Lu Q. Le

Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome

Mice with a targeted disruption of the gene encoding a lymphoid-expressed orphan G protein-coupled receptor, G2A, demonstrate a normal pattern of T and B lineage differentiation through young adulthood. As G2A-deficient animals age, they develop secondary lymphoid organ enlargement associated with abnormal expansion of both T and B lymphocytes. Older G2A-deficient mice (>1 year) develop a slowly progressive wasting syndrome, characterized by lymphocytic infiltration into various tissues, glomerular immune complex deposition, and anti-nuclear autoantibodies. G2A-deficient T cells are hyperresponsive to TCR stimulation, exhibiting enhanced proliferation and a lower threshold for activation. Our findings demonstrate that G2A plays a critical role in controlling peripheral lymphocyte homeostasis and that its ablation results in the development of a novel, late-onset autoimmune syndrome.

Tumor microenvironment and neurofibromatosis type I: connecting the GAPs

The human disease von Recklinghausens neurofibromatosis (Nf1) is one of the most common genetic disorders. It is caused by mutations in the NF1 tumor suppressor gene, which encodes a GTPase activating protein (GAP) that negatively regulates p21-RAS signaling. Dermal and plexiform neurofibromas as well as malignant peripheral nerve sheath tumors and other malignant tumors, are significant complications in Nf1. Neurofibromas are complex tumors and composed mainly of abnormal local cells including Schwann cells, endothelial cells, fibroblasts and additionally a large number of infiltrating inflammatory mast cells. Recent work has indicated a role for the microenvironment in plexiform neurofibroma genesis. The emerging evidence points to mast cells as crucial contributors to neurofibroma tumorigenesis. Therefore, further understanding of the molecular interactions between Schwann cells and their environment will provide tools to develop new therapies aimed at delaying or preventing tumor formation in Nf1 patients.

Cell of Origin and Microenvironment Contribution for NF1-Associated Dermal Neurofibromas

The tumor predisposition disorder neurofibromatosis type I (NF1) is one of the most common genetic disorders of the nervous system. It is caused by mutations in the Nf1 tumor-suppressor gene, which encodes a GTPase-activating protein (GAP) that negatively regulates p21-RAS. Development of malignant nerve tumors and neurofibromas occurs frequently in NF1. However, little is known about the molecular mechanisms mediating the initiation and progression of these complex tumors, or the identity of the specific cell type that gives rise to dermal or cutaneous neurofibromas. In this study, we identify a population of stem/progenitor cells residing in the dermis termed skin-derived precursors (SKPs) that, through loss of Nf1, form neurofibromas. We propose that SKPs, or their derivatives, are the cell of origin of dermal neurofibroma. We also provide evidence that additional signals from nonneoplastic cells in the tumor microenvironment play essential roles in neurofibromagenesis.

Genome-wide siRNA-based functional genomics of pigmentation identifies novel genes and pathways that impact melanogenesis in human cells.

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinsons disease), auditory disorders (Waardenburgs syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.

CXCR4/CXCL12 mediate autocrine cell- cycle progression in NF1-associated malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with neurofibromatosis type 1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G-protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in nontransformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell-cycle progression through PI3-kinase (PI3K) and β-catenin signaling. Suppression of CXCR4 activity either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development and identify a therapeutic target.

Positron emission tomography imaging analysis of G2A as a negative modifier of lymphoid leukemogenesis initiated by the BCR-ABL oncogene

G2A is a lymphocyte-expressed G protein-coupled receptor whose genetic ablation results in the development of autoimmunity. Using HSV-TK reporter gene directed positron emission tomography (PET), we demonstrate that prior to any indication of the onset of illness, mice transplanted with BCR-ABL transduced G2A-deficient bone marrow harbor expanded populations of leukemic cells compared to recipients of wild-type bone marrow. The target cell type and anatomical locations of leukemia development are indistinguishable in animals transplanted with G2A+/+ or G2A-/- cells. Shorter disease latency in the G2A-deficient background is associated with an increased rate of cellular expansion. PET can be successfully applied to the temporal and spatial analysis of Bcr-Abl driven leukemic progression and should have utility for the study of other leukemias and lymphomas.

Cells of Origin in the Embryonic Nerve Roots for NF1-Associated Plexiform Neurofibroma

Neurofibromatosis type 1 is a tumor-predisposing genetic disorder. Plexiform neurofibromas are common NF1 tumors carrying a risk of malignant transformation, which is typically fatal. Little is known about mechanisms mediating initiation and identity of specific cell type that gives rise to neurofibromas. Using cell-lineage tracing, we identify a population of GAP43(+) PLP(+) precursors in embryonic nerve roots as the cells of origin for these tumors and report a non-germline neurofibroma model for preclinical drug screening to identify effective therapies. The identity of the tumor cell of origin and facility for isolation and expansion provides fertile ground for continued analysis to define factors critical for neurofibromagenesis. It also provides unique approaches to develop therapies to prevent neurofibroma formation in NF1 patients.

Skin-derived Precursors as a Source of Progenitors for Cutaneous Nerve Regeneration†‡§

Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain, and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth‐permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells dedifferentiate to a progenitor/stem cell phenotype to promote axonal regrowth. In this study, we show that skin‐derived precursors (SKPs), a recently identified neural crest‐related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro three‐dimensional cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis. STEM Cells2012;30:2261–2270

Preclinical Therapeutic Efficacy of a Novel Pharmacologic Inducer of Apoptosis in Malignant Peripheral Nerve Sheath Tumors

Neurofibromatosis type I (NF1) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNST), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. Patients with MPNST may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically engineered mouse model of MPNST that recapitulates human NF1-associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining nontoxic to normally dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compounds potential as a novel chemotherapeutic agent.

A case of simultaneously occurring lichen sclerosus and segmental vitiligo: connecting the underlying autoimmune pathogenesis

A 41‐year‐old African‐American woman reported a 6‐month history of asymptomatic white macules and patches that started on her left foot, and then spread proximally up her leg to her thigh, and then to her buttocks. She also noted that several of her pubic hairs had turned white, but only on the left side of the midline. She also complained of concomitant severe burning and itching in the vaginal area and that her vaginal skin had turned whitish pink. The patient denied contact with industrial or hazardous chemicals, and her medical history included childhood iron deficiency anemia only.