 cells were rounder and less spread out (Figs. 2C, 3D). These findings suggest that lucanthone treatment disturbs TM formation in GSC.

Fig. 2.

Fig. 2.

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Lucanthone reduces TM formation in GSC in vitro. GBM43 (A) and GBM9 (C) cultures treated with medium or 3 µM lucanthone for 24 hours visualized by phalloidin Texas Red stain. Quantification of TM length, TM per cell, and cell circularity of GBM43 (B) and GBM9 (D) cultures. **P < 0.01, ****P < 0.0001, Kolmogorov–Smirnov test comparing distributions to control-treated cultures.

Fig. 3.

Fig. 3.

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Lucanthone slows the growth of TMZ-resistant gliomas in vivo. (A) GLUC2 cells selected for TMZ resistance express the stemness marker CD133. (B) Treatment scheme used for the study. (C) Mice were segregated into treatment groups with statistically similar baseline tumor luminescence. (D) Fold increase in luminescence of each mouse at Day 27 versus Day 7. (E) Tumor luminescence of 6 mice per treatment group. (F) Representative examples of tumors of control- and lucanthone-treated mice. (G) Tumor volume quantifications. Bars are mean +/− SEM. N = 9 or more mice per group. *P < 0.05, ***P < 0.001, t test.

Lucanthone Slows the Growth of TMZ-Resistant Gliomas.

Our culture data have indicated that lucanthone may serve as a potent adjuvant to overcome cytoprotective autophagy induction after DNA damage elicited by TMZ (Radin et al., 2022). Unfortunately, the clinical utility of lysosomal autophagy inhibitors, such as chloroquine, is very limited because they augment the systemic toxicity of TMZ and limit the clinical utility of this dual treatment regimen (Compter et al., 2021). To examine whether lucanthone would still be able to maintain its efficacy in gliomas that exhibit resistance to TMZ, we used cells that had been selected to grow in high TMZ concentrations. We have reported that GLUC2 cells that grow in high concentrations of TMZ exhibit increased levels of expression of the stemness marker CD133 (Radin et al., 2022) (Fig. 3A), suggesting a dynamic response to this continued TMZ treatment. GLUC2 spheres were subjected to five cycles of 500 μM repeated TMZ treatment. The spheres were dissociated and implanted into the striatum of mice and allowed to form tumors for 1 week (Fig. 3B). Tumor engraftment was assessed by bioluminescent (IVIS) imaging. Animals with similar tumor luminescent intensity were matched (Fig. 3C) and then treated either with vehicle-control or with 50 mg/kg/d lucanthone for approximately 3 weeks. Tumor burden was assessed by IVIS imaging over time. Twenty-seven days post-inoculation, relative luminescence of lucanthone-treated mice was approximately 50% of that displayed by control-treated mice (Figs. 3, D and E). To measure the tumor size more accurately, the animals were euthanized and the brain sections subjected to H&E stain (Fig. 3F). Quantification of tumor volume revealed that lucanthone reduced tumor growth by ∼50% (Fig. 3G). Our results demonstrate that even after the GBM cells adapt to persistent TMZ treatment in vivo, they may still be sensitive to lucanthone, likely because it acts through a mechanism distinct from TMZ.

Lucanthone Targets Lysosomes, Inhibits Autophagy, and Reduces Olig2 Levels In Vivo in TMZ-Resistant Gliomas.

To examine whether the in vivo function of lucanthone was still to inhibit autophagy in the TMZ-resistant gliomas, tumor sections were stained for the autophagosome marker P62, the lysosomal protease Cathepsin D, and the lysosomal membrane marker LAMP1. In control-treated tumors, P62 levels, as well as levels of Cathepsin D and LAMP1 were low (Fig. 4, A and B). In lucanthone-treated tumors, P62 was significantly increased as was Cathepsin D and LAMP1 (Fig. 4, A and B). These findings confirm that in vivo as well lucanthone exerted its oncolytic effects by inhibiting autophagy through targeting lysosomes.

Fig. 4.

Fig. 4.

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Lucanthone inhibits autophagy and reduces Olig2 in TMZ-resistant gliomas. (A) Representative immunohistochemical images of P62, Cathepsin D, and LAMP1 in tumors of control- and lucanthone-treated mice. (B) Quantification of P62, Cathepsin D, and LAMP1 intensity in control- and lucanthone treated tumors. Bars are mean +/− S.E.M, N = 5 animals per group. **P < 0.01, ***P < 0.001, t test. (C) Representative immunohistochemical images of Olig2 in tumors of control and lucanthone-treated mice. (D) Quantification of Olig2 intensity and Olig2+ area in control and lucanthone-treated tumors. Bars are mean +/− S.E.M, N = 6 animals per group. **P < 0.01, Mann–Whitney test, ***P < 0.001, t test.

