ALBERT

Description: Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function of the tumor suppressor gene, NF1. 50% of NF1 patients have osseous manifestations, including short stature, scoliosis, and reduced bone mineral density. Osteoclasts are hematopoietic stem cell-derived cells that function to resorb bone. We recently reported that osteoclasts derived from NF1 patients and Nf1 heterozygous (Nf1+/−) mice have elevated migration, adhesion, and bone resorption and our studies indicate that the gain-of-function of the Nf1+/− osteoclasts is, at least in part, caused by hyperactivation of macrophage colony-stimulating factor (M-CSF)-stimulated Ras, phosphoinositol-3-kinase (PI3K), and Erk. Rho GTPases function downstream of Ras and PI3K and act as binary switches, cycling between an inactive (GDP-bound) and active (GTP-bound) state, to regulate osteoclast actin ring formation, bone resorption, and development of filamentous actin structures associated with migration and adhesion. We hypothesized that hyperactivation of Rac1, Rac2, or both Rac1 and Rac2 contribute to the increased osteoclast function observed in Nf1+/− mice and NF1 patients. To examine this hypothesis, we intercrossed Nf1+/− mice with conditional Rac1flox/floxMxcre+ mice or with Rac2−/− mice to generate WT, Nf1+/−, Rac1−/−, Nf1+/−;Rac1−/−, Rac2−/−, and Nf1+/−;Rac2−/− mice. Genetic disruption of Rac1, but not of Rac2, restored the increased colony forming unit-macrophage (CFU-M), tatrate resistant acid phosphate+ (TRAP+) CFU-M, osteoclast migration, and bone resorption observed in Nf1+/− cultures. Osteoclast bone resorbing capacity is dependent on the organization of the actin cytoskeleton into a large f-actin-rich structure referred to as the sealing zone. The podosome belt evolves into the sealing zone in actively resorbing osteoclasts. A significantly higher level of belt formation, seen in mature osteoclasts, was observed in Nf1+/− cultures as compared to WT. Upon genetic deletion of Rac1, the Nf1+/−;Rac1−/− osteoclasts demonstrated belt formation at a similar level to that of WT osteoclasts. These data indicate that Rac1 plays an essential role in functional f-actin organization and suggest that inhibition of Rac1 in the setting of Nf1 haploinsufficiency is able to normalize osteoclast hyperactivity by correcting the cytoskeletal organization of f-actin-based structures. Mechanistically, Rac1 deficiency normalized M-CSF-stimulated phospho-Erk and phospho-Akt and pharmacologic inhibition of MEK and PI3K using PD98059 or Ly294002, respectively, normalized Nf1+/− osteoclast development and maturation. The critical role of Rac1, but not of Rac2, in osteoclast function is significant as it suggests that the Rac GTPases contribute non-redundant functions in various myeloid cell types and imply that blocking Rac1 function, while sparing that of Rac2, may provide a level of specificity to therapeutics for skeletal diseases. Collectively, these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1 and imply that Rac1 may be a rational therapeutic target for dysplastic and erosive bone diseases.

Description: Fanconi Anemia (FA) is an autosomal recessive DNA repair disorder clinically characterized by congenital abnormalities, progressive bone marrow (BM) failure and a propensity for malignancies. It is advantageous to bank FA patients’ cells for autologous treatment of pancytopenia, transplantation following chemotherapy or future gene therapy trials. Because the amount of cells able to be collected by BM harvesting is limited and the process is often problematic in children, mobilization of stem cells into the circulation would be an ideal means of collecting repopulating hematopoietic stem and progenitor cells (HSPC). Traditional mobilization protocols utilizing granulocyte colony-stimulating factor (G-CSF) as a mobilizing agent result in poor yields of phenotypic HSPC in FA patients. We have shown that analogous to the human patients with FA, administration of recombinant G-CSF is not sufficient to effectively mobilize myeloid progenitors from the BM of Fancc −/− mice (Broxmeyer et al, JEM, 2005). Antagonists to chemokine receptors, together with G-CSF, can enhance mobilization of murine wild type (WT) HSPC. AMD3100 is a selective antagonist of CXCR4, which is thought to function in the retention of HSPC in the BM. We hypothesized that the combination of AMD3100 and G-CSF would enhance mobilization of HSPC in Fanca −/− and Fancc −/− mice. Peripheral blood was collected to assess the number of myeloid progenitors and HSPC repopulating ability. Myeloid progenitor numbers were assessed using progenitor assays. Either G-CSF or AMD3100 alone resulted in poor mobilization of hematopoietic progenitors. In contrast, administration of AMD3100 and G-CSF together increased the number of colonies of myeloid progenitors up to 18 fold above that of either G-CSF or AMD3100 alone in both Fanca −/− and Fancc −/− mice. The stem cell activity of mobilized peripheral blood cells was evaluated using competitive repopulation assays, in which a common pool of isogenic ‘competitor cells’ and ‘test cells’ from mice of different genotypes or treatment conditions are co-transplanted. Fanca −/− and Fancc −/− cells mobilized with both G-CSF and AMD3100 yielded a 3–4 fold increase in peripheral blood chimerism as compared to the cells mobilized with G-CSF or AMD3100 alone. The latter mice also had a decreased survival. Phenotypic analysis of the peripheral blood of primary recipients confirmed the engraftment of multi-lineage test cells. Transplantation of chimeric bone marrow into secondary recipients maintained the test cell chimerism, confirming that long-term repopulating cells were mobilized. Collectively, these data from two FA murine models demonstrate that the AMD3100 and G-CSF protocol is an effective strategy to mobilize FA deficient repopulating HSPC. This has potential implications for more efficient banking of FA patients’ HSPC for future use or for use in gene transfer protocols.

