Adult humans have about 25 trillion red blood cells (RBCs), and each second we recycle about 5 million RBCs by erythrophagocytosis (EP) in macrophages of the reticuloendothelial system. Despite the ...central role for EP in mammalian iron metabolism, the molecules and pathways responsible for heme trafficking during EP remain unknown. Here, we show that the mammalian homolog of HRG1, a transmembrane heme permease in C. elegans, is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during EP. HRG1 is strongly expressed in macrophages of the reticuloendothelial system and specifically localizes to the phagolysosomal membranes during EP. Depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment. Importantly, missense polymorphisms in human HRG1 are defective in heme transport. Our results reveal HRG1 as the long-sought heme transporter for heme-iron recycling in macrophages and suggest that genetic variations in HRG1 could be modifiers of human iron metabolism.
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► Humans have about 25 trillion RBCs, each containing over a billion heme moieties ► Five million senescent RBCs are recycled per second by erythrophagocytosis in macrophages to make new RBCs ► HRG1, the mammalian homolog of C. elegans heme transporter, recycles heme-iron from the macrophage phagolysosome ► Missense polymorphisms in human HRG1 may be genetic modifiers of iron metabolism
Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6-NTRK3 gene fusion. However, a number of case reports have identified variant ...fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4-NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6-NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4-NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4-NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4-NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.
Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To ...determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34
lineage
hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.
A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles ...arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.
Pyogenic granuloma, also called lobular capillary hemangioma, is a condition usually occurring in skin or mucosa and often related to prior local trauma or pregnancy. However, the etiopathogenesis of ...pyogenic granuloma is poorly understood and whether pyogenic granuloma being a reactive process or a tumor is unknown. In an attempt to clarify this issue, we performed genome-wide transcriptional profiling of laser-captured vessels from pyogenic granuloma and from a richly vascularized tissue, placenta, as well as, from proliferative and involutive hemangiomas. Our study identified a gene signature specific to pyogenic granuloma. In the serial analysis of gene expression (SAGE) database, this signature was linked to 'white blood cells monocytes'. It also demonstrated high enrichment for gene ontology terms corresponding to 'vasculature development' and 'regulation of blood pressure'. This signature included genes of the nitric oxide pathway alongside genes related to hypoxia-induced angiogenesis and vascular injury, three conditions biologically interconnected. Finally, one of the genes specifically associated with pyogenic granuloma was FLT4, a tyrosine-kinase receptor related to pathological angiogenesis. All together, these data advocate for pyogenic granuloma to be a reactive lesion resulting from tissue injury, followed by an impaired wound healing response, during which vascular growth is driven by FLT4 and the nitric oxide pathway.
Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and ...therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.
Limited progress has been made in the treatment of advanced-stage pediatric solid tumors despite the accelerated pace of cancer discovery over the last decade. Tyrosine kinase inhibition is one ...tractable therapeutic modality for treating human malignancy. However, little is known about the kinases critical to the development or maintenance of many pediatric solid tumors such as Ewing sarcoma. Using a fluorescent, bead-based technology to profile activated tyrosine kinases, we identified focal adhesion kinase (FAK, PTK2) as a candidate target in Ewing sarcoma. FAK is a tyrosine kinase critical for cellular adhesion, growth, and survival. As such, it is a compelling target for cancer-based therapy. In this study, we have shown that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by short hairpin RNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, treatment of Ewing sarcoma cell lines with PF-562271 induced apoptosis and led to downregulation of AKT/mTOR and CAS activity. Finally, we showed that small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo. With FAK inhibitors currently in early-phase clinical trials for adult malignancies, these findings may bear immediate relevance to patients with Ewing sarcoma.
Infantile hemangiomas are characterized by rapid capillary growth during the first year of life followed by involution during early childhood. The natural history of these lesions creates a unique ...opportunity to study the changes in gene expression that occur in the vessels of these tumors as they proliferate and regress. Here we use laser capture microdissection and genome-wide transcriptional profiling of vessels from proliferating and involuting hemangiomas to identify differentially expressed genes. Relative to normal placental vessels, proliferating hemangiomas were characterized by increased expression of genes involved in endothelial-pericyte interactions, such as angiopoietin-2 ( ANGPT2 ), jagged-1 ( JAG1 ), and notch-4 ( NOTCH4 ), as well as genes involved in neural and vascular patterning, such as neuropilin-2 ( NETO2 ), a plexin domain containing receptor ( plexinC1 ), and an ephrin receptor ( EPHB3 ). Insulin-like growth factor binding protein-3 ( IGFBP3 ) was down-regulated in proliferating hemangiomas. Involuting hemangiomas were characterized by the expression of chronic inflammatory mediators, such as the chemokine, stromal cell-derived factor-1 ( SDF-1 ), and factors that may attenuate the angiogenic response, such as a member of the Down syndrome critical region ( DSCR ) family. The identification of genes differentially expressed in proliferating and involuting hemangiomas in vivo will contribute to our understanding of this vascular lesion, which remains a leading cause of morbidity in newborn children.
We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. ...First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling path-way are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.
Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP ...tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.