We have previously shown that lucanthone’s effects on the glioma tumors results in reduced Olig2 levels (Radin et al., 2022). As shown in Fig. 4, C and D, control-treated tumors exhibited widespread Olig2 positivity, particularly near the edge of tumors. However, in lucanthone-treated tumors, Olig2 intensity as well as Olig2 area positivity were significantly decreased (Fig. 4, C and D), illustrating that lucanthone still retained its ability to reduce Olig2 levels in TMZ-resistant GLUC2 gliomas.

Lucanthone May Act as a PPT1 Inhibitor.

Our previous results indicate that lucanthone targeted lysosomes and disrupted autophagic flux in glioma cells cultured in serum-rich medium and in culture conditions that enhanced the stem-like qualities of tumor cells (Radin et al., 2022). Drugs with autophagy inhibition activity, similar to lucanthone, have been shown to modulate the activity of palmitoyl protein thioesterase 1 (PPT1): the structurally related autophagy inhibitor chloroquine was recently shown to act as an inhibitor of PPT1, which can enhance antitumor efficacy of anti-programmed cell death protein 1 treatment in melanoma (Sharma et al., 2020). We sought to examine whether PPT1 may be a relevant marker in GBM and whether lucanthone may act on PPT1. The prognostic value of PPT1 has been evaluated in peripheral cancers previously (Rebecca et al., 2019). To examine its potential prognostic utility in gliomas, we leveraged available data from The Cancer Genome Atlas. Combining survival times of GBM and low-grade glioma, we found that patients with elevated PPT1 expression had a median survival of 33.7 months compared with 93.2 months for patients whose tumors exhibit low PPT1 expression (Supplemental Fig. 1A). We also found that PPT1 expression increases with tumor grade (Supplemental Fig. 1B) and is most elevated in the mesenchymal subtype of GBM (Supplemental Fig. 1C). Using the Ivy Glioblastoma atlas (Puchalski et al., 2018), we determined that PPT1 seems to be uniformly expressed among multiple different areas of the tumor (Supplemental Fig. 1D).

To investigate the interaction between PPT1 and lucanthone, we modeled the preferred bound form of lucanthone. Bovine PPT1, which is 97.5% similar to human PPT1, assumes the same overall structure in the presence (PDB 1eh5) or absence (PDB 1ei9) of palmitate (Bellizzi et al., 2000). Only a single structure of human PPT1 is available (PDB 3gro) (Dobrovetsky et al., 2009), which has a Cα RMSD of 0.48 Å to 1eh5 excluding residues R151-A169 (PDB numbering); these residues hinge toward the palmitate-binding cleft (Fig. 5A). The functional relevance of the 3gro conformation is unclear as it would not permit substrate binding (Fig. 5B). Additionally, the 3gro conformation may be an artifact of crystal packing; R151-A169 are at the monomer interface and contribute to extensive intermolecular contacts.

Fig. 5.

Fig. 5.

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Lucanthone is a potential PPT1 inhibitor. (A) 1eh5 (gray, R151-A169 dark green ribbon) accommodates palmitate (light green surface) while 3gro (light blue, R151-A169 dark blue) occludes the palmitate binding site. (B) Views of R151-A169 highlight local conformational divergence and incompatibility between structure 3gro and the location of palmitate in 1eh5. Three views are presented: without palmitate, with palmitate in the same orientation, and with palmitate in a second orientation. (C) Lucanthone preferentially occupies a hydrophobic pocket in PPT1 distal to N-glycan modifications in the palmitate-binding groove. N-glycan modifications are highlighted in teal and palmitate in green. Top insert: hydrophobic residues are colored yellow, acidic residues are colored red, and basic residues are colored blue. Bottom insert: residues within 5Å of lucanthone are highlighted with numbering according to 1eh5. For clarity, hydrogen atoms on lucanthone are not illustrated. (D) GBM43 and GB9 cells were pretreated with 1 mM NtBuHA or media for 30 minutes prior to treatment with 10 or 5 μM lucanthone for 72 hours, respectively, after which an MTT assay was performed. Bars are mean +/− S.E.M. N = 7 independent experiments **P < 0.01, t test. (E) Representative Western blot of three biologic replicates showing relative PPT1, LC3B-I, LC3B-II, and β-actin levels in GBM9 cells. From left to right treatment conditions: DMSO, lucanthone (5 μM), NtBuHA (1 mM), and the combination; treatment time: 72 hours. (F) Quantification of PPT1 and (G) of LC3-II protein levels of three separate experiments described in (E), normalized to loading control (β-actin) and normalized to control treatment condition. One-way ANOVA and Tukey multiple comparisons tests were performed. Bars are mean +/− S.E.M. N = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

