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B Introduction: b Post allograft transplant patients are prone to develop a continuum of lympho-proliferative diseases (PTLD) that exhibit distinct clinicopathological forms that range from benign to malignant. However, a mutation of Linker I Histone 1 (H1-4) i gene is detected in the Burkitt PTLD mass and this recurrent polymorphic PTLD tonsil. [Extracted from the article]

Central nervous system germ cell tumors (CNS-GCT) are malignant neoplasms that arise predominantly during adolescence and young adulthood. These tumors are typically sensitive to treatment, but resulting long-term health deficits are common. Additional clinical challenges include surgical risks associated with tumor biopsy, and need to determine treatment response for adapting radiotherapy protocols. The aim of this study was to establish the detectability of circulating-tumor DNA (ctDNA) from cerebrospinal fluid (CSF) of children with CNS-GCT as a potential biomarker. We obtained CSF from patients with CNS-GCT by lumbar puncture or intra-operatively. Cell-free DNA (cfDNA) was extracted and subjected to low-pass whole genome sequencing (LP-WGS). Copy-number alterations (CNAs) were inferred and served as a marker of measurable residual disease (MRD). Comparisons with imaging findings and tumor marker levels were made. A total of 29 CSF samples from 21 patients (16 with germinoma, 5 with non-germinomatous GCT) were sequenced. Twenty samples from 19 patients were collected at diagnosis, and 9 samples from 7 patients were collected during or after therapy. Among the diagnostic samples, CNAs were detected in samples from 17/19 patients (89%), which included 8 with marker-negative tumors. Specific clinical scenarios suggested that serial cfDNA analysis may carry utility in tracking treatment responses as well as clarifying indeterminate imaging findings. Our results provide evidence for the high-sensitivity in detecting ctDNA from CSF of CNS-GCT patients using LP-WGS, with potential utility for non-invasive diagnosis and disease monitoring in upcoming CNS-GCT studies., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Hospital for Sick Children Research Ethical Board (REB # 1000071241) and the Hong Kong Children’s Hospital Research Ethics Committee (HKCH-REC-2020-068). Written informed consent was obtained from all patients, their parents or guardians. Consent for publication: Written informed consent for the publication was obtained from all patients participants, their parents or guardians. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Diffuse intrinsic pontine glioma (DIPG) remains a significant therapeutic challenge due to the lack of effective and safe treatment options. This study explores the potential of combining histone deacetylase (HDAC) and carbonic anhydrase 9 (CA9) inhibitors in treating DIPG. Analysis of RNA sequencing data and tumor tissue from patient samples for the expression of the carbonic anhydrase family and hypoxia signaling pathway activity revealed clinical relevance for targeting CA9 in DIPG. A synergy screen was conducted using CA9 inhibitor SLC-0111 and HDAC inhibitors panobinostat, vorinostat, entinostat, and pyroxamide. The combination of SLC-0111 and pyroxamide demonstrated the highest synergy and was selected for further analysis. Combining SLC-0111 and pyroxamide effectively inhibited DIPG cell proliferation, reduced cell migration and invasion potential, and enhanced histone acetylation, leading to decreased cell population in S Phase. Additionally, the combination therapy induced a greater reduction in intracellular pH than either agent alone. Data from this study suggest that the combination of SLC-0111 and pyroxamide holds promise for treating experimental DIPG, and further investigation of this combination therapy in preclinical models is warranted to evaluate its potential as a viable treatment for DIPG., (© 2024. The Author(s), under exclusive licence to The Japan Society of Brain Tumor Pathology.)

With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine., (© 2024. The Author(s).)

