The Role of MicroRNAs in Chronic Lymphocytic Leukemia

Comprehensive Review The Role of MicroRNAs in Chronic Lymphocytic Leukemia Sabina Chiaretti,1 Valerio Fulci,2 Giuseppe Macino,2 Robin Foà1 Abstract Chronic lymphocytic leukemia (CLL) derives from the clonal expansion of small, mature B lymphocytes and is characterized by a very heterogeneous clinical course, with some patients having a relatively indolent disease and others requiring therapeutic intervention shortly after diagnosis. Although the reasons for such a different behavior have been, at least in part, elucidated, the first oncogenic hit in CLL is not yet known, and novel fields of investigation are required. Recently, microRNAs (miRNAs) have emerged as key players in several biologic processes, including differentiation and cancer, and are regarded as an appealing field of research. Indeed, several reports point to a possible role of these tiny RNAs in CLL, suggesting that they are potential candidates for disease initiation as well as putative prognostic markers. In this review, an overall view of the role of miRNAs in hematopoiesis will be provided; furthermore, the most relevant works linking miRNAs to CLL will be extensively described. Clinical Leukemia, Vol. 1, No. 5, 287-291, 2007 Key words: CD5, CD18, Immunoglobulin variable region genes, Lymphoproliferative disorders, Polymerase chain reaction, ZAP70

Chronic Lymphocytic Leukemia: The State of the Art Chronic lymphocytic leukemia (CLL) represents the most frequent leukemia in the Western countries, where it accounts for approximately 30% of all leukemias1,2 and is usually diagnosed in the seventh decade of life. This disease is derived from the clonal expansion of aberrant CD5+CD19+ small lymphocytes that have already entered the germinal center. In the past decade, there has been increasing interest in this disease that has progressively enabled acquisition of biologic information of prognostic significance that is now an important tool for patient stratification and therapeutic management. Among the most important biologic findings that have prognostic significance is the mutational status of the immunoglobulin variable region (IgVH) genes. In fact, patients with CLL can be distinguished into 2 major subgroups according to their clinical behavior. Since the introduction of IgVH screening, it has become clear that this behavior is mostly sustained by the presence of somatic mutations of the IgVH genes3-5: patients who display > 2% mutations have a significantly better outcome than those who have ≤ 2% mutations. In addition, by gene expression profiling, it was possible to identify a small set of genes, in particular ZAP70 and lipoprotein lipase (LPL), whose expression is strictly associated with the IgVH mutational status. Furthermore, the different IgVH families have been correlated with clinical outcome: the VH3-21 family is associated with an adverse prognosis, regardless of the presence of hypermutations6,7; similarly, patients with VH1-69 appear to have an unfavorable outcome. Cytogenetic aberrations have prognostic significance in CLL: the most recurrent aberration is represented by the 13q deletion (13q14),8 detected in approximately 50% of cases. Because of the high incidence of this deletion, several groups have extensively analyzed this region to identify a putative tumor suppressor gene whose loss could be involved in the pathogenesis of CLL; nevertheless, such a gene has not been recognized thus far.

1Division

of Hematology of Genetics Department of Cellular Biotechnologies and Hematology, University “La Sapienza,” Rome, Italy

2Division

Submitted: August 20, 2007; Revised: September 17, 2007; Accepted: September 17, 2007 Address for correspondence: Robin Foà, MD, Division of Hematology, University “La Sapienza,” Via Benevento 6, 00161 Rome, Italy Fax: 39-06-85795792; e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1931-6925, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.

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MicroRNAs in Chronic Lymphocytic Leukemia MicroRNA: A Novel Class of Small Non-Coding RNAs MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs.9 These short RNA molecules (20-23 nucleotides) are negative regulators of messenger RNA (mRNA) expression at the posttranscriptional level. In animals, miRNAs recognize their target mRNAs by base-pairing, inducing mRNA degradation or translation inhibition.10 The exact mechanisms of miRNA-mediated downregulation are still under investigation; however, the current model postulates that these small molecules can recruit the RNA-induced silencing complex onto their target mRNAs and target them to the P-bodies,11 where mRNAs are stored or degraded. Several lines of evidence suggest that miRNAs have a deep impact on global gene expression. Indeed, approximately 450 miRNAs have been identified in humans so far, with an average of approximately 200 putative targets for each miRNA,12 resulting in > 1 transcript in 3 being under control of ≥ 1 miRNAs. The current view supports the idea that these tiny RNAs can coordinately regulate a large number of transcripts,13 suggesting that they are likely candidates for the role of tissue identity specification. In fact, miRNAs have been shown to be involved in virtually every biologic process; more specifically, they play a role in development,14 cancer,15 and metabolism.

