Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27-/- mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40-/- cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

GSK-3 modulates SHH-driven proliferation in postnatal cerebellar neurogenesis and medulloblastoma.

Cerebellar development requires regulated proliferation of cerebellar granule neuron progenitors (CGNPs). Inadequate CGNP proliferation causes cerebellar hypoplasia whereas excessive CGNP proliferation can cause medulloblastoma, the most common malignant pediatric brain tumor. Although sonic hedgehog (SHH) signaling is known to activate CGNP proliferation, the mechanisms downregulating proliferation are less defined. We investigated CGNP regulation by GSK-3, which downregulates proliferation in the forebrain, gut and breast by suppressing mitogenic WNT signaling in mouse. In striking contrast to these systems, we found that co-deleting Gsk3a and Gsk3b blocked CGNP proliferation, causing severe cerebellar hypoplasia. The GSK-3 inhibitor CHIR-98014 similarly downregulated SHH-driven proliferation. Transcriptomic analysis showed activated WNT signaling and upregulated Cdkn1a in Gsk3a/b-deleted CGNPs. Ctnnb co-deletion increased CGNP proliferation and rescued cerebellar hypoproliferation in Gsk3a/b mutants, demonstrating physiological control of CGNPs by GSK-3, mediated through WNT. SHH-driven medulloblastomas similarly required GSK-3, as co-deleting Gsk3a/b blocked tumor growth in medulloblastoma-prone SmoM2 mice. These data show that a GSK-3/WNT axis modulates the developmental proliferation of CGNPs and the pathological growth of SHH-driven medulloblastoma. The requirement for GSK-3 in SHH-driven proliferation suggests that GSK-3 may be targeted for SHH-driven medulloblastoma therapy.

Projected t-SNE for batch correction.

MOTIVATION: Low-dimensional representations of high-dimensional data are routinely employed in biomedical research to visualize, interpret and communicate results from different pipelines. In this article, we propose a novel procedure to directly estimate t-SNE embeddings that are not driven by batch effects. Without correction, interesting structure in the data can be obscured by batch effects. The proposed algorithm can therefore significantly aid visualization of high-dimensional data. RESULTS: The proposed methods are based on linear algebra and constrained optimization, leading to efficient algorithms and fast computation in many high-dimensional settings. Results on artificial single-cell transcription profiling data show that the proposed procedure successfully removes multiple batch effects from t-SNE embeddings, while retaining fundamental information on cell types. When applied to single-cell gene expression data to investigate mouse medulloblastoma, the proposed method successfully removes batches related with mice identifiers and the date of the experiment, while preserving clusters of oligodendrocytes, astrocytes, and endothelial cells and microglia, which are expected to lie in the stroma within or adjacent to the tumours. AVAILABILITY AND IMPLEMENTATION: Source code implementing the proposed approach is available as an R package at https://github.com/emanuelealiverti/BC_tSNE, including a tutorial to reproduce the simulation studies. CONTACT: aliverti@stat.unipd.it.

Poly(2-oxazoline) nanoparticle delivery enhances the therapeutic potential of vismodegib for medulloblastoma by improving CNS pharmacokinetics and reducing systemic toxicity.

We report a nanoparticle formulation of the SHH-pathway inhibitor vismodegib that improves efficacy for medulloblastoma, while reducing toxicity. Limited blood-brain barrier (BBB) penetration and dose-limiting extitle/citraneural toxicities complicate systemic therapies for brain tumors. Vismodegib is FDA-approved for SHH-driven basal cell carcinoma, but implementation for medulloblastoma has been limited by inadequate efficacy and excessive bone toxicity. To address these issues through optimized drug delivery, we formulated vismodegib in polyoxazoline block copolymer micelles (POx-vismo). We then evaluated POx-vismo in transgenic mice that develop SHH-driven medulloblastomas with native vasculature and tumor microenvironment. POx-vismo improved CNS pharmacokinetics and reduced bone toxicity. Mechanistically, the nanoparticle carrier did not enter the CNS, and acted within the vascular compartment to improve drug delivery. Unlike conventional vismodegib, POx-vismo extended survival in medulloblastoma-bearing mice. Our results show the broad potential for non-targeted nanoparticle formulation to improve systemic brain tumor therapy, and specifically to improve vismodegib therapy for SHH-driven cancers.

