Search

Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.

Background: Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children. All previous studies examining the role of systemic agents have failed to demonstrate a survival benefit; the only standard of care is radiation therapy (RT). Successful implementation of radiosensitization strategies in DMG remains an essential and promising avenue of investigation. We explore the use of Napabucasin, an NAD(P)H quinone dehydrogenase 1 (NQO1)-bioactivatable reactive oxygen species (ROS)-inducer, as a potential therapeutic radiosensitizer in DMG., Methods: In this study, we conduct in vitro and in vivo assays using patient-derived DMG cultures to elucidate the mechanism of action of Napabucasin and its radiosensitizing properties. As penetration of systemic therapy through the blood-brain barrier (BBB) is a significant limitation to the success of DMG therapies, we explore focused ultrasound (FUS) and convection-enhanced delivery (CED) to overcome the BBB and maximize therapeutic efficacy., Results: Napabucasin is a potent ROS-inducer and radiosensitizer in DMG, and treatment-mediated ROS production and cytotoxicity are dependent on NQO1. In subcutaneous xenograft models, combination therapy with RT improves local control. After optimizing targeted drug delivery using CED in an orthotopic mouse model, we establish the novel feasibility and survival benefit of CED of Napabucasin concurrent with RT., Conclusions: As nearly all DMG patients will receive RT as part of their treatment course, our validation of the efficacy of radiosensitizing therapy using CED to prolong survival in DMG opens the door for exciting novel studies of alternative radiosensitization strategies in this devastating disease while overcoming limitations of the BBB., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

We identify a population of Protogenin-positive (PRTG +ve ) MYC high NESTIN low stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RL VZ ). Oncogenic transformation of early Prtg +ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG +ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RL VZ , a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTG high compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RL VZ PRTG +ve stem cells inhabiting a specific perivascular niche. Targeting the PRTG high compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB., Competing Interests: Declaration of interests The authors declare no competing interests. A patent related to this work was submitted by University of Pittsburgh (University Docket No. 05700/F&R ref. 48881-0028P01), US 63/275,326, “Molecules that bind to Protogenin polypeptides.”, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life., (©2024 American Association for Cancer Research.)

Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma (MB) formation and progression. We have identified an lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) MBs. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium-oxide-nanoparticle-coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40%-50% in an intracranial MB xenograft mouse model. Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy. Thus, the lnc-HLX-2-7-HLX-MYC axis is important for regulating G3 MB progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor with a need for novel therapies. So far, monotherapies have failed to prolong survival for these patients, and combinatorial strategies have often shown severe, dose-limiting toxicities. In this issue of the JCI, Duchatel, Jackson, and colleagues address this challenge by introducing a drug combination that mitigates side effects and overcomes resistance. After identifying the PI3K/mTOR pathway as a therapeutic vulnerability, they treated DIPG-bearing mice with paxalisib and saw responses but also observed hyperglycemia as a severe side effect. Combining paxalisib with metformin mitigated this toxicity, but also upregulated protein kinase C (PKC) signaling. To tackle this mechanism of resistance, the authors added the PKC inhibitor enzastaurin to their drug combination and showed that this triple therapy led to improved survival. This approach paves the way for improved outcomes for patients with DIPG and other brain tumors.

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development., (© 2023. The Author(s).)

