ALBERT

Description: Von Recklinghausen’s neurofibromatosis type 1 (NF1) is the most common genetic disease in man with a predisposition to cancer, a disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase for p21ras. Affected individuals are predisposed to plexiform neurofibromas, congenital tumors that arise from cranial and peripheral nerves for which no effective therapies are available. We have recently utilized a genetically engineered murine model to demonstrate that mast cells and the c-kit/kitL pathway have a key role in plexiform neurofibroma formation. In the present study, we utilized this model to determine whether imatinib mesylate, an FDA approved drug that targets the c-kit ligand in mast cells and other cell lineages in the tumor microenvironment in inducing tumor regression. Krox20; Nf1flox/− mice begin to develop plexiform neurofibromas by 6 months of age, especially in the dorsal root ganglia involving the sciatic nerve. Fluoridinated PET/CT images were utilized to verify that experimental mice had developed plexiform neurofibromas and to evaluate tumor progression. Following a 12 week treatment, Krox20; Nf1flox/− mice who received the vehicle had an increase in FDG uptake in the tumor growth area. In contrast, mice received imatinib mesylate had a dramatic reduction in the FDG uptake. At autopsy, the spinal cords were examined and sections were prepared for histologic examination. Grossly, tumors from the vehicle control treated mice were consistent with plexiform neurofibromas, as previously described (Zhu, Science 2002). In contrast, dissection of the spinal cords from Gleevec treated mice revealed that the mean volume of the tumor was significantly reduced. In addition, though there is still hyperplasia observed in the dorsal root ganglia from imatinib mesylate treated mice, the overall architecture of the nerve is improved as compared to the hematoxylin and eosin stained sections from the placebo treated control. Serial sections from 10 tumors isolated from placebo treated mice and serial sections from all dorsal root ganglia in the FDG positive affected regions of imatinib mesylate treated sections were scored. There is variability in the number of mast cells from the placebo treated tumors. However, there is a consistent, significant reduction in the number of mast cells scored in the dorsal root ganglia from the imatinib mesylate treated mice, suggesting that imatinib mesylate is inhibiting the c-kit receptor kinase and thus reducing mast cell numbers in the neurofibroma. Finally, since there are anatomic reductions in tumor size of imatinib mesylate treated mice as compared to placebo control treated animals, we hypothesized that the FDG affected regions would have an increase in apoptosis as compared to the amount of apoptosis observed in tumors from placebo treated mice. While only rare TUNEL positive cells were observed in tumors treated with the placebo, up to 10% of cells were TUNEL positive in tumors received imatinib mesylate. Collectively, these studies provide direct evidence that targeting the microenvironment induces regression of a plexiform neurofibromas and provides the basis for conducting clinical trials for a tumor that affects approximately 10–15 thousand patients in the US alone.

Description: The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade and the Ras-PI3-K-Akt pathways are intricately regulated and evolutionarily conserved pathways that have been implicated in specialized cellular functions including proliferation, differentiation, survival and degranulation. Recent data suggest that the strength and duration of these signals is maintained by extracellular growth factors and integrin stimuli as well as intracellular protein scaffolds. In the present study, we investigated the role of Kinase suppressor of Ras (KSR), a scaffold that appears to regulate both Ras-Erk and Ras-PI3-K activity in influencing mast cell function. In vivo, KSR−/− mice have a 2–3 fold reduction of resident mast cells in multiple organs including the peritoneum and the skin as evaluated by scoring Alcian blue positive cells. To evaluate the mechanistic underpinnings of these in vivo observations, bone marrow derived mast cells (BMMCs) were generated and proliferation, survival, degranulation, and migration was examined. A 3–4 fold reduction in kit-ligand mediated proliferation as measured by [3H]thymidine incorporation was observed in KSR−/− BMMCs as compared to WT BMMCs. In addition, a 50% increase in apoptosis was observed in KSR−/− mast cells as compared to that in WT cells as measured by flow cytometeric analysis using Annexin/PI staining. Given that Erk and Akt are established molecular targets control proliferation and survival, respectively; we next performed western blots to evaluate if the changes in biological activity was associated with these signaling pathways. Importantly, a reduction in phosphorylation of ERK and phosphorylation of AKT was observed in the KSR −/− BMMCs as compared to that in WT BMMCs. Given the role of PI3-K signals in mediating cytoskeletal organization in mast cells, we next tested whether the reduction in PI3-K signals was associated with a reduction in degranulation and migration. Following stimulation with kit-ligand and cross-linking of the IgE receptor, KSR−/− mast cells were found to have a 30–50% decrease in b-hexosaminidase release. Moreover, KSR−/− mast cells have up to a 5 fold reduction in migration to kit-ligand as measured over a range of kit-ligand concentrations. Collectively, the in vivo and in vitro studies suggest that KSR is an important regulatory kinase that may be a viable molecular target for modulating inflammatory mast cell functions.

