Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world and remains an ‘enigma’ of modern hematology. Notwithstanding intensive research, it is largely unclear what aberrations are associated with the disease onset and its strikingly heterogeneous clinical course. While some patients require treatment immediately after diagnosis, others have an indolent disease that might never require therapy. Interestingly, there is no unifying gene mutation, chromosomal aberration or translocation present in CLL. The most frequent aberration is a deletion of the 13q14 region present in >50% of CLL cases and in other B-cell malignancies including mantle cell lymphoma and multiple myeloma. Researchers were unsuccessfully trying to identify the tumor suppressor(s) located in this region for two decades. The work of Croce and Calin published in 2002 enlightened the field and detailed the first evidence of miRNAs involvement in cancer by indicating that the 13q14 region contains two miRNA genes (miR-15a-16-1) Citation[1]. These miRNAs were shown to target one of the anti-apoptotic molecules (Bcl-2) that supports the survival of malignant B cells, which fits well with the scenario of CLL pathogenesis and progression. A subsequent publication not only implicated the potential use of miRNAs as prognostic markers in CLL but also described inherited germline mutations in miR-16-1 in two CLL cases Citation[2]. Finally, the causality and direct effect of miR-15a-16-1 was documented in a recent publication reporting a CLL-like phenotype in mice with deletion of these two miRNAs Citation[3]. These studies show a remarkable consistency in data supporting the contribution of miR-16 aberrations to CLL pathogenesis. Surprisingly, large-scale sequencing analysis in >150 CLL cases revealed that mutations in miR-16-1 or other miRNAs are extremely rare in CLL (<0.5%) Citation[4], and in most cases, only one miR-15a-16-1 allele is affected by deletion. Significantly, the case of del13q14 was not completely ‘closed’, as it was shown that the genes surrounding miR-15a-16-1 also contributed to a phenotype in transgenic mice Citation[3] and that approximately half of CLL patients do not harbor deletion or downregulation of miR-15a-16-1. Additionally, a homologous cluster (miR-15b-miR-16-2) was found on chromosome 3, and its contribution to the regulation of Bcl-2 and CLL biology is yet unclear.
These pioneering studies of miR-15a-16-1 were followed and accompanied by numerous comparisons of miRNA expression in CLL cases with favorable versus unfavorable prognosis (reviewed in Citation[5]). It is well known that patients with aggressive disease generally have leukemia cells that express unmutated immunoglobulin heavy-chain variable subgenes (IGHV) and/or higher levels of tyrosine kinase ZAP-70, which are both engaged in the BCR-signaling pathway. Deregulation of BCR signaling, a pathway governing the fate of B cells, is probably a universal event in the pathogenesis of CLL and other B-cell lymphomas. Several studies published between 2005 and 2009 identified approximately 18 miRNAs differentially expressed between CLL subtypes defined based on IGHV status and/or ZAP-70 expression (reviewed in Citation[5]). However, results of these studies were only partially overlapping and have not reported functional implications of such miRNA profiles. Authors have repeatedly described higher expression of miR-29 in cases with good prognosis. This miRNA was shown to regulate Mcl-1 and Tcl-1 Citation[5], whose higher expression was described in aggressive disease. The differences in miRNA levels in CLL cases with more active BCR signaling (unmutated IGHV and/or ZAP-70 positive) suggested that they might directly or indirectly influence this pathway. Only recently such data were presented, demonstrating the direct role of miR-155 in the regulation of SHIP-1 phosphatase, which balances BCR signaling in CLL Citation[6]. MicroRNA-155 is one of the primary candidates in this regard, since its levels are highly upregulated by BCR stimulation, and its overexpression is known to cause B-cell lymphomas in the mouse model Citation[7,8]. This corresponds well with higher miR-155 levels in aggressive CLL, which can be partially explained by its MYB-dependent regulation Citation[9].
BCR signaling is associated with CLL biology to such an extent that specific variable (V) segments of immunoglobulin genes are preferentially utilized by CLL cells, which also affects the disease prognosis. It is believed that such immunoglobulin genes bind autoantigens, which in turn supports the survival of malignant B cells. Considering the significant functions of immunoglobulin genes (and miRNAs) in normal and malignant B cells, it is notable that the human locus for immunoglobulin genes encodes a miRNA. The miR-650 gene and its homologs are localized in several light chain (IgL) variable subgenes of the V2 family. We have shown that miR-650 expression is regulated by coupled expression with its host gene for IgL and is thus strongly expressed in cases with a V2 family of variable IgL subgenes Citation[10]. miR-650 expression is associated with CLL prognosis and influences B-cell proliferation through regulation of several target genes. Another study presented at the 2012 EHA conference (14–17 June, Amsterdam, The Netherlands) also suggested that CLL cases with stereotype IGHV preferentially express particular miRNAs Citation[11]. These data altogether implicate the miRNAs contribution to the deregulation of BCR signaling in CLL cells.
