bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–07–16
23 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase.
  2. Isoform-selective targeting of PI3K: time to consider new opportunities?
  3. An integrated mechanistic and data-driven computational model predicts cell responses to high- and low-affinity EGFR ligands.
  4. Partial inhibition of class III PI3K VPS-34 ameliorates motor aging and prolongs health span.
  5. Towards effective adoption of novel image analysis methods.
  6. Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization.
  7. An engineered biosensor enables dynamic aspartate measurements in living cells.
  8. Analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform.
  9. MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia.
  10. Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24.
  11. RAS-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy.
  12. PTEN deficiency facilitates gemcitabine efficacy in cancer by modulating the phosphorylation of PP2Ac and DCK.
  13. Addgene AAV Data Hub: a platform for sharing AAV experimental data.
  14. Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.
  15. Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.
  16. Pathogenesis of cancers derived from thyroid follicular cells.
  17. CMOT: Cross-Modality Optimal Transport for multimodal inference.
  18. Transmorph: a unifying computational framework for modular single-cell RNA-seq data integration.
  19. Engineering RNA export for measurement and manipulation of living cells.
  20. A Data-Driven Signaling Network Inference Approach for Phosphoproteomics.
  21. Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth.
  22. Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.
  23. Fetal manipulation of maternal metabolism is a critical function of the imprinted Igf2 gene.
  1. J Biol Chem. 2023 Jul 07. pii: S0021-9258(23)02050-1. [Epub ahead of print] 105022
    Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase.
    Grace L Waddell, Emma E Drew, Henry P Rupp, Scott D Hansen.
      Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by phosphoinositide-3-kinase (PI3K). Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate PI(3,4)P2. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single molecule TIRF microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that localization of the central catalytic domain of SHIP1 is insensitive to dynamic changes in PI(3,4,5)P3 and PI(3,4)P2 both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine (PS) and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal SH2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor derived phosphopeptides presented either in solution or conjugated to a membrane. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid binding specificity, protein-protein interactions, and the activation of autoinhibited SHIP1.
    Keywords:  SHIP1; Src homology 2 domain (SH2 domain); phosphatase; phosphatidylinositol; phosphatidylinositol phosphatase; phosphatidylinositol signaling; phosphatidylinositol-3-kinase
    DOI:  https://doi.org/10.1016/j.jbc.2023.105022
  2. Trends Pharmacol Sci. 2023 Jul 10. pii: S0165-6147(23)00129-3. [Epub ahead of print]
    Isoform-selective targeting of PI3K: time to consider new opportunities?
    Davide Cirillo, Marta Diceglie, Marc Nazaré.
      Phosphoinositide-3-kinases (PI3Ks) are central to several cellular signaling pathways in human physiology and are potential pharmacological targets for many pathologies including cancer, thrombosis, and pulmonary diseases. Tremendous efforts to develop isoform-selective inhibitors have culminated in the approval of several drugs, validating PI3K as a tractable and therapeutically relevant target. Although successful therapeutic validation has focused on isoform-selective class I orthosteric inhibitors, recent clinical findings have indicated challenges regarding poor drug tolerance owing to sustained on-target inhibition. Hence, additional approaches are warranted to increase the clinical benefits of specific clinical treatment options, which may involve the employment of so far underexploited targeting modalities or the development of inhibitors for currently underexplored PI3K class II isoforms. We review recent key discoveries in the development of isoform-selective inhibitors, focusing particularly on PI3K class II isoforms, and highlight the emerging importance of developing a broader arsenal of pharmacological tools.
    Keywords:  PI3K; cancer; immunotherapy; isoform-selective inhibitors; thrombosis; toxicity
    DOI:  https://doi.org/10.1016/j.tips.2023.06.002
  3. bioRxiv. 2023 Jun 26. pii: 2023.06.25.543329. [Epub ahead of print]
    An integrated mechanistic and data-driven computational model predicts cell responses to high- and low-affinity EGFR ligands.
    Paul J Myers, Sung Hyun Lee, Matthew J Lazzara.
      The biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal transduction and may eventually be leveraged to understand receptor signaling system performance depends on cell context.
    One-sentence summary: Integrated kinetic and data-driven EGFR signaling model identifies the specific signaling mechanisms that dictate cell responses to EGFR activation by different ligands.
    DOI:  https://doi.org/10.1101/2023.06.25.543329
  4. PLoS Biol. 2023 07;21(7): e3002165
    Partial inhibition of class III PI3K VPS-34 ameliorates motor aging and prolongs health span.
