The TΑp63/BCL2 axis represents a novel mechanism of clinical aggressiveness in chronic lymphocytic leukemia

Abstract

The TA-isoform of the p63 transcription factor (TAp63) has been reported to contribute to clinical aggressiveness in chronic lymphocytic leukemia (CLL) in a hitherto elusive way. Here, we sought to further understand and define the role of TAp63 in the pathophysiology of CLL. First, we found that elevated TAp63 expression levels are linked with adverse clinical outcomes, including disease relapse and shorter time-to-first treatment and overall survival. Next, prompted by the fact that TAp63 participates in an NF-κB/TAp63/BCL2 antiapoptotic axis in activated mature, normal B cells, we explored molecular links between TAp63 and BCL2 also in CLL. We documented a strong correlation at both the protein and the messenger RNA (mRNA) levels, alluding to the potential prosurvival role of TAp63. This claim was supported by inducible downregulation of TAp63 expression in the MEC1 CLL cell line using clustered regularly interspaced short palindromic repeats (CRISPR) system, which resulted in downregulation of BCL2 expression. Next, using chromatin immunoprecipitation (ChIP) sequencing, we examined whether BCL2 might constitute a transcriptional target of TAp63 and identified a significant binding profile of TAp63 in the BCL2 gene locus, across a genomic region previously characterized as a super enhancer in CLL. Moreover, we identified high-confidence TAp63 binding regions in genes mainly implicated in immune response and DNA-damage procedures. Finally, we found that upregulated TAp63 expression levels render CLL cells less responsive to apoptosis induction with the BCL2 inhibitor venetoclax. On these grounds, TAp63 appears to act as a positive modulator of BCL2, hence contributing to the antiapoptotic phenotype that underlies clinical aggressiveness and treatment resistance in CLL.

Graphical abstract

Figure 1.

Elevated levels of TAp63 are associated with adverse clinical course and chemoresistance in CLL. (A) TAp63 mRNA expression levels in different subgroups of CLL patients. TAp63 mRNA expression is significantly higher in U-CLL (n = 89) compared with M-CLL (n = 77), and the same pattern is observed between TAp63^high (n = 49) and TAp63^low (n = 117) cases. (B-C) Kaplan Meier curves for time to first treatment (TTFT) and overall survival (OS). TAp63^high CLL patients exhibit shorter TTFT (B) (P = .03) (TAp63^highmedian TTFT, 1.58 years; TAp63^lowmedian TTFT, 4.07) and shorter OS (C) (P = .04) (TAp63^highmedian OS, 7.825 years; TAp63^lowmedian OS, undefined due to the large number of censored cases) compared with TAp63^low cases. (D) TAp63 mRNA expression levels over the disease course. CLL patients at disease express higher TAp63 mRNA levels compared with the timepoint of diagnosis (FD, 3.47; P = .02). *P ≤ .05, ****P ≤ .0001.

Figure 2.

TAp63 and BCL2 expression profiles are correlated at both the protein and the mRNA level. (A) Scatter plot for correlation in mRNA level in 56 U-CLL cases before treatment showing a modest but statistically significant correlation between TAp63 and BCL2 (ρ = 0.31, P = .01). (B) TAp63 protein levels (measured by flow cytometry) are highly correlated with the corresponding BCL2 protein levels (measured by western blotting and normalized to b-actin) after RNAi silencing of the TP63 gene in 4 TP63^high cases using 3 different siRNAs (ρ = 0.78, P = .003). All comparisons were assessed using as a control cells transfected with a siRNA nonhomologous to any known mammalian gene. (C-D) Inducible downregulation of TAp63 expression (gRNA1: FD, 1.7; P = .03; gRNA2: FD, 1.53) resulted in downregulation of BCL2 expression (gRNA1: FD, 1.34; P = .05; gRNA2: FD, 1.12) in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells (C), with strong correlation (ρ = 0.97, P < .0001) between their expression levels (D). The assay was performed in triplicate. *P ≤ .05. RNAi, RNA interference.

Figure 3.

Analysis of the TAp63 binding landscape. (A) Overlap between TAp63 binding sites in primary cells from 1 CLL case as well as MEC1 cell line as identified using a stringent MACS peak calling cutoff q = 0.00001. One hundred and twenty common genes were found between the 2 comparison groups. (B) Genomic distribution of TAp63 binding sites in both samples. Promoter binding corresponds to 17% and 11.4% in CLL and MEC1 cells, respectively. (C) De novo motif analysis for the TAp63 binding sites in both samples identified a motif resembling the p63 (matrix id in the JASPAR motif database: MA0525.2)-p53 (matrix id: MA0106.3)-p73 (matrix id: MA0861.1) family of motifs as the most statistically significant (e-value: CLL cells, 2.5e-146; MEC1, 8.7e-476). (D) IGV genomic browser snapshots depicting the TAp63 binding profile and epigenomics marks on the MDM2 locus. A promoter-proximal strong TAp63 binding was found at the MDM2 gene, confirming its characterization as a well-described positive TAp63 transcriptional target. (E) IGV genomic browser snapshots illustrating the TAp63 binding events and epigenomics marks at the BCL2 locus in a region previously characterized as a super-enhancer in CLL. (F-G) Co-depiction of TAp63 binding signals with publicly available ATAC-seq (GEO no. GSM3382058) and H3K27ac (GEO no. GSM3382050) data around its total binding sites in both samples using an aggregate plot (top half of the scheme) and a heatmap (bottom half of the scheme) analysis TAp63 binding sites coexist with ATAC-seq and H3K27ac signals, corresponding to accessible and active chromatin. MACS, model-based analysis of ChIP-seq.

Figure 4.

TAp63^high CLL cases are resistant to the BCL2 inhibitor venetoclax. Bar graph illustrating the normalized viability (to DMSO-treated cells), measured by flow cytometry, in TAp63^high (n = 8) vs TAp63^low (n = 6) cases after 24 and 48 hours of venetoclax treatment. TAp63^high cases exhibit no statistically significant decrease in cell viability, whereas cell viability in TAp63^low cases was significantly reduced at both the 24-hour (A) and the 48-hour (B) timepoints. Because normalized viability is depicted in the graph, the value of each DMSO samples is equal to 1. **P ≤ .01. ABT-199, venetoclax.