In addition to the contribution of BCR signaling to CLL aggressiveness, the most unfavorable CLL subtype is associated with aberrations in the p53 tumor-suppressor that leads to resistance to chemo- and chemoimmuno therapy (median survival: approximately 3 years). We previously compared the expression of miRNAs in cases with p53 deletion and/or mutation and revealed downregulation of several miRNAs (miR-34a, miR-29c and miR-17-5p) Citation[12]. The downregulation of miR-34a was also repeatedly described by other groups Citation[13,14] and is in concordance with miR-34a being a downstream target of p53 Citation[15]. Remarkably, miR-34a induction can be observed in vivo during the administration of chemo-immuno therapy in CLL and correlates with in vitro sensitivity to drugs used in CLL treatment Citation[16].
CLL patients would clearly benefit from early identification of p53 aberration, and the challenge is to develop clinically useful tests of p53-pathway function. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (TP53 mutation not accompanied by p53 deletion), and miR-34a downregulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease Citation[16]. This can be accomplished without laborious treatment of cells with γ-irradiation, which had been previously used to identify p53-pathway functional impairment in CLL. Although numerous publications mention the prognostic significance of miRNAs in CLL, quantification of miR-34a might, practically, be most beneficial because of its potential to discriminate cases with very aggressive disease.
The Croce group not only implicated the deletion of miR-15a-16-1 cluster in Bcl-2 regulation but also recently presented a model with miRNAs being central to the pathogenesis of CLL cases with 11q deletion (approximately 20% of CLL cases) Citation[17]. The deletion of 11q affects several protein-coding genes (including ATM) and two miRNAs (miR-34b and miR-34c). This study provided some evidence that miR-34b/c regulate BCR signaling through targeting ZAP-70 Citation[17]. Moreover, authors also claim that miR-15a-16-1 are downstream targets of p53 and, in turn, regulate its expression. This model thus includes genes located at loci linked to three of four most common chromosomal aberrations in CLL (del13q14, del17p13 and del11q23), which are altogether present in approximately 70% of CLL cases. However, some controversy surrounds these findings since miR-34b/c were reported to be hardly detectable in any CLL cells Citation[12,13]. Additionally, cases with del11q should have a higher expression of ZAP-70 based on this model, which was not the case in previously published studies.
Future directions
Despite numerous developments, we are far from understanding of the whole gene networks regulated by miRNAs in B cells. The majority of published research only tested the role of a specific miRNA in the regulation of few targets, selected for either their computational predictions of strong complementarity to mRNAs or known relevance of the possible target (like Bcl-2, Mcl-1 and Tcl-1). The unbiased identification of disease-relevant targets for studied miRNAs will require the use of high throughput genomic techniques such as microarrays or next-generation sequencing approaches to compare gene expression and miRNA expression patterns in CLL cells. This is essential for identification of preferential targets for a miRNA in the context of other mRNAs in the specific cancer cell type. It is most interesting to unravel to what extent specific miRNAs contribute to the biological differences in the two main CLL subtypes (with mutated and unmutated IGHV) and in normal B cells, and also the progression of CLL. Moreover, this could lead to identification of protein-coding genes responsible for the malignant phenotype of cells. This was demonstrated by Pallasch et al. by comparing the expression of miRNAs in normal and malignant B cells, which led to identification of several downregulated miRNAs Citation[18]. By applying bioinformatic tools, authors noticed that the gene PLAG1 is an overrepresented target of these miRNAs, suggesting that it might be a novel oncogene in CLL.
MicroRNAs not only arouse interest among researchers studying malignant B-cell physiology but also they theoretically represent therapeutic targets. Sampath et al. detailed how expression of miR-106 can be activated by inhibitors of histone deacetylases, which in turn downregulates ubiquitin ligase ITCH and induces p53-independent apoptosis in CLL cells Citation[19]. It has also been demonstrated that aberrant methylation might be a general mechanism for deregulated expression of miRNAs in CLL Citation[18,20]. This partially uncovers the mechanism of action for chromatin structure-modifying drugs that are currently tested in clinical trials. Significantly, miRNAs have the potential for direct use as therapeutics owing to their small size and easy delivery to cells. Recently, a group from the Dana-Farber Cancer Institute, Boston, MA, USA utilized antisense oligonucleotide against miR-155 to inhibit the growth of Waldenström macroglobulinemia and CLL cells in vitro and in Waldenström macroglobulinemia-transplanted mice Citation[21]. They administered the locked nucleic acid oligonucleotides against miR-155 without additional carriers and proved that they are delivered to cancer cells in vivo. However, many challenges remain with translating miRNAs to clinical therapies or their use as prognostic markers. Targets for most of the studied miRNAs are, primarily, poorly characterized and publications are mainly piling up indirect evidence regarding miRNAs role in CLL biology. The field would greatly benefit from unbiased approaches in identifying miRNA targets, which would foster better understanding of the real extent of their relevance for CLL B-cell biology.
Acknowledgements
M Mraz thanks TJ Kipps (UCSD) for inspiring discussions.
Financial & competing interest disclosure
The work was supported by IGA MZCR NT11218-6/2010, OPVK project SuPReMMe-CZ.1.07/2.3.00/20.0045 and VaVPI project CEITEC – CZ.1.05/1.1.00/02.0068. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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