Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin

Abstract

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 up-regulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by up-regulation 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.

Fig. 1.

Hierarchical cluster analysis of NK-AML patients. Overview of two-way (genes against samples) hierarchical cluster (Euclidean distance) of 85 AML samples using the genes that varies the most between samples. Mean centered signal intensities of gene-expression are depicted by a log-transformed (2 scale). Color areas indicate relative expression of each gene with respect to the gene median expression (red above, green below the median value and black, samples with signal intensity to background of 2 or less). Some miRNAs are represented by more than one probe. As shown, the clustering is mainly determined by the presence of cytoplasmic nucleophosmin (NPMc+). Among the NPMc+ positive samples, we identified also many subgroups. Patients with FLT3-ITD+ segregated among the different clusters.

Fig. 2.

MiR-204 targets HOXA10 and MEIS1 in AML. (A) Western blotting showing HOXA10 protein using whole cell lysates from MEG-01 and OCI-AML3 cell lines transfected with scrambled oligonucleotides or miR-139, miR-128a, and miR-204. The blots were stripped and reprobed with β-actin for loading control. (B) Western blotting showing MEIS1 protein expression in OCI-AML3 cell lines transfected with scrambled oligonucleotides or sense miR-204 and miR-101. (C) Western blotting showing HOXA10 and MEIS1 protein expression in MEG-01 and K562 cells transfected with scrambled oligonucleotides or antisense oligonucleotides against miR-204. The numbers below the immunoblot images represent the intensity of the HOXA-10/MEIS bands relative to the β-actin expression. (D) Relative luciferase activity in MEG01 cells transiently cotransfected with the luciferase reporter vector containing the 3′ UTR of MEIS1 with miR-204 or scrambled oligonucleotides. The results are presented as a fold difference in the luciferase/Renilla ratios with respect to the luciferase reporter constructs transfected with scrambled oligonucleotides.

Fig. 3.

MiR-125a targets CD34. (A) Western blotting showing CD34 protein expression in KG1a cells 48 h after transfection with scrambled oligonucleotides or sense miR-125a, mIR-24, miR-9, or all combined oligonucleotides (B) Relative luciferase activity in KG1a cells transiently cotransfected with the luciferase reporter vector containing the 3′ UTR of CD34 predicted to interact with miR-125a or scrambled oligonucleotides. The results are presented as a fold difference in the luciferase/Renilla ratios with respect to the empty reporter (pGl3 alone).

Fig. 4.

MiR-155 is overexpressed in FLT3-ITD+ AML. (A) Average miR-155 expression in 32 NK-AML patients with FLT3-ITD+ (n = 10) and FLT3-wt (n = 22) after normalization with 18s and 2ΔC_t calculations. The average miR-155 expression values between the two groups were compared by using t test. (B) IP/Western blot. MV4-11 cells were incubated for 24 and 48 h with SU14813 (300 nM) or DMSO (vehicle control). Lysates were generated and an immunoprecipitation was performed with an antibody for FLT3. SDS/PAGE was performed, followed by a Western blot with an antibody for phospho-tyrosine. Subsequently, the blot was stripped and reprobed with anti-FLT3. (C) Northern blotting of miR-155 in MV4-11 cells incubated for 24 and 48 h with SU14813 (300 nM) or DMSO (vehicle control). Total RNA was obtained with TRIzol and Northern blotting was performed with a probe antisense to miR-155. The blot was stripped and reprobed with U6 for loading control. (D) IP/Western blot. BaF3 and BaF3 FLT3-ITD cells were incubated for 24 h with SU14813 (300 nM) or DMSO (vehicle control). Lysates were generated and an immunoprecipitation was performed with an antibody for FLT3. SDS/PAGE was performed, followed by a Western blot with an antibody for phospho-tyrosine. Subsequently, the blot was stripped and reprobed with anti-FLT3. (E) Northern blotting for miR-155 in BaF3 and Baf3 FLT3-ITD clones incubated for 24 and 48 h with SU14813 or DMSO (control) as described. The blot was stripped and reprobed with U6 for loading control.