We determined the most favorable pose of lucanthone using the flexible DOCK6 protocol (Mukherjee et al., 2010). Prior to docking, bovine-specific residues in 1eh5 were converted to the analogous human residues. Importantly, we did not preselect any particular binding locations on the receptors and included all prepared DOCK6 binding spheres. The highest-scoring lucanthone pose across both PPT1 conformations was obtained with the PPT1 model generated from 1eh5. In this pose, lucanthone occupies a hydrophobic cavity distal to N-glycan modifications that is an extension of the palmitate-binding groove (Fig. 5C). To our surprise, this cavity was previously identified as a predicted binding site for PPT1 inhibitors (Vergoten and Bailly, 2022). For example, PPT1 is a direct target of and potently inhibited by compound DC661 (Rebecca et al., 2019). The hydrophobic cavity occupied by lucanthone is also occupied by one headgroup of DC661 in the most energetically favorable pose of DC661 bound to PPT1 (Vergoten and Bailly, 2022). Together, the observations that the highest-scoring lucanthone pose occurs in a functionally associated PPT1 conformation, and that the predicted lucanthone binding cavity has also been described for known PPT1 inhibitors, support the hypothesis that PPT1 may be a target of lucanthone.

To gain additional insight into the molecular action of lucanthone and whether its effects are mediated by PPT1, we used N-tert butylhydroxylamine (NtBuHA), a hydroxylamine derivative that has been shown to act as a chemical mimetic of PPT1 (Sarkar et al., 2013; Rebecca et al., 2017, 2019; Nicastri et al., 2018; Sharma et al., 2020; Brun et al., 2022). Previous studies have shown that NtBuHA reduces apoptosis of PPT1-deficient cells and alleviates motor and exploratory behavioral deficits exhibited by these mutant mice (Sarkar et al., 2013).

Glioma cells (GBM9 and GBM43) were plated in a 96-well plate and allowed to adhere overnight. The following day, cultures were treated with 1 mM NtBuHA for 30 minutes followed by 10 µM (for GBM43) or 5 µM (for GBM9) lucanthone for 3 days. Cultures were then subjected to MTT assay (Radin et al., 2022). Lucanthone treatment alone reduced viability by 40% to 70% in the glioma lines. While NtBuHA alone had no effect on cell viability, its combination with lucanthone resulted in significant reversal of lucanthone’s oncolytic activity (by 33%–45%, Fig. 5D). This rescue of cell viability provides evidence that lucanthone may act as an inhibitor of PPT1 and that lucanthone-treated cells are amenable to rescue by NtBuHA. Further mechanistic evidence was obtained by assessing levels of autophagy and PPT1 after treating GBM9 cells with lucanthone in the presence and absence of NtBuHA. As expected, lucanthone treatment increased the amount of LC3B-II in GBM9 tumor cells (Fig. 5, E and G), indicating a significant modulation of autophagy. We also found that lucanthone decreased the levels of PPT1 in GBM9 cells. (Fig. 5, E and F). Treatment with NtBuHA alone subtly increased PPT1 and did not alter LC3B-II levels. However, NtBuHA restored PPT1 reductions induced by lucanthone to control-treated levels (Fig. 5, E and F). Additionally, when GBM9 tumor cells were treated with lucanthone and NtBuHA, we observed a reduction in the elevation of LC3B-II induced by lucanthone alone (Fig. 5, E and G). These data, in line with viability data, demonstrate that a PPT1 mimetic is able to partially alleviate the oncolytic effects of lucanthone, further supporting its function as a PPT1 inhibitor.

To examine whether lucanthone was able to affect PPT1 levels in vivo, tumor sections from animals inoculated with TMZ-resistant glioma (as in Fig. 4) were stained for PPT1 and Cathepsin D. In control-treated tumors, PPT1 levels were high whereas levels of Cathepsin D were low (Supplemental Fig. 2). In lucanthone-treated tumors, PPT1 was significantly decreased while Cathepsin D levels increased. These findings suggest that treatment of glioma with lucanthone in vivo resulted in reduced PPT1 levels.

