Differentially expressed miRNAs in cytogenetic and molecular subtypes of pediatric acute myeloid leukemia.

BACKGROUND: miRNAs regulate gene expression, and thus play an important role in critical cellular processes. Aberrant miRNA expression patterns have been found in various types of cancer. So far, information about the expression of miRNAs in pediatric acute myeloid leukemia is limited. PROCEDURE: We studied expression of miR-29a, -155, -196a, and -196b by stem-loop based RT-qPCR in 82 pediatric acute myeloid leukemia patients selected to represent relevant cytogenetic and molecular subgroups. RESULTS: High miR-196a and -b expression was observed in patients carrying MLL gene rearrangements (P < 0.001), NPM1 mutations (P < 0.001), or FLT3-ITD in a cytogenetically normal background (P ≤ 0.02), compared to all other patients. In contrast, CEBPA mutated cases had a low expression of miR-196a and -b (P ≤ 0.001). Expression of miR-196a and -b was correlated with expression of neighboring HOXA and HOXB genes (Spearman's r = 0.46-0.82, P < 0.01). Expression of miR-155 was not related to cytogenetic features but high expression of miR-155 was observed in FLT3-ITD (P = 0.001) and NPM1-mutated cases (P = 0.04). Lower miR-29a expression was mainly observed in MLL-rearranged pediatric acute myeloid leukemia, specifically in cases carrying t(10;11) (P < 0.001). CONCLUSIONS: We show aberrant expression of specific miRNAs in clinically relevant cytogenetic and molecular subgroups of pediatric acute myeloid leukemia, suggesting a role for these miRNAs in the underlying biology in these specific subgroups.

miR-128b is a potent glucocorticoid sensitizer in MLL-AF4 acute lymphocytic leukemia cells and exerts cooperative effects with miR-221.

MLL-AF4 acute lymphocytic leukemia (ALL) has a poor prognosis. MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. Our analysis of previously published data showed that expression of miR-128b and miR-221 is down-regulated in MLL-rearranged ALL relative to other types of ALL. Reexpression of these miRNAs cooperatively sensitizes 2 cultured lines of MLL-AF4 ALL cells to glucocorticoids. Target genes down-regulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes; miR-221 down-regulates CDKN1B. These results demonstrate that down-regulation of miR-128b and miR-221 is implicated in glucocorticoid resistance and that restoration of their levels is a potentially promising therapeutic in MLL-AF4 ALL.

MicroRNA characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia.

BACKGROUND: MicroRNA regulate the activity of protein-coding genes including those involved in hematopoietic cancers. The aim of the current study was to explore which microRNA are unique for seven different subtypes of pediatric acute lymphoblastic leukemia. DESIGN AND METHODS: Expression levels of 397 microRNA (including novel microRNA) were measured by quantitative real-time polymerase chain reaction in 81 cases of pediatric leukemia and 17 normal hematopoietic control cases. RESULTS: All major subtypes of acute lymphoblastic leukemia, i.e. T-cell, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid acute lymphoblastic leukemia, with the exception of BCR-ABL-positive and 'B-other' acute lymphoblastic leukemias (defined as precursor B-cell acute lymphoblastic leukemia not carrying the foregoing cytogenetic aberrations), were found to have unique microRNA-signatures that differed from each other and from those of healthy hematopoietic cells. Strikingly, the microRNA signature of TEL-AML1-positive and hyperdiploid cases partly overlapped, which may suggest a common underlying biology. Moreover, aberrant down-regulation of let-7b (~70-fold) in MLL-rearranged acute lymphoblastic leukemia was linked to up-regulation of oncoprotein c-Myc (P(FDR)<0.0001). Resistance to vincristine and daunorubicin was characterized by an approximately 20-fold up-regulation of miR-125b, miR-99a and miR-100 (P(FDR)≤0.002). No discriminative microRNA were found for prednisolone response and only one microRNA was linked to resistance to L-asparaginase. A combined expression profile based on 14 microRNA that were individually associated with prognosis, was highly predictive of clinical outcome in pediatric acute lymphoblastic leukemia (5-year disease-free survival of 89.4%±7% versus 60.8±12%, P=0.001). CONCLUSIONS: Genetic subtypes and drug-resistant leukemic cells display characteristic microRNA signatures in pediatric acute lymphoblastic leukemia. Functional studies of discriminative and prognostically important microRNA may provide new insights into the biology of pediatric acute lymphoblastic leukemia.

Expression of miR-196b is not exclusively MLL-driven but is especially linked to activation of HOXA genes in pediatric acute lymphoblastic leukemia.

