Mir-125 in Normal and Malignant Hematopoiesis

SPOTLIGHT REVIEW MiR-125 in normal and malignant hematopoiesis

L Shaham1,2, V Binder3,4,NGefen1,5, A Borkhardt3 and S Izraeli1,5

MiR-125 is a highly conserved microRNA throughout many different species from nematode to humans. In humans, there are three homologs (hsa-miR-125b-1, hsa-miR-125b-2 and hsa-miR-125a). Here we review a recent research on the role of miR-125 in normal and malignant hematopoietic cells. Its high expression in hematopoietic stem cells (HSCs) enhances self-renewal and survival. Its expression in speciﬁc subtypes of myeloid and lymphoid leukemias provides resistance to apoptosis and blocks further differentiation. A direct oncogenic role in the hematopoietic system has recently been demonstrated by several mouse models. Targets of miR-125b include key proteins regulating apoptosis, innate immunity, inﬂammation and hematopoietic differentiation.

Leukemia (2012) 26, 2011–2018; doi:10.1038/leu.2012.90 Keywords: microRNA; hematopoiesis; hematological malignancies; acute myeloid leukemia; acute lymphoblastic leukemia

MicroRNAs (miRNAs) are 21–23-nucleotide non-coding RNAs that nucleotides with the seed region of miR-125b (ebv-miR-BART21-5p, have crucial roles in fundamental biological processes by ebv-miR-BART8 and rlcv-miR-rL1-25). In humans, as in most of the regulating the levels of multiple proteins. They are transcribed genomes, there are two paralogs (hsa-miR-125b-1 on chromosome as primary miRNAs and processed in the nucleus by the RNase III 11 and hsa-miR-125b-2 on chromosome 21), coding for the same endonuclease DROSHA to liberate 70-nucleotide stem loops, the mature sequence. There is no obvious correlation between precursor miRNA. The precursor miRNAs are then exported from the number of paralogs and the complexity of the genome, as the nucleus to the cytoplasm by Exportin 5/RanGTP, where further the genome of the zebrafish (Danio rerio) contains three paralogs cleavage by the second RNase III enzyme DICER releases the of miR-125b, whereas there is only one copy in chimpanzees (Pan mature miRNAs.1–3 MiRNAs bind mainly to the 30untranslated paniscus), pufferfish (Tetraodon nigroviridis) or worms (Schistosoma region of mRNAs by direct base-pairing to the 50miRNA japonicum, Schmidtea mediterranea). seed region, resulting either in repression of gene translation1 In addition to paralogs that code for the same mature or induction of mRNA degradation.2 Recent data reveals, sequence, there are homologs that code for different mature however, that miRNA regulation entails a far more complex sequences, which contain the same seed region and therefore posttranscriptional control than initially anticipated.3 might have similar functions. In the human genome, the homolog Many miRNAs are genomically organized as clusters and are co- miR-125a is encoded on chromosome 19. In some other species transcribed from a single promoter as a single polycistronic (Danio rerio), there are three homologs encoded on different transcript, although inconsistent expression of members can be chromosomes (miR-125a, -125b and -125c), all sharing the same detected in some miRNA clusters.4,5 seed region (Figure 1a). Interestingly, miR-351, which is encoded Here, we review the role of miR-125 in normal and malignant only in the mouse and rat genome, has the same seed region as hematopoiesis. MiR-125, a highly conserved miRNA transcribed miR-125b. from three different clusters, has recently emerged as a key In humans, miR-125b-2 is organized in a cluster with miR-99a regulator of HSCs. It has been shown to be dysregulated in and let-7c. Hsa-miR-125b-2 is encoded B50 kb downstream of multiple malignancies and to affect cell survival, differentiation hsa-miR-99a and hsa-let-7c, which are located about 650 bp from SPOTLIGHT and proliferation through the suppression of multiple targets. each other. Although the distance of 50 kb is greater than the These fascinating recent discoveries raise many questions regard- standard distance for a cluster, analysis of various genomes shows ing its regulation, interactions with additional co-transcribed that this region is highly conserved in its organization, and miRNAs and mechanisms of activity. therefore this region is considered to be a cluster. A second cluster (on chromosome 11 in humans) contains the paralog miR-125b-1 with let-7a-2 and miR-100, which both differ in only one GENOMIC ORGANIZATION OF THE MIR-125B CLUSTER nucleotide outside of the seed region of their family members, MiR-125b is highly conserved among mammals, vertebrates let-7c and miR-99a, respectively. The genomic organization of and nematodes. Although the nomenclature sometimes differ this second cluster is similar to that of the first. A third cluster (for example cel-lin-4 in Caenorhabditis elegans), the seed region is (on chromosome 19 in humans) contains miR-99b, let-7e and the same throughout all species containing miR-125b (Figure 1a). miR-125a. Here, all three miRNAs are encoded close to each In viral genomes, there is a maximum concordance of only six other (o1 kb).

