All-trans retinoic acid induces durable tumor immunity in IDH-mutant gliomas by rescuing transcriptional repression of the CRBP1-retinoic acid axis.

Diffuse gliomas are epigenetically dysregulated, immunologically cold, and fatal tumors characterized by mutations in isocitrate dehydrogenase (IDH). Although IDH mutations yield a uniquely immunosuppressive tumor microenvironment, the regulatory mechanisms that drive the immune landscape of IDH mutant (IDHm) gliomas remain unknown. Here, we reveal that transcriptional repression of retinoic acid (RA) pathway signaling impairs both innate and adaptive immune surveillance in IDHm glioma through epigenetic silencing of retinol binding protein 1 (RBP1) and induces a profound anti-inflammatory landscape marked by loss of inflammatory cell states and infiltration of suppressive myeloid phenotypes. Restorative retinoic acid therapy in murine glioma models promotes clonal CD4 + T cell expansion and induces tumor regression in IDHm, but not IDH wildtype (IDHwt), gliomas. Our findings provide a mechanistic rationale for RA immunotherapy in IDHm glioma and is the basis for an ongoing investigator-initiated, single-center clinical trial investigating all-trans retinoic acid (ATRA) in recurrent IDHm human subjects.

Meningioma: current updates on genetics, classification, and mouse modeling.

Meningiomas, the most common primary brain tumors in adults, are often benign and curable by surgical resection. However, a subset is of higher grade, shows aggressive growth behavior as well as brain invasion, and often recurs even after several rounds of surgery. Increasing evidence suggests that tumor classification and grading primarily based on histopathology do not always accurately predict tumor aggressiveness and recurrence behavior. The underlying biology of aggressive treatment-resistant meningiomas and the impact of specific genetic aberrations present in these high-grade tumors is still only insufficiently understood. Therefore, an in-depth research into the biology of this tumor type is warranted. More recent studies based on large-scale molecular data such as whole exome/genome sequencing, DNA methylation sequencing, and RNA sequencing have provided new insights into the biology of meningiomas and have revealed new risk factors and prognostic subtypes. The most common genetic aberration in meningiomas is functional loss of NF2 and occurs in both low- and high-grade meningiomas, whereas NF2-wildtype meningiomas are enriched for recurrent mutations in TRAF7, KLF4, AKT1, PI3KCA, and SMO and are more frequently benign. Most meningioma mouse models are based on patient-derived xenografts and only recently have new genetically engineered mouse models of meningioma been developed that will aid in the systematic evaluation of specific mutations found in meningioma and their impact on tumor behavior. In this article, we review recent advances in the understanding of meningioma biology and classification and highlight the most common genetic mutations, as well as discuss new genetically engineered mouse models of meningioma.

Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples.

Hypertranscription is widespread in aggressive human cancers. However detection relies on mRNAs, which are heavily processed and have variable half-lives, and on accurate cell number estimations. Previously we introduced FFPE-CUTAC, a genome-wide method for mapping RNA Polymerase II in formalin-fixed paraffin-embedded (FFPE) sections. Here we apply FFPE-CUTAC on slides and curls to demonstrate hypertranscription at regulatory elements and replication-coupled histone genes. We find that hypertranscription differs between transgene-driven mouse gliomas and scales with enhanced proliferation and reduced mitochondrial DNA. We also apply FFPE-CUTAC to identify tumor-specific patterns in assorted human tumor-normal pairs. We analyze the top-ranked 100 annotated regulatory elements that are hypertranscribed in most of the tumors and identify multiple loci around ERBB2 on Chromosome 17q12-21 in the breast and colon cancer samples, mapping likely HER2 amplifications punctuated by selective sweeps. Our results demonstrate that FFPE-CUTAC measurement of hypertranscription provides an affordable and sensitive genome-wide strategy for cancer diagnosis.

ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models.

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we developed a series of genetically engineered mouse models of treatment-naïve and -experienced NTRK1/2/3 fusion-driven gliomas. Both the TRK kinase domain and the N-terminal fusion partners influenced tumor histology and aggressiveness. Treatment with TRK kinase inhibitors significantly extended survival of NTRK fusion-driven glioma mice in a fusion- and inhibitor-dependent manner, but tumors ultimately recurred due to the presence of treatment-resistant persister cells. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools for preclinical testing of novel inhibitors and to study the cellular responses of NTRK fusion-driven gliomas to therapy.

