The Role of Microrna in Human Leukemia: a Review

The role of microRNA in human leukemia: a review

S Yendamuri1 and GA Calin2

1Department of Thoracic Surgery, Roswell Park Cancer Institute, Buffalo, NY, USA and 2Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX, USA

MicroRNAs (miRNAs or miRs) are 18–22-nucleotide non-coding target. If the sequence is a perfect match, the final result seems RNAs that have emerged as a new paradigm of epigenetic to be target mRNA degradation in a manner similar to siRNA- regulation in both normal development and cellular function, and in the pathogenesis of human disease including cancer. induced gene expression silencing. This seems to be the This review summarizes the current literature of mechanism of predominant mechanism in plants. A less than perfect match gene regulation by miRNA and their role in hematopoiesis and may result in inefficient translation leading to effective down- leukemogenesis. An understanding of these processes sug- regulation of the gene, a mechanism more common in gests further avenues for research to understand gene regula- animals.7–9 Isolated examples of miRNA leading to upregulation tion and miRNA-based therapeutic approaches. of genes have been reported.10 Leukemia (2009) 23, 1257–1263; doi:10.1038/leu.2008.382; The mechanism of translational repression is known only published online 15 January 2009 11 Keywords: microRNA; non-coding RNA; microarray; partially. The mRNA segments that miRNAs bind seem to be mostly in the 30UTRs of genes. In these regions, typically, several miRNA-binding sequences seem to act in concert. The combined effect of this binding seems to depend on the distance between the binding sequences and the number of binding 12 0 MicroRNAs regulate gene expression sequences. However, if these sequences are placed in the 5 end or even in the exons, repression still takes place, suggesting 1 that sequence rather than location may be the over-riding factor. Discovered in Caenorhabditis elegans in 1993, microRNAs 30UTR binding may affect translation both at the initiation step (miRNAs) have emerged as a major mechanism of epigenetic and at the post-initiation translation. It has been shown that regulation in forms of life as diverse as algae and human beings. reporter genes with the m7G-capped mRNA are repressed by Although the number of miRNAs in lower life forms is about miRNAs but not those with internal ribosome entry site 100–200, B1000 miRNAs are predicted to be present in sequences, suggesting some effect of miRNAs on initiation.13 humans. Bioinformatics predicts that about one-third of all However, some miRNAs cause repression of mRNA with protein-coding genes are regulated by miRNAs. Therefore, it is internal ribosome entry site sequences.14 Also, polysomes co- no surprise that miRNAs are being shown to regulate all facets of sediment with miRNAs and their target mRNAs, suggesting an normal and abnormal cellular activity. influence on the elongation process of translation.15,16 Some MicroRNA precursors (pre-miRNA) are transcripts that often evidence also exists that miRNA may regulate repression by contain several miRNAs. These are processed by Drosha (a preventing 60S subunit joining.17 Ribonuclease III) to form pre-miRNAs, B70 nucleotide hairpin Early in the investigation of miRNA-mediated gene regulation, structures. Drosha partners include DiGeorge syndrome critical 2 the over-riding theme invoked was that miRNAs alter gene region 8 (DGCR8). After being transported to the cytoplasm by expression in animals purely by translational repression. Exportin-5, these pre-miRNAs are processed by Dicer (another 3 However, an increasing body of evidence suggests that Ribonuclease III) into dsRNA with two nucleotide overhangs. 4 miRNA–mRNA interaction leads to a de-stabilization of mRNA Dicer partners include TAR RNA binding protein. Single-strand leading to alteration in mRNA levels, albeit modest. Although RNAs from these dsRNA associate with several proteins to form

enough evidence of alteration of transcript levels exists in SPOTLIGHT the RNA induced silencing complex (RISC), which is thought to 18 5 microarray experiments, the exact mechanism has only been be responsible for regulation of translation of the target genes. partially elucidated in Drosophila melanogaster and zebra- Prominent components of the RISC include the argonaute family 19,20 6 fish. Experiments have shown that the P body protein GW of proteins. The final effect of the miRNA on a specific target is 182 marks the mRNA for decay. This is based on an interaction both sequence specific and context specific. Although a with Argonaute 1, a key player in miRNA activity. Also, miR- minimum of six nucleotides need to be a perfect match 430 induces deadenylation and decay of several mRNA during (nucleotide 2–8), the preceding and succeeding nucleotides zebrafish embryogenesis. Therefore, the mechanism of regula- need not be so. In fact, in some instances, a mismatch ‘bulge’ tion of gene expression by miRNAs is multi-faceted and still region seems to be required. Following the bulge, complemen- being elucidated. tarity at nucleotide 13–16 seems to enhance repression of the

