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 phosphoinositide 3-kinase (PI3K)-AKT pathway is a relevant signal transduction axis which regulates survival, growth and proliferation of hematopoietic cells through a variety of downstream targets. Constitutive activation of the PI3K-AKT pathway is observed in up to 70% of acute myelogenous leukemia (AML), primarily due to activating mutations in receptor tyrosine kinases (e.g. c-KIT) or RAS and is also present in T-cell-non-Hodgkins-lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Hodgkins Lymphoma and myelodysplastic syndrome (MDS). We analyzed the effect of activating point mutations in the catalytic subunit p110alpha of class IA PI3K (PIK3CA) in hematopoietic cells. We transfected early hematopoietic, IL-3 dependent cells (Ba/F3 cell line) with point mutations in the helical (exon 9, E542K, E545A) and kinase domain (exon 20, H1047R) of the p110alpha catalytic subunit and determined the effect of PI3K-AKT pathway activation on cellular proliferation, survival and leukemogenic expansion in vitro and in vivo. We demonstrate that the p110alpha point mutations constitutively activated the PI3K-AKT pathway and resulted in factor-independent growth of Ba/F3 cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not anti-apoptotic signaling of mutant PIK3CA. In a syngenic mouse model, hematopoietic cells expressing mutant p110alpha induced a leukemia-like disease. FACS analysis demonstrated a median chimerism of 68% in the bone marrow and 35% in peripheral blood of diseased mice, which were characterized by anemia, leukemic infiltration of hematopoietic organs, and 90% mortality within 5 weeks. No substantial differences were observed between E542K, E545A and H1047R. Mice, which were transplanted with an activating c-KIT point mutation (D814V), showed a significantly reduced survival and 100% died within 9 days. In conclusion our data show, that PIK3CA point mutations, by activating the PI3K-AKT pathway, confer factor-independence to hematopoietic cells in vitro and induce leukemogenic activity in vivo. As mutant c-KIT (D814V) showed enhanced leukemogenic potential, signaling through c-KIT may involve additional downstream pathways which cooperate with activated PI3K in leukemia progression. Our model is useful for the differential investigation of the leukemogenic relevance of the PI3K-AKT pathway and pharmacological inhibitor studies.

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: Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.