bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024‒07‒14
sixteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU



  1. Nat Commun. 2024 Jul 08. 15(1): 5707
      The recent technological and computational advances in mass spectrometry-based single-cell proteomics have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell remains challenging. To address some of those limitations, we present a dedicated sample preparation chip, the proteoCHIP EVO 96 that directly interfaces with the Evosep One. This, in combination with the Bruker timsTOF demonstrates double the identifications without manual sample handling and the newest generation timsTOF Ultra identifies up to 4000 with an average of 3500 protein groups per single HEK-293T without a carrier or match-between runs. Our workflow spans 4 orders of magnitude, identifies over 50 E3 ubiquitin-protein ligases, and profiles key regulatory proteins upon small molecule stimulation. This study demonstrates that the proteoCHIP EVO 96-based sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.
    DOI:  https://doi.org/10.1038/s41467-024-49651-w
  2. Res Sq. 2024 Jun 27. pii: rs.3.rs-4021885. [Epub ahead of print]
      Type 2 Diabetes (T2D) is a condition that is often associated with obesity and defined by reduced sensitivity of PI3K signaling to insulin (insulin resistance), hyperinsulinemia and hyperglycemia. Molecular causes and early signaling events underlying insulin resistance are not well understood. Insulin activation of PI3K signaling causes mTOR dependent induction of PTEN translation, a negative regulator of PI3K signaling. We speculated that insulin resistance is due to insulin dependent induction of PTEN protein that prevent further increases in PI3K signaling. Here we show that in a diet induced model of obesity and insulin resistance, PTEN levels are increased in fat, muscle and liver tissues. Onset of hyperinsulinemia and PTEN induction in tissue is followed by hyperglycemia, hepatic steatosis and severe glucose intolerance. Treatment with a PTEN phosphatase inhibitor prevents and reverses these phenotypes, whereas an mTORC1 kinase inhibitor reverses all but the hepatic steatosis. These data suggest that induction of PTEN by increasing levels of insulin elevates feedback inhibition of the pathway to a point where downstream PI3K signaling is reduced and hyperglycemia ensues. PTEN induction is thus necessary for insulin resistance and the type 2 diabetes phenotype and a potential therapeutic target.
    DOI:  https://doi.org/10.21203/rs.3.rs-4021885/v1
  3. Mol Genet Genomics. 2024 Jul 09. 299(1): 66
      PIK3CA-related overgrowth spectrum (PROS) is an umbrella term to describe a diverse range of developmental disorders. Research to date has predominantly emerged from Europe and North America, resulting in a notable scarcity of studies focusing on East Asian populations. Currently, the prevalence and distribution of PIK3CA variants across various genetic loci and their correlation with distinct phenotypes in East Asian populations remain unclear. This study aims to elucidate the phenotype-genotype correlations of PROS in East Asian populations. We presented the phenotypes and genotypes of 82 Chinese patients. Among our cohort, 67 individuals carried PIK3CA variants, including missense, frameshift, and splice variants. Six patients presented with both PIK3CA and an additional variant. Seven PIK3CA-negative patients exhibited overlapping PROS manifestations with variants in GNAQ, AKT1, PTEN, MAP3K3, GNA11, or KRAS. An integrative review of the literature pertaining to East Asian populations revealed that specific variants are uniquely associated with certain PROS phenotypes. Some rare variants were exclusively identified in cases of megalencephaly and diffuse capillary malformation with overgrowth. Non-hotspot variants with undefined oncogenicity were more common in CNS phenotypes. Diseases with vascular malformation were more likely to have variants in the helical domain, whereas phenotypes involving adipose/muscle overgrowth without vascular abnormalities predominantly presented variants in the C2 domain. Our findings underscore the unique phenotype-genotype patterns within the East Asian PROS population, highlighting the necessity for an expanded cohort to further elucidate these correlations. Such endeavors would significantly facilitate the development of PI3Kα selective inhibitors tailored for the East Asian population in the future.
    Keywords:   PIK3CA variant; PIK3CA-related overgrowth spectrum; East Asian; Genotype; PROS; Phenotype
    DOI:  https://doi.org/10.1007/s00438-024-02159-w
  4. Nature. 2024 Jul 10.
      Advancements in precision oncology over the past decades have led to new therapeutic interventions, but the efficacy of such treatments is generally limited by an adaptive process that fosters drug resistance1. In addition to genetic mutations2, recent research has identified a role for non-genetic plasticity in transient drug tolerance3 and the acquisition of stable resistance4,5. However, the dynamics of cell-state transitions that occur in the adaptation to cancer therapies remain unknown and require a systems-level longitudinal framework. Here we demonstrate that resistance develops through trajectories of cell-state transitions accompanied by a progressive increase in cell fitness, which we denote as the 'resistance continuum'. This cellular adaptation involves a stepwise assembly of gene expression programmes and epigenetically reinforced cell states underpinned by phenotypic plasticity, adaptation to stress and metabolic reprogramming. Our results support the notion that epithelial-to-mesenchymal transition or stemness programmes-often considered a proxy for phenotypic plasticity-enable adaptation, rather than a full resistance mechanism. Through systematic genetic perturbations, we identify the acquisition of metabolic dependencies, exposing vulnerabilities that can potentially be exploited therapeutically. The concept of the resistance continuum highlights the dynamic nature of cellular adaptation and calls for complementary therapies directed at the mechanisms underlying adaptive cell-state transitions.