Correspondence: Dr GA Calin, Department of Experimental Therapeutics Unit 36, The University of Texas MD Anderson Cancer Technologies to investigate the miRNA expression in normal Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. and malignant tissues E-mail: [email protected] or Dr S Yendamuri, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. E-mail: [email protected] The most commonly used high-throughput technique for the Received 20 October 2008; revised 28 November 2008; accepted 8 genome-wide assessment of miRNA expression levels in a large December 2008; published online 15 January 2009 number of samples is represented by miRNA microarrays.21–23 The role of microRNA in human leukemia S Yendamuri and GA Calin 1258 Several technical variants of miRNA arrays have been indepen- uses on the slide application of the Klenow fragment of the DNA dently developed in the past few years, and the main differences polymerase I to extend miRNAs hybridized to DNA probes.29 between them are the oligo-probe design, the probe immo- Generation of small RNA libraries and deep sequencing using a bilization chemistry, the sample labeling and the microarray high-throughput approach is another way of identification and chip signal detection method. miRNA expression analysis can relative quantification of miRNAs.30 Each of these techniques also be assessed by alternative methodologies. For example, the has its strengths and weaknesses (Table 1), and the confirmatory bead-based flow cytometric technique,24 characterized by use of a second independent technique is mandatory at the highly specific solution-phase probe/target hybridization present time to identify false-positive/negative data for all the kinetics, is able to profile only limited subsets of miRNAs different assays undertaken. (o100 miRNAs/profile) due to the limitations of the color- decoding system per sample; quantitative RT-PCR specific either miRNAs regulate hematopoiesis for precursor miRNA25 or for active miRNA26 is a highly sensitive methodology, requiring small amounts of starting The hematopoietic system is a good model because of the material for the rapid and quantitative analysis of a subset of principle that a common progenitor cell can give rise to cells of miRNAs, and is excellent for microarray data validation or for different lineages. This process of commitment is governed by the study of a limited number of miRNAs. The miRNA serial the alteration in expression of several genes together. Given the analysis of gene expression (miRAGE) approach, the genome- ability of a single miRNA to control the expression of several wide miRNA analysis with serial analysis of gene expression,27 genes, it is conceptually attractive to propose that miRNAs is used for both the analysis of known miRNAs and discovery of SPOTLIGHT regulate this process (Figure 1). unknown miRNAs with large amounts of computational and bioinformatics efforts for the analysis of the obtained tagged sequences. The invader assay directly detects specific RNA Lymphoid development molecules using an isothermal signal amplification process with a fluorescence resonance energy transfer-based fluorescence The first evidence that miRNAs may be involved in regulation readout.28 The RNA primed, array-based Klenow enzyme assay of hematopoiesis came from Chen et al.31 In a series of

Table 1 Comparison of the methods used for the identiﬁcation and detection of microRNAs

Technique Sensitivity Speciﬁcity Throughput Reproducibility Steps in method

miRna microarrays Medium Low Very high Very high Medium Real-time PCR High High Medium High Medium Bead-based ﬂow cytometry Low High High Medium Many Northern blotting Low Very low Low Medium Many In situ hybridization Low High Low Medium Few Deep sequencing Medium High High High Many RAKEa Low Low High Very high Few Invader assay Medium Medium Low Low Few aRNA primed array-based Klenow enzyme assay.

Hematopoeitic stem cell

Common lymphoid progenitor Common myeloid progenitor miR-223 miR-142 miR150

Double negative pro-B Erythrocyte megakaryocyte Granulocyte monocyte progenitor progenitor miR221 miR223 miR222 miR223 miR424 miR17~92 miR150 miR150 miR155 miR146 Double positive pre-B Erythroid Megakaryocyte Granulocyte Monocyte progenitor progenitor progenitor progenitor

miR-181

CD8 CD4 Mature B RBC Platelets Granulocytes Monocyte/ Macrophage

Figure 1 Known microRNA (miRNA) that regulate hematopoeisis. miRNA in bold italics promote differentiation along the pathway. Conversely, miRNA in regular font inhibit differentiation along the pathway.