1Department of Pediatric Hemato-Oncology, Functional Genomics and Childhood Leukemia Research, Cancer Research Center, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel; 2The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel; 3Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University, Du¨sseldorf, Germany; 4Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA and 5Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Correspondence: Professor S Izraeli, Department of Pediatric Hemato-Oncology, Functional Genomics and Childhood Leukemia Research, Cancer Research Center, Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel. E-mail [email protected] The study is partial fulﬁllment of the requirements for completing a PhD degree (Lital Shaham) for Bar-Ilan University, Ramat Gan, Israel. Received 27 April 2011; revised 12 March 2012; accepted 13 March 2012; accepted article preview online 29 March 2012; advance online publication, 24 April 2012 Mir 125 in hematopoiesis and leukemia L Shaham et al 2012

Seed region

Host gene‚ MiR-100-let-7a-2 cluster host gene‘

1 2

[-] Chr 11 Hsa-miR-100 Hsa-let-7a-2 Hsa-miR-125b-1

50 kb 6 kb 47 kb Host gene‚ LINC00478‘

3 4

[+] Chr 21 SPOTLIGHT Hsa-miR-99a Hsa-let-7c Hsa-miR-125b-2

470 kb <1 kb 50 kb

[+] Chr 19 Hsa-miR-99b Hsa1-let-7e Hsa-miR-125a

<1 kb <1 kb Figure 1. (a) The seed region of homologs and orthologs of miR-125 is highly conserved throughout different species. (b) Genomic organization of the miR-cluster containing miR-125, shown together with their host genes and potential promoter regions. 1: A putative promoter predicted by Long et al.,7 Wang et al.,9 Ozsolak et al.8 and Corcoran et al.6 2: A putative promoter predicted by Long et al.,7 Wang et al.,9 and Corcoran et al. 6 3: A putative promoter predicted by Long et al.,7 Wang et al.,9 Ozsolak et al.,8 Corcoran et al.,6 and Montey et al.11 4: A putative promoter predicted by Corcoran et al.,6 and Ozsolak et al.8 5: A putative promoter predicted by Long et al.,7 Wang et al.,9 Ozsolak et al.,8 and Corcoran et al.6 Dashed lines indicate structures that were annotated only in a single study.

This composition suggests that the cluster containing miR-125a the miR-125b clusters. Whereas most of the studies agree on probably has only one promoter and is transcribed as one hsa-miR-99a, hsa-let-7c and hsa-miR-125b-2 being intragenic transcript, whereas the cluster containing miR-125b may (host gene LINC00478, chr 21: 17.442.842–17.982.094; contain an alternative promoter, making it possible to transcribe NC_000021.8, NCBI build 37.2) and hsa-miR-100, hsa-let-7a-2, miR-125b solely (Figure 1b).6–9 This hypothesis is supported by hsa-miR-125b-1, hsa-miR-99b, hsa-let-7e and hsa-miR-125a being recent research predicting miRNA promoters by combining intergenic, Corcoran et al.6 claim that hsa-miR-100 and hsa-let-7a-2 bioinformatic analysis of nearby upstream DNA regulatory are also intragenic (host gene, non-protein coding, chr 11: elements (transcription start sites (TSS), conserved transcription 121.959.811–122.073.770; NC_000011.9, AK091713). Several factor-binding sites, TATA boxes, A/B boxes) and epigenetic independent putative promoters have been described modifications (CpG islands, histone modifications, nucleosome (Figure 1b). One that drives co-expression of hsa-miR-100 and positioning patterns) with chromatin immunopreciptiation hsa-let-7a-2 independent of their potential host gene,6–9 one that screens or reporter assays.6–11 drives co-expression of hsa-miR-99a and hsa-let-7c independent of It seems that about half of the miRNAs located in introns their host gene,6–9,11 and another that drives co-expression of protein-coding genes (B60%) contain putative promoters of hsa-miR-99b, hsa-let-7c and hsa-miR-125a.6–9 Hsa-miR-125b- regulating transcription independent of their host gene.7 This is 1(refs 6,7,9) and hsa-miR-125b-2(refs 6,8) are predicted to be expressed also supported by the observed discrepancy in expression of from an independent promoter. several miRNAs and their host genes.12,13 Ozsolak et al.8 showed In summary, all three human homologs of miR-125 are involved that intronic miRNAs having independent transcription initiation in a cluster containing members of the miR-99 and let-7 family. regions had the shortest TSS-to-miRNA distance with a mean The genomic organization of these three clusters is highly distance of 4.2±3.5 kb. These miRNAs were located significantly conserved as well as the seed regions of the different paralogs farther (mean distance of 57 kb) from their host gene TSSs in and orthologs. This underscores the hypothesis that these three comparison with intronic miRNAs utilizing host gene TSSs. These miRNA families are partly co-regulated and have cooperative results suggest that certain intronic miRNAs located far away from biological functions. their host TSSs may have evolved to adopt nearby independent novel TSSs in order to make the transcription of primary miRNA faster and more efficient.8 REGULATORS OF MIR-125B EXPRESSION These issues on how the regulation of miRNAs expression is Relatively little is known about the regulation of miR-125b dependent on their genomic localization are highly relevant for expression. The regulatory sequences (promoter, enhancer) of

