Characterization of Long Non-Coding RNAs in Chronic Lymphocytic Leukemia: Evidence for Association with Disease Progression in Trisomy 12 Patients

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a heterogeneous B cell malignancy with patients being categorized into disease subsets based on several key biologic parameters, e.g., mutation status (mutated, M; or unmutated, UM) of the immunoglobulin heavy chain variable region (IGHV), acquired chromosomal abnormalities, and expression of CD38 and CD49d. Furthermore, about one third of CLL patients express stereotyped B cell receptors and/or may acquire high risk common mutations in genes such as NOTCH1 and SF3B1 suggesting ongoing genetic evolution as drivers of disease development. Critical to this concept, those CLL patients with trisomy 12 (T12) defects have a higher incidence of mutations in NOTCH1 and often have a stereotyped receptor. However, T12 patients may have a variable clinical course that appears to be unrelated to these 2 drivers suggesting an additional, possibly non-coding genetic component that may further impact disease progression in these patients. One potentially relevant genetic factor that could influence T12 clinical course is long non-coding RNAs (lncRNAs). LncRNAs are transcripts longer than 200 nucleotides that can affect a number of cellular processes. Importantly, lncRNAs have been implicated in various cancers including malignant hematopoiesis indicating they could be therapeutic targets and/or clinically useful biomarkers.

METHODS: To pursue a role for lncRNAs in T12 we used v3.0 Arraystar Human LncRNA Microarrays to assess the global profile of lncRNA expression in CLL with an emphasis on patients with T12. Two cohorts of 6 patients with T12 were selected for comparison: one defined as progressive with a short time to treatment (TTT) (treatment ≤1 year after diagnosis) and one as indolent (no treatment > 5 years after diagnosis). Each cohort included 3 patients with M and 3 with UM IGHV status. RNA from normal CD5+ and CD5- B cells was included as a control. To compensate for the small sample size in each cohort, a significant difference in lncRNA expression between the groups was defined as a fold change (FC) ≥5.0, p-value ≤0.05 and false discovery rate (FDR) ≤ 0.05.

RESULTS: An initial global comparison of CD5+/CD5- normal B cells vs all CLL samples found that 609 lncRNAs were differentially expressed using the criteria listed above with 158 lncRNAs having a FC>10. Notable lncRNAs in this group included: LOC541472 (down in CLL and associated with the IL-6 gene), D63785 (up in CLL and associated with TBC1D3C, an oncoprotein), CTC-459I6.1 (up in CLL and associated with RASGRF2) and AC002480.5 (down in CLL and associated with STEAP1B, shown to be overexpressed in prostate cancer).

We next evaluated T12 samples and identified 90 candidate lncRNAs that may discriminate between progressive and indolent T12 cases. Within this group were 11 lncRNAs with a FC > 10, 5 of which have no known associated gene. Of those associated with known genes, 3 were ultra-conserved region encoding lncRNAs down-regulated in progressive T12 patients (TTT ≤1 yr) and linked to hephaestin-like protein 1 precursor, pannexin-1, and tubulin beta-3 chain isoform 1. Of potential high relevance we found that the lncRNA LPP-AS1 was down-regulated in progressive T12 patients (TTT ≤1 yr) and known to be associated with the LIM-containing lipoma preferred partner (LPP) gene (p=0.028; FDR=0.03 and FC=18.3). Looking specifically at IGHV M progressive T12 patients (T12M≤1 yr) vs IGHV M indolent T12 patients (T12M>5 yrs), we again found the LPP-AS1 lncRNA was highly down-regulated in T12M≤1 (p=0.00046; FDR=0.006 and FC=34.5) but it was not found to be differentially expressed in the UM T12≤1 yr vs UM T12>5 yr comparison. The LPP gene has been shown to play a role in cell-cell adhesion, motility and signaling, and is often the fusion partner for the mixed lineage leukemia (MLL) gene in secondary acute leukemia. Furthermore, LLP may play a role in breast cancer cell invasion. LPP-AS1 may be participating in IGHV M T12 progression by affecting LPP and thus influencing migration through the lymph node microenvironment.

CONCLUSION: While candidate lncRNAs in T12 CLL need to be validated, the LPP-AS1 lncRNA shows promise as a possible marker and potential treatment target for those patients with T12 and M IGHV that may progress rapidly. Further studies are needed to evaluate the impact of lncRNAs on clinical outcome of T12 CLL patients.