A New Way to Treat Brain Tumors: Targeting Proteins Coded by Microcephaly Genes?: Brain tumors and microcephaly arise from opposing derangements regulating progenitor growth. Drivers of microcephaly could be attractive brain tumor targets.

New targets for brain tumor therapies may be identified by mutations that cause hereditary microcephaly. Brain growth depends on the repeated proliferation of stem and progenitor cells. Microcephaly syndromes result from mutations that specifically impair the ability of brain progenitor or stem cells to proliferate, by inducing either premature differentiation or apoptosis. Brain tumors that derive from brain progenitor or stem cells may share many of the specific requirements of their cells of origin. These tumors may therefore be susceptible to disruptions of the protein products of genes that are mutated in microcephaly. The potential for the products of microcephaly genes to be therapeutic targets in brain tumors are highlighted hereby reviewing research on EG5, KIF14, ASPM, CDK6, and ATR. Treatments that disrupt these proteins may open new avenues for brain tumor therapy that have increased efficacy and decreased toxicity.

ATR maintains chromosomal integrity during postnatal cerebellar neurogenesis and is required for medulloblastoma formation.

Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. We report that ATR, which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. Our data show that p53-driven apoptosis and cell cycle arrest - and, in the absence of p53, non-apoptotic cell death - redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition.

Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice.

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.

Tonic activation of Bax primes neural progenitors for rapid apoptosis through a mechanism preserved in medulloblastoma.

Commitment to survival or apoptosis within expanding progenitor populations poses distinct risks and benefits to the organism. We investigated whether specialized mechanisms regulate apoptosis in mouse neural progenitors and in the progenitor-derived brain tumor medulloblastoma. Here, we identified constitutive activation of proapoptotic Bax, maintained in check by Bcl-xL, as a mechanism for rapid cell death, common to postnatal neural progenitors and medulloblastoma. We found that tonic activation of Bax in cerebellar progenitors, along with sensitivity to DNA damage, was linked to differentiation state. In cerebellar progenitors, active Bax localized to mitochondria, where it was bound to Bcl-xL. Disruption of Bax:Bcl-xL binding by BH3-mimetic ABT 737 caused rapid apoptosis of cerebellar progenitors and primary murine medulloblastoma cells. Conditional deletion of Mcl-1, in contrast, did not cause death of cerebellar progenitors. Our findings identify a mechanism for the sensitivity of brain progenitors to typical anticancer therapies and reveal that this mechanism persists in medulloblastoma, a malignant brain tumor markedly sensitive to radiation and chemotherapy.

Selective Posterior Cerebral Artery Wada Better Predicts Good Memory and Naming Outcomes Following Selective Stereotactic Thermal Ablation for Medial Temporal Lobe Epilepsy Than Internal Carotid Artery Wada.

The conventional intracarotid amobarbital (Wada) test has been used to assess memory function in patients being considered for temporal lobe epilepsy (TLE) surgery. Minimally invasive approaches that target the medial temporal lobe (MTL) and spare neocortex are increasingly used, but a knowledge gap remains in how to assess memory and language risk from these procedures. We retrospectively compared results of two versions of the Wada test, the intracarotid artery (ICA-Wada) and posterior cerebral artery (PCA-Wada) approaches, with respect to predicting subsequent memory and language outcomes, particularly after stereotactic laser amygdalohippocampotomy (SLAH). We included all patients being considered for SLAH who underwent both ICA-Wada and PCA-Wada at a single institution. Memory and confrontation naming assessments were conducted using standardized neuropsychological tests to assess pre- to post-surgical changes in cognitive performance. Of 13 patients who initially failed the ICA-Wada, only one patient subsequently failed the PCA-Wada (p=0.003, two-sided binomial test with p 0 =0.5) demonstrating that these tests assess different brain regions or networks. PCA-Wada had a high negative predictive value for the safety of SLAH, compared to ICA-Wada, as none of the patients who underwent SLAH after passing the PCA-Wada experienced catastrophic memory decline (0 of 9 subjects, p <.004, two-sided binomial test with p 0 =0.5), and all experienced a good cognitive outcome. In contrast, the single patient who received a left anterior temporal lobectomy after failed ICA- and passed PCA-Wada experienced a persistent, near catastrophic memory decline. On confrontation naming, few patients exhibited disturbance during the PCA-Wada. Following surgery, SLAH patients showed no naming decline, while open resection patients, whose surgeries all included ipsilateral temporal lobe neocortex, experienced significant naming difficulties (Fisher's exact test, p <.05). These findings demonstrate that (1) failing the ICA-Wada falsely predicts memory decline following SLAH, (2) PCA-Wada better predicts good memory outcomes of SLAH for MTLE, and (3) the MTL brain structures affected by both PCA-Wada and SLAH are not directly involved in language processing.