Leukemia The role of microRNA in human leukemia S Yendamuri and GA Calin 1259 experiments they showed that different hematopoietic tissues miR-424, which is located on chromosome X.37 The expression had different levels of expression of three miRNAs: miR-181, of miR-424 is also controlled by PU.1. miR-424 acts at least miR-223 and miR-142. Although miR-181 was strongly ex- partially by repression of NFIA.38 pressed in tissues rich in lymphocytes, miR-223 was exclusively expressed in the bone marrow. miR-142 was expressed in all hematopoietic tissues, with no expression in non-hematopoietic Megakaryocyte development tissues. On further expression studies in lineage-specific cells, it Garzon et al.39 showed that differentiation into the mega- was clear that miR-181 was highly expressed in B lymphocytes karyocytic lineage is associated with the downregulation of compared with non-committed cells. miR-223 expression was several miRNAs. Prominent among them are miR-10-a and restricted to cells of myeloid origin. miR-142 was highest in miR-130a. They also showed that these miRNAs downregulated B-lymphoid and myeloid lineages. In order to elucidate the mafB and HOXA-1, respectively, providing a mechanism for this cause and effect versus association, each of the above miRNAs biological effect. Using a novel method of miRNA profiling was overexpressed in non-committed cells. With the over- capable of generating a usable profile with as low as 10 ng of expression of miR-181, as expected there was an increase in the RNA, Lu et al.40 showed an overexpression of miR-150 in cells proportion of cells with the B-lymphoid lineage. However, with differentiated into the megakaryocytic lineage. Overexpression miR-142 and miR-223, there was an increase in the proportion of miR-150 in megakaryocyte–erythrocyte progenitor cells led to of T-lymphoid cells and a modest alteration in the proportion a preferential differentiation toward the megakaryocytic lineage of myeloid cells. The results with miR-181 were duplicated both in vitro and in vivo. This effect is mediated by repression in vivo, giving further supportive evidence of their role. In-vivo of MYB, which is a known critical target of miR-150. Labbaye experiments showed not only an increase in the proportion of et al.41 have dissected out another molecular pathway control- B lymphocytes, but also a decrease in T lymphocytes, ling megakaryopoiesis. They have shown that the PLZF particularly in the CD8 þ subpopulation. The actual mechanism transcription factor downregulates miR-146a. Downregulation of action of miR-181 was elucidated in other experiments, of miR-146a leads to overexpression of the CXCR4 protein (an which revealed that this effect is achieved by alterations in the SDF-1 receptor), which enhances megakaryocytic proliferation, level of Bcl-2, CD69 and TCR-alpha.32 Further influences on the differentiation and maturation. development of lymphocytes have been proposed on the basis of transgenic mouse models. Overexpression of miR-150 led to the blockade of B lymphocytes in experiments reported by Erythrocyte development Xiao et al.33 Also, selective deletion of the miR17–92 cluster Felli et al.42 studied miRNA expression patterns during prevents the transition of pro-B to pre-B cells by an increase in erythrocyte differentiation. Downregulation of miR-221 and the pro-apoptotic factor Bim.34 miR-222, which leads to increased kit, expression encouraged Similar differences in the expression profiles were found expansion of erythrocyte progenitors. Bruchova et al.43 showed during the development of other hematopoietic cells. In several that as differentiation proceeds along the erythrocyte differentia- of these, specific alterations in the miRNAs have been tion pathway, there is a downregulation of miR-221, miR-222, associated with alterations in their targets, and distinct pathways miR-155 and miR150, and an upregulation of miR-16 and miR- controlling the regulation of this process have been identified. 451. These results were confirmed in a study by Masaki et al.44 These are summarized below.

