ALBERT

Description: Neurofibromatosis type 1 (NF1) is the most common genetic disorder with a predisposition to malignancy and affects 1 in 3500 persons worldwide. NF1 is caused by a mutation in the NF1 tumor suppressor gene that encodes the protein neurofibromin. Patients with NF1 have cutaneous, diffuse, and plexiform neurofibromas, tumors comprised primarily of Schwann cells, blood vessels, fibroblasts, and mast cells. Studies from human and murine models that closely recapitulate human plexiform neurofibroma formation indicate that tumorigenesis necessitates NF1 loss of heterozygosity in the Schwann cell. In addition, our most recent studies with bone marrow transplantation and pharmacologic experiments implicate haploinsufficiency of Nf1 (Nf1+/−) and c-kit signaling in the hematopoietic system as required and sufficient for tumor progression. Here, we review recent studies implicating the hematopoietic system in plexiform neurofibroma genesis, delineate the physiology of stem cell factor–dependent hematopoietic cells and their contribution to the neurofibroma microenvironment, and highlight the application of this research toward the first successful, targeted medical treatment of a patient with a nonresectable and debilitating neurofibroma. Finally, we emphasize the importance of the tumor microenvironment hypothesis, asserting that tumorigenic cells in the neurofibroma do not arise and grow in isolation.

Description: Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1282 Ezrin is a member of the ERM (ezrin, moesin and radixin) protein family that links plasma membrane proteins to the actin cytoskeleton. Ezrin in other in vitro cell systems has been hypothesized to participate in cell-cell contact and could have a role in stem/ progenitor cell mobilization and adhesion. To test this hypothesis, we crossed ezrinflox/flox mice with Mx1 cre transgenic mice to generate an inducible ezrin knock out mouse model. Inducible disruption of the ezrin gene in hematopoietic cells was achieved by the administration of polyIC. Ezrin knock out HSPCs exhibited a 30–40% decrease in baseline and chemokine stromal cell-derived factor-1 (SDF-1) stimulated motility in transwell migration assays in vitro. In addition, loss of ezrin led to a 60% decrease in the homing capacity of HSPCs in lethally irradiated recipient mice following transplantation. There was a 40–55% decrease in colony forming cells in peripheral blood and spleen of the mice following ezrin knock out, suggesting that ezrin knock out HSPCs may be deficient in egressing out of the bone marrow. To further understand the cause of the impaired motility of ezrin knock out HSPCs, we examined F-actin level of HSPCs at baseline and in response to SDF-1. Ezrin knock out HSPCs displayed 1.5 to 2 fold higher level of F-actin at baseline when compared with wild type cells. Following stimulation with SDF-1, wild type HSPCs that migrated to the bottom compartment of the transwell demonstrated a 2 time greater decrease in F-actin level when compared with ezrin knock out cells, suggesting that ezrin may participate in the regulation of F-actin depolymerization in HSPCs. In summary, we demonstrate that ezrin modulates HSPC migration and homing likely through its regulation on F-actin organization. Disclosures: No relevant conflicts of interest to declare.

Description: Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc−/−;Fancg−/− mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.

Description: Abstract 18 Eosinophils are increasingly recognized as important myeloid effector cells in the inflammatory environment of many human diseases. Although eosinophils critically contribute to chronic asthmatic inflammation, few therapies directly target these cells. Eosinophils rapidly migrate to eotaxin elicited by allergic sensitization and challenge, a chemokine that ligates the CCR3 receptor. Eotaxin:CCR3 signaling critically regulates allergen-induced eosinophil infiltration in murine models by activating the Rho-family proteins. In several cell systems, the Rho proteins Rac and CDC42 activate p21-activated kinase 1 (PAK1), which we have previously shown to regulate F-actin dynamics and histamine release in the degranulating mast cell. In these studies, we examined eotaxin-induced eosinophil migration using genetic and hematopoietic ablation of Pak1 (Pak1−/−) in a murine asthma model. Using an in vitro transwell migration assay system, we evaluated the migration of bone marrow derived eosinophils of both genotypes to eotaxin (N=10). Pak1−/− eosinophils exhibited profoundly diminished eotaxin-induced chemotaxis in vitro relative to wild-type (Pak1+/+) eosinophils (p 〈 0.0001) with a 30% overall decrease in migrating Pak1−/− compared to Pak1+/+ eosinophils. Furthermore, we compared the eotaxin-induced localization and arrangement of F-actin in eosinophils of both genotypes by fluorescence cytometry and deconvolution confocal microscopy of fluorescently-tagged phalloidin in seeking to explain this migration defect. Preliminary findings suggest decreased F-actin polymerization in eotaxin-treated Pak1−/− eosinophils. In an independent line of experiments designed to compare eotaxin-mediated eosinophil recruitment in vivo we injected mice of both genotypes with an intraperitoneal dose of eotaxin or saline. Pak1+/+ mice showed an 8 fold eotaxin-mediated increase in eosinophil recruitment over control whereas Pak1−/− mice demonstrated only a modest 3–4 fold increase (p〈 0.05). Finally we pursued PAK1's function in an experimental disease model in which the eosinophil's key role in pathogenesis is well documented. In 3 cohorts of 7 age, gender and strain matched Pak1+/+ and Pak1−/− ova albumin (OVA)-sensitized and challenged mice, we scored lung eosinophilic inflammation by histology and compared eosinophil counts and eotaxin concentrations in broncho-alveolar lavage fluid (BALF) by fluorescence cytometry and ELISA respectively. We also assessed OVA-specific T-cell subset cytokine secretion in our asthma mice by ELISA. Lung-parenchymal eosinophilic inflammation was diminished in Pak1−/− ova-sensitized mice versus Pak1+/+'s (p

