Upregulation of Human Pim-2 in B-CLL, Involves Interactions between Oct-1, Oct-2, Bob-1 and NF-kB.

hPim-2 is a proto-oncogene that encodes a serine/threonine kinase and inhibits apoptosis by phosphorylation of BAD. We have shown that hPim is upregulated in human non-Hodgkin’s lymphomas (NHL) and in chronic lymphocytic leukemia (B-CLL) and its cellular transcript levels in B-CLL correlates with lymphocyte doubling time. We found no mutations in the promoter region of hPim-2 in B-cells of 30 patients with CLL (~2000 bp upstream). The proximal promoter region of hPim-2 (600 bp) contains two adjacent NF-kB-binding elements, two adjacent Oct-binding elements and an SP1 element by bioinformatic analysis. Studies have recently shown that the transcription factor Oct-2 and the B-cell specific Oct cofactor Bob-1 are overexpressed in certain large B-cell lymphomas, whereas increased expression of Bob-1 has also been observed in T-cell neoplasms. Shift assays (EMSA) analysis, using nuclear extracts from B-CLL cells and various fragments of hPim-2 promoter region used as probes, revealed that complexes containing an Oct elements were consistently heavier in B-CLL extracts compared with control B-cells. Accordingly, Oct-1, Oct-2 and Bob-1 protein levels were significantly higher in B-CLL compared to healthy extracts. Moreover, chromatin immunoprecipitation (Chip) assays confirmed that in-vivo Oct-1+2 and Bob-1 are indeed physically attached to the hPim-2 promoter, and that this interaction is significantly more intensive in B-CLL cells than in control B-cells. Furthermore, we have found in addition that the p52 isoform subunit of NF-kB predominates the interaction with the kB element in the hPim-2 promoter in B-CLL cells, as compared to the p50 isoform observed in control B-cells. To determine whether these interactions are transcriptionaly significant, we fused the luciferase reporter gene to various promoter fragments, and monitored luciferase expression in-vitro after incubation with either B-CLL or normal B-cell extracts. Luciferase expression was consistently higher when Oct element-containing fragment was incubated with B-CLL cell extracts. Together, these results suggest that the upregulation of hPim-2 in B-CLL is due to enhanced expression and transcriptional activity of the Oct-1+2 and Bob-1 complex and that it might synergistically act with the p52 containing NF-kB transcription factor.