Transforming Growth Factor-β directly induces PUMA during the rapid induction of apoptosis in Myc-driven B-cell lymphomas.

c-MYC transformed human Burkitts Lymphoma (BL) cells are highly sensitive to Transforming Growth Factor-beta (TGF-ß-induced apoptosis. Previously we demonstrated that TGF-ß-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway which is dependent on the activation of BAX and/or BAK. TGF-ß directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-XL, but has no effect on the direct BAX/BAK activators BIM or BID (tBID). Here we show that TGF-ß induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-ß also induced PUMA in normal germinal centre CD77 positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-ß target gene in B cells and we identify a putative SMAD binding region (SBR) within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-ß signalling. Consitutive activity of the isolated SBR in luciferase reporter assays was dependent on SMAD consensus sequences and was partially dependent on endogenous TGF-ßsignalling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-ß-mediated apoptotic response. Analysis of Eµ-Myc cell lines demonstrated that c-myc driven murine lymphomas are also sensitive to TGF-ß-mediated apoptosis. Moreover Puma-/- Eµ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-ß therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.