Zebrafish-A Suitable Model for Rapid Translation of Effective Therapies for Pediatric Cancers.

Pediatric cancers are the leading cause of disease-related deaths in children and adolescents. Most of these tumors are difficult to treat and have poor overall survival. Concerns have also been raised about drug toxicity and long-term detrimental side effects of therapies. In this review, we discuss the advantages and unique attributes of zebrafish as pediatric cancer models and their importance in targeted drug discovery and toxicity assays. We have also placed a special focus on zebrafish models of pediatric brain cancers-the most common and difficult solid tumor to treat.

The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

BACKGROUND: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. METHODS: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. RESULTS: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. CONCLUSIONS: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.

A road map for the treatment of pediatric diffuse midline glioma.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Single-molecule systems for detection and monitoring of plasma circulating nucleosomes and oncoproteins in Diffuse Midline Glioma.

The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of cancer patients potentiates a new generation of non-invasive diagnostics and treatment monitoring approaches. However, confident detection of these tumor-originating markers is challenging, especially in the context of brain tumors, in which extremely low amounts of these analytes circulate in the patient's plasma. Here, we applied a sensitive single-molecule technology to profile multiple histone modifications on millions of individual nucleosomes from the plasma of Diffuse Midline Glioma (DMG) patients. The system reveals epigenetic patterns that are unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly capture and quantify the tumor-originating oncoproteins, H3-K27M and mutant p53, from the plasma of children diagnosed with DMG. This single-molecule system allows for accurate molecular classification of patients, utilizing less than 1ml of liquid-biopsy material. Furthermore, we show that our simple and rapid detection strategy correlates with MRI measurements and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the utility of this approach for non-invasive treatment monitoring of DMG patients. This work underscores the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.

Rational combination platform trial design for children and young adults with Diffuse Midline Glioma: a report from PNOC.

Diffuse midline glioma (DMG) is a devastating pediatric brain tumor unresponsive to hundreds of clinical trials. Approximately 80% of DMGs harbor H3K27M oncohistones, which reprogram the epigenome to increase the metabolic profile of the tumor cells. We have previously shown preclinical efficacy of targeting both oxidative phosphorylation and glycolysis through treatment with ONC201, which activates the mitochondrial protease ClpP, and paxalisib, which inhibits PI3K/mTOR, respectively. This combination treatment aimed at inducing metabolic distress led to the design of the first DMG-specific platform trial PNOC022 (NCT05009992). Here, we expand on the PNOC022 rationale and discuss various considerations, including liquid biome, microbiome, and genomic biomarkers, quality-of-life endpoints, and novel imaging modalities, such that we offer direction on future clinical trials in DMG.

Bridging the age gap: a review of molecularly informed treatments for glioma in adolescents and young adults.

Gliomas are the most common primary central nervous system (CNS) tumors and a major cause of cancer-related mortality in children (age <15 years), adolescents and young adults (AYA, ages 15-39 years), and adults (age >39 years). Molecular pathology has helped enhance the characterization of these tumors, revealing a heterogeneous and ever more complex group of malignancies. Recent molecular analyses have led to an increased appreciation of common genomic alterations prevalent across all ages. The 2021 World Health Organization (WHO) CNS tumor classification, 5th edition (WHO CNS5) brings forward a nomenclature distinguishing "pediatric-type" and "adult-type" gliomas. The spectrum of gliomas in AYA comprises both "pediatric-like" and "adult-like" tumor entities but remains ill-defined. With fragmentation of clinical management between pediatric and adult centers, AYAs face challenges related to gaps in medical care, lower rates of enrollment in clinical trials and additional psychosocial and economic challenges. This calls for a rethinking of diagnostic and therapeutic approaches, to improve access to appropriate testing and potentially beneficial treatments to patients of all ages.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation.

H3K27M Mutant Glioma: Disease Definition and Biological Underpinnings.

High-grade glioma (HGG) is the most common cause of cancer death in children, and the most common primary central nervous system (CNS) tumor in adults. While pediatric HGG was once thought to be biologically similar to the adult form of disease, research has shown these malignancies to be significantly molecularly distinct, necessitating distinct approaches to their clinical management. However, emerging data have shown shared molecular events in pediatric and adult HGG including the histone H3K27M mutation. This somatic missense mutation occurs in genes encoding one of two isoforms of the Histone H3 protein, H3F3A (H3.3) or HIST1H3B (H3.1), and is detected in up to 80% of pediatric diffuse midline gliomas and in up to 60% of adult diffuse gliomas. Importantly, the H3K27M mutation is associated with poorer overall survival and response to therapy compared to patients with H3 wild-type tumors. Here, we review the clinical features and biological underpinnings of pediatric and adult H3K27M mutant glioma, offering a groundwork for understanding current research and clinical approaches for the care of patients suffering with this challenging disease.

Pan-cancer atlas of somatic core and linker histone mutations.