Role of MicroRNAs in Hematopoietic Differentiation The first study involving miRNAs in hematopoietic differentiation was published in 2004 by Chen et al.16 With a molecular biology approach, Bartel et al showed that experimental overexpression of miR-181 in lymphoid progenitor cells resulted in an increase of B-lineage cells in vitro and in vivo. Indeed, a recent work by Li and colleagues elegantly showed that miR-181 is required to modulate the T-cell receptor signaling strength, thus playing a critical role in the development and selection of T lymphocytes.17 miR-150 also plays a role in the differentiation of lymphoid lineage. Zhou et al reported that miR-150 is highly expressed in mature B-cell stages, and in mice, enforced expression of miR-150 in lymphoid progenitors blocks development beyond the pro– B-cell stage without significantly affecting T-cell development.18 MicroRNAs have also been shown to play a role in myeloid lineage differentiation. In 2005, Fazi et al reported that the expression of miR-223 is induced upon granulocytic differentiation in vitro; moreover, they showed that blockade of miR-223 activity impairs granulopoiesis, while its ectopic expression enhances granulocytic differentiation.19

MicroRNAs in Hematologic Malignancies Aberrant expression of miRNAs has been shown to be a common feature of several different malignancies, and leukemias have thus become natural candidates for an extensive profiling approach to describe miRNAs’ content of different classes of malignancies. The very first miRNA that has been shown to play a role in lymphoma is miR-155. Indeed, this non-coding RNA was identified as overexpressed in lymphomas by Tam and colleagues even before the discovery of miRNAs in animals.20 At that time, it was defined as BIC non-coding transcript. Several laboratories have focused their attention on this miRNA: Costinean et al have

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shown that transgenic mice overexpressing miR-155 in B lymphocytes display an abnormal proliferation of these cells, which ultimately results in leukemia development.21 More recently, 2 articles have tried to address the biologic role of miR-155 in the immune system by a knock-out strategy, showing that lack of miR-155 results in the impairment of adaptive immune response in mice.22,23 Another example of miRNAs involved in lymphomas is the cluster encoding for miR-17-92, which has been shown to act as an oncogene.24,25 Whereas these works were focused on the role of a single miRNA or a cluster of miRNAs in the development of the disease, other approaches have tried to describe miRNA signatures associated with disease and, in some cases, with different classes of patients, aiming at the identification of miRNAs of diagnostic and/or prognostic value. Profiling of miRNA expression is a challenging task because of the intrinsic characteristics of these tiny RNAs. Such short molecules are difficult to detect by microarray-based technologies, which are prone to mishybridization artifacts; moreover, other approaches such as cloning or quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) might not be practical on a large scale. Nevertheless, several studies addressing the role of miRNA in hematopoietic malignancies have been published. In this context, one of the most extensive analyses was performed by Lu et al in 2005.26 They profiled the expression of 218 miRNAs in 334 samples, including 72 acute lymphocytic leukemias. However, rather than identifying miRNAs, whose expression correlated with prognosis or disease progression, their work highlighted that the miRNA signature of each tumor is strictly associated with one of its healthy counterparts.

MicroRNAs and Chronic Lymphocytic Leukemia MicroRNAs are Potentially Involved in Chronic Lymphocytic Leukemia Initiation Because the primary oncogenic hit in CLL has not yet been elucidated, in recent years miRNAs have represented an appealing field of research. A great amount of research has been performed by Calin et al. They first reported that miR-15 and miR-16, which are located on the q14 region of chromosome 13, are deleted in 68% of patients with CLL, suggesting that these 2 miRNAs might play a role in disease initiation. Furthermore, the authors showed that the downmodulation of these miRNAs was not induced by mutations, dysfunction in the methylation pattern, or in the miRNA processing machinery.27 Thereafter, the same authors took advantage of a microarray-based approach, using a chip containing 245 miRNAs (161 from humans and 84 from mice) to evaluate the miRNA profile of 38 patients with CLL as well as samples obtained from blood mononuclear cells and tonsil CD5+ B lymphocytes.28 Of note, in this work, the authors again reported the downregulation of miR-15 and miR-16, although in a smaller proportion of cases (25% and 45% of cases, respectively). Among the identified miRNAs, some can play an important role in oncogenesis, eg, miR-155, previously reported to be overexpressed in Burkitt lymphoma; miR-21, known to regulate the apoptotic machinery; miR-26a, located at the 3p21.3 region and often deleted in epithelial cancers; and miR-92-1 and miR-17, located at the 13q32 region, that is amplified in malignant lymphoma.

