S.E. and J.K.K. as well as J.H.K. and M.M.H.E. contributed equally to this study.
MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and in the development of leukemia. In addition, modulation of miRNA expression can be exploited therapeutically. To identify tumor suppressive miRNAs in pediatric acute myeloid leukemia (AML), we performed a large-scale miRNA expression profiling in 90 cytogenetically characterized, de novo AML cases using a RT-qPCR platform.
In total, 253 miRNAs were significantly differentially expressed between patients with MLL rearrangements, t(8;21), inv(16), t(7;12), and t(15;17). Hierarchical clustering of patient samples using these sets of miRNA values showed that t(15;17) samples clearly cluster away from the other pediatric AML samples while t(7;12) patients cluster closely to core binding factor AMLs, (t(8;21) and inv(16). These three groups largely cluster away from the majority of MLL rearranged samples. Eight miRNAs specifically downregulated in MLL rearranged, t(8;21) and inv(16) AMLs were functionally evaluated in vitro using three cell lines representing those cytogenetic groups: THP-1 (MLL rearranged), KASUMI-1 (t[8;21]) and ME-1 (inv). Two of two miRNAs tested in KASUMI-1 cells (miR-9 and miR-582), two of three miRNAs tested in ME-1 (miR-192/194 bicistron and miR-660) and one of three miRNAs tested in THP-1 (miR-181a/b bicistron) reduced cell growth and colony-forming capacity upon ectopic expression. In KASUMI-1 cells, one miRNA was identified, miR-9, that not only reduced cell growth and colony forming capacity but also strongly induced monocytic differentiation in concert with calcitriol without affecting apoptosis. During normal hematopoiesis miR-9 is only expressed in macrophages.The effects on cell growth, colony-forming capacity and differentiation were confirmed in a second AML cell line with t(8;21), SKNO-1. The differentiation induction was restricted to t(8;21) leukemic cell lines, while its growth inhibitory function was also evident in normal CD34+ hematopoietic stem and progenitor cells. Most strikingly, miR-9 exerted a tumor suppressive function in primary leukemic blasts from patients with t(8;21) (n=2), but not in patients with MLL rearrangements (n=3). Using global gene expression studies upon ectopic miR-9 expression, we identified and validated LIN28B and HMGA2 as high fidelity target genes of miR-9 by RT-qPCR, western blotting and luciferase reporter assays. LIN28B is known to suppress let-7 processing. Indeed, miR-9 overexpresion increased the levels of mature let-7 family members, also leading to HMGA2 downregulation. ShRNA-mediated downregulation of LIN28B or HMGA2 partially recapitulated the effects of miR-9 on proliferation and differentiation of t(8;21) cell lines.
Thus, miR-9 is a tumor suppressor-miR in t(8;21) de novo pediatric AML, that acts in a stringent cell context dependent manner in concert with let-7 family members by repressing the oncogenic LIN28B/HMGA2 axis.
This work was supported by grants to J.H.K. from the German National Academic Foundation (KL-2374/2-1) and to J.K.K., L.V., A.D.vO, C.M.Z. and M.M.H.-E. from the Children Cancer Free Foundation (KIKA, project 49).
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.