To interrogate whether PPT1 mRNA expression is altered in vivo in tumors treated with lucanthone, mice were inoculated with GLUC2 GSC. After determining using luminescent imaging that these mice had tumors (day 7), they were subjected to daily administration of vehicle or 50 mg/kg lucanthone for 2 weeks. At the end of the treatment period, tumors were harvested and PPT1 levels were evaluated. Reverse transcription quantitative polymerase chain reaction data indicated that the mRNA expression of PPT1 was not significantly altered by lucanthone treatment (Supplemental Fig. 1E). However, lucanthone did reduce Olig2 mRNA expression in tumors (Supplemental Fig. 1E), in agreement with our previous in vitro and in vivo findings (Radin et al., 2022). This result indicates that lucanthone treatment may principally alter PPT1 protein levels without significantly modifying PPT1 mRNA levels.

Discussion

Our understanding of the cell-intrinsic and -extrinsic inputs underpinning the universal lethality of GBM has increased substantially over the past several years, yet they have not yet resulted in new therapies against GBM. The standard-of-care modalities are not sufficient to mitigate recurrence, which has very poor outcomes (Hegi et al., 2005; Stupp et al., 2005, 2017). One possible explanation as to why novel, experimental treatments are not effective in recurrent gliomas is that they are only tested on treatment-naïve GBM cells/tumors, which have not been subjected to TMZ/radiation. When treatments that are shown to work in treatment-naïve preclinical settings are applied to the recurrent tumors, they often target glioma cells that have been selected for higher levels of, for example, drug efflux pumps. Augmented levels of drug efflux pumps may hinder the efficacy of novel drug modalities even if those modalities operate in a manner distinct to that of TMZ (Veringa et al., 2013).

In this study, murine glioma GLUC2 cells were treated with exceedingly high doses of TMZ to select for cells with resistance to this chemotherapeutic and then inoculated mice with the surviving cells. Since TMZ preferentially targets rapidly proliferating cells, it is not surprising that tumors arising from these cells grew at a slower rate: as a way of comparison, tumors arising from these TMZ-resistant cells at day 27 were 40% smaller than tumors arising from parental GLUC2 GSC at 21 days post-inoculation (Radin et al., 2022), suggesting that these tumors were affected by high-dose TMZ well after discontinuing the exposure to TMZ. Though these tumors grew at a slower rate, they were still susceptible to lucanthone treatment (Fig. 3, F and G). Prior work has illustrated that dimeric quinacrines, mechanistically similar to lucanthone, which were shown to be potent PPT1 inhibitors, are effective as monotherapies against pancreatic tumors that are derived from tumors refractory to radiation and anti-cytotoxic T lymphocyte-associated protein 4 therapy (Rebecca et al., 2017). Here we provide evidence that lucanthone may also act as an inhibitor of PPT1. Our molecular docking studies indicate that lucanthone binds PPT1 in a manner similar to that of dimeric quinacrines (Fig. 5C). Pretreatment of cultures with the PPT1 chemical mimetic NtBuHA attenuates the oncolytic effects of lucanthone (Fig. 5D) and reverses lucanthone’s autophagy inhibition (Fig. 5, E–G), supporting the idea that PPT1 protein may be a molecular target of lucanthone. We will conduct appropriate experiments to biochemically validate the inhibitory effect of lucanthone on PPT1 using either cellular thermal shift assays or surface plasmon resonance.

To our knowledge, we are the first group to look at changes of PPT1 levels at the protein and mRNA level after treatment with a putative PPT1 inhibitor. We found that while acute treatment with lucanthone reduces PPT1 levels in culture (Fig. 5F), PPT1 mRNA levels in tumors remain relatively unchanged after 2 weeks of treatment. While PPT1 protein levels may decrease, there is evidence to suggest that there are PPT1-independent mechanisms that may confer resistance to PPT1 inhibitors. For example, recent evidence underpins the role of UDP-glucose ceramide glucosyltransferase in mediating resistance to oncolytic agents that exert their effects by targeting lysosomes (Jain et al., 2023). Treatment of tumor cells with PPT1 inhibitors resulted in increased glycosphingolipids, cholesterol, and sphingolipids in lysosomes and plasma membranes, while UDP-glucose ceramide glucosyltransferase and PPT1 inhibition synergistically repressed tumor growth in vivo (Jain et al., 2023). This resistance mechanism may also be active in glioma and warrants further examination. A cell-extrinsic mechanism of lucanthone resistance may be due to pro-tumorigenic modulation of the tumor microenvironment.

