Pro‐apoptotic TP53 homolog TAp63 is repressed via epigenetic silencing and B‐cell receptor signalling in chronic lymphocytic leukaemia

Abstract

Summary Chronic lymphocytic leukaemia (CLL) is an accumulative disorder marked by deficient apoptosis. The TP53 homolog TAp63 promotes apoptosis and chemosensitivity in solid tumours and its deregulation may contribute to CLL cell survival. We found that TAp63α was the most prevalent TP63 isoform in CLL. Compared to healthy B cells, TAp63 mRNA was repressed in 55.7% of CLL samples. TP63 promoter methylation was high in CLL and inversely correlated with TP63 protein expression in B-cell lymphoma cell lines. siRNA-mediated knockdown of TP63 resulted in partial protection from spontaneous apoptosis accompanied by reductions in PMAIP1 (NOXA), BBC3 (PUMA), and BAX mRNA in CLL cells and increased proliferation of Raji lymphoma cells. TAp63 mRNA levels were higher in CLL with unmutated IGHV. B cell receptor (BCR) engagement led to repression of TP63 mRNA expression in malignant B cells, while pharmacological inhibition of BCR signalling prevented TP63 downregulation. MIR21, known to target TAp63, correlated inversely with TAp63 expression in CLL, and BCR-mediated downregulation of TP63 was accompanied by MIR21 upregulation in most CLL samples. Our data illustrate the pro-apoptotic function of TP63, provide insights into the mechanisms of BCR-targeting agents, and establish a rationale for designing novel approaches to induce TP63 in CLL and B-cell lymphoma.

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia

“…Although TP63 gene contains two transcriptional start sites that enable the generation of transcripts containing (TAp63) or lacking (ΔNp63) the N‐terminal transactivation domain, we found that only the TAp63 isoform was expressed in CLL, in line with previous studies . Generally, most CLL cases expressed low levels of TAp63 mRNA and p63 protein in accordance with previous reports, with the striking exception of subset #8, where p63 was overexpressed compared to the majority of other U‐CLL or M‐CLL cases.…”

“…While in solid tumors TP63 mainly plays a pro‐apoptotic and tumor‐suppressive role, its role in B cell neoplasia is unclear. Particularly for CLL, the two published studies reported opposing results, variably proposing TP63 as pro‐apoptotic 51 or anti‐apoptotic . In our study TP63 was found consistently differentially methylated and expressed between the two subsets.…”

The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells

“…CLL cell apoptosis was measured in duplicate as previously described using the ApoScreen Annexin V apoptosis Kit (19). Briefly, cells were resuspended in 150 μl of Annexin V binding buffer containing 1 μl of Annexin V-PE, 1 μl of 7-AAD and 1 μl of CD19- or CD3-FITC mAbs (Southern Biotech, Birmingham, AL) followed by flow cytometry on a FACSCalibur (Becton Dickinson, Palo Alto, CA).…”

“…Proteins were analyzed by immunoblotting as previously described (19). The following antibodies were used: Bcl-2, Bcl-xL, Bim, phospho-Bim, phospho-IκBα, cleaved PARP, Mcl-1, p65/RelA, p52/p100, XIAP, FOXO3A (Cell Signaling), Bcl2-A1 (Abcam, Cambridge, MA), NEDD8 (Epitomics, Burlingame, CA), p27 (Santa Cruz, Biotechnology, Santa Cruz, CA), Noxa (Imgenex, San Diego, CA), β-actin (Sigma Aldrich), horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (BioRad).…”

BCR/ABL mRNA Targeting Small Interfering RNA Effects on Proliferation and Apoptosis in Chronic Myeloid Leukemia

“…But as is often the case with competitors in the same field: it may be that only their combined efforts will help to overcome the numerous problems associated with drug resistance in cancer treatment. The promising power of RNAi for therapeutic purposes and gives new hope that a potent tool for the reversion of an oncogenic phenotype is now in our hands (Humphries et al, 2013). BCR/ABL protein is considered relatively stable to the antisense attack (half-life, 48 hours), and it is difficult to influence its level by a single application of anti-BCR/ABL substances (Kanduri et al, 2013).…”