Glioblastoma induces the recruitment and differentiation of hybrid neutrophils from skull bone marrow.

Tumor-associated neutrophil (TAN) effects on glioblastoma biology remain under-characterized. We show here that 'hybrid' neutrophils with dendritic features - including morphological complexity, expression of antigen presentation genes, and the ability to process exogenous peptide and stimulate MHCII-dependent T cell activation - accumulate intratumorally and suppress tumor growth in vivo . Trajectory analysis of patient TAN scRNA-seq identifies this phenotype as a polarization state which is distinct from canonical cytotoxic TANs and differentiates intratumorally from immature precursors absent in circulation. Rather, these hybrid-inducible immature neutrophils - which we identified in patient and murine glioblastomas - arise from local skull marrow. Through labeled skull flap transplantation and targeted ablation, we characterize calvarial marrow as a potent contributor of antitumoral myeloid APCs, including hybrid TANs and dendritic cells, which elicit T cell cytotoxicity and memory. As such, agents augmenting neutrophil egress from skull marrow - such as intracalvarial AMD3100 whose survival prolonging-effect in GBM we demonstrate - present therapeutic potential.

A single-cell atlas of glioblastoma evolution under therapy reveals cell-intrinsic and cell-extrinsic therapeutic targets.

Recent longitudinal studies of glioblastoma (GBM) have demonstrated a lack of apparent selection pressure for specific DNA mutations in recurrent disease. Single-cell lineage tracing has shown that GBM cells possess a high degree of plasticity. Together this suggests that phenotype switching, as opposed to genetic evolution, may be the escape mechanism that explains the failure of precision therapies to date. We profiled 86 primary-recurrent patient-matched paired GBM specimens with single-nucleus RNA, single-cell open-chromatin, DNA and spatial transcriptomic/proteomic assays. We found that recurrent GBMs are characterized by a shift to a mesenchymal phenotype. We show that the mesenchymal state is mediated by activator protein 1. Increased T-cell abundance at recurrence was prognostic and correlated with hypermutation status. We identified tumor-supportive networks of paracrine and autocrine signals between GBM cells, nonmalignant neuroglia and immune cells. We present cell-intrinsic and cell-extrinsic targets and a single-cell multiomics atlas of GBM under therapy.

ATRX regulates glial identity and the tumor microenvironment in IDH-mutant glioma.

BACKGROUND: Recent single-cell transcriptomic studies report that IDH-mutant gliomas share a common hierarchy of cellular phenotypes, independent of genetic subtype. However, the genetic differences between IDH-mutant glioma subtypes are prognostic, predictive of response to chemotherapy, and correlate with distinct tumor microenvironments. RESULTS: To reconcile these findings, we profile 22 human IDH-mutant gliomas using scATAC-seq and scRNA-seq. We determine the cell-type-specific differences in transcription factor expression and associated regulatory grammars between IDH-mutant glioma subtypes. We find that while IDH-mutant gliomas do share a common distribution of cell types, there are significant differences in the expression and targeting of transcription factors that regulate glial identity and cytokine elaboration. We knock out the chromatin remodeler ATRX, which suffers loss-of-function alterations in most IDH-mutant astrocytomas, in an IDH-mutant immunocompetent intracranial murine model. We find that both human ATRX-mutant gliomas and murine ATRX-knockout gliomas are more heavily infiltrated by immunosuppressive monocytic-lineage cells derived from circulation than ATRX-intact gliomas, in an IDH-mutant background. ATRX knockout in murine glioma recapitulates gene expression and open chromatin signatures that are specific to human ATRX-mutant astrocytomas, including drivers of astrocytic lineage and immune-cell chemotaxis. Through single-cell cleavage under targets and tagmentation assays and meta-analysis of public data, we show that ATRX loss leads to a global depletion in CCCTC-binding factor association with DNA, gene dysregulation along associated chromatin loops, and protection from therapy-induced senescence. CONCLUSIONS: These studies explain how IDH-mutant gliomas from different subtypes maintain distinct phenotypes and tumor microenvironments despite a common lineage hierarchy.

