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. 2017 Aug 7;214(8):2453-2470.
doi: 10.1084/jem.20161595.

miR-99 regulates normal and malignant hematopoietic stem cell self-renewal

Mona Khalaj  1   2 , Carolien M Woolthuis  1 , Wenhuo Hu  1 , Benjamin H Durham  1 , S Haihua Chu  3 , Sarah Qamar  1   2 , Scott A Armstrong  3 , Christopher Y Park  4
Affiliations

miR-99 regulates normal and malignant hematopoietic stem cell self-renewal

Mona Khalaj et al. J Exp Med. .

Abstract

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9-driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99's role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.

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Figures

Figure 1.
Figure 1.
miR-99 is highly expressed in hematopoietic stem and progenitors and suppresses myeloid differentiation in vitro. (A–C) Normalized expression levels of miR-99b, miR-99a, and miR-100 as determined by quantitative RT-PCR using miRNA TaqMan probes in mouse hematopoietic cell populations: hematopoietic stem cell (HSC), multipotent progenitor (MPP) Flk−, MPP Flk+, common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cells. Expression was normalized against mmu-mir-16. Error bars denote SEM. Representative data from five independent experiments are shown. (D) miR-99 is down-regulated 48 h post-transduction of HSCs with the lentiviral anti–miR-99 vector as shown by quantitative RT-PCR. Expression was normalized against U6 (Student’s t test; n = 3). Representative data from two independent experiments are shown. (E) Comparable number of colonies form after miR-99 KD in first plating, with an increase in the number of CFU macrophage (CFU-M) colonies. 100 GFP+ HSC cells were cultured in methylcellulose. The colonies were scored after 7 d. Data represent mean percentage ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (F) Smaller colonies were observed after second plating of GFP+ cells derived from miR-99 KD HSCs. Representative data of three independent experiments are shown. (G) Colony-forming capacity of HSCs is reduced after miR-99 KD in a second plating. 15,000 GFP+ cells were replated 7 d after the first plating. Colony types were scored after 7 to 10 d. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (H) miR-99 KD in HSCs induces granulocytic differentiation in methylcellulose colony assays. 7 d after plating, colonies were analyzed for expression of myeloid differentiation markers by flow cytometry. Mean percentage ± SEM (Student’s t test; n = 2). Representative data of three independent experiments are shown. (I) Flow cytometry analysis of LSK cells transduced with anti–miR-99 or Scr vectors and maintained in liquid culture for 8 d reveals a decrease in the absolute number of GFP+Lin−Sca-1+c-Kit+CD150+ HSCs. FSCw denotes forward scatter-width. The data shown are gated on LSK cells. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
Figure 2.
miR-99 inhibition impairs HSC reconstitution capacity in vivo by inducing differentiation and increased cell cycling. (A) GFP+ chimerism of mice transplanted with miR-99 KD HSCs. HSCs were transduced with anti–miR-99 or scramble (Scr) control vectors, and 48 h later, 5,000 GFP+ cells were transplanted into lethally irradiated recipients along with 300,000 cells from Sca-1–depleted helper BM. Peripheral blood GFP chimerism was analyzed every 4 wk. Data represent mean percentages ± SEM (Student’s t test; n = 11 for Scr and n = 13 for miR-99 KD mice) and are representative of two independent experiments. Post tx, post-transplantation. (B) Flow cytometry analysis of the peripheral blood every 4 wk after transplantation of HSCs transduced with anti–miR-99 or Scr vectors. Data represent mean percentages ± SEM (Student’s t test; n = 11 for Scr and n = 13 for miR-99 KD mice) and are representative of two independent experiments. (C–E) Absolute number of GFP+Lin−c-Kit+Sca-1+CD48−CD150+ HSCs (C), CD48+CD150+ MPPa’s (multipotent progenitors a; (D), and CD48+ CD150neg MPPb's (multipotent progenitors b; E) in bilateral long bones and hips 16 wk post-transplant of HSCs. Data represent mean count ± SEM (Student’s t test; n = 