High-throughput in vitro drug screening and in vivo studies identify fenretinide as a brain-penetrant DMG therapeutic

Dannielle H Upton; Jie Liu; Sandra M George; Santosh Valvi; Caitlin Ung; Benjamin S Rayner; Anjana Gopalakrishnan; Ruby Pandher; Aaminah Khan; Pooja Venkat; Chelsea Mayoh; Holly Holliday; Nicole Yeung; Hieu Nguyen; Laura Franshaw; Anahid Ehteda; Han Shen; Giovanna Farruggia; Isabella Orienti; C Patrick Reynolds; Maria Tsoli; David S Ziegler

doi:10.1093/neuonc/noaf035

. 2025 Feb 12;27(7):1813–1828. doi: 10.1093/neuonc/noaf035

High-throughput in vitro drug screening and in vivo studies identify fenretinide as a brain-penetrant DMG therapeutic

Dannielle H Upton ^1,², Jie Liu ³, Sandra M George ⁴, Santosh Valvi ⁵, Caitlin Ung ⁶, Benjamin S Rayner ^7,⁸, Anjana Gopalakrishnan ⁹, Ruby Pandher ^10,¹¹, Aaminah Khan ^12,¹³, Pooja Venkat ¹⁴, Chelsea Mayoh ^15,¹⁶, Holly Holliday ^17,¹⁸, Nicole Yeung ¹⁹, Hieu Nguyen ²⁰, Laura Franshaw ²¹, Anahid Ehteda ²², Han Shen ²³, Giovanna Farruggia ²⁴, Isabella Orienti ²⁵, C Patrick Reynolds ²⁶, Maria Tsoli ^27,^28,^#, David S Ziegler ^29,^30,^31,^#,^✉

¹ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

² Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

³ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

⁴ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

⁵ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

⁶ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

⁷ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

⁸ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

⁹ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹⁰ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

¹¹ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹² School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

¹³ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹⁴ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹⁵ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

¹⁶ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹⁷ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

¹⁸ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

¹⁹ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²⁰ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²¹ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²² Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²³ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²⁴ Alma Mater Studiorum University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy

²⁵ Alma Mater Studiorum University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy

²⁶ Cancer Center, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, USA

²⁷ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

²⁸ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

²⁹ Kid’s Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia

³⁰ School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia

³¹ Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

Maria Tsoli and David S Ziegler contributed equally to this work..

^✉

Corresponding Author: David S. Ziegler, MBBS, MD (Res), Kids Cancer Centre, Sydney Children’s Hospital, High St, Randwick, NSW 2031, Australia (d.ziegler@unsw.edu.au).

PMCID: PMC12417829 PMID: 39935375

Abstract

Background

Diffuse midline glioma, characterized by H3K27 alteration (DMG), is the predominant high-grade glioma in children. It commonly originates in the brainstem, yet effective treatments for these patients remain elusive.

Methods

To identify novel therapies for DMG, we conducted high-throughput drug screens (HTS) using biologically active, clinically approved compounds against DMG neurospheres. Multiple primary DMG cultures were utilized in conjunction with in vitro cytotoxicity and clonogenic assays to validate the efficacy of top compounds. Molecularly diverse patient-derived and transgenic DMG orthotopic models were employed to assess therapeutic efficacy alongside pharmacokinetic and immunohistochemical analyses. Mechanistic studies, including RNA sequencing, western blotting, and flow cytometry, were conducted to elucidate the antitumor efficacy of the most promising compound, fenretinide, in DMG cells.

Results

Through HTS, 6 compounds were identified and validated for their potent cytotoxic activity. However, most of these compounds failed to improve survival in an orthotopic Diffuse Midline Glioma (DMG) model due to limited blood-brain barrier (BBB) penetration. In contrast, fenretinide exhibited effective BBB penetration, significantly enhancing the survival of tumor-bearing animals. Mechanistic studies revealed that fenretinide increased reactive oxygen species (ROS) generation and induced apoptosis by inhibiting PDGFRα. RNA-sequencing further elucidated that fenretinide upregulates the Unfolded Protein Response (UPR) and endoplasmic reticulum (ER) stress pathways while downregulating neurogenesis. The in vivo antitumor efficacy of 2 fenretinide formulations was demonstrated in PDGFRα-amplified and transgenic DMG models.

Conclusion

This comprehensive study has identified new DMG therapeutic vulnerabilities and highlights fenretinide as a brain-penetrant, anti-DMG agent.

Keywords: DMG, fenretinide, high-throughput screening, PDX, therapeutic

Key Points.

Identified and validated 6 compounds with potent cytotoxic activity through HTS.
Highlighted the limited BBB penetration of most agents in DMG models, hampering their efficacy.
Demonstrated significant survival enhancement in DMG models with fenretinide.

Importance of the Study.

DMGs are the most aggressive of all childhood cancers. This study demonstrated that despite in vitro activity, most drugs fail in vivo due to inadequate BBB permeation. In contrast, fenretinide successfully penetrated the BBB, exhibiting significant anti-tumor activity by inhibiting multiple oncogenic pathways, including PDGFRα, with promise for clinical translation.

Diffuse midline glioma, H3K27 altered (DMG), is a malignant childhood brain tumor and the most common form of high-grade glioma affecting children.¹ DMG most commonly originates in the brainstem and is usually not amenable to surgical resection due to its location and diffuse infiltrative nature. Despite multiple clinical trials, radiotherapy remains the only available standard treatment, transiently improving neurological function and temporarily halting disease progression in up to 70% of patients. However, the prognosis of DMG remains dismal, with over 90% of children dying within 2 years of diagnosis, demonstrating the urgent need to identify better treatment options.²

Molecularly, DMGs are characterized by K27M mutations in H3.3 or H3.1 (78%); and TP53 mutations (77%) which are not targetable and inactivating mutations of ATRX.^3–5 Other alterations include ACVR1 activating mutations (in 20–32% of cases); gene amplifications in receptor tyrosine kinases (RTKs), such as platelet-derived growth factor receptor (PDGFR)^6,7 and mutations of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) cascade⁸ and loss of phosphatase and tensin (PTEN) homolog in up to 40% of cases.⁵ Recent studies have also identified that most H3K27M mutant DMGs resemble oligodendroglial precursor cells, which exhibit higher proliferation and overexpression of PDGFRα independently of a genetic mutation or amplification, suggesting the potential for therapeutic intervention against PDGFRα.⁹ Despite these targetable molecular aberrations, trials of specific inhibitors of PDGFRα, such as imatinib, dasatinib, and ponatinib have not demonstrated clinical efficacy.¹⁰ One potential reason for the failure of targeted agents in DMG is the blood-brain barrier (BBB), which has been shown to remain intact and restrict the delivery of systemically administered therapies, leading to ineffective concentrations in the tumor.¹¹ Thus, despite the in vitro activity of PDGFR inhibitors against DMG, most clinical trials have shown no survival benefit, likely due to their poor brain penetration.^12,13

Drug repurposing of already clinically approved therapeutics can overcome the lengthy and expensive process of developing targeted therapies.¹⁴ High-throughput screening (HTS), an efficient method of screening large numbers of drug candidates, can determine the activity of clinically approved and available pharmacological compounds against tumor samples.¹⁴ For example, HTS in cell lines derived from genetically engineered DMG mouse models using the RCAS/tv-a system identified BMS-754807 as a potential therapeutic agent.¹⁵ Similarly, a targeted screen of 83 drugs selected by pediatric neuro-oncologists on patient-derived DMG cell cultures identified panobinostat as a potential therapeutic option.¹⁶

Here we conducted a large, unbiased HTS utilizing biologically active, FDA-approved pharmaceutical compounds encompassing diverse activities, targets, and mechanisms of action. Through screening a panel of DMG primary cultures, we identified fenretinide as a promising therapeutic against DMG. Our findings reveal that fenretinide acts via multiple therapeutic vulnerabilities, including inhibition of PDGFRα, upregulation of the unfolded protein response (UPR)/endoplasmic reticulum (ER) stress, and downregulation of neurogenesis pathways. Moreover, fenretinide demonstrates the critical ability to penetrate the blood-brain barrier (BBB), making it a promising antitumor agent for DMG treatment.