Achieving adequate exposure of the free therapeutic agent at the target is a critical determinant of efficacious chemotherapy. With this in mind, a major challenge in developing therapies for central nervous system (CNS) tumors is to overcome barriers to delivery, including the blood-brain barrier (BBB). Panobinostat is a nonselective pan-histone deacetylase inhibitor that is being tested in preclinical and clinical studies, including for the treatment of pediatric medulloblastoma, which has a propensity for leptomeningeal spread and diffuse midline glioma, which can infiltrate into supratentorial brain regions. In this study, we examined the rate, extent, and spatial heterogeneity of panobinostat CNS distribution in mice. Transporter-deficient mouse studies show that panobinostat is a dual substrate of P-glycoprotein (P-gp) and breast cancer resistant protein (Bcrp), which are major efflux transporters expressed at the BBB. The CNS delivery of panobinostat was moderately limited by P-gp and Bcrp, and the unbound tissue-to-plasma partition coefficient of panobinostat was 0.32 and 0.21 in the brain and spinal cord in wild-type mice. In addition, following intravenous administration, panobinostat demonstrated heterogeneous distribution among brain regions, indicating that its efficacy would be influenced by tumor location or the presence and extent of leptomeningeal spread. Simulation using a compartmental BBB model suggests inadequate exposure of free panobinostat in the brain following a recommended oral dosing regimen in patients. Therefore, alternative approaches to CNS delivery may be necessary to have adequate exposure of free panobinostat for the treatment of a broad range of pediatric brain tumors. SIGNIFICANCE STATEMENT: This study shows that the central nervous system (CNS) penetration of panobinostat is limited by P-gp and Bcrp, and its efficacy may be limited by inadequate distribution to the tumor. Panobinostat has heterogeneous distribution into various brain regions, indicating that its efficacy might depend on the anatomical location of the tumors. These distributional parameters in the mouse CNS can inform both preclinical and clinical trial study design and may guide treatment for these devastating brain tumors in children., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA., (© 2023. The Author(s).)

Identifying the cells from which cancers arise is critical for understanding the molecular underpinnings of tumor evolution. To determine whether stem/progenitor cells can serve as cells of origin, we created a Msi2-Cre ERT2 knock-in mouse. When crossed to CAG-LSL-Myc T58A mice, Msi2-Cre ERT2 mice developed multiple pancreatic cancer subtypes: ductal, acinar, adenosquamous, and rare anaplastic tumors. Combining single-cell genomics with computational analysis of developmental states and lineage trajectories, we demonstrate that MYC preferentially triggers transformation of the most immature MSI2 + pancreas cells into multi-lineage pre-cancer cells. These pre-cancer cells subsequently diverge to establish pancreatic cancer subtypes by activating distinct transcriptional programs and large-scale genomic changes, and enforced expression of specific signals like Ras can redirect subtype specification. This study shows that multiple pancreatic cancer subtypes can arise from a common pool of MSI2 + cells and provides a powerful model to understand and control the programs that shape divergent fates in pancreatic cancer., Competing Interests: Declaration of interests T.R. is a founder and member of the Board of Directors, and holds executive roles at Tiger Hill Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations., (© 2023. The Author(s).)

Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. circ_63706 was identified as sonic hedgehog (SHH) group-specific, with its expression confirmed by RNA-FISH analysis in clinical tissue samples. The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target., (© 2023. The Author(s).)

Detection of tumor progression in patients with glioblastoma remains a major challenge. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of patients with glioblastoma. In our study, we evaluated the potential of serum-derived EVs from glioblastoma patients to serve as biomarker for tumor progression. EVs from serum of glioblastoma patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EV markers were defined by using a proximity-extension assay and bead-based flow cytometry. Tumor progression was defined according to modified RANO criteria. EVs from the serum of glioblastoma patients (n = 67) showed an upregulation of CD29, CD44, CD81, CD146, C1QA and histone H3 as compared to serum EVs from healthy volunteers (P value range: <.0001 to .08). For two independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44 and histone H3 upon tumor progression, but not in patients with stable disease. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA and histone H3 correlated with RANO-defined tumor progression with an AUC of 0.76. Measurement of CD29, CD44, CD81, C1QA and histone H3 in serum-derived EVs of glioblastoma patients, along with standard MRI assessment, has the potential to improve detection of true tumor progression and thus could be a useful biomarker for clinical decision making., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse., Significance: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice., Experimental Design: HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease., Results: A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models., Conclusions: Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207., (©2022 American Association for Cancer Research.)

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB., (© 2022. The Author(s).)

