Role of Lncrnas in Early Stage Immunoglobulin Heavy Chain Variable Region (IGHV) Unmutated CLL Disease Progression

BACKGROUND: Chronic lymphocytic leukemia (CLL) remains a heterogeneous disease despite significant advances in disease classification based upon cellular and molecular markers. Rai stage, immunoglobulin heavy chain variable region mutation status (IGHV), cytogenetic abnormalities, beta 2 microglobulin levels, expression of ZAP-70, CD38 and CD49d provide useful guides alone or in prognostic models for progression risk assessment but variability in clinical outcome in early Rai stage (Rai 0/I) unmutated (UM) IGHV patients persists. This variability in clinical outcome suggests that there are yet-to-be defined biologic/genetic factors that contribute to disease heterogeneity. Long non-coding RNAs (lncRNAs) display a range of diverse activities including regulation of gene transcription, post translational regulation and epigenetic regulation and represent an intriguing prospect for influencing CLL disease progression. LncRNAs have been implicated in diagnosis, prognosis and therapy of various cancers however their role in influencing disease progression in early Rai stage UM CLL has not yet been studied.

Methods: To investigate a role for lncRNAs in UM Rai 0/I CLL patients we employed the Arraystar Human lncRNA Microarray v4.0. Using blood samples collected within 3 months of diagnosis, two cohorts of UM Rai 0/I patients were compared: one defined as progressive disease with time to first treatment (TTT) ≤ 2 years after diagnosis (n=34) and one as indolent disease with TTT ≥5 years after diagnosis (n=29). Differentially expressed lncRNAs were identified using Agilent GeneSpring GX Software v12.1. To be considered for further analysis, the difference in lncRNA expression had to have a fold change (FC) ≥ 2.0, a t-test p-value ≤ 0.05 and a false discovery rate (FDR) ≤ 0.05.

Results: Over 1100 lncRNAs were found to be differentially expressed between the progressive and indolent UM CLL cohorts. Greater than 150 of these lncRNAs have known associated genes and are well annotated and validated. Among the differentially expressed lncRNAs, several are of particular interest and potentially relevant to CLL disease biology. For example, the lncRNA AL833181 was overexpressed in the progressive group. The associated gene for AL833181 is BCL11A and encodes a zinc finger protein that interacts with BCL6 and may itself serve as a proto-oncogene. Translocations involving BCL11A have been shown to identify a very aggressive subset of CLL (Am J Pathology, 2009). Therefore it is possible this lncRNA may be playing a role in deregulated expression of BCL11A.

Within the indolent disease group, CTD-2566J3.1 was overexpressed >10-fold as compared to the progressive disease group. Its associated gene RAD51B is essential for DNA repair, suggesting the possibility that CTD-2566J3 may protect CLL cells from further DNA damage and disease progression. Also overexpressed in the indolent disease group were 2 lncRNAs that are associated with genes that encode proteins involved in the ubiquitin-proteasome system. USP2-AS1 lncRNA is associated with the USP2 gene and encodes an ubiquitin-specific protease required for TNF-alpha induced NF-kB signaling and is a specific deubiquitinase for cyclin D1, a known proto-oncogene. Furthermore, USP2-AS1 may also be linked to MYC. RP11-522I20.3 is associated with the QBLN1gene which encodes a protein that mediates the proteasomal degradation of misfolded or accumulated proteins. Thus, the associated lncRNAs may be acting as post-transcriptional gene silencers.

Although CLL is not viewed as an "invasive" cancer, there were 5 lncRNAs overexpressed in the indolent group that are associated with Ankyrin genes which play a role in cell motility, activation and proliferation along with the lncRNA RP11-477D19 that is associated with theTIAM2 gene. TIAM2is a Rac guanine nucleotide exchange factor that can promote invasion and motility of cells. The respective lncRNAs may be down-regulating these genes and prohibiting malignant cell expansion.

Conclusions: Our study reveals that there are indeed specific lncRNAs that are expressed at different levels in progressive versus indolent early Rai stage UM CLL and have the potential to impact a number of relevant biological processes and pathways. While these data are preliminary, the lncRNA AL833181 and its associated gene BCL11A may prove to be a potential marker and therapeutic target for aggressive disease.