Medical Biology - Research Publications
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ItemLoss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1)(WILEY, 2016-09)Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.
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ItemCritical B-lymphoid cell intrinsic role of endogenous MCL-1 in c-MYC-induced lymphomagenesis(SPRINGERNATURE, 2016-03)Evasion of apoptosis is critical for tumorigenesis, and sustained survival of nascent neoplastic cells may depend upon the endogenous levels of pro-survival BCL-2 family members. Indeed, previous studies using gene-targeted mice revealed that BCL-XL, but surprisingly not BCL-2, is critical for the development of c-MYC-induced pre-B/B lymphomas. However, it remains unclear whether another pro-survival BCL-2 relative contributes to their development. MCL-1 is an intriguing candidate, because it is required for cell survival during early B-lymphocyte differentiation. It is expressed abnormally high in several types of human B-cell lymphomas and is implicated in their resistance to chemotherapy. To test the B-cell intrinsic requirement for endogenous MCL-1 in lymphoma development, we conditionally deleted Mcl-1 in B-lymphoid cells of Eμ-Myc transgenic mice. We found that MCL-1 loss in early B-lymphoid progenitors delayed MYC-driven lymphomagenesis. Moreover, the lymphomas that arose when MCL-1 levels were diminished appeared to have been selected for reduced levels of BIM and/or increased levels of BCL-XL. These results underscore the importance of MCL-1 in lymphoma development and show that alterations in the levels of other cell death regulators can compensate for deficiencies in MCL-1 expression.
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ItemRAG-induced DNA lesions activate proapoptotic BIM to suppress lymphomagenesis in p53-deficient mice(ROCKEFELLER UNIV PRESS, 2016-09)Neoplastic transformation is driven by oncogenic lesions that facilitate unrestrained cell expansion and resistance to antiproliferative signals. These oncogenic DNA lesions, acquired through errors in DNA replication, gene recombination, or extrinsically imposed damage, are thought to activate multiple tumor suppressive pathways, particularly apoptotic cell death. DNA damage induces apoptosis through well-described p53-mediated induction of PUMA and NOXA. However, loss of both these mediators (even together with defects in p53-mediated induction of cell cycle arrest and cell senescence) does not recapitulate the tumor susceptibility observed in p53(-/-) mice. Thus, potentially oncogenic DNA lesions are likely to also trigger apoptosis through additional, p53-independent processes. We found that loss of the BH3-only protein BIM accelerated lymphoma development in p53-deficient mice. This process was negated by concomitant loss of RAG1/2-mediated antigen receptor gene rearrangement. This demonstrates that BIM is critical for the induction of apoptosis caused by potentially oncogenic DNA lesions elicited by RAG1/2-induced gene rearrangement. Furthermore, this highlights the role of a BIM-mediated tumor suppressor pathway that acts in parallel to the p53 pathway and remains active even in the absence of wild-type p53 function, suggesting this may be exploited in the treatment of p53-deficient cancers.
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ItemThe BH3-only proteins BIM and PUMA are not critical for the reticulocyte apoptosis caused by loss of the pro-survival protein BCL-XL(NATURE PUBLISHING GROUP, 2017-07)Anaemia is a major global health problem arising from diverse causes and for which improved therapeutic strategies are needed. Erythroid cells can undergo apoptotic cell death and loss of pro-survival BCL-XL is known to trigger apoptosis during late-stage erythroid development. However, the mechanism by which loss or pharmacological blockade of BCL-XL leads to erythroid cell apoptosis remains unclear. Here we sought to identify the precise stage of erythropoiesis that depends on BCL-XL. We also tested whether deficiency of BIM or PUMA, the two main pro-apoptotic antagonists of BCL-XL, could prevent reticulocyte death and anaemia caused by BCL-XL loss. Using an in vivo mouse model of tamoxifen-inducible Bclx gene deletion and in vitro assays with a BCL-XL-selective inhibitor, we interrogated each stage of erythrocyte differentiation for BCL-XL dependency. This revealed that reticulocytes, but not orthochromatic erythroblasts, require BCL-XL for their survival. Surprisingly, concurrent loss of BIM or PUMA had no significant impact on the development of anemia following acute BCL-XL deletion in vivo. However, analysis of mixed bone marrow chimaeric mice revealed that loss of PUMA, but not loss of BIM, partially alleviated impaired erythropoiesis caused by BCL-XL deficiency. Insight into how the network of pro-survival and pro-apoptotic proteins works will assist the development of strategies to mitigate the effects of abnormal cell death during erythropoiesis and prevent anaemia in patients treated with BCL-XL-specific BH3-mimetic drugs.
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ItemImpact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK(NATURE PUBLISHING GROUP, 2015-10)It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.
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ItemCell cycle progression dictates the requirement for BCL2 in natural killer cell survival(ROCKEFELLER UNIV PRESS, 2017-02)Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.