We report a case of a primary cardiac spindle cell neoplasm with concerning histological features and a rare PDGFRA::USP8 gene fusion in a 3 year old boy. The patient presented with a large cardiac mass predominantly in the right ventricle, originating from the ventricular septum. The mass was resected with grossly negative margins. Pathology revealed an unclassified spindle cell neoplasm with a PDGFRA::USP8 gene fusion. This gene fusion has only been previously reported twice in the medical literature, one in a pediatric cardiac sarcoma and the other in an abdominal soft tissue tumor in an adult woman. The patient is alive and well with no evidence of recurrence 11 months after excision., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Molecular characterization is key to optimally diagnose and manage cancer. The complexity and cost of routine genomic analysis have unfortunately limited its use and denied many patients access to precision medicine. A possible solution is to rationalize use-creating a tiered approach to testing which uses inexpensive techniques for most patients and limits expensive testing to patients with the highest needs. Here, we tested the utility of this approach to molecularly characterize pediatric glioma in a cost- and time-sensitive manner., Methods: We used a tiered testing pipeline of immunohistochemistry (IHC), customized fusion panels or fluorescence in situ hybridization (FISH), and targeted RNA sequencing in pediatric gliomas. Two distinct diagnostic algorithms were used for low- and high-grade gliomas (LGGs and HGGs). The percentage of driver alterations identified, associated testing costs, and turnaround time (TAT) are reported., Results: The tiered approach successfully characterized 96% (95 of 99) of gliomas. For 82 LGGs, IHC, targeted fusion panel or FISH, and targeted RNA sequencing solved 35% (29 of 82), 29% (24 of 82), and 30% (25 of 82) of cases, respectively. A total of 64% (53 of 82) of samples were characterized without targeted RNA sequencing. Of 17 HGG samples, 13 were characterized by IHC and four were characterized by targeted RNA sequencing. The average cost per sample was more affordable when using the tiered approach as compared with up-front targeted RNA sequencing in LGG ($405 US dollars [USD] v $745 USD) and HGGs ($282 USD v $745 USD). The average TAT per sample was also shorter using the tiered approach (10 days for LGG, 5 days for HGG v 14 days for targeted RNA sequencing)., Conclusion: Our tiered approach molecularly characterized 96% of samples in a cost- and time-sensitive manner. Such an approach may be feasible in neuro-oncology centers worldwide, particularly in resource-limited settings.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Next generation sequencing (NGS) has increased the detection of fusion genes in cancer. NGS has found multiple fusions in single tumor samples; however, the incidence of this in pediatric soft tissue and bone tumors (PSTBTs) is not well documented. The aim of this study is to catalogue the incidence of multiple fusions in a series of PSTBTs, and apply a modified gene fusion classification system to determine clinical relevance., Methodology: RNA from 78 bone and soft tissue tumors and 7 external quality assessment samples were sequenced and analyzed using recently-described Metafusion (MF) software and classified using a modification of previously-published schema for fusion classification into 3 tiers: 1, strong clinical significance; 2, potential clinical significance; and 3, unknown clinical significance., Results: One-hundred forty-five fusions were detected in 85 samples. Fifty-five samples (65%) had a single fusion and 30 (35%) had more than 1 fusion. No samples contained more than 1 tier 1 fusion. There were 40 tier 1 (28%), 36 tier 2 (24%), and 69 (48%) tier 3 fusions., Conclusions: A significant percentage of PSTBTs harbor more than 1 fusion, and by applying a modified fusion classification scheme, the potential clinical relevance of such fusions can be determined., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Oncogenic fusion genes may be identified from next-generation sequencing data, typically RNA-sequencing. However, in a clinical setting, identifying these alterations is challenging against a background of nonrelevant fusion calls that reduce workflow precision and specificity. Furthermore, although numerous algorithms have been developed to detect fusions in RNA-sequencing, there are variations in their individual sensitivities. Here this problem was addressed by introducing MetaFusion into clinical use. Its utility was illustrated when applied to both whole-transcriptome and targeted sequencing data sets. MetaFusion combines ensemble fusion calls from eight individual fusion-calling algorithms with practice-informed identification of gene fusions that are known to be clinically relevant. In doing so, it allows oncogenic fusions to be identified with near-perfect sensitivity and high precision and specificity, significantly outperforming the individual fusion callers it uses as well as existing clinical-grade software. MetaFusion enhances clinical yield over existing methods and is able to identify fusions that have patient relevance for the purposes of diagnosis, prognosis, and treatment., Competing Interests: Disclosure Statement None declared., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted., Methods: Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected., Results: Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial., Conclusions: H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target., (© 2022. The Author(s).)

Chromatin structure, transcription, DNA replication, and repair are regulated via locus-specific incorporation of histone variants and posttranslational modifications that guide effector chromatin-binding proteins. Here we report unbiased, quantitative interactomes for the replication-coupled (H3.1) and replication-independent (H3.3) histone H3 variants based on BioID proximity labeling, which allows interactions in intact, living cells to be detected. Along with a significant proportion of previously reported interactions detected by affinity purification followed by mass spectrometry, three quarters of the 608 histone-associated proteins that we identified are new, uncharacterized histone associations. The data reveal important biological nuances not captured by traditional biochemical means. For example, we found that the chromatin assembly factor-1 histone chaperone not only deposits the replication-coupled H3.1 histone variant during S-phase but also associates with H3.3 throughout the cell cycle in vivo. We also identified other variant-specific associations, such as with transcription factors, chromatin regulators, and with the mitotic machinery. Our proximity-based analysis is thus a rich resource that extends the H3 interactome and reveals new sets of variant-specific associations., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