Description: Interactions between tumorigenic cells and the microenvironment are increasingly recognized as integral to tumor progression in a range of human malignancies. However, the specific cellular mechanisms that are required to initiate these multistage processes are incompletely understood. Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1, a pandemic autosomal dominant genetic disorder of the nervous system characterized by the development of neurofibromas. Neurofibromas are complex tumors composed of Schwann cells, fibroblasts, endothelial cells, and high concentrations of degranulating mast cells. Though neurofibromas are generally benign, plexiform neurofibromas can progress to malignancy. Genetic studies in cre/lox mice indicate that nullizygous loss of Nf1 in the tumorigenic Schwann cells (Krox20; Nf1flox/flox) is necessary, but not sufficient for neurofibroma formation when the microenvironment is wildtype. However, neurofibromas form with 100% penetrance in Krox20; Nf1flox/− mice that are heterozygous at Nf1 in all lineages of the tumor microenvironment (Science, 2002). Here, we addressed the role of the hematopoietic system in the tumor microenvironment by using adoptive transfer. Syngeneic Nf1+/− or wildtype (WT) bone marrow was transplanted into lethally irradiated Krox20;Nf1flox/flox mice. Krox20; Nf1flox/flox recipients transplanted with WT bone marrow (n=25) did not develop plexiform neurofibromas and had a normal lifespan. In contrast, Krox20; Nf1flox/flox mice transplanted with Nf1+/− bone marrow (n=25) consistently developed neurofibromas infiltrated with Nf1+/− mast cells. These mice had a 90% mortality at 14 months following transplantation. In complementary experiments, WT bone marrow was transplanted into irradiated Krox20; Nf1flox/− mice. Despite the remainder of the tumor microenvironment being heterozygous, WT bone marrow was sufficient to prevent tumor progression in Krox20; Nf1flox/− mice. To specifically assess the role of the mast cell compartment in tumor progression, Nf1+/− mice were intercrossed with two strains of naturally occurring W mutant mice that have variably diminished c-kit activity and mast cell function. Mice homozygous at the Wv locus have a greater than 90% reduction in c-kit activity, while W41/W41 mutants have approximately a 65–75% reduction in c-kit activity. Importantly, while Krox20;Nf1flox/flox mice transplanted with Nf1+/− bone marrow consistently develop plexiform neurofibromas, adoptive transfer of Nf1+/−; Wv/Wv or Nf1+/−; W41/W41 bone marrow cells into Krox20; Nf1flox/flox mice was sufficient to prevent neurofibroma formation. Collectively, these studies provide genetic evidence that the hematopoietic system and specifically mast cells are integral to plexiform neurofibroma formation in genetically engineered mice. These studies have therapeutic implications for NF1 since molecular therapies directed at the haploinsufficient hematopoietic cells, particularly the c-kit receptor tyrosine kinase, may have an important role in treating or preventing plexiform neurofibromas.