Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

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.

Transcriptomic Profiles of Normal Pituitary Cells and Pituitary Neuroendocrine Tumor Cells.

The pituitary gland is one of the most cellularly diverse regions of the brain. Recent advancements in transcriptomic biology, such as single-cell RNA sequencing, bring an unprecedented glimpse into the molecular composition of the pituitary, both in its normal physiological state and in disease. Deciphering the normal pituitary transcriptomic signatures provides a better insight into the ontological origin and development of five types of endocrine cells, a process involving complex cascades of transcription factors that are still being established. In parallel with these observations about normal pituitary development, recent transcriptomic findings on pituitary neuroendocrine tumors (PitNETs) demonstrate both preservations and changes in transcription factor expression patterns compared to those seen during gland development. Furthermore, recent studies also identify differentially expressed genes that drive various tumor behaviors, including hormone hypersecretion and tumor aggression. Understanding the comprehensive multiomic profiles of PitNETs is essential in developing molecular profile-based therapies for PitNETs not curable with current treatment modalities and could eventually help align PitNETs with the breakthroughs being made in applying precision medicine to other tumors.

IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish.

Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.

Kapd Is Essential for Specification of the Dopaminergic Neurogenesis in Zebrafish Embryos.

To define novel networks of Parkinson's disease (PD) pathogenesis, the substantia nigra pars compacta of A53T mice, where a death-promoting protein, FAS-associated factor 1 was ectopically expressed for 2 weeks in the 2-, 4-, 6-, and 8-month-old mice, and was subjected to transcriptomic analysis. Compendia of expression profiles and a hierarchical clustering heat map of differentially expressed genes associated with PD were bioinformatically generated. Transcripts level of a particular gene was fluctuated by 20, 60, and 0.75 fold in the 4-, 6-, and 8-month-old mice compared to the 2 months old. Because the gene contained Kelch domain, it was named as Kapd (Kelch-containing protein associated with PD). Biological functions of Kapd were systematically investigated in the zebrafish embryos. First, transcripts of a zebrafish homologue of Kapd, kapd were found in the floor plate of the neural tube at 10 h post fertilization (hpf), and restricted to the tegmentum, hypothalamus, and cerebellum at 24 hpf. Second, knockdown of kapd caused developmental defects of DA progenitors in the midbrain neural keel and midbrain? hindbrain boundary at 10 hpf. Third, overexpression of kapd increased transcripts level of the dopaminergic immature neuron marker, shha in the prethalamus at 16.5 hpf. Finally, developmental consequences of kapd knockdown reduced transcripts level of the markers for the immature and mature DA neurons, nkx2.2, olig2, otx2b, and th in the ventral diencephalon of the midbrain at 18 hpf. It is thus most probable that Kapd play a key role in the specification of the DA neuronal precursors in zebrafish embryos.

Trim46 contributes to the midbrain development via Sonic Hedgehog signaling pathway in zebrafish embryos.

TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis.

march5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos.

MARCH5 is a RING finger E3 ligase involved in mitochondrial integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function of MARCH5 during development, we assessed transcript expression in zebrafish embryos. We found that march5 transcripts were of maternal origin and evenly distributed at the 1-cell stage, except for the mid-blastula transition, with expression predominantly in the developing central nervous system at later stages of embryogenesis. Overexpression of march5 impaired convergent extension movement during gastrulation, resulting in reduced patterning along the dorsoventral axis and alterations in the ventral cell types. Overexpression and knockdown of march5 disrupted the organization of the developing telencephalon and diencephalon. Lastly, we found that the transcription of march5 was tightly regulated by the transcriptional regulators CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results demonstrate the essential role of March5 in the development of zebrafish embryos.

Znf76 is associated with development of the eyes, midbrain, MHB, and hindbrain in zebrafish embryos.

ZNF76 is a transcriptional repressor that targets the TATA-binding protein (TBP) and plays an essential role during brain development; however, its function during embryogenesis remains unclear. Here, we report the expression pattern and potential functions of znf76 in zebrafish embryos. Maternal transcripts of znf76 were detected at low levels in embryos at the 1-cell stage, with zygotic transcripts appearing at the sphere stage. At the bud stage, the distribution of znf76 transcripts was polarized to the anterior and posterior regions of the embryos, and znf76 transcripts were further restricted to the trigeminal placode and proctodeum posterior gut of the embryos at 18 h postfertilization (hpf). znf76 transcripts were localized to the midbrain-hindbrain boundary (MHB), hindbrain, and developing eyes at 24 hpf. Ectopic expression of znf76 with 5'-capped znf76 mRNA microinjected into embryos at the 1-cell stage caused phenotypic defects in the eyes, MHB, hindbrain, and spinal cord. Overexpression of znf76 resulted in a drastic reduction of pax2a, fgf8a, and rx1 transcripts in the optic stalk, MHB, and eyes, respectively. Taken together, these data indicate that Znf76 governs developmental processes in the MHB, hindbrain, and eyes in zebrafish embryos. We also discuss the Fgf8 signaling networks associated with the Znf76 function.