Another result that has become evident after lucanthone administration is on tumor microtubes. TMs and the extraordinary extent to which glioma cells are connected in vitro and in vivo are a relatively newly described process in the glioma literature. With the hypothesis that TMs may convey resistance to radiation, efforts are ongoing to target them using gap junction inhibitors such as meclofenamate (Osswald et al., 2015; Weil et al., 2017; Venkataramani et al., 2019; Horne et al., 2021; Xie et al., 2021; Becker and Eisenmann, 2022; Becker et al., 2022; Joseph et al., 2022; Zeyen et al., 2022). The relationship between lysosomes/autophagy and TMs has yet to be investigated, though there is literature to suggest that lysosomal targeting may affect the genesis of TMs. In human retinal pigmented epithelial cells, chloroquine inhibits microtubule nucleation by reducing p150glued, the largest subunit of dynactin (Osswald et al., 2015). This finding suggests that it might be possible for lysosomal targeting agents to prevent TM formation, which may subsequently sensitize GSC to radiation. Another possible explanation of lucanthone’s effects on TM formation includes downstream cellular consequences of lysosomal autophagy inhibition. In tumor cells, lysosome membrane permeabilization can cause zinc to spill out into the cytoplasm resulting in mitochondrial dysfunction (Du et al., 2021) and the release of reactive oxygen species (Vera-Ramirez et al., 2018; Chen et al., 2021). The release of reactive oxygen species from damaged mitochondria has a wide array of cytoplasmic targets (Joseph et al., 2022), including the collapse of actin polymers into aggregates (Varland et al., 2019), which may explain lucanthone’s effects on TM formation in GSC.

The finding that lucanthone retains efficacy in TMZ-resistant gliomas is particularly encouraging considering there are no established therapeutic protocols that significantly extend survival of GBM patients who are resistant to TMZ. Lucanthone still retains the ability to slow tumor growth in animals inoculated with TMZ-resistant glioma cells, inhibiting lysosomal autophagy (evident by elevated p62, Cathepsin D, and LAMP1). Further, lucanthone-mediated reduction in the numbers and distribution of Olig2-expressing cells is promising and warrants pairing lucanthone and radiation in preclinical models of recurrent or TMZ-resistant GBM, as it may be a safe and effective additional therapeutic. Further studies with mouse models harboring different driver mutations are necessary to generalize the results. These experiments set the basis to examine in the future if lucanthone-mediated autophagy inhibition may enhance the effectiveness of immune therapies including those that modulate the immunosuppressive tumor microenvironment.

Acknowledgments

The authors would like to thank members of the Tsirka laboratory and John Haley, Kenneth Shroyer, and Yusuf Hannun for thoughtful discussions.

Data Availability

The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Material.

Abbreviations

GBM

glioblastoma

GSC

glioma cancer stem cell

IVIS

in vivo imaging system

LAMP1

lysosomal associated membrane protein 1

NtBuHA

N-tert butylhydroxylamine

PFA

paraformaldehyde

PPT1

palmitoyl protein thioesterase I

TBST

Tris-buffered saline with 0.1% Tween 20

TM

tumor microtube

TMZ

temozolomide

TTF

tumor-treating fields

Authorship Contributions

Participated in research design: Radin, Shifman, Outhwaite, Tsirka.

Conducted experiments: Radin, Shifman, Outhwaite, Sharma.

Contributed new reagents: Bases.

Performed data analysis: Radin, Shifman, Outhwaite, Seeliger, Tsirka.

Wrote or contributed to the writing of the manuscript: Radin, Shifman, Outhwaite, Sharma, Seeliger, Tsirka.

Footnotes

This work was partially supported by National Institutes of Health National Cancer Institute [Grant F30CA257677] (to D.P.R.) and National Institute of General Medical Sciences [Grant T32GM008444] (to D.P.R., S.S., and I.R.O.), [Grant R35GM119437] (to M.A.S.), and [Grant T32GM136572] (to I.R.O.); SBU URECA (to A.S.); and a Stony Brook University OVPR Seed Grant (to S.E.T.).

The authors have declared that no conflict of interest exists.

Inline graphicThis article has supplemental material available at jpet.aspetjournals.org.

