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Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Supplementary Figures, legends and References

Supplementary Table S1. GBM-sLCR features

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro. Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2–M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo. We propose this combinatorial treatment as a potential approach for patients with GBM.

Screening results U251 TMZ

Combined supplementary Figures

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment.Significance:Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521

Supplementary Tables

Supplementary Figures 1-6

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma.Significance:These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

Extended experimental details to supplement the main article file methods section

XLS file - 102K, TCGA data for TP53BP1 in the GBM dataset

PDF file - 132K

PDF file - 130K, Cell cycle alteration in 53BP1 silenced cells, upon exposure to IR

PDF file - 310K, 53BP1 null mouse cells, but not heterozygous, and robust silencing in GBM cells lead to high sensitivity to IR

XLS file - 66K, Co-occurrence of TP53BP1 and TP53 alterations in the TCGA dataset for GBM

PDF file - 205, 53BP1 modulates NHEJ in glioma cell lines

PDF file - 22K, 53BP1 silencing does not increase TMZ sensitivity in T98G cells

PDF file, 906K, Supplementary Materials and Methods Flow cytometry. Colony formation assay. Supplementary Tables Supplementary Table 1. siRNAs and shRNAs constructs used in this study. Supplementary Table 2. Expression primers and ChIP primers used in this study. Supplementary Table 3. Antibodies used in this study. Supplementary Figures Supplementary Figure S1. Analysis of siRNA screening for acetyltransferases. Supplementary Figure S2. Validation of candidate genes from the siRNA screening. Supplementary Figure S3. Stable depletion of Pcaf does not affect DNA synthesis in NIH3T3 cells. Supplementary Figure S4. Inhibition of PCAF expression affects neurosphere formation and proliferation of GBM543 cells. Supplementary Figure S5. Depletion of PCAF and inhibitors to PCAF induce apoptosis. Supplementary Figure S6. Inhibition of Pcaf expression affects proliferation of tNSCs.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro. Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2–M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo. We propose this combinatorial treatment as a potential approach for patients with GBM.

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma. Significance: These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

BACKGROUND Currently approved Poly ADP-Ribose Polymerase (PARP) inhibitors (PARPi) selectively inhibit and trap both PARP1 and PARP2 (PARP1/2) at sites of single strand (ss) deoxyribonucleic acid (DNA) (ssDNA) damage, preventing ssDNA repair and leading to replication-dependent DNA double strand breaks. Recent data suggest that only inhibition of PARP1 is required for anti-proliferative effects, while PARP2 functions in the survival of haematopoietic stem and progenitor cells. These observations suggest that the inhibition and trapping of PARP 2 is not needed for anti-cancer effects, and may be a major driver of haematological toxicity observed. AZD9574 is a novel brain-penetrant PARPi that potently and selectively inhibits and traps PARP1, with the goal of delivering efficacious, less toxic, and more combinable PARPi. Furthermore, owing to its central nervous system penetration capability, AZD9574 may provide a new treatment option for patients with CNS malignancies or patients with brain metastases characterized by homologous recombination deficiency (HRD). METHODS This is a first-in-human modular study primarily designed to evaluate the safety and tolerability of AZD9574 as monotherapy and in combination with anti-cancer agents at increasing dose levels in patients with advanced solid malignancies, followed by expansion cohorts in specific indications. The study will also characterize the pharmacokinetics of AZD9574 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers and anti-tumour activity. Module 1 will enrol patients with advanced breast, ovarian, pancreatic or prostate tumours harbouring homologous recombination deficiencies. Module 2 will enrol patients with isocitrate dehydrogenase (IDH)1/2 mutated glioma.

