bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–03–03
fifteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.
  2. Clonal differences underlie variable responses to sequential and prolonged treatment.
  3. Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements.
  4. Deciphering cancer cell state plasticity with single-cell genomics and artificial intelligence.
  5. Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.
  6. Changes in cell-cycle rate drive diverging cell fates.
  7. Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity.
  8. The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition.
  9. Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.
  10. Integrating single-cell multi-omics and prior biological knowledge for a functional characterization of the immune system.
  11. HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.
  12. The mTOR pathway genes MTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.
  13. Engineering self-deliverable ribonucleoproteins for genome editing in the brain.
  14. On standardization of controls in lifespan studies.
  15. Characterizing the impacts of dataset imbalance on single-cell data integration.
  1. Sci Signal. 2024 Feb 27. 17(825): eadf2670
    Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.
    Emily C Erickson, Inchul You, Grace Perry, Aurelien Dugourd, Katherine A Donovan, Claire Crafter, Jeffrey W Johannes, Stuart Williamson, Jennifer I Moss, Susana Ros, Robert E Ziegler, Simon T Barry, Eric S Fischer, Nathanael S Gray, Ralitsa R Madsen, Alex Toker.
      More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.
    DOI:  https://doi.org/10.1126/scisignal.adf2670
  2. Cell Syst. 2024 Feb 14. pii: S2405-4712(24)00032-2. [Epub ahead of print]
    Clonal differences underlie variable responses to sequential and prolonged treatment.
    Dylan L Schaff, Aria J Fasse, Phoebe E White, Robert J Vander Velde, Sydney M Shaffer.
      Cancer cells exhibit dramatic differences in gene expression at the single-cell level, which can predict whether they become resistant to treatment. Treatment perpetuates this heterogeneity, resulting in a diversity of cell states among resistant clones. However, it remains unclear whether these differences lead to distinct responses when another treatment is applied or the same treatment is continued. In this study, we combined single-cell RNA sequencing with barcoding to track resistant clones through prolonged and sequential treatments. We found that cells within the same clone have similar gene expression states after multiple rounds of treatment. Moreover, we demonstrated that individual clones have distinct and differing fates, including growth, survival, or death, when subjected to a second treatment or when the first treatment is continued. By identifying gene expression states that predict clone survival, this work provides a foundation for selecting optimal therapies that target the most aggressive resistant clones within a tumor. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  barcoding; cancer systems biology; clonal tracing; drug resistance; scRNA-seq
    DOI:  https://doi.org/10.1016/j.cels.2024.01.011
  3. bioRxiv. 2024 Feb 17. pii: 2024.02.16.580760. [Epub ahead of print]
    Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements.
    Abhineet Ram, Michael Pargett, Yongin Choi, Devan Murphy, Markhus Cabel, Nont Kosaisawe, Gerald Quon, John Albeck.
      The Ras/ERK pathway drives cell proliferation and other oncogenic behaviors, and quantifying its activity in situ is of high interest in cancer diagnosis and therapy. Pathway activation is often assayed by measuring phosphorylated ERK. However, this form of measurement overlooks dynamic aspects of signaling that can only be observed over time. In this study, we combine a live, single-cell ERK biosensor approach with multiplexed immunofluorescence staining of downstream target proteins to ask how well immunostaining captures the dynamic history of ERK activity. Combining linear regression, machine learning, and differential equation models, we develop an interpretive framework for immunostains, in which Fra-1 and pRb levels imply long term activation of ERK signaling, while Egr-1 and c-Myc indicate recent activation. We show that this framework can distinguish different classes of ERK dynamics within a heterogeneous population, providing a tool for annotating ERK dynamics within fixed tissues.
    DOI:  https://doi.org/10.1101/2024.02.16.580760
  4. Genome Med. 2024 Feb 26. 16(1): 36
    Deciphering cancer cell state plasticity with single-cell genomics and artificial intelligence.
    Emily Holton, Walter Muskovic, Joseph E Powell.