References

  1. Abdul Rahim SADirkse AOudin ASchuster ABohler JBarthelemy VMuller AVallar LJanji BGolebiewska A, et al. (2017) Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A. Br J Cancer 117:813–825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amaravadi RK, Winkler JD (2012) Lys05: a new lysosomal autophagy inhibitor. Autophagy 8:1383–1384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ausman JI, Shapiro WR, Rall DP (1970) Studies on the chemotherapy of experimental brain tumors: development of an experimental model. Cancer Res 30:2394–2400. [PubMed] [Google Scholar]
  4. Bases RE, Mendez F (1997) Topoisomerase inhibition by lucanthone, an adjuvant in radiation therapy. Int J Radiat Oncol Biol Phys 37:1133–1137. [DOI] [PubMed] [Google Scholar]
  5. Becker KN, Eisenmann KM (2022) New targets in the glioblastoma tumor microtube multiverse: Emerging roles for the TGF-β/TSP1 signaling axis. Neuro-oncol 24:554–555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Becker KN, Pettee KM, Sugrue A, Reinard KA, Schroeder JL, Eisenmann KM (2022) The cytoskeleton effectors rho-kinase (ROCK) and mammalian diaphanous-related (mDia) formin have dynamic roles in tumor microtube formation in invasive glioblastoma cells. Cells 11:1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bellizzi JJ 3rd, Widom J, Kemp C, Lu JY, Das AK, Hofmann SL, Clardy J (2000) The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc Natl Acad Sci USA 97:4573–4578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brun SBestion ERaymond EBassissi FJilkova ZMMezouar SRachid MNovello MTracz JHamaï A, et al. (2022) GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions. Autophagy 18:678–694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caponegro MD, Oh K, Madeira MM, Radin D, Sterge N, Tayyab M, Moffitt RA, Tsirka SE (2021) A distinct microglial subset at the tumor-stroma interface of glioma. Glia 69:1767–1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carew JS, Espitia CM, Esquivel JA 2nd, Mahalingam D, Kelly KR, Reddy G, Giles FJ, Nawrocki ST (2011) Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. J Biol Chem 286:6602–6613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen J, Li Y, Yu TS, McKay RM, Burns DK, Kernie SG, Parada LF (2012) A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488:522–526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chen J, Pan Q, Bai Y, Chen X, Zhou Y (2021) Hydroxychloroquine induces apoptosis in cholangiocarcinoma via reactive oxygen species accumulation induced by autophagy inhibition. Front Mol Biosci 8:720370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Compter IEekers DBPHoeben ARouschop KMAReymen BAckermans LBeckervordersantforth JBauer NJCAnten MMWesseling P, et al. (2021) Chloroquine combined with concurrent radiotherapy and temozolomide for newly diagnosed glioblastoma: a phase IB trial. Autophagy 17:2604–2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dassonneville L, Bailly C (1999) Stimulation of topoisomerase II-mediated DNA cleavage by an indazole analogue of lucanthone. Biochem Pharmacol 58:1307–1312. [DOI] [PubMed] [Google Scholar]
  15. Del Rowe JD, Bello J, Mitnick R, Sood B, Filippi C, Moran J, Freeman K, Mendez F, Bases R (1999) Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone. Int J Radiat Oncol Biol Phys 43:89–93. [DOI] [PubMed] [Google Scholar]
  16. DeVorkin LPavey NCarleton GComber AHo CLim JMcNamara EHuang HKim PZacharias LG, et al. (2019) Autophagy regulation of metabolism is required for CD8+ T cell anti-tumor immunity. Cell Rep 27:502–513.e5. [DOI] [PubMed] [Google Scholar]
  17. Dobrovetsky E, Seitova A, Tong Y, Tempel W, Dong A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, BochKarev A,, et al. (2009) Human palmitoyl-protein thioesterase 1. DOI: 10.2210/pdb3GRO/pdb. [DOI] [Google Scholar]
  18. Du W, Gu M, Hu M, Pinchi P, Chen W, Ryan M, Nold T, Bannaga A, Xu H (2021) Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma. Cell Rep 37:109848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hegi MEDiserens ACGorlia THamou MFde Tribolet NWeller MKros JMHainfellner JAMason WMariani L, et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352:997–1003. [DOI] [PubMed] [Google Scholar]
  20. Hori YSHosoda RAkiyama YSebori RWanibuchi MMikami TSugino TSuzuki KMaruyama MTsukamoto M, et al. (2015) Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells. J Neurooncol 122:11–20. [DOI] [PubMed] [Google Scholar]
  21. Horne EADiaz PCimino PJJung EXu CHamel EWagenbach MKumasaka DWageling NBAzorin DD, et al. (2021) A brain-penetrant microtubule-targeting agent that disrupts hallmarks of glioma tumorigenesis. Neurooncol Adv 3:vdaa165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hu YL, DeLay M, Jahangiri A, Molinaro AM, Rose SD, Carbonell WS, Aghi MK (2012) Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 72:1773–1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ito H, Daido S, Kanzawa T, Kondo S, Kondo Y (2005) Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 26:1401–1410. [PubMed] [Google Scholar]
