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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 to 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. Knock down of p21 in MLL transformed cells phenocopied over-expression 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 expression of p21, a known regulator of normal stem cell function.
MicroRNAs are small ~22-nt regulatory RNAs (1) that serve important roles in a variety of normal and pathologic processes in a wide range of organisms. They function in association with the RNA-inducing silencing complex to control gene expression at the post-transcriptional level by mediating mRNA translational repression and/or triggering RNA degradation (2, 3). MicroRNAs may regulate expression of approximately 25-50% of the mRNA transcriptome (4).
MicroRNAs have diverse and important roles in animals. They are differentially expressed during development of multiple cellular lineages and respond to a variety of extracellular signals. Furthermore, mis-regulation of miRNAs has been implicated in the pathogenesis of various diseases, including hematopoietic malignancies (5). For example, miR-15 and miR-16, which are located in the commonly deleted region of chromosome 11 in B-cell chronic lymphocytic leukemia (CLL), are potential tumor suppressors whose decreased expression levels are associated with increased expression of their predicted target BCL2 (6), a suppressor of apoptosis. MiRNAs also function as potent oncogenes, or so-called oncomirs. For example, enforced expression of miR-155 in hematopoietic progenitors leads to myeloproliferative disease and in transgenic mice induces B-cell lymphoma (7, 8).
The first oncomir to be characterized was miR-17-92 (referred to as miR-17 polycistron), which encodes seven mature microRNAs: miR-17 5p, miR-17 3p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92 1. Increased expression of the miR-17 polycistron is a feature of lymphomas with amplification of the C13orf25 genes at chromosome 13q31.3. Experimental over-expression of the miR-17 polycistron in a c-Myc transgenic mouse model accelerates lymphoma onset, which was subsequently shown to result from miR-19a repression of Pten to promote survival (9-11). Moreover, enforced expression of the miR-17 polycistron promotes proliferation in CML and B-cell lymphoma cell lines by targeting expression of the cyclin-dependent kinase inhibitor p21 (12, 13). Increased dosage of individual members of the polycistron, including miR-17-5p, miR-20a or miR-106a, which share the same seed sequences, effectively blocks human monocytic differentiation through suppression of the AML1 proto-oncogene (14). Taken together, these studies demonstrate that the contribution of individual miR-17 microRNA(s) and their respective targets vary in different hematologic lineage neoplasms. More recently, miR-17 polycistron miRNAs have been shown to be highly expressed in human MLL leukemias, although the functional consequence of this remains to be determined (24, 25).
Here, we demonstrate that the miR-17 polycistron regulates leukemia stem cell (LSC) potential in a mouse model of MLL-associated acute myeloid leukemia (AML) by modulating expression of the cyclin-dependent kinase inhibitor p21. Polycistron expression is down-regulated upon exit of LSCs from the self-renewing compartment, whereas forced expression blocks myeloid leukemia cell differentiation, enhances proliferation, and significantly decreases leukemia latency in vivo. Knockdown of p21 phenocopies miR-17 polycistron over-expression in MLL leukemia cells, validating p21 as a biologically relevant in vivo target and a downstream rate-limiting regulator of LSC potential.
C57BL/6 mice congenic for CD45 (Ly5.1/Ly5.2) were employed for transplant studies. All experiments on mice in this study were performed with the approval of and in accordance with Stanford University’s Administrative Panel on Laboratory Animal Care.
Retroviral constructs encoding MLL-AF10 and the miR-17-19b polycistron have been previously described (9, 15, 16). Knockdown constructs for p21 (TRCN0000042583 and TRCN0000042586) were purchased from Open Biosystems (Huntsville, AL). pBabe-c-myc-zeo was reported previously (17). Retroviral transductions and in vitro replating assays were performed essentially as described previously using primary murine myeloid progenitors harvested from bone marrow (16). Cells transduced with retroviral vectors were selected for stable transduction in methylcellulose medium containing the appropriate antibiotic (1 μg/ml puromycin and/or 1mg/ml neomycin and/or 0.3 mg zeocin/ml). The estrogen inducible MLL-ENL system was used as described (18).