Leukemia (2012) 2011 – 2018 & 2012 Macmillan Publishers Limited Mir 125 in hematopoiesis and leukemia L Shaham et al 2013 miR-125b and its associated miRNAs located in its cluster have not yet been fully characterized. Contradictory data exists regarding the regulation of miR-125b. It has been shown to be induced 14 Premature B Plasma B by CDX2, induced or repressed by nuclear factor kappa beta BCL6 (NF-kb)15–17 and P53,18,19 and to be repressed by myelocyto- cell cell matosis viral oncogene homolog (MYC)20 and AKT1.21 It has been suggested that miR-125b is directly regulated by ? androgens. In prostate cancer cells, androgen stimulation was ? shown to induce an enhanced abundance of miR-125b in an c-Myc androgen receptor-dependent manner. It was observed that the IRF-4 androgen receptor was recruited to the 5’DNA region of miR- 125b-2 in the presence of R1881 (synthetic androgen). This ? stimulation was androgen-speciﬁc, as estradiol did not induce this BLIMP-1/PRMD1 22 effect. The upregulation of miR-125b was shown to be miR-125b associated with the reduction of BCL2-antagonist/killer (BAK1), P53 and PUMA levels, all of which are known targets of miR-125b.22,23 This is further supported by the fact that lowering Figure 2. Schematic diagram of miR-125b regulating the inhibition miR-125b expression by androgen withdrawal was associated with 30,31 an increase in BAK1, P53 and PUMA expression.23 of PC differentiation.

MIR-125B IN HEMATOPOIESIS The ability of a single hematopoietic stem cell (HSC) to LPS reconstitute the entire blood system for the lifetime of an organism is made possible by the balance of factors that regulate self-renewal and fate-commitment decisions. The expression pattern of miR-125b suggests that it has a specialized role in regulating hematopoiesis at the stem-cell level. Analysis of miRNA expression profiles in multiple hematopoietic subpopulations demonstrated that one of the most expressed miRNAs in HSCs, miR-125b compared with all other progenitor populations, is miR-125b 24,25 IkB (Bsevenfold more than total bone marrow (BM)). Further NKIRAS2 analysis of lineage-negative cells revealed that miR-125b is one of NF-kB IkB TNF- the miRNAs that was specifically enriched in the long-term HSC population.24 Whereas the levels of miR-125b drop significantly in committed progenitors, the levels in common lymphoid Cell type dependent: progenitors are higher than in common myeloid progenitors.25 Repression/Activation HSCs transduced with miR-125b demonstrate an improved NF-kB miR-125b-1 engraftment and increased self-renewal as shown by serial transplantation experiments.24–26 Importantly, miR-125b was Pathogen clearance shown to affect mainly long-term multilineage engraftment, being consistent with a putative function of regulating stem-cell Figure 3. Schematic diagram of LPS activating pathogen clearance homeostasis.24,26 There are contradictory findings regarding the response, together with miR-125b known activities.15–17,35 lineage that is expanded because of the overexpression of miR- 125b. Ooi et al.25 demonstrated preferential expansion of the lymphoid lineage whereas other studies demonstrated a miR-125b may be the result of a negative feedback loop in the 24 preferential expansion of the myeloid lineage. granulocyte colony-stimulating factor pathway. This interesting SPOTLIGHT Contrasting results from different studies might be related to model awaits confirmation in primary cells. the varying levels of miR-125b achieved during transduction MiR-125b is also involved in inflammation and innate immunity. experiments. Indeed, a major limitation of these studies is that all In a recent publication by Chaudhuri et al.,33 miR-125b was of them are overexpression experiments performed in cells found to be enriched in macrophage cells and to enhance already expressing relatively high levels of endogenous miR-125b. their activity including antigen presentation, T-cell activation and Expression of miR-125b results not only in the expansion antitumorigenic potential. Targeting IRF-4, a negative regulator of of both myeloid24,26–28 and lymphoid25,26 progenitors but also in macrophage proinflammatory pathways, was suggested to be the differentiation arrest.29–32 High expression of miR-125b is mechanism for these phenotypes.33 observed in germinal center (GC) centroblasts during human In B-lymphocytes and macrophages, signals originating in B-lineage differentiation. It is suggested that miR-125b is an response to the Gram-negative product Lipopolysaccharides inhibitor of premature plasma cell (PC) differentiation, as it (LPS) trigger Toll-like receptor 4 and activate NF-kb. NF-kb induces targets the transcription factors B-lymphocyte-induced maturation the expression of several proinflammatory cytokines, including protein-1 (BLIMP-1) and interferon regulatory protein-4 tumor necrosis factor a, which is involved in pathogen clearance (IRF-4)30,31(Figure 2). It may also regulate T-lymphoid differentia- (Figure 3). tion as it has recently been demonstrated to be expressed in MiR-125b acts as a negative regulator of this pathway by human naı¨ve T-cells and to target several T-cell differentiation reducing the levels of its target tumor necrosis factora15,34 and, genes.32 unusual for miRNAs, by enhancing the stability of the NF-kb In 32D mouse myeloid cells, miR-125b was shown to be induced inhibitor NKIRAS2 (KBRAS2)35 (Figure 3). Enhancement of the by granulocyte colony-stimulating factor and to negatively stability of a target gene transcript was described in several regulate granulocyte colony-stimulating factor signaling.27 recent publications.15,36 One of the suggested mechanisms for the Therefore, the block in myeloid differentiation29 caused by enhanced stability of a transcript is the binding of an miRNA with

