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. 2010 May 1;70(9):3833-42.
doi: 10.1158/0008-5472.CAN-09-3268. Epub 2010 Apr 20.

The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression

Piu Wong  1 , Masayuki Iwasaki, Tim C P Somervaille, Francesca Ficara, Christine Carico, Christopher Arnold, Chang-Zheng Chen, Michael L Cleary
Affiliations

The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression

Piu Wong et al. Cancer Res. .

Abstract

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function.

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Conflict of interest statement

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. miR-17 family microRNAs are aberrantly expressed in MLL LSCs
(A) MiRNA expression levels were determined by quantitative RT-PCR of LSC-enriched (c-kit+) versus LSC-depleted (c-kit-) cell populations of MLL-AF10 AML. Data are expressed relative to miRNA levels in the c-kit- fraction (±SEM of triplicate analyses). (B) Expression levels of miR-17-5p are shown for c-kit+ versus c-kit- spleen cells isolated from leukemic mice. Data are expressed relative to levels observed in c-kit- leukemia cells (±SEM of triplicate analyses). (C) miR-17-5p, miR-106a and miR-20a levels in myeloid progenitors transduced by MLL-ENL-ER were determined at day 5 following withdrawal of tamoxifen (4OHT). Data are expressed relative to miR levels at day 0 (±SEM of triplicate analyses). (D) Expression levels of miR-17-5p and miR-20a in normal and leukemic progenitors were determined by RT-PCR. Bar chart displays data relative to levels in LKS cells (±SEM of triplicate analyses).
Figure 2
Figure 2. Over-expression of the miR-17-19b construct accelerates MLL-mediated leukemia
(A) The miR-17 polycistron is schematically shown (top) with the miR-17-19b construct indicated by brackets. Respective microRNA expression levels indicated below were determined by quantitative RT-PCR of c-kit enriched myeloid progenitors (c-kit+) co-transduced with MLL-AF10 and miR-17-19b. Data are expressed relative to the miRNA levels observed in cells co-transduced with MLL-AF10 plus vector (±SEM of triplicate analyses). (B) Survival curves are shown for cohorts of mice transplanted with myeloid progenitors co-transduced with MLL-AF10 and miR-17-19b or empty vector. Acute leukemia was confirmed by peripheral blood leukocyte counts, FACS analyses, and/or necropsy.
Figure 3
Figure 3. Forced expression of miR-17-19b blocks myeloid differentiation and increases LSC frequencies in MLL leukemias
(A) FACS profiles show representative expression levels for c-kit and Mac1 on splenocytes from mice with AML induced by transplantation of cells transduced with the indicated genes. Bar graph depicts mean (±SEM) expression levels (n=5 in each cohort). (B) The cytologic features are shown for splenocytes from leukemic mice transplanted with cells transduced with the indicated genes. Bar graph shows the mean number of cells with the indicated morphologic features (n=5 in each cohort). 500 cells were counted in total. Blasts are morphologically defined by increased cell size and enlarged nucleus with significantly reduced cytoplasmic content. (C) Bar graph depicts the CFC frequencies determined for Mac1+ splenocytes FACS-purified from leukemic mice and cultured in methylcellulose medium. Data are the CFC numbers (±SEM) observed in each round of two serial platings (n=5).
Figure 4
Figure 4. Increased cell proliferation induced by co-expression of miR-17-19b in MLL-AF10 leukemia cells is mediated through p21 suppression
(A) DNA content was determined by FACS analysis of explanted leukemia cells cultured in methylcellulose medium for 5 d. Bar graph summarizes data (±SEM) for three mice in each cohort. (B) Sequence alignment of the miR-17-5p seed region with a portion of the p21 mRNA 3’UTR. (C) Bar graph shows expression levels of the indicated transcripts as determined by Q-PCR analysis of splenocytes isolated from leukemic mice. Results are the mean (±SEM) of three leukemias in each category, and are expressed relative to transcript levels in leukemia cells transformed by MLL-AF10 alone. Right panel shows western blot analysis for p21 in leukemia cells induced by co-transduction of the genes indicated at the top of the panel. Tubulin served as a loading control.
Figure 5
Figure 5. p21 is a critical target of the miR-17 polycistron in MLL leukemia
(A) p21 levels were determined by quantitative RT-PCR of c-kit enriched myeloid progenitors (c-kit+) co-transduced with MLL-AF10 and the p21 knockdown vectors. Data are expressed relative to the miRNA levels observed in cells co-transduced with MLL-AF10 plus vector (±SEM of triplicate analyses). Right panel shows western blot analysis for p21 in cells transduced by the genes indicated at the top of the panel. Tubulin served as a loading control. (B) Survival curves are shown for cohorts of mice transplanted with myeloid progenitors co-transduced with MLL-AF10 and p21 knockdown or empty vectors. Acute leukemia was confirmed by peripheral blood leukocyte counts, FACS analyses, and/or necropsy. (C) DNA content analysis was performed by FACS analysis of explanted leukemia cells cultured in methylcellulose medium for 5 d. Bar graph summarizes data (±SEM) for three mice in each cohort. (D) Luciferase reporter assays in MV4;11 cells transfected with the indicated constructs. Error bars represent the mean (± SD) of 3 independent experiments.
Figure 6
Figure 6. c-myc is a critical upstream regulator of the miR-17 polycistron in MLL leukemia
(A) Bar graph depicts the CFC frequencies determined for progenitor cells (c-kit+) transduced with MLL-AF10 + c-myc or MLL-AF10 alone and cultured in methylcellulose medium. Data are the mean CFC numbers (±SEM) observed in each round of serial plating (n=3). (B) Bar graph shows expression levels of the indicated transcripts as determined by Q-PCR analysis of transformed cells. Results are the mean (±SEM) of three samples in each category, and are expressed relative to transcript levels in cells transformed by MLL-AF10. (C) c-myc transcript levels are shown for c-kit+ versus c-kit- spleen cells isolated from leukemic mice. Data are expressed relative to levels observed in c-kit- leukemia cells (±SEM of triplicate analyses).
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