Transplantation experiments were performed as described previously (19) with the following minor modifications. For co-transduction experiments, transduced progenitors were incubated in 0.9% methycellulose medium containing cytokines (20 ng/ml SCF, 10 ng/ml IL-6, 10 ng/ml IL-3) in the presence of puromycin (1 μg/ml) and neomycin (1 mg/ml) for five days, and then transplanted (1 × 105 cells) together with a radioprotective dose of total bone marrow cells (1 × 105) into the retro-orbital venous sinus of 6-12 week-old syngeneic C57BL/6 mice that had been lethally irradiated with 9.0 Gy of total body γ irradiation (135Cs). When transplanted mice exhibited signs of ill health (shortness of breath, lethargy and hunched posture) they were euthanized. Donor and recipient cells were distinguished by FACs analysis of CD45 congenic marker expression. Necropsy tissues were fixed in buffered formalin, sectioned, and stained with hematoxylin and eosin (H&E) for histological analysis.
Bone marrow and spleen cells were stained with fluorochrome-conjugated monoclonal antibodies against c-kit (2B8 clone), Mac-1 (M1/70 clone) and Gr-1 (RB6-8C5 clone) for analysis of leukemic cell differentiation using antibodies purchased from PharMingen or eBioscience and procedures described previously (16). FACS sorting conditions used to obtain highly purified populations for LSK (Lin-/c-kithigh/Sca-1high), CMP (Lin-/c-kithigh/Sca-1-/CD34+/FcγRlow), and GMP (Lin-/c-kithigh/Sca-1-/CD34high/FcγRhigh) were described previously (20). LSC-enriched and LSC-depleted leukemia cell populations were isolated by FACS based on c-kit expression using conditions described previously (21). DNA content analysis was performed by PI staining and analyzed by FACS. Apoptosis was quantified using an annexin V apopotosis detection kit according to the manufacturer’s instructions (BD Pharmingen).
p21 wild type and 3’UTR mutant luciferase constructs were made as previously described (13). Two million MV4;11 cells were electroporated with 1μg miR-17-19b DNA, 0.8 μg of the firefly luciferase reporter DNA, and 0.16 μg pRL-TK control DNA (Promega) using the Amaxa nucleofection technology™ (Amaxa, Koeln, Germany) program A-30. Forty-eight hours later, firefly and Renilla luciferase activities were measured using dual-luciferase assays (Promega). Three independent experiments were carried out.
Leukemia cells were harvested and lysed in 250 μL 2× sample buffer. Proteins from approximately 20 μL lysate were fractionated by electrophoresis through 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (BioRad, Richmond, CA) using Tris-glycine sodium dodecyl sulfate transfer buffer. After blocking with 5% milk, proteins were detected using an anti-p21 polyclonal antibody (c-19, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-tubulin monoclonal antibody (DM-1a, Sigma, St. Louis, MO).
RT-PCR profiling of individual miRNAs was performed according to the manufacturer’s instructions (Applied Biosystems) using FACS purified c-kit+ and c-kit- cells from murine leukemias. cDNA was synthesized and subjected to real-time PCR using commercially prepared reagents according to the manufacturer’s instructions. Taqman probes for the following miRNAs and genes were purchased from Applied Biosystems: has-miR142-5p (465); has-miR-14-23p (1189); has-miR17-5p (393); has-miR18a (2422); has-miR19a (395); has-miR19b (396); has-miR-20a (580); has-miR-16 (391); mmu-miR-106a (1128); has-miR10a (2288); has-miR196b (496); has-miR155 (479); has-miR-204 (508); snoRNA202 (1232); p19Arf (Mm01257348); Cdkn1a (p21) (Mm01303209_m1); Cdkn1b (p27) (Mm00438167_g1); Cdkn2a (Mm00494449_m1); Cdkn2c (Mm00483243_m1); Cdkn4b p15 (Mm00483241_m1); c-myc (Mm00501741_m1); E2F1 (Mm432939_m1); E2F3 (Mm01138833_m1); β-Actin (Mm00607939_s1). Primers for mouse p16Ink4a were designed previously (22) and purchased from Applied Biosystems. Expression levels of target mRNA transcripts and miRNAs were normalized using β-Actin and snoRNA 202, respectively, based on the Ct values.