& 2012 Macmillan Publishers Limited Leukemia (2012) 2011 – 2018 Mir 125 in hematopoiesis and leukemia L Shaham et al 2014 the AU-rich elements of an mRNA. This interaction, mediated leukemia associated with Down’s syndrome (DS). Klusmann through the FXR1–AGO2 complex, results in enhancement of et al.28 showed that knockdown of miR-125b in AMKL cell lines translation of the target gene.37 Interestingly, NF-kb can either decreased their proliferation. Ectopic expression of the miRNA in repress or activate pri-miR-125b transcription depending on the murine and human progenitors enhanced the size and number of cell type and on the amount of time after encountering the megakaryocyte and megakaryocytic erythroid progenitor colonies. pathogen, thereby either further enhancing or suppressing the Consistent with the putative role of miR-125b in HSC self-renewal, inﬂammatory response.15–17,35 MKs and MEPs transduced with miR-125b-2 displayed a Hence, miR-125b has been suggested to have a remarkably proliferative advantage and the ability for self-renewal as diverse role in the hematopoietic system. It enhances survival and shown by serial replating assays. Interestingly, unlike the proliferation of some early hematopoietic progenitors and blocks granulocytic and monocytic differentiation arrest observed in their terminal differentiation. It is also involved in inﬂammation by myeloid progenitors,27–29 impairment of differentiation of regulation of the NF-kb pathway and by promotion of differentia- megakaryocyte transduced with miR-125b was not observed.28 tion and activation of macrophages. Finally, miR-125b was shown to collaborate with mutated GATA1, However, we suggest evaluating these results with extra caution typically expressed in DS acute megakaryocytic leukemia, in the as most of this data is based on overexpression experiments. The transformation of murine megakaryocytic progenitors.28 Together, true and non-redundant functions of this miRNA in the biology of these observations suggest a major pro-proliferative role for miR- hematopoietic cells will be determined only by future loss-of- 125b in AMKL. function studies.

MIR-125B IN HEMATOPOIETIC MALIGNANCIES MIR-125B IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) Leukemias and other hematopoietic neoplasms are often caused 38 Similar to AMLs, the observation suggesting a direct role for by somatic aberrations in hematopoietic developmental genes. miR-125b in ALL is its upregulation caused by the insertion of miR- Given its expression pattern and the putative role of miR-125b in 125b-1 into the Immunoglobulin heavy chain locus (IgH) enhancer HSCs, it is reasonable to hypothesize that dysregulation of its 42,45–47 SPOTLIGHT locus in ALLs harboring the translocation t(11;14)(q24;q32) expression may be associated with hematopoietic malignancies. (Table 1). Indeed, such a dysregulation has recently been described in the We observed that increased expression of miR-125b, together myeloid and lymphoid malignancies and a direct role as a with miR-99a and let-7c from the cluster on chromosome 21 (up hematopoietic oncomiR has been experimentally demonstrated. to 20-fold overexpression compared with other ALLs), is speciﬁ- cally associated with TEL-AML1 (ETV6-RUNX1) ALL, the most MIR-125B IN MYELOID MALIGNANCIES common childhood ALL48 (Table 1). This has recently been A direct role for miR-125b in acute myeloid leukemia (AML) and in conﬁrmed by Schotte et al.49 Our original hypothesis was that the myelodysplastic syndrome is thought to exist owing to the miRNA cluster encoded on chromosome 21 would be involvement of miR-125b in the rare recurrent chromosomal overexpressed in DS and in hyperdiploid ALL, two conditions translocation t(2;11)(p21;q23), leading to an overexpression having additional copies of chromosome 21. To our surprise, (up to 90-fold) of the miRNA in comparison with normal BM.29 overexpression was observed only in the control group TEL-AML1 Overexpression of miR-125b was shown to cause myeloid ALL that had a structural aberration of chromosome 21. The basis differentiation arrest in human CD34 þ cells,28,29 in several for the association between this fusion translocation and leukemic cell lines and in 32D cells treated with granulocyte overexpression of the miR-125b cluster is unclear as miR-125b colony-stimulating factor.27 does not seem to be regulated by the TEL-AML1 fusion. Increased expression of miR-125b has been observed in a Overexpression studies in lymphoid progenitors and knock- variety of myeloid malignancies. The expression levels varied down studies in the REH leukemia cell line (TEL-AML1-positive) depending on the methodologies used for expression and demonstrated that miR-125b provides a survival advantage in the controls, but are generally up to 20-fold compared with normal presence of various pro-apoptotic stimuli in a P53-independent BM. These malignancies include myelodysplastic syndrome manner. This anti-apoptotic function was also demonstrated for involving the del (5q),39,40 AMLs harboring the AML1/ETO primary TEL-AML1 ALL cells treated with vincristine49 and in Jurkat translocation,41 AMLs associated with the FLT3 mutation,41 a few T-ALL cells or primary mouse lymphoid progenitors transduced cases of chronic myelogenous leukemia, myeoproliferative with miR-125b.25 As TEL-AML1 and miR-125b expression appear as neoplasms42 and acute promyelocytic leukemia43,44 (Table 1). independent events, it is tempting to speculate that they may The most investigated hematopoietic malignancy with cooperate in the leukemogenic process.48 A similar increased increased expression of miR-125b is acute megakaryocytic expression of miR-125b was also described in BCR-ABL ALL and

