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miRNA in Richter syndrome
CLL.9 The levels of expression of miR-150 and miR-26a were found to be significantly downregulated in post- transfer samples compared with pre-transfer samples, independently of the IGHV mutated/unmutated status of the pre-transfer CLL samples (Figure 6B). This reduction was concordant with what we observed in the analyzed human cohorts (Figure 2A). Among the other six miRNA, we identified the same variation trends in miR-21 (2/5 cases) and miR-181b (3/5 cases) although statistical signif- icance was not reached (Online Supplementary Figure 6A). Among canonical pathways common to both the miR- 150 and miR-26a validated targets, we identified mostly cancer-related pathways, including the TP53 pathway (Online Supplementary Figure 6B).
Since no RS cell lines have been reported before, while performing these studies, we used two different CLL cell lines (MEC1 and HG3) and one diffuse large B-cell lym- phoma (DLBCL) cell line (HB) (Figure 6C) to investigate the role of miRNA from the “restricted signature” in cell proliferation and colony formation: only the ectopic over- expression of miR-21 through a miR-mimic significantly increased cell proliferation in all three cell lines, when compared with scrambled controls (Figure 6C). We fur- ther examined the long-term effects (up to 21 days) of miR-21 on CLL and DLBCL cell proliferation using a soft agar colony-formation assay. Overexpression of miR-21 significantly increased colony formation in all cell models when compared with scrambled controls (Figure 6C). There have been reports that aberrant expression of miR- 21 is associated with poor clinical outcome in patients with CLL.32 These data support a functional effect of miR- 21 in both CLL and DLBCL cells that needs to be tested in future RS cell models.
Discussion
By using a stringent training/validation workflow on multiple, independent CLL/RS cohorts from several insti- tutions, analyzed by three different expression profiling methods, we identified a miRNA signature that is involved in the process of transformation of CLL to RS. This signature is composed of the overexpressed miR-21, miR-146b and miR-181b, and by the downregulated miR- 150. These miRNA are SDE at the time of Richter trans- formation, but not after the same time of evolution of CLL in patients who never underwent Richter transfor- mation (“control” group). miR-21 is a well-known onco- gene, overexpressed and associated with poor prognosis in CLL,32,33 and highly expressed in patients who do not respond to fludarabine treatment.34 In addition, miR-21 transgenic mice spontaneously develop a pre-B-cell lym- phoblastic lymphoma/leukemia.23 miR-150, on the other hand, is lowly expressed in CLL proliferation centers35,36 and shows reduced expression in poor prognosis CLL.37 Recently, Balatti et al. reported that a signature of 23 miRNA is differentially expressed in CLL samples which developed RS compared with CLL samples which did not develop RS. In their study, it was demonstrated that miR- 125a-5p is highly expressed, while miR-34a-5p is down- regulated in pre-RS samples compared with samples from a control group and that deregulation of miR-34a and miR-125a-5p can predict ~50% of RS with a false positive rate of ~9%.38 The different results we obtained can be explained by the fact that we focused on miRNA which
are deregulated in the stage of RS versus pre-RS (group 1b versus group 1a) and not on miRNA that can predict Richter transformation (group 1a versus group 2a).
Of interest, the same four miRNA from this “restricted” signature are reported in literature as being differentially expressed in another fatal condition, sepsis (Online Supplementary Table S6), in the same way as in RS, making this signature similar to that of an acute infectious disease characterized by multiple organ failure. For example, tumor-secreted miR-21 can bind to toll-like receptors (TLR) on immune cells, triggering an inflammatory response that may contribute to tumor growth and metastasis.39,40 In addition, miR-21 suppresses T-cell apop- tosis,41 and promotes Th17 cell differentiation, which is important for the development of multiple autoimmune diseases.42,43 miR-146b modulates the TLR4 signaling pathway through targeting of TLR4, MyD88, IRAK-1 and TRAF6, and has anti-inflammatory activity by reducing several pro-inflammatory cytokines, such as interleukin- 6, tumor necrosis factor-a, interleukin-8 and CCL2/3/7.44,45 Furthermore, miR-150 is important for the development of mature natural killer cells, which are of primordial importance for the induction of adaptive immune responses, and defense against pathogens.46 miR- 150 can also decrease the production of inflammatory cytokines, such as interleukin-2 and tumor necrosis fac- tor-a, through disruption of CD28/B7 co-stimulatory sig- nal transduction, resulting in immune tolerance.47 In the context of acute lung injury, miR-181b may stimulate inflammation through activation of nuclear factor-κB,48 a pathway commonly deregulated in CLL, which is cur- rently being investigated as a therapeutic target for the treatment of CLL and RS.49
To address our miRNA signature in the setting of markedly enhanced CLL B-cell proliferation, as occurs in patients with Richter transformation, we used a CLL pri- mary xenograft mouse model and found that the levels of expression of miR-26a and miR-150 were significantly reduced in proliferating CLL cells purified from the spleen of xenografted mice compared to the levels in pre-transfer cells. miR-26a reduces the expansion/accumulation of leukemic cells of Em-TCL1 mice and in vivo administration of miR-26a promotes apoptosis in mice.50
Further support for this new view of the pathogenesis of RS, as a disease related to both malignant and immune- related processes, comes from the network analysis we performed. miRNA networks in cancer have been described to be disjointed and composed of multiple sub- networks.51 In contrast, the miRNA network after Richter transformation (group 1b) contains a significantly higher number of edges, with almost all nodes connected to the main network.
A noteworthy observation is that the expression of viral miRNA, detected by the Firefly assay, shows a very high level of correlation, leading to the construction of a highly connected viral miRNA network. In all four networks (groups 1a, 1b, 2a, and 2b, see Online Supplementary Figure S5) the viral miRNA are all part of the main graph and miR-4286 and miR-1260a (a tRNA-derived miRNA)52 are always joining this main network. This observation leads us to the supposition that there is an unknown mecha- nism that controls the expression of the different viral miRNA simultaneously. Moreover, it has recently been shown in an in vivo model that immunosuppressive thera- py leads to the reactivation of Epstein-Barr virus in CLL
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