Expression profiling was performed to identify miRNAs that are preferentially expressed in MLL LSCs. LSC-enriched sub-populations in AMLs initiated by the MLL-AF10 oncogene were prospectively isolated by flow cytometry based on their differential expression of c-kit (16, 21). High throughput RT-PCR profiling of more than 200 individual microRNAs revealed that ~20% were expressed at more than 2 fold (p<0.001) higher levels in LSC-enriched (c-kit+) versus LSC-depleted (c-kit-) AML cell fractions, whereas ~10% displayed the opposite profile (C. A. & C-Z. C., in preparation). The upregulated miRNAs included several (miR-10a, miR-20a, miR-17-5p, miR-106a, miR-155, miR-181a and miR-196b) (Figure 1A) that have previously been implicated in regulating normal and aberrant hematopoiesis (23, 24). MiR-17-5p and miR-20a are both encoded within the miR-17-92 polycistron, which has been implicated in B-cell lymphoma and modulation of myeloid differentiation (9, 14). MiR-17 cluster miRNAs are also highly expressed in human MLL leukemias (25, 26), but the functional consequences are unknown. Therefore, we focused on the contributions of miR-17 cluster miRNAs in MLL-mediated leukemias.
Analysis of additional molecular subtypes of MLL leukemia (MLL-LAF4 and MLL-GAS7) showed a 2-3 fold higher level of miR-17-5p in c-kit+ versus c-kit- MLL leukemia cells (Figure 1B) demonstrating that miR-17 family microRNAs are consistently expressed at higher levels in the LSC-enriched fraction irrespective of the MLL fusion partner.
Using an estrogen-inducible system for controlling the activity of MLL-ENL, the levels of miR-17-5p, miR-106a and miR-20a decreased dramatically (4-16 fold) 5 days after inactivation of MLL-ENL following withdrawal of tamoxifen, which caused loss of self-renewal and terminal differentiation (Figure 1C). Thus, miR-17 miRNAs are preferentially expressed in self-renewing MLL leukemia cells.
Quantitative RT-PCR analysis was performed on prospectively isolated normal progenitor and differentiated myeloid cell populations to assess miRNA expression profiles within the hematopoietic compartment. Expression of miR-17-5p and miR-20a progressively increased with differentiation from multipotent progenitors (Lin- Sca-1+ c-kit+) through CMP and peaked at the GMP stage. Expression levels dropped dramatically with further myeloid differentiation and were lowest in cells with the most mature myeloid immunophenotype (c-kit- Gr1+ Mac1+) (Figure 1D). The mid-myeloid peak in expression was similar to that of genes critical for maintenance of LSC self-renewal suggesting that microRNAs of the miR-17 polycistron may be involved in regulating transient self-renewal of normal myeloid progenitors as well as the enhanced self-renewal of MLL LSCs (27). Furthermore, the expression levels of miR-17-5p and miR-20a in the c-kit positive leukemia cell population were 3-4 fold higher than in normal GMPs (Figure 1D), indicating that abnormal expression of the miR-17 cluster is a feature of MLL LSCs.
To explore the possibility that the miR-17 polycistron may contribute to MLL-associated leukemia, a construct encoding all of its constituent miRNAs except miR-92b (miR-17-19b) was co-expressed along with MLL-AF10 to determine whether its enhanced expression affects MLL leukemogenesis. Myeloid progenitors (c-kit+) co-transduced with MLL-AF10 plus miR-17-19b (denoted MA10/miR17) exhibited miR-17-5p and miR-20a transcript levels that were 2-3 fold higher than progenitors co-transduced with MLL-AF10 plus empty vector (MA10/v) (Figure 2A). The LSC non-specific miRNAs within the cluster (miR-18a, miR-19a, miR-19b) showed minimal fold induction (<1.5), therefore this construct faithfully expressed MLL LSC-specific miRNAs at a high level (Figure 2A). Following transplantation of 100,000 co-transduced cells with an equal number of total bone marrow radio-protective cells into lethally irradiated recipient mice, the MA10/miR17 cohort developed leukemia with substantially shortened latencies (median 36 days) compared to mice transplanted with MA10/v co-transduced cells (median latency 60 days) (p<0.001) (Figure 2B). Mice in both cohorts displayed similar pathologies with bone marrow effacement by blasts, enlarged spleens, and infiltration of the liver by leukemic cells (data not shown). Therefore, the miR-17 polycistron serves a rate-limiting role in the progression of MLL-associated leukemia.
FACS analysis demonstrated that all leukemias derived from transplanted cells exclusively displayed myeloid phenotypes (Mac1+ Gr1+), indicating that the miR-17 polycistron did not play an instructive role in influencing the lineage derivation of leukemia. However, MA10/miR17 leukemia cells displayed a more undifferentiated phenotype (high levels of c-kit and lower levels of Mac1) suggesting that the miR-17 polycistron blocked myeloid differentiation in MLL-induced leukemia (Figure 3A).
