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.