Description: Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML) associated with loss of the normal NF1 allele. Genetically engineered mice containing nullizygous loss of Nf1 in the hematopoietic system develop a leukemia with complete penetrance that is similar to JMML, including hepatosplenomegaly, elevated peripheral blood cell counts, and elevated numbers of myeloid progenitors that are hypersensitive to multiple cytokines, particularly GM-CSF. Though we and others have found that p21ras is hyperactivated in Nf1−/− myeloid progenitors, inhibiting Ras specifically as a therapeutic target has been challenging. Therefore, identification of alterations in distinct p21ras effector pathways that control leukemia progression in Nf1-deficient cells is critical for understanding disease pathogenesis and identifying therapeutic targets. Here we intercrossed MxCre; Nf1flox/flox mice with mice that are deficient in the small Rho GTPases Rac1 or Rac2 to generate syngeneic progeny that were MxCre; Nf1flox/flox, MxCre; Nf1flox/flox;Rac1flox flox or MxCre; Nf1flox/flox; Rac2 −/−. Consistent with previous studies (Le, Blood 2004) MxCre; Nf1flox/flox mice develop a progressive myeloproliferative disease with 100% penetrance 6 months following interferon inducible induction of the MxCre transgene to disrupt the Nf1flox alleles. Eighty percent of MxCre; Nf1flox/flox die by 9 months after inactivation. Genetic disruption of Rac2 was not sufficient to diminish the onset or severity of the characteristic myeloproliferative disease of MxCre; Nf1flox/flox mice. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice followed for 11 months all survived and had normal bone marrow cellularity, spleen weight and splenic architecture. MxCre; Nf1flox/flox mice have elevated numbers of both HPP-CFC and LPP-CFC in the bone marrow and spleen. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice had myeloid progenitor numbers that were comparable to wildtype, age-matched controls. Further, though myeloid progenitors from MxCre; Nf1flox/flox mice are hypersensitive to GM-CSF, myeloid progenitors from MxCre; Nf1flox/flox; Rac1flox/flox have a sensitivity to GM-CSF comparable to wildtype controls. The correction in the myeloproliferative phenotype isolated from MxCre; Nf1flox/flox; Rac1flox/flox mice was associated with the reduction in Rac-GTP and prolonged Erk phosphorylation, a MAPK effector that is characteristically elevated in Nf1−/− myeloid progenitors. Collectively, these genetic data identify Ras-Rac1 signaling pathway as a key axis in the genesis of juvenile myelomonocytic leukemia and provide evidence that Rac1 is a therapeutic molecular target for this myeloproliferative disease that currently has no effective therapies.

Description: Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a GTPase activating protein for Ras called neurofibromin. NF1 is a genetic disorder that affects approximately 250,000 individuals in the US, Europe, and Japan alone. Neurofibromas, the hallmark of NF1, are complex tumors characterized by tumorigenic Schwann cells, neoangiogenesis, fibrosis, and degranulating mast cells. Studies in experimental models have emphasized the role of inflammatory cells in altering the microenvironment and facilitating malignant outgrowth. Similarly, Parada (Science, 2002) found that nullizygosity of Nf1 in Schwann cells of conditional knockout mice (Krox20;Nf1flox/flox) was necessary but not sufficient for neurofibroma formation and haploinsufficiency of Nf1 in lineages within the tumor microenvironment was required for neurofibroma progression. We previously provided the first genetic, cellular, and biochemical evidence that haploinsufficiency of Nf1 alters Ras activity and cell fates in mast cells (JEM, 2000, 2001) and identified a mechanism underlying the recruitment of mast cells to tumorigenic Schwann cells (JCI 2003). However, it remains unclear whether Nf1 +/− bone marrow derived hematopoietic cells can directly contribute to neurofibroma formation in vivo. To address this question, Nf1+/− or wildtype (WT) EGFP+ bone marrow (BM) was adoptively transferred into lethally irradiated Krox20;Nf1flox/flox mice and cohorts were followed prospectively for tumor formation using positron emission tomography and computerized axial tomography. Mice transplanted with Nf1+/− but not WT BM developed progressive enlargement of the trigeminal nerve, dorsal root ganglia, peripheral nerves, and motor paralysis similar to Krox20;Nf1flox/− mice that are haploinsufficient at Nf1 in all lineages of the tumor microenvironment. Postmortem analysis revealed that Krox20;Nf1flox/flox mice transplanted with Nf1+/− BM had cellular neurofibromas containing Schwann cells, fibroblasts, blood vessels and mast cells, which closely resembled the cellular architecture of human neurofibromas. Mice transplanted with WT BM did not develop neurofibromas. These studies establish that recruitment of Nf1 +/− BM derived cells to the neurofibroma microenvironment is directly linked to neurofibroma formation and progression. Given our observations, therapies which prevent both the recruitment and the tumor promoting functions of Nf1 +/− hematopoietic cells in neurofibroma formation are currently being tested in vivo as pre-clinical trials.

