Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.
Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc؉ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19 -25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc؉ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/ mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc؉ mutated (n ؍ 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n ؍ 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX upregulation observed in NPMc؉ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD؉ samples were characterized by upregulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc؉ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations. FLT3-ITD ͉ HOX ͉ NPM1A cute myeloid leukemia (AML) arises from multiple and sequential genetic alterations involving hematopoietic precursors (1). In Ϸ25% of cases, specific chromosomal translocations like the t(8;21), inv(16) or t(15;17) represent the initial events leading to malignant transformation (1) and are associated with a good outcome. In contrast, 40-50% of AMLs have normal karyotype by conventional banding analysis and are characterized by great molecular and clinical heterogeneity (2). Recent work has identified novel molecular abnormalities in normal karyotype AML (NK-AML) that has improved the classification and risk stratification of this large subgroup of patients. Among them, internal tandem duplications in the juxta-membrane domain or mutations in the second tyrosine kinase domain (TKD) of the FLT3 gene have been found in 30-45% of NK-AML (3). Both types of mutations constitutively activate FLT3 and FLT3-ITD mutations have been associated with increased risk of relapse (4). Mutations in the myeloid transcription factor CEBPA have been detected in 10-15% of NK-AML (5) and are associated with favorable prognosis (5, 6).Mutations of the nucleophosmin (NPM1) gene, usually occurring at exon-12 (7) and more rarely at exon-11 (8) represent the most common genetic alteration in AML-NK (50-60% of cases) and account for about one-third of all adult AML (7). This gene encodes for a ubiquitously expressed...
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 AMLassociated 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. IntroductionIn acute myeloid leukemia (AML), a clinically and molecularly heterogeneous disease, 1 recurrent cytogenetic abnormalities help define subgroups with different prognosis, and identify patients who might benefit from targeted therapies. 1 However, almost half adult AMLs display normal karyotype at conventional cytogenetics, 2 and the clinical and molecular features of this large subgroup of patients are still poorly understood. [3][4][5][6][7] We recently observed that about 60% of adult AML with normal karyotype display aberrant cytoplasmic expression of nucleophosmin (NPM). 8 A multifunctional protein [9][10][11][12][13][14][15] that characteristically shuttles between the nucleus and the cytoplasm, 16 NPM is found mainly in the nucleolus, [17][18][19] where it is one of the most abundant of the approximately 700 proteins so far identified by proteomic techniques. 20 Cytoplasmic NPM identifies a distinct subgroup of AML, named NPMc ϩ AML, that accounts for about 35% of all adult AML and is characterized by wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD mutations, absence of CD34, and relatively good response to induction therapy. 8 NPMc ϩ AML also has a distinct gene expression profile 21 and carries mutations in exon-12 of the NPM gene 8 that serve as predictor of favorable prognosis in AML with normal karyotype, [22][23][24] and as a marker for monitoring of minimal residual disease. 25 In spite of the close association between the aberrant cytoplasmic expression of NPM and exon-12 NPM mutations, 8 the mechanism underlying cytoplasmic accumulation of NPM in leukemic cells and its interference with wild-type NPM protein remaine...
