Upregulation of Mir-99a Is Associated with Poor Prognosis of Acute Myeloid Leukemia and Promotes Myeloid Leukemia Cell Expansion

Acute myeloid leukemia (AML) is a hereditarily and clinically heterogeneous disease driven by a subpopulation of leukemic stem cells (LSCs) that generate the bulk of leukemic cells. LSCs can resist available treatments and result in disease progression and relapse. High number of LSCs or expression of a LSC related gene signature is independently associated with poor prognosis in AML, supporting the notion for LSCs as important targets for therapeutic intervention. It is now evident that miRNAs contribute significantly to the leukemogenic phenotype. To dissect the miRNA expression profile of LSCs, we performed miRNA array analysis in enriched LSCs cells and non-LSCs (leukemic blast cells) from bone marrow (BM) cells of the same AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a were upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients. The cohort were divided into two groups based on the median expression level of miR-99a in LSCs reference to non-LSCs, and correlation analysis indicated that higher expression levels of miR-99a in LSCs were significantly correlated with worse overall survival and event-free survival in these AML patients. Compared with cord blood CD34⁺ HSPCs, miR-99a was significantly highly expressed in LSCs from AML patients. The expression level of miR-99a was also higher in K562/A02 (multidrug resistant counterpart of K562 cell line) and K562/G01 (Gleevec resistant counterpart of K562 cell line) than the parental K562 cells. These results point to the importance of a novel role of miR-99a in LSC activity and disease progression.

By gain-of-function studies via stable lentiviral transduction, we demonstrated that miR-99a functionally regulated the growth and survival of leukemic cells in vitro and in vivo. Enforced miR-99a expression significantly increased the clonogenic capacity in primary AML LSCs. Further studies in myeloid leukemia cell lines revealed that ectopic miR-99a led to accelerated cell-cycle progression and cell proliferation. MiR-99a overexpression (OE) also promotes leukemic cell survival after exposure to chemotherapeutic agents in AML cell lines. In vivo study in a subcutaneous xenograft model showed that a significant lower tumor inhibition rate was observed overtime in the miR-99a/OE cell injected mice compared to the Ctrl cell injected mice when treated with Ara-C. All these results demonstrated that miR-99a, the miRNA enriched from LSCs at relapse stage, plays an important role in myeloid leukemic cell survival and expansion.

To explore the mechanism underlying the enhanced proliferation and chemoresistance meditated by miR-99a, we identified differential gene expression profiles by microarray analysis. The results provide a potential regulatory pathway that links the downregulated targets of miR-99a (i.e., PPP3CA) to the dysregulated key genes associated with leukemic cell expansion. Overexpression of miR-99a led to upregulation of CCNE1, and downregulation of CDKN2A which mediate G1 cell cycle arrest by stabilizing the structure of p53 or inducing p53 activation. All these results suggest an important role of miR-99a in cell cycle entry and progression, leading to the accelerated proliferation and overcoming of chemotherapeutic-agents mediated cell cycle arrest.

Collectively, our study provides strong evidence that miR-99a plays an important role in altering biological properties, which may lead to the pathogenesis of myeloid leukemia and chemoresistance induction. The levels of miR-99a may be used as a biomarker for the prognosis and progression of AML.