miRNAs regulate leukemopoiesis Myeloid development The decision regarding whether a common myeloid progenitor Given the extensive regulation of normal hematopoietic will differentiate into a granulocyte or a monocyte seems to be development by miRNA, it is no surprise that miRNAs are controlled by miR-223. miR-223 is highly expressed in the extensively involved in the pathogenesis of hematopoietic granulocyte fraction of the myeloid cells and seems to be malignancies. In fact, the first convincing evidence of the role repressed in the monocyte component.35 Using an acute pro- of miRNAs in human cancer was found in the pathogenesis of myelocytic leukemia cell-line model, an auto-regulation model chronic lymphocytic leukemia (CLL).45 The following section of miR-223 has been proposed. A C/EBPa-binding site has been summarizes the known findings of miRNAs in hematopoietic located upstream of the miR-223 gene in this model. C/EBPa malignancies. SPOTLIGHT competes with another transcription factor, NFIA (a CCAAT-box binding transcription factor), for this site. Retinoic acid, which induces a progenitor cell to differentiate into a granulocyte, Lymphomas leads to the replacement of NFIA with C/EBPa, leading to MiR-155 has been extensively studied in lymphomas. Apart activation of miR-223. Given that NFIA is in itself a target of from being overexpressed in Hodgkin’s, primary mediastinal miR-223, an auto-regulatory loop can be conceived. An and B-cell lymphomas,46,47 other lines of evidence points to its alternative regulatory mechanism for this process has also been oncogenic role. MiR-155 seems to be generated by alternative delineated by Fukao et al.36 These investigators found a PU.1 splicing of the non-protein coding BIC region. In transgenic and C/EBPb-binding site capable of activating miR-223 trans- mice overexpressing miR-155, there is a polyclonal expansion cription in mice. The PU.1-binding site is conserved in humans, of pre-B cells followed by B-cell malignancy, presumably by a making this a strong candidate for the control of miR-223 second transforming event that the miR-155 overexpression expression. miR-223 seems to act through the regulation of predisposes to.48 MiR-155 expression is highest in the activated expression of MEF2c, which is a known promoter of granulocyte B-cell type of diffuse large B-cell lymphomas and overexpres- differentiation. sion is associated with downregulation of 42 genes, 11 of which TPA-induced monocyte/macrophage differentiation of the cell are predicted by miRNA prediction algorithms. Included in line HL60 induces a specific miRNA expression pattern. One these genes are KLRK1, AKAP10 and CSNK1G2, which have of the miRNAs overexpressed during this differentiation is been linked to the malignant phenotype of diffuse B-cell

