Mir-99 Is a Regulator of Hematopoietic and Leukemic Stem Cell Differentiation

MicroRNAs play an important role in the regulation of normal and malignant stem cells of the hematopoietic system.  Analysis of microRNA-Seq data from 168 acute myeloid leukemia (AML) patients in the TCGA database reveals widespread dysregulation of miRNA expression, including miR-99b.  Interestingly, miR-99b expression  inversely correlates with differentiation status of the AMLs as determined by the French-American-British classification scheme, suggesting miR-99 is a negative regulator of differentiation.  We also have shown that miR-99a and miR-99b are highly expressed in mouse hematopoietic stem cells (HSCs) compared to their more differentiated progeny. Thus, we hypothesized that miR-99 is a regulator of differentiation in normal and leukemic stem cells.

To test the function of miR-99 in normal hematopoiesis, we knocked down miR-99a or miR-99b in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+) using lentiviral vectors, resulting in ~3.5 fold reduction in the number of colonies formed in methylcellulose assays upon secondary plating (P=0.01), with the average colony being approximately 2 times smaller as measured by the number of cells (P=0.03). Consistently, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of HSCs upon miR-99 knockdown (KD) at 16 weeks (P=0.0004). Analysis of peripheral blood of the transplanted mice revealed ~3-fold increase in the proportion of donor-derived myeloid cells, suggesting that miR-99 may regulate the self-renewal and/or limit differentiation of HSPCs (P=0.01). Consistent with these observations, gene set enrichment analysis (GSEA) using the RNA-sequencing data generated from hematopoietic stem and progenitor cells (HSPCs) with miR-99 KD revealed significant enrichment for the myeloid differentiation gene signature (Lindstedt-dendritic-cell-maturation, Nominal p value =0.0, FDR q value=0.02). Moreover, analysis of GFP+ miR-99 KD cells after second plating revealed a 3-fold increase in apoptosis as measured by caspase 3 activation. To better understand the mechanism mediating the reduced reconstitution capacity of miR-99 KD HSCs, we analyzed the composition of progenitors in the bone marrow of transplanted mice.  miR-99 KD resulted in a highly significant decrease in the percentage of myeloid progenitors of donor-derived cells 16 weeks post transplantation (60% decrease, P=0.01), consistent with miR-99 KD inducing HSPC differentiation. Overall, these data suggest that miR-99 is a negative regulator of HSPC differentiation.

Consistent with a role in maintaining LSCs, miR-99 KD in the AML cell lines MonoMac6 and U937 induced expression of myeloid differentiation markers including CD15, CD14 and CD13. This was accompanied by a 2-50 fold reduction in cell growth depending on the AML cell line tested and a significant increase in apoptosis; the levels of miR-99 expression correlated with the degree of the growth defect. In addition, concomitant expression of miR-99b KD and MLL-AF9 overexpression vectors in HSPCs followed by serial methocult assays lead to decreased colony formation in third platings.  We also observed a 50% reduction in the percentage of Lin-Sca-1-c-Kit+ myeloid progenitors in secondarily plated cells (P value=0.01). These data suggest in MLL-AF9+ leukemia, miR-99 acts to restrain cell growth and maintain the undifferentiated state of leukemic blasts. Together, these data indicate that miR-99a/b regulates the proliferation of normal and leukemic stem cells by negatively regulating apoptosis and maintaining the stem cell state.