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Fanconi anemia (FA) is a complex genetic disorder characterized by congenital abnormalities, bone marrow failure, and myeloid malignancies. Identification of 13 FA genes has been instrumental to explore gene transfer technologies aimed at correction of autologous FA deficient stem cells. To date, three human FA stem cell gene therapy trials with standard 4-day transduction protocols using gammaretroviral vectors failed to provide clinical benefit. In addition, 2-4 day ex vivo manipulation of bone marrow from mice containing a disruption of the homologue of human FANCC (Fancc) results in a time-dependent increase in apoptosis and a risk for malignant transformation of hematopoietic cells. Here, we show that a 14-hour transduction period allows a foamyviral vector construct expressing the human FANCC cDNA to efficiently transduce murine FA stem cells with 1-2 proviral integrations per genome. Functionally, the repopulating activity of Fancc-/- stem cells from reconstituted mice expressing the recombinant FANCC transgene was comparable to wildtype controls. Collectively, these data provide evidence that short term transduction of c-kit+ cells with a foamyviral vector is sufficient for functional correction of a stem cell phenotype in a murine FA model. These data could have implications for future gene therapy trials for FA patients.

In an ENU mutagenesis screen using Mpl^-/- mice, we isolated a semi-dominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional co-regulator p300 and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl^-/- p300^Plt6/+ mice displayed elevated platelet counts relative to Mpl^-/- p300^+/+ controls, while mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300^Plt6 mutation, or recipients of Mpl^+/+ p300^Plt6/Plt6 bone marrow also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl^-/- thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial TPO-independent platelet production.

The mutagenic enzyme activation-induced cytidine deaminase (AID) is absolutely required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B-cells. Deregulated expression of AID is associated with various B-cell malignancies and currently, it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B-cells and there is limited evidence that this also occurs in normal blood B-cells. Importantly, the functional significance of these splice variants remains unknown. Here we show that normal GC human B-cells and blood memory B-cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B-cells as well as malignant B-cells from B-cell chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly, our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B-cells.

The malignant Hodgkin-/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (HL) are derived from mature B cells, but have lost a considerable part of the B cell-specific gene expression pattern. Consequences of such a lineage infidelity for lymphoma pathogenesis are currently not defined. Here, we report that HRS cells aberrantly express the common cytokine-receptor -chain (_c) cytokine IL-21, which is usually restricted to a subset of CD4⁺ T cells, and the corresponding IL-21 receptor. We demonstrate that IL-21 activates STAT3 in HRS cells, up-regulates STAT3 target genes, and protects HRS cells from CD95 death receptor-induced apoptosis. Furthermore, IL-21 is involved in up-regulation of the CC chemokine macrophage-inflammatory protein-3 (MIP-3) in HRS cells. MIP-3 in turn attracts CCR6⁺CD4⁺CD25⁺FoxP3⁺CD127^lo regulatory T cells towards HRS cells, which might favour their immune escape. Together, these data support the concept that aberrant expression of B lineage-inappropriate genes plays an important role for the biology of HL tumor cells.

During developmental hematopoiesis, multi-lineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multi-lineage hematopoietic potential. Furthermore, we have begun to define the molecular signals that regulate their development. We found that the formation of yolk sac hemogenic endothelium and its hematopoietic potential were significantly impaired in the absence of RA signaling, and could be restored in RA-deficient (Raldh2^-/-) embryos by provision of exogenous RA in utero. Thus, we identify a novel, critical role for retinoic acid (RA) signaling in the development of hemogenic endothelium that contributes to definitive hematopoiesis.

Specific inhibitors of PI3K-isoforms are currently evaluated for their therapeutic potential in leukemia. We found that bcr/abl-positive human leukemic cells express PI3K and therefore explored its impact on leukemia development. Using PI3K-deficient mice we define a dual role of PI3K in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in NK-cell-mediated tumor surveillance: Abelson-transformed PI3K-deficient cells induced leukemia in RAG2-deficient mice with an increased latency indicating that PI3K accelerated leukemia progression in vivo. However, the absence of PI3K also affected NK-cell-mediated tumor surveillance. PI3K-deficient NK-cells failed to lyse a large variety of target cells because of defective degranulation as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3K-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both - leukemic cells and NK-cells - lack PI3K. Other tumor models confirmed that PI3K-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3K in the NK-compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when employing PI3K-inhibitors as anti-leukemic agents in clinical trials.

Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplanted recipients. These observations suggested that previous conclusions of HSCs dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LV), we have investigated whether the LV-marking of mouse HSCs induces a competitive repopulation advantage in serially transplanted recipients. As deduced from analyses conducted in primary and secondary recipients we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By means of LAM-PCR analysis we characterized LV- targeted genes in HSC clones that engrafted up to quaternary recipients. While nine clones harboured integrations close to defined Retroviral Insertion Sites, none of them was characterized as a Common Integration Site, and none of them was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a mono or oligoclonal hematopoiesis in transplanted recipients.

Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder characterized by recurring fever episodes and results from disturbed isoprenoid biosynthesis. LPS-stimulated peripheral blood mononuclear cells (PBMC) from MKD patients secrete high levels of IL-1β, due to the presence of hyperactive caspase-1 and this has been proposed to be the primary cause of recurring inflammation. Here we show that inhibition of HMG-CoA reductase by simvastatin treatment, mimicking MKD, results in increased IL-1β secretion in a Rac1/PI3K-dependent manner. Simvastatin treatment was found to activate PKB/c-akt, a primary effector of PI3K, and ectopic expression of constitutively active PKB was sufficient to induce IL-1β release. The small GTPase Rac1 was also found to be activated by simvastatin and this was required for both PKB activation and IL-1β secretion. IL-1β release is dependent on processing by caspase-1, and simvastatin treatment resulted in increased caspase-1 activity in a Rac1/PI3K-dependent manner. These data suggest that in MKD, dysregulated isoprenoid biosynthesis activates Rac1/PI3K/PKB resulting in caspase-1 activation with increased IL-1β processing and release. Importantly, inhibition of Rac1 in PBMC isolated from MKD patients resulted in a dramatic reduction in IL-1β release. These data suggest that pharmacological inhibition of Rac1 could provide a novel therapeutic strategy for treatment of MKD.

Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered as an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the two daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal since accumulation of non muscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearing of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible of the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by non-ionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains two PDZ-motifs, binds PDZ domains 2^nd, 3^rd, and 5^th of FAP-1 (Fas-associated phosphatase 1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 co-immunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.