A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene.

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency.

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila.

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.

Identification of Variants in Alpha-1-Antitrypsin by High Resolution Melting.

BACKGROUND: Alpha-1-antitrypsin deficiency (AATD) is one of the most common hereditary disorders occurring in populations of European origin and is due to variants in SERPINA1, which encodes a protease inhibitor of neutrophil elastase, limiting lung damage from this enzyme. The World Health Organization has recommended that individuals with chronic obstructive pulmonary disease and asthma be tested for AATD. The development of inexpensive and simple genetic testing will help to meet this goal. METHODS: Primers and synthetic SERPINA1 gene fragments (gBlocks) were designed for 5 AATD-associated variants. PCR was run on a CFX96 Thermal Cycler with High Resolution Melting (HRM) capacity and data analyzed using the supplied HRM-analysis software. Genomic DNA from individuals (n = 86) genotyped for the S and Z variants were used for validation. HRM-analysis was performed on 3 additional samples with low alpha-1-antitrypsin levels inconsistent with the genotype determined in our clinical laboratory. RESULTS: Unique normalized melt curve and difference curve patterns were identified for the AAT variants Z, S, I, F, and MMalton using gBlocks. Similar curve shapes were seen when these primers were used to analyze the gDNA samples. HRM identified the genotypes of the gDNA correctly with 100% concordance. The curve shapes of some samples did not match the melting patterns of the targeted variant. Sequencing was used to identify the variants, including rare AATD variants c.1108_1115delinsAAAAACA (p.Glu370Lysfs*31) and c.1130dup (p.Leu377fs). CONCLUSION: We developed a rapid and inexpensive HRM-analysis method for genotyping of Z, S, MMalton, I, and F variants that was also capable of detecting other variants.

Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method.

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.

A role for Hemolectin in coagulation and immunity in Drosophila melanogaster.

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

Hemolymph coagulation and phenoloxidase in Drosophila larvae.

Hemolymph coagulation is a first response to wounding in insects. Although studies have been performed in large-bodied insects such as the moth Galleria mellonella, less is known about clotting in Drosophila melanogaster, the insect most useful for genetic and molecular analyses of innate immunity. Here we show the similarities between clots in Drosophila and Galleria by light- and electron microscopy. Phenoloxidase changes the Drosophila clot's physical properties through cross-linking and melanization, but it is not necessary for preliminary soft clot formation. Bacteria associate with the clot, but this alone does not necessarily kill them. The stage is now set for rapid advances in our understanding of insect hemolymph coagulation, its roles in immune defense and wound healing, and for a more comprehensive grasp of the insect immune system in general.

Allogeneic tumor-dendritic cell fusion vaccines for generation of broad prostate cancer T-cell responses.

Antigen-loaded dendritic cells (DC) are considered potent stimulators of protective immunity. In some studies, DC hybridized with tumor cells have shown promising antitumor responses in mice as well as in humans. A practical drawback of this approach is the limited availability of autologous tumor samples. We investigated the possibility of hybridizing allogeneic prostate cancer cells with human-monocyte-derived DC, and thereby combine the wide repertoire of antigens from the tumor cells with the costimulatory features of the autologous antigen-presenting cells. Three tumor cell lines were used for hybridization using polyethylene glycol (PEG). We show that all three cell lines formed hybrids with DC to the same extent and without significant loss of viability. Restimulation of autologous T cells with these hybrids resulted in generation of tumor-specific IFNgamma-producing T cells with all three tumor cell lines. Similar tumor specificity could not be obtained if T cells were stimulated using a mixture of non-PEG-fused tumor cells and DC. Moreover, these T cells were capable of specific recognition of other tumor cells of prostate cancer origin and autologous DC pulsed with lysate from these prostate cancer cell lines, while a panel of unrelated EBV-transformed B cell lines were not recognized. These results are strongly indicative of the true tumor specificity of these T cells. Our results suggest that DC hybridized with allogeneic prostate cancer cell lines are potent stimulators of a broad prostate-tumor-specific response.

Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors.

The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. We tested how PO is activated following injury in 2 insects, i.e. the fruit fly Drosophila melanogaster and the wax moth Galleria mellonella. Rapid PO activation in Drosophila was limited to discrete areas of the hemolymph clot which forms after injury. Surprisingly, unlike systemic PO activation during bacterial sepsis, clot melanization was not sensitive to microbial elicitors in our assay. Instead, Drosophila clot melanization was activated by endogenous signals such as apoptotic cells and was superinduced by phosphatidylserine, a negatively charged phospholipid normally found on the inner surface of the plasma membrane and exposed during apoptosis. In contrast, melanization in G. mellonella hemolymph was stronger and more uniform and was sensitive to peptidoglycan. This shows that both exogenous and endogenous signals can trigger the same immune mechanism in species- and context-dependent ways. Our findings have implications for the evolutionary dynamics of immune mechanisms and are in agreement with recent comparisons of insect immune transcriptomes.

Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger.

The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.