Lin Tai

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family and its receptor, Fas, are critical for shutdown of chronic immune responses1-3 and prevention of autoimmunity4,5. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding10,11. We generated gene-targeted mice that selectively lack either secreted FasL (ΔsFasL) or membrane-bound FasL (ΔmFasL) to resolve which of these forms is required for cell killing and to explore their hypothetical non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, (on a C57BL/6 background) FasLΔm/Δm mice succumbed to SLE-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasLΔm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

The BH3-Only Protein Bid Is Dispensable for DNA Damage- and Replicative Stress-Induced Apoptosis or Cell-Cycle Arrest

Bid, a caspase-activated proapoptotic BH3-only protein, is essential for Fas-induced hepatocyte destruction. Recent studies published in Cell produced conflicting results, indicating that loss of Bid either protects or enhances apoptosis induced by DNA damage or replicative stress. To resolve this controversy, we generated novel Bid-deficient mice on an inbred C57BL/6 background and removed the drug-selection cassette from the targeted locus. Nine distinct cell types from these Bid-deficient mice underwent cell-cycle arrest and apoptosis in a manner indistinguishable from control WT cells in response to DNA damage or replicative stress. Moreover, we found that even cells from the original Bid-deficient mice responded normally to these stimuli, indicating that differences in genetic background or the presence of a strong promoter within the targeted locus are unlikely to explain the differences between our results and those reported previously. We conclude that Bid has no role in DNA damage- or replicative stress-induced apoptosis or cell-cycle arrest.

Puma and to a lesser extent Noxa are suppressors of Myc-induced lymphomagenesis.

Evasion of apoptosis contributes importantly to c-Myc-induced tumorigenesis. The BH3-only Bcl-2 family members Puma and Noxa are critical pro-apoptotic transcriptional targets of p53, a major mediator of Myc-induced apoptosis and suppressor of Myc-induced tumorigenesis. Hence, we have explored the impact of their individual or combined loss on myc-driven lymphomagenesis. Notably, Puma deficiency both increased B-lineage cells and accelerated the development of B lymphoma, accompanied by leukaemia, but not of pre-B lymphoma. Noxa deficiency alone also increased B-lineage cells but did not accelerate lymphomagenesis. However, its deficiency combined with loss of one puma allele produced more rapid onset of both pre-B and B lymphomas than did loss of a single puma allele alone. Nevertheless, the acceleration evoked by loss of both genes was not as marked as that caused by p53 heterozygosity. These results show that Puma imposes a significant, and Noxa a minor barrier to c-Myc-driven lymphomagenesis. They also indicate that additional BH3-only proteins probably also drive Myc-induced apoptosis and that non-apoptotic functions of p53 may contribute substantially to its tumour suppressor role.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok−/− mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok−/− mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok−/− mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eμ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated.

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein

The BH3‐only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo‐binding sites within the Bim promoters (BimΔFoxo/ΔFoxo). Contrary to Bim‐deficient mice, BimΔFoxo/ΔFoxo mice had a normal hematopoietic system. Moreover, cytokine‐dependent haematopoietic cells from BimΔFoxo/ΔFoxo and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.

Characterisation of a novel A1-specific monoclonal antibody.

