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Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction., SPI1 fusion genes in T-cell acute lymphoblastic leukemia (T-ALL) are commonly found with co-occurring NRAS mutations. Here, the authors show that the combination of these oncogenes is necessary to drive T-ALL in a murine model and that the oncogenic activity of the SPI1 fusion is dependent on β-catenin.

Peripheral neuroblastic tumors (PNTs) are the most common extracranial solid tumors in early childhood. They represent a spectrum of neural crest derived tumors including neuroblastoma, ganglioneuroblastoma and ganglioneuroma. PNTs exhibit heterogeneity due to interconverting malignant cell states described as adrenergic/nor-adrenergic or mesenchymal/neural crest cell in origin. The factors determining individual patient levels of tumor heterogeneity, their impact on the malignant phenotype, and the presence of other cell states are unknown. Here, single-cell RNA-sequencing analysis of 4267 cells from 7 PNTs demonstrated extensive transcriptomic heterogeneity. Trajectory modelling showed that malignant neuroblasts move between adrenergic and mesenchymal cell states via a novel state that we termed a “transitional” phenotype. Transitional cells are characterized by gene expression programs linked to a sympathoadrenal development, and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature was highly predictive of poor prognosis when compared to adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identified a similar transitional H3K27-acetylation super-enhancer landscape, supporting the concept that PNTs have phenotypic plasticity and transdifferentiation capacity. Additionally, examination of PNT microenvironments, found that neuroblastomas contained low immune cell infiltration, high levels of non-inflammatory macrophages, and low cytotoxic T lymphocyte levels compared with more benign PNT subtypes. Modeling of cell-cell signaling in the tumor microenvironment predicted specific paracrine effects toward the various subtypes of malignant cells, suggesting further cell-extrinsic influences on malignant cell phenotype. Collectively, our study reveals the presence of a previously unrecognized transitional cell state with high malignant potential and an immune cell architecture which serve both as potential biomarkers and therapeutic targets.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cellsin vivoand on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a–C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENTThe complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the Huntingtin gene, where excessive (≥ 36) CAG repeats encode for glutamine expansion in the huntingtin protein. Research using mouse models and human pathological material has indicated dysfunctions in a myriad of systems, including mitochondrial and ubiquitin/proteasome complexes, cytoskeletal transport, signaling, and transcriptional regulation. Here, we examined the earliest biochemical and pathways involved in HD pathology. We conducted a proteomics study combined with immunocytochemical analysis of undifferentiated HD-affected and unaffected human embryonic stem cells (hESC). Analysis of 1883 identifications derived from membrane and cytosolic enriched fractions revealed mitochondria as the primary dysfunctional organ in HD-affected pluripotent cells in the absence of significant differences in huntingtin protein. Furthermore, on the basis of analysis of 645 proteins found in neurodifferentiated hESC, we show a shift to transcriptional dysregulation and cytoskeletal abnormalities as the primary pathologies in HD-affected cells differentiating along neural lineages in vitro. We also show this is concomitant with an up-regulation in expression of huntingtin protein in HD-affected cells. This study demonstrates the utility of a model that recapitulates HD pathology and offers insights into disease initiation, etiology, progression, and potential therapeutic intervention.