bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒10‒08
twelve papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

  1. Trends Cell Biol. 2023 Sep 28. pii: S0962-8924(23)00194-0. [Epub ahead of print]
      Receptor tyrosine kinase (RTK)-mediated signal transduction is fundamental to cell function and drives important cellular outcomes which, when dysregulated, can lead to malignant tumour growth and metastasis. The initiation of signals from plasma membrane-bound RTKs is subjected to multiple regulatory mechanisms that control downstream effector protein recruitment and function. The high propensity of RTKs to condense via liquid-liquid phase separation (LLPS) into membraneless organelles with downstream effector proteins provides a further fundamental mechanism for signal regulation. Herein we highlight how this phenomenon contributes to cancer signalling and consider the potential impact of LLPS on outcomes for cancer patients.
    Keywords:  cancer signalling; kinase signalling; liquid–liquid phase separation; protein condensate
  2. J Allergy Clin Immunol Pract. 2023 Sep 28. pii: S2213-2198(23)01046-2. [Epub ahead of print]
      The phosphoinositide 3-kinase (PI3K) pathway regulates diverse cellular processes, with finely tuned PI3Kδ activity being crucial for immune cell development and function. Genetic hyperactivation of PI3Kδ causes the inborn error of immunity activated PI3Kδ syndrome (APDS). Several PI3Kδ inhibitors have been investigated as treatment options for APDS, but only leniolisib has shown both efficacy and tolerability. In contrast, severe immune-mediated adverse events such as colitis, neutropenia, and hepatotoxicity have been observed with other PI3Kδ inhibitors, particularly those indicated for hematologic malignancies. We propose that leniolisib is distinguished from other PI3Kδ inhibitors due to its structure, specific inhibitory properties selectively targeting the δ isoform without overinhibition of the δ or γ isoforms, and the precise match between APDS mechanism of disease and drug mechanism of action.
    Keywords:  APDS; PI3K; PI3Kδ; PI3Kδ inhibitor; activated PI3Kδ syndrome; leniolisib; mechanism of action
  3. Liver Int. 2023 Oct 07.
      BACKGROUND & AIMS: The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation.APPROACH & RESULTS: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression.
    CONCLUSIONS: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.
    Keywords:  EMT; extracellular-matrix; stemness; tumour growth; vasculature development
  4. Mol Cell. 2023 Oct 05. pii: S1097-2765(23)00701-3. [Epub ahead of print]83(19): 3533-3545.e5
      CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
    Keywords:  CRISPR/Cas9; Cell editing; DNA topology; Gene editing; Genome-wide off-target; Negative supercoiling; Next Generation Sequencing; Optical-tweezers; Single-molecule microscopy; Transcription-coupled off-targets
  5. JCI Insight. 2023 Oct 03. pii: e167829. [Epub ahead of print]
      MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in CD8+ T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T cells expressing a phospho-silencing mutant TSC2-S1365A (SA) retain normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, SA CD8+ T cells display greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T cells used as adoptive cell therapy display greater anti-tumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhances both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.
    Keywords:  Adaptive immunity; Cancer immunotherapy; Cell Biology; Immunology; T cells
  6. Geroscience. 2023 Oct 06.
    RAP PAC Investigators
      Treatment with rapamycin, an inhibitor of the mechanistic Target Of Rapamycin Complex One (mTORC1) protein kinase, has been repeatedly demonstrated to extend lifespan and prevent or delay age-related diseases in diverse model systems. Concerns over the risk of potentially serious side effects in humans, including immunosuppression and metabolic disruptions, have cautiously limited the translation of rapamycin and its analogs as a treatment for aging associated conditions. During the last decade, we and others have developed a working model that suggests that while inhibition of mTORC1 promotes healthy aging, many of the negative side effects of rapamycin are associated with "off-target" inhibition of a second mTOR complex, mTORC2. Differences in the kinetics and molecular mechanisms by which rapamycin inhibits mTORC1 and mTORC2 suggest that a therapeutic window for rapamycin could be exploited using intermittent dosing schedules or alternative rapalogs that may enable more selective inhibition of mTORC1. However, the optimal dosing schedules and the long-term efficacy of such interventions in humans are unknown. Here, we highlight ongoing or upcoming clinical trials that will address outstanding questions regarding the safety, pharmacokinetics, pharmacodynamics, and efficacy of rapamycin and rapalogs on several clinically oriented outcomes. Results from these early phase studies will help guide the design of phase 3 clinical trials to determine whether rapamycin can be used safely to inhibit mTORC1 for the treatment and prevention of age-related diseases in humans.
    Keywords:  Aging; Everolimus; Metabolism; Muscle; Sirolimus; mTOR
  7. Cytometry A. 2023 Oct 03.
      Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.
    Keywords:  Cell Painting; image-based profiling; phenotypic screening; quantitative imaging
  8. Nat Commun. 2023 Oct 04. 14(1): 6201
      Endonuclease G (ENDOG), a nuclear-encoded mitochondrial intermembrane space protein, is well known to be translocated into the nucleus during apoptosis. Recent studies have shown that ENDOG might enter the mitochondrial matrix to regulate mitochondrial genome cleavage and replication. However, little is known about the role of ENDOG in the cytosol. Our previous work showed that cytoplasmic ENDOG competitively binds with 14-3-3γ, which released TSC2 to repress mTORC1 signaling and induce autophagy. Here, we demonstrate that cytoplasmic ENDOG could also release Rictor from 14-3-3γ to activate the mTORC2-AKT-ACLY axis, resulting in acetyl-CoA production. Importantly, we observe that ENDOG could translocate to the ER, bind with Bip, and release IRE1a/PERK to activate the endoplasmic reticulum stress response, promoting lipid synthesis. Taken together, we demonstrate that loss of ENDOG suppresses acetyl-CoA production and lipid synthesis, along with reducing endoplasmic reticulum stress, which eventually alleviates high-fat diet-induced nonalcoholic fatty liver disease in female mice.
  9. Pediatr Dermatol. 2023 Oct 06.
      High-flow vascular malformations have been associated with multiple syndromes including capillary malformation-arteriovenous malformation (CM-AVM) syndrome, hereditary hemorrhagic telangiectasia syndrome, and less commonly, phosphatase and tensin homolog hamartoma tumor syndrome (PHTS). We present a series of three patients with clinically challenging complex AVMs who were found to have underlying PHTS. In all patients, diagnosis was delayed, and the presence of the AVM prompted sampling and genetic testing for PHTS in the absence of other clinical features of the condition. This series highlights the importance of screening for PHTS in the setting of high-flow vascular malformations.
    Keywords:  PTEN hamartoma tumor syndrome; arteriovenous malformation; embolization; sclerotherapy; vascular anomalies
  10. bioRxiv. 2023 Sep 19. pii: 2023.06.02.543453. [Epub ahead of print]
      Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2- kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP3. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.
  11. PLoS One. 2023 ;18(10): e0292317
      Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.