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Sabina Chiaretti et al

Table 1

Summary of Publications Related to MicroRNA in Chronic Lymphocytic Leukemia Study Population

Methodologic Approach

Results

Calin et al27

60 Patients with CLL, tonsil CD5+ B cells

Northern blotting RT-PCR Western blotting

miR-15 and miR-16 downmodulation in 68% of patients with CLL

Calin et al28

38 CLL samples, 6 normal controls (from lymph node, tonsils, and mononuclear cells from peripheral blood)

Microarray-based (368 probes)

Distinctive signature of CLL; correlation between miR expression and ZAP70, IgVH, mutational status, and 13q14 deletion

Study

Calin et al31

94 Patients with CLL (training set), Microarray-based (368 probes) 50 patients with CLL (test set)

Identification of 13 miRs that are associated with ZAP70, IgVH, and time to treatment; identification of somatic or germline mutations in a set of miRs

Cimmino et al29

26 Patients with CLL, 2 tonsil CD5+ B cells, MEG-01 cell line

Northern blotting Microarray-based (368 probes)

Confirmation of miR-15 and miR-16 downmodulation in patients with CLL; evidence of Bcl-2 regulation by miR-15 and miR-16; induction of apoptosis after miR-15/miR-16 transfection

Pekarsky et al33

80 Patients with CLL

Microarray-based (368 probes)

Description of a differential expression of TCL1 in the ZAP70+, IgVH unmutated patients; correlation with aggressiveness of disease; inverse correlation between miR-29 and miR-181 expression and TCL1 protein levels

Raveche et al32

NZB mice

PCR

Downmodulation of miR-16 downmodulation in NZB mouse spleens; induction of apoptosis after mir-15/mir-16 transfection

51 Patients with CLL, 7 healthy controls

Cloning Quantitative PCR approach

Identification of differentially expressed miRs between donors and patients with CLL; identification of 3 miRs differentially expressed between IgVH mutated and unmutated cases

Fulci et

al34

Because from these studies miR-15a and miR-16 appeared to play an important role in CLL, Calin et al sought to elucidate their putative targets.28 To support the hypothesis that miR-15a and miR-16 could play a role in CLL initiation, they first described an inverse correlation between miR-15a and miR-16 and the Bcl-2 protein. Secondly, they reported that transfection of miR-15a/miR16 into the MEG-01 cell line, a leukemia cell line characterized by high levels of Bcl-2 expression and no detectable levels of miR-15a and miR-16, results in a high reduction of Bcl-2 levels and that the effect is exerted by each miRNA. In addition, Bcl-2 downmodulation induced an activation of the intrinsic apoptotic pathway.29 On the other hand, Linsley and colleagues, upon transfection of miR-16 in a panel of different cell lines, did not observe any downmodulation of Bcl-2 at the RNA and protein levels.30 Furthermore, Calin et al detected in CLL a germ-line or somatic mutation in 5 miRNAs, namely miR-16, miR-27b, miR-29b-2, miR-187, and miR-206, as well as 13q14.3 monoallelic deletions.31 Raveche and colleagues recently reported that, in the New Zealand black (NZB) mice, the presence of a mutation that occurs in a nearly identical location of the flanking region of miR-16, which is associated with the dramatic decrease of miR-16 levels in NZB spleens; furthermore, they confirmed that mir-16 transfection induces apoptosis and reduction of cells in the S-phase, again pointing to a role of miR-16 in CLL.32 Finally, another finding originates from the fact that T-cell leukemia/lymphoma 1 (TCL1), an oncogene located on the q31.2 region of chromosome 14, has been shown to be more highly expressed in patients with unmutated IgVH and to correlate with ZAP70 expression levels. Intriguingly, it has been shown that miR-29 and miR181 are regulators of TCL1, and an inverse correlation between these 2 miRNAs and TCL1 has also been demonstrated.33

Our group adopted a completely different approach to characterize the miRNA expression profile in CLL. A combined cloning and quantitative RT-PCR strategy was followed, obtaining the expression profile of 21 miRNAs in 51 patients with CLL, 5 CD19+ controls from healthy donors and 2 CD5+ B cells from cord blood. The 21 miRNAs profiled were the most abundant miRNAs in these cell types, accounting for ≥ 90% of the total miRNAs cloned. We were able to identify a set of miRNAs differentially expressed between CLL samples and normal B lymphocytes, miR-21, miR-150, and miR-155 being upregulated in patients with CLL.34 This is consistent with previous reports showing the correlation between miR-155 overexpression and lymphoproliferative disorders21,35 and the antiapoptotic properties of miR-21.36-38 On the other hand, some of our findings were in disagreement with previous reports. One of the major differences is represented by the number of patients with CLL in which a substantial decrease of miR-15a is observed: whereas Calin’s group observed a decrease of miR-15a expression in 68% of patients,27 we could detect such behavior (20-fold decrease on average) in 11% of patients; intriguingly, 3 of the 6 cases had a biallelic deletion of 13q14, thus suggesting that this phenomenon is detectable only when both alleles are lost.34 Furthermore, in line with the results reported by Linsley et al,30 our group did not observe an inverse correlation between miR-15a/miR16 levels and Bcl-2 protein expression.

