Schwann Cells Are Key Regulators of Corneal Epithelial Renewal.

Purpose: Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly, enabling vision. In other organs, Schwann cells (SCs) associated with tissue-innervating axon terminals mediate tissue regeneration. This study defines the critical role of the corneal axon-ensheathing SCs in homeostatic and regenerative corneal epithelial cell renewal. Methods: SC localization in the cornea was determined by in situ hybridization and immunohistochemistry with SC markers. In vivo SC visualization and/or ablation were performed in mice with inducible corneal SC-specific expression of tdTomato and/or Diphtheria toxin, respectively. The relative locations of SCs and LSCs were observed with immunohistochemical analysis of harvested genetically SC-prelabeled mouse corneas with LSC-specific antibodies. The correlation between cornea-innervating axons and the appearance of SCs was ascertained using corneal denervation in rats. To determine the limbal niche cellular composition and gene expression changes associated with innervation-dependent epithelial renewal, single-cell RNA sequencing (scRNA-seq) of dissociated healthy, de-epithelized, and denervated cornea limbi was performed. Results: We observed limbal enrichment of corneal axon-associated myelinating and non-myelinating SCs. Induced local genetic ablation of SCs, although leaving corneal sensory innervation intact, markedly inhibited corneal epithelial renewal. scRNA-seq analysis (1) highlighted the transcriptional heterogenicity of cells populating the limbal niche, and (2) identified transcriptional changes associated with corneal innervation and during wound healing that model potential regulatory paracrine interactions between SCs and LSCs. Conclusions: Limbal SCs are required for innervation-dependent corneal epithelial renewal.

Nail-associated mesenchymal cells contribute to and are essential for dorsal digit tip regeneration.

Here, we ask why the nail base is essential for mammalian digit tip regeneration, focusing on the inductive nail mesenchyme. We identify a transcriptional signature for these cells that includes Lmx1b and show that the Lmx1b-expressing nail mesenchyme is essential for blastema formation. We use a combination of Lmx1bCreERT2-based lineage-tracing and single-cell transcriptional analyses to show that the nail mesenchyme contributes cells for two pro-regenerative mechanisms. One group of cells maintains their identity and regenerates the new nail mesenchyme. A second group contributes specifically to the dorsal blastema, loses their nail mesenchyme phenotype, acquires a blastema transcriptional state that is highly similar to blastema cells of other origins, and ultimately contributes to regeneration of the dorsal but not ventral dermis and bone. Thus, the regenerative necessity for an intact nail base is explained, at least in part, by a requirement for the inductive nail mesenchyme.

Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma.

Survival for high-risk neuroblastoma remains poor. Most patients who recur, present with metastatic disease, and few targetable pathways that govern spread to distant sites are currently known. We previously developed a metastatic mouse model to select cells with enhanced ability to spread to the bone and brain and identified a signature based on differentially expressed genes, which also predicted patient survival. To discover new neuroblastoma therapies, we utilized the Connectivity Map to identify compounds that can reverse this metastatic transcriptional signature and found calcipotriol, a vitamin D3 analog, to be a compound that selectively targets cell lines with enhanced metastatic potential. Calcipotriol treatment of enhanced metastatic, but not parental, cells reduces proliferation and survival via vitamin D receptor (VDR) signaling, increases the expression of RASSF2, a negative regulator of the Hippo signaling pathway, and reduces the levels of the Hippo pathway effectors YAP and TAZ. RASSF2 is required for the effects of calcipotriol and for the reduction of levels and nuclear localization of YAP/TAZ. Migration of the enhanced metastatic cells and YAP/TAZ levels are reduced after calcipotriol treatment and YAP overexpression reduces calcipotriol sensitivity. Furthermore, metastatic cells that overexpress VDR also showed lower tumor burden in vivo. IMPLICATIONS: This newly identified link between VDR signaling and the Hippo pathway could inform treatment strategies for metastatic neuroblastoma.

The DEAD-box helicase DDX56 is a conserved stemness regulator in normal and cancer stem cells.

