John R. Jeffers

Puma is an essential mediator of p53-dependent and -independent apoptotic pathways.

Puma encodes a BH3-only protein that is induced by the p53 tumor suppressor and other apoptotic stimuli. To assess its physiological role in apoptosis, we generated Puma knockout mice by gene targeting. Here we report that Puma is essential for hematopoietic cell death triggered by ionizing radiation (IR), deregulated c-Myc expression, and cytokine withdrawal. Puma is also required for IR-induced death throughout the developing nervous system and accounts for nearly all of the apoptotic activity attributed to p53 under these conditions. These findings establish Puma as a principal mediator of cell death in response to diverse apoptotic signals, implicating Puma as a likely tumor suppressor.

Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis.

While activation of BAX/BAK by BH3-only molecules (BH3s) is essential for mitochondrial apoptosis, the underlying mechanisms remain unsettled. Here we demonstrate that BAX undergoes stepwise structural reorganization leading to mitochondrial targeting and homo-oligomerization. The alpha1 helix of BAX keeps the alpha9 helix engaged in the dimerization pocket, rendering BAX as a monomer in cytosol. The activator BH3s, tBID/BIM/PUMA, attack and expose the alpha1 helix of BAX, resulting in secondary disengagement of the alpha9 helix and thereby mitochondrial insertion. Activator BH3s remain associated with the N-terminally exposed BAX through the BH1 domain to drive homo-oligomerization. BAK, an integral mitochondrial membrane protein, has bypassed the first activation step, explaining why its killing kinetics are faster than those of BAX. Furthermore, death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis. Accordingly, deficiency of Bim/Puma impedes ER stress-induced BAX/BAK activation and apoptosis. Our study provides mechanistic insights regarding the spatiotemporal execution of BAX/BAK-governed cell death.

BID, BIM, and PUMA Are Essential for Activation of the BAX- and BAK-Dependent Cell Death Program

Deadly Trio The proteins BAX and BAK act as a key decision point, regulating apoptosis by controlling the permeability of the mitochondrial outer membrane. Evidence has been presented for two mechanisms of activation of BAX and BAK: an indirect mechanism where proapoptotic proteins neutralize the antiapoptotic effects of the protein BCL-2 and its relatives; or direct activation of BAX and BAK by BIM, BID, or PUMA. Analysis of the situation in vivo is complicated by the overlapping function of BIM, BID, and PUMA. Ren et al. (p. 1390; see the Perspective by Martin) thus analyzed triple-knockout mice lacking BIM, BID, and PUMA. Apoptosis during mouse development required a direct effect of one of these proteins to activate BAX or BAK, thereby promoting cell death. Proapoptotic proteins act directly on mitochondrial “gatekeeper” proteins to initiate apoptotic events during mouse development. Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are associated with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c–mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.

Selection against PUMA gene expression in Myc-driven B-cell lymphomagenesis.

ABSTRACT The p53 tumor suppressor pathway limits oncogenesis by inducing cell cycle arrest or apoptosis. A key p53 target gene is PUMA, which encodes a BH3-only proapoptotic protein. Here we demonstrate that Puma deletion in the Eμ-Myc mouse model of Burkitt lymphoma accelerates lymphomagenesis and that ∼75% of Eμ-Myc lymphomas naturally select against Puma protein expression. Furthermore, approximately 40% of primary human Burkitt lymphomas fail to express detectable levels of PUMA and in some tumors this is associated with DNA methylation. Burkitt lymphoma cell lines phenocopy the primary tumors with respect to DNA methylation and diminished PUMA expression, which can be reactivated following inhibition of DNA methyltransferases. These findings establish that PUMA is silenced in human malignancies, and they suggest PUMA as a target for the development of novel chemotherapeutics.

Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B

Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

The relative contribution of pro-apoptotic p53 target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53-/- mice. To our surprise, Dr5-/-; Puma-/- mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.

Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Reprogramming of the somatic state to pluripotency can be induced by a defined set of transcription factors including Oct3/4, Sox2, Klf4, and c‐Myc [Cell 2006;126:663‐676]. These induced pluripotent stem cells (iPSCs) hold great promise in human therapy and disease modeling. However, tumor suppressive activities of p53, which are necessary to prevent persistence of DNA damage in mammalian cells, have proven a serious impediment to formation of iPSCs [Nat Methods 2011;8:409‐412]. We examined the requirement for downstream p53 activities in suppressing efficiency of reprogramming as well as preventing persistence of DNA damage into the early iPSCs. We discovered that the majority of the p53 activation occurred through early reprogramming‐induced DNA damage with the activated expression of the apoptotic inducer Puma and the cell cycle inhibitor p21. While Puma deficiency increases reprogramming efficiency only in the absence of c‐Myc, double deficiency of Puma and p21 has achieved a level of efficiency that exceeded that of p53 deficiency alone. We further demonstrated that, in both the presence and absence of p21, Puma deficiency was able to prevent any increase in persistent DNA damage in early iPSCs. This may be due to a compensatory cellular senescent response to reprogramming‐induced DNA damage in pre‐iPSCs. Therefore, our findings provide a potentially safe approach to enhance iPSC derivation by transiently silencing Puma and p21 without compromising genomic integrity. STEM CELLS 2012;30:888–897