Morphologic assessment of splenocytes obtained at necropsy of MA10/miR17 leukemic mice revealed an approximately two fold higher proportion of blast cells and substantially fewer differentiated macrophages and neutrophils (Figure 3B) compared to splenocytes from MA10/v mice. Furthermore, in semi-solid culture assays, the frequencies of colony forming cells (CFCs), which correspond to LSCs (21, 27), in the spleens of leukemic mice were approximately 2 fold more abundant for MA10/miR17 leukemias (Figure 3C). Leukemia cells transduced by MA10/miR17 continued to self-renew at a higher rate generating almost 3 fold more colonies in a subsequent passage than cells transduced with MA10/v (Figure 3C). Thus, MLL leukemias over-expressing the miR-17 polycistron exhibit less differentiation as well as a higher frequency of clonogenic leukemia cells with features of LSCs.
Cell cycle analysis of explanted leukemia cells growing in methylcellulose cultures revealed approximately a 15% increase in the proportion of cells in S/G2/M for MA10/miR17 versus MA10/v leukemias (Figure 4A). The higher fraction of cells in cycle correlated with substantially lower transcript and protein levels for the CDK inhibitor p21, but not other cell cycle regulators, across three pairwise, independently generated leukemias (Figure 4C). The apoptosis rate as measured by annexin V and 7-AAD staining was indistinguishable between MA10/miR17 versus MA10/v leukemias (data not shown). Since the seed regions (2-7 nucleotides of 5’ microRNA) of miR-17-5p and miR-20a share perfect complementarities with the 3’ untranslated region of p21 mRNA (Figure 4B), this suggested that these microRNAs may directly regulate p21 to modulate cell cycle progression in MLL leukemia cells.
p21 expression was knocked down in MLL transduced cells to assess whether its silencing recapitulates the effect of mir-17-19b construct over-expression in MLL-mediated leukemia. Myeloid progenitors (c-kit+) co-transduced with an MLL oncogene plus p21 knockdown construct (MA10/p21kd) exhibited 40-60% lower p21 transcript and protein levels than progenitors co-transduced with the MLL-AF10 oncogene plus empty vector (MA10/v) (Figure 5A). Following transplantation of 50,000 co-transduced cells with 100,000 total bone marrow radio-protective cells into lethally irradiated recipient mice, the MA10/p21kd cohort developed leukemia with substantially shortened latencies (median 58 days) compared to mice in the MA10/v cohort (median 90 days) (p<0.001) (Figure 5B). Leukemias in both cohorts displayed similar pathologic features (data not shown), but explanted leukemia cells co-transduced with p21 kd constructs showed 10-15 % increase in the percentage of cells in S/G2/M when compared to MA10/v leukemias (Figure 5C). Moreover, reporter assays performed in the MV4;11 MLL leukemia cell line showed that expression of a luciferase reporter containing the p21 3’UTR was reduced by 40% when co-expressed with the miR-17-19b polycistron compared to the vector alone. The observed reduction was highly specific since mutations of the miR-17 seed sequences in the p21 3’UTR prevented comparable reporter activity reduction (Figure 5D). Thus, p21 is a critical in vivo and direct target of the miR-17 polycistron in mediating MLL leukemias.
c-myc has been shown to directly bind and activate expression of the miR-17 polycistron in a mouse lymphoma model (10). Therefore, we investigated if enforced expression of c-myc together with an MLL fusion oncogene would induce enhanced oncogenic properties through the miR-17 pathway. In a serial replating assay, co-expression of c-myc with MLL-AF10 resulted in at least two-fold higher colony numbers in the second and third rounds of plating when compared to the MLL-AF10 + vector control transduced cells (Figure 6A). Moreover, the levels of MLL LSC-specific microRNAs miR-17-5p and miR-20a were upregulated by more than 3-fold (Figure 6B). This is consistent with the finding that c-myc levels were 2-fold higher in LSC-enriched versus LSC-depleted leukemia cell populations (Figure 6C). These results suggest that MLL fusion oncogenes interface with the c-myc network to de-regulate expression of the miR-17 cluster and maintain LSC self-renewal.