Across the animal kingdom, adult tissue homeostasis is regulated by adult stem cell activity, which is commonly dysregulated in human cancers. However, identifying key regulators of stem cells in the milieu of thousands of genes dysregulated in a given cancer is challenging. Here, using a comparative genomics approach between planarian adult stem cells and patient-derived glioblastoma stem cells (GSCs), we identify and demonstrate the role of DEAD-box helicase DDX56 in regulating aspects of stemness in four stem cell systems: planarians, mouse neural stem cells, human GSCs, and a fly model of glioblastoma. In a human GSC line, DDX56 localizes to the nucleolus, and using planarians, when DDX56 is lost, stem cells dysregulate expression of ribosomal RNAs and lose nucleolar integrity prior to stem cell death. Together, a comparative genomic approach can be used to uncover conserved stemness regulators that are functional in both normal and cancer stem cells.

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

LRIG1-Mediated Inhibition of EGF Receptor Signaling Regulates Neural Precursor Cell Proliferation in the Neocortex.

Here, we ask how neural stem cells (NSCs) transition in the developing neocortex from a rapidly to a slowly proliferating state, a process required to maintain lifelong stem cell pools. We identify LRIG1, known to regulate receptor tyrosine kinase signaling in other cell types, as a negative regulator of cortical NSC proliferation. LRIG1 is expressed in murine cortical NSCs as they start to proliferate more slowly during embryogenesis and then peaks postnatally when they transition to give rise to a portion of adult NSCs. Constitutive or acute loss of Lrig1 in NSCs over this developmental time frame causes stem cell expansion due to increased proliferation. LRIG1 controls NSC proliferation by associating with and negatively regulating the epidermal growth factor receptor (EGFR). These data support a model in which LRIG1 dampens the stem cell response to EGFR ligands within the cortical environment to slow their proliferation as they transition to postnatal adult NSCs.

(H)Elping nerve growth factor: Elp1 inhibits TrkA's phosphatase to maintain retrograde signaling.

Nerve growth factor (NGF) regulates many aspects of neuronal biology by retrogradely propagating signals along axons to the targets of those axons. How this occurs when axons contain a plethora of proteins that can silence those signals has long perplexed the neurotrophin field. In this issue of the JCI, Li et al. suggest an answer to this vexing problem, while exploring why the Elp1 gene that is mutated in familial dysautonomia (FD) causes peripheral neuropathy. They describe a distinctive function of Elp1 as a protein that is required to sustain NGF signaling by blocking the activity of its phosphatase that shuts off those signals. This finding helps explain the innervation deficits prominent in FD and reveals a unique role for Elp1 in the regulation of NGF-dependent TrkA activity.

Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

Comprehensive genomic profiling of glioblastoma tumors, BTICs, and xenografts reveals stability and adaptation to growth environments.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for Tropomyosin Receptor Kinases.

The tropomyosin receptor kinase TrkA/B/C family is responsible for human neuronal growth, survival, and differentiation from early nervous system development stages onward. Downregulation of TrkA/B/C receptors characterizes numerous neurological disorders including Alzheimer's disease (AD). Abnormally expressed Trk receptors or chimeric Trk fusion proteins are also well-characterized oncogenic drivers in a variety of neurogenic and non-neurogenic human neoplasms and are currently the focus of intensive clinical research. Previously, we have described the clinical translation of a highly selective and potent carbon-11-labeled pan-Trk radioligand and the preclinical characterization of the optimized fluorine-18-labeled analogue, [18F]TRACK, for in vivo Trk positron emission tomography (PET) imaging. We describe herein central nervous system selectivity assessment and first-in-human study of [18F]TRACK.

Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging.

The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer's disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted.

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration.

Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.

ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts.

BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

Interleukin-6 Regulates Adult Neural Stem Cell Numbers during Normal and Abnormal Post-natal Development.