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is classified into four primary molecular subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3 (G3), and Group 4 (G4), and further genomic and proteomic subtypes have been reported. Subgroup heterogeneity and few actionable mutations have hindered the development of targeted therapies, especially for G3 MB, which has a particularly poor prognosis. To identify novel therapeutic targets for MB, we performed mass spectrometry-based deep expression proteomics and phosphoproteomics in 20 orthotopic patient-derived xenograft (PDX) models of MB comprising SHH, G3, and G4 subgroups. We found that the proteomic profiles of MB PDX tumors are closely aligned with those of primary human MB tumors illustrating the utility of PDX models. SHH PDXs were enriched for NFκB and p38 MAPK signaling, while G3 PDXs were characterized by MYC activity. Additionally, we found a significant association between actinomycin D sensitivity and increased abundance of MYC and MYC target genes. Our results highlight several candidate pathways that may serve as targets for new MB therapies. Mass spectrometry data are available via ProteomeXchange with identifier PXD035070.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1-4 . Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5-8 . By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10 . However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4 . Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES + KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target., Methods: We investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing., Results: We identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2-4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed., Conclusion: Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors., Competing Interests: Competing interests: TS is currently employed by Spark Therapeutics. KK is currently employed by BioNTech and is an inventor on a patent related to use of nucleoside-modified mRNA. DMB is currently employed by Tmunity Therapeutics. JBF, DMB, JMM, and KRB hold patents for the discovery and development of immunotherapies for cancer, including patents related to glypican 2 (GPC2)-directed immunotherapies. KRB and JMM receive research funding from Tmunity for research on GPC2-directed immunotherapies and JBF, DMB, JMM, and KRB receive royalties from Tmunity for licensing of GPC2-related intellectual property. JMM is a founder of both Tantigen Bio and Hula Therapeutics, focused on cellular therapies for childhood cancers, but neither are working on GPC2-directed therapeutics. All other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Although some of the regulatory genes, signaling pathways, and gene regulatory networks altered in medulloblastomas (MB) are known, the roles of non-coding RNAs, particularly long non-coding RNAs (lncRNAs), are poorly described. Here we report that the lncRNA SPRIGHTLY ( SPRY4-IT1 ) gene is upregulated in group 4 medulloblastoma (G4 MB)., Methods: SPRIGHTLY expression was assessed in MB subgroup patient-derived xenografts, cell lines, and patient samples. The effect of SPRIGHTLY hemizygous deletion on proliferation, invasion, apoptosis, and colony formation were assessed in vitro and on tumor growth in vivo. dChIRP pull-down assays were used to assess SPRIGHTLY- binding partners, confirmed by immunoprecipitation. SMYD3 ΔE5 transcripts were examined in cell lines and publicly available RNA-seq data. Pathway analysis was performed by phospho-kinase profiling and RNA-seq., Results: CRISPR/Cas9 deletion of SPRIGHTLY reduced cell viability and invasion and increased apoptosis in G4 MB cell lines in vitro. SPRIGHTLY hemizygous-deleted G4 MB cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells expressing both copies of SPRIGHTLY . SPRIGHTLY lncRNA bound to the intronic region of the SMYD3 pre-mRNA transcript. SPRIGHTLY also interacted with PTPB1 protein to regulate SMYD3 exon skipping to produce an aberrant protein. SPRIGHTLY -driven SMYD3 regulation enhanced the expression of EGFR pathway genes in G4 MB cell lines and activated cell coagulation/hemostasis-related gene expression, suggesting a novel oncogenic role in G4 MB., Conclusions: These results demonstrate the importance of SPRIGHTLY lncRNA as a promoter of G4 MB and the role of the SPRIGHTLY-SMYD3-PTPB1 axis as an important oncogenic regulator in MB., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC., (© 2022. The Author(s).)

Medulloblastoma (MB) is the most common primary malignant pediatric brain cancer. We recently identified novel roles for the MEK/MAPK pathway in regulating human Sonic Hedgehog (SHH) MB tumorigenesis. The MEK inhibitor, selumetinib, decreased SHH MB growth while extending survival in mouse models. However, the treated mice ultimately succumbed to disease progression. Here, we perform RNA sequencing on selumetinib-treated orthotopic xenografts to identify molecular pathways that compensate for MEK inhibition specifically in vivo. Notably, the JAK/STAT3 pathway exhibits increased activation in selumetinib-treated tumors. The combination of selumetinib and the JAK/STAT3 pathway inhibitor, pacritinib, further reduces growth in two xenograft models and also enhances survival. Multiplex spatial profiling of proteins in drug-treated xenografts reveals shifted molecular dependencies and compensatory changes following combination drug treatment. Our study warrants further investigation into MEK and JAK/STAT3 inhibition as a novel combinatory therapeutic strategy for SHH MB., (© 2022. The Author(s).)

