The Regulation, Targets and Clinical Relevance of Microrna Mir-650 in Chronic Lymphocytic Leukemia (CLL),

Abstract 3874

It is known that the biology of CLL and other B-cell malignancies is driven by processes dependent on immunoglobulin structure and BCR-signaling. Recently, our group and others have described the importance of miRNAs in CLL and their involvement in BCR-signaling, IgG production and V(D)J recombination. Considering the important functions of miRNAs it is remarkable that the human locus for immunoglobulin lambda light chain (IgL) contains a miR gene. The miR-650 gene is localized in exon 1 of IgL variable subgene (1st exon of V2 family members). The aim of this study was to reveal the regulation and expression of miR-650 in CLL and its relation to disease prognosis.

CLL samples were separated by RosetteSep Human B Cell Enrichment Cocktail (obtained purity ≥95% of CD5+19+ cells). The expression of light chain surface immunoglobulin chain (lambda vs kappa) was determined by flow cytometry. The utilized IgL variable (V) segment was determined using BIOMED-2 protocol and sequencing. To study the relation of miR-650 expression and IgL rearrangement the surface expression of Ig light chain and the utilized V segment was determined in a CLL cohort containing 47 patients (λ n=27, κ n=19, λ+κ n=1). The gene expression was analyzed by TaqMan Assays (ABI) for mature miR-650 and protein-coding genes: CDK1, EBF3 and ING4. The western-blot for miR-650 targets was performed after transfection (48hrs/96hrs) of B-cell lines (NALM-6, MEC-1) with a short RNA mimicking miR-650 (Dharmacon).

The analyses of miR-650 expression revealed that cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=14) had ∼10 fold higher expression of miR-650 (p<0.005) when compared to samples utilizing different V lambda family (n=13) or expressing kappa Ig (n=20). This suggests a unique mechanism for coordinate expression of miR-650 and immunoglobulin light chain (for IgL utilizing the V2 family members). This observation is partially surprising because miR-650 was originally identified in colorectal and breast cells and it was believed to be regulated independently of immunoglobulin genes. Our data demonstrate that miR-650 expression is likely regulated at least partially by immunoglobulin light chain promoter.

We next studied the possible targets for miR-650 in B-cells. Recently, two targets were identified in solid tumors - ING4 (Inhibitor of Growth 4) and CDK1 (cyclin dependent kinase 1) (Zhang, 2010; Chien, 2010). It has been demonstrated that miR-650 is involved in the p16INK4-mediated pathway and directly regulates the CDK1. This publication suggested that up-regulation of miR-650 leads to inhibition of cell cycle progression. Moreover, the putative targets predicted by software tools (TargetScan, miRanda) include genes important for B-cell biology like EBF3 (early B-cell factor3), CLLU1, Bcl2 and cyclin D1. We therefore studied correlation between miR-650 expression and the expression of mRNAs for CDK1, ING4 and EBF3 (a predicted target with the highest score). The expression of miR-650 was not significantly associated with the expression of any of these genes on mRNA level. The lack of available material did not allow us to study the expression of CDK1, ING4, EBF3 protein levels in the original cohort. However, the transfection of B-cells with short RNA mimicking miR-650 led to down-regulation of protein levels of CDK1 and EBF3. This confirms the relevance of CDK1 as a target in B-cells and identifies a new target - EBF3, which is known to be important for B-cells development. Moreover, the expression of miR-650 was associated with overall survival (OS) and treatment free survival (TFS) in CLL (n=82). In this analysis patients were divided in two groups (based on the median of miR-650 expression). The higher expression of miR-650 was associated with statistically significant (p<0.05) longer OS (not-reached vs. 161 months) and TFS (60 vs. 34 months). This is in line with the observation that miR-650 inhibits CDK1 and cell cycle progression.

In conclusion, we have described a mechanism regulating miR-650 expression, identified its relevant targets in B-cells and demonstrated the association of miR-650 expression with CLL prognosis.

Supported by IGA MZCR NT11218-6/2010, MSMTMSM0021622430, NS10439-3/2009, FR-TI2/254