Materials and Methods

Human DMG Neurosphere-Forming Culture

Primary DMG lines and murine DMG cells were grown as previously reported in Khan et al. (59) and du Chatinier et al. (60), respectively.

Inducible Lentiviral PDGFRα shRNA DMG Cells

Experimental details are provided in the Supplementary information.

High-Throughput Screening

A collection of 3570 compounds from the Prestwick Chemical Library, LOPAC, and TocriScreen Total libraries was screened on SU-DIPGVI and HSJD-DIPG007 cells. Cells were seeded in 96- or 384-well plates and treated with compounds at 5 µM (HSJD-DIPG007) or 10 µM (SU-DIPGVI) for 72 h. Cell viability was measured using the Alamar Blue assay and analyzed with ActivityBase software, with raw data normalized to the DMSO control. More experimental details are provided in the Supplementary information.

Proliferation Assay

The cytotoxicity assays were as outlined in Khan et al (59). Cells were seeded at 2500–3500 cells/100 µl.

Soft Agar Colony Assays

Experimental details are provided in the Supplementary information.

Lentiviral Experiments

Inducible lentiviral PDGFRα transduced RA055 cells were seeded at 800 cells/well in media containing 5% FBS with or without 2 µg/ml doxycycline with or without 1.6 µM fenretinide in 6-well plates and maintained for a period of 3 weeks before staining and analysis. More experimental details are provided in the Supplementary information.

Intracranial DMG Cell Injection, Animal Monitoring, and Endpoint

Intracranial injections were undertaken as outlined in Khan et al. (59) and du Chatinier et al. (60). All survival study mice were monitored daily for signs of tumor development by assessing neurological and clinical symptoms. Mice exhibiting clinical signs of neurologic declines, such as ataxia, circling, and head tilting with or without 20% weight loss were humanely euthanased for histological analysis of tumors.

In Vivo Efficacy Drug Treatments

Experimental details are provided in the Supplementary information.

Pharmacokinetic Analysis of In Vivo Brain samples

Experimental details are provided in the Supplementary information.

Histological Analysis of Brain Tissue

Experimental details are provided in Supplementary Information, while the analysis was performed using Andy’s Algorithms on the image analysis pipeline FIJI (61).

Apoptosis, Mitochondrial Function, and DNA damage assays

Reactive oxygen species production assay.—Experiments were performed using MitoSox Red staining (Thermo-Fisher) and flow cytometric analysis. Experimental details are provided in the Supplementary information.

Mitochondrial Depolarization

Experiments were performed with JC-1 staining (Thermo-Fisher) and flow cytometric analysis. Experimental details are provided in the Supplementary information.

LOcation-Specific DNA Repair and Damage Quantification (LORD-Q)

Measurement of mitochondrial and nuclear DNA damage was performed using the LORD-q assay as established by literature.¹⁷ A detailed experimental protocol is provided in the Supplementary information.

Caspase Activity Assay

Experimental details are provided in the Supplementary information.

Annexin V FITC/Propidium Iodide Assay

HSJD-DIPG007 cells were treated with fenretinide, harvested after 24–48 h, and stained with Annexin V FITC and Propidium Iodide. Samples were analyzed using a BD FACSCanto II flow cytometer. More experimental details are provided in Supplementary Information.

Molecular Analysis: Proteome Profiler Array

Human Phospho-RTK Array Kit from R&D systems was used as per the manufacturer’s guidelines. Array was imaged using the ChemiDoc Touch Imaging System (Bio-Rad).

Protein Kinase Activity Measurement

Inhibition of protein kinase (PDGFRα) activity was investigated by using ADP-Glo Kinase Assay (Promega). Luminescence was measured with Wallac 1420 Victor2 Microplate Reader (Perkin Elmer Life Sciences).

Real-time Quantitative Polymerase Chain Reaction (qPCR)

Experimental details are provided in the Supplementary information.

Western Blot Analysis

Experimental details are provided in the Supplementary information.

RNA Sequencing and Analysis

HSJD-DIPG007 and RA055 cells were treated with fenretinide or vehicle control for 24-72 h, followed by RNA extraction and sequencing. RNA quality was assessed using spectrophotometry and the TapeStation RNA assay. Libraries were prepared, normalized, pooled, and sequenced on the NovaSeq 6000. Reads were aligned to the human genome using STAR, quantified with RSEM, (63) and differential expression was analyzed with edgeR. Enrichment analysis of significant genes was conducted using enrichR, ranking gene sets by adjusted p-value and combined score. (64). More experimental details are provided in the Supplementary information.

Statistics

Data were analyzed with GraphPad Prism 8 using an ordinary one-way ANOVA with multiple comparisons. P values less than 0.05 were considered statistically significant, and P values and statistical significance were noted on graphs when necessary. Cytotoxic assay and soft agar colony-forming assay results are displayed as average with error shown as SEM. The data from the qPCR runs were in the form of cycle threshold (Ct) values. The expression differences between samples were calculated using 2^−ΔΔCT method. Survival analysis of orthotopic xenograft models was performed with MantelCox log-rank analysis. The data presented in this manuscript have been verified using ImageTwin to ensure accuracy and integrity.

Study Approval

All animal experiments were performed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes under the Animal Research Regulation of New South Wales (Australia) and under a protocol approved by the Animal Use and Care Committees of the University of New South Wales.

Data Availability

The data generated in this study are available upon request from the corresponding author.

Results

High Throughput Screening Identifies Compounds of Interest in DMG

We performed a high throughput screen of 3570 biologically active, clinically approved pharmaceutical compounds, each at a single concentration of 5 µM for HSJD-DIPG007 and 10 µM for SU-DIPGVI cells¹⁸ (Figure 1A, B). This identified 90 drug hits that reduced DMG cell viability to less than 10% compared to untreated cells (Supplementary Table S1). Of these 90 compounds, 29 were selected for further investigation based on a comprehensive literature review for each compound that suggested their potential for BBB permeation. Twenty-one of the 29 compounds were excluded after validation testing showed reduced cytotoxic effects, with IC50s > 10 µM against HSJD-DIPG007 and/or SU-DIPGVI cells or similar activity against DMG cells compared with nontumor MRC5 cells (Supplementary Table S2, Supplementary Figure S1). Of the 8 remaining drugs, Lanatoside C was selected over digoxin (both cardiac glycosides), and mefloquine hydrochloride was selected over quinacrine dihydrochloride (both antimalarials) for further investigation due to improved performance and lower IC50 values in the validation testing.

Graphs and data visualizing the results of a high-throughput screening analysis of 3,570 compounds against DMG cells. Subfigures include a cell viability scatter plot for SU-DIPGVI, highlighting top hits in red and other compounds in black, along with a similar scatter plot for the HSJD-DIPG007 DMG culture. Additional subfigures present dose-response cytotoxicity curves for six compounds, Lanatoside C, Ivermectin, Fenretinide, SAHA, Mefloquine hydrochloride, and Parthenolide, across a panel of DMG and normal cells. — High-throughput screening and identification of new compounds with anti-DMG activities. Primary screening results of 3,570 compounds against DMG, which were arranged in order of percentage cell viability. Red data points indicate the top 90 hits identified through HTS. **(A)** SU-DIPGVI cells treated with 10 µM of each compound and **(B)** HSJD-DIPG007 cells treated with 5 µM of each compound. Drug cytotoxicity was assessed in SU-DIPGVI, HSJD-DIPG007, and MRC5 cells. All drugs examined showed a dose-dependent cell viability reduction (C) Lanatoside C (D) Ivermectin (E) Fenretinide (F) SAHA (G) Mefloquine hydrochloride (H) Parthenolide. (N = 3) **= P < 0.005*** = P < 0.001.