Table 1. miR-125b dysregulation in hematological malignancies

ALL AML MM Other

ETV6/RUNX1(refs 48,49) DS-AMKL28 TC classiﬁcation#5 t(14;16) CML (accelerated phase and (q32;q23)51 B-lymphoid blast crisis)42 t(11;14)(q24;q32)45–47 t(2;11)(p21;q23)29 TC classiﬁcation #5 MDS involving the del (5q)39,40,42 t(14;20)(q32;q11)51 BCR/ABL42 AML1/ETO41 High-risk MM50 Associated with the FLT3 mutation41 APL with the translocation PML/RARA43,44 Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia; DS-AMKL, Down’s syndrome acute megakaryocytic leukemia; MDS, myelodysplastic syndrome; MM, multiple myeloma; TC, translocation/cyclin.

Leukemia (2012) 2011 – 2018 & 2012 Macmillan Publishers Limited Mir 125 in hematopoiesis and leukemia L Shaham et al 2015 cooperation between the miRNA and BCR-ABL has been shown 21.48,50,51 Thus, there is no correlation between the expression of in vivo26,42 (Table 1). miR-125b and its genomic copy.

MIR-125B CAUSES AML AND ALL IN MOUSE MODELS Two transplantation models of murine leukemias caused by miR- TARGETS AND PATHWAYS REGULATING CELL SURVIVAL, 125b were recently described. In one study, mice were PROLIFERATION AND DIFFERENTIATION reconstituted with BM progenitors transduced with three different One of the greatest challenges in miRNA research is the expression levels of miR-125b.24 Two months after reconstitution, identification and functional analysis of their targets. These all mice expressing miR-125b, regardless of the construct used, challenges stem from (a) the poor predictability of targets based had a subtle elevation in total white blood cell counts. Mice on sequence alignment to 30untranslated regions; (b) the reconstituted with BM expressing high levels of miR-125b magnitude of change in RNA and protein levels of the target, a exhibited a clear myeloproliferative neoplasm that was change that is, in the case of proteins, often below the sensitivity characterized by an increased absolute number of monocytes of current routine protein analysis methodologies and (c) the and neutrophils, and decreased levels of lymphocytes, red blood multiplicity of targets affected by each miRNA. Indeed, as miRNAs cells and platelets. These mice transformed to fatal AML. are thought to affect the expression of multiple proteins, it is Interestingly, transplantations with cells expressing the lowest difficult to calculate and model the network effects of these levels of miR-125b were associated with an increase in lymphoid perturbations. cells, similar to the observations reported by Ooi et al.25 In another With these important caveats in mind, several putative targets transplantation model, transduction of lineage-negative fetal liver of miR-125b regulating cell survival, proliferation and differentia- hematopoietic cells with a retrovirus-expressing miR-125b (700- tion have been suggested and experimentally confirmed (Table 2). fold overexpression) lead to fatal hematopoietic malignancies The P53 protein regulates multiple cellular pathways in consisting of myeloproliferative neoplasms, B- and T-ALL.26 response to stress, in particular in response to genotoxic stress. To examine whether overexpression of miR-125b is the cause of Le et al.56 showed that miR-125b downregulates the expression of ALL in cases having the translocation t(11;14)(q24;q32), transgenic P53 and its targets in zebrafish and human cells. However, in mice, mice expressing miR-125b under the immunoglobulin enhancer where the binding site is not conserved, P53 was not found to be (Em/miR-125b-TG mice) were recently generated. These mice targeted by miR-125b.48 In addition, it was suggested that miR- developed B-cell clonal lymphoid leukemias resistant to apoptosis, 125b targets five additional genes that are upstream regulators of similar to