    DOI:  https://doi.org/10.1038/s41586-024-07690-9
  5. EMBO J. 2024 Jul 12.
      Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.
    Keywords:  HaloTag; Neighbour Labelling; PUFFFIN; Pluripotency; Synthetic Signalling
    DOI:  https://doi.org/10.1038/s44318-024-00154-w
  6. Cell Commun Signal. 2024 Jul 12. 22(1): 360
      Inhibitory phosphatases, such as the inositol-5-phosphatase SHIP1 could potentially contribute to B-cell acute lymphoblastic leukemia (B-ALL) by raising the threshold for activation of the autoimmunity checkpoint, allowing malignant cells with strong oncogenic B-cell receptor signaling to escape negative selection. Here, we show that SHIP1 is differentially expressed across B-ALL subtypes and that high versus low SHIP1 expression is associated with specific B-ALL subgroups. In particular, we found high SHIP1 expression in both, Philadelphia chromosome (Ph)-positive and ETV6-RUNX1-rearranged B-ALL cells. As demonstrated by targeted knockdown of SHIP1 by RNA interference, proliferation of B-ALL cells in vitro and their tumorigenic spread in vivo depended in part on SHIP1 expression. We investigated the regulation of SHIP1, as an important antagonist of the AKT signaling pathway, by the B-cell-specific transcription factor Ikaros. Targeted restoration of Ikaros and pharmacological inhibition of the antagonistic casein kinase 2, led to a strong reduction in SHIP1 expression and at the same time to a significant inhibition of AKT activation and cell growth. Importantly, the tumor suppressive function of Ikaros was enhanced by a SHIP1-dependent additive effect. Furthermore, our study shows that all three AKT isoforms contribute to the pro-mitogenic and anti-apoptotic signaling in B-ALL cells. Conversely, hyperactivation of a single AKT isoform is sufficient to induce negative selection by increased oxidative stress. In summary, our study demonstrates the regulatory function of Ikaros on SHIP1 expression in B-ALL and highlights the relevance of sustained SHIP1 expression to prevent cells with hyperactivated PI3K/AKT/mTOR signaling from undergoing negative selection.
    Keywords:  Acute lymphoblastic leukemia; Inositol 5-phosphatase; PI3K/AKT-signaling; ROS; SHIP1; Targeted hyperactivation
    DOI:  https://doi.org/10.1186/s12964-024-01732-5
  7. Nat Commun. 2024 Jul 07. 15(1): 5690
      Omics techniques generate comprehensive profiles of biomolecules in cells and tissues. However, a holistic understanding of underlying systems requires joint analyses of multiple data modalities. We present DPM, a data fusion method for integrating omics datasets using directionality and significance estimates of genes, transcripts, or proteins. DPM allows users to define how the input datasets are expected to interact directionally given the experimental design or biological relationships between the datasets. DPM prioritises genes and pathways that change consistently across the datasets and penalises those with inconsistent directionality. To demonstrate our approach, we characterise gene and pathway regulation in IDH-mutant gliomas by jointly analysing transcriptomic, proteomic, and DNA methylation datasets. Directional integration of survival information in ovarian cancer reveals candidate biomarkers with consistent prognostic signals in transcript and protein expression. DPM is a general and adaptable framework for gene prioritisation and pathway analysis in multi-omics datasets.
    DOI:  https://doi.org/10.1038/s41467-024-49986-4
  8. Dis Model Mech. 2024 Jul 01. pii: dmm050947. [Epub ahead of print]17(7):
      Human stem cell-based modeling systems are valuable tools that can greatly improve the clinical translation of basic research. Importantly, the successful application of human stem cell-based models to biomedical research depends on the widespread adoption of ethical principles and practical standards. To achieve this outcome, the International Society for Stem Cell Research (ISSCR) provides a comprehensive set of recommendations that aim to promote the ethical usage of human stem cells and to ensure rigor and reproducibility within the field. Understanding and implementing these recommendations should be a top priority for investigators around the world.
    Keywords:  Disease modeling; ISSCR; Stem cells
    DOI:  https://doi.org/10.1242/dmm.050947
  9. Nat Cancer. 2024 Jul 11.
      Despite tremendous progress in precision oncology, adaptive resistance mechanisms limit the long-term effectiveness of molecularly targeted agents. Here we evaluated the pharmacological profile of MTX-531 that was computationally designed to selectively target two key resistance drivers, epidermal growth factor receptor and phosphatidylinositol 3-OH kinase (PI3K). MTX-531 exhibits low-nanomolar potency against both targets with a high degree of specificity predicted by cocrystal structural analyses. MTX-531 monotherapy uniformly resulted in tumor regressions of squamous head and neck patient-derived xenograft (PDX) models. The combination of MTX-531 with mitogen-activated protein kinase kinase or KRAS-G12C inhibitors led to durable regressions of BRAF-mutant or KRAS-mutant colorectal cancer PDX models, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to the hyperglycemia commonly seen with PI3K inhibitors. Here, we show that MTX-531 acts as a weak agonist of peroxisome proliferator-activated receptor-γ, an attribute that likely mitigates hyperglycemia induced by PI3K inhibition. This unique feature of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors.