These 6 drugs were next validated against an extended panel of DIPG cells to establish a dose-response curve and IC50 for each agent in each DIPG neurosphere culture (Figure 1C-H, Supplementary Table S3). All 6 candidate drugs exhibited cytotoxic activity in at least 6 of the 8 primary patient-derived DIPG neurosphere-forming cultures, with lower IC50s in DMG cultures in comparison with normal healthy astrocytes (NHAs) (Supplementary Table S4). The 6 candidate drugs were further tested in soft agar colony-forming assays. All drugs decreased colony formation in a dose-dependent manner at low micromolar concentrations in HSJD-DIPG007 cells (Supplementary Figure S2A–F), with similar findings observed across all drug candidates tested in SU-DIPGVI cells (Supplementary Figure S2G–L).

Validation of Anti-tumor Efficacy of Drug Candidates in DMG Orthotopic Animals

In vitro activity does not always correspond with in vivo efficacy, particularly in DMG models where the intact BBB can prevent drug penetration into the tumor.¹⁹ Therefore, to further prioritize the top hits, we next examined the in vivo efficacy of each of the 6 candidate drugs in aggressive orthotopic DMG models (Figure 2). We did not observe any significant weight loss or other evidence of systemic toxicity associated with any individual drug treatments (Supplementary Figure S3). Treatment with LYM-X-SORB-fenretinide, a formulation designed to improve intestinal absorption, led to significant survival benefits (Figure 2C) (median survival of vehicle 53 days vs fenretinide 58 days, P = 0.009) and a significant decrease in Ki67 positive staining (P = 0.0393) (Figure 2D).²⁰ In contrast, lanatoside C, ivermectin, SAHA, mefloquine hydrochloride, and parthenolide treatments showed no improvement in survival compared to vehicle-treated controls. (Figure 2A, B, F–H, Supplementary Figure S4). To understand why these active agents failed to improve survival in vivo, we next measured the intra-tumoural concentrations of each compound in each in vivo model (Supplementary Figure S4)). While brain penetration was confirmed through LC-MS/MS analysis for fenretinide and mefloquine hydrochloride (Figure 2E, Supplementary Figure S4E) lanatoside C, ivermectin, SAHA, and parthenolide each failed to penetrate the BBB with undetectable drug concentrations in brain and tumor tissue (Supplementary Figure S4A–B, D, F).

Kaplan-Meier survival curves and bar graphs show the effects of various treatments on DMG-engrafted animals. Subfigures display survival percentages for mice treated with Lanatoside C, Ivermectin, LYM-X-SORB fenretinide, SAHA, Mefloquine hydrochloride, and Parthenolide, compared to vehicle-treated controls. Other subfigures display immunohistochemical quantification for marker KI67 in LYM-X-SORB fenretinide-treated animals and the fenretinide drug concentrations in animal brains. — Fenretinide increases survival in an in vivo HSJD-DIPG007 orthotopic model. Kaplan-Meier plot of percentage survival of HSJD-DIPG007-xenografted BalbC/Nude mice treated with **(A)** lanatoside C **(B)** ivermectin **(C)** LYM-X-SORB fenretinide. (D) Quantification of Ki67 staining following treatment with LYM-X-SORB fenretinide, * = p < 0.05. (E) Pharmacokinetic analysis following fenretinide treatment. Kaplan-Meier plot of percentage survival of HSJD-DIPG007-xenografted BalbC/Nude mice treated with **(F)** SAHA **(G)** mefloquine hydrochloride **(H)** parthenolide.

Fenretinide is the Only Active Retinoid When Tested in DMG Cells

Fenretinide belongs to a class of compounds called “retinoids,” some of which are commonly used in children and in other childhood cancers.^21,22 We, therefore, sought to establish whether the highly potent effect of fenretinide against DIPG was a class effect that could be extended to other retinoids. Cytotoxicity assays performed in both SU-DIPGVI and HSJD-DIPG007 cells, using 9-cis retinoic acid, 13-cis retinoic acid, ATRA, and fenretinide, revealed that the potent cytotoxicity was specific to fenretinide. Treatment with other retinoids did not affect tumor cell viability, even at concentrations greater than 10 µM in both HSJD-DIPG007 and SU-DIPGVI cells (Supplementary Figure 5A, B).

Fenretinide induces the production of reactive oxygen species (ROS), caspase activation, and apoptosis in HSJD-DIPG007 cells in vitro.

Fenretinide has been shown to induce apoptosis by stimulating signaling molecules such as ceramide²³ and caspases,²⁴ as well as elevating ROS levels.²⁵ To investigate whether fenretinide induces ROS production and, subsequently, apoptosis in DMG, a MitoSOX Red assay was used to assess ROS levels. Fenretinide treatment led to potent ROS production in DMG cells starting as early as 1 hour following treatment (Figure 3A). Using JC-1 staining, we assessed mitochondrial depolarization in fenretinide-treated DIPG cells and observed a significant reduction in the red/green fluorescence ratio, indicative of impaired mitochondrial integrity in 2 primary DMG cultures (Supplementary Figure 6A, B). Consistent with previous studies, fenretinide treatment resulted in a significant increase in phospho-H2AX levels, indicating activation of the DNA damage response in 2 DMG cultures (Supplementary Figure 6C,D). Given the observed increase in ROS production and phospho-H2AX levels, we hypothesized that fenretinide-induced oxidative stress might directly contribute to DNA damage. To investigate this, we employed the LOcation-specific DNA Repair and Damage Quantification (LORD-Q) assay, which estimates DNA damage rates through real-time polymerase chain reaction (PCR).¹⁷ DNA damage is quantified by observing changes in the delta Ct (ΔCt) between long and short DNA fragments during PCR amplification. Interestingly, fenretinide did not induce detectable mitochondrial or nuclear DNA damage in HSJD-DIPG007 cells after 24 or 48 h of treatment. In contrast, RA055 cells showed a significant increase in nuclear DNA lesions and a decrease in mitochondrial DNA lesions after 48 hours, highlighting cell line-specific effects (Supplementary Figure 6E, F). It is important to note that our assessment was limited to the COL1A1 gene, and damage may occur in other genes that were not evaluated in this study. To further understand the effect of fenretinide on the induction of apoptosis, we next investigated the activation of caspases in DMG cells. Treatment with fenretinide led to significant increases in caspase 3/7, caspase 8, and caspase 9, indicating induction of both intrinsic and extrinsic apoptotic pathways (Figure 3B, Supplementary Figure S7A, B). Fenretinide treatment subsequently increased protein levels of cleaved caspase and cleaved PARP protein expression over time (Figure 3C, Supplementary Figure S6G–J). The effect on apoptosis occurred in a dose-dependent manner as measured by the impact of fenretinide on Annexin-V and 7AAD staining (Figure 3D). Similarly, we evaluated the effects of fenretinide treatment in murine glioma cells (H3K27M). Fenretinide was similarly cytotoxic as primary DMG cells and enhanced DNA damage response PH2AX and apoptotic markers cleaved caspase 3 and cleaved PARP (Supplementary Figure S8A–H).

Graphs characterising the effects of fenertinide on apoptosis, ROS and key oncogenic targets. Subfigures indicate that fenretinide induces ROS and enhances caspase3/7 activation and apoptosis. Other subfigures indicate gene expression analysis, western blot images and quantification for markers like PDGFRa. — Fenretinide induces the production of reactive oxygen species (ROS), apoptosis and affects PDGFRα in DMG. **(A)** ROS production in HSJD-DIPG007 fenretinide-treated cells over time (30 minutes to 4 hours). Antimycin was used as a positive control. *** = P < 0.001. (N = 3) **(B)** Luminescence quantification of induction of caspase 3/7, in HSJD-DIPG007 cells treated with fenretinide. (N = 2) **(C)** Western blot of HSJD-DIPG007 treated with fenretinide for 24 and 48 h. (N = 3) **(D)** Quantification of apoptosis in HSJD-DIPG007 cells treated with fenretinide. Columns represent apoptotic events at 24 h at different concentrations of fenretinide (mean ± SEM). * = P < 0.05, ***=P < 0.001. (N = 2). **(E)** Western blot of HSJD-DIPG007 treated with fenretinide for 24 and 48 h. (N = 3). The same GAPDH is included for C and E as these blots originate from the same protein lysates. **(F)** Expression of PDGFRα mRNA as quantified by real-time PCR in HSJD-DIPG007 cells treated with fenretinide for 24 or 48 h. Expression levels of PDGFRα were normalized to the GAPDH control, and expression was compared between the untreated and treated samples (N = 2). **(G)** Western blot of HSJD-DIPG007 treated with fenretinide for 24 and 48 h (N = 3). **(H)** Western blot of RA055 cells treated with fenretinide for 24 and 48 h (N = 3). **(I)** Western blot of RA055 cells treated with fenretinide for 24 and 48 h (N = 3.) The same GAPDH is included for H and I as these blots originate from the same protein lysates. **(J)** Western blot of RA055 cells treated with fenretinide for 24 and 48 hours. (N = 3).