MicroRNA Profiles as a Tool for Patient Classification The approach of Calin et al led to the identification of an miRNA signature that is discriminative of patients with CLL and includes a modulation of a set miRNAs, some of which are known to be located at fragile sites.28,31 In addition, they concluded that

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MicroRNAs in Chronic Lymphocytic Leukemia it was possible to identify a signature associated with ZAP70 levels. Calin and colleagues also identified an miRNA signature associated with 13q14 deletions that is characterized by the downregulation of miR-16, miR-24-2, miR-195, miR-203, miR-220, and miR-221 and by the upregulation of miR-7-1, miR-19a, miR-136, miR-154, miR-217, and the precursor of miR-218-2; finally, by miRNA profiling, it was possible to discriminate IgVH mutated from unmutated cases. Again using a microarray-based approach, the same group evaluated an extended cohort of patients with CLL (94 included in a training set, 50 included in a test set for validation analysis) and was able to prove that a signature based on 13 miRNAs, including miR-15a and miR-16, was discriminative of IgVH mutation status, ZAP70 expression, and time to treatment.31 The approach followed by our group also allowed the identification of a set of miRNAs that are more highly expressed in IgVH mutated cases, namely miR-223, miR-150, and miR-29, partly in agreement with the results of Calin et al.31 A summary of the most relevant publications related to miRNAs in CLL is provided in Table 1.27-29,31-34

Comparison Between the MicroRNA Expression Profile of Chronic Lymphocytic Leukemia and Other Related Malignancies A recent paper by Landgraf et al describes an atlas of miRNAs expression in mammals as assessed by miRNAs cloning and sequencing.39 Among the > 250 libraries cloned, there are libraries from patients with CLL, marginal zone lymphoma (MZL), follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, and mantle cell lymphoma (MCL). Although the numbers of patients analyzed are relatively small, this allows comparison of CLL miRNA content with that of these other lymphoprolipherative disorders. This analysis suggests a similarity between CLL, MCL, follicular lymphoma, and MZL. This is not surprising because, as already mentioned, it is well-known that these akin lymphoproliferative disorders are sometimes difficult to discriminate, and have often been misclassified in the past. At variance, Burkitt lymphoma and DLBCL appear to be more distant; the difference among CLL, Burkitt lymphoma, and DLBCL is indeed expected, because these diseases are characterized by a dramatically different clinical scenario but could be of great interest for the identification of miRNAs that play a role in cell cycle progression in B-cell lymphoproliferative disorders, because Burkitt lymphoma has an impressive proliferation rate. Moreover, it is interesting to point out that comparison of the data from patients with CLL with these lymphomas highlights a dramatic upregulation of miR-150 in CLL, much more significant than the one observed when comparing CLL and normal CD19+ samples, and an almost complete absence of miR-126 in CLL, suggesting that these latter miRNAs should be extensively investigated.

Conclusion In this review, we report the most relevant scientific contributions that have the definition of the miRNAs that are involved in the hematopoietic development as well as in leukemogenesis processes, showing that some miRNAs are deregulated in different types

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of leukemias and, in particular, CLL. Yet, the greatest open challenge remains the identification of the specific target(s) of a given miRNA. So far, in fact, the putative targets are mostly based on computational analyses that are extremely useful, but do not provide any experimental evidence. Nevertheless, some putative targets can be determined. As an example, miR-150 is known to be highly expressed in mature B cells: Myb, a target of miR-150, is found expressed at low levels in patients with CLL or MCL (Chiaretti et al unpublished observation), suggesting a direct interaction between these 2 transcripts. Another example comes from miR-29 and LPL. In fact, as discussed, the latter gene is found differentially expressed between IgVH unmutated and mutated CLL cases (high expression in IgVH unmutated cases), but the reason for such difference and its role in the disease is not clear; it is remarkable to highlight that LPL is predicted to be a target of miR-29. Another approach is to identify genes by oligonucleotide arrays that are perturbed upon down- or upmodulation of a specific miRNA. This approach has the advantage of providing a large amount of information in a single experiment, but because miRNAs usually act at the posttranscriptional level, this approach requires confirmation at the protein level, and this can generate misleading results. In conclusion, to date, it has been possible to identify a number of miRNAs that are deregulated in leukemias and lymphomas, including CLL, but the exact targets need to be defined in an experimental fashion.

Acknowledgements This review was supported by Associazione Italiana per la Ricerca sul Cancro, Ministero dell’Istruzione, Università e Ricerca (MIUR), COFIN and FIRB projects, and Fondazione Internazionale di Ricerca in Medicina Sperimentale.

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