Anissa M. Jabbour

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma−/− cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-xL and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide–substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Myeloid progenitor cells lacking p53 exhibit delayed up-regulation of Puma and prolonged survival after cytokine deprivation

Loss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)-dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53(-/-), and FoxO3a(-/-) FDM cells and found that p53(-/-) but not FoxO3a(-/-) cells were protected against IL-3 withdrawal. Loss of p21(cip/waf), which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53(-/-) hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53(-/-) cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis.

Caspase-2 is resistant to inhibition by inhibitor of apoptosis proteins (IAPs) and can activate caspase-7

Caspases are a family of cysteine proteases with roles in cytokine maturation or apoptosis. Caspase‐2 was the first pro‐apoptotic caspase identified, but its functions in apoptotic signal transduction are still being elucidated. This study examined the regulation of the activity of caspase‐2 using recombinant proteins and a yeast‐based system. Our data suggest that for human caspase‐2 to be active its large and small subunits must be separated. For maximal activity its prodomain must also be removed. Consistent with its proposed identity as an upstream caspase, caspase‐2 could provoke the activation of caspase‐7. Caspase‐2 was not subject to inhibition by members of the IAP family of apoptosis inhibitors.

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

Triggering of apoptosis by Puma is determined by the threshold set by prosurvival Bcl-2 family proteins.

Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF(3) cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF(3) cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation.

Cytoplasmic p53 is not required for PUMA-induced apoptosis

The p53 upregulated modifier of apoptosis, PUMA, was originally identified as a gene product that was transcriptionally upregulated by p53. Unlike thymocytes from wild-type mice, which undergo marked apoptosis following irradiation, thymocytes from mice genetically mutant for puma or p53 are profoundly resistant, indicating that both PUMA and p53 are required for radiation-induced apoptosis in this cell type. Thus, in thymocytes, PUMA is an essential mediator of p53induced apoptosis. PUMA is a proapoptotic ‘BH3-only’ member of the Bcl-2 family of apoptotic regulators (reviewed in Strasser). Like other BH3-only proteins, PUMA induces apoptosis by binding to the prosurvival members of the Bcl-2 family, for example, Bcl-2, Bcl-x, Mcl-1, thereby relieving the inhibitory effect of the prosurvival proteins on the proapoptotic proteins, Bax and Bak. This results in activation of Bax and Bak, subsequent release of cytochrome c from the mitochondria, and ultimately, cell death. For example, DNA damage caused by ionizing radiation results in the accumulation and activation of p53, which upregulates puma transcription. Newly synthesized PUMA is then able to antagonize Bcl-2 family prosurvival proteins resulting in the activation of Bax and Bak and eventual cell death. Although it was initially proposed that p53 acts solely in the nucleus to transactivate genes such as puma to induce cell death, a recent report by Chipuk et al. proposed an additional, radically different role for p53 in PUMA-mediated apoptosis. In this model, p53 is not only needed to transactivate puma, it must also accumulate in the cytoplasm, where it initially binds to Bcl-x. As the level of PUMA rises, it displaces p53 from Bcl-x. Liberated p53 is then able to bind and activate Bax, thereby inducing mitochondrial outer membrane permeabilization and cytochrome c release leading to cell death. According to this model, PUMA is unable to induce cell death in the absence of p53. Indeed, Chipuk et al. stated that ‘PUMA is not sufficient for apoptosis or sensitization to UVinduced apoptosis in the absence of p53,’ and consistent with this, they found that puma expression did not cause apoptosis in HCT116 p53 / cells, or sensitize them to apoptosis after exposure to UV. To verify this model, in which cytoplasmic p53 is required for PUMA-mediated apoptosis, we used a 4-hydroxy tamoxifen (4HT)-inducible lentiviral system to express wild-type PUMA in p53 / cells (Figure 1a). Using this system we generated 4HT-inducible PUMA lines of mouse embryonic fibroblasts (MEFs) and IL-3-dependent myeloid cell lines from p53 / mice. Rather than confirming that p53 was necessary for the induction of cell death by PUMA, in both p53-null cell types, expression of PUMA efficiently caused cells to undergo apoptosis (Figure 1b–d). Importantly, apoptosis occurred at levels of PUMA expression that were below the level of detection by Western blotting in MEFs and in IL-3-dependent cells (data not shown and Supplementary Figure S1), and the induction of apoptosis was specific, because PUMA expression failed to induce death of bax/bak double knockout (DKO) MEFs and myeloid cells (data not shown). These results are not consistent with a mechanism in which PUMA acts by liberating p53 from Bcl-x so that it can bind and activate Bax on the mitochondria, but support a model in which p53 is solely required for the transcriptional activation of puma following irradiation. Although our experiments were in MEFs and IL-3-dependent myeloid cell lines, whereas Chipuk et al. examined HCT116 cells, the differences in requirement for p53 are not likely to be due to cell type, because other investigators have looked at the effects of PUMA expression in other p53 WT, mutant and null cell lines. For example, the Vousden and Vogelstein laboratories reported that PUMA expression was sufficient to cause apoptosis in several p53 mutant cell lines. Furthermore, although Chipuk et al. also claimed that PUMA was unable to cause death in p53þ /þ HCT116 cells, Yu et al. found that PUMA could efficiently kill p53þ /þ HCT116 cells. Because Chipuk et al. did not report either the efficiency of transfection nor the level of PUMA expressed in either HCT116 p53þ /þ or p53 / cells, it is possible that PUMA did not cause apoptosis in their hands because too few cells were transfected, or the amount of PUMA expressed was insufficient. Our results demonstrate that PUMA can cause apoptosis independently of p53 in both p53 null fibroblasts and in p53 null IL-3-dependent myeloid cell lines, and are consistent with earlier observations both in WT and p53 / HCT116 cells and in several other p53 mutant cell lines. Several studies using cells from puma-deficient mice have also implicated PUMA in forms of apoptosis known to be p53independent, namely that caused by cytokine withdrawal or treatment with dexamethasone. Collectively these results indicate that PUMA can cause apoptosis in the absence of p53. Although we did not investigate whether cytosolic p53 can directly activate Bax to induce cell death, it is clear that once puma is transactivated by p53, there is no requirement for cytoplasmic p53 for PUMA to cause apoptosis. Cell Death and Differentiation (2008) 15, 213–219 & 2008 Nature Publishing Group All rights reserved 1350-9047/08

Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death

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Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1.

P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.

Hoxb8 regulates expression of microRNAs to control cell death and differentiation

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action is still incompletely understood. In the nematode Caenorhabditis elegans, 131 of 1090 somatic cells undergo programmed cell death during development. Transgenic expression of human Bcl-2 reduced cell death during nematode development, and partially complemented mutation of ced-9, indicating that Bcl-2 can functionally interact with the nematode cell death machinery. Identification of the nematode target(s) of Bcl-2 inhibition would help clarify the mechanism by which Bcl-2 suppresses apoptosis in mammalian cells. Exploiting yeast-based systems and biochemical assays, we analysed the ability of Bcl-2 to interact with and regulate the activity of nematode apoptosis proteins. Unlike CED-9, Bcl-2 could not directly associate with the caspase-activating adaptor protein CED-4, nor could it inhibit CED-4-dependent yeast death. By contrast, Bcl-2 could bind the C. elegans pro-apoptotic BH3-only Bcl-2 family member EGL-1. These data prompt us to hypothesise that Bcl-2 might suppress nematode cell death by preventing EGL-1 from antagonising CED-9, rather than by inhibiting CED-4.