    DOI:  https://doi.org/10.1038/s43018-024-00781-6
  10. Cancer Lett. 2024 Jul 08. pii: S0304-3835(24)00507-X. [Epub ahead of print]598 217112
      Although anti-HER2 therapy has made significant strides in reducing metastasis and relapse in HER2-positive breast cancer, resistance to agents like trastuzumab, pertuzumab, and lapatinib frequently develops in patients undergoing treatment. Previous studies suggest that the hyperactivation of the PI3K-AKT signaling pathway by PIK3CA/PTEN gene mutations is implicated in HER2 resistance. In this study, we introduce a novel PI3K-p110α Proteolysis TAargeting Chimera (PROTAC) that effectively inhibits the proliferation of breast cancer cells by degrading PI3K-p110α. When tested in two lapatinib-resistant cell lines, JIMT1 and MDA-MB-453, both of which harbor PIK3CA mutations, the PI3K PROTAC notably reduced cell proliferation and induced G1 phase cell cycle arrest. Importantly, even at very low concentrations, PI3K PROTAC restored sensitivity to lapatinib. Furthermore, the efficacy of PI3K PROTAC surpassed that of Alpelisib, a selective PI3K-p110α kinase inhibitor in clinic. The superior performance of PI3K PROTAC was also confirmed in lapatinib-resistant breast cancer xenograft tumors and patient-derived breast cancer organoids (PDOs). In conclusion, this study reveals that the novel PI3K PROTAC we synthesized could serve as an effective agent to overcome lapatinib resistance.
    Keywords:  AKT; Alpelisib; HER2 resistance; Lapatinib; PI3K; PIK3CA mutation; PROTAC
    DOI:  https://doi.org/10.1016/j.canlet.2024.217112
  11. Cell Commun Signal. 2024 Jul 09. 22(1): 356
      BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.
    RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.
    CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
    Keywords:  Antioxidant response; Cardiomyocyte; Cardiovascular disease; Cell survival; Extracellular vesicles; Heart regeneration; Hypoxia; Induced pluripotent stem cells; NRF2
    DOI:  https://doi.org/10.1186/s12964-024-01722-7
  12. J Biol Chem. 2024 Jul 04. pii: S0021-9258(24)02036-2. [Epub ahead of print] 107535
      Bruton's tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.
    Keywords:  B-cell receptor; BTK; Cell signaling; Inositol Hexakisphosphate; Kinase
    DOI:  https://doi.org/10.1016/j.jbc.2024.107535
  13. bioRxiv. 2024 Jun 27. pii: 2024.06.22.600201. [Epub ahead of print]
      Background: The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG.Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters.
    Results: On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation.
    Conclusion: Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance.
    SIGNIFICANCE STATEMENT: The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.
    DOI:  https://doi.org/10.1101/2024.06.22.600201
  14. Nat Commun. 2024 Jul 08. 15(1): 5706
      Haematopoietic stem cells (HSCs) possess unique physiological adaptations to sustain blood cell production and cope with stress responses throughout life. To maintain these adaptations, HSCs rely on maintaining a tightly controlled protein translation rate. However, the mechanism of how HSCs regulate protein translation remains to be fully elucidated. In this study, we investigate the role of transfer RNA (tRNA) m1A58 'writer' proteins TRMT6 and TRMT61A in regulating HSCs function. Trmt6 deletion promoted HSC proliferation through aberrant activation of mTORC1 signaling. TRMT6-deficient HSCs exhibited an impaired self-renewal ability in competitive transplantation assay. Mechanistically, single cell RNA-seq analysis reveals that the mTORC1 signaling pathway is highly upregulated in HSC-enriched cell populations after Trmt6 deletion. m1A-tRNA-seq and Western blot analysis suggest that TRMT6 promotes methylation modification of specific tRNA and expression of TSC1, fine-tuning mTORC1 signaling levels. Furthermore, Pharmacological inhibition of the mTORC1 pathway rescued functional defect in TRMT6-deficient HSCs. To our knowledge, this study is the first to elucidate a mechanism by which TRMT6-TRMT61A complex-mediated tRNA-m1A58 modification regulates HSC homeostasis.
    DOI:  https://doi.org/10.1038/s41467-024-50110-9
  15. Dev Cell. 2024 Jul 03. pii: S1534-5807(24)00383-6. [Epub ahead of print]
      CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
    Keywords:  ALFA; CRISPR; Cas9; Drosophila; SEED; SSA; conditionally stable; knockin; nanobody; tissue specific
    DOI:  https://doi.org/10.1016/j.devcel.2024.06.004
  16. Nat Commun. 2024 Jul 10. 15(1): 5796
      Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.
    DOI:  https://doi.org/10.1038/s41467-024-50128-z