Medulloblastoma is an aggressive brain tumor that occurs predominantly in children. Despite intensive therapy, many patients die of the disease, and novel therapies are desperately needed. Although immunotherapy has shown promise in many cancers, the low mutational burden, limited infiltration of immune effector cells, and immune-suppressive microenvironment of medulloblastoma have led to the assumption that it is unlikely to respond to immunotherapy. However, emerging evidence is challenging this view. Here we review recent preclinical and clinical studies that have identified mechanisms of immune evasion in medulloblastoma, and highlight possible therapeutic interventions that may give new hope to medulloblastoma patients and their families., (© 2022 Eisemann and Wechsler-Reya; Published by Cold Spring Harbor Laboratory Press.)

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc -driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1 -deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1 -deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1 + macrophages, and we show that CD45 high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abbas, George, Ancliffe, Howlett, Jones, Kuchibhotla, Wechsler-Reya, Gottardo and Endersby.)

Background: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models., Methods: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups., Results: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB., Conclusions: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glioblastomas are universally fatal cancers and contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anticancer drug discovery based on in vitro cultures tends to identify targets with poor therapeutic indices and fails to accurately model the effects of the tumor microenvironment. Here, leveraging in vivo genetic screening, we identified the histone H3 lysine 4 trimethylation (H3K4me3) regulator DPY30 (Dpy-30 histone methyltransferase complex regulatory subunit) as an in vivo–specific glioblastoma dependency. On the basis of the hypothesis that in vivo epigenetic regulation may define critical GSC dependencies, we interrogated active chromatin landscapes of GSCs derived from intracranial patient-derived xenografts (PDXs) and cell culture through H3K4me3 chromatin immunoprecipitation and transcriptome analyses. Intracranial-specific genes marked by H3K4me3 included FOS , NF κ B , and phosphodiesterase (PDE) family members. In intracranial PDX tumors, DPY30 regulated angiogenesis and hypoxia pathways in an H3K4me3-dependent manner but was dispensable in vitro in cultured GSCs. PDE4B was a key downstream effector of DPY30, and the PDE4 inhibitor rolipram preferentially targeted DPY30-expressing cells and impaired PDX tumor growth in mice without affecting tumor cells cultured in vitro. Collectively, the MLL/SET1 (mixed lineage leukemia/SET domain-containing 1, histone lysine methyltransferase) complex member DPY30 selectively regulates H3K4me3 modification on genes critical to support angiogenesis and tumor growth in vivo, suggesting the DPY30-PDE4B axis as a specific therapeutic target in glioblastoma.

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by imatinib (IM), a tyrosine kinase inhibitor (TKI). However, IM does not cure GIST, and adjuvant therapy only delays recurrence in high-risk tumors. We hypothesized that GIST contains cells with primary IM resistance that may represent a reservoir for disease persistence. Here, we report a subpopulation of CD34 + KIT low human GIST cells that have intrinsic IM resistance. These cells possess cancer stem cell-like expression profiles and behavior, including self-renewal and differentiation into CD34 + KIT high progeny that are sensitive to IM treatment. We also found that TKI treatment of GIST cell lines led to induction of stem cell-associated transcription factors ( OCT4 and NANOG ) and concomitant enrichment of the CD34 + KIT low cell population. Using a data-driven approach, we constructed a transcriptomic-oncogenic map (Onco-GPS) based on the gene expression of 134 GIST samples to define pathway activation during GIST tumorigenesis. Tumors with low KIT expression had overexpression of cancer stem cell gene signatures consistent with our in vitro findings. Additionally, these tumors had activation of the Gas6/AXL pathway and NF-κB signaling gene signatures. We evaluated these targets in vitro and found that primary IM-resistant GIST cells were effectively targeted with either single-agent bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor), as well as with either agent in combination with IM. Collectively, these findings suggest that CD34 + KIT low cells represent a distinct, but targetable, subpopulation in human GIST that may represent a novel mechanism of primary TKI resistance, as well as a target for overcoming disease persistence following TKI therapy., (©2021 American Association for Cancer Research.)

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias., (© 2021 by The American Society of Hematology.)

