B-Cell Receptor Signaling Suppresses TAp63-Induced Apoptosis Via PI3-K/mTOR Pathway and Upregulation of MiR-21 in Chronic Lymphocytic Leukemia (CLL)

Abstract 562

p63, an ancestral homolog of p53, encodes two major variants that have variable expression and context-specific functions in malignant tissues. We and others have shown that N-terminally truncated ΔNp63 promotes tumor growth in carcinomas. Meanwhile, the full-length TAp63 variant, which predominates in lymphoid malignancies, is anti-oncogenic in solid tumor models, where it mediates Ras-induced cellular senescence, suppresses anchorage-independent growth, and induces apoptosis. In hepatoma cells, TAp63 activates both extrinsic and intrinsic apoptosis pathways and enhances chemosensitivity. CLL clonal B cells have a low proliferative potential and disrupted apoptotic mechanism as a result of intrinsic defects and interaction with the microenvironment. At the crossroads of those pathways, the B-cell receptor (BCR) serves as a key survival molecule in CLL. Little is known about whether p63 regulates B-cell survival in CLL. Here we sought to investigate the role of TAp63 in regulation of apoptosis in CLL B cells and lymphoma cell lines and determined whether B-cell receptor signaling modulates p63.

Forty-eight patients with B-CLL were enrolled in this study. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and co-cultured with M210B4 murine stroma cell line layers in RPMI supplemented with 15% fetal bovine serum (FBS). B-cell lymphoma cell lines Daudi, DOHH, Raji, OCI-LY3, OCI-LY19, SU DHL-4 and SUDHL-10 were maintained in RPMI with 10% FBS. CLL B cells and Raji cells were transfected with TAp63α expression vector or with siRNAs targeting p63 or miR-21 using Lonza Nucleofector with B-cell nucleofection solution (CLL B cells) and Solution V (Raji cells). Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry. B-cell receptor was engaged with 5 mg/mL (Raji cells) or 10 mg/mL IgM (CLL B cells). Expression of p63 and miR-21 was evaluated by immunoblotting and real-time RT-PCR.

Median age of patients was 63 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (80%). TAp63α was the predominantly expressed p63 variant in CLL cells and 7 lymphoma cell lines. Compared with normal B cells, TAp63 mRNA transcript levels were low in 28 CLL patient samples (using an arbitrary cutoff of <10% normal; 58.3%) and normal/elevated in 20 samples (41.7%). Patients with high expression of p63 were more likely to exhibit unmutated IGHV (U-CLL; p=0.016). siRNA-mediated knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis of CLL cells cultured on M210B4 murine bone marrow stroma (p<0.01) and was accompanied by a reduction in Noxa, Puma and Bax transcript levels. By contrast, enforced expression of TAp63α enhanced apoptosis. p63 knockdown in the Raji lymphoma cells resulted in increased proliferation and metabolic activity (p<0.05). B-cell receptor engagement suppressed p63 expression in CLL cells and Raji lymphoma cells. Pre-incubation of Raji cells with Syk inhibitor R406 and inhibitors of the PI-3K/mTOR pathway (LY294002, rapamycin, and CAL-101) resulted in the reversal of this phenomenon. Meanwhile, chemical inhibition of MEK, Erk, JNK, and p38 and siRNA-mediated knockdown of the transcription factor FOXO (a downstream targets of PI-3K) had no effect on p63 expression. Since TAp63α is a known target of miR-21, a microRNA that has been implicated in the pathogenesis of CLL, we examined their relationship in CLL and lymphoma. We found that TAp63 transcript levels inversely correlated with the expression of miR-21 in CLL B cells, but not in lymphoma cell lines. BCR stimulation led to increased miR-21 levels in CLL B cells, whereas knockdown of miR-21 resulted in upregulation of TAp63 in 3 out of 5 tested samples.

TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells and B-cell lymphoma cell lines, where it modulates the apoptosis program. BCR signaling repressed TAp63α via PI-3K/mTOR pathway and via upregulation of miR-21. This may be particularly relevant in U-CLL, where baseline p63 levels were higher. These data provide additional insights and rationale for targeting the BCR pathway and miR-21 in CLL and lymphoma.