Description: Fanconi anemia (FA) is a heterogenous genome instability syndrome with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair. However, it is incompletely understood why FA-deficient cells also develop gross aneuploidy and multinucleation, which are symptoms of error-prone chromosome segregation. Emerging evidence indicates that the FA signaling network functions as a guardian of the genome throughout the cell cycle, including chromosome segregation during mitosis. However, the mechanistic aspects of the critical role of the FA signaling in mitosis remain poorly understood. We have recently shown that the FA signaling network localizes to the mitotic apparatus to control the spindle assembly checkpoint and centrosome maintenance (J Clin Invest 2013, in press). The spindle assembly checkpoint (SAC) is a complex tumor suppressor signaling network that prevents premature separation of sister chromatids by delaying the metaphase-to-anaphase transition until all the kinetochores are properly attached to the mitotic spindle. Since weakened SAC promotes stochastic chromosome segregation, mutagenesis and cancer, these findings shed new light on the role of FA signaling in maintenance of genomic stability. We found the subcellular localization of FA proteins to the mitotic apparatus is spatiotemporally regulated as cells divide. Our new data revealed the pathways connecting the FANCA protein with canonical mitotic phosphosignaling networks. We have employed unbiased kinome-wide phospho-mass spectrometry to compare the landscape of abnormalities of mitotic signaling pathways in primary FANCA-/- patient cells and gene-corrected isogenic cells. These experiments led us to identify and quantify a wide range of phosphorylation abnormalities of multiple FANCA-dependent centrosome-, kinetochore- and chromosome-associated regulators of mitosis. Our data illuminated the role for FA signaling in three critical stages of cell division: (1) the spindle assembly checkpoint, (2) anaphase and (3) cytokinesis. Thus, we employed live phase-contrast imaging of primary FANCA-/- patient cells in comparison to gene-corrected cells to separately quantify aberrations in (1) chromosome congression and metaphase-anaphase transition (SAC malfunction), (2) execution of anaphase and (3) completion of cytokinesis. Our findings further our understanding of human cell cycle control and provide new insights into the origins of genomic instability in Fanconi anemia by establishing mechanistic connection between the FANCA protein and key mitotic signaling networks. The identification of cell division pathways regulated by FANCA has implications for future targeted drug development in Fanconi anemia and FA-deficient malignancies in the general population. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4178 Fanconi anemia (FA) is a rare recessive disease manifested by progressive bone marrow (BM) failure, congenital abnormalities and a predisposition to cancer. The only curative treatment for the pancytopenia to date is allogeneic stem cell transplantation. However, because allogeneic grafts are available to only about 30% of patients and mismatch grafts have a higher incidence of sequelae, autologous gene transfer into hematopoietic stem cells (HSCs) is a potential viable therapy. Major obstacles for gene therapy in FA include: low numbers of HSCs, an increased frequency of apoptosis and propensity for clonal selection following prolonged culture and the risks of all gene transfer technology including transactivation of proto-oncogenes from potent viral promoters such as the spleen focus-forming virus promoter (SFFV). We have recently found that a lentiviral (LV) vector containing SFFV promotor rendered a high functional and biochemical expression of the transgene in human and murine repopulating HSCs. However, given the recent concerns in the use of potent viral promoters like SFFV, in this study we developed LV vectors harboring a mammalian cell endogenous promoter human phosphoglycerate kinase (hPGK) to drive the expression of human FANCA and tested the ability of this construct to correct the phenotypes of human FANCA−/− fibroblasts and murine Fanca−/− hematopoietic stem and progenitor cells. Viral-mediated functional correction of FANCA was first evidenced by the restoration of FANCD2 mono-ubiquitination and correction of mitomycin C (MMC)-induced G2/M cell cycle arrest in human FANCA−/− fibroblast cells. In addition, the survival of genetically modified murine Fanca−/− clonogenic progenitors in the presence of 5nM MMC was improved from 30% (transduced with EGFP reporter) to 80% (transduced with FANCA cDNA), nearly equivalent to the survival of WT progenitors (90%) at that concentration. In addition, in a competitive repopulation assay, the long-term repopulating ability of Fanca−/− stem cells in the lethally-irradiated recipient mice was dramatically increased from 10% test cell chimerism (transduced with EGFP reporter) to 55% test cell chimerism (transduced with FANCA cDNA). Furthermore, the chimerism was maintained over 6 months in the primary recipients and an additional 6 months in the secondary recipients. Finally, resistance to MMC was retained in progenitors cultured from FACS sorted bone marrow test cells from the primary and secondary recipients. Collectively, these data suggest that the lentiviral construct using a mammalian cell endogenous promoter (hPGK) is sufficient to correct the phenotypes of murine FA HSC/progenitor cells. Studies are now ongoing to examine LV mediated FANCA expression in human primitive progenitor/ SCID/NOD repopulating cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 704 Neurofibromatosis type 1 predisposes individuals to the development of juvenile myelomonocytic leukemia, a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene which encodes a Ras GTPase activating protein, results in hyperactivity of Raf-Mek-Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with plethoric mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2's differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek-Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption does not grossly compromise myelopoiesis, dual Erk1/2 ablation rapidly ablates granulocyte and monocyte production, diminishes progenitor cell number, and prevents HSPC proliferation. Intriguingly, genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre+Nf1flox/floxErk1−/−Erk2flox/flox) fully protects against the genesis of MPD. Collectively, we identify a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly-selective Mek-Erk inhibitors for the attenuation of a leukemia that is otherwise resistant to traditional therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Fancc suppresses cross-linker–induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)–deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.