During mammalian brain development, neural progenitor cells proliferate extensively but can ensure the production of correct numbers of various types of mature cells by balancing symmetric proliferative versus asymmetric differentiative cell divisions. This process of cell fate determination may be harnessed for developing cancer therapy. Here, we test this idea by targeting KIF20A, a mitotic kinesin crucial for the control of cell division modes, in a genetic model of medulloblastoma (MB) and human MB cells. Inducible Kif20a knockout in both normal and MB-initiating granule neuron progenitors (GNPs) causes early cell cycle exit and precocious neuronal differentiation without causing cytokinesis failure and suppresses the development of Sonic Hedgehog (SHH)-activated MB. Inducible KIF20A knockdown in human MB cells inhibits proliferation both in cultures and in growing tumors. Our results indicate that targeting the fate specification process of nascent daughter cells presents a novel avenue for developing anti-proliferation treatment for malignant brain tumors.

Background: Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the noncoding RNA genome, in particular long noncoding RNAs (lncRNAs), contributes to MB subgrouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in Group 3 MBs., Methods: Publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, lnc-HLX-2-7 was deleted by CRISPR/Cas9 in the MB cell line. Intracranial injected tumors were further characterized by bulk and single-cell RNA-seq., Results: Lnc-HLX-2-7 is highly upregulated in Group 3 MB cell lines, patient-derived xenografts, and primary MBs compared with other MB subgroups as assessed by quantitative real-time, RNA-seq, and RNA fluorescence in situ hybridization. Depletion of lnc-HLX-2-7 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. Lnc-HLX-2-7-deleted cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX-2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MYC oncogene regulated lnc-HLX-2-7, and the small-molecule bromodomain and extraterminal domain family‒bromodomain 4 inhibitor Jun Qi 1 (JQ1) reduced lnc-HLX-2-7 expression., Conclusions: Lnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in Group 3 MBs., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function., Methods: Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence., Results: HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution., Conclusions: Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC's transactivating and transrepressing functions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Medulloblastoma (MB) comprises four subtypes of which group 3 MB are the most aggressive. Although overall survival for MB has improved, the outcome of group 3 MB remains dismal. C- MYC (MYC) amplification or MYC overexpression which characterizes group 3 MB is a strong negative prognostic factor and is frequently associated with metastases and relapses. We previously reported that MYC expression alone promotes highly aggressive MB phenotypes, in part via repression of thrombospondin-1 (TSP-1), a potent tumor suppressor., Methods: In this study, we examined the potential role of TSP-1 and TSP-1 peptidomimetic ABT-898 in MYC- amplified human MB cell lines and two distinct murine models of MYC-driven group 3 MBs., Results: We found that TSP-1 reconstitution diminished metastases and prolonged survival in orthotopic xenografts and promoted chemo- and radio-sensitivity via AKT signaling. Furthermore, we demonstrate that ABT-898 can recapitulate the effects of TSP-1 expression in MB cells in vitro and specifically induced apoptosis in murine group 3 MB tumor cells., Conclusion: Our data underscore the importance of TSP-1 as a critical tumor suppressor in MB and highlight TSP-1 peptidomimetics as promising novel therapeutics for the most lethal subtype of MB., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely, MYC -amplified Group 3 and TP53 -mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that cell cycle checkpoint kinase (CHK1/2) inhibition synergistically enhanced the cytotoxic activity of clinically used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The glioma-associated family of transcription factors (GLI) have emerged as a promising therapeutic target for a variety of human cancers. In particular, GLI1 plays a central role as a transcriptional regulator for multiple oncogenic signaling pathways, including the hedgehog (Hh) signaling pathway. We undertook a computational screening approach to identify small molecules that directly bind GLI1 for potential development as inhibitors of GLI-mediated transcription. Through these studies, we identified compound 1 , which is an 8-hydroxyquinoline, as a high-affinity binder of GLI1. Compound 1 inhibits GLI1-mediated transcriptional activity in several Hh-dependent cellular models, including a primary model of murine medulloblastoma. We also performed a series of computational analyses to define more clearly the mechanism(s) through which 1 inhibits GLI1 function after binding. Our results strongly suggest that binding of 1 to GLI1 does not prevent GLI1/DNA binding nor disrupt the GLI1/DNA complex, but rather, it induces specific conformational changes in the overall complex that prevent proper GLI function. These results highlight the potential of this compound for further development as an anti-cancer agent that targets GLI1., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis., (©2020 American Association for Cancer Research.)

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-β receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)