the human leukemias.42 P53: PPP1CA, PPP2CA, PRKRA, PLK3 and PLAGL1.56,57 These findings Together, the observations in human leukemias and in mouse were not confirmed in human leukemia cells (REH) with models demonstrate that miR-125b (both miR-125b-1 and miR- endogenous expression of miR-125b,48 and therefore the anti- 125b-2) is a leukemogenic oncomiR. apoptotic effects of miR-125b in these cells are P53 independent. Whether or not P53 is a bona-fide target in all cell types and species, miR-125b was demonstrated to target other components MIR-125B IN MULTIPLE MYELOMA (MM) of the P53 pro-apoptotic network, including BAK1, PUMA, BMF, MM is a malignant proliferation of BM PCs, characterized by a TRP53INP1 and Kruppel-like factor 13 (KLF13).22,23,25,27,42,56–59 profound genomic instability involving both numerical and All were shown to be direct targets of miR-125b in more than structural chromosomal aberrations with potential prognostic one system and cell type (Figure 4). relevance. A few cases of MM were found to express miR-125b-2, Inhibition of the three targets, DICER1, ST18(ref. 28) and in particular the translocation/cyclin classification #5, involving SUV39H1(ref. 60) was suggested to be associated with the pro- either the t(14;16)(q32;q23) or t(14;20)(q32;q11) translocations (up proliferation phenotype endowed by the overexpression of to 14-fold upregulation)50,51 (Table 1). Interestingly, similar to ALL, miR-125b. Small hairpin RNAs directed against DICER1 or ST18 in miR-125b-2 is not overexpressed in MM with trisomy 21. human CD34 þ hematopoietic stem and progenitor cells (HSPCs) Not much is known about the function of miR-125b in MM, increased the number of CFUs by 1.4-fold.28 The possibility that except the suggested inactivation of P53.52 On the contrary, the DICER, a key protein in miRNA physiology, may be a target of U266 MM cell line transduced with miR-125b exhibited an miR-125b is intriguing, as it would suggest that some of the exaggerated death rate compared with control cells by effects observed by miR-125b expression might be because of the SPOTLIGHT downregulation of its targets BLIMP-1 and IRF-4. The inhibition of other miRNAs. introduction of miR-125b also led to the reduced expression of SYNDECAN-1 (CD138), a cell surface molecule expressed by myeloma and primary PCs.30 Hence, it is unclear whether miR- 125b has an oncogenic role in MM. As MM is an extremely Table 2. Experimentally confirmed cellular targets of miR-125b heterogeneous malignancy, the role of miR-125b needs to be further examined in the specific subtypes of MM in which it is BAK1(refs 22,23,27,57,59) IRF-4(refs 30,31,33) SUV39H1(ref. 60) overexpressed. BCL3(ref. 71) c-Jun27 Tef18 BLIMP-1(refs 30,31) Jund27 TNF-a15 BMF25,58 KLF13(ref. 25) TRP53INP1(refs 42,56,57) MIR-125B AND TRISOMY 21 CBFb14 MUCIN1(ref. 74) Vitamin D receptor78 CDC25A77 P53(refs 56,57) As miR-125b-2 is located on chromosome 21, it is reasonable to (ref. 77) (refs 56,57) hypothesize that it could be upregulated in DS or in leukemias CDK6 PLAGL1 DICER28 PLK3(refs 56,57) with somatic polysomy of chromosome 21. Indeed, upregulation ERBB2,3(refs 73) PPP1CA/2CA56,57 of all five miRNAs encoded on chromosome 21 was detected in (refs 79) (ref. 32) 53,54 ETS1 PRDM1 the brain and hearts of fetuses with DS. However, the FGFR2(ref. 80) PRKRA56,57 association between germline or somatic trisomy 21 and the IFNG32 PUMA23 expression of miR-125b-2 in leukemias is not straightforward. MiR- IL2RB32 ST18(ref. 28) 125b-2 is overexpressed in the myeloid leukemias28,55 but not in IL10RA32 Stat3(ref. 27) 48 the lymphoid leukemias associated with DS. Moreover, it is Abbreviation: KLF13, Kruppel-like factor 13; TNF-a, tumor necrosis factor a. hardly expressed in ALL or myelomas with somatic trisomy