High-grade diffuse glioma (HGG) is the leading cause of brain tumour death. While the genetic drivers of HGG have been well described, targeting these has thus far had little impact on survival suggesting other mechanisms are at play. Here we interrogate the alternative splicing landscape of pediatric and adult HGG through multi-omic analyses, uncovering an increased splicing burden compared with normal brain. The rate of recurrent alternative splicing in cancer drivers exceeds their mutation rate, a pattern that is recapitulated in pan-cancer analyses, and is associated with worse prognosis in HGG. We investigate potential oncogenicity by interrogating cancer pathways affected by alternative splicing in HGG; spliced cancer drivers include members of the RAS/MAPK pathway. RAS suppressor neurofibromin 1 is differentially spliced to a less active isoform in >80% of HGG downstream from REST upregulation, activating the RAS/MAPK pathway and reducing glioblastoma patient survival. Overall, our results identify non-mutagenic mechanisms by which cancers activate oncogenic pathways which need to accounted for in personalized medicine approaches., (© 2022. The Author(s).)

Motivation: Current fusion detection tools use diverse calling approaches and provide varying results, making selection of the appropriate tool challenging. Ensemble fusion calling techniques appear promising; however, current options have limited accessibility and function., Results: MetaFusion is a flexible metacalling tool that amalgamates outputs from any number of fusion callers. Individual caller results are standardized by conversion into the new file type Common Fusion Format. Calls are annotated, merged using graph clustering, filtered and ranked to provide a final output of high-confidence candidates. MetaFusion consistently achieves higher precision and recall than individual callers on real and simulated datasets, and reaches up to 100% precision, indicating that ensemble calling is imperative for high-confidence results. MetaFusion uses FusionAnnotator to annotate calls with information from cancer fusion databases and is provided with a Benchmarking Toolkit to calibrate new callers., Availability and Implementation: MetaFusion is freely available at https://github.com/ccmbioinfo/MetaFusion., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The RAS/MAPK pathway is an emerging targeted pathway across a spectrum of both adult and pediatric cancers. Typically, this is associated with a single, well-characterized point mutation in an oncogene. Hypermutant tumors that harbor many somatic mutations may obscure the interpretation of such targetable genomic events. We find that replication repair-deficient (RRD) cancers, which are universally hypermutant and affect children born with RRD cancer predisposition, are enriched for RAS/MAPK mutations ( P = 10 -8 ). These mutations are not random, exist in subclones, and increase in allelic frequency over time. The RAS/MAPK pathway is activated both transcriptionally and at the protein level in patient-derived RRD tumors, and these tumors responded to MEK inhibition in vitro and in vivo . Treatment of patients with RAS/MAPK hypermutant gliomas reveals durable responses to MEK inhibition. Our observations suggest that hypermutant tumors may be addicted to oncogenic pathways, resulting in favorable response to targeted therapies. SIGNIFICANCE: Tumors harboring a single RAS/MAPK driver mutation are targeted individually for therapeutic purposes. We find that in RRD hypermutant cancers, mutations in the RAS/MAPK pathway are enriched, highly expressed, and result in sensitivity to MEK inhibitors. Targeting an oncogenic pathway may provide therapeutic options for these hypermutant polyclonal cancers. This article is highlighted in the In This Issue feature, p. 1307 ., (©2021 American Association for Cancer Research.)

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair-deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. SIGNIFICANCE: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction. This article is highlighted in the In This Issue feature, p. 995 ., (©2020 American Association for Cancer Research.)

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo , independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole -driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE -driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy. See related commentary by Wisdom and Kirsch p. 5459 ., (©2020 American Association for Cancer Research.)

Histone H3 lysine 27 (H3K27M) mutations represent the canonical oncohistone, occurring frequently in midline gliomas but also identified in haematopoietic malignancies and carcinomas. H3K27M functions, at least in part, through widespread changes in H3K27 trimethylation but its role in tumour initiation remains obscure. To address this, we created a transgenic mouse expressing H3.3K27M in diverse progenitor cell populations. H3.3K27M expression drives tumorigenesis in multiple tissues, which is further enhanced by Trp53 deletion. We find that H3.3K27M epigenetically activates a transcriptome, enriched for PRC2 and SOX10 targets, that overrides developmental and tissue specificity and is conserved between H3.3K27M-mutant mouse and human tumours. A key feature of the H3K27M transcriptome is activation of a RAS/MYC axis, which we find can be targeted therapeutically in isogenic and primary DIPG cell lines with H3.3K27M mutations, providing an explanation for the common co-occurrence of alterations in these pathways in human H3.3K27M-driven cancer. Taken together, these results show how H3.3K27M-driven transcriptome remodelling promotes tumorigenesis and will be critical for targeting cancers with these mutations.

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1 G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3b K27M and Pik3ca H1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1 G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs., Competing Interests: Declarations of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs., Methods: Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts., Results: Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo., Conclusions: Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. For commercial re-use, please contact journals.permissions@oup.com.)