Fenretinide Inhibits PDGFRα in DMG Cells

Since fenretinide has previously been shown to affect kinases such as FAK²⁶ and mTOR,²⁷ we next sought to investigate whether it can affect receptor tyrosine kinases in DMG. We first evaluated whether fenretinide affects the phosphorylation status of receptor tyrosine kinases using a 96-well phospho-kinase array as previously described.²⁸ Fenretinide treatment significantly reduced phospho-PDGFRα signal intensities compared to untreated HSJD-DIPG007 cells (Supplementary Figure S9). Western blotting analysis confirmed significant decreases in levels of total and phospho-PDGFRα (Figure 3E, Supplementary Figure S10A–C) but no changes in PI3K, AKT, and mTOR following treatment with 2 µM of fenretinide at 48 h (Supplementary Figure S10D–K). Given the effect of fenretinide on both total and phospho-PDGFRα, we next assessed whether fenretinide acts as a direct kinase inhibitor. Protein kinase activity measurement assays were thus employed using the ADP-Glo kinase assay. We found that fenretinide did not affect PDGFRα activity through direct binding compared to the established PDGFR inhibitor, ponatinib, as a positive control (Supplementary Figure S11). Therefore, to investigate whether the effect on PDGFRα occurs at the transcriptional level, we evaluated PDGFRA mRNA expression levels by real-time qPCR. Compared to untreated cells, PDGFRA mRNA expression levels were significantly lower in fenretinide-treated DMG cells at both 24 and 48 hours (Figure 3F). Together, these results suggest that fenretinide does not directly target kinase activity but inhibits PDGFRA through suppression of gene transcription. Given the effect on PDGFRA gene transcription, we next sought to determine whether fenretinide affected epigenetic drivers. We found that fenretinide treatment increased H3K27 acetylation, trimethylation at 24 and 48 h and total H3 levels in total protein lysates at 48 h (Figure 3G, Supplementary Figure S12A-C) with no changes observed when examined in nuclear histone extracts (Supplementary Figure S13A). Similarly, total lysates from murine glioma cells treated with fenretinide exhibited significantly higher levels of H3K27 trimethylation and total H3 levels (Supplementary Figure S13B–E).

Given the effects of fenretinide on PDGFRA gene expression and protein levels, we sought to investigate its effects on PDGFRA over-expressing RA055 DMG cells. Western blotting analysis confirmed an increase in cleaved parp and caspase (Figure 3H, Supplementary Figure S14) and a decrease in p-PDGFRα and p-PI3Kp85 when treated with 4 µM fenretinide at 24 and 48 hours (Figure 3I, Supplementary Figure S15, Supplementary Figure S16). However, no downstream effects were seen on AKT or mTOR (Supplementary Figure S16). Similar to results shown in HSJD-DIPG007 cells, fenretinide treatment increased H3K27 acetylation, trimethylation, and total H3 in total protein lysates at 24 and 48 h in PDGFRα-amplified RA055 DMG cells (Figure 3J, Supplementary Figure S17). To validate whether fenretinide exerts its effect at least in part through PDGFRα inhibition and whether its activity could be enhanced with further PDGFRα suppression, we performed knockdown experiments. Knockdown of PDGFRα in RA055 cells using doxycycline-inducible shRNA significantly decreased colony formation, which was further enhanced by treatment with fenretinide (Supplementary Figure S18).

Fenretinide Treatment Upregulates the ER Stress and UPR Pathways while Also Downregulating Neurogenesis

Given that fenretinide affects PDGFRA at the transcriptional level, and that fenretinide was active across the broader DMG panel, and that fenretinide was active in cells without PDGFRA as a driver, we next sought to determine the broader impact of fenretinide on the transcriptome through bulk RNA-sequencing. Significant gene expression changes were observed in both HSJD-DIPG007 and RA055 (PDGFRα amplified) cultures when treated with fenretinide for 24 hours (Supplementary Figure S19, 20). Gene ontology (GO) enrichment analysis of biological processes performed on the upregulated genes showed enrichment and a high number of genes in gene sets related to ER stress and the UPR in both HSJD-DIPG007 and RA055. (Figure 4A, Supplementary Figure S21). Gene expression analysis confirmed increased expression of UPR components ATF4 (Figure 4B), CHOP (Figure 4C), BIP (Figure 4D), and XBP1 (Figure 4E), with levels of both CHOP and BIP reaching statistical significance in HSJD-DIPG007 cells (Figure 4C, E; P = 0.0156, P = 0.0096, respectively) but not RA055 cells. Protein analysis confirmed a significant increase in CHOP, BiP, and XPB1 in HSJD-DIPG007 and ATF4, CHOP, BiP, and XPB1 RA055 cells after 24 hours of treatment with fenretinide (Supplementary Figure S22). To examine whether these effects were independent of, or occurred secondary to induction of ROS, we tested the impact of antioxidant supplementation. Treatment with neither n-acetylcysteine (NAC) nor Mito-tempo inhibited fenretinide-induced UPR-mediated cell death (Supplementary Figure 23), suggesting an independent effect.

Graphs depicting the effects of fenretinide on pathways associated with ER stress and unfolded protein response. Subfigures show upregulated gene ontology pathways and compare gene expression levels related to ER stress and the unfolded protein response such as ATF4, CHOP, BiP and XBP1 in fenretinide-treated DMG cells versus controls. — Fenretinide treatment upregulates ER stress and unfolded protein response in DMG cells. **(A)** Top 20 upregulated gene ontology (biological processes) identified through RNA sequencing analysis in HSJD-DIPG007 cells treated with 2 µM fenretinide for 24 h. Expression of **(B)** Atf4, **(C)** Chop, **(D)** Bip and **(E)** sXbp1 mRNA as quantified by real-time PCR in HSJD-DIPG007 and RA055 cells treated with fenretinide for 24 h. Gene expression levels were normalized to the 18s/B2m control, and expression was compared between the untreated and treated samples. * = P < 0.05, **=P < 0.005.

GO enrichment analysis of biological processes performed on the downregulated genes showed enrichment with an overlap of significant genes in gene sets related to neurogenesis in both the HSJD-DIPG007 and RA055 cells (Figure 5A, Supplementary Figure S24). Gene expression analysis confirmed significantly decreased levels of ASCL1, OLIG2, and DLL1 in RA055 cells (P = 0.009, P = 0.0076, P = 0.0117, respectively, Figure 5B, G, H), with ASCL1 also significantly decreased in HSJD-DIPG007 cells (P = 0.0013, Figure 5B). No significant changes in gene expression were observed in DIPG007 or RA55 cells in NOTCH1, HES1, LEF1, or NESTIN (Figure 5C-F) Proteomic analysis confirmed a significant decrease in ASCL1, C-Notch1, Notch1, and Olig2 in HSJD-DIPG007 and APC, ASCL1, C-Notch1, Notch1, LEF1, Nestin, Olig2 and DLL1 in RA055 cells after 24 hours treatment with fenretinide (Supplementary Figure 25). Together these results suggest a disparate effect of fenretinide on ER stress, UPR, and neurogenesis pathways.