Leukemia The role of microRNA in human leukemia S Yendamuri and GA Calin 1260 lymphomas.49 Detection of miR-155 in the sera of patients has clear example of how miRNA can have far-reaching influence also been proposed as a diagnostic test.50 on the overall function of the cell secondary to its ability to Overexpression of the miR17–92 cluster in mice leads to the influence several target mRNA by various mechanisms. Small development of a lymphoproliferative disease and death. This is RNA analysis of CLL cells reveals other consistent alterations of secondary to the downregulation of PTEN and Bim.51 This miRNA that may be important in the pathogenesis of CLL. These region is commonly amplified in B-cell lymphoma patients.52 include downregulation of miR-181a, let-7a and miR-30d. The promoter region of this polycistronic cluster has E2F Overexpression of miR-155 was also observed. Other novel transcription factor binding sites that bind primarily to E2F3. miRNAs located in chromosome 1 were reported in the same This leads to a potential model in which the miR-17–92 is analysis.62 Another miRNA profile analysis of CLL patients placed in a regulatory loop between its regulator E2F3 and its confirms the overexpression of miR-155, but also shows target E2F1, which is a pro-apoptotic factor. Therefore, over- downregulation of miR-150 and miR-92.63 Although such expression of the cluster can move the cells toward a profiles have been correlated with prognosis, their functional proliferative mode in this balance.53 The overexpression of this and mechanistic significance remains to be elucidated. miR-29 polycistronic cassette and the myc oncogene leads to a and miR-181 have been shown to be involved in CLL synergistic cancer development.54 pathogenesis by altering TCL1 expression.64 In Reed–Sternberg cells characteristic of Hodgkin’s cells, it has been shown that miR-9 and miR-7a lead to a downregulation of PRDM1/blimp-1 activity and may contribute to the maintenance of the malignant phenotype of these cells.55 Acute lymphoblastic leukemia SPOTLIGHT Chromosomal analysis of a fragile site in chromosome 12 in The first evidence of the involvement of miRNAs in acute radiation-induced T-cell lymphomas has shown that this region lymphoblastic leukemia (ALL) was demonstrated when an is the home to 52 miRNAs. At least one of these miRNAs seems insertion of miR-125-1, a human homologue of lin-4, to be mechanistically involved in the pathogenesis of these was seen in a rearranged heavy-chain immunoglobulin gene tumors. MiR-203 seems to be regulated by hypermethylation. locus in a patient with B-cell ALL.65 Zanette et al.66 have Increased miR-203 leads to repression of ABL1, a well-known carried out a miRNA profiling of ALL and have shown the oncogene, suggesting a potential therapeutic target.56 New upregulation of miR-128b, miR-204, miR218, miR-331 and miRNAs have also been found at the PVT1 locus, which is found miR-181b-1, apart from miR-17-92 cluster. Mi et al.67 have in 20% of Burkitt’s lymphoma. This region has seven miRNAs shown a distinct miRNA signature in patients with ALL. The (including mmu-miR-1204 and mmu-miR-1206), all of which functional significance of these alterations has yet to be are expressed at a higher level in late-stage B cells, suggesting explored. potential roles in lymphoma.57 The let-7a miRNA has also been implicated in the maintenance of the Burkitt’s lymphoma phenotype by influencing myc expression.58 Downregulation of miR-143 and miR-145 has also been shown in Burkitt’s Acute myeloid leukemia lymphoma tissues and may contribute to their pathogenesis An association between miRNA signatures and cytogenetics was through their effect on ERK5.59 shown by Garzon et al.68 In this study miRNA expression patterns were correlated with molecular abnormalities like t(11q23), trisomy 8 and FLT3-ITD mutations. Specific miRNA Chronic lymphocytic leukemia alterations were also correlated with prognosis. Patients Chronic lymphocytic leukemia is a disease that has shown with a high expression of miR-191 and miR-199a had worse distinct alterations in the 13q14 region, suggesting the presence overall and event-free survival. A different signature was also of a tumor suppressor in this region. There is also an generated and validated by Marcucci et al.69 This analysis overexpression of bcl-2 in these cells. Although this over- generated a miRNA summary value that proved to be a expression is explained in some patients by the juxtaposition of significant independent predictor of survival. Another study the bcl-2 gene to the immunoglobin regions, this translocation reported recently by Dixon-McIver et al.70 reported specific is responsible for bcl-2 overexpression in only about 5% of miRNA signatures that correlate with specific karyotypic the cases. Given the presence of miR-15a and miR-16-1 in the alterations in acute myeloid leukemia, apart from a general set 13q14 region and that bcl-2 is a predicted target of this cluster, of miRNA that were deregulated across the entire leukemic set. the association was further explored.45 Cimmino et al.60 did For example, t(15;17) translocation was associated with an indeed find that this cluster regulated bcl2 expression.Further upregulation of miR-127, miR-154, miR-154, miR-299, miR- exploration of the genome-wide effects of the cluster over- 323, miR-368 and miR-370. Apart from distinct miRNA expression in a CLL cell line by analysis of the transcriptome and signatures for diagnosis and prognosis, the functional effects of the proteome after overexpression revealed several interesting some alterations have also been shown. For example, with results.61 Overexpression of the miR-15a/mR-16-1 cluster led to overexpression of miR-155, mice developed an expansion of the a downregulation of 3307 genes and an upregulation of 265 granulocyte/monocyte population with pathological features genes. As AU rich elements (ARE)-mediated instability has been characteristic of myeloid neoplasia.71 This clinically correlates implicated in the regulation of gene expression by miR-16-1, the with FLT3-ITD þ samples that show an upregulation of miR- prevalence of AREs in the downregulated genes was explored. In 155.72 Also, acute myeloid leukemia patients carrying NPM1 all, 20.1% (666/3307) of the downregulated genes had ARE mutations show a downregulation of miR-204 (among other elements. Analysis of the proteomic changes involved revealed specific alterations). The investigators also showed that miR-204 27 different proteins that showed at least a four-fold change. targets HOXA10 and MEIS1, suggesting that the HOX upregula- Prominent among these changes are BCL-2 and WT1, in tion seen in these patients may be secondary to miRNA accordance with prediction patterns and earlier evidence. Other alterations. Some evidence linking ALL1 fusion protein to proteins found are involved in cell-cycle control, ontogenesis, miRNA alterations through targeting DROSHA has also been tumor-suppressor genes and anti-apoptotic processes. This is a postulated.73