& 2012 Macmillan Publishers Limited Leukemia (2012) 2011 – 2018 Mir 125 in hematopoiesis and leukemia L Shaham et al 2016 expression levels in common lymphoid progenitors compared with common myeloid progenitors.25 These results suggest that miR-125b Cell type the miR-125b-2 polycistron may be regulated by dependent Trp53inp1 posttranscriptional mechanisms in early hematopoiesis. Similar to miR-125b-2, miR-99a was found to be upregulated in AMKL cell 61 41 P53 Bak1 lines, in AMLs with the translocation AML1/ETO, in APL involving PML/RARA44 and in resistant ALLs.49 However, in more Puma differentiated B-cells, a correlation in the expression level could not be demonstrated between miR-125b-2 and miR-99a or let-7c. Klf13 An increased expression of miR-125b was found in GC Bmf centroblasts during human B-lineage differentiation, whereas miR-99a and let-7c were overexpressed in macrophages and T-cells, respectively.30 Although not much is known about the added function of the Apoptosis whole cluster, beyond the activity of miR-125b, in hematopoietic malignancies and in normal hematopoiesis, coordinated regulation of miR-125b and let-7a (a let-7 family member) in 16,17,35,60,62 Figure 4. Schematic diagram of suggested functional interactions inﬂammation was demonstrated. For example, between P53, its targets and miR-125b. activation of macrophage cells with LPS mediated the repression of miR-125b levels and the induction of let-7a expression. This Another mechanism, through which miR-125b may exert its coordinated regulation resulted in the activation of NF-kb and pro-proliferative effect on hematopoietic cells, is by targeting induction of the inﬂammatory response. the histone methyltransferase SUV39H1.60 The downregulation of SUV39H1 by miR-125b, in two different non-hematopoietic

SPOTLIGHT systems, led to derepression of key inflammatory chemokines MIR-125A and cytokine genes such as interleukin (IL)-6 and monocyte MiR-125a, which shares the same seed region as miR-125b chemoattractant protein-1 (MCP-1), by the decrease of the (Figure 1), was also demonstrated to have a similar expression repressive H3K9me3 chromatin mark at their promoter pattern. Indeed, miR-125a is enriched in HSCs (up to 23-fold regions.60 IL-6 is a major cytokine-driving hematopoietic cell over total BM), particularly in long-term HSCs (up to 6-fold)24,63,64 proliferation, as shown in MM in which miR-125b is sometimes and downregulated upon differentiation.64 MiR-125a was overexpressed. demonstrated to have a role in HSCs, increasing their number MiR-125b was demonstrated to inhibit cell differentiation of in vivo and in vitro,63,64 and causing a myeloproliferative granulocytes and monocytes in the myeloid lineage27–29 and of phenotype upon transplantation.64 Whereas Guo et al.63 showed PCs30,31 and naı¨ve T-cells32 in the lymphoid lineage. MiR-125b, one amplification of the HSC pool upon sustained miR-125a of the signature miRNAs of naı¨ve CD4 þ T-cells, was found to overexpression, Gerrits et al.64 reported a decline of the regulate a network of gene-encoding molecules involved in T-cell primitive BM compartment and loss of competitive advantage differentiation into effector cells, including IFNG, IL2RB, IL10RA and upon secondary transplantation. PRDM1.32 The inhibition of B-cell differentiation into mature PCs is These phenotypes could be explained by the reduction of thought to be caused by the inhibition of BLIMP-1 and IRF-4.30,31 apoptosis in the immature hematopoietic progenitors, possibly Although not directly demonstrated, it was suggested that BCL-6 through targeting pro-apoptotic genes. BAK1 was demonstrated mediates the overexpression of miR-125b in GC B-cells through to be directly targeted by miR-125a.63,64 the repression of C-MYC. C-MYC has a consensus binding The similarity to miR-125b is not restricted to the stem cell site 12 kb upstream of human miR-125b and was shown to population, as miR-125a and its cluster members, miR-99b and let- downregulate miR-125b.20,30 It has been suggested that miR-125b 7e were found to be preferentially expressed by the centroblasts facilitates BCL-6 maintenance of the GC phenotype by inhibiting in the GC. However, as opposed to miR-125b, miR-125a was not BLIMP-1 and IRF-4 expression (essential for PC differentiation) and able to negatively regulate the 3’untranslated region of BLIMP-1 by creating a feed forward regulation of BCL-6(ref. 30) (Figure 2). and IRF-4.30 Notwithstanding the similar function, the expression of miR- 125a does not always correlate with miR-125b expression. MiR- THE ROLE OF THE CLUSTER MIR-125B-2, MIR-99A AND 125a was found to have a role in activated T-cells,65 as opposed to LET-7C IN LEUKEMIA/HEMATOPOIESIS miR-125b in naı¨ve T-cells.32 This was accomplished by targeting As described earlier, miR-125b-2 resides in a cluster together with the KLF13, which resulted in the reduction of RANTES levels, let-7c and miR-99a on chromosome 21 (Figure 1b). Few reports through a feedback loop.65 have studied the cluster as one functional unit. They indicate a Moreover, miR-125a and its cluster members were found to be common regulation of the cluster members in different types of expressed at a higher level in myeloma translocation/cyclin hematopoietic malignancies. However, nothing is known on the classification #4 involving the t(4;14)(p16.3;q32) translocation, added function of the other cluster members. which is a different MM classification subtype than miR-125b.51 Expression of all members of the cluster was demonstrated in naı¨ve T-cells,32 B-cell precursor (BCP)-ALL,48 myelodysplastic syndrome39 and myeloma.50,51 C-MYC was found to regulate the MIR-125B IN SOLID TUMORS—ONCOMIR OR TUMOR cluster as one functional unit.20 However, only let-7c and miR- SUPPRESSOR? 125b were found to be upregulated in response to androgens.22 Dysregulation of miR-125b has been reported to occur in multiple Although the expression of let-7c varies, miR-125b is almost human cancer types, including cancers of the stomach,66 colon,67 always co-expressed with miR-99a. This correlation was observed pancreas,68 bladder69,70 and ovary,71 as well as in gliomas,72 breast in early hematopoietic progenitors. MiR-99a demonstrates an cancer,59,73,74 melanomas75 and prostate cancer.22,23 expression profile similar to miR-125b, with highest expression Clinical prostate cancer samples were found to over- levels in HSCs and gradually decreasing levels as the progenitors express miR-125b.76 Furthermore, enforced expression of miR- differentiate. Moreover, miR-99a also demonstrates higher 125b promoted tumor growth through mediation of a broad