Recent genomic data points to a growing role for somatic mutations altering core histone and linker histone-encoding genes in cancer. However, the prevalence and the clinical and biological implications of histone gene mutations in malignant tumors remain incompletely defined. To address these knowledge gaps, we analyzed somatic mutations in 88 linker and core histone genes across 12,743 tumors from pediatric, adolescent and young adult (AYA), and adult cancer patients. We established a pan-cancer histone mutation atlas contextualized by patient age, survival outcome, and tumor location. Overall, 11% of tumors harbored somatic histone mutations, with the highest rates observed among chondrosarcoma (67%), pediatric high-grade glioma (pHGG, >60%), and lymphoma (>30%). Previously unreported histone mutations were discovered in pHGG and other pediatric brain tumors, extending the spectrum of histone gene alterations associated with these cancers. Histone mutation status predicted patient survival outcome in tumor entities including adrenocortical carcinoma. Recurrent pan-cancer histone mutation hotspots were defined and shown to converge on evolutionarily conserved and functional residues. Moreover, we studied histone gene mutations in 1700 pan-cancer cell lines to validate the prevalence and spectrum of histone mutations seen in primary tumors and derived histone-associated drug response profiles, revealing candidate drugs targeting histone mutant cancer cells. This study presents the first-of-its-kind atlas of both core and linker histone mutations across pediatric, AYA, and adult cancers, providing a framework by which specific cancers may be redefined in the context of histone and chromatin alterations.

A review of the anti-tumor potential of current therapeutics targeting the mitochondrial protease ClpP in H3K27-altered, diffuse midline glioma.

Diffuse midline gliomas (DMGs) are devastating pediatric brain tumors recognized as the leading cause of cancer-related death in children. DMGs are high-grade gliomas (HGGs) diagnosed along the brain's midline. Euchromatin is the hallmark feature of DMG, caused by global hypomethylation of H3K27 either through point mutations in histone H3 genes (H3K27M), or by overexpression of the enhancer of zeste homolog inhibitory protein (EZHIP). In a clinical trial for adults with progressive HGGs, a 22-year-old patient with a thalamic H3K27-altered DMG, showed remarkable clinical and radiological responses to dordaviprone (ONC201). This response in a H3K27-altered HGG patient, coupled with the lack of response of patients harboring wildtype-H3 tumors, has increased the clinical interest in dordaviprone for the treatment of DMG. Additional reports of clinical benefit have emerged, but research defining mechanisms of action (MOA) fall behind dordaviprone's clinical use, with biomarkers of response unresolved. Here, we summarize dordaviprone's safety, interrogate its preclinical MOA- identifying the mitochondrial protease 'ClpP' as a biomarker of response, and discuss other ClpP-agonists, expanding the arsenal of potential weapons in the fight against DMG. Finally, we discuss combination strategies including ClpP-agonists, and its immunomodulatory effects suggestive of a role for the tumor microenvironment in DMG patients' response.

OpenPBTA: The Open Pediatric Brain Tumor Atlas.

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children's Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and TP53 loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community.

PNOC015: Repeated convection-enhanced delivery of MTX110 (aqueous panobinostat) in children with newly diagnosed diffuse intrinsic pontine glioma.

BACKGROUND: The objective of this study was to determine the safety, tolerability, and distribution of MTX110 (aqueous panobinostat) delivered by convection-enhanced delivery (CED) in patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG) who completed focal radiation therapy (RT). METHODS: Patients with DIPG (2-21 years) were enrolled after RT. CED of MTX110 combined with gadoteridol was completed across 7 dose levels (DL) (30-90 µM; volumes ranging from 3 mL to 2 consecutive doses of 6 mL). An accelerated dose escalation design was used. Distribution of infusate was monitored with real-time MR imaging. Repeat CED was performed every 4-8 weeks. Quality-of-life (QoL) assessments were obtained at baseline, every 3 months on therapy, and end of therapy. RESULTS: Between May 2018 and March 2020, 7 patients who received a total of 48 CED infusions, were enrolled (median age 8 years, range 5-21). Three patients experienced dose-limited toxicities. Four grade 3 treatment-related adverse events were observed. Most toxicities were transient new or worsening neurologic function. Median overall survival (OS) was 26.1 months (95% confidence interval: 14.8-not reached). Progression-free survival was 4-14 months (median, 7). Cumulative percentage of tumor coverage for combined CED infusions per patient ranged from 35.6% to 81.0%. Increased CED infusions were negatively associated with self-reported QoL assessments. CONCLUSION: Repeat CED of MTX110 with real-time imaging with gadoteridol is tolerable for patients with DIPG. Median OS of 26.1 months compares favorably with historical data for children with DIPG. The results support further investigation of this strategy in a larger cohort.

LIN28B and Let-7 in Diffuse Midline Glioma: A Review.

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

Optical genome mapping identifies a novel pediatric embryonal tumor with a ZNF532::NUTM1 fusion.

The molecular characteristics of pediatric brain tumors have not only allowed for tumor subgrouping but have led to the introduction of novel treatment options for patients with specific tumor alterations. Therefore, an accurate histologic and molecular diagnosis is critical for optimized management of all pediatric patients with brain tumors, including central nervous system embryonal tumors. We present a case where optical genome mapping identified a ZNF532::NUTM1 fusion in a patient with a unique tumor best characterized histologically as a central nervous system embryonal tumor with rhabdoid features. Additional analyses including immunohistochemistry for NUT protein, methylation array, whole genome, and RNA-sequencing was done to confirm the presence of the fusion in the tumor. This is the first description of a pediatric patient with a ZNF532::NUTM1 fusion, yet the histology of this tumor is similar to that of adult cancers with ZNF::NUTM1 fusions reported in the literature. Although rare, the distinct pathology and underlying molecular characteristics of the ZNF532::NUTM1 tumor separates this from other embryonal tumors. Therefore, screening for this or similar NUTM1 rearrangements should be considered for all patients with unclassified central nervous system tumors with rhabdoid features to ensure accurate diagnosis. Ultimately, with additional cases, we may be able to better inform therapeutic management for these patients. © 2023 The Pathological Society of Great Britain and Ireland.