Graphs illustrating the effects of fenretinide on neural stem cell markers in DMG cells. Subfigures show downregulated gene ontology pathways and reduced mRNA expression of key markers associated with stem cells such as Ascl1, Notch1, Hes1, Lef1, Nestin, Olig2, and Dll1 in fenretinide-treated cells compared to controls. — Fenretinide treatment down-regulates markers of neural stem cells in DMG cells. (A) Top downregulated gene ontology (biological processes) identified through RNA sequencing analysis in HSJD-DIPG007 cells treated with 2 µM fenretinide for 24 h Expression of **(B)** Ascl1, **(C)** Notch1 **(D)** Hes1 **(E)** Lef1, **(F)** Nestin, **(G)** Olig2, **(H)** Dll1 mRNA as quantified by real-time PCR in HSJD-DIPG007 and RA055 cells treated with fenretinide for 24 h. Gene expression levels were normalized to the 18s/B2M control, and expression was compared between the untreated and treated samples. * = P < 0.05, **=P < 0.005.

Fenretinide Treatment Upregulates the Mitophagy and Autophagy

Pathways related to mitochondrial dysfunction, including mitophagy, were identified, supporting the ROS-mediated effects of fenretinide (Supplementary Table 5). Gene expression analysis revealed cell line-specific responses to treatment. In RA055 cells, mitophagy-related markers such as PARKIN expression increased significantly after 48 h, while PINK1 remained unchanged. HSJD-DIPG007 cells showed no changes in PARKIN or PINK1 expression (Supplementary Figure S26A,B). Autophagy markers in RA055 cells showed elevated WIPI1, LC3, and SQSTM1 expression, whereas HSJD-DIPG007 cells exhibited increased LC3 and SQSTM1 but no changes in WIPI1 (Supplementary Figure S26C–E). Proteomic analysis confirmed significant effects in HSJD-DIPG007 at 48 h, with increased levels of PARKIN, LC3A/B, WIPI1, and SQSTM1 (Supplementary Figure S27A–F). In RA055 cells, PINK1 and WIPI1 showed changes at 48 h, with no alterations in other mitophagy or autophagy mediators at the protein level (Supplementary Figure S27G-K).

Fenretinide is Active In Vivo in PDGFRα-Amplified DMG Models

We next sought to examine the in vivo efficacy of fenretinide in PDGFRα-amplified RA055 DMG orthotopic models using 2 different formulations (LYM-X-SORB and nanomicellar fenretinide). The LYM-X-SORB-fenretinide formulation was designed to improve the intestinal absorption of fenretinide as described previously.²⁰ The nanomicellar fenretinide formulation has been more recently developed to allow a nano-targeted delivery of fenretinide²⁹ to solid tumors. RA055 engrafted mice were treated with vehicle control or fenretinide formulations. The median survival was 48 days for vehicle-treated, and LYM-X-SORB fenretinide-treated mice showed a significant survival benefit with a median survival of 60 days (P = 0.0149) compared to vehicle-treated mice (Figure 6A). RA055 engrafted mice were treated with nanomicelle control and nanomicellar fenretinide. The median survival was significantly enhanced in nanomicellar fenretinide-treated mice with a median survival of 66 days compared to 55 days in the nanomicelle control (P = 0.0264) (Figure 6B). In addition, we employed an immunocompetent murine model of DMG featuring a constitutively active PDGFRa to assess the efficacy of the LYM-X-SORB-fenretinide formulation. Our findings revealed a significant increase in median survival for animals treated with fenretinide (58 days) compared to the vehicle-treated group (54 days, P = 0.0082) (Figure 6C). Pharmacokinetic analysis further validated the presence of fenretinide in the brains of the engrafted animals (Figure 6D). The observed differences in drug levels between the PDX and transgenic models, despite moderate efficacy in both, may be attributed to their distinct tumor growth patterns, with PDX models showing diffuse growth similar to patient biopsies and transgenic models displaying a glioblastoma-like structure with densely packed, highly nuclear regions.³⁰

Graphs and images depicting fenretinide activity in PDGFRα-amplified DMG models. Kaplan-Meier survival plots compare LYM-X-SORB fenretinide and nanomicellar fenretinide-treated versus control in animals engrafted with patient-derived or murine DMG cells. Additional subfigures present immunohistochemical images and quantification of Ki-67 and PDGFRa expression in fenretinide-treated and control groups. — Fenretinide is active against PDGFRα-amplified DMG models.(A) Kaplan-Meier plot of percentage survival of PDGFRa-amplified DMG -xenografted BalbC/Nude mice treated with LYM-X-SORB fenretinide or vehicle control. **(B)** Kaplan-Meier plot of percentage survival of PDGFRa-amplified DMG-xenografted BalbC/Nude mice treated with nanomicellar fenretinide or nanomicelles. **(C)** Kaplan-Meier plot of percentage survival of murine DMG cells (H3.3/K27M, TP53loss, PDGFRA/D842V) engrafted in C57/BL6 animals treated with LYM-X-SORB fenretinide or vehicle. **(D)** Pharmacokinetic analysis performed in the brains of animals post-treatment with fenretinide. **(E)** Representative Ki67 staining images and resulting quantification of DMG tumors post LYM-X-SORB fenretinide treatment. **(F)** Representative Ki67 staining images and resulting quantification of DMG tumors post nanomicellar fenretinide treatment. **(G)** Representative PDGFRα staining images and resulting quantification of DMG tumors post LYM-X-SORB fenretinide treatment. **(H)** Representative PDGFRα staining images and resulting quantification of DMG tumors post nanomicellar fenretinide treatment. The black scale bar on each image indicates 50 µm.

Ki-67 staining confirmed the presence of tumor cells in the brainstem of intracranially injected mice, with a significant decrease (P < 0.0001) in Ki67 staining observed in the brains of mice treated with LYM-X-SORB fenretinide (Figure 6E, Supplementary Figure S28A) or nanomicellar fenretinide (P = 0.0003) (Figure 6F, Supplementary Figure S28E). However, no significant changes were observed in cleaved caspase-3 staining with either fenretinide formulation (Supplementary Figure S29A, B, Supplementary Figure S28B,F).

PDGFRα quantification with immunohistochemistry confirmed that treatment with fenretinide inhibited PDGFR expression in vivo, with a significant reduction in PDGFRα-positive cells observed in the brains of mice treated with LYM-X-SORB fenretinide (P = 0.0053) (Figure 6G, Supplementary Figure S28C) and a significant decrease in mice treated with nanomicellar fenretinide (P = 0.014) (Figure 6H, Supplementary Figure S28G). These findings demonstrate the antitumor efficacy of both fenretinide formulations and their potential as brain penetrant modalities for targeting PDGFRα in a PDGFRα-amplified DMG model. Further immunohistochemical analysis revealed that neither fenretinide formulation restored H3K27me3 levels in the RA055 PDX model (Supplementary Figure S28D,H, Supplementary Figure S29C,D). Similarly, no effects on trimethylation were observed in the HSJD-DIP007 PDX model following LYM-X-SORB fenretinide treatment (Supplementary Figure S30).

Fenretinide Combination with Irradiation Does Not Enhance Therapeutic Benefit In Vivo

Given that radiotherapy is currently the only standard therapeutic option for DMG patients, we evaluated whether combining it with fenretinide could enhance its effectiveness. Clonogenic assays were performed in HSJD-DIPG007 cells, treating them with a low dose of fenretinide (0.2 µM) and 2 Gy irradiation. The combination treatment resulted in a significantly lower number of colonies compared to the individual treatments (Supplementary Figure S31A). Building on these promising findings, we assessed the therapeutic efficacy of the LYM-X-SORB fenretinide formulation in combination with irradiation in the HSJD-DIPG007 orthotopic DMG model. Engrafted mice were divided into 4 treatment groups: vehicle control, LYM-X-SORB fenretinide formulation, irradiation (8 Gy), and the combination. The median survival for vehicle-treated mice was 53 days, while LYM-X-SORB fenretinide-treated mice showed a median survival of 58 days, as previously reported (P = 0.009). Although the irradiation-treated group demonstrated improved survival (65 days, P < 0.0001 vs vehicle), the combination therapy did not result in further survival benefit (61 days) (Supplementary Figure S31B).

Discussion

Active treatments are urgently needed for DMG. HTS allows the evaluation of large numbers of pharmaceutical compounds with various biological activities, targets, and mechanisms of action. A considerable advantage of using an HTS approach is that it does not rely on inhibiting known oncogenic targets and can potentially unveil novel targets for further investigation. However, one of the limitations is that it is primarily an in vitro technique that does not consider the many barriers and interactions that occur in vivo. Such screening has been used to identify therapies for other cancers,^31,32 but limited screening has been undertaken to date for DMG.³³ We have identified potential novel therapies using our DMG cell panel in vitro and our unique in vivo animal models.