The evolution of alternative splicing in glioblastoma under therapy.

BACKGROUND: Alternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy. RESULT: We profile 37 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type-specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells. CONCLUSION: These studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development.

Ensemble learning for classifying single-cell data and projection across reference atlases.

SUMMARY: Single-cell data are being generated at an accelerating pace. How best to project data across single-cell atlases is an open problem. We developed a boosted learner that overcomes the greatest challenge with status quo classifiers: low sensitivity, especially when dealing with rare cell types. By comparing novel and published data from distinct scRNA-seq modalities that were acquired from the same tissues, we show that this approach preserves cell-type labels when mapping across diverse platforms. AVAILABILITY AND IMPLEMENTATION: https://github.com/diazlab/ELSA. CONTACT: aaron.diaz@ucsf.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Phenotypes of Proliferating Glioblastoma Cells Reside on a Single Axis of Variation.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631.

Intranasal delivery of targeted polyfunctional gold-iron oxide nanoparticles loaded with therapeutic microRNAs for combined theranostic multimodality imaging and presensitization of glioblastoma to temozolomide.

The prognosis for glioblastoma (GBM) remains depressingly low. The biological barriers of the brain present a major challenge to achieving adequate drug concentrations for GBM therapy. To address this, we explore the potential of the nose-to-brain direct transport pathway to bypass the blood-brain barrier, and to enable targeted delivery of theranostic polyfunctional gold-iron oxide nanoparticles (polyGIONs) surface loaded with therapeutic miRNAs (miR-100 and antimiR-21) to GBMs in mice. These nanoformulations would thus allow presensitization of GBM cells to the systemically delivered chemotherapy drug temozolomide (TMZ), as well as in vivo multimodality molecular and anatomic imaging of nanoparticle delivery, trafficking, and treatment effects. First, we synthesized GIONs coated with ß-cyclodextrin-chitosan (CD-CS) hybrid polymer, and co-loaded with miR-100 and antimiR-21. Then we decorated their surface with PEG-T7 peptide using CD-adamantane host-guest chemistry. The resultant polyGIONs showed efficient miRNA loading with enhanced serum stability. We characterized them for particle size, PDI, polymer functionalization, charge and release using dynamic light scattering analysis, TEM and qRT-PCR. For in vivo intranasal delivery, we used U87-MG GBM cell-derived orthotopic xenograft models in mice. Intranasal delivery resulted in efficient accumulation of Cy5-miRNAs in mice treated with T7-targeted polyGIONs, as demonstrated by in vivo optical fluorescence and MR imaging. We measured the therapeutic response of these FLUC-EGFP labelled U87-MG GBMs using bioluminescence imaging. Overall, there was a significant increase in survival of mice co-treated with T7-polyGIONs loaded with miR-100/antimiR-21 plus systemic TMZ, compared to the untreated control group, or the animals receiving non-targeted polyGIONs-miR-100/antimiR-21, or TMZ alone. Once translated clinically, this novel theranostic nanoformulation and its associated intranasal delivery strategy will have a strong potential to potentiate the effects of TMZ treatment in GBM patients.

Restoring guardianship of the genome: Anticancer drug strategies to reverse oncogenic mutant p53 misfolding.

p53 is a transcription factor that activates numerous genes involved in essential maintenance of genetic stability. P53 is the most frequently mutated gene in human cancer. One third of these mutations are structural, resulting in mutant p53 with a disrupted protein conformation. Here we review current progress in a relatively underexplored aspect of p53-targeted drug development, that is, strategies to reactivate wild-type function of misfolded mutant p53. Unfortunately, most p53-targeted drugs are still at early stages of development and many of them are progressing slowly toward clinical implementation. Significant challenges need to be addressed before clinical translation of new anti-misfolding p53-targeted drugs.

Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model.

Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.

A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells.

Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.

Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission.

Neural information processing depends on precisely timed, Ca2+-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Drep-2 is a novel synaptic protein important for learning and memory.

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.