MicroRNA Profiling Reveals Aberrant MicroRNA Expression in Adult ETP-ALL and Functional Studies Demonstrate a Role of Mir-222 in Acute Leukemia,

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup in pediatric acute T-lymphoblastic leukemia (T-ALL). ETP-ALLs originate from early thymic progenitors that retain stem cell features and are characterized by a specific immunophenotype and by a specific gene expression profile. Recently, we have characterized ETP-ALL as subgroup of early T-ALL in adults. To unravel the underlying mechanisms for the aberrant and distinct gene expression profile in ETP-ALL we now explored the expression of microRNAs (miRNAs) as gene regulators in ETP-ALL and further investigated their functional role in acute leukemia. Patients and methods: We screened expression levels of 667 miRNAs in newly diagnosed T-ALL patients (n=14: ETP-ALL n=8, non-ETP early T-ALL n=3, thymic T-ALL n=3) and 2 healthy controls (CD3 selected T cells) using a Taqman Low Density Array (TLDA; Applied Biosystems). The expression levels of miR-221 and miR-222 were validated in a large cohort of adult T-ALL (n=178: ETP-ALL n=66 and typical T-ALL n=112 including early-, thymic-, and mature T-ALLs) and healthy controls (n=6) by real-time-PCR (Taqman microRNA assays; Applied Biosystems). These patients were enrolled on the GMALL study 07/03. Functional analysis was performed by AMAXA transfection of pSUPER vector containing the miR-221 and miR-222 DNA sequences in Jurkat (T-lineage ALL) and KG1a (AML) cells. Viability was determined by cell proliferation reagent WST-1, and BrdU ELISA. Apoptosis was measured by annexin-V/7AAD staining with subsequent flow cytometric analysis. Results: In our screen 229 (34%) of the 667 miRNAs represented by the TLDA were expressed at detectable levels in at least two-thirds of the T-ALL samples and the controls. Of these we identified 55 miRNAs to be differentially expressed in T-ALL samples compared to healthy controls: 49 miRNAs were upregulated and 6 miRNAs were downregulated in the patient samples. Of these, miR-221 and miR-222 were the only miRNAs significantly upregulated in the ETP-ALL compared to the combined early and thymic T-ALL subgroups (8.0-fold, P<0.01; 5.0-fold, P<0.01, respectively). Therefore, we further validated miR-221 and miR-222 expression in a larger set of 178 T-ALL samples and observed a 3.3-fold higher expression of miR-221 (P<0.0001) and 3.9-fold higher expression of miR-222 (P<0.0001) in ETP-ALL (n=66) compared to typical T-ALL (n=112). For the correlation of miR-221 and miR-222 expression with molecular features, samples were divided into groups (low and high) according to the median of the miRNA expression levels. High miR-222 expression was associated with an immature phenotype of early T-ALL (P<0.01) and cell surface expression of CD34 (mean: 32%) compared to low miR-222 expressers (mean: 8%; P<0.01). Moreover, high miR-222 expression was associated with expression of myeloid markers CD33 (mean: 23% compared to 4% in low miRNA expressers, P<0.01) and CD13 (mean: 18% vs. 4%, P<0.01). Similar results were obtained for miR-221. In vitro studies revealed that overexpression of miR-222 inhibited proliferation of Jurkat (58% reduction of proliferation compared to cells transfected with the empty vector, P<0.001) and KG1a cells (50% reduction of proliferation compared to control cells, P<0.01) determined by WST-1. A reduction of the DNA synthesis was detected by BrdU incorporation after miR-222 overexpression in Jurkat (37% reduction compared to control cells) and in KG1a cells (54% reduction compared to control cells). Overexpression of miR-222 induced a 1.3- (P=0.02) and 3.0-fold (P<0.01) increase in apoptosis in Jurkat and KG1a cells, respectively. No significant changes were observed for miR-221 transfected cells in these functional assays. Conclusion: In summary, our study revealed aberrant expression of miRNAs in ETP-ALL, with miR-221 and miR-222 as the most overexpressed miRNAs. Functional analysis demonstrated that miR-222 impaired the proliferation and induced apoptosis, indicating a potential role for miR-222 in acute leukemia. Importantly, miR-222 targets such as the proto-oncogene c-KIT and the ETS1oncogene are contained in the gene expression profile of ETP-ALL. Thus, aberrant expression of miR-222 may directly impact leukemogenesis by altering expression of key oncogenes in acute leukemia.

Goekbuget:Micromet: Consultancy.