scRNA-seq for Microcephaly Research [IV]: Dirichlet Regression for Single-Cell Population Differences.

Analysis of single-cell RNA sequencing typically includes the clustering of cells and subsequent determination of the population size of each cluster, relative to the whole. In an experimental setting, two or more conditions are compared to assess changes in cellular composition of the sampled tissue. Cluster populations are frequently normalized to the total number of cells from each replicate in order to facilitate comparisons. After normalization, they become interdependent fractions and therefore cannot be compared using individual t-tests. Here we describe the use of Dirichlet regression to compare changes in cellular composition between two or more conditions when multiple biological replicates (three or more) are sampled under each condition. We provide an example of R code to conduct a similar analysis and interpret the results.

Automated Immunofluorescence Staining for Analysis of DNA Damage and Apoptosis in Brain Sections.

Neural progenitors show a strong tendency to undergo apoptosis in response to DNA damage, and both impaired DNA repair and increased neural progenitor apoptosis are associated with microcephaly. Here we present an immunohistochemistry-based method for assessing DNA damage and apoptosis in the neonatal mouse brain. These methods are suitable for determining in specific experimental conditions the fractions of cells with DNA double-strand breaks, the fractions of cells undergoing apoptosis, or both. While DNA damage in neural progenitors can trigger apoptosis, inappropriate apoptosis may also result from other processes. Simultaneous analysis of DNA damage and apoptosis in mouse models of microcephaly can determine how genetic instability and cell death contribute to the observed phenotype.

Processing Neonatal Mouse Brains for Immunohistochemical Analysis: As Required for Including Spindle Orientation Analysis and Analysis of DNA Damage and Apoptosis.

Analyzing sections of neonatal mouse brain using immunohistochemistry can inform microcephaly pathogenesis, but obtaining and staining high-quality sections can be challenging. The neonatal brain shows less structural integrity than the adult brain. As a result, embedding technique must be optimized to allow sections without cracks or other anatomic disruptions. Moreover, paraffin embedding, which maximized tissue preservation, can reduce antigenicity of proteins in the embedded tissues. We describe an optimized embedding technique and antigen recovery technique that allows successful sectioning and immunohistochemical staining.

IGFBP2 promotes proliferation and cell migration through STAT3 signaling in Sonic hedgehog medulloblastoma.

Medulloblastoma (MB) is the most common pediatric brain malignancy and is divided into four molecularly distinct subgroups: WNT, Sonic Hedgehog (SHHp53mut and SHHp53wt), Group 3, and Group 4. Previous reports suggest that SHH MB features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB tumor cells interact with and potentially modify their microenvironment, we performed cytokine array analysis of culture media from freshly isolated MB patient tumor cells, spontaneous SHH MB mouse tumor cells and mouse and human MB cell lines. We found that the SHH MB cells produced elevated levels of IGFBP2 compared to non-SHH MBs. We confirmed these results using ELISA, western blotting, and immunofluorescence staining. IGFBP2 is a pleiotropic member of the IGFBP super-family with secreted and intracellular functions that can modulate tumor cell proliferation, metastasis, and drug resistance, but has been understudied in medulloblastoma. We found that IGFBP2 is required for SHH MB cell proliferation, colony formation, and cell migration, through promoting STAT3 activation and upregulation of epithelial to mesenchymal transition markers; indeed, ectopic STAT3 expression fully compensated for IGFBP2 knockdown in wound healing assays. Taken together, our findings reveal novel roles for IGFBP2 in SHH medulloblastoma growth and metastasis, which is associated with very poor prognosis, and they indicate an IGFBP2-STAT3 axis that could represent a novel therapeutic target in medulloblastoma.

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth.

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.

Chronic AMPK inactivation slows SHH medulloblastoma progression by inhibiting mTORC1 signaling and depleting tumor stem cells.

We show that inactivating AMPK in a genetic medulloblastoma model depletes tumor stem cells and slows progression. In medulloblastoma, the most common malignant pediatric brain tumor, drug-resistant stem cells co-exist with transit-amplifying cells and terminally differentiated neuronal progeny. Prior studies show that Hk2-dependent glycolysis promotes medulloblastoma progression by suppressing neural differentiation. To determine how the metabolic regulator AMPK affects medulloblastoma growth and differentiation, we inactivated AMPK genetically in medulloblastomas. We bred conditional Prkaa1 and Prkaa2 deletions into medulloblastoma-prone SmoM2 mice and compared SmoM2-driven medulloblastomas with intact or inactivated AMPK. AMPK-inactivation increased event-free survival (EFS) and altered cellular heterogeneity, increasing differentiation and decreasing tumor stem cell populations. Surprisingly, AMPK-inactivation decreased mTORC1 activity and decreased Hk2 expression. Hk2 deletion similarly depleted medulloblastoma stem cells, implicating reduced glycolysis in the AMPK-inactivated phenotype. Our results show that AMPK inactivation disproportionately impairs medulloblastoma stem cell populations typically refractory to conventional therapies.

Radiotherapy-Induced Neurocognitive Impairment Is Driven by Heightened Apoptotic Priming in Early Life and Prevented by Blocking BAX.

Although external beam radiotherapy (xRT) is commonly used to treat central nervous system (CNS) tumors in patients of all ages, young children treated with xRT frequently experience life-altering and dose-limiting neurocognitive impairment (NI) while adults do not. The lack of understanding of mechanisms responsible for these differences has impeded the development of neuroprotective treatments. Using a newly developed mouse model of xRT-induced NI, we found that neurocognitive function is impaired by ionizing radiation in a dose- and age-dependent manner, with the youngest animals being most affected. Histologic analysis revealed xRT-driven neuronal degeneration and cell death in neurogenic brain regions in young animals but not adults. BH3 profiling showed that neural stem and progenitor cells, neurons, and astrocytes in young mice are highly primed for apoptosis, rendering them hypersensitive to genotoxic damage. Analysis of single-cell RNA sequencing data revealed that neural cell vulnerability stems from heightened expression of proapoptotic genes including BAX, which is associated with developmental and mitogenic signaling by MYC. xRT induced apoptosis in primed neural cells by triggering a p53- and PUMA-initiated, proapoptotic feedback loop requiring cleavage of BID and culminating in BAX oligomerization and caspase activation. Notably, loss of BAX protected against apoptosis induced by proapoptotic signaling in vitro and prevented xRT-induced apoptosis in neural cells in vivo as well as neurocognitive sequelae. On the basis of these findings, preventing xRT-induced apoptosis specifically in immature neural cells by blocking BAX, BIM, or BID via direct or upstream mechanisms is expected to ameliorate NI in pediatric patients with CNS tumor. SIGNIFICANCE: Age- and differentiation-dependent apoptotic priming plays a pivotal role in driving radiotherapy-induced neurocognitive impairment and can be targeted for neuroprotection in pediatric patients.

Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179.

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.

Spatiotemporal analysis of induced neural stem cell therapy to overcome advanced glioblastoma recurrence.

Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer glioblastoma (GBM); however, treatment durability remains a major challenge. We sought to define the events that contribute to dynamic adaptation of GBM during treatment with human skin-derived induced NSCs releasing the pro-apoptotic agent TRAIL (iNSC-TRAIL) and develop strategies that convert initial tumor kill into sustained GBM suppression. In vivo and ex vivo analysis before, during, and after treatment revealed significant shifts in tumor transcriptome and spatial distribution as the tumors adapted to treatment. To address this, we designed iNSC delivery strategies that increased spatiotemporal TRAIL coverage and significantly decreased GBM volume throughout the brain, reducing tumor burden 100-fold as quantified in live ex vivo brain slices. The varying impact of different strategies on treatment durability and median survival of both solid and invasive tumors provides important guidance for optimizing iNSC therapy.

Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib.

The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo-treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.

OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma.

PURPOSE: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. EXPERIMENTAL DESIGN: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. RESULTS: We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. CONCLUSIONS: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.