Sarah Burgin

P21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W(sh)/W(sh) mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

Pak1 regulates multiple c-Kit mediated Ras-MAPK gain-in-function phenotypes in Nf1+/− mast cells

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 locus, which encodes neurofibromin, a negative regulator of Ras. Patients with NF1 develop numerous neurofibromas, which contain many inflammatory mast cells that contribute to tumor formation. Subsequent to c-Kit stimulation, signaling from Ras to Rac1/2 to the MAPK pathway appears to be responsible for multiple hyperactive mast cell phenotypes; however, the specific effectors that mediate these functions remain uncertain. p21-activated kinase 1 (Pak1) is a downstream mediator of Rac1/2 that has been implicated as a positive regulator of MAPK pathway members and is a modulator of cell growth and cytoskeletal dynamics. Using an intercross of Pak 1(-/-) mice with Nf1(+/-) mice, we determined that Pak1 regulates hyperactive Ras-dependent proliferation via a Pak1/Erk pathway, whereas a Pak1/p38 pathway is required for the increased migration in Nf1(+/-) mast cells. Furthermore, we confirmed that loss of Pak1 corrects the dermal accumulation of Nf1(+/-) mast cells in vivo to levels found in wild-type mice. Thus, Pak1 is a novel mast cell mediator that functions as a key node in the MAPK signaling network and potential therapeutic target in NF1 patients.

Nf1-/- Schwann cell-conditioned medium modulates mast cell degranulation by c-Kit-mediated hyperactivation of phosphatidylinositol 3-kinase

Neurofibromatosis type 1 (NF1) is a common genetic disorder and is characterized by both malignant and nonmalignant neurofibromas, which are composed of Schwann cells, degranulating mast cells, fibroblasts, and extracellular matrix. We and others have previously shown that hyperactivation of the c-Kit pathway in an Nf1 haploinsufficient microenvironment is required for both tumor formation and progression. Mast cells play a key role in both tumorigenesis and neoangiogenesis via the production of matrix metalloproteinases, heparin, and a range of different growth factors. In the present study, we show that tumorigenic Schwann cells derived from Nf1(-/-) embryos promote increased degranulation of Nf1(+/-) mast cells compared with wild-type mast cells via the secretion of the Kit ligand. Furthermore, we used genetic intercrosses as well as pharmacological agents to link the hyperactivation of the p21(Ras)-phosphatidylinositol 3-kinase (PI3K) pathway to the increased degranulation of Nf1(+/-) mast cells both in vitro and in vivo. These studies identify the p21(Ras)-PI3K pathway as a major regulator of the gain in Nf1(+/-) mast cell degranulation in neurofibromas. Collectively, these studies identify both c-Kit and PI3K as molecular targets that modulate mast cell functions in cases of NF1.

Kinase Suppressor of Ras (KSR1) modulates multiple Kit-ligand-dependent mast cell functions

The intricately regulated Ras pathway coordinates multiple kit-ligand-induced mast cell functions, including chemotaxis, proliferation, and degranulation. However, the intracellular proteins that modulate the intensity and duration of stem cell factor-induced signals and the consequent cellular response are incompletely understood. Scaffolding proteins coordinate the spatial organization of mitogen-activated protein kinase proteins that may potentiate and/or inhibit cell functions. The kinase suppressor of Ras (KSR1) protein is known to function as a molecular scaffold and coordinates the organization of Raf/Mek/Erk in response to receptor tyrosine kinases. However, the impact of KSR1 in myeloid mast cell functions and in response to stem cell factor remains unknown. In the present study, we investigated the role of KSR1 in regulating cellular functions of bone marrow-derived mast cells of KSR1-deficient ((-/-)) mice. Genetic disruption of KSR1 resulted in both striking reductions in kit-ligand-mediated proliferation and degranulation, which are commonly attributed to mitogen-activated protein kinase signals. Surprisingly, disruption of the KSR1 scaffold also resulted in a decline in migration that is generally not linked to Raf-Erk signals. We found that loss of KSR1 does impact the biochemical activation of p21-activated kinase, a kinase that is known to modulate Raf-Erk signals and also F-actin polymerization key to mast cell migration. Collectively, these studies demonstrate that the scaffolding protein KSR1 has an important role in multiple kit-ligand-mediated mast cell functions. This study elucidates varied mast cell physiological functions for KSR1, including those related to cytoskeletal organization, and it suggests a novel molecular target for attenuating mast cell-mediated inflammation.

Chondroblastoma of the sphenoid sinus

Chondroblastoma is a rare, benign chondroid tumor, responsible for less than one percent of all primary bone tumors. They typically occur in the long bones of the lower extremity, although they also uncommonly occur in the temporal bone, the most frequent head and neck site. There is one reported case of chondroblastoma occurring in the maxillary sinus. To our knowledge, there are no reports of chondroblastoma involving the sphenoid sinus. We present a case of a 30-year-old woman with a sphenoid sinus chondroblastoma. We obtained institutional review board approval from our institution in order to present this case.

Pathogenetic Analysis of Sinonasal Teratocarcinosarcomas Reveal Actionable β-catenin Overexpression and a β-catenin Mutation

Objective Sinonasal teratocarcinosarcomas are rare, aggressive tumors of the skull base. Treatment options are limited and outcomes are poor. Little is known in regard to the genetic factors regulating these tumors. Characterization of actionable molecular alterations in these tumors could provide potentially successful therapeutic options. Methods We performed targeted exome sequencing on an index sinonasal teratocarcinosarcoma specimen to identify potential driver mutations. We performed immunohistochemical stains for &bgr;‐catenin on paraffin‐embedded tissue on the index tumor and a subsequent teratocarcinosarcoma. Online databases of cancer mutations (Catalogue of Somatic Mutations in Cancer and The Cancer Genome Atlas) were accessed. Results We identified an activating p.S45F mutation in &bgr;‐catenin in our index sinonasal teratocarcinosarcoma. This mutation results in constitutive signaling in the Wnt/&bgr;‐catenin pathway. We confirmed &bgr;‐catenin overexpression and nuclear localization via immunohistochemistry in the index tumor and a second patient. The p.S45F activating mutation was found in a variety of solid tumors, and accounts for 3.3 to 10.4% of all known &bgr;‐catenin mutations. Conclusion We identified a potential driver mutation in &bgr;‐catenin in a sinonasal teratocarcinosarcoma, resulting in &bgr;‐catenin overexpression. These findings suggest a role for the Wnt/&bgr;‐catenin pathway in sinonasal teratocarcinosarcoma tumorigenesis and a role for anti‐&bgr;‐catenin targeted therapy.