Haploinsufficiency of phosphodiesterase 10A activates PI3K/AKT signaling independent of PTEN to induce an aggressive glioma phenotype.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

Phase separation of YAP-MAML2 differentially regulates the transcriptome.

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag.

For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma.

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ T cells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Visualizing genomic characteristics across an RNA-Seq based reference landscape of normal and neoplastic brain.

In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-Seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA-Seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Nuclear condensates of YAP fusion proteins alter transcription to drive ependymoma tumourigenesis.

Nuclear localization of HIPPO-YAP fusion proteins has been implicated in supratentorial ependymoma development. Here, unexpectedly, we find that liquid-liquid phase separation, rather than nuclear localization, of recurrent patient-derived YAP fusions, YAP-MAMLD1 and C11ORF95-YAP, underlies ependymoma tumourigenesis from neural progenitor cells. Mutagenesis and chimaera assays demonstrate that an intrinsically disordered region promotes oligomerization of the YAP fusions into nuclear, puncta-like, membrane-less condensates. Oligomerization and nuclear condensates induced by YAP fusion with a coiled-coil domain of transcriptional activator GCN4 also promote ependymoma formation. YAP-MAMLD1 concentrates transcription factors and co-activators, including BRD4, MED1 and TEAD, in condensates while excluding transcriptional repressive PRC2, and induces long-range enhancer-promoter interactions that promote transcription and oncogenic programmes. Blocking condensate-mediated transcriptional co-activator activity inhibits tumourigenesis, indicating a critical role of liquid phase separation for YAP fusion oncogenic activity in ependymoma. YAP fusions containing the intrinsically disordered region features are common in human tumours, suggesting that nuclear condensates could be targeted to treat YAP-fusion-induced cancers.

An RNA seq-based reference landscape of human normal and neoplastic brain.

In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape.

Functional genomic analysis of adult and pediatric brain tumor isolates.

Background: Adult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. Methods: We performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. Results: We found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. Conclusions: The results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the Study: Our results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.

An RNA seq-based reference landscape of human normal and neoplastic brain.

In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape.

Multiparametric Longitudinal Profiling of RCAS-tva-Induced PDGFB-Driven Experimental Glioma.

Glioblastomas are incurable primary brain tumors harboring a heterogeneous landscape of genetic and metabolic alterations. Longitudinal imaging by MRI and [18F]FET-PET measurements enable us to visualize the features of evolving tumors in a dynamic manner. Yet, close-meshed longitudinal imaging time points for characterizing temporal and spatial metabolic alterations during tumor evolution in patients is not feasible because patients usually present with already established tumors. The replication-competent avian sarcoma-leukosis virus (RCAS)/tumor virus receptor-A (tva) system is a powerful preclinical glioma model offering a high grade of spatial and temporal control of somatic gene delivery in vivo. Consequently, here, we aimed at using MRI and [18F]FET-PET to identify typical neuroimaging characteristics of the platelet-derived growth factor B (PDGFB)-driven glioma model using the RCAS-tva system. Our study showed that this preclinical glioma model displays MRI and [18F]FET-PET features that highly resemble the corresponding established human disease, emphasizing the high translational relevance of this experimental model. Furthermore, our investigations unravel exponential growth dynamics and a model-specific tumor microenvironment, as assessed by histology and immunochemistry. Taken together, our study provides further insights into this preclinical model and advocates for the imaging-stratified design of preclinical therapeutic interventions.

Oncogenic role of a developmentally regulated NTRK2 splice variant.

Temporally regulated alternative splicing choices are vital for proper development, yet the wrong splice choice may be detrimental. Here, we highlight a previously unidentified role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis, and forced overexpression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges as the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring tropomyosin receptor kinase fusions. Affinity purification-mass spectrometry proteomic analysis reveals distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, transforming growth factor-ß, Sonic Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

Both YAP1-MAML2 and constitutively active YAP1 drive the formation of tumors that resemble NF2 mutant meningiomas in mice.

YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.

Olverembatinib inhibits SARS-CoV-2-Omicron variant-mediated cytokine release in human peripheral blood mononuclear cells.

The N-terminus domain (NTD) of the SARS-CoV-2 Omicron variant spike protien strongly induces multiple inflammatory molecules in human peripheral blood mononuclear cells, unaffected by the mutations observed in the NTD. Olverembatinib, a clinical-stage multi-kinase inhibitor, potently inhibits Omicron NTD-mediated cytokine release.