    Zhongliang Hu, Yamei Luo, Yuting Liu, Yaru Luo, Liangce Wang, Shengsong Gou, Yuling Peng, Rui Wei, Da Jia, Yuan Wang, Shangbang Gao, Yan Zhang.
      Global increase of life expectancy is rarely accompanied by increased health span, calling for a greater understanding of age-associated behavioral decline. Motor independence is strongly associated with the quality of life of elderly people, yet the regulators for motor aging have not been systematically explored. Here, we designed a fast and efficient genome-wide screening assay in Caenorhabditis elegans and identified 34 consistent genes as potential regulators of motor aging. Among the top hits, we found VPS-34, the class III phosphatidylinositol 3-kinase that phosphorylates phosphatidylinositol (PI) to phosphatidylinositol 3-phosphate (PI(3)P), regulates motor function in aged but not young worms. It primarily functions in aged motor neurons by inhibiting PI(3)P-PI-PI(4)P conversion to reduce neurotransmission at the neuromuscular junction (NMJ). Genetic and pharmacological inhibition of VPS-34 improve neurotransmission and muscle integrity, ameliorating motor aging in both worms and mice. Thus, our genome-wide screening revealed an evolutionarily conserved, actionable target to delay motor aging and prolong health span.
    DOI:  https://doi.org/10.1371/journal.pbio.3002165
  5. Nat Methods. 2023 Jul;20(7): 971-972
    Towards effective adoption of novel image analysis methods.
    Talley Lambert, Jennifer Waters.
      
    DOI:  https://doi.org/10.1038/s41592-023-01910-2
  6. Nat Commun. 2023 Jul 14. 14(1): 3994
    Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization.
    Marine Berquez, Zhiyong Chen, Beatrice Paola Festa, Patrick Krohn, Svenja Aline Keller, Silvia Parolo, Mikhail Korzinkin, Anna Gaponova, Endre Laczko, Enrico Domenici, Olivier Devuyst, Alessandro Luciani.
      Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.
    DOI:  https://doi.org/10.1038/s41467-023-39261-3
  7. bioRxiv. 2023 Jun 27. pii: 2023.06.27.546775. [Epub ahead of print]
    An engineered biosensor enables dynamic aspartate measurements in living cells.
    Kristian Davidsen, Jonathan S Marvin, Abhi Aggarwal, Timothy A Brown, Lucas B Sullivan.
      Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.
    DOI:  https://doi.org/10.1101/2023.06.27.546775
  8. Nat Commun. 2023 07 12. 14(1): 4154
    Analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform.
    Fengchao Yu, Guo Ci Teo, Andy T Kong, Klemens Fröhlich, Ginny Xiaohe Li, Vadim Demichev, Alexey I Nesvizhskii.
      Liquid chromatography (LC) coupled with data-independent acquisition (DIA) mass spectrometry (MS) has been increasingly used in quantitative proteomics studies. Here, we present a fast and sensitive approach for direct peptide identification from DIA data, MSFragger-DIA, which leverages the unmatched speed of the fragment ion indexing-based search engine MSFragger. Different from most existing methods, MSFragger-DIA conducts a database search of the DIA tandem mass (MS/MS) spectra prior to spectral feature detection and peak tracing across the LC dimension. To streamline the analysis of DIA data and enable easy reproducibility, we integrate MSFragger-DIA into the FragPipe computational platform for seamless support of peptide identification and spectral library building from DIA, data-dependent acquisition (DDA), or both data types combined. We compare MSFragger-DIA with other DIA tools, such as DIA-Umpire based workflow in FragPipe, Spectronaut, DIA-NN library-free, and MaxDIA. We demonstrate the fast, sensitive, and accurate performance of MSFragger-DIA across a variety of sample types and data acquisition schemes, including single-cell proteomics, phosphoproteomics, and large-scale tumor proteome profiling studies.
    DOI:  https://doi.org/10.1038/s41467-023-39869-5
  9. Elife. 2023 07 10. pii: e82597. [Epub ahead of print]12
    MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia.
    Courtney A Copeland, Benjamin A Olenchock, David Ziehr, Sarah McGarrity, Kevin Leahy, Jamey D Young, Joseph Loscalzo, William M Oldham.
      Hypoxia requires metabolic adaptations to sustain energetically demanding cellular activities. While the metabolic consequences of hypoxia have been studied extensively in cancer cell models, comparatively little is known about how primary cell metabolism responds to hypoxia. Thus, we developed metabolic flux models for human lung fibroblast and pulmonary artery smooth muscