Leukaemia hijacks a neural mechanism to invade the central nervous system.

Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.

Formin 2 Regulates Lysosomal Degradation of Wnt-Associated ß-Catenin in Neural Progenitors.

Although neural progenitor proliferation along the ventricular zone is regulated by ß-catenin through Wnt signaling, the cytoskeletal mechanisms that regulate expression and localization of these proteins are not well understood. Our prior studies have shown that loss of the actin-binding Filamin A (FlnA) and actin-nucleating protein Formin 2 (Fmn2) impairs endocytosis of low-density-lipoprotein-receptor-related protein 6 (Lrp6), thereby disrupting ß-catenin activation, resulting in decreased brain size. Here, we report that activated RhoA-GTPase disengages Fmn2 N- to C-terminal binding to promote Fmn2 activation and redistribution into lysosomal vesicles. Fmn2 colocalizes with ß-catenin in lysosomes and promotes its degradation. Further, Fmn2 binds the E3 ligase Smurf2, enhances Smurf2-dependent ubiquitination, and degradation of Dishevelled-2 (Dvl2), thereby initiates ß-catenin degradation. Finally, Fmn2 overexpression disrupts neuroepithelial integrity, neuronal migration, and proliferation-phenotypes in E13 mouse embryos, as seen with loss of Fmn2+FlnA function. Conversely, co-expression of Dvl2 with Fmn2 rescues the proliferation defect due to Fmn2 overexpression in mouse embryos. These findings suggest that there is a homeostatic feedback mechanism in the cytoskeletal-dependent regulation of neural proliferation within the cerebral cortex. Upstream, Fmn2 promotes proliferation by stabilizing the Lrp6 receptor, leading to ß-catenin activation. Downstream, RhoA-activated Fmn2 promotes lysosomal degradation of Dvl2, leading to ß-catenin degradation.

Filamin A- and formin 2-dependent endocytosis regulates proliferation via the canonical Wnt pathway.

Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. Here, we report that the actin-binding protein filamin A (FlnA) physically interacts with the actin-nucleating protein formin 2 (Fmn2). Loss of FlnA and Fmn2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FlnA interacts with the Wnt co-receptor Lrp6. Loss of FlnA and Fmn2 impairs Lrp6 endocytosis, downstream Gsk3ß activity, and ß-catenin accumulation in the nucleus. The proliferative defect in Flna and Fmn2 null neural progenitors is rescued by inhibiting Gsk3ß activity. Our findings thus reveal a novel mechanism whereby actin-associated proteins regulate proliferation by mediating the endocytosis and transportation of components in the canonical Wnt pathway. Moreover, the Fmn2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology protein Daam. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation.

Clonality assessment of cutaneous B-cell lymphoid proliferations: a comparison of flow cytometry immunophenotyping, molecular studies, and immunohistochemistry/in situ hybridization and review of the literature.

Cutaneous lymphoid infiltrates are diagnostically challenging. Although ancillary techniques to assess clonality can help distinguish between reactive lymphoid hyperplasia and lymphoma, one of the most widely used techniques in hematopathology, flow cytometry immunophenotyping (FCI), has not been routinely applied to skin specimens. We performed FCI on 73 skin specimens from 67 patients clinically suspected of having a cutaneous B-cell lymphoma (CBCL) and compared the results with those obtained from immunoglobulin heavy chain (IGH) gene molecular studies (58 cases, primarily by polymerase chain reaction) and either immunohistochemistry (IHC) or in situ hybridization to evaluate for light chain restriction (22 and 2 cases, respectively). Sufficient quantity of CD45 (leukocyte common antigen)-positive cells and staining quality were achieved in 88% of cases by FCI, and clonality was detected in 68% of CBCLs versus molecular studies showing sufficient DNA quality in 74% and only 39% clonality detection, and interpretable/contributory IHC results in 84% of cases with 55% clonality detection. Clonality was documented more frequently in secondary rather than primary CBCLs by all 3 techniques. Therefore, FCI is feasible and appears to be more reliable than molecular studies or IHC/in situ hybridization for detecting clonality in CBCLs and can provide additional prognostically and therapeutically relevant information. The exception is cases with plasmacytic differentiation such as marginal zone lymphoma for which IHC might be a superior tool. We have also shown that a large subset of primary cutaneous follicle center lymphomas express CD10 and/or BCL2 by FCI. Recent advances in FCI beg the question of applicability to cutaneous T-cell and NK-cell lymphomas.

RNA Sequencing Identifies Novel NRG1 Fusions in Solid Tumors that Lack Co-Occurring Oncogenic Drivers.

NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n = 7), breast (n = 2), colorectal (n = 3), esophageal (n = 2), ovarian (n = 1), pancreatic (n = 1), and unknown primary (n = 1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n = 8) and previously unreported (n = 11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development.

BACKGROUND: Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled. RESULTS: We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate. CONCLUSIONS: This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.

Beta-catenin signaling levels in progenitors influence the laminar cell fates of projection neurons.

The mechanisms underlying the timing of the laminar fate decisions during cortical neurogenesis remain poorly understood. Here we show that beta-catenin signaling in cortical neural precursors can regulate the laminar fate of their daughters. In ventricular zone neural precursors, beta-catenin signaling is higher when deep-layer neurons are being generated and lower when upper-layer neurons are being generated. Overactivation of beta-catenin in cortical precursors midway through corticogenesis increased the relative production of deep-layer neurons, while inhibition of signaling increased the relative production of upper-layer neurons. Furthermore, in late-gestation upper-layer precursors, overactive beta-catenin signaling was able to partially restore production of deep-layer neurons. These observations suggest that increased beta-catenin signaling can reset the timing of cortical precursors to promote the production of deep-layer neurons, while inhibition of beta-catenin signaling advances the timing to promote upper-layer production.

Focal reduction of alphaE-catenin causes premature differentiation and reduction of beta-catenin signaling during cortical development.

Cerebral cortical precursor cells reside in a neuroepithelial cell layer that regulates their proliferation and differentiation. Global disruptions in epithelial architecture induced by loss of the adherens junction component alphaE-catenin lead to hyperproliferation. Here we show that cell autonomous reduction of alphaE-catenin in the background of normal precursors in vivo causes cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate, while normal neighboring precursors are unaffected. Mechanistically, alphaE-catenin likely regulates cortical precursor differentiation by maintaining beta-catenin signaling, as reduction of alphaE-catenin leads to reduction of beta-catenin signaling in vivo. These results demonstrate that, at the cellular level, alphaE-catenin serves to maintain precursors in the proliferative ventricular zone, and suggest an unexpected function for alphaE-catenin in preserving beta-catenin signaling during cortical development.

Lis1-Nde1-dependent neuronal fate control determines cerebral cortical size and lamination.

Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

Nestin reporter transgene labels multiple central nervous system precursor cells.

Embryonic neuroepithelia and adult subventricular zone (SVZ) stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP) specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes.

The simple life (of cortical progenitors).

Asymmetric cell division plays a major role in the generation of cell diversity during development. In this issue of Neuron, Sun and colleagues present evidence that the epidermal growth factor receptor is asymmetrically distributed in mitotic cerebral cortical precursors, and the resulting unequal inheritance generates offspring with different responsiveness to growth factor and unique cell fates.

Cell-autonomous beta-catenin signaling regulates cortical precursor proliferation.

Overexpression of beta-catenin, a protein that functions in both cell adhesion and signaling, causes expansion of the cerebral cortical precursor population and cortical surface area enlargement. Here, we find that focal elimination of beta-catenin from cortical neural precursors in vivo causes premature neuronal differentiation. Precursors within the cerebral cortical ventricular zone exhibit robust beta-catenin-mediated transcriptional activation, which is downregulated as cells exit the ventricular zone. Targeted inhibition of beta-catenin signaling during embryonic development causes cortical precursor cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate. These results show that beta-catenin-mediated transcriptional activation functions in the decision of cortical ventricular zone precursors to proliferate or differentiate during development, and suggest that the cell-autonomous signaling activity of beta-catenin can control the production of cortical neurons and thus regulate cerebral cortical size.

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain.

We previously demonstrated that chemokine receptors are expressed by neural progenitors grown as cultured neurospheres. To examine the significance of these findings for neural progenitor function in vivo, we investigated whether chemokine receptors were expressed by cells having the characteristics of neural progenitors in neurogenic regions of the postnatal brain. Using in situ hybridization we demonstrated the expression of CCR1, CCR2, CCR5, CXCR3, and CXCR4 chemokine receptors by cells in the dentate gyrus (DG), subventricular zone of the lateral ventricle, and olfactory bulb. The pattern of expression for all of these receptors was similar, including regions where neural progenitors normally reside. In addition, we attempted to colocalize chemokine receptors with markers for neural progenitors. In order to do this we used nestin-EGFP and TLX-LacZ transgenic mice, as well as labeling for Ki67, a marker for dividing cells. In all three areas of the brain we demonstrated colocalization of chemokine receptors with these three markers in populations of cells. Expression of chemokine receptors by neural progenitors was further confirmed using CXCR4-EGFP BAC transgenic mice. Expression of CXCR4 in the DG included cells that expressed nestin and GFAP as well as cells that appeared to be immature granule neurons expressing PSA-NCAM, calretinin, and Prox-1. CXCR4-expressing cells in the DG were found in close proximity to immature granule neurons that expressed the chemokine SDF-1/CXCL12. Cells expressing CXCR4 frequently coexpressed CCR2 receptors. These data support the hypothesis that chemokine receptors are important in regulating the migration of progenitor cells in postnatal brain.

Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.

Neuroblasts migrate long distances in the postnatal subventricular zone (SVZ) and rostral migratory stream (RMS) to the olfactory bulbs. Many fundamental features of SVZ migration are still poorly understood, and we addressed several important questions using two-photon time-lapse microscopy of brain slices from postnatal and adult eGFP(+) transgenic mice. 1) Longitudinal arrays of neuroblasts, so-called chain migration, have never been dynamically visualized in situ. We found that neuroblasts expressing doublecortin-eGFP (Dcx-eGFP) and glutamic acid decarboxylase-eGFP (Gad-eGFP) remained within arrays, which maintained their shape for many hours, despite the fact that there was a wide variety of movement within arrays. 2) In the dorsal SVZ, neuroblasts migrated rostrocaudally as expected, but migration shifted to dorsoventral orientations throughout ventral regions of the lateral ventricle. 3) Whereas polarized bipolar morphology has been a gold standard for inferring migration in histologic sections, our data indicated that migratory morphology was not predictive of motility. 4) Is there local motility in addition to long distance migration? 5) How fast is SVZ migration? Unexpectedly, one-third of motile neuroblasts moved locally in complex exploratory patterns and at average speeds slower than long distance movement. 6) Finally, we tested, and disproved, the hypothesis that all motile cells in the SVZ express doublecortin, indicating that Dcx is not required for migration of all SVZ cell types. These data show that cell motility in the SVZ and RMS is far more complex then previously thought and involves multiple cell types, behaviors, speeds, and directions.

Afadin controls cell polarization and mitotic spindle orientation in developing cortical radial glia.

BACKGROUND: In developing tissues, cell polarity and tissue architecture play essential roles in the regulation of proliferation and differentiation. During cerebral cortical development, adherens junctions link highly polarized radial glial cells in a neurogenic niche that controls their behavior. How adherens junctions regulate radial glial cell polarity and/or differentiation in mammalian cortical development is poorly understood. RESULTS: Conditional deletion of Afadin, a protein required for formation and maintenance of epithelial tissues, leads to abnormalities in radial glial cell polarity and subsequent loss of adherens junctions. We observed increased numbers of obliquely-oriented progenitor cell divisions, increased exit from the ventricular zone neuroepithelium, and increased production of intermediate progenitors. CONCLUSIONS: Together, these findings indicate that Afadin plays an essential role in regulating apical-basal polarity and adherens junction integrity of radial glial cells, and suggest that epithelial architecture plays an important role in radial glial identity by regulating mitotic orientation and preventing premature exit from the neurogenic niche.

Differential expression of alpha-E-catenin and alpha-N-catenin in the developing cerebral cortex.

Alpha (alpha) catenin proteins can regulate both cell adhesion and cell proliferation. Here, we characterize the expression of two forms of alpha-catenin, alphaE-catenin and alphaN-catenin, in the developing cerebral cortex and primary cortical cultures. In situ hybridization and immunofluorescence studies reveal that alphaE-catenin is highly expressed in neuroepithelial precursor cells in the developing cortical ventricular zone, with markedly reduced expression in the cortical plate; in contrast, alphaN-catenin expression is low in the ventricular zone and high in the developing cortical plate. In the ventricular zone, immunoreactivity for both alphaE-catenin and alphaN-catenin is enriched in rings at the lumenal surface, reflecting localization at adherens junctions together with beta-catenin. Expression of alphaE-catenin in primary cortical precursor cultures is initially robust, but declines as neural precursors differentiate into neurons. Reflecting its expression pattern in vivo, alphaN-catenin is expressed in both neural precursors as well in neurons differentiated in culture. These differential expression patterns of alphaE-catenin and alphaN-catenin suggest both distinct and overlapping functions during cortical development.

A cell behavior screen: identification, sorting, and enrichment of cells based on motility.

BACKGROUND: Identifying and isolating cells with specific behavioral characteristics will facilitate the understanding of the molecular basis regulating these behaviors. Although many approaches exist to characterize cell motility, retrieving cells of specific motility following analysis remains challenging. RESULTS: Cells migrating on substrates coated with fluorescent microspheres generate non-fluorescent tracks as they move and ingest the spheres. The area cleared by each cell allows for quantitation of single cell and population motility; because individual cell fluorescence is proportional to motility, cells can be sorted according to their degree of movement. Using this approach, we sorted a glioblastoma cell line into high motility and low motility populations and found stable differences in motility following sorting. CONCLUSION: We describe an approach to identify, sort, and enrich populations of cells possessing specific levels of motility. Unlike existing assays of cell motility, this approach enables recovery of characterized cell populations, and can enable screens to identify factors that might regulate motility differences even within clonal population of cells.

The role of adherens junctions in the developing neocortex.

The disproportional enlargement of the neocortex through evolution has been instrumental in the success of vertebrates, in particular mammals. The neocortex is a multilayered sheet of neurons generated from a simple proliferative neuroepithelium through a myriad of mechanisms with substantial evolutionary conservation. This developing neuroepithelium is populated by progenitors that can generate additional progenitors as well as post-mitotic neurons. Subtle alterations in the production of progenitors vs. differentiated cells during development can result in dramatic differences in neocortical size. This review article will examine how cadherin adhesion proteins, in particular α-catenin and N-cadherin, function in regulating the neural progenitor microenvironment, cell proliferation, and differentiation in cortical development.

AKT activation by N-cadherin regulates beta-catenin signaling and neuronal differentiation during cortical development.

BACKGROUND: During cerebral cortical development, neural precursor-precursor interactions in the ventricular zone neurogenic niche coordinate signaling pathways that regulate proliferation and differentiation. Previous studies with shRNA knockdown approaches indicated that N-cadherin adhesion between cortical precursors regulates ß-catenin signaling, but the underlying mechanisms remained poorly understood. RESULTS: Here, with conditional knockout approaches, we find further supporting evidence that N-cadherin maintains ß-catenin signaling during cortical development. Using shRNA to N-cadherin and dominant negative N-cadherin overexpression in cell culture, we find that N-cadherin regulates Wnt-stimulated ß-catenin signaling in a cell-autonomous fashion. Knockdown or inhibition of N-cadherin with function-blocking antibodies leads to reduced activation of the Wnt co-receptor LRP6. We also find that N-cadherin regulates ß-catenin via AKT, as reduction of N-cadherin causes decreased AKT activation and reduced phosphorylation of AKT targets GSK3ß and ß-catenin. Inhibition of AKT signaling in neural precursors in vivo leads to reduced ß-catenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic cell death. CONCLUSIONS: These results show that N-cadherin regulates ß-catenin signaling through both Wnt and AKT, and suggest a previously unrecognized role for AKT in neuronal differentiation and cell survival during cortical development.