Immunotherapy is a promising treatment modality for highly invasive gliomas; however, clinical trials thus far have failed to provide significant clinical benefit to most GBM patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells even though greater than 40% of glioma cells are composed of immune cells in some patients. The majority of immune cells in GBM are immune-suppressive myeloid cells that block T cell infiltration and/or activation. To elucidate the mechanisms of immune evasion and to understand how the immune system interacts with glioma and stromal cells that shape the immune-suppressive landscape in GBM, there is an urgent need for immune-competent preclinical models that recapitulate the human disease. Human GBM is divided into three molecular subtypes (proneural:PR, classical:CL, and mesenchymal:MES) based on specific gene expression patterns and signature mutational profiles. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent all three human GBM molecular subtypes. We performed whole-exome sequencing as well as STR fingerprinting of each primary tumorsphere line and also performed immune phenotyping of each glioma model with flow cytometry. To gain molecular insights and determine cellular heterogeneity, we also performed single-cell RNA sequencing from the six models. Glioma cell analysis at the single-cell level revealed that cell-of-origin rather than the oncogenic driver (such as EGFRviii) plays a dominant role in determining the molecular phenotypes of glioma cells, driving their classification into a molecular subtype defined by human studies. In addition, we identified eight molecular subtypes of glioma-associated myeloid (GAMs), seven different subtypes of T cells, and provide molecular definitions of glioma-associated pericytes and endothelial cells in murine gliomas. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between humans and mice at the single-cell level. Furthermore, we report qualitative and quantitative differences in the cell-to-cell communication among different stromal cells and glioma cells in each model, and propose that these interactions shape the local niche and functional neighborhoods. Finally, we leverage these preclinical models to elucidate underlying molecular mechanisms that drive differential sensitivities of each model to immunotherapies: anti-PD1, CTLA4, and 4-1BB in vivo. In summary, we report deeply characterized mouse models of human GBM subtypes and highlight their utility as preclinical models for immunotherapy evaluation and foundational tumor immunology studies. Citation Format: Caiyi Wang, Jose Maldonado, Thomas D. Gallup, Nourhan Abdelfattah, Jia-Shiun Leu, Nithin Joshy, joshy George, Jihye Paik, Massimo Squatrito, Kyuson Yun. Elucidating cell-to-cell communication and immunotherapy responses in deeply characterized mouse models of human glioma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1331.

Simple Summary The standard of care for patients with newly diagnosed glioblastoma (GBM) comprises surgery followed by radio- and chemotherapy. In addition, dexamethasone is used to manage the development of inflammation within the brain in general, and particularly during treatment. The effects of dexamethasone on patient survival however remain controversial because several clinical studies suggest that dexamethasone could potentially restrict effective radiotherapy. With the idea to improve GBM therapy, we set out to identify small molecule inhibitors that could improve the killing of GBM cells when applied together with radiotherapy. We have identified a novel dexamethasone-induced mechanism that can directly protect GBM cells from radiotherapy and thus may contribute to the adverse effects observed in the clinic. Strikingly, this mechanism also sensitises GBM cells to tyrosine kinase inhibitors, thus encouraging the revision of the use of these inhibitors for the treatment of GBM, potentially in an adjuvant setting. Abstract (1) Background: Despite the indisputable effectiveness of dexamethasone (DEXA) to reduce inflammation in glioblastoma (GBM) patients, its influence on tumour progression and radiotherapy response remains controversial. (2) Methods: We analysed patient data and used expression and cell biological analyses to assess effects of DEXA on GBM cells. We tested the efficacy of tyrosine kinase inhibitors in vitro and in vivo. (3) Results: We confirm in our patient cohort that administration of DEXA correlates with worse overall survival and shorter time to relapse. In GBM cells and glioma stem-like cells (GSCs) DEXA down-regulates genes controlling G2/M and mitotic-spindle checkpoints, and it enables cells to override the spindle assembly checkpoint (SAC). Concurrently, DEXA up-regulates Platelet Derived Growth Factor Receptor (PDGFR) signalling, which stimulates expression of anti-apoptotic regulators BCL2L1 and MCL1, required for survival during extended mitosis. Importantly, the protective potential of DEXA is dependent on intact tyrosine kinase signalling and ponatinib, sunitinib and dasatinib, all effectively overcome the radio-protective and pro-proliferative activity of DEXA. Moreover, we discovered that DEXA-induced signalling creates a therapeutic vulnerability for sunitinib in GSCs and GBM cells in vitro and in vivo. (4) Conclusions: Our results reveal a novel DEXA-induced mechanism in GBM cells and provide a rationale for revisiting the use of tyrosine kinase inhibitors for the treatment of GBM.