Of the 6 HTS drug hits, we found that 5 drugs—lanatoside C, ivermectin, SAHA, mefloquine hydrochloride, and parthenolide, despite showing in vitro activity, had no impact on animal survival and cellular proliferation. Previous in vitro studies have demonstrated that lanatoside C,^34–36 ivermectin,^37–39 SAHA,⁴⁰ mefloquine hydrochloride⁴¹ and parthenolide⁴² all have anticancer potential due to their mechanisms of action and molecular pathways targets. Lanatoside C⁴³ and ivermectin^37,44 have been examined in glioblastoma (GBM), nevertheless, the employment of subcutaneous models did not allow for testing of BBB permeability. Parthenolide has previously been examined in an orthotopic GBM model, decreasing tumor volume, however, the effect on overall survival was not reported.⁴⁵ Despite the strong in vitro activity and sufficient levels of mefloquine in animal brains, we observed no therapeutic efficacy. This lack of efficacy may be attributed to the absence of key targets identified for mefloquine, such as beta-catenin,⁴⁶ lysosomes,⁴⁷ and lipid peroxidation (LPCAT3)⁴⁸ in the in vivo setting. The absence of these targets in the animal models could explain the failure to translate the observed in vitro effect into a therapeutic benefit. Generally, we demonstrated that the lack of therapeutic efficacy observed in vivo is likely due to an inability to achieve sufficient drug concentration in the tumor to have a potent effect. These comprehensive negative results highlight the intact BBB as a key mechanism of failure of therapies in DMG, and the necessity of comprehensive preclinical testing in vitro and in vivo before clinical trials are initiated.

Fenretinide is a semisynthetic retinoid derivative with potential antineoplastic and chemo-preventive activities,^20,27 and a previous study has shown in vivo efficacy in medulloblastoma.⁴⁹ We found that fenretinide significantly increased survival in 2 orthotopic patient-derived DMG animal models and one immunocompetent DMG model using 2 unique formulations of fenretinide (LYM-X-SORB and nanomicellar fenretinide), indicating that fenretinide can penetrate the BBB and is active in DMG. Similar to reports in other cancer types, we showed that treatment with fenretinide increased ROS production and induced mitochondrial depolarization and apoptosis in DMG cells.^25,50,51 Our experiments have uncovered new mechanisms of action for fenretinide. We found that treatment with fenretinide inhibits PDGFRα gene expression, protein, and phosphorylation levels. We confirmed that fenretinide upregulates ER stress and UPR^52–54 autophagy and concurrently identified a novel downregulation of neurogenesis. Furthermore, we confirmed that fenretinide is also effective in DMG models exhibiting PDGFRα amplification, a driver that can result in resistance to RTK inhibitors.⁵⁵ The loss of H3K27 trimethylation, driven by the H3K27M mutation, is a hallmark of DMG tumors. In vitro, fenretinide treatment led to a significant increase in H3K27me3, likely linked to higher total H3 levels. However, RNA sequencing did not show upregulation of histone-related genes, suggesting the change is not due to new histone synthesis. A possible explanation is histone leakage from the nucleus into the cytoplasm, forming micronuclei with altered H3K27me3.⁵⁶ Further studies are needed to explore whether these changes reflect a stress response or a role for histones as danger signals.⁵⁷ In vivo, fenretinide treatment did not result in significant changes, indicating that the drug concentrations reaching the brain may not induce strong epigenetic effects.

Recently Filbin et al.⁹ found that H3K27M mutant gliomas contain cells that resemble oligodendrocyte precursor cells (OPC), which exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are in part sustained by PDGFRα signaling. This suggests that the oligodendrocyte precursor-like cells are the predominant subpopulation of H3K27M tumors and may be susceptible to therapeutic strategies that target lineage-defined cellular programs such as PDGFRα.⁹ Although our experiments indicate effective targeting of PDGFRα, further experiments may determine if fenretinide can directly influence OPC-like cells.

Retinoid compounds and their synthetic analogs have long been studied as possible anticancer agents in clinical trials for adult and pediatric malignancies.⁵⁸ However, vitamin-A-associated toxicity has limited its long-term use, thus, other compounds with reduced side effects have been developed, such as fenretinide.⁵⁸ Multiple studies have indicated that fenretinide has an improved toxicity profile with accumulation in fatty tissues such as adipose tissue, mammary gland, and brain rather than the liver.^59,60 We have shown that fenretinide is a PDGFRα inhibitor with BBB permeability with promise for clinical translation. Until recently, clinical trials of PDGFR inhibitors in CNS tumors have failed due to toxicity, low BBB permeability, and limited accumulation at the tumor site.^61,62 PDGFRα inhibitor avapritinib is currently in a clinical trial (NCT04773782) and has demonstrated high BBB penetration making it a promising therapeutic for CNS tumors, supported by case reports suggesting some clinical activity in DMG.⁶³ Unfortunately, in our studies, combining fenretinide with irradiation did not offer any additional survival benefit over either treatment alone. Future studies are needed to determine whether this potential therapeutic efficacy can be enhanced through combinatorial strategies. Transcriptomic analyses, such as bulk RNA-seq or single-cell RNA-seq in the brains from DMG PDX or transgenic models, would be beneficial in identifying further mechanisms of action, assessing the effects on the microenvironment, and determining synergistic combinations.

Here we utilized a diverse panel of DMG primary cultures for high-throughput drug screening, revealing the critical impact of blood-brain barrier (BBB) permeability on drug efficacy in vivo. Among the identified compounds, fenretinide emerged as a drug that warrants further investigation as a potential combination therapy for DMG. Mechanistic studies demonstrated that fenretinide enhanced apoptotic cell death by inhibiting PDGFRα transcription, leading to downregulation of PDGFRa, increasing ROS production, with induction of apoptosis, increasing ER stress, UPR and downregulating neurogenesis, revealing new oncogenic dependencies in this disease. Notably, fenretinide represents a novel treatment approach targeting PDGFRα in DMG, overcoming the limitation of many PDGFR inhibitors with poor BBB penetration. With its BBB permeability and potent anti-DMG activity, fenretinide holds promising clinical potential as a PDGFRα inhibitor for the treatment of DMG.

Supplementary material

Supplementary material is available online at Neuro-Oncology (https://academic.oup.com/neuro-oncology).

noaf035_suppl_Supplementary_Materials

noaf035_suppl_supplementary_materials.docx^{(30.7MB, docx)}

Acknowledgements

We thank Prof Michelle Monje, Dr Angel Montero Carcaboso, and Dr Esther Hulleman for generously supplying the SU-DIPG, HSJD-DIPG, and VUMC-DIPG10 cells, respectively and the Core Services Group at Children’s Cancer Institute for their technical support. We thank Dr Barry Maurer for generously supplying the Fenretinide LYM-X-SORB formulation. Mass spectrometric results were obtained at the Bioanalytical Mass Spectrometry Facility within the Mark Wainwright Analytical Centre of the University of New South Wales. We thank the Biological Resources Imaging Laboratory (BRIL) at the University of New South Wales for technical assistance. RNA sequencing was performed at the Ramaciotti Centre for Genomics at the University of New South Wales, Sydney, Australia.

Contributor Information

Dannielle H Upton, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Jie Liu, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Sandra M George, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Santosh Valvi, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Caitlin Ung, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Benjamin S Rayner, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Anjana Gopalakrishnan, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Ruby Pandher, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Aaminah Khan, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Pooja Venkat, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Chelsea Mayoh, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Holly Holliday, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Nicole Yeung, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Hieu Nguyen, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Laura Franshaw, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Anahid Ehteda, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Han Shen, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Giovanna Farruggia, Alma Mater Studiorum University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy.

Isabella Orienti, Alma Mater Studiorum University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy.

C Patrick Reynolds, Cancer Center, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, USA.

