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Diffuse Midline Gliomas (DMGs) are devastating and incurable pediatric brain tumours. DMGs are characterised by epigenetic aberrations in the form of histone modifications, coupled with mitotic abnormalities, which together drive a stem-like transcriptional cell state that is critically dependent on the cell cycle. Polo-like kinase 1 (PLK1) is a pivotal regulator of mitosis and cell cycle progression. To determine an effective therapeutic strategy against DMG, we characterised the anti-tumour potential of PLK1 inhibition, either alone or in combination with radiotherapy. We show that targeting PLK1 with small molecule inhibitors BI2536 and BI6727 (volasertib) reduced DMG colony formation and cell proliferation, and induced G2/M checkpoint arrest and apoptosis in vitro at physiologically achievable, clinically relevant concentrations. When combined with radiotherapy, PLK1 inhibitors acted as potent radiosensitisers with synergistic suppression of DMG survival. We further show that PLK1 inhibitors exert potent anti-tumour effects in highly aggressive orthotopic DMG models, effectively extending the median survival of tumour harbouring mice both as a single agent and enhanced in combination with radiotherapy. We further demonstrate a potential novel role for PLK1 in defining an oncogenic transcriptional state in DMG by acting as a conduit between cell cycle progression and the DMG epigenome through modulation of polycomb repressive complex 2 (PRC2) activity and its resultant histone methyltransferase function. Together, these data indicate that targeting PLK1 is a highly promising therapeutic strategy to arrest DMG tumorgenicity. This data has formed the basis for a planned international Phase 1/2 trial of the PLK1 inhibitor volasertib in combination with radiotherapy in paediatric patients with DMG. Citation Format: Elisha Hayden, Laura Franshaw, Dannielle Upton, Jie Liu, Hieu Nguyen, Swapna Joshi, Maria Tsoli, Emmy Dolman, Benjamin Rayner, David Ziegler. Preclinical examination of PLK1 inhibitors for the treatment of diffuse midline gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1561.

Diffuse Intrinsic Pontine Gliomas (DIPG) are a subset of Diffuse Midline Gliomas (DMG) and are the most devastating of all brain tumours. There are currently no effective treatments. ACT001 is a novel anti-cancer agent in clinical development that is blood-brain-barrier permeable. Previous studies undertaken in adult glioblastoma suggest that ACT001 exerts an anti-tumour effect via induction of oxidative stress and inhibition of NF-kB and STAT3 pathways. In DIPG, we have found that ACT001 demonstrated potent cytotoxic activity against a panel of DIPG neurospheres and also inhibited colony formation. Flow cytometric analysis confirmed the induction of apoptosis in vitro. Interestingly, we also observed increased expression of some markers of the ROS-dependant NRF2 endogenous antioxidant pathway within DIPG neurospheres suggesting an alternative mechanism of inhibition of DIPG cell proliferation involving ROS generation. In vivo testing of ACT001 in a highly aggressive DIPG-orthotopic model showed that ACT001 was well tolerated and significantly improved survival. ACT001 treatment of HSJD-DIPG007 tumour-bearing animals extended median survival from 59 to 78 days (p=0.0005). ACT001 treated mice had significantly decreased proliferating DIPG cells, analysed by Ki67 staining (p=0.01), and significantly increased H3K27me3 staining (p=0.0479). Given the significant effects on H3K27me3 we evaluated two potential drug combinations with known, clinically available epigenetic modifers. Combination of ACT001 with the FACT inhibitor CBL0137 or the HDAC inhibitor SAHA each significantly and synergistically decreased colony formation. These combinations are currently being evaluated in vivo in orthotopic models of DIPG. ACT001 is currently in a Phase 1 paediatric trial for children with DIPG/DMG. Clinical activity has been demonstrated in DIPG/DMG patients, including a reduction in tumour burden and clinical response. These combined preclinical and clinical results suggest that ACT001 is a viable therapy for patients with DIPG/DMG and preclinical combination therapy testing may guide further clinical trials.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG., Diffuse intrinsic pontine glioma (DIPG) is an almost incurable malignant childhood brain tumor. Here, the authors show that the polyamine synthetic pathway is activated in DIPG and that the dual targeting of polyamine synthesis and uptake results in prolonged survival in animal models.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12-months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,570 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds - auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine - that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in vitro. To determine whether the in vitro efficacy could be replicated in vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide, auranofin and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different fenretinide formulations which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found that there were high over-expression levels of polyamine synthetic enzymes from DIPG primary patient samples and neurosphere cultures. Using alamar blue cytotoxicity and soft-agar clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2 and subsequently uptake of polyamines. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation in vitro. Consistent with the in vitro results, the combination of DFMO and AMXT 1501 significantly prolonged the survival of mice bearing 3 different DIPG orthografts with at least 2/3 of the animals surviving up to 160 days. Addition of irradiation further improved the survival of mice treated with DFMO and AMXT 1501. Differential expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG and other paediatric brain tumours including high grade gliomas compared with normal brain tissue. Our results suggest that DIPG tumours are exquisitely sensitive to polyamine inhibitors, and that dual blockade of polyamine synthesis and transport is a promising novel therapeutic strategy. AMXT 1501 is currently in clinical development, and following completion of an adult Phase 1 trial, a clinical trial of AMXT 1501 + DFMO for DIPG patients is planned through the CONNECT consortium.

DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found high expression levels of synthetic enzymes in the polyamine pathway in primary patient samples and cultures. Using cytotoxicity and clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Gene expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG compared with all other high-risk childhood cancers. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation. Consistent with the in vitro results, the combination treatment significantly prolonged the survival of mice bearing 3 different DIPG orthografts with 2/3 of the animals surviving up to 160 days. Addition of irradiation further improved the survival of mice treated with DFMO and AMXT 1501. Our results suggest that DIPG tumours are exquisitely sensitive to polyamine inhibitors and that dual blockade of polyamine synthesis and transport is a promising novel therapeutic strategy. AMXT 1501 is currently in clinical development for adult cancers (NCT03536728). A clinical trial for DIPG patients is planned through the CONNECT consortium.

Diffuse Intrinsic Pontine Glioma (DIPG) is the most aggressive pediatric high-grade glioma with median survival of only 12 months from diagnosis. Current therapies are essentially palliative. The blood-brain barrier (BBB) is a major obstacle, limiting delivery of effective chemotherapeutics into the brain. We hypothesized that tumors in the brainstem region have a BBB less permeable than tumors in other brain regions. We have confirmed the presence of an intact BBB in three orthotopic models of DIPG by Evans Blue extravasation assay. Immunohistochemical staining of CD13+ pericytes and CD34+ endothelial cells in healthy mouse brain compared to orthotopic DIPG model showed higher levels of both components in brainstem compared to cortical region. Single-cell RNA sequencing experiments are currently being undertaken to investigate region-specific differences in BBB cell populations and the impact of DIPG on signaling pathways that govern permeability. To determine if tumor location impacts therapeutic outcome, we performed in vivo efficacy studies with DIPG orthotopically injected into cortical region or brainstem region and treated with SAHA, HDAC inhibitor, or temsirolimus, mTOR inhibitor. Temsirolimus or SAHA was ineffective at extending survival in mice injected with DIPG in the brainstem compared to control. However, temsirolimus led to a significant improvement in survival in mice injected with DIPG cells in cortical region (median survival 85 days) compared to control (median survival 69 days (P≤0.01)). This suggests that the same tumor in cortical region may respond to systemic therapy that is ineffective in the brainstem and that the intact BBB in the brainstem is a major reason for treatment failure in DIPG. In conclusion, the BBB in the brainstem and in the presence of DIPG may be altered, changing signaling pathways that affect permeability. Understanding the brainstem cerebrovasculature may potentially lead to a novel strategy to treat DIPG as well as other brain tumors.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are a subset of Diffuse Midline Gliomas (DMG) and are the most devastating of all brain tumors. There are no effective treatments and all children die of their tumor within 12-months. We performed a high-throughput drug screen with 3,570 biologically active, clinically approved compounds against a panel of DIPG cultures. Parthenolide, a compound derived from the herb T.parthenium, was found to be one of the most effective drugs tested, demonstrating significant anti-tumor activity. However, parthenolide also affected healthy cell viability and showed no in-vivo efficacy. ACT001 is a novel agent in clinical development that is a fumarate salt form of dimethylamino-micheliolide, which is semi-synthesized from parthenolide. ACT001 is blood-brain-barrier permeable and exerts an anti-tumor effect via inhibition of NF-κB and STAT3 pathways. ACT001 demonstrated potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells and inhibited colony formation in-vitro. To determine whether this activity could be replicated in-vivo, we tested ACT001 in a DIPG-orthotopic model. ACT001 was well tolerated and significantly improved survival of tumor-bearing animals, extending survival by 33% in ACT001 treated mice. We have initiated a Phase 1 paediatric trial of ACT001 for children with relapsed/refractory solid or CNS tumors, with an expansion cohort planned for patients with DIPG/DMG. Eleven patients have been enrolled, and the dose escalated from dose level-1 at 188mg/m2 bd to dose level-4 at 700mg/m2 bd. To date, no dose limiting or Grade 3/4 toxicities have been observed. At the highest dose level, clinical activity has been demonstrated in two patients, one with DIPG with a reduction in tumor burden, and another with DMG with H3K27M mutation with an objective radiographic and clinical response. These combined preclinical and clinical results suggest that ACT001 is an active novel therapy for patients with DIPG/DMG.