The molecular basis underlying glioblastoma (GBM) heterogeneity and plasticity is not fully understood. Using transcriptomic data of human patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a key regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the neurofibromatosis type 1 gene (NF1), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature and reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research., Tropomyosin receptor kinase B (TrkB), encoded by the neurotrophic tyrosine receptor kinase 2 (NTRK2) gene, exhibits intricate splicing patterns and post-translational modifications. Here, the authors perform whole gene and transcript-level analyses and report the TrkB.T1 splice variant enhances PDGF-driven gliomas in vivo and augments PI3K signaling cascades in vitro.

The last decade’s progress unraveling the mutational landscape of all age groups of cancer has uncovered mutations in histones as vital contributors of tumorigenesis. Here we review three new aspects of oncogenic histones: first, the identification of additional histone mutations potentially contributing to cancer formation; second, tumors expressing histone mutations to study the crosstalk of post-translational modifications, and; third, development of sophisticated biological model systems to reproduce tumorigenesis. At the outset, we recapitulate the firstly discovered histone mutations in pediatric and adolescent tumors of the brain and bone, which still remain the most pronounced histone alterations in cancer. We branch out to discuss the ramifications of histone mutations, including novel ones, that stem from altered protein-protein interactions of cognate histone modifiers as well as the stability of the nucleosome. We close by discussing animal models of oncogenic histones that reproduce tumor formation molecularly and morphologically and the prospect of utilizing them for drug testing, leading to efficient treatment and cure of deadly cancers with histone mutations.

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance, and following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here, we found that astrocytes, when pre-irradiated, increased stemness and survival of co-cultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. We identified extracellular matrix derived from irradiated astrocytes as a major driver of this phenotype, and astrocyte-derived transglutaminase 2 (TGM2) as a promoter of glioma stemness and radioresistance. TGM2 levels were increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard of care radiotherapy by reducing stemness in glioma.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection. Influenza A virus (IAV) infection induces transcription termination defects in host cells, an effect modulated by SUMOylation of an intrinsically disordered region of the influenza NS1 protein expressed by the 1918 pandemic IAV strain.

The highest-grade gliomas invariably recur as incurable tumors following standard of care comprising surgery, radiotherapy, and chemotherapy. The majority of the recurrent tumors form within the area of the brain receiving high-dose irradiation during treatment of the primary tumor, indicating that the recurrent tumor forms in an irradiated microenvironment. The tumor microenvironment has been demonstrated to influence the therapeutic response and stemness characteristics of tumor cells, but the influence of radiation on the microenvironment and its subsequent consequences for tumor cells are incompletely understood. Here, we used genetically engineered glioma mouse models and human glioma samples to characterize the impact of standard of care radiotherapy on the brain tumor microenvironment. We found that tumor-associated astrocytes subjected to radiation in vitro could enhance tumor cell stemness and survival of co-cultured glioma cells. More aggressive gliomas formed in vivo when mouse brains were irradiated prior to tumor cell implantation, suggesting that the irradiated brain microenvironment supports tumor growth. We isolated the effect of irradiated astrocytes to extracellular matrix secreted by these cells, and specifically found that astrocyte-derived transglutaminase 2 (TGM2) is a stromal promoter of glioma stemness and radioresistance. TGM2 levels were increased after radiation in glioma mouse models. Recombinant TGM2 enhanced, and TGM2 inhibitors blocked, glioma cell stemness. In human GBM tissue, TGM2 levels were increased in recurrent vs. primary tumors. In summary, in addition to supporting TGM2 as a potential therapeutic target in glioma, our data indicate that radiotherapy results in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ., Chemotherapy resistance in recurrent gliomas is a large hurdle for successful therapy. Here, the authors show that some recurrent gliomas harbour O-6-methylguanine-DNA methyltransferase (MGMT) genomic rearrangements, and in vitro and in vivo these contribute to temozolomide resistance.

Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival., LIF is a pleiotropic cytokine that promotes an immunosuppressive microenvironment and has critical functions in embryonic development. Here, the authors show that LIF regulates CD8+ T cell tumor infiltration in cancer by repressing CXCL19 and promoting the presence of protumoral macrophages and thatLIF inhibition, via neutralizing antibodies, promotes T cell infiltration and synergizes with immune checkpoint inhbitors resulting in tumor regression and immunological memory.

