Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

There is no benefit to the treatment of high-grade astrocytomas in children with eight-drugs-in-1-day chemotherapy compared with CCNU, vincristine, and prednisone. Extent of tumor resection and histopathologic diagnosis are significant prognostic variables. The overall outcome for children with high-grade astrocytomas remains poor.

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells underexpressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemiaassociated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Diamond Blackfan anemia (DBA)5 is a severe hypoplastic anemia that generally presents early in infancy (1, 2). Other clinical features of DBA are heterogeneous with some patients presenting craniofacial abnormalities, growth failure, predisposition to cancer, and other congenital abnormalities. Most cases of DBA arise spontaneously, with only a small proportion exhibiting familial transmission typically showing autosomal dominant inheritance. Approximately 25% of the DBA cases have been linked to mutations in the gene encoding ribosomal protein S19 (RPS19), and in these cases haploinsufficiency for this ribosomal protein gives rise to the disease (3, 4). The remaining cases are of unknown etiology.The Rps19 protein is a component of the small ribosomal subunit, and as such, defects in some aspect of ribosome structure, function, or synthesis may be an underlying cause of DBA. The human Rps19 protein belongs to a family of ribosomal proteins restricted to eukaryotes and archea. Rps19 does not have a homolog in the eubacterial ribosome where the properties of individual ribosomal proteins have been most extensively studied. As such, little is known regarding the function of members of the eukaryotic Rps19 family. In prokaryotes, ribosomal proteins play critical roles in ribosomal assembly through their interactions with each other and rRNA (5). These interactions promote both steps in rRNA processing important for subunit maturation and the formation of active sites in mature subunits necessary for ribosome function. The precise functions of the ribosomal proteins in the production of the subunits in eukaryotes are poorly characterized, which calls for a more detailed investigation of the role played by Rps19 in ribosome synthesis and function.The yeast Saccharomyces cerevisiae has proven to be an outstanding system to investigate factors involved in eukaryotic ribosome synthesis...

Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.

Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)-CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF-1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF-LIF receptor (LIF-R) axis affects the biology of rhabdomyosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: (a) phosphorylation of mitogenactivated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and (c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that (a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF-LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma. [Cancer Res 2007;67(5):2131-40]

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A minority of children with CNS AT/RT treated on HS III may be long-term survivors without irradiation. More effective therapies are desperately needed.

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