IntroductionAcute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. Cytogenetic and/or molecular studies are used to assign 30% to 40% of AML cases carrying specific genetic lesions (eg, t(15;17), t(8;21) or Inv(16)) to different prognostic subgroups in order to monitor minimal residual disease and to select patients who could benefit from targeted therapies. 1 However, they cannot be applied to 40% to 50% of patients with AML who at conventional cytogenetics exhibit a normal karyotype. 1,2 We recently found exon-12 nucleophosmin (NPM) gene mutations in approximately 60% of AMLs with normal karyotype (about one third of all adult AML) that are characterized by distinct morphologic, phenotypic, and molecular features, 3 as well as distinct gene expression profile signature. 4 NPM mutations predict good response to induction therapy and favorable prognosis, 5-8 and could serve to monitor minimal residual disease. 9 Thus, analysis of NPM mutations emerges as a major new step in the diagnostic/prognostic work-up of patients with AML with a normal karyotype.Reverse-transcriptase-polymerase chain reaction (RT-PCR), mutational analysis, or gene expression profiling is usually used for detecting genetic abnormalities or specifically overexpressed genes with diagnostic/prognostic significance. However, demand is growing for simple, inexpensive, and specific immunologic tests 10 that can serve as a surrogate to, or used in integration with, molecular studies. Examples include immunohistochemical detection of proteins such as anaplastic lymphoma kinase (ALK) in CD30 ϩ anaplastic large cell lymphoma, 10 Zap-70 in chronic lymphocytic leukemia, 11 annexin were involved in biochemical studies; I.N. and R.M. were involved in the confocal microscopic analysis of cells; R.P., A.T., and E.T. were involved in the immunohistochemical study of AML samples; C.M. was involved in the identification of NPM mutations; R.B., D.D., G.R., R.R., M.A., L.L., S.V., L.B., E.G., A.G., G.S., F.P., F.L.-C., and P.-G.P. were involved in the analysis of NPM gene mutations in the GIMEMA-EORTC trial; and F.M., S.A., G.S., and M.F.M. were involved in organizing the clinical trial.M.P.M. and N.B. contributed equally to this work.Reprints: Brunangelo Falini, Institute of Hematology, Policlinico Monteluce, 06122 Perugia, Italy; e-mail: faliniem@unipg.it.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on April 8, 2019. by guest www.bloodjournal.org From A1 in hairy cell leukemia, 12 myeloid leukemia factor-1 (MLF1) in myelodysplasia/AML with t(3;5), 13 and promyelocytic leukemia (PML) in acute promyelocytic leukemia carrying the t(15;17). 14 In a small group of patients with AML, we showed that aberrant cytoplasmic expression of NPM, a multifunctional 15-20 nucleocytoplasmic shuttling protein 21 with restricted nucleolar localization, 10...
Nucleophosmin (NPM) is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. Many findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles of this molecule. The gene NPM1 that encodes for nucleophosmin (NPM1) is translocated or mutated in various lymphomas and leukemias, forming fusion proteins (NPM-ALK, NPM-RARalpha, NPM-MLF1) or NPM mutant products. Here, we review the structure and functions of NPM, as well as the biological, clinical and pathological features of human hematologic malignancies with NPM1 gene alterations. NPM-ALK indentifies a new category of T/Null lymphomas with distinctive molecular and clinico-pathological features, that is going to be included as a novel disease entity (ALK+ anaplastic large cell lymphoma) in the new WHO classification of lymphoid neoplasms. NPM1 mutations occur specifically in about 30% of adult de novo AML and cause aberrant cytoplasmic expression of NPM (hence the term NPMc+ AML). NPMc+ AML associates with normal karyotpe, and shows wide morphological spectrum, multilineage involvement, a unique gene expression signature, a high frequency of FLT3-internal tandem duplications, and distinctive clinical and prognostic features. The availability of specific antibodies and molecular techniques for the detection of NPM1 gene alterations has an enormous impact in the biological study diagnosis, prognostic stratification, and monitoring of minimal residual disease of various lymphomas and leukemias. The discovery of NPM1 gene alterations also represents the rationale basis for development of molecular targeted drugs.
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 þ ). NPMc þ 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 þ 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 þ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc þ 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 þ 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 þ AML.
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. [Cancer Res 2007;67(13):6230-7]
isoforms that have been associated with cellular resistance to ionizing radiation. 3 We want to bring attention to some significant unsolved issues. Frequency of this new mutation in AML population is unknown at present, since all large studies where NPM1 mutations were determined only involved exon 12 amplification from genomic DNA or use of specific probes for known mutations precluding exon 11 region 4-7 (see references cited in Mrozek et al .1 ), which let in all cases this new truncation mutation go unnoticed. Even recommended immunohistochemical determinations 2 may have failed detecting this alteration, due to its mixed staining pattern. New analyses of the thousands of samples available in large leukaemia study groups would allow to obtain the frequency for this new type of NPM1 mutation, thus opening the possibility of redefining new subclasses of AML-NK based in these molecular findings. At this point, it is interesting to add that in their recent review 2 Falini et al. mentioned an abstract where the presence of an exon 11 mutation in an AML patient is described (Albiero et al. Haematologica 2006; 91: 237, abstract). The reported mutation at exon 11 is the same kind of eight nucleotide insertion as the one we have found and reported here, but the inserted nucleotides are different in sequence, although both alterations produce a similar truncated protein. These data reinforce the possibility that a new hot spot for insertional mutation in NPM1 might have been located and strengthen the need for establishing the frequency of this type of mutation in AML patients as well as the biological consequences of this new finding.
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