Maria Tsoli, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

David S Ziegler, Kid’s Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia; School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia; Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia.

Conflict of interest statement: DSZ reports consulting / advisory board fees from Bayer, Astra Zeneca, Accendatech, Novartis, Day One, FivePhusion, Amgen, Alexion, and Norgine and research support from Accendatech.

Funding

National Health and Medical Research Council (D.S.Z., A.K.) Synergy Grant #2019056. National Health and Medical Research Council, Investigator Grant APP2017898 (D.S.Z.). Cancer Institute New South Wales Program Grant (TPG2037) (D.S.Z.). Cancer Australia, Cancer Council NSW (D.S.Z.). Cure Brain Cancer Foundation (D.S.Z., M.T.). The Cure Starts Now Australia, The Cure Starts Now Foundation, Hope for Caroline Foundation, Julian Boivin Courage for Cures Foundation, Abbie's Army, Michael Mosier Defeat DIPG Foundation, Reflections of Grace Foundation, Brooke Healey Foundation, Soar With Grace Foundation, Jeffrey Thomas Hayden Foundation, The Jones Family Foundation, Musella Foundation, Pray, Hope Believe Foundation, Smiles for Sophie Foundation, Benny's World, Love Chloe Foundation, Aiden's Avengers, A Cure from Caleb Society, The Operation Grace White Foundation, Ryan's Hope, Wayland Villars DIPG Foundation, American Childhood Cancer Organization, Juliana Rose Donnelly Trust, Sheila Jones & Friends, The Ellie Kavalieros DIPG Research Fund, Voices Against Brain Cancer and The DIPG Collaborative (M.T., D.S.Z.). Levi’s Project (M.T., D.S.Z.). Benny Wills Foundation (D.S.Z.).

Author contributions

Conceptualization: DU, MT. DSZ. Methodology: IO, GF, PR. Investigation: DU, JL, SG, SV, NY, CU, BR, AG, RP, AK, LF, AE, PV, CM, MT. Funding acquisition: MT, DSZ. Supervision: MT, DSZ. Writing—original draft: DU. Writing—review & editing: DU, MT, DSZ.

Data Availability

All data are available in the main text or the supplementary materials

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28. Rani S, O’Driscoll L. Analysis of changes in phosphorylation of receptor tyrosine kinases: antibody arrays. In: Germano S, ed. Receptor Tyrosine Kinases: Methods and Protocols. New York, NY: Springer New York; 2015:15–23. [DOI] [PubMed] [Google Scholar]
29. Orienti I, Farruggia G, Nguyen F, et al. Nanomicellar lenalidomide-fenretinide combination suppresses tumor growth in an MYCN amplified neuroblastoma tumor. Int J Nanomedicine. 2020;15:6873–6886. [DOI] [PMC free article] [PubMed] [Google Scholar]
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35. Durmaz I, Guven EB, Ersahin T, et al. Liver cancer cells are sensitive to Lanatoside C induced cell death independent of their PTEN status. Phytomedicine. 2016;23(1):42–51. [DOI] [PubMed] [Google Scholar]
36. Chao M-W, Chen T-H, Huang H-L, et al. Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells. Sci Rep. 2017;7(1):46134. [DOI] [PMC free article] [PubMed] [Google Scholar]
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38. Dou Q, Chen H-N, Wang K, et al. Ivermectin induces cytostatic autophagy by blocking the PAK1/Akt axis in breast cancer. Cancer Res. 2016;76(15):4457–4469. [DOI] [PubMed] [Google Scholar]
39. Juarez M, Schcolnik-Cabrera A, Dueñas-Gonzalez A. The multitargeted drug ivermectin: from an antiparasitic agent to a repositioned cancer drug. Am J Cancer Res. 2018;8(2):317–331. [PMC free article] [PubMed] [Google Scholar]
40. Lohitesh K, Saini H, Srivastava A, et al. Autophagy inhibition potentiates SAHA‑mediated apoptosis in glioblastoma cells by accumulation of damaged mitochondria. Oncol Rep. 2018;39(6):2787–2796. [DOI] [PubMed] [Google Scholar]
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42. Anderson KN, Bejcek BE. Parthenolide induces apoptosis in glioblastomas without affecting NF-kappaB. J Pharmacol Sci. 2008;106(2):318–320. [DOI] [PubMed] [Google Scholar]
43. Badr CE, Wurdinger T, Nilsson J, et al. Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway. Neuro-Oncology. 2011;13(11):1213–1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
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47. Das S, Dielschneider R, Chanas-LaRue A, Johnston JB, Gibson SB. Antimalarial drugs trigger lysosome-mediated cell death in chronic lymphocytic leukemia (CLL) cells. Leuk Res. 2018;70:79–86. [DOI] [PubMed] [Google Scholar]
48. Tao Q, Liu N, Wu J, et al. Mefloquine enhances the efficacy of anti-PD-1 immunotherapy via IFN-gamma-STAT1-IRF1-LPCAT3-induced ferroptosis in tumors. J ImmunoTher Cancer. 2024;12(3):e008554. [DOI] [PMC free article] [PubMed] [Google Scholar]
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51. Rehman F, Shanmugasundaram P, Schrey MP. Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity. Br J Cancer. 2004;91(10):1821–1828. [DOI] [PMC free article] [PubMed] [Google Scholar]
52. Anding AL, Jones JD, Newton MA, Curley RW, Jr, Clagett-Dame M. 4-HPR is an endoplasmic reticulum stress aggravator and sensitizes breast cancer cells resistant to TRAIL/Apo2L. Anticancer Res. 2018;38(8):4403–4416. [DOI] [PubMed] [Google Scholar]
53. Lai WL, Wong NS. The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells. Exp Cell Res. 2008;314(8):1667–1682. [DOI] [PubMed] [Google Scholar]
54. Kadara H, Lacroix L, Lotan D, Lotan R. Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-hydroxyphenyl)retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells. Cancer Biol Ther. 2007;6(5):705–711. [DOI] [PubMed] [Google Scholar]
55. Chen Y-f, Fu L-w. Mechanisms of acquired resistance to tyrosine kinase inhibitors. Acta Pharmaceutica Sinica B. 2011;1(4):197–207. [DOI] [PMC free article] [PubMed] [Google Scholar]
56. Agustinus AS, Al-Rawi D, Dameracharla B, et al. Epigenetic dysregulation from chromosomal transit in micronuclei. Nature. 2023;619(7968):176–183. [DOI] [PMC free article] [PubMed] [Google Scholar]
57. Hadnagy A, Beaulieu R, Balicki D. Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics. Mol Cancer Ther. 2008;7(4):740–748. [DOI] [PubMed] [Google Scholar]
58. Dobrotkova V, Chlapek P, Mazanek P, Sterba J, Veselska R. Traffic lights for retinoids in oncology: molecular markers of retinoid resistance and sensitivity and their use in the management of cancer differentiation therapy. BMC Cancer. 2018;18(1):1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
59. Mehta RG, Moon RC, Hawthorne M, Formelli F, Costa A. Distribution of fenretinide in the mammary gland of breast cancer patients. Eur J Cancer. 1991;27(2):138–141. [DOI] [PubMed] [Google Scholar]
60. McIlroy GD, Delibegovic M, Owen C, et al. Fenretinide treatment prevents diet-induced obesity in association with major alterations in retinoid homeostatic gene expression in adipose, liver, and hypothalamus. Diabetes. 2013;62(3):825–836. [DOI] [PMC free article] [PubMed] [Google Scholar]
61. Nabors LB, Supko JG, Rosenfeld M, et al. ; New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium. Phase I trial of sorafenib in patients with recurrent or progressive malignant glioma. Neuro-Oncology. 2011;13(12):1324–1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
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63. Mayr L, Trissal M, Schwark K, et al. DDDR-22. Translation of the PDGFRA/KIT inhibitor Avapritinib for pediatric High-Grade Glioma. Neuro-Oncology. 2022;24(Supplement_7):vii103–vii103. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

noaf035_suppl_Supplementary_Materials

noaf035_suppl_supplementary_materials.docx^{(30.7MB, docx)}

Data Availability Statement

The data generated in this study are available upon request from the corresponding author.