Summary The molecular basis underlying Glioblastoma (GBM) heterogeneity and plasticity are not fully understood. Using transcriptomic data of patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a master regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the Neurofibromatosis type 1 gene ( NF1 ), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature, reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations. Significance Glioblastoma (GBM) is a very heterogenous disease for which multiple transcriptional subtypes have been described. Among these subtypes, the Mesenchymal (MES) GBMs have the worst prognosis. Here we provide the first causal evidence linking Neurofibromatosis type 1 gene (NF1) signalling and the acquisition of a MES gene expression program through the regulation of the AP-1 transcription factor FOSL1 . Using patient expression datasets, combined with in vitro and in vivo gain- and loss-of function mouse models, we show that FOSL1 is an important modulator of GBM that is required and sufficient for the activation of a MES program. Our work sheds light on the mechanisms that control the tumorigenicity of the most aggressive adult brain tumor type.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells

The occurrence and importance of gene fusions in glioma has been appreciated only recently, largely due to high-throughput technologies, and gene fusions have been indicated as one of the major genomic abnormalities in glioblastoma (GBM), the most frequent and aggressive glioma subtype. The functional role of the majority of these alterations is completely unexplored. Recurrent gene fusions involving the Tropomyosin Receptor tyrosine Kinases (TRK) receptor family have been recently described in a variety of tumors, including both pediatric and adult low-grade (LGG) and high-grade gliomas (HGG). Strikingly, 40% of non-brain stem pediatric HGGs in infants have been shown to carry TRK gene fusions, making Trk inhibition an important potential therapeutic intervention in patients in which the current treatment modalities have devastating side effects. For the in vivo study of genomic rearrangements, we have recently generated an innovative mouse model that combines the genome editing capability of the CRISPR/Cas9 system with the somatic gene delivery of the RCAS/tv-a system. By searching the scientific literature we have identified approximately 30 different fusion involving NTRK1, NTRK2 and NTRK3 genes. To discriminate those fusions with tumorigenic potential in gliomas we have performed an in vivo gRNA pair screening. Four different RCAS-gRNA library pools have been transduced into p53-null TVA-Cas9 neural stem cells (NSCs) and subsequently transplanted into NOD/SCID mice. So far, half of the mice rapidly developed (1–2 months) quite aggressive tumors. We are currently characterizing those tumors to identify the most potent oncogenic NTRK fusions. NTRK gene rearrangements are emerging as novel targets across multiple tumor types, because of the increasing availability of new drugs with anti-Trk activity. As part of our studies, we are investigating the response and the resistance mechanism to Trk inhibitors that are currently used in different clinical trials.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway., The epidermal growth factor receptor (EGFR) signalling is regulated at multiple levels. Here the authors show that the importin RanBP6 acts as a tumor suppressor in Glioblastoma and regulates EGFR signalling through promoting translocation of STAT3 to the nuclei and repressing EGFR transcription.

Glioblastoma expression subtypes have been previously been associated with genomic abnormalities, treatment response, and differences in tumor microenvironment. We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical, a revision of the previously reported TCGA subtypes. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and the presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Comparison of matching primary and recurrent gliomas elucidated treatment-induced phenotypic tumor evolution, including expression subtype switching, in 45% of our cohort as well as associations between microenvironmental components and treatment response. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence was associated with CD8+ T cell enrichment. Frequency of M2 macrophage detection was associated with short-term relapse after radiation therapy. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild-type glioblastoma during treatment modulated tumor evolution. Characterization of the evolving glioblastoma transcriptome and tumor microenvironment aids in designing more effective immunotherapy trials.

It has been gradually established that the vast majority of human tumors are extraordinarily heterogeneous at a genetic level. To accurately recapitulate this complexity, it is now evident that in vivo animal models of cancers will require to recreate not just a handful of simple genetic alterations, but possibly dozens and increasingly intricate. Here, we have combined the RCAS/TVA system with the CRISPR/Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells (NSCs) of a variety of known tumor suppressor genes (Trp53, Cdkn2a and Pten), in combination with the expression of an oncogene driver, leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs (gRNAs) we generated different gene fusions, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk), and we show that they have transforming potential in vitro and in vivo. Lastly, using homology-directed-repair (HDR), we also produced tumors carrying the Braf V600E mutation, frequently identified in a variety of subtypes of gliomas. In summary, we have developed an extremely powerful and versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ Tcell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.