A single microRNA may regulate more than 100 transcripts and therefore could serve as a potential master regulator of the oncogenetic program of cancers, including leukemia (28). To identify candidate oncomirs that may contribute to MLL leukemogenesis, we screened for microRNAs differentially expressed in AML sub-populations enriched versus depleted for LSCs in a mouse model that recapitulates many of the pathologic and molecular features of human AML. Of several differentially expressed microRNAs, the miR-17 polycistron was found to be a rate-limiting regulator of MLL LSC potential through modulation of p21 levels. Although the miR-17 polycistron has previously been associated with hematologic cancers and implicated in targeting of p21, our current studies link this oncomir and CDKI in the molecular regulation of LSC self-renewal in AML for the first time.
In AMLs induced by MLL oncoproteins, the c-kit+ fraction of leukemia cells is highly enriched for LSCs (21). In addition to marked differences in leukemogenic, clonogenic, and morphologic properties, over 5,000 genes are differentially expressed in the c-kit+ versus c-kit- cell fractions from AMLs induced by MLL-AF10, and several have been validated as critical for LSC maintenance (27). Therefore, we hypothesized that these functionally and molecularly distinct subpopulations might also differentially express miRNAs that contribute to LSC biology. Consistent with the substantial differences in gene expression, almost one-third of analyzed microRNAs displayed 2-fold or greater differences in expression (data not shown). One of the differentially expressed miRNAs is miR-155 (6 fold higher in the c-kit+ fraction), which has been shown to induce a myeloproliferative disease when sustainably expressed in HSCs (8). Its differential expression in LSCs in our model and high level expression in a subset of human AMLs including those with MLL aberrations (8), strongly support a pathogenic role in MLL leukemias. The largest group of differentially expressed miRNAs, however, comprised members of the miR-17 family, which are encoded in paralogous polycistrons such as miR-17-92. Although originally characterized for its role as an oncomir in human and mouse lymphomas, our studies implicate miR-17-92 and related microRNAs in AML pathogenesis consistent with their high expression in human leukemias containing MLL chromosomal aberrations (25, 26).
Previous studies have shown that expression of miR-17 family members, including miR-17-5p, 20a and 106a, decreases with progressive differentiation along the myeloid, megakaryocytic, and monocytic lineages (14, 29). Our studies further refine the normal expression profiles by showing that levels of miR-17-5p and miR-20a peak in GMPs and committed myeloid precursors, then rapidly decrease with terminal differentiation. This suggests that miR-17 family microRNAs may normally regulate the transient self-renewal and proliferative expansion of mid-myeloid progenitors/precursors. However, expression of the miR-17-19b cluster alone in myeloid progenitors failed to induce immortalization in vitro or leukemia when transplanted in vivo (data not shown). Interestingly, the downstream progenitors that normally express the miR-17 polycistron constitute the normal cellular counterparts of MLL LSCs, which maintain their aberrant self-renewal following conversion of a mid-myeloid gene expression program into an LSC maintenance program (27). Thus, miR-17 family microRNAs may represent additional components of the LSC maintenance program rather than direct transcriptional targets of MLL oncoproteins.
Ectopic expression of the miR-17-19b construct leads to reduction of endogenous p21 levels in MLL leukemia cells as well as reduced expression of a transfected luciferase reporter gene harboring the p21 3’UTR region. Furthermore, knock down of p21 partially recapitulates the phenotype of miR-17-19b polycistron over-expression, thus validating p21 as a physiologic and direct target in regulating LSC frequency and accelerating MLL leukemia. Since MLL oncoproteins can efficiently transform fetal liver progenitors harvested from miR-17 polycistron knockout mice, the polycistron is dispensable for MLL leukemogenesis (data not shown). MiR-17-5p family members miR-20a and miR-106b, which share an identical seed sequence with miR-17-5p, have also been shown to target p21 (30) and are both differentially expressed in MLL LSCs. As a CDKI, p21 inhibits the activity of cyclin-CDK2 or CDK4 complexes, thus regulating cell cycle progression at G1. Expression of p21 is normally induced during p53-dependent cell cycle arrest in response to a variety of stress stimuli such as DNA damage, however MLL oncoproteins were reported to suppress p53-mediated p21 induction in response to ionizing radiation (31). MiRNAs of the miR-17 polycistron may therefore provide another level (post-transcriptional) of p21 suppression in addition to MLL oncoprotein blockade of p53 whose levels were not detectably altered comparing MA10/v- and MA10/miR17-induced leukemias (data not shown). p21 has recently been implicated in promoting LSC self-renewal through modulation of quiescence and DNA repair (32). However, its role in MLL LSCs is to antagonize self-renewal by inhibiting cell cycle progression. Recent studies demonstrate that miR-19 within the miR-17 polycistron is responsible for accelerating disease onset in the c-myc mediated lymphoma mouse model (10, 11). Other proposed targets of miR-17 family members, including AML1 (14), were not down-regulated in MLL-AF10 leukemias over-expressing the miR-17-19b polycistron (data not shown). Taken together, the findings suggest that different microRNAs within the cluster contribute to its oncogenicity and regulate different targets depending on cell type and the specific oncogenic stimulus.