Leukemia (2012) 2011 – 2018 & 2012 Macmillan Publishers Limited Mir 125 in hematopoiesis and leukemia L Shaham et al 2017 attenuation of the intrinsic apoptosis pathway by targeting P53, 4 Guo L, Lu Z. Global expression analysis of miRNA gene cluster and family based 23 PUMA and BAK1. MiR-125b was also found to stimulate on isomiRs from deep sequencing data. Comput Biol Chem 2010; 34: 165–171. 22 androgen-independent growth of prostate cancer cells in vitro. 5 Yu J, Wang F, Yang GH, Wang FL, Ma YN, Du ZW et al. Human microRNA High expression of miR-125b has also been observed clusters: genomic organization and expression profile in leukemia cell lines. in oligodendroglial gliomas.72 Overexpression of miR-125b in Biochem Biophys Res Commun 2006; 349: 59–68. human glioma cell lines promotes proliferation and inhibits 6 Corcoran DL, Pandit KV, Gordon B, Bhattacharjee A, Kaminski N, Benos PV. apoptosis through targeting BMF58 or through the reduction of Features of mammalian microRNA promoters emerge from polymerase II P53 protein levels and targets (P21 and BAX).56 chromatin immunoprecipitation data. PLoS One 2009; 4: e5279. 7 Long YS, Deng GF, Sun XS, Yi YH, Su T, Zhao QH et al. Identification of the In contrast, the upregulation of miR-125b expression specifically transcriptional promoters in the proximal regions of human microRNA genes. Mol resulted in a proliferation defect in the CD133-positive glioma cells Biol Rep 2011; 38: 4153–4157. (a putative stem cell marker in normal and malignant 8 Ozsolak F, Poling LL, Wang Z, Liu H, Liu XS, Roeder RG et al. Chromatin structure 77 brain tissues). Consistent with this observation, in breast analyses identify miRNA promoters. Genes Dev 2008; 22: 3172–3183. cancer, miR-125b might be a tumor suppressor. MiR-125b levels 9 Wang X, Xuan Z, Zhao X, Li Y, Zhang MQ. High-resolution human core-promoter were demonstrated to decrease, as compared with normal breast prediction with CoreBoost_HM. Genome Res 2009; 19: 266–275. tissue. Furthermore, miR-125b was found to suppress the 10 Jansen BJ, Sama IE, Eleveld-Trancikova D, van Hout-Kuijer MA, Jansen JH, oncoproteins MUC1, ERBB2 and ERBB3, inhibiting the growth of Huynen MA et al. MicroRNA genes preferentially expressed in dendritic cells breast cancer cells and promoting their sensitivity to genotoxic contain sites for conserved transcription factor binding motifs in their promoters. 73,74 BMC Genomics 2011; 12: 330. anticancer agents. 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BMC Genomics 2007; 8: 166. 125b-1 and miR-125b-2 contribute to oncogenesis or function as 14 Lin KY, Zhang XJ, Feng DD, Zhang H, Zeng CW, Han BW et al. miR-125b, tumor suppressors. a target of CDX2, regulates cell differentiation through repression of the core binding factor in hematopoietic malignancies. J Biol Chem 2011; 286: 38253–38263. FUTURE PERSPECTIVES 15 Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B et al. Modulation of The functional analysis of the clusters of miR-125b-2 and 125b-1 is miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation only one of the questions that require further research. A large and their possible roles in regulating the response to endotoxin shock. J Immunol body of recent research has suggested that miR-125b is a key 2007; 179: 5082–5089. regulator of normal and malignant hematopoiesis. However, in the 16 Zhou R, Hu G, Liu J, Gong AY, Drescher KM, Chen XM. 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