p53 Homolog TAp63 elicits apoptosis in Chronic Lymphocytic Leukemia (CLL) B-Cells,

Abstract 3894

p63 is a p53 homolog whose function depends on the cellular context. The full-length TAp63 variant may carry an anti-oncogenic potential in solid tumor models, where it mediates Ras-induced cellular senescence, antagonizes tumorigenesis and suppresses the development of metastases. In hepatoma cells TAp63 is involved in activation of both extrinsic and intrinsic apoptosis pathways. By contrast, ΔNp63, an amino-terminally truncated p63 variant, is oncogenic in tumors of squamous cell origin. TAp63 is the predominantly expressed p63 isoform in lymphoid malignancies. Increased expression of TAp63 in diffuse large B-cell lymphoma confers an unfavorable prognosis. In CLL TAp63 was shown to mediate B-cell homing to the bone marrow, thus possibly contributing to apoptosis evasion. We studied expression of p63 in CLL and determined whether p63 plays a role in CLL B-cell survival and sensitivity to chemotherapy.

We enrolled 25 previously untreated subjects with B-CLL at the Norris Cotton Cancer Center (Lebanon, NH). CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique and purified using a B-cell (CLL) Isolation Kit. Small interfering RNA against p63 were delivered using Lonza Nucleofector with transfection efficiency of 20–50% and viability of 60–80% at 24 h. Viability was enhanced when cells were cocultured with “feeder cells”. To test sensitivity to chemotherapy, cells were treated with 10 μM chlorambucil and 5 μM fludarabine for 48–72 h. For apoptosis analysis cells were stained with Annexin V and 7-AAD and assayed by flow cytometry. Five lymph node tissues were analyzed for p63 expression by immunohistochemistry using a mouse monoclonal p63 antibody (clone 4A4).

Of 25 CLL patients 15 were males (60%). Median age was 61 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (92%). TAp63 mRNA transcripts were expressed in all CLL samples, while ΔNp63 variant was not detected. TAp63α was the predominantly expressed splicing variant. A C-terminally truncated TAp63γ variant was detected at a low level. TAp63 mRNA transcript levels were higher in CLL B-cells than in normal B-cells, but 10- to 100-fold lower than in high-grade lymphoma cell lines. Of five lymph nodes analyzed, weak nuclear p63 staining was detected in one. In this small cohort, p63 expression in the peripheral blood CLL B-cells did not correlate with ZAP-70 expression. CD38-positive (>30%) CLL samples expressed higher TAp63 mRNA transcript levels (p<0.05). siRNA knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis at 24–48 h, which persisted when cells were cultured on a “feeder layer”. Furthermore, p63 knockdown conferred protection against chlorambucil and fludarabine. B-cell receptor crosslinking with IgM resulted in a 2.5-fold repression of TAp63 transcription, consistent with the pro-survival role of a B-cell receptor in CLL.

Thus, TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells. TAp63a contributes to the intrinsic apoptosis program in CLL and may play a role in sensitizing CLL B-cells to standard chemotherapy drugs.