Circular RNA signature of aggressive CLL with t(14;19)(q32;q13). An ERIC study

Abstract

In Chronic Lymphocytic Leukemia (CLL), t(14;19)(q32;q13), leading to the overexpression of BCL3, is found in ∼1% of cases and is associated with an aggressive disease. In this study, leveraging a large CLL patient cohort collected thanks to an international collaboration, we investigate for the first time the circular transcriptome (circRNAome) associated with the rare t(14;19), in comparison with CLL without t(14;19) and B cells of age-matched healthy donors. We described the circRNAs commonly dysregulated in CLL, including circCSNK1G3 and circEXOC6B(3–5), which were depleted, and circZNF609 and circLPAR3, which were overexpressed in malignant cells. Of importance, we disclosed the circRNA signature of CLL with t(14;19), formed by circRNAs with expression significantly altered specifically in link with this lesion, ectopically expressed like circCDK14(3–4), circCORO1C, circCLEC2D, and circEMB, or downregulated like circCEP70(3–6). Several of these molecules were previously shown to be dysregulated or play a role in cancer, whereas most of the signature circRNAs deserve further investigation. CLL patients with high circCORO1C and circCLEC2D expression had significantly worse clinical outcomes, with shorter time to first treatment and overall survival. This study disclosed new molecular features of the aggressive CLL subtype with t(14;19).

Supplementary Information: The online version contains supplementary material available at 10.1186/s13045-025-01725-y.

Keywords: BCL3; CLL; Circular RNA; T(14;19).

Fig. 1

CircRNA expression variation in tCLL, oCLL, and B cells, as a normal counterpart. A) Principal component analysis of circRNA expression profiles, normalized and adjusted to remove batch effects using surrogate variables methods; B) Number of circRNA differentially (Limma-Voom, Benjamini–Hochberg adjustment for multiple testing, adj. p ≤ 0.05) expressed, overexpressed or less expressed, in pairwise comparisons of tCLL, oCLL and B cell samples (UP, overexpressed; DW, less expressed); C) Overlap of circRNAs differentially expressed in pairwise comparisons of tCLL, oCLL and B cell samples; D) Volcano plot of circRNAs significantly differentially expressed in tCLL compared to B cells (the x axis reports the Log of the Fold Change (LFC) in tCLL vs. B cell samples, the y axis the negative Log10 of the adj. P-value, the dot color indicates the expression level percentile in tCLL according to the color scale in the legend; E) Heatmap (Manhattan distance metric, Ward D2 clustering method) with sample clustering of the 1,961 circRNAs differentially expressed in at least one of the in pairwise comparisons of tCLL, oCLL and B cell samples

Fig. 2

Validation of circRNA commonly dysregulated in tCLL and oCLL and the circRNA signature of tCLL. A) RT-qPCR quantification of circLPAR3 and circCSNK1G3 in tCLL, oCLL and B cell samples confirmed a similar dysregulation in both CLL groups (relative amounts determined with the ΔΔCt method and normalized on GAPDH expression; Tukey’s multiple comparisons test, *, ** and **** indicate adj. p.value ≤ 0.05, ≤ 0.01, and ≤ 0.0001; each circRNA has been quantified in 5 tCLL and 5 oCLL cases, from an extended cohort); B) Heatmap of 80 circRNAs differentially expressed in all the pairwise comparisons of tCLL vs. B cell samples, and in TCLL vs. oCLL, representing the circRNA signature of tCLL (Manhattan distance metric, Ward D2 clustering method; the color bar on the left indicates, according to the legend: i) the signature circRNAs also altered in oCLL compared to B cells but to a significant lesser extent than in tCLL, and those altered in oCLL, plus, according to the literature, ii) the circRNAs associated to tumor suppressor or oncogenic functions, iii) expressed by genes being known transcriptional targets of BCL3 and iv) highly expressed (average expression among the 20% highest in tCLL or B cell samples); C) RT-qPCR quantification of selected tCLL signature circRNAs (circCDK14(3–4), circCORO1C, circCLEC2D, circEMB, circCEP70(3–6)) validating RNA-seq based findings (relative amounts determined with the ΔΔCt method and normalized on GAPDH expression; Tukey’s multiple comparisons test, *, ** and **** indicate adj. p.value ≤ 0.05, ≤ 0.01, and ≤ 0.0001; each circRNA has been quantified in 5 tCLL and 5 oCLL cases, from an extended cohort); D) Average circular-to-linear expression proportion (CLP; bars; vertical axis on the left) and scaled expression relative to the B cell expression mean (vertical axis on the right) of the circular (solid lines, round dots) and linear (dashed lines, square dots) transcript of the CSNK1G3, LPAR3, PTK2, and TNFSF10 genes; error bars indicate the standard error from the average CLP; E) Kaplan-Meier plots of time to first treatment (TTFT) and overall survival (OS) of CLL patients stratified by circRNA expression (high, over the average; low, below the average; Log Rank test)