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Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.

Aquaporin 2 (AQP2) phosphorylation at Ser-256 by protein kinase A (PKA) is a key signal for vasopressin-stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser-256 in regulating AQP2 maturation. AQP2-transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p-AQP2) at Ser-256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin-regulated vesicular compartment occurred with a concomitant decrease in p-AQP2 at Ser-256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256. Conversely, impaired constitutive phosphorylation in a Golgi-associated compartment occurring in cells expressing mutated S256A-AQP2 or E258K-AQP2 causes phosphorylation-defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K-AQP2, in which the phenotype is caused by an impaired routing of AQP2.

Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplasmic shuttling protein that shows a restricted nucleolar localization. Mutations of NPM1 gene leading to aberrant cytoplasmic dislocation of nucleophosmin (NPMc+) occurs in about one third of acute myeloid leukaemia (AML) patients that exhibit distinctive biological and clinical features. We discuss the latest advances in the molecular basis of nucleophosmin traffic under physiological conditions, describe the molecular abnormalities underlying altered transport of nucleophosmin in NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukaemogenesis. We then outline how a highly specific immunohistochemical assay can be exploited to diagnose NPM1-mutated AML and myeloid sarcoma in paraffin-embedded samples by looking at aberrant nucleophosmin accumulation in cytoplasm of leukaemic cells. This procedure is also suitable for detection of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surrogate for molecular analysis can serve as first-line screening in AML and should facilitate implementation of the 2008 World Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc+ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleophosmin transport in AML with mutated NPM1.

BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14(ARF). Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14(ARF) in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14(ARF)) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14(ARF)) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.