  24. Jain VHarper SLVersace AMFingerman DBrown GSBhardwaj MCrissey MASGoldman ARRuthel GLiu Q, et al. (2023) Targeting UGCG overcomes resistance to lysosomal autophagy inhibition. Cancer Discov 13:454–473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jawhari S, Ratinaud MH, Verdier M (2016) Glioblastoma, hypoxia and autophagy: a survival-prone “ménage-à-trois.” Cell Death Dis 7:e2434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Joseph JVMagaut CRStorevik SGeraldo LHMathivet TLatif MARudewicz JGuyon JGambaretti MHaukas F, et al. (2022) TGF-β promotes microtube formation in glioblastoma through thrombospondin 1. Neuro-oncol 24:541–553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:448–457. [DOI] [PubMed] [Google Scholar]
  28. Kim SCho HHong SOOh SJLee HJCho EWoo SRSong JSChung JYSon SW, et al. (2021) LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells. Autophagy 17:1978–1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M, Kaina B (2013) Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One 8:e55665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee SW, Kim HK, Lee NH, Yi HY, Kim HS, Hong SH, Hong YK, Joe YA (2015) The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells. Cancer Lett 360:195–204. [DOI] [PubMed] [Google Scholar]
  31. Liang XDe Vera MEBuchser WJRomo de Vivar Chavez ALoughran PBeer Stolz DBasse PWang TVan Houten BZeh HJ 3rd, et al. (2012) Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression. Cancer Res 72:2791–2801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lin CJ, Lee CC, Shih YL, Lin CH, Wang SH, Chen TH, Shih CM (2012) Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells. PLoS One 7:e38706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lomonaco SL, Finniss S, Xiang C, Decarvalho A, Umansky F, Kalkanis SN, Mikkelsen T, Brodie C (2009) The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells. Int J Cancer 125:717–722. [DOI] [PubMed] [Google Scholar]
  34. Luo M, Kelley MR (2004) Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone. Anticancer Res 24:2127–2134. [PubMed] [Google Scholar]
  35. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput 11:3696–3713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McAfee QZhang ZSamanta ALevi SMMa XHPiao SLynch JPUehara TSepulveda ARDavis LE, et al. (2012) Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. Proc Natl Acad Sci USA 109:8253–8258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mendez F, Goldman JD, Bases RE (2002) Abasic sites in DNA of HeLa cells induced by lucanthone. Cancer Invest 20:983–991. [DOI] [PubMed] [Google Scholar]
  38. Miyauchi JT, Caponegro MD, Chen D, Choi MK, Li M, Tsirka SE (2018) Deletion of neuropilin 1 from microglia or bone marrow-derived macrophages slows glioma progression. Cancer Res 78:685–694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Miyauchi JT, Chen D, Choi M, Nissen JC, Shroyer KR, Djordevic S, Zachary IC, Selwood D, Tsirka SE (2016) Ablation of neuropilin 1 from glioma-associated microglia and macrophages slows tumor progression. Oncotarget 7:9801–9814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mukherjee S, Balius TE, Rizzo RC (2010) Docking validation resources: protein family and ligand flexibility experiments. J Chem Inf Model 50:1986–2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mulkearns-Hubert EETorre-Healy LASilver DJEurich JTBayik DSerbinowski EHitomi MZhou JPrzychodzen BZhang R, et al. (2019) Development of a Cx46 targeting strategy for cancer stem cells. Cell Rep 27:1062–1072.e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Naidu MDAgarwal RPena LACunha LMezei MShen MWilson DM 3rdLiu YSanchez ZChaudhary P, et al. (2011) Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1) by direct protein binding. PLoS One 6:e23679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nicastri MC, Rebecca VW, Amaravadi RK, Winkler JD (2018) Dimeric quinacrines as chemical tools to identify PPT1, a new regulator of autophagy in cancer cells. Mol Cell Oncol 5:e1395504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Osswald MJung ESahm FSolecki GVenkataramani VBlaes JWeil SHorstmann HWiestler BSyed M, et al. (2015) Brain tumour cells interconnect to a functional and resistant network. Nature 528:93–98. [DOI] [PubMed] [Google Scholar]
  45. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. [DOI] [PubMed] [Google Scholar]