Transcription of the miR-17 cluster is directly regulated by c-myc (33). Interestingly, c-myc is critical for MLL-mediated transformation (34). Indeed, c-myc expression is down-regulated more than 2-fold in LSC-enriched versus LSC-depleted MLL leukemia cell populations consistent with comparable down-regulation of the miR-17-19b cluster in these respective populations. Although E2F1 and E2F3 have been reported to be direct targets of the miR-17 polycistron, and are capable of directly activating transcription of these miRNAs creating a negative feedback loop (33, 35, 36), levels of E2F1 and E2F3 were not down-regulated in MLL-AF10 leukemias over-expressing the miR-17-19b polycistron (data not shown). These observations suggest a model in which MLL oncoproteins sustain the expression of c-myc, which in turn activates the miR-17 polycistron to help maintain LSC self-renewal.
Hox proteins and their cofactors are integral components of the MLL leukemogenic pathway, therefore microRNAs that regulate or are co-expressed with Hox genes are candidates for pathogenic roles in MLL leukemogenesis. Previous studies have shown that miR-204 targets Meis1 and Hoxa10, and its low expression levels inversely correlate with high levels of these targets in AMLs carrying NPM1 mutations (37). Our microRNA expression analyses are consistent with these observations since the levels of miR-204 were extremely low (Ct value >35) in MLL leukemia cells expressing high levels of Meis1 and Hoxa10, but 2 fold higher in the cell fraction (c-kit-) depleted of LSCs (Figure 1) that displays modest down-regulation of Hox and Meis expression. We have shown previously that Meis1 is a critical and rate-limiting regulator of MLL LSC potential (16). MiR-10a, on the other hand, is highly co-expressed with Hox genes in AML with normal karyotypes (38). MiR-10a was the most differentially upregulated miRNA (~20 fold) in MLL LSCs (Figure 1), despite the fact that its neighboring HoxB genes were minimally expressed. MiR-10a has been implicated in affecting transformation susceptibility by positively controlling protein synthesis through stimulating ribosomal mRNA translation (39). MiR-196b, located between Hoxa9 and Hoxa10, is a common retroviral integration site and its expression is upregulated in MLL LSCs, consistent with its high level expression in leukemias with MLL translocations and its ability to induce proliferation and a partial block of myeloid cell differentiation (40). Based on their prior oncogenic associations and expression profiles, further studies are warranted to establish whether miR-10a and/or miR-204 may directly contribute to a broader genetic program that promotes or maintains MLL/Hox-mediated leukemogenesis.
In summary, miRNA expression profiling of MLL LSCs identified differentially expressed miRNAs, most notably members of the miR-17 family, which modulate differentiation state, self-renewal, cell cycle status, and LSC frequency at least in part through direct suppression of p21. This defines the mechanistic basis by which a hematopoietic oncomir influences LSC potential by modulating expression of a known regulator of stem cell self-renewal.
We acknowledge M Ambrus, C. Nicolas and K. Ochis for technical support. We thank Lin He for providing the miR-17-19b construct. We are very grateful to Irene Blat, Andrea Ventura and Tyler Jacks for providing and genotyping miR-17 knock-out fetal liver cells. These studies were supported by the Children’s Health Initiative of the Packard Foundation, grants from the National Institutes of Health (CA55029) and the Leukemia and Lymphoma Society, and in part by a Croucher Foundation Research Grant to P. Wong.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
P. Wong designed, performed and analyzed research and wrote the paper, M. Iwasaki, T.C.P. Somervaille, F. Ficara, C. Carico, and C. Arnold performed research, C-Z. Chen analyzed research, and M.L. Cleary analyzed research and edited the paper.