Medical Biology - Research Publications

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    Author Correction: Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance.
    Deng, Y ; Diepstraten, ST ; Potts, MA ; Giner, G ; Trezise, S ; Ng, AP ; Healey, G ; Kane, SR ; Cooray, A ; Behrens, K ; Heidersbach, A ; Kueh, AJ ; Pal, M ; Wilcox, S ; Tai, L ; Alexander, WS ; Visvader, JE ; Nutt, SL ; Strasser, A ; Haley, B ; Zhao, Q ; Kelly, GL ; Herold, MJ (Springer Science and Business Media LLC, 2022-08-25)
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    Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance
    Deng, Y ; Diepstraten, ST ; Potts, MA ; Giner, G ; Trezise, S ; Ng, AP ; Healey, G ; Kane, SR ; Cooray, A ; Behrens, K ; Heidersbach, A ; Kueh, AJ ; Pal, M ; Wilcox, S ; Tai, L ; Alexander, WS ; Visvader, JE ; Nutt, SL ; Strasser, A ; Haley, B ; Zhao, Q ; Kelly, GL ; Herold, MJ (NATURE PORTFOLIO, 2022-08-12)
    CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.
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    Removal of BFL-1 sensitises some melanoma cells to killing by BH3 mimetic drugs
    Gangoda, L ; Schenk, RL ; Tai, L ; Szeto, P ; Cheung, JG ; Strasser, A ; Lessene, G ; Shackleton, M ; Herold, MJ (SPRINGERNATURE, 2022-04-04)
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    In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis
    Heitink, L ; Whittle, JR ; Vaillant, F ; Capaldo, BD ; Dekkers, JF ; Dawson, CA ; Milevskiy, MJG ; Surgenor, E ; Tsai, M ; Chen, H-R ; Christie, M ; Chen, Y ; Smyth, GK ; Herold, MJ ; Strasser, A ; Lindeman, GJ ; Visvader, JE (WILEY, 2022-01-26)
    Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.
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    Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection
    Doerflinger, M ; Deng, Y ; Whitney, P ; Salvamoser, R ; Engel, S ; Kueh, AJ ; Tai, L ; Bachem, A ; Gressier, E ; Geoghegan, ND ; Wilcox, S ; Rogers, KL ; Garnham, AL ; Dengler, MA ; Bader, SM ; Ebert, G ; Pearson, JS ; De Nardo, D ; Wang, N ; Yang, C ; Pereira, M ; Bryant, CE ; Strugnell, RA ; Vince, JE ; Pellegrini, M ; Strasser, A ; Bedoui, S ; Herold, MJ (CELL PRESS, 2020-09-15)
    Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
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    Mutually exclusive regulation of T cell survival by IL-7R and antigen receptor-induced signals
    Koenen, P ; Heinzel, S ; Carrington, EM ; Happo, L ; Alexander, WS ; Zhang, J-G ; Herold, MJ ; Scott, CL ; Lew, AM ; Strasser, A ; Hodgkin, PD (NATURE PUBLISHING GROUP, 2013-04-01)
    Two major processes govern T cell proliferation and survival: interleukin-7-mediated homeostasis and antigen-induced selection. How cells transit between the two states is unknown. Here we show that T cell receptor ligation actively inhibits homeostatic survival signals while initiating a new, dominant survival programme. This switch is mediated by a change in the expression of pro- and anti-apoptosis proteins through the downregulation of Bcl-2 and the induction of Bim, A1 and Bcl-xL. Calcineurin inhibitors prevent the initiation of the new survival programme, while permitting the dominant repression of Bcl-2. Thus, in the presence of these drugs the response to antigen receptor ligation is cell death. Our results identify a molecular switch that can serve as an attractive target for inducing antigen-specific tolerance in treating autoimmune disease patients and transplant recipients.
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    Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice
    Herold, MJ ; Stuchbery, R ; Merino, D ; Willson, T ; Strasser, A ; Hildeman, D ; Bouillet, P (NATURE PUBLISHING GROUP, 2014-10-01)
    The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.
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    Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA
    Herold, MJ ; O'Reilly, LA ; Lin, A ; Srivastava, R ; Doerflinger, M ; Bouillet, P ; Strasser, A ; Puthalakath, H (NATURE PUBLISHING GROUP, 2014-07-01)
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    Characterisation of a novel A1-specific monoclonal antibody
    Lang, MJ ; Brennan, MS ; O'Reilly, LA ; Ottina, E ; Czabotar, PE ; Whitlock, E ; Fairlie, WD ; Tai, L ; Strasser, A ; Herold, MJ (NATURE PUBLISHING GROUP, 2014-12-01)
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    RAG-induced DNA lesions activate proapoptotic BIM to suppress lymphomagenesis in p53-deficient mice
    Delbridge, ARD ; Pang, SHM ; Vandenberg, CJ ; Grabow, S ; Aubrey, BJ ; Tai, L ; Herold, MJ ; Strasser, A (ROCKEFELLER UNIV PRESS, 2016-09-01)
    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.