Abstract
Acute myeloid leukemia (AML) is composed of functionally heterogeneous cells including leukemic stem cells (LSCs), which exhibit the ability to self-renew and propagate disease. It is thought that failure of common chemotherapy regimens is due to insufficient eradication of LSCs. However, the mechanisms that maintain stem cell function in the hematopoietic system are not well understood. MicroRNAs play an important role in the regulation of normal and malignant hematopoietic stem cells. Our studies showed that miR-99, a miRNA highly expressed in AML patient cell populations enriched for LSC activity, is among the most highly expressed miRNAs in hematopoietic stem cells (HSCs), suggesting that miR-99 plays a role in regulating normal HSCs as well as LSCs.
To test the role of miR-99 in normal hematopoiesis, we knocked down (KD) miR-99 in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+), which resulted in ~3 fold reduced methylcellulose colony formation upon secondary plating (P=0.01), as well as accelerated granulopoiesis as demonstrated by increased Gr1+Mac1+ cells 7 days after culture initiation (P<0.01), suggesting that miR-99 functions to suppresses differentiation. Consistent with this model, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of miR-99 KD compared to scrambled controls (P=0.0004). In addition, Ki-67/DAPI staining of stably engrafted miR-99 KD hematopoietic stem and progenitor cells (HSPCs) showed increased cell cycling, demonstrating that miR-99 also maintains HSPC quiescence. Gene set enrichment analysis (GSEA) of RNA-sequencing data generated from stably engrafted Lin-Sca-1+c-Kit+ cells revealed that miR-99 KD induces significant depletion of LT-HSC gene signatures (P<0.001) and induction of a late progenitor signature (P<0.001), providing further evidence that miR-99 normally functions to maintain HSPCs in the undifferentiated state.
To test whether miR-99 maintains LSCs, we performed miR-99 KD experiments using the MLL-AF9 retroviral mouse model. miR-99 KD resulted in a significant extension in survival in secondary transplants compared to scrambled controls (median 92 days vs. 48 days, P<0.001). Evaluation of the bone marrow at the time of death revealed ~2.5 fold decrease in the frequency of LSCs (P<0.01) and ~2 fold increase in the percentage of cycling LSCs (in SG2M) (P<0.001). Analysis of RNA-seq data from miR-99 KD LSCs revealed induction of a differentiated normal progenitor signature (P<0.001) and depletion of a shared HSC/LSC gene signature (P=0.05). Giemsa staining of peripheral blood showed miR-99 KD also induced a significant increase in the number of differentiated myeloid precursors in the peripheral blood (P<0.001), reminiscent of AML differentiation-inducing agents used in the clinic such as ATRA. Consistent with a role in regulating leukemic blast differentiation, microRNA-Seq data from the 153 AML patients in the TCGA database revealed that miR-99 expression inversely correlated with their French-American-British classifications, with low expression levels associated with M4 and M5 subtypes. Compatible with a role in maintaining LSCs, miR-99 KD in a primary AML sample reduced long-term engraftment upon xenotransplantation into NSG mice, and the engrafting cells displayed increased CD14 expression. Together, these data demonstrate that similar to normal HSPCs, miR-99 maintains LSCs function.
As miR-99 functions to maintain both LSCs and HSCs, we asked which miR-99 target genes mediate miR-99 KD phenotypes. To address this question, we performed a shRNA library-based forward genetic screen designed to rescue the reduced HSC function following miR-99 KD. We designed 180 shRNAs against 45 predicted miR-99 targets that we identified as upregulated upon acute miR-99 KD in mouse HSPCs. Among the conserved miR-99 targets, Hoxa1, a member of the Hox family of transcription factors, was among the top hits, with all 4 shRNAs being enriched compared to controls. Ectopic expression of Hoxa1 in MonoMac6 AML cells was sufficient to induce differentiation, a phenotype similar to miR-99 KD. These data indicate that Hoxa1 is an important downstream mediator of miR-99 function.
Disclosures
No relevant conflicts of interest to declare.
Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.