4) and are representative of two independent experiments. (F) Ki-67/DAPI staining of donor-derived GFP+Lin−c-Kit+ HSPCs 6 mo post-transplant of miR-99 KD or Scr HSCs. Data represent mean percentage ± SEM (Student’s t test; n = 7 for Scr and n = 8 for miR-99 KD) and are representative of two independent experiments. (G) RNA-seq analysis of LSK cells FACS sorted from BM of mice transplanted with miR-99 KD or scramble control HSCs 3 mo after transplant (n = 2). (H and I) Gene set enrichment analysis for differentially expressed genes in stably engrafted miR-99 KD versus Scr LSK cells. FDR, false discovery rate; NES, normalized enrichment score. (J) Annexin V staining of total GFP+ cells in the BM 6 mo post-transplant. Data represent mean percentage ± SEM (Student’s t test; n = 7 for Scr and n = 8 for miR-99 KD) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
Figure 3.
miR-99 KD improves survival in an MLL-AF9 model of leukemia by depleting leukemia stem cells. (A) miRNA-sequencing data from 153 AML patients in the TCGA database. Sum of read counts of all miR-99 family members (miR-99a, miR-99b, and miR-100) is graphed as a function of the French–American–British (FAB) classification. (B) Schematic for miR-99 KD experiments in the MLL-AF9 mouse model of AML. LSK cells were cotransduced with GFP+ miR-99 KD and tdTom+ MLL-AF9 overexpressing vectors, and GFP+ tdTom+ cells were transplanted into sublethally irradiated mice. BM from transplanted animals was used for secondary transplants and methylcellulose replating assays. (C) Methylcellulose colony plating assay of post-transformed BM from primary AML recipients with miR-99 KD. 500 GFP+ tdTom+ cells were plated into methylcellulose media and replated every 7 d. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (D) Kaplan–Meier curves of recipients of secondary leukemia transplants. 300 GFP+ tdTom+ leukemic BM cells from primary recipients were transplanted into sublethally irradiated C57BL/6 recipients (Mantel–Cox test; n = 12 for Scr and n = 11 for miR-99 KD). Representative data from four independent experiments are shown. (E and F) Flow cytometry analysis of L-GMPs from the BM of secondary recipients. Gates were drawn on the GFP+ tdTom+ CD16/32+ population. Mean percentage ± SEM (Student’s t test; n = 12 for Scr and n = 9 for miR-99 KD). Representative data from four independent experiments are shown. (G) GFP+ tdTom+ cells were FACS sorted from the BM of primary recipients and transplanted in limiting dilutions into secondary recipients. Limiting dilution analysis was performed using ELDA software. See also Fig. S3 D. Representative data two independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4.
Figure 4.
miR-99 KD depletes LSCs in the MLL-AF9 mouse model of AML by inducing differentiation. (A) Representative flow cytometry graph and the corresponding bar graph for the cell cycle staining performed on L-GMPs FACS sorted from the BM of secondary MLL-AF9 transplantation recipients. Data represent mean percentage ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (B) White blood cell (WBC) count of the secondary transplantation recipients. Data represent mean count ± SEM (Student’s t test; n = 6) and are representative of three independent experiments. (C) Wright–Giemsa staining of the peripheral blood from secondary transplant recipients, and summary of cytological features based on a 200-cell manual count per condition (bars, 25 µm). (D) miR-99 KD increases the absolute number of normal c-Kit− GFP+ tdTom+ cells in the BM. Data represent mean count ± SEM (Student’s t test; n = 5 for Scr and n = 3 for miR-99 KD) and are representative of three independent experiments. (E) Heat map of RNA-seq data generated from L-GMPs derived from the BM of secondary transplantation recipients with or without miR-99 KD at the time of death. (n = 2). (F) GSEA of differentially expressed genes in miR-99 KD and scramble control L-GMPs shows induction of a more differentiated signature. (G) TaqMan quantitative RT-PCR analysis of Met and Irf8 in miR-99 KD and Scr L-GMPs sorted from secondary recipients. Expression data are normalized to Actb (Student’s t test; n = 3) and are representative of two independent experiments. (H) GSEA of miR-99 KD and scramble control L-GMPs reveals induction of genes present in a signature up-regulated in normal GMPs compared with L-GMP (Table S1). **, P < 0.01; ***, P < 0.001.