Leukemia The role of microRNA in human leukemia S Yendamuri and GA Calin 1261 Chronic myelogenous leukemia Conclusion Venturini et al.74 showed that the miR17–92 cluster is regulated by the BCR–ABL and c-MYC. Knockdown of c-MYC miRNAs control several critical cellular processes and have in cell lines leads to a decrease in expression of the emerged as a fundamental epigenetic regulatory mechanism. cluster. Upregulation of the cluster occurs in both chronic This is true in normal and abnormal cellular states, including myelogenous leukemia (CML) and chronic-phase patients but cancer. In the past few years there has been a significant not in blast-crisis CML. Also, in CML, loss of a 7-Mb region expansion of the understanding of the role of these small that encodes 12% of all genomic miRNAs has been found. molecules in the pathogenesis of leukemia. Further studies are This region also includes miR-203, the loss of which upregulates needed to define specific processes and molecular interaction to ABL-1.56 harness this knowledge to improve patient care. These studies have the potential of improving diagnosis, prognostication, provision of biomarkers and the creation of novel therapeutic agents for the treatment of leukemia. miRNAs provide a potential therapeutic opportunity

The oncologist has to face a double dilemma: how to improve Future directions the life expectancy of cancer patients while the majority of chemotherapeutic regimens have side effects and limited New miRNAs and new non-coding RNAs will be discovered efficacy, and the molecular approach (such as antisense by a variety of methods involving microarrays, deep oligonucleotides, small-molecule inhibitors, antibodies, vac- sequencing and cloning. cines, etc.) is for the most part experimental and at an early Non-coding RNA networks will be discovered and found to clinical stage. One solution is to rely on new discoveries, be important in the pathogenesis of all types of leukemias and believing that siRNAs or miRNAs or other yet undiscovered also in various therapy-related aspects, such as resistance to biological molecules could solve the issue. To exemplify one therapy. such possible strategy, we will use B-CLL, the most frequent The non-coding RNAs will represent a new category of adult leukemia in the Western world75 and one of the best diagnostic markers for cancer patients and for leukemia studied models of interplay between coding and non-coding patients in particular. RNAs in human cancers.76 Human B-cell CLL is characterized Non-coding RNA-based gene therapy will be investigated in by predominantly non-dividing malignant B cells overexpres- the near future and, as the delivery issues are less significant, sing the anti-apoptotic Bcl2 protein. Deletion or downregulation leukemias will be among the first cancers to be tested of miR-15a/miR-16-1 cluster located at chromosome 13q14.3 initially. represents an early event directly involved in the regulation of BCL2 expression.60 During the evolution of malignant clones, other miRNAs are deleted (such as miR-29) or overexpressed Acknowledgements (such as miR-21, miR-155 or the cluster miR-221/miR-222), contributing to the aggressiveness of B-CLL. Such abnormalities Dr Calin is supported as a Fellow of The University of Texas MD influence the expression of other protein-coding genes, as was Anderson Research Trust, as a Fellow of University of Texas reported that miR-221 and miR-222 directly downmodulate System Regents Research Scholar and as a Ladjevardian Regents c-KIT receptor and miR-29 regulated the levels of TCL1 Research Scholar Fund. This study was also funded by an 64 oncogene or by targeting other non-coding RNAs such as Institutional Research Grant and by a CCSG Award to George A 77 non-coding UCGs uc.160 and uc.78. The consequences of Calin. this steady accumulation of abnormalities are represented initially by a low apoptosis and high survival of malignant B cells and lately by the evolution of more aggressive clones with References a higher proliferative capacity and at a molecular level by the overexpression of BCL-2 and TCL1 oncogenes. Therefore, a 1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic mixed strategy of targeting both these oncogenes at the same gene lin-4 encodes small RNAs with antisense complementarity to time by using combined RNA drugs could be proposed. For lin-14. Cell 1993; 75: 843–854. SPOTLIGHT example, using a miRNA that will target both BCL-2 and TCL1 2 Berezikov E, Chung WJ, Willis J, Cuppen E, Lai EC. Mammalian could be an option. 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