In response to the dynamic intra-tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia-associated transcription factor (MITF). The response to hypoxia is driven by hypoxia-inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA-binding specificity, it is unclear whether they co-regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up-regulated by HIF1α and co-regulates a subset of HIF targets including VEGFA. Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo-hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma., (© 2019 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons Ltd.)

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome., (© 2017 Falletta et al.; Published by Cold Spring Harbor Laboratory Press.)

Paediatric brain tumours arising in the thalamus present significant diagnostic and therapeutic challenges to physicians due to their sensitive midline location. As such, genetic analysis for biomarkers to aid in the diagnosis, prognosis and treatment of these tumours is needed. Here, we identified 64 thalamic gliomas with clinical follow-up and characterized targeted genomic alterations using newly optimized droplet digital and NanoString-based assays. The median age at diagnosis was 9.25 years (range, 0.63-17.55) and median survival was 6.43 (range, 0.01-27.63) years. Our cohort contained 42 and 22 tumours reviewed as low and high grade gliomas, respectively. Five (12 %) low grade and 11 (50 %) high grade gliomas were positive for the H3F3A/HIST1H3B K27M (H3K27M) mutation. Kaplan-Meier survival analysis revealed significantly worse overall survival for patients harbouring the H3K27M mutation versus H3F3A/HIST1H3B wild type (H3WT) samples (log-rank p < 0.0001) with a median survival of 1.02 vs. 9.12 years. Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations or fusion events were detected in 44 % of patients, and was associated with long-term survival in the absence of H3K27M (log-rank p < 0.0001). Multivariate analysis demonstrated H3K27M status and high grade histology to be the most significant independent predictors of poor overall survival with hazard ratios of 6.945 and 7.721 (p < 0.0001), respectively. In contrast, MAPK pathway activation is a predictor of favourable patient outcome, although not independent of other clinical factors. Importantly, we show that low grade malignancies may harbour H3K27M mutations and that these tumours show a dismal survival compared to low grade H3WT cases. Our data strongly supports the inclusion of targeted genetic testing in childhood thalamic tumours to most accurately stratify patients into appropriate risk groups.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITF(low)/c-Jun(high) syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITF(low)/c-Jun(high) melanomas to counteract their growth-promoting and immunosuppressive functions.

Pediatric high-grade astrocytomas (pHGA) and diffuse intrinsic pontine gliomas (DIPG) are devastating malignancies for which no effective therapies exist. We investigated the therapeutic potential of PARP1 inhibition in preclinical models of pHGA and DIPG. PARP1 levels were characterized in pHGA and DIPG patient samples and tumor-derived cell lines. The effects of PARP inhibitors veliparib, olaparib, and niraparib as monotherapy or as radiosensitizers on cell viability, DNA damage, and PARP1 activity were evaluated in a panel of pHGA and DIPG cell lines. Survival benefit of niraparib was examined in an orthotopic xenograft model of pHGA. About 85% of pHGAs and 76% of DIPG tissue microarray samples expressed PARP1. Six of 8 primary cell lines highly expressed PARP1. Interestingly, across multiple cell lines, some PARP1 protein expression was required for response to PARP inhibition; however, there was no correlation between protein level or PARP1 activity and sensitivity to PARP inhibitors. Niraparib was the most effective at reducing cell viability and proliferation (MTT and Ki67). Niraparib induced DNA damage (γH2AX foci) and induced growth arrest. Pretreatment of pHGA cells with a sublethal dose of niraparib (1 μmol/L) before 2 Gy of ionizing radiation (IR) decreased the rate of DNA damage repair, colony growth, and relative cell number. Niraparib (50 mg/kg) inhibited PARP1 activity in vivo and extended survival of mice with orthotopic pHGA xenografts, when administered before IR (20 Gy, fractionated), relative to control mice (40 vs. 25 days). Our data provide in vitro and in vivo evidence that niraparib may be an effective radiosensitizer for pHGA and DIPG., (©2015 American Association for Cancer Research.)

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis., (© 2015. Published by The Company of Biologists Ltd.)

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage. In contrast, although Adprt1b was dispensable for PARylation at DSBs, Adprt1a and, to a lesser extent, Adprt2 were required for this event. Disruption of adprt2 had a subtle impact on the ability of cells to perform NHEJ. However, disruption of adprt1a decreased the ability of cells to perform end joining with a concomitant increase in homologous recombination. PAR-dependent regulation of NHEJ was achieved through promoting recruitment and/or retention of Ku at DSBs. Furthermore, a PAR interaction motif in Ku70 was required for this regulation and efficient NHEJ. These data illustrate that PARylation at DSBs promotes NHEJ through recruitment or retention of repair factors at sites of DNA damage.