All data are available in the main text or the supplementary materials

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[CIT0038] 38. Dou Q, Chen H-N, Wang K, et al. Ivermectin induces cytostatic autophagy by blocking the PAK1/Akt axis in breast cancer. Cancer Res. 2016;76(15):4457–4469. [DOI] [PubMed] [Google Scholar]

[CIT0039] 39. Juarez M, Schcolnik-Cabrera A, Dueñas-Gonzalez A. The multitargeted drug ivermectin: from an antiparasitic agent to a repositioned cancer drug. Am J Cancer Res. 2018;8(2):317–331. [PMC free article] [PubMed] [Google Scholar]

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[CIT0043] 43. Badr CE, Wurdinger T, Nilsson J, et al. Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway. Neuro-Oncology. 2011;13(11):1213–1224. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CIT0045] 45. Nakabayashi H, Shimizu K. Involvement of Akt/NF-κB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo. BMC Cancer. 2012;12(1):453. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0046] 46. Li YH, Yang SL, Zhang GF, Wu J-C, Gong L-L, Ming-Zhong, Lin R-X. Mefloquine targets beta-catenin pathway and thus can play a role in the treatment of liver cancer. Microb Pathog. 2018;118:357–360. [DOI] [PubMed] [Google Scholar]

[CIT0047] 47. Das S, Dielschneider R, Chanas-LaRue A, Johnston JB, Gibson SB. Antimalarial drugs trigger lysosome-mediated cell death in chronic lymphocytic leukemia (CLL) cells. Leuk Res. 2018;70:79–86. [DOI] [PubMed] [Google Scholar]

[CIT0048] 48. Tao Q, Liu N, Wu J, et al. Mefloquine enhances the efficacy of anti-PD-1 immunotherapy via IFN-gamma-STAT1-IRF1-LPCAT3-induced ferroptosis in tumors. J ImmunoTher Cancer. 2024;12(3):e008554. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CIT0050] 50. Lovat PE, Corazzari M, Goranov B, Piacentini M, Redfern CP. Molecular mechanisms of fenretinide-induced apoptosis of neuroblastoma cells. Ann N Y Acad Sci. 2004;1028(1):81–89. [DOI] [PubMed] [Google Scholar]

[CIT0051] 51. Rehman F, Shanmugasundaram P, Schrey MP. Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity. Br J Cancer. 2004;91(10):1821–1828. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0052] 52. Anding AL, Jones JD, Newton MA, Curley RW, Jr, Clagett-Dame M. 4-HPR is an endoplasmic reticulum stress aggravator and sensitizes breast cancer cells resistant to TRAIL/Apo2L. Anticancer Res. 2018;38(8):4403–4416. [DOI] [PubMed] [Google Scholar]

[CIT0053] 53. Lai WL, Wong NS. The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells. Exp Cell Res. 2008;314(8):1667–1682. [DOI] [PubMed] [Google Scholar]

[CIT0054] 54. Kadara H, Lacroix L, Lotan D, Lotan R. Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-hydroxyphenyl)retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells. Cancer Biol Ther. 2007;6(5):705–711. [DOI] [PubMed] [Google Scholar]

[CIT0055] 55. Chen Y-f, Fu L-w. Mechanisms of acquired resistance to tyrosine kinase inhibitors. Acta Pharmaceutica Sinica B. 2011;1(4):197–207. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0056] 56. Agustinus AS, Al-Rawi D, Dameracharla B, et al. Epigenetic dysregulation from chromosomal transit in micronuclei. Nature. 2023;619(7968):176–183. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0057] 57. Hadnagy A, Beaulieu R, Balicki D. Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics. Mol Cancer Ther. 2008;7(4):740–748. [DOI] [PubMed] [Google Scholar]

[CIT0058] 58. Dobrotkova V, Chlapek P, Mazanek P, Sterba J, Veselska R. Traffic lights for retinoids in oncology: molecular markers of retinoid resistance and sensitivity and their use in the management of cancer differentiation therapy. BMC Cancer. 2018;18(1):1059. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0059] 59. Mehta RG, Moon RC, Hawthorne M, Formelli F, Costa A. Distribution of fenretinide in the mammary gland of breast cancer patients. Eur J Cancer. 1991;27(2):138–141. [DOI] [PubMed] [Google Scholar]

[CIT0060] 60. McIlroy GD, Delibegovic M, Owen C, et al. Fenretinide treatment prevents diet-induced obesity in association with major alterations in retinoid homeostatic gene expression in adipose, liver, and hypothalamus. Diabetes. 2013;62(3):825–836. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0061] 61. Nabors LB, Supko JG, Rosenfeld M, et al. ; New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium. Phase I trial of sorafenib in patients with recurrent or progressive malignant glioma. Neuro-Oncology. 2011;13(12):1324–1330. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0062] 62. Wetmore C, Daryani VM, Billups CA, et al. Phase II evaluation of sunitinib in the treatment of recurrent or refractory high-grade glioma or ependymoma in children: a children’s Oncology Group Study ACNS1021. Cancer Med. 2016;5(7):1416–1424. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0063] 63. Mayr L, Trissal M, Schwark K, et al. DDDR-22. Translation of the PDGFRA/KIT inhibitor Avapritinib for pediatric High-Grade Glioma. Neuro-Oncology. 2022;24(Supplement_7):vii103–vii103. [Google Scholar]

PERMALINK

High-throughput in vitro drug screening and in vivo studies identify fenretinide as a brain-penetrant DMG therapeutic

Dannielle H Upton

Jie Liu

Sandra M George

Santosh Valvi

Caitlin Ung

Benjamin S Rayner

Anjana Gopalakrishnan

Ruby Pandher

Aaminah Khan

Pooja Venkat

Chelsea Mayoh

Holly Holliday

Nicole Yeung

Hieu Nguyen

Laura Franshaw

Anahid Ehteda

Han Shen

Giovanna Farruggia

Isabella Orienti

C Patrick Reynolds

Maria Tsoli

David S Ziegler

Abstract

Background

Methods

Results

Conclusion

Key Points.

Importance of the Study.

Materials and Methods

Human DMG Neurosphere-Forming Culture

Inducible Lentiviral PDGFRα shRNA DMG Cells

High-Throughput Screening

Proliferation Assay

Soft Agar Colony Assays

Lentiviral Experiments

Intracranial DMG Cell Injection, Animal Monitoring, and Endpoint

In Vivo Efficacy Drug Treatments

Pharmacokinetic Analysis of In Vivo Brain samples

Histological Analysis of Brain Tissue

Apoptosis, Mitochondrial Function, and DNA damage assays

Mitochondrial Depolarization

LOcation-Specific DNA Repair and Damage Quantification (LORD-Q)

Caspase Activity Assay

Annexin V FITC/Propidium Iodide Assay

Molecular Analysis: Proteome Profiler Array

Protein Kinase Activity Measurement

Real-time Quantitative Polymerase Chain Reaction (qPCR)

Western Blot Analysis

RNA Sequencing and Analysis

Statistics

Study Approval

Data Availability

Results

High Throughput Screening Identifies Compounds of Interest in DMG

Figure 1.

Validation of Anti-tumor Efficacy of Drug Candidates in DMG Orthotopic Animals

Figure 2.

Fenretinide is the Only Active Retinoid When Tested in DMG Cells

Fenretinide induces the production of reactive oxygen species (ROS), caspase activation, and apoptosis in HSJD-DIPG007 cells in vitro.

Figure 3.

Fenretinide Inhibits PDGFRα in DMG Cells

Fenretinide Treatment Upregulates the ER Stress and UPR Pathways while Also Downregulating Neurogenesis

Figure 4.

Figure 5.

Fenretinide Treatment Upregulates the Mitophagy and Autophagy

Fenretinide is Active In Vivo in PDGFRα-Amplified DMG Models

Figure 6.

Fenretinide Combination with Irradiation Does Not Enhance Therapeutic Benefit In Vivo

Discussion

Supplementary material

Acknowledgements

Contributor Information

Funding

Author contributions

Data Availability

References

Associated Data