Simple Summary Diffuse intrinsic pontine gliomas (DIPGs) are the most aggressive high-grade gliomas known to affect children. Due to the infiltrative nature of the DIPG tumours in the brainstem, they are very difficult to treat. Unfortunately, children succumb to their disease within 1–2 years from their diagnosis. Novel therapeutic treatments are, thus, urgently needed. Using primary cultures and orthotopic models of DIPG, we evaluated the therapeutic efficacy of passionfruit like nanoarchitectures functionalized with human serum albumin and loaded with doxorubicin (NA-HSA-Dox). We found that NA-HSA-Dox were significantly effective at penetrating DIPG spheroids and subsequently at reducing the proliferation and colony formation in DIPG cells. Although an antitumour effect was not observed in orthotopic models of DIPG, NA-HSA-Dox was well tolerated. These study demonstrates the importance of employing brain tumour orthotopic models for the identification of novel therapies and the need to develop treatments that effectively cross the blood brain barrier. Abstract Diffuse Intrinsic Pontine Gliomas (DIPGs) are highly aggressive paediatric brain tumours. Currently, irradiation is the only standard treatment, but is palliative in nature and most patients die within 12 months of diagnosis. Novel therapeutic approaches are urgently needed for the treatment of this devastating disease. We have developed non-persistent gold nano-architectures (NAs) functionalised with human serum albumin (HSA) for the delivery of doxorubicin. Doxorubicin has been previously reported to be cytotoxic in DIPG cells. In this study, we have preclinically evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoarchitectures (NAs-HSA-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and doxorubicin-loaded NAs. Colony formation assays demonstrated greater potency of NAs-HSA-Dox on colony formation compared to doxorubicin. Western blot analysis indicated increased apoptotic markers cleaved Parp, cleaved caspase 3 and phosphorylated H2AX in NAs-HSA-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of NAs-HSA-Dox into DIPG neurospheres compared to doxorubicin alone. Despite the potency of the NAs in vitro, treatment of an orthotopic model of DIPG showed no antitumour effect. This disparate outcome may be due to the integrity of the blood-brain barrier and highlights the need to develop therapies to enhance penetration of drugs into DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumour for which new treatments are needed. A high throughput drug screen of 3500 pharmaceutical compounds identified anti-malarials, including quinacrine as having potent activity against DIPG neurospheres. CBL0137, a compound modelled on quinacrine, is an anti-cancer compound which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. CBL0137 effectively crosses the blood-brain barrier and is currently in Phase I trials in adult cancer. CBL0137 induced apoptosis in DIPG neurospheres in vitro and had profound cytotoxic activity against a panel of DIPG cultures. In a DIPG orthotopic model, treatment with CBL0137 significantly improved survival. We found that treatment with CBL0137 up-regulated TP53 and increased histone H3.3 acetylation and tri-methylation in DIPG cells. We therefore examined the interaction between CBL0137 and the HDAC inhibitor, panobinostat. In vitro experiments showed that the two agents had profound synergistic activity against DIPG neurospheres in clonogenic assays and enhanced apoptosis. Transcriptomic analysis and immunoblotting indicated that combination treatment activated signalling pathways controlled by Retinoblastoma (RB)/E2F1 and subsequently increased phosphorylation and enzymatic activity of enhancer of zeste homolog 2 (EZH2). Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged the survival of two orthotopic models of DIPG, while histological analysis showed increased H3K27me3 and decreased Ki67 positive cells. Given these promising results, a paediatric trial of CBL0137 is planned to open through the Children’s Oncology Group with an expansion cohort for DIPG patients.

There are two errors and one omission in the original article. Author Gottardo's correct name is Nicholas G. Gottardo, author Hulleman's correct affiliation is no. 3 (VUMC, Amsterdam), and the Acknowledgements should include the following sentence: "We would like to thank Dr Angel Montero Carcaboso (Hospital Sant Joan de Deu, Barcelona, Spain) for generously supplying the HSJD-DIPG007 cells."