  46. Portnow J, Badie B, Chen M, Liu A, Blanchard S, Synold TW (2009) The neuropharmacokinetics of temozolomide in patients with resectable brain tumors: potential implications for the current approach to chemoradiation. Clin Cancer Res 15:7092–7098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Puchalski RBShah NMiller JDalley RNomura SRYoon JGSmith KALankerovich MBertagnolli DBickley K, et al. (2018) An anatomic transcriptional atlas of human glioblastoma. Science 360:660–663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Radin DP, Smith G, Moushiaveshi V, Wolf A, Bases R, Tsirka SE (2022) Lucanthone targets lysosomes to perturb glioma proliferation, chemoresistance and stemness, and slows tumor growth in vivo. Front Oncol 12:852940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rebecca VWNicastri MCFennelly CChude CIBarber-Rotenberg JSRonghe AMcAfee QMcLaughlin NPZhang GGoldman AR, et al. (2019) PPT1 promotes tumor growth and is the molecular target of chloroquine derivatives in cancer. Cancer Discov 9:220–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rebecca VWNicastri MCMcLaughlin NFennelly CMcAfee QRonghe ANofal MLim CYWitze EChude CI, et al. (2017) A unified approach to targeting the lysosome’s degradative and growth signaling roles. Cancer Discov 7:1266–1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Reilly KM, Loisel DA, Bronson RT, McLaughlin ME, Jacks T (2000) Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nat Genet 26:109–113. [DOI] [PubMed] [Google Scholar]
  52. Sarkar C, Chandra G, Peng S, Zhang Z, Liu A, Mukherjee AB (2013) Neuroprotection and lifespan extension in Ppt1(-/-) mice by NtBuHA: therapeutic implications for INCL. Nat Neurosci 16:1608–1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sharma GOjha RNoguera-Ortega ERebecca VWAttanasio JLiu SPiao SLee JJNicastri MCHarper SL, et al. (2020) PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma. JCI Insight 5:e133235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shteingauz APorat YVoloshin TSchneiderman RSMunster MZeevi EKaynan NGotlib KGiladi MKirson ED, et al. (2018) AMPK-dependent autophagy upregulation serves as a survival mechanism in response to tumor treating fields (TTFields). Cell Death Dis 9:1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Stupp RMason WPvan den Bent MJWeller MFisher BTaphoorn MJBelanger KBrandes AAMarosi CBogdahn U, et al. ; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996. [DOI] [PubMed] [Google Scholar]
  56. Stupp RTaillibert SKanner ARead WSteinberg DLhermitte BToms SIdbaih AAhluwalia MSFink K, et al. (2017) Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: a randomized clinical trial. JAMA 318:2306–2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Valdor R, García-Bernal D, Riquelme D, Martinez CM, Moraleda JM, Cuervo AM, Macian F, Martinez S (2019) Glioblastoma ablates pericytes antitumor immune function through aberrant up-regulation of chaperone-mediated autophagy. Proc Natl Acad Sci USA 116:20655–20665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Varland S, Vandekerckhove J, Drazic A (2019) Actin post-translational modifications: the Cinderella of cytoskeletal control. Trends Biochem Sci 44:502–516. [DOI] [PubMed] [Google Scholar]
  59. Venkataramani VTanev DIStrahle CStudier-Fischer AFankhauser LKessler TKörber CKardorff MRatliff MXie R, et al. (2019) Glutamatergic synaptic input to glioma cells drives brain tumour progression. Nature 573:532–538. [DOI] [PubMed] [Google Scholar]
  60. Vera-Ramirez L, Vodnala SK, Nini R, Hunter KW, Green JE (2018) Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence. Nat Commun 9:1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vergoten G, Bailly C (2022) Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated forms: a computational approach. J Biomol Struct Dyn 40:8197–8205. [DOI] [PubMed] [Google Scholar]
  62. Veringa SJBiesmans Dvan Vuurden DGJansen MHWedekind LEHorsman IWesseling PVandertop WPNoske DPKaspers GJ, et al. (2013) In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma. PLoS One 8:e61512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weil S, Osswald M, Solecki G, Grosch J, Jung E, Lemke D, Ratliff M, Hänggi D, Wick W, Winkler F (2017) Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas. Neuro-oncol 19:1316–1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Xie RKessler TGrosch JHai LVenkataramani VHuang LHoffmann DCSolecki GRatliff MSchlesner M, et al. (2021) Tumor cell network integration in glioma represents a stemness feature. Neuro-oncol 23:757–769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Ye H, Chen M, Cao F, Huang H, Zhan R, Zheng X (2016) Chloroquine, an autophagy inhibitor, potentiates the radiosensitivity of glioma initiating cells by inhibiting autophagy and activating apoptosis. BMC Neurol 16:178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yi L, Zhou C, Wang B, Chen T, Xu M, Xu L, Feng H (2013) Implantation of GL261 neurospheres into C57/BL6 mice: a more reliable syngeneic graft model for research on glioma-initiating cells. Int J Oncol 43:477–484. [DOI] [PubMed] [Google Scholar]
  67. Zeyen TPotthoff ALNemeth RHeiland DHBurger MCSteinbach JPHau PTabatabai GGlas MSchlegel U, et al. (2022) Phase I/II trial of meclofenamate in progressive MGMT-methylated glioblastoma under temozolomide second-line therapy-the MecMeth/NOA-24 trial. Trials 23:57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zou Y, Wang Q, Li B, Xie B, Wang W (2014) Temozolomide induces autophagy via ATM-AMPK-ULK1 pathways in glioma. Mol Med Rep 10:411–416. [DOI] [PubMed] [Google Scholar]

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