Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology

Takeda, Andrew J.; Maher, Timothy J.; Zhang, Yu; Lanahan, Stephen M; Bucklin, Molly L.; Compton, Susan R; Tyler, Paul M; Comrie, William A.; Matsuda, M.; Olivier, Kenneth N.; Pittaluga, Stefania; McElwee, Joshua; Priel, Debra A. Long; Kuhns, Douglas B.; Williams, Roger L.; Mustillo, Peter; Wymann, Matthias P.; Rao, V. Koneti; Lucas, Carrie L.

doi:10.1038/s41467-019-12311-5

Cited by 70 publications

(72 citation statements)

References 60 publications

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“…Early results from these clinical trials confirm enhanced proinflammatory responses resulting from use of the inhibitor in human subjects (De Henau and Rausch et al 2016). This is reflected in other studies using mouse models (Kaneda and Messer et al 2016) and in humans (Takeda and Maher et al 2019) where PI3K is inactivated through genetic ablation or mutation. The application of PI3K inhibitors in cancer for manipulating TLR-mediated signalling affirms the importance of these pathways and as potential targets in inflammatory disease.…”

Section: Significance and Overviewsupporting

confidence: 54%

Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

Tong¹

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Small GTPases of the Rab family have wide ranging and essential roles in recruiting effectors to mediate membrane trafficking and receptor signalling events in mammalian cells. Nucleotide loading and activation/deactivation of the Rabs themselves are regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and other accessory proteins. While Rabs have important roles in innate immune cells, for hostpathogen interactions, receptor signalling and membrane trafficking, in many cases their GEFs and other accessory proteins have not been elucidated. Pathogen-activated, Toll-like receptors (TLRs) initiate and modulate transcription of inflammatory cytokines and other innate immune responses which are important for immune defence but can also contribute to chronic disease. Macrophages are key cells of the innate immune system and previous work from our laboratory established a role for promiscuous Rab family member, Rab8a, in TLR signalling. Rab8a is activated in TLR pathways to recruit phosphoinositide 3-kinase gamma (PI3K) as an effector which upregulates TLR-induced Akt/mTOR signalling to drive a biased program of anti-inflammatory cytokines. TLRassociated PI3K has now emerged as a key mediator of macrophage programming (or polarisation) in inflammation and cancer and its cognate GTPase, Rab8a, has a prominent role in immunity and disease. Rab8a is itself recruited to macrophage ruffles and macropinosomes through its association with the TLR crosstalk-activated endocytic receptor LRP1. However, it is not yet known how Rab8a is activated in TLR pathways. This project set out to identify the essential Rab8a GEF(s) needed to activate Rab8a as part of the LRP1 complex. Two well-known Rab8 GEFs, GRAB and Rabin8, which previously were uncharacterised in macrophages, were investigated in this project. GRAB was identified as part of the low density lipoprotein receptor-related protein 1 (LRP1) complex in pull-downs analysed by mass spectrometry. Co-immunoprecipitation and fluorescence microscopy showed that both GRAB and the structurally similar GEF, Rabin8, undergo LPS-inducible binding to Rab8a and are localised at sites of Rab8a enrichment. To carry out functional studies, stable knockouts (KOs) of Rabin8, GRAB, as well as a double KO were produced via CRISPR-Cas9 gene editing in macrophage cell lines. Nucleotide activation assays were developed for this project and live-cell imaging with KO cell lines showed that both GEFs contribute additively to TLR4-induced Rab8a GTP-loading, but they are not needed for Rab8a iii membrane recruitment. Analysis of TLR signalling after double KO of both GEFs suggested redundant roles for Rabin8 and GRAB in activating Rab8a for PI3K-dependent Akt/mTOR signalling. Live cell imaging utilising a fluorescent Akt1 reporter confirmed that LPS/TLRinduced Akt signalling is generated on macropinosomes and requires GRAB and Rabin8 GEF function. Next, to investigate possible regulators of the Rab8 GEFs, pull-downs and mass spectrometry were performed and identif...

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Section: Significance and Overviewsupporting

confidence: 54%

Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

Tong¹

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“…Like other class I catalytic subunits (p110-α, p110-β, and p110-δ), p110-γ binds a p85 regulatory subunit to form PI3K, which phosphorylate inositol lipids and is involved in the immune response. p110-γ is highly expressed in leukocytes and is important for restraining inflammation and promoting appropriate adaptive immune responses in both humans and mice (Takeda et al 2019). p110-γ null mice show defective thymocyte development and T cell activation, as well as neutrophil migration and oxidative burst (Sasaki et al 2000).…”

Section: Irf7mentioning

confidence: 99%

A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation

Weyden

Swiaţkowska²,

Iyer³

et al. 2020

G3 Genes|Genomes|Genetics

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Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonization (15 increased and 19 decreased; P < 0.005 and genotype effect <-55 or >+55). While several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonization, which may represent potential therapeutic targets.

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“…It would be expected that these mutations are activating, although this has not been fully explored. Intriguingly, PI3Kg loss of function mutations in the C-terminal regulatory motif (R1021P, N1085S) have been identified in patients with immunodeficiencies [46,47] (Fig. 1B+C).…”

Section: Introductionmentioning

confidence: 96%

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

Rathinaswamy

Gaieb

Fleming

et al. 2020

Preprint

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Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110γ subunit playing a key role in immune signalling. PI3Kγ is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3Kγ kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

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Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology

Cited by 70 publications

References 60 publications

Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

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