Tp63 Contributes to the Apoptosis Resistant Phenotype in Aggressive Chronic Lymphocytic Leukemia

Konference: 2015 57th ASH Annual Meeting - účast ČR

Kategorie: Maligní lymfomy a leukémie; Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: 641. CLL: Biology and Pathophysiology, excluding Therapy: Poster

Číslo abstraktu: 4142

Autoři: Stavroula Ntoufa; Nikos Papakonstantinou; Despoina Papazoglou; Maria Tsagiopoulou; prof. RNDr. Šárka Pospíšilová, Ph.D.; Dr. Achilles Anagnostopoulos; Prof. MD Richard Rosenquist (Brandell), PhD; M.D. Paolo Ghia, Ph.D.; MD Kostas Stamatopoulos

Alterations in the expression of TP63, a TP53 gene family member, are known to critically affect tumorigenesis though the gene is almost never mutated. In cancer, the following two TP63 isoforms have been extensively studied with contrasting/opposite functions: DNp63, known to promote oncogenesis, and TAp63, which instead has been shown to promote apoptosis. Conflicting information is available regarding the role of TP63 in chronic lymphocytic leukemia (CLL), in particular its effects on cell death (pro-apoptotic or anti-apoptotic?). In a previous DNA methylation profiling study of aggressive CLL subgroups with unmutated B-cell receptor immunoglobulin (BcR IG), we identified TP63 among the differentially methylated and expressed genes. In more detail, TP63 was overexpressed and hypomethylated in stereotyped subset #8 (IGHV4-39/IGKV1(D)-39; IgG-switched), while the opposite was seen for subset #6 (IGHV1-69/IGKV3-20); a finding that might be clinically relevant since subset #8 displays the highest risk for Richter’s transformation among all CLL. Given recent evidence that the increased propensity for RT in subset #8 might be associated with intense B-cell receptor (BcR) signaling capacity, here we investigated potential links between BcR stimulation, TP63 expression and CLL cell survival. To this end, we studied primary leukemic cells negatively isolated prior to treatment from 14 cases assigned to subset #6 (n=5), subset #8 (n=4) or non-subset CLL cases with unmutated BcR IG (U-CLL, n=5). RT-PCR analysis detected mRNA of the TAp63 isoform only, whereas the DNp63 isoform was not expressed in any case. As revealed by Western blotting analysis, BcR stimulation for 21 hours with anti-IgM (in subset #6 and non-subset cases) or anti-IgG (exclusively in subset #8) had differential effects on TP63 expression in the different CLL subgroups analyzed. More specifically, whereas TP63 levels remained unaltered in subset #6, a significant up-regulation (Fold=2.8; p<0.05) was seen in subset #8 cases and, in contrast, downregulation was observed in non-subset cases (Fold=-1.6; p<0.05). Next, we investigated the functional impact of TP63 expression on CLL cell viability at 48 hours after BcR stimulation by flow cytometry analysis, using Annexin V and Propidium iodide staining. As expected, no changes were seen in stimulated versus unstimulated (control) subset #6 CLL cells. In contrast, TP63 upregulation after BcR stimulation of subset #8 cases (n=4) was followed by significantly augmented CLL cell survival (Fold=1.6; p<0.05), whereas the opposite was seen in non-subset U-CLL cases (Fold=-1.5; p<0.05), where p63 downregulation was associated with reduced CLL cell survival (p<0.001). In order to investigate whether the observed effects on cell viability were regulated by TP63, we downregulated TP63 expression in primary cells from 5 CLL cases using TP63-specific siRNA. This treatment led to a significant (Fold=-1.8; p<0.05) reduction in cell viability, further supporting a pro-survival function of TP63. In conclusion, we show that TP63 expression can be regulated by BcR stimulation, but the observed effect differs and can be opposite in CLL subgroups with distinct immunogenetic features. In particular, we highlight p63 as a novel pro-survival factor in CLL subset #8, thus identifying another player in the complex pathophysiology of this very aggressive subset with a high risk for Richter's transformation.

Datum přednesení příspěvku: 7. 12. 2015