The Aberrantly Expressed Long Noncoding RNA, TRERNA1, Predicts for Aggressive Disease in Chronic Lymphocytic Leukemia

Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression, and dysregulated expression of lncRNAs has been reported in many cancers. One of the most documented ways by which lncRNAs can contribute to cancer aggressiveness is by altering gene expression through associations with chromatin modifying proteins. However, the vast majority of lncRNAs dysregulated in cancer remain to be functionally characterized. Specifically, there have been few studies investigating the role of lncRNAs in chronic lymphocytic leukemia (CLL). Numerous studies have been published documenting the role of micro RNAs (miRs) in CLL, indicating that non-coding RNAs can play a significant role in this disease. Therefore we hypothesize that dysregulated lncRNA expression in CLL contributes to aggressive disease. We performed microarray analysis using Arraystar Human LncRNA Array v2.0, a platform that analyzes over 30,000 lncRNA transcripts in addition to 30,000 coding transcripts. We found that many lncRNAs are aberrantly expressed in CLL compared to healthy donor B cells. We identified the lncRNA treRNA (TRERNA1) as overexpressed in CLL cells (p = .0014). treRNA has been previously described to have enhancer-like function (Ørom et al., 2010) as well as translational regulatory functions (Gummireddy et al., 2013). It has been reported as overexpressed in breast cancer lymph-node metastases and colon cancer (Gummireddy et al., 2013). In addition to expressing spliced treRNA, CLL cells contain a transcript that retains the intron between the two coding exons due to insufficient splicing. Therefore we investigated the prognostic significance of both spliced and retained intron treRNA (ri-treRNA) in subsequent studies. We obtained 144 well-characterized CLL patient samples from the CLL Research Consortium (CRC) and measured transcript expression by quantitative reverse transcription PCR (qRT-PCR). We separated patients into high or low expressers of treRNA relative to the overall median expression and found that patients with higher expression of treRNA have significantly shorter time to treatment (p = .04). High expression of treRNA also correlates with the poor prognostic indicators unmutated IGHV (p < .0001) and low ZAP70 methylation (p <.001). We validated the correlation with unmutated IGHV in a second cohort of 147 previously untreated CLL samples collected prior to starting therapy in a clinical trial (E2997; Grever et al., 2007) comparing fludarabine to the combination of fludarabine plus cyclophosphamide (FC).  We found that high expression (relative to the median) of spliced, but not ri-treRNA, independently predicts for shorter progression free survival in patients receiving FC (HR 3.14, 95% CI 1.61-6.14, p < .001). Since the data from this clinical study suggests a role for treRNA in mediating DNA damage response, we established a stable retroviral system to further study this observation in vitro. We used the CLL cell line established in our lab (OSUCLL; Hertlein et al., 2013) to express empty vector or treRNA. Expression of treRNA does not alter viability, proliferation, or migration. However, OSUCLL expressing treRNA display modest resistance to FC treatment. This correlates with less induction of the DNA damage indicator, γH2AX, as well as the DNA damage response protein, TP53, although these changes were not statistically significant. Consistent with the clinical data, ri-treRNA did not show a differential response to in vitro FC treatment. In summary, we have identified a lncRNA in CLL which may play a role in DNA damage response, and serve as a biomarker predictive of aggressive disease.