The long noncoding RNA, treRNA, decreases DNA damage and is associated with poor response to chemotherapy in chronic lymphocytic leukemia

Abstract

The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have investigated lncRNAs in chronic lymphocytic leukemia (CLL). We have identified one particular lncRNA, treRNA, which is overexpressed in CLL B-cells. We measured transcript expression in 144 CLL patient samples and separated samples into high or low expression of treRNA relative to the overall median. We found that high expression of treRNA is significantly associated with shorter time to treatment. High treRNA also correlates with poor prognostic indicators such as unmutated IGHV and high ZAP70 protein expression. We validated these initial findings in samples collected in a clinical trial comparing the nucleoside analog fludarabine alone or in combination with the alkylating agent cyclophosphamide in untreated CLL samples collected prior to starting therapy (E2997). High expression of treRNA was independently prognostic for shorter progression free survival in patients receiving fludarabine plus cyclophosphamide. Given these results, in order to study the role of treRNA in DNA damage response we generated a model cell line system where treRNA was over-expressed in the human B-CLL cell line OSU-CLL. Relative to the vector control line, there was less cell death in OSU-CLL over-expressing treRNA after exposure to fludarabine and mafosfamide, due in part to a reduction in DNA damage. Therefore, we suggest that treRNA is a novel biomarker in CLL associated with aggressive disease and poor response to chemotherapy through enhanced protection against cytotoxic mediated DNA damage.

Keywords: DNA damage; chronic lymphocytic leukemia; lncRNA; prognostic factor; treRNA.

Figure 1. LncRNAs are aberrantly expressed in CLL

a. Relative expression of lncRNAs in primary CLL cells compared to normal B cells measured by qRT-PCR. P-values were determined by linear mixed effects models. b. Relative expression of treRNA and ENST00000413901 in IGHV unmutated (black) compared to IGHV mutated (red) primary CLL cells. Gray dots are samples with unknown mutational status. DeltaCT = (lncRNA CT - housekeeping gene CT). P-values were determined by two-sided t-tests; p = 0.0395 for ENST00000413901 and p = 0.0125 for treRNA.

Figure 2. TreRNA is associated with time to treatment in CLL

Time to treatment in the a. CRC patient set b. CD38 negative patients, and c. ZAP70 protein expression negative patients. P-values were determined by the log-rank test.

Figure 3. TreRNA is associated with progression-free survival in CLL

a. Progression-free survival, b. overall survival, and c. progression-free survival by treatment arm and by treRNA group. P-values were determined by the log-rank test.

Figure 4. TreRNA expression impairs DNA damage following treatment with fludarabine and mafosfamide

a. Viability, measured by MTS, of OSU-CLL treRNA and OSU-CLL empty vector after 48 hours of fludarabine treatment. Data are shown with individual replicates and best fit lines. P-values were determined by linear mixed effects models. *, p = 0.002; **, p < 0.001. b. Representative immunoblot of γH2AX induction in OSU-CLL-treRNA versus empty vector cell lines following 24 hour treatment with fludarabine (F, 10uM), mafosfamide (M, 2.36uM) or a combination of fludarabine plus mafosfamide (F+M). Actin probed as a loading control. Image cropped to show relevant cell lines and controls. c. Representative images from comet assay after one hour of drugging with F, M, or F+M in OSU-CLL empty vector and OSU-CLL treRNA. d. Quantitation of comet assay using olive tail moment for 200 cells per each condition. P-values were determined by linear mixed effects models.