Circulating systemic factors can regulate adult neural stem cell (NSC) biology, but the identity of these circulating cues is still being defined. Here, we have focused on the cytokine interleukin-6 (IL-6), since increased circulating levels of IL-6 are associated with neural pathologies such as autism and bipolar disorder. We show that IL-6 promotes proliferation of post-natal murine forebrain NSCs and that, when the IL-6 receptor is inducibly knocked out in post-natal or adult neural precursors, this causes a long-term decrease in forebrain NSCs. Moreover, a transient circulating surge of IL-6 in perinatal or adult mice causes an acute increase in neural precursor proliferation followed by long-term depletion of adult NSC pools. Thus, IL-6 signaling is both necessary and sufficient for adult NSC self-renewal, and acute perturbations in circulating IL-6, as observed in many pathological situations, have long-lasting effects on the size of adult NSC pools.

Identification of [18F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging.

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first 18F-labeled optimized lead suitable for in vivo imaging of Trk, [18F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity.

A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria.

Axon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor wt and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting. Foretinib delayed chemotherapy-induced and Wallerian axonal degeneration in culture by preventing axotomy-induced local energy deficit and preserving mitochondria, and peripheral Wallerian degeneration in vivo. These findings identify a new axon degeneration pathway and a potentially clinically useful therapeutic drug.

RNAi screen identifies essential regulators of human brain metastasis-initiating cells.

Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-ß ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human.

The proto-oncogenes NTRK1/2/3 encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [11C]-(R)-3 ([11C]-(R)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (R)-3 is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [11C]-(R)-3 readily crosses the blood-brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer's disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [11C]-(R)-3 in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human.

A Metastatic Mouse Model Identifies Genes That Regulate Neuroblastoma Metastasis.

Metastatic relapse is the major cause of death in pediatric neuroblastoma, where there remains a lack of therapies to target this stage of disease. To understand the molecular mechanisms mediating neuroblastoma metastasis, we developed a mouse model using intracardiac injection and in vivo selection to isolate malignant cell subpopulations with a higher propensity for metastasis to bone and the central nervous system. Gene expression profiling revealed primary and metastatic cells as two distinct cell populations defined by differential expression of 412 genes and of multiple pathways, including CADM1, SPHK1, and YAP/TAZ, whose expression independently predicted survival. In the metastatic subpopulations, a gene signature was defined (MET-75) that predicted survival of neuroblastoma patients with metastatic disease. Mechanistic investigations demonstrated causal roles for CADM1, SPHK1, and YAP/TAZ in mediating metastatic phenotypes in vitro and in vivo Notably, pharmacologic targeting of SPHK1 or YAP/TAZ was sufficient to inhibit neuroblastoma metastasis in vivo Overall, we identify gene expression signatures and candidate therapeutics that could improve the treatment of metastatic neuroblastoma. Cancer Res; 77(3); 696-706. ©2017 AACR.

Transcript signatures that predict outcome and identify targetable pathways in MYCN-amplified neuroblastoma.

BACKGROUND: In the pediatric cancer neuroblastoma (NB), patients are stratified into low, intermediate or high-risk subsets based in part on MYCN amplification status. While MYCN amplification in general predicts unfavorable outcome, no clinical or genomic factors have been identified that predict outcome within these cohorts of high-risk patients. In particular, it is currently not possible at diagnosis to determine which high-risk neuroblastoma patients will ultimately fail upfront therapy. EXPERIMENTAL DESIGN: We analyzed the prognostic potential of most published gene expression signatures for NB and developed a new prognostic signature to predict outcome for patients with MYCN amplification. Network and pathway analyses identified candidate therapeutic targets for this MYCN-amplified patient subset with poor outcome. RESULTS: Most signatures have a high capacity to predict outcome of unselected NB patients. However, the majority of published signatures, as well as most randomly generated signatures, are highly confounded by MYCN amplification, and fail to predict outcome in subpopulations of high-risk patients with MYCN-amplified NB. We identify a MYCN module signature that predicts patient outcome for those with MYCN-amplified tumors, that also predicts potential tractable therapeutic signaling pathways and targets including the DNA repair enzyme Poly [ADP-ribose] polymerase 1 (PARP1). CONCLUSION: Many prognostic signatures for NB are confounded by MYCN amplification and fail to predict outcome for the subset of high-risk patients with MYCN amplification. We report a MYCN module signature that is associated with distinct patient outcomes, and predicts candidate therapeutic targets in DNA repair pathways, including PARP1 in MYCN-amplified NB.