Figure 5.
Figure 5.
miR-99 functionally suppresses human AML differentiation. (A) Normalized expression levels of miR-99a, miR-99b and miR-100 by quantitative PCR using miRNA TaqMan probes in human HSPCs, including hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs). Expression was normalized to sno-R2. Data represent mean ± SEM and are representative of five independent experiments. (B) The colony-forming capacity of CD34+ human cord blood cells is reduced after miR-99 KD. CD34+ cells were transduced with lentiviral anti–miR-99 or scramble control. GFP+ cells were isolated and cultured in complete methylcellulose, and the colonies were scored after 14 d. Data represent mean ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (C) Flow cytometric evaluation of myeloid differentiation marker expression on MonoMac6 AML cells 5 d after transduction with anti–miR-99 or scramble control. Data represent mean ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (D) Wright–Giemsa stains of cytospin preparations of MonoMac6 cells 8 d post-transduction with lentiviral anti–miR-99 or Scr reveals induction of differentiation upon miR-99 KD (bars, 25 µm). (E) Overview of the xenotransplantation experiment performed on MonoMac6 AMLs. Cells were transduced with anti–miR-99 or Scr. After 48 h, GFP+ cells were flow sorted, and 800,000 cells were transplanted into sublethally irradiated NSGs. BM was analyzed 4 wk after the transplant. (F) miR-99 KD reduces the of GFP+ engraftment of MonoMac6 cells in the BM of recipients 4 wk post-transplantation. Data represent mean percentage ± SEM (Student’s t test; n = 4) and are representative of two independent experiments. (G) Representative histogram and aggregated data from flow cytometric evaluation of CD14 expression on GFP+ xenografted cells in the BM of the recipient animals 4 wk post-transplantation. Data represent mean percentage ± SEM (Student’s t test; n = 4) and are representative of two independent experiments. (H) Flow cytometry analysis of three AML patient samples after miR-99 KD. Patient samples were transduced with anti–miR-99 or scramble control and analyzed for the expression of differentiation markers 5–8 d later. Data represent mean percentage ± SEM (Student’s t test; n = 2) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 6.
Figure 6.
Forward genetic screen identifies miR-99 target genes. (A) Heat map shows genes differentially expressed between scramble control and miR-99 KD LSK cells. RNA-Seq was performed on LSK cells 48 h after transduction with miR-99 KD or scramble control lentivirus. Infected cells were FACS sorted based on GFP expression (n = 3 for control and n = 2 for miR-99 KD). (B) Functional annotation of genes up-regulated in LSK cells after miR-99 KD using the database for annotation, visualization, and integrated discovery (DAVID). The top functional groups are depicted as a function of −log (p-value). (C) GSEA of the differentially expressed genes in LSK cells after miR-99 KD reveals induction of a differentiation gene signature. The gene set denotes genes down-regulated in CD133+ cells (hematopoietic stem cells [HSCs]) compared with CD133− cells. (D) Top up-regulated miR-99 target genes identified from RNA-seq experiments in LSK cells transduced with miR-99 KD or scramble control vectors. FC, fold change. (E) Schematic representation of the shRNA library screen experiment designed to identify miR-99 target genes that rescue the hematopoietic phenotype induced by miR-99 KD. LSK cells were cotransduced with lentiviral anti–miR-99 and the retroviral shRNA library. 48 h later, the resulting GFP+mCherry+ cells were FACS sorted into complete methylcellulose and allowed to form colonies. 