Description: Abstract SCI-33 Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Neurofibromas form in association with peripheral nerves and are composed of Schwann cells, blood vessels, fibroblasts and degranulating mast cells. Genetic studies in engineered mice indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation in a genetically engineered murine model and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Recent studies in our group have focused on evaluating the role of bone marrow-derived cells, and particularly mast cells, in the tumor microenvironment. Previous work by our group established that human and murine Nf1 deficient Schwann cells secrete high concentrations of kit-ligand. Kit-ligand has a central role in multiple mast cell functions including chemotaxis, proliferation and degranulation. In a series of bone marrow transplantation studies, we established that Nf1 haploinsufficiency in bone marrow is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling diminishes neurofibroma initiation and progression. Collectively, the studies implicate mast cells as active participants in tumor formation and identify therapeutic targets for human phase 1-2 clinical trials. Disclosures Off Label Use: The drug imatinib mesylate was used to treat plexiform neurofibromas in genetically engineered mice, in a child with a plexiform neurofibroma, and it is currently being tested in a phase 2 clinical trial.

Description: Abstract 3629 Poster Board III-565 Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients. Disclosures: No relevant conflicts of interest to declare.

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.

Description: Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase activating protein for Ras. Though nullizygous loss of NF1 is associated with the development of malignancies, haploinsufficient phenotypes are now being increasingly recognized to alter cell fates and functions in a number of tissues resulting in nonmalignant disease manifestations. Bone manifestations, including skeletal dysplasias, scoliosis, and osteoporosis occur in 30–60% of all NF1 patients and osteoporosis is an increasingly recognized health problem for women with NF1. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone resorbing cells of the skeleton. Using an established murine model of NF1 developed using homologous recombination, we found that Nf1+/− mice contain elevated numbers of multinucleated osteoclasts and osteoclast progenitors per femur in vivo. Both osteoclasts and osteoclast progenitors from Nf1+/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL, growth factors that are integral to osteoclast maturation and activation. M-CSF stimulated p21ras-GTP and Akt phosphorylation was elevated in Nf1+/− osteoclasts associated with gains in function in survival and proliferation. Bone resorption by osteoclasts is linked to the migration and adherence of these cells to a local bone surface. Purified populations of Nf1+/− osteoclasts were initially placed in the upper chamber of a transwell coated with vitronectin and haptotaxis to M-CSF was determined. Nf1+/− osteoclasts had a 2–3 fold increase in migration as compared to syngeneic wildtype cells. A similar increase in adhesion of Nf1+/− osteoclasts to the integrin avb3 was also observed. Following adhesion, osteoclasts form a specialized cell-extracellular matrix to initiate degradation of bone matrix by secreting proteinases. Nf1+/− osteoclasts had significantly increased bone resorption as measured by scoring the number and area of individual bone resorbing “pits” on dentine slices and by scoring the total area of resorption. These collective increases in osteoclast function were validated in vivo by the observation that serum TRAP5b activity, a sensitive measure of osteoclast lytic activity was 2.5 fold higher in Nf1+/− mice as compared to WT mice. Furthermore, we performed ovariectomy, an established model of osteoporosis associated with an increase in osteoclast function. In two independent experiments, we found that Nf1+/− mice had a significantly greater reduction in bone mineral density following ovariectomy as compared to syngeneic wildtype mice. We hypothesized that hyperactivation of class1A-PI3-K may contribute to these gains in cellular function. We found that intercrossing Nf1+/− and Class1A PI3-K deficient mice (p85a) restores elevated PI3-K activity, and Nf1+/− osteoclast functions to wildtype levels. Furthermore, in vitro differentiated osteoclasts from NF1 patients also display elevated Ras-PI3-K activity and increased lytic activity analogous to murine Nf1+/− osteoclasts. Collectively, we identify a novel cellular and biochemical NF1 haploinsufficient phenotype in osteoclasts that has potential implications in the pathogenesis of NF1 bone disease.