      Cancer stem cell plasticity refers to the ability of tumour cells to dynamically switch between states-for example, from cancer stem cells to non-cancer stem cell states. Governed by regulatory processes, cells transition through a continuum, with this transition space often referred to as a cell state landscape. Plasticity in cancer cell states leads to divergent biological behaviours, with certain cell states, or state transitions, responsible for tumour progression and therapeutic response. The advent of single-cell assays means these features can now be measured for individual cancer cells and at scale. However, the high dimensionality of this data, complex relationships between genomic features, and a lack of precise knowledge of the genomic profiles defining cancer cell states have opened the door for artificial intelligence methods for depicting cancer cell state landscapes. The contribution of cell state plasticity to cancer phenotypes such as treatment resistance, metastasis, and dormancy has been masked by analysis of 'bulk' genomic data-constituted of the average signal from millions of cells. Single-cell technologies solve this problem by producing a high-dimensional cellular landscape of the tumour ecosystem, quantifying the genomic profiles of individual cells, and creating a more detailed model to investigate cancer plasticity (Genome Res 31:1719, 2021; Semin Cancer Biol 53: 48-58, 2018; Signal Transduct Target Ther 5:1-36, 2020). In conjunction, rapid development in artificial intelligence methods has led to numerous tools that can be employed to study cancer cell plasticity.
    DOI:  https://doi.org/10.1186/s13073-024-01309-4
  5. Cell Commun Signal. 2024 Feb 28. 22(1): 154
    Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.
    Guy Nadel, Zhong Yao, Avital Hacohen-Lev-Ran, Ehud Wainstein, Galia Maik-Rachline, Tamar Ziv, Zvi Naor, Arie Admon, Rony Seger.
       BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known.
    METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting.
    RESULTS: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCβ) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis.
    CONCLUSION: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery.
    Keywords:  Apoptosis; JNK; PKC; PP2A phosphorylation; PP2A switch
    DOI:  https://doi.org/10.1186/s12964-024-01536-7
  6. Nat Rev Genet. 2024 Mar 01.
    Changes in cell-cycle rate drive diverging cell fates.
    Kate E Galloway.
      
    DOI:  https://doi.org/10.1038/s41576-024-00714-0
  7. bioRxiv. 2024 Feb 13. pii: 2024.02.13.580131. [Epub ahead of print]
    Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity.
    Jonathan Kuhn, Parijat Banerjee, Andrew Haye, Douglas N Robinson, Pablo A Iglesias, Peter N Devreotes.
      To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in Dictyostelium and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.
    DOI:  https://doi.org/10.1101/2024.02.13.580131
  8. Transl Oncol. 2024 Feb 26. pii: S1936-5233(23)00243-7. [Epub ahead of print]43 101857
    The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition.
    Claudia Kalla, German Ott, Francesca Finotello, Karolina Niewola-Staszkowska, Giusy Di Conza, Michael Lahn, Lars van der Veen, Julia Schüler, Roger Falkenstern-Ge, Joanna Kopecka, Chiara Riganti.
      Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.
    Keywords:  PI3/AKT/mTOR inhibition; Phosphoinositide 3-kinase delta (PI3K-δ); apoptosis; combinatorial therapy; malignant pleural mesothelioma; tumor induced-immunosuppression
    DOI:  https://doi.org/10.1016/j.tranon.2023.101857
  9. Cell Rep. 2024 Feb 24. pii: S2211-1247(24)00165-7. [Epub ahead of print]43(3): 113837
    Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.
    Jose Luis Herrera, Masanobu Komatsu.
      Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.
    Keywords:  AKT3; CP: Cell biology; Cell quiescence; Cell-cell interaction; JAG1; Juxtracrine signaling; Notch; RRAS; UNC5B; VE-cadherin; Vascular permeability
    DOI:  https://doi.org/10.1016/j.celrep.2024.113837
  10. Nat Immunol. 2024 Mar;25(3): 405-417
    Integrating single-cell multi-omics and prior biological knowledge for a functional characterization of the immune system.
    Philipp Sven Lars Schäfer, Daniel Dimitrov, Eduardo J Villablanca, Julio Saez-Rodriguez.
      The immune system comprises diverse specialized cell types that cooperate to defend the host against a wide range of pathogenic threats. Recent advancements in single-cell and spatial multi-omics technologies provide rich information about the molecular state of immune cells. Here, we review how the integration of single-cell and spatial multi-omics data with prior knowledge-gathered from decades of detailed biochemical studies-allows us to obtain functional insights, focusing on gene regulatory processes and cell-cell interactions. We present diverse applications in immunology and critically assess underlying assumptions and limitations. Finally, we offer a perspective on the ongoing technological and algorithmic developments that promise to get us closer to a systemic mechanistic understanding of the immune system.
