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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...

Hematopoietic transcription factors are involved in chromosomal translocations, which generate fusion proteins contributing to leukemia pathogenesis. Analysis of patient's primary leukemia blasts revealed that those carrying the t(8;21) generating AML1/ETO, the most common acute myeloid leukemia-associated fusion protein, display low levels of a microRNA-223 (miR-223), a regulator of myelopoiesis. Here, we show that miR-223 is a direct transcriptional target of AML1/ETO. By recruiting chromatin remodeling enzymes at an AML1-binding site on the pre-miR-223 gene, AML1/ETO induces heterochromatic silencing of miR-223. Ectopic miR-223 expression, RNAi against AML1/ETO, or demethylating treatment enhances miR-223 levels and restores cell differentiation. Here, we identify an additional action for a leukemia fusion protein linking the epigenetic silencing of a microRNA locus to the differentiation block of leukemia.

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...

Rigorous sequential RQ-PCR monitoring provides the strongest predictor of RFS in APL and, when coupled with pre-emptive therapy, provides a valid strategy to reduce rates of clinical relapse. This provides a model for development of a more individualized approach to management of other molecularly defined subtypes of acute leukemia.

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Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed 430 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: o100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.