Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Transcriptome analysis of primary adult B-cell lineage acute lymphoblastic leukemia identifies pathogenic variants and gene fusions, and predicts subtypes for in depth molecular diagnosis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) is classified into subgroups based on known driver oncogenes and molecular lesions, including translocations and recurrent mutations. However, the current diagnostic tests do not identify subtypes or oncogenic lesions for all B-ALL samples, creating a heterogeneous B-ALL group of unknown subtypes. METHODS: We sorted primary adult B-ALL cells and performed transcriptome analysis by bulk RNA sequencing (RNA-seq). RESULTS: Transcriptomic analysis of an adult B-ALL cohort allowed the classification of four patient samples with subtypes that were not previously revealed by standard gene panels. The leukemia of two patients were of the DUX4 subtype and two were CRLF2+ Ph-like B-ALL. Furthermore, single nucleotide variant analysis detected the oncogenic NRAS-G12D, KRAS-G12D, and KRAS-G13D mutations in three of the patient samples, presenting targetable mutations. Additional oncogenic variants and gene fusions were uncovered, as well as multiple variants in the PDE4DIP gene across five of the patient samples. CONCLUSION: We demonstrate that RNA-seq is an effective tool for precision medicine in B-ALL by providing comprehensive molecular profiling of leukemia cells, identifying subtype and oncogenic lesions, and stratifying patients for appropriate therapy.

Ikaros Regulates microRNA Networks in Acute Lymphoblastic Leukemia.

The hematopoietic transcription factor Ikaros (IKZF1) regulates normal B cell development and functions as a tumor suppressor in precursor B cell acute lymphoblastic leukemia (B-ALL). MicroRNAs (miRNAs) are small regulatory RNAs that through post-transcriptional gene regulation play critical roles in intracellular processes including cell growth in cancer. However, the role of Ikaros in the regulation of miRNA expression in developing B cells is unknown. In this study, we examined the Ikaros-regulated miRNA targets using human IKZF1-mutated Ph+ B-ALL cell lines. Inducible expression of wild-type Ikaros (the Ik1 isoform) caused B-ALL growth arrest and exit from the cell cycle. Global miRNA expression analysis revealed a total of 31 miRNAs regulated by IK1, and ChIP-seq analysis showed that Ikaros bound to several Ik1-responsive miRNA genes. Examination of the prognostic significance of miRNA expression in B-ALL indicate that the IK1-regulated miRNAs hsa-miR-26b, hsa-miR-130b and hsa-miR-4649 are significantly associated with outcome in B-ALL. Our findings establish a potential regulatory circuit between the tumor-suppressor Ikaros and the oncogenic miRNA networks in IKZF1-mutated B-ALL. These results indicate that Ikaros regulates the expression of a subset of miRNAs, of which several may contribute to B-ALL growth.

Higher-Order Chromatin Structures of Chromosomally Integrated HHV-6A Predict Integration Sites.

Human herpesvirus -6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes. Viral integration can occur in several cell types, including germinal cells, resulting in individuals that harbor the viral genome in every cell of their body. The integrated genome is efficiently silenced but can sporadically reactivate resulting in various clinical symptoms. To date, the integration mechanism and the subsequent silencing of HHV-6A/B genes remains poorly understood. Here we investigate the genome-wide chromatin contacts of the integrated HHV-6A in latently-infected cells. We show that HHV-6A becomes transcriptionally silent upon infection of these cells over the course of seven days. In addition, we established an HHV-6-specific 4C-seq approach, revealing that the HHV-6A 3D interactome is associated with quiescent chromatin states in cells harboring integrated virus. Furthermore, we observed that the majority of virus chromatin interactions occur toward the distal ends of specific human chromosomes. Exploiting this finding, we established a 4C-seq method that accurately detects the chromosomal integration sites. We further implement long-read minION sequencing in the 4C-seq assay and developed a method to identify HHV-6A/B integration sites in clinical samples.

p38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. In males, microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. In females, the effect of p38α deficiency was divergent, exhibiting a unique transcriptional profile that included an upregulation of tissue protective genes, and a small subset of inflammatory genes that were also upregulated in males. Taken together, these results reveal a p38α-dependent sex-specific molecular pathway in microglia that is protective in CNS autoimmunity in males, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus suggesting design of future sex-specific therapeutic approaches.

Epigenetic encoding, heritability and plasticity of glioma transcriptional cell states.

Single-cell RNA sequencing has revealed extensive transcriptional cell state diversity in cancer, often observed independently of genetic heterogeneity, raising the central question of how malignant cell states are encoded epigenetically. To address this, here we performed multiomics single-cell profiling-integrating DNA methylation, transcriptome and genotype within the same cells-of diffuse gliomas, tumors characterized by defined transcriptional cell state diversity. Direct comparison of the epigenetic profiles of distinct cell states revealed key switches for state transitions recapitulating neurodevelopmental trajectories and highlighted dysregulated epigenetic mechanisms underlying gliomagenesis. We further developed a quantitative framework to directly measure cell state heritability and transition dynamics based on high-resolution lineage trees in human samples. We demonstrated heritability of malignant cell states, with key differences in hierarchal and plastic cell state architectures in IDH-mutant glioma versus IDH-wild-type glioblastoma, respectively. This work provides a framework anchoring transcriptional cancer cell states in their epigenetic encoding, inheritance and transition dynamics.

Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.

Seizure prophylaxis in an animal model of epilepsy by dietary fluoxetine supplementation.

Clinical and animal model evidence suggests that selective serotonin reuptake inhibitors (SSRIs) act as anticonvulsants. The present studies tested the possibility that the El mouse model of genetically predisposed/handling-triggered epilepsy would exhibit fewer seizures following SSRI treatment via dietary fluoxetine adulteration. In particular, potential bioenergetic and neural mechanisms for anticonvulsant efficacy of fluoxetine were explored using food intake/body weight monitoring and quantification of brain serotonin transporter protein. El mice consuming a chow diet ad libitum or yoked in quantity to fluoxetine diet intake exhibited seizure incidence of 40% in response to tail-suspension handling, whereas seizures were abolished (0%) among El mice consuming a fluoxetine-adultered diet over 7 days. A 3 day period of fluoxetine administration was insufficient to exert anticonvulsant efficacy and all treatment groups exhibited the same circadian locomotor activity patterns at the time of seizure susceptibility testing. Bioenergetic factors could not account for the anticonvulsant efficacy of fluoxetine since yoked diet controls with matched food intake, body weight change and blood glucose levels exhibited the same 40% seizure incidence as ad libitum chow controls. Importantly, the 7 day period of dietary fluoxetine exposure was effective in selectively reducing cell density in the parietal cortex and increasing serotonin transporter protein content in the nucleus accumbens. Taken together, these results suggest that dietary fluoxetine supplementation abolishes handling-induced seizure susceptibility in El mice via a neural remodeling mechanism independent of energy balance.