Dear Editor, A1/BFL-1 is the least studied pro-survival BCL-2 family member. This can be largely attributed to the lack of proper tools to study A1/BFL-1 function. Owing to the genomic organisation of the A1 locus in mice (three expressed A1 genes and one pseudo-gene, interspersed by unrelated genes)1 a knockout is challenging. We generated shRNA transgenic mice in which all functional A1 isoforms were knocked down. In accordance with A1 mRNA expression studies, we found that A1 is critical for the development and survival of lymphocytes and granulocytes.2 As the A1/BFL-1 protein is regulated by ubiquitin-dependent proteasomal degradation, the A1 mRNA expression data may not truly reflect the A1/BFL-1 protein levels. Previous attempts to generate A1-specific antibodies have failed and commercially available antibodies do not reliably detect the endogenous protein. To generate A1-specific monoclonal antibodies, we immunised rats with a truncated/mutated A1 protein (delta-C20, P104K)3 together with two KLH-conjugated peptides corresponding to central and C-terminal residues of the A1 protein (aa71–84; aa129–154). Screening by ELISA and western blotting identified one monoclonal antibody that detected overexpressed A1-a, A1-b and A1-d, and to a lesser extent overexpressed human homologue BFL-1 (data not shown and Figure 1a). To test whether this antibody could reliably detect endogenous A1, we used the mouse WEHI-231 B lymphoma cells, known to express high levels of this protein.4 Western blotting revealed a single band of the molecular weight expected for A1 in untreated WEHI-231 cells (Figure 1b, first lane). Overexpressed A1 protein is highly unstable due to ubiquitin-dependent proteasomal degradation.5 To further verify the specificity of the A1 antibody, we tested the impact of protein synthesis inhibition or proteasome inhibition on the protein detected in WEHI-231 cells. As expected, the protein synthesis inhibitor cyclohexamide (CHX) decreased the intensity of the protein band, whereas the proteasome inhibitor (MG132) increased it substantially (Figure 1b). Furthermore, we were able to show that this antibody can be used to immunoprecipitate endogenous A1 protein from lysates of WEHI-231 cells (Figure 1c). Next we examined whether this antibody could also detect endogenous A1 in primary mouse cells. In accordance with previous reports on A1 mRNA expression,1 we could reliably detect A1 protein in haematopoietic tissues, such as the lymph nodes and spleen but not in the heart, kidney, liver or lungs (Figure 1d). Immunohistochemical staining using this antibody showed strong A1 protein staining within cell foci in the germinal centres of lymph nodes of non-immunised mice (Figure 1e). No staining with this antibody against A1 was observed in non-haematopoietic tissues, such as the pancreas or the heart (data not shown). To further validate the specificity of this A1 antibody in primary cells, mouse spleen cells were treated with crosslinking IgM antibodies, a stimulus known to upregulate A1 mRNA levels in B lymphocytes.6 Such BCR (B-cell receptor) stimulation increased the protein band detected by our A1 antibody and its density was further augmented when cells were additionally treated with the proteasome inhibitor MG132 during the last hour of the stimulation (Figure 1f). A1 mRNA levels are upregulated when bone marrow cells are treated with GM-CSF or when mast cells are stimulated with the calcium ionophore ionomycin.7, 8 These stimuli caused strong upregulation of the protein band detected by the A1 antibody and the density of this protein band was further increased by the addition of MG132 during the last hour of stimulation (Figures 1g and h). Finally, we validated the specificity of the antibody by using our A1 knockdown mice. In cells from these animals high GFP levels indicate high levels of A1 shRNA expression and thus low levels of endogenous A1 protein.2 We therefore FACS-sorted GFP-positive and GFP-negative spleen cells and treated them with concanavalin A (ConA), a stimulus known to upregulate A1 mRNA levels in T cells.9 As expected, our antibody detected a protein band of the molecular weight predicted for A1 in ConA-stimulated GFP-negative cells but not in the GFP-positive (i.e. A1 shRNA expressing) splenocytes (Figure 1i). This confirms the specificity of our A1 antibody. Figure 1 The newly developed A1 antibody reliably detects the endogenous levels of the pro-survival BCL-2 family member A1. (a) EYZ (control), A1-a, -b, -d and BFL-1 expression vectors were transiently transfected into 293T cells and protein lysates (total protein ... In conclusion, we present here for the first time a mouse A1-specific monoclonal antibody capable of detecting endogenous A1 protein in cell lines as well as in primary mouse cells. Unfortunately, this antibody does not recognise endogenous levels of human BFL-1 (data not shown). This antibody will be made available commercially.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

RAG-induced DNA lesions activate proapoptotic BIM to suppress lymphomagenesis in p53-deficient mice

Delbridge, Strasser, and collaborators show that potentially oncogenic RAG1/2-dependent DNA lesions trigger apoptosis through the induction of BIM, which functions as an efficient tumor suppressor.