10 d later, colonies were resuspended, and cells were replated a second time and cultured for an additional 7 d. gDNA from the resulting GFP+ mCherry+ cells was deep sequenced to identify integrated shRNAs. Representative data from three independent experiments are shown. (F) Pooled shRNA library screen identifies genes that are positively selected in LSKs transduced with anti–miR-99 KD. The y axis depicts normalized enrichment scores, which is defined as the enrichment score for each shRNA divided by its enrichment score at T0, calculated as the mean of three independent experiments, and shown in descending order. The x axis denotes genes from the screen that are predicted to be targeted by miR-99 both in mouse and human. Positive controls included shRNAs targeting Tet2 and Tgif1. Average data from three independent experiments are shown. (G) Normalized enrichments scores and the number of independent enriched shRNAs for each gene, depicted for the top genes identified in the shRNA screen. Average data from three independent experiments are shown.
Figure 7.
Figure 7.
HOXA1 mediates miR-99 function in normal and malignant hematopoiesis. (A) TaqMan quantitative RT-PCR for HOXA1, BMPR2, and RAVER2 expression upon miR-99 overexpression in MonoMac6 AML cells 48 h post-transduction. Expression was normalized to ACTB (Student’s t test; n = 3). Representative data from two independent experiments are shown. (B) TaqMan quantitative RT-PCR for HOXA1, BMPR2, and RAVER2 expression upon miR-99 KD in MonoMac6 AML cells 48 h post-transduction. Expression was normalized to ACTB (Student’s t test; n = 3). Representative data from two independent experiments are shown. (C and D) Flow cytometry analysis of MonoMac6 cells 4 d after transduction with HOXA1-overexpressing virus. miR-99 KD induces the myeloid differentiation markers CD15 (C) and CD14 (D). Data represent mean percentage ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (E) Wright–Giemsa staining of MonoMac6 cells 4 d post-transduction with miR-99 KD (bars, 25 µm). (F) Growth curve for MonoMac6 cells as a function of time after transduction. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (G) Representative flow cytometry graph and the corresponding diagram depicting Annexin V apoptosis assay with HOXA1 overexpression in MonoMac6 cells. Data represent mean percentage ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (H) TaqMan quantitative RT-PCR for Hoxa1 expression 48 h after shHoxa1 transduction of LSK cells. Expression was normalized to Actb (Student’s t test; n = 3). Representative data from two independent experiments are shown. (I) Hoxa1 KD partially rescues colonies reduced upon miR-99 KD. LSK cells were infected with anti–miR-99 (GFP+) and shHoxa1 (mCherry+) viruses. 2 d post-transduction, the resulting GFP+ mCherry+ cells were sorted into Methocult M3434 and replated after 7 d. Shown are the results from secondary colonies, which were scored 10 d after plating. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative data of two independent experiments. (J) Simultaneous KD of Hoxa1 in miR-99 KD LSKs increases the size of miR-99 KD colonies. Shown are representative colonies from secondary platings. (K) Schematic for Hoxa1 KD LSC rescue experiments. miR-99 KD MLL-AF9 (GFP+ tdTom+) BM blasts from primary recipients were transduced with shHoxa1 (mCherry+), and the resulting GFP+ tdtom+ mCherry+ cells were transplanted into sublethally irradiated mice. (L) Kaplan–Meier curves of mice transplanted with MLL-AF9 blasts. 100,000 BM blasts from primary recipients of the indicated genotypes were transplanted into sublethally irradiated secondary recipients (Mantel–Cox test; n = 8 per condition). Representative data from two independent experiments are shown. (M) Flow cytometry analysis of L-GMPs from the BM of secondary recipients. Cells were pregated on GFP+ tdTom+ mCherry+ CD16/32+ cells. Data represent mean percentage ± SEM (Student’s t test; n = 3 for Scr and shHoxa1#1 and n = 4 for miR-99 KD and shHoxa1#2) and are representative of two independent experiments. (N) TaqMan quantitative RT-PCR for Hoxa1 expression in L-GMPs sorted from secondary recipients at the time of death. Expression was normalized to Actb. Data represent mean ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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