p53 and COX-2 Inhibitors Control the Replication and Viability of Non-Transformed and CLL B Lymphocytes

Innate immune system-driven replication of antigen receptor-triggered, non-transformed human B2 cells depends upon BAFF-mediated survival signals. The latter promote a COX-2--PGE2--Mcl-1 pathway within the dividing subpopulation and significantly reduce activation-induced apoptosis within clonal progeny (J. Immunol. 176:6736, 2006). Mcl-1 is known to counter mitochondria-dependent apoptosis induced by Foxo-upregulated Bim and by p53-upregulated Bax, Bak, Bid and Bad. To gain insight into mechanism(s) responsible for this activation-induced apoptosis, levels of the above pro-apoptotic molecules, as well as the transcription factor p53, were monitored in cultures of CFSE-labeled, non-transformed human B cells stimulated by surrogate C3dg-bound antigen, IL-4 and BAFF. The latter typically undergo 5–6 divisions over a week of culture. Based on the preferentially upregulated expression of p53 and its downstream pro-apoptotic molecules, and the reversing effects of p53 siRNA, it was concluded that the activation-induced cell death involves a p53-mediated apoptosis program. The day 2 administration of Nutlin, a pharmacologic inhibitor of p53 interaction with its negative regulator, MdM2, resulted in further augmented p53 expression and activity. This was revealed by diminished cycling and increased apoptosis within the replicating population. To examine whether positive/negative modulation of the COX-2--PGE-2--Mcl-1 pathway can alter the sensitivity to Nutlin (10 mM), activated cultures were additionally supplemented with either exogenous PGE2 (10 nM) or the COX-2 inhibitor, CAY10404 (30 mM). While a pulse with PGE2 reversed the pro-apoptotic effects of Nutlin, a concurrent pulse with COX-2 inhibitor augmented the degree of apoptosis observed. For example, while absolute cell yield (viable + apoptotic) in cultures with

• DMSO vehicle control,

• Nutlin,

• Nutlin + COX-2 inhibitor, or

• Nutlin + PGE2 was 167 ×10³, 72 ×10³, 41 ×10³, and 96 ×10³, respectively, the percent viability within the divided fraction was 61%, 48%, 39%, and 71%, respectively.

The data suggest that a COX-2--PGE2--Mcl-1 pathway may override the effects of an active p53 pathway in cycling, non-transformed human B lymphocytes. Ongoing studies are exploring whether proliferating CLL clones are likewise reciprocally regulated by p53 and COX-2 pathways. CFSE-labeled CLL B cells were stimulated for 8 days with TLR9 ligand, ODN-2006 (0.2 mM) + IL15 (15 ng/ml), yielding up to 7–8 divisions. A day 5 pulse with Nutlin resulted in significant day 8 apoptosis of both the cycling and few non-cycling cells within activated CLL cultures (e.g. undivided cells = 64% and 10% viability, and divided cells = 45% and 2%, within control and Nutlin cultures, respectively). Preliminary results indicate that COX-2 inhibitor significantly impairs CLL replication and viability in a manner dependent upon the timed addition of inhibitor. A day 2 pulse with CAY10404 reduced the day 8 yield of CLL cells with ≥ 4 divisions, as well as slightly reducing undivided cell viability. A day 5 pulse impaired the yield of cells with ≥ 5 divisions and the viability of both non-cycling and dividing CLL. Present studies are evaluating whether low concentrations of Nutlin and COX-2 inhibitor may exhibit synergy in blocking the growth/viability of CLL clones. A two-pronged therapeutic approach at augmenting p53 levels and diminishing COX-2 levels might be warranted in the treatment of CLL.