    DOI:  https://doi.org/10.1038/s41590-024-01768-2
  11. Genome Biol. 2024 Feb 26. 25(1): 58
    HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.
    Shiqian Li, Yafei Wang, Miesje van der Stoel, Xin Zhou, Shrinidhi Madhusudan, Kristiina Kanerva, Van Dien Nguyen, Nazli Eskici, Vesa M Olkkonen, You Zhou, Taneli Raivio, Elina Ikonen.
      Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.
    Keywords:  Auxin-inducible degron 2; Chemogenetics; Functional inactivation; Homology-directed repair enhancers; Homozygous knock-in; Human embryonic stem cell (hESC); hESC-derived neurons
    DOI:  https://doi.org/10.1186/s13059-024-03187-w
  12. Elife. 2024 Feb 27. pii: RP91010. [Epub ahead of print]12
    The mTOR pathway genes MTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.
    Lena H Nguyen, Youfen Xu, Maanasi Nair, Angelique Bordey.
      Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activator genes, Rheb or MTOR, or biallelic inactivation of the mTORC1 repressor genes, Depdc5, Tsc1, or Pten in the mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.
    Keywords:  HCN channels; Ih current; action potential; epilepsy; malformation of cortical development; mouse; neuroscience; seizures
    DOI:  https://doi.org/10.7554/eLife.91010
  13. Nat Commun. 2024 Feb 26. 15(1): 1727
    Engineering self-deliverable ribonucleoproteins for genome editing in the brain.
    Kai Chen, Elizabeth C Stahl, Min Hyung Kang, Bryant Xu, Ryan Allen, Marena Trinidad, Jennifer A Doudna.
      The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.
    DOI:  https://doi.org/10.1038/s41467-024-45998-2
  14. Aging (Albany NY). 2024 Feb 27. 16
    On standardization of controls in lifespan studies.
    Olga Spiridonova, Dmitrii Kriukov, Nikolai Nemirovich-Danchenko, Leonid Peshkin.
      The search for interventions to slow down and even reverse aging is a burgeoning field. The literature cites hundreds of supposedly beneficial pharmacological and genetic interventions in model organisms: mice, rats, flies and worms, where research into physiology is routinely accompanied by lifespan data. However, when experimental animals from one article live as long as controls from another article, comparing the results of interventions across studies can yield misleading outcomes. Theoretically, all lifespan data are ripe for re-analysis: we could contrast the molecular targets and pathways across studies and help focus the further search for interventions. Alas, the results of most longevity studies are difficult to compare. This is in part because there are no clear, universally accepted standards for conducting such experiments or even for reporting such data. The situation is worsened by the fact that the authors often do not describe experimental conditions completely. As a result, works on longevity make up a set of precedents, each of which might be interesting in its own right, yet incoherent and incomparable at least for the reason that in a general context, it may indicate, for example, not prolonging the life of an average organism, but compensating for any genetic abnormalities of a particular sample or inappropriate living conditions. Here we point out specific issues and propose solutions for quality control by checking both inter- and intra-study consistency of lifespan data.
    Keywords:  aging; animal disease models; data standardization; survival modeling
    DOI:  https://doi.org/10.18632/aging.205604
  15. Nat Biotechnol. 2024 Mar 01.
    Characterizing the impacts of dataset imbalance on single-cell data integration.
    Hassaan Maan, Lin Zhang, Chengxin Yu, Michael J Geuenich, Kieran R Campbell, Bo Wang.
      Computational methods for integrating single-cell transcriptomic data from multiple samples and conditions do not generally account for imbalances in the cell types measured in different datasets. In this study, we examined how differences in the cell types present, the number of cells per cell type and the cell type proportions across samples affect downstream analyses after integration. The Iniquitate pipeline assesses the robustness of integration results after perturbing the degree of imbalance between datasets. Benchmarking of five state-of-the-art single-cell RNA sequencing integration techniques in 2,600 integration experiments indicates that sample imbalance has substantial impacts on downstream analyses and the biological interpretation of integration results. Imbalance perturbation led to statistically significant variation in unsupervised clustering, cell type classification, differential expression and marker gene annotation, query-to-reference mapping and trajectory inference. We quantified the impacts of imbalance through newly introduced properties-aggregate cell type support and minimum cell type center distance. To better characterize and mitigate impacts of imbalance, we introduce balanced clustering metrics and imbalanced integration guidelines for integration method users.
    DOI:  https://doi.org/10.1038/s41587-023-02097-9
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