Alessandra Pucciarini

BRAF Mutations in Hairy-Cell Leukemia

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.).

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

Born to Be Exported: COOH-Terminal Nuclear Export Signals of Different Strength Ensure Cytoplasmic Accumulation of Nucleophosmin Leukemic Mutants

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.

IRTA1 is selectively expressed in nodal and extranodal marginal zone lymphomas

Falini B, Agostinelli C, Bigerna B, Pucciarini A, Pacini R, Tabarrini A, Falcinelli F, Piccioli M, Paulli M, Gambacorta M, Ponzoni M, Tiacci E, Ascani S, Martelli M P, Dalla Favera R, Stein H & Pileri S A 
(2012) Histopathology 61, 930–941

A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF

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 p14ARF. 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 p14ARF in a ‘dose-dependent tug of war’ fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14ARF) 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 p14ARF) 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.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): a comparison with NPMc+ AML.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc+ AML

Cytoplasmic mutated nucleophosmin is stable in primary leukemic cells and in a xenotransplant model of NPMc+ acute myeloid leukemia in SCID mice

Findings of this study indicate that NPM1 mutations are stable in patients with acute myeloid leukemia, providing a rationale for monitoring of minimal residual disease. We investigated the NPM1 mutation status or subcellular expression of NPM protein (nuclear vs. aberrant cytoplasmic) at diagnosis and relapse in 125 patients with acute myeloid leukemia from Italy and Germany. All 52 patients with acute myeloidleukemia carrying at diagnosis mutated or cytoplasmic NPM (NPMc+ acute myeloid leukemia) retained this feature at relapse. Notably, cytoplasmic mutated NPM has now been retained for eight years in a xenotransplant model of NPMc+ acute myeloid leukemia in immunodeficient mice. None of 73 acute myeloid leukemia patients carrying at diagnosis wild-type NPM1 gene or showing at immunohistochemistry nucleus-restricted expression of nucleophosmin (NPMc− acute myeloid leukemia), which is predictive of NPM1 gene in germline configuration, acquired cytoplasmic mutated NPM at relapse. This finding further confirms that NPMc+ acute myeloid leukemia represents a primary event rather than a transformation stage of NPMc− acute myeloid leukemia. The stability of cytoplasmic mutated NPM in patients with acute myeloid leukemia, even at relapse in extramedullary sites, and in a xenotransplant model, suggest this event is crucial for leukemogenesis and represents the rationale for monitoring minimal residual disease and molecular targeted therapy in NPMc+ acute myeloid leukemia.

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

Absence of BRAF-V600E in the human cell lines BONNA-12, ESKOL, HAIR-M, and HC-1 questions their origin from hairy cell leukemia

To the editor: Hairy cell leukemia (HCL) shows distinct clinicopathologic, immunophenotypic, and gene expression features.[1][1][⇓][2]–[3][3] We previously identified the BRAF -V600E mutation as the disease-defining genetic event in HCL.[4][4] This mutation is present in virtually all cases of

Human monocyte-derived dendritic cells exposed to hyperthermia show a distinct gene expression profile and selective upregulation of IGFBP6

Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.