BACKGROUND: Deregulation of microRNA may contribute to hematopoietic malignancies. MicroRNA-196b (miR-196b) is highly expressed in MLL-rearranged leukemia and has been shown to be activated by MLL and MLL-fusion genes. DESIGN AND METHODS: In order to determine whether high expression of miR-196b is restricted to MLL-rearranged leukemia, we used quantitative stem-loop reverse transcriptase polymerase chain reaction to measure the expression of this microRNA in 72 selected cases of pediatric acute lymphoblastic leukemia i.e. MLL-rearranged and non-MLL-rearranged precursor B-cell and T-cell acute lymphoblastic leukemias. We also determined the expression of HOXA-genes flanking miR-196 by microarray and real-time quantitative polymerase chain reaction. Furthermore, we used CpG island-arrays to explore the DNA methylation status of miR-196b and HOXA. RESULTS: We demonstrated that high expression of miR-196b is not unique to MLL-rearranged acute lymphoblastic leukemia but also occurs in patients with T-cell acute lymphoblastic leukemia patients carrying CALM-AF10, SET-NUP214 and inversion of chromosome 7. Like MLL-rearrangements, these abnormalities have been functionally linked with up-regulation of HOXA. In correspondence, miR-196b expression in these patients correlated strongly with the levels of HOXA family genes (Spearman's correlation coefficient ≥ 0.7; P≤0.005). Since miR-196b is encoded on the HOXA cluster, these data suggest co-activation of miR-196b and HOXA genes in acute lymphoblastic leukemia. Up-regulation of miR-196b coincides with reduced DNA methylation at CpG islands in the promoter regions of miR-196b and the entire HOXA cluster in MLL-rearranged cases compared to in cases of non-MLL precursor B-cell acute lymphoblastic leukemia and normal bone marrow (P<0.05), suggesting an epigenetic origin for miR-196b over-expression. Although patients with MLL-rearranged acute lymphoblastic leukemia are highly resistant to prednisolone and L-asparaginase, this resistance was not attributed to miR-196b expression. CONCLUSIONS: High expression of miR-196b is not exclusively MLL-driven but can also be found in other types of leukemia with aberrant activation of HOXA genes. Since miR-196b has been shown by others to exert oncogenic activity in bone marrow progenitor cells, the findings of the present study imply a potential role for miR-196b in the underlying biology of all HOXA-activated leukemias.

Protein geranylgeranylation is critical for the regulation of survival and proliferation of lymphoma tumor cells.

PURPOSE: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. The following study was conducted to examine the importance of protein farnesylation and geranylgeranylation for the regulation of lymphoma cell survival and proliferation. EXPERIMENTAL DESIGN: Lymphoma cells were treated with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor lovastatin, which inhibits protein farnesylation and geranylgeranylation by the depletion of intracellular pools of farnesylpyrophosphate and geranylgeranylpyrophosphate. In addition, farnesyl transferase and geranylgeranyl transferase activities were specifically inhibited by FTI-277 and GGTI-298, respectively. RESULTS: Only inhibition of geranylgeranylation by lovastatin led to reduction of cell viability in lymphoma cell lines and purified tumor cells from lymphoma patients in a time- and dose-dependent way. Reduction in the number of viable cells was mediated by both induction of apoptosis and inhibition of proliferation. In addition, GGTI-298 was more effective in induction of apoptosis and inhibition of proliferation than FTI-277. Apoptosis induced by inhibition of protein geranylgeranylation was associated with a reduction of Mcl-1 protein levels, collapse of the mitochondrial transmembrane potential, and caspase-3 activation. Inhibition of proliferation resulted from the induction of G(1) arrest. Furthermore, lovastatin at low concentrations sensitized lymphoma cells to dexamethasone, including cells resistant to this drug. CONCLUSION: These results indicate that protein geranylgeranylation is critical for the regulation of lymphoma tumor cell survival and proliferation and that pharmacological agents such as lovastatin or geranylgeranyl transferase inhibitors, alone or in combination with other drugs, may be useful in the treatment of lymphoma.

A novel mutation in the miR-128b gene reduces miRNA processing and leads to glucocorticoid resistance of MLL-AF4 acute lymphocytic leukemia cells.

MLL-AF4 acute lymphocytic leukemia has a poor prognosis, and the mechanisms by which these leukemias develop are not understood despite intensive research based on well-known concepts and methods. MicroRNAs (miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate expression of target mRNA transcripts. We recently reported that ectopic expression of miR-128b together with miR-221, two of the miRNAs downregulated in MLL-AF4 ALL, restores glucocorticoid resistance through downregulation of the MLL-AF4 chimeric fusion proteins MLL-AF4 and AF4-MLL that are generated by chromosomal translocation t(4;11). Here we report the identification of new mutations in miR-128b in RS4;11 cells, derived from MLL-AF4 ALL patient. One novel mutation significantly reduces the processing of miR-128b. Finally, this base change occurs in a primary MLL-AF4 ALL sample as an acquired mutation. These results demonstrate that the novel mutation in miR-128b in MLL-AF4 ALL alters the processing of miR-128b and that the resultant downregulation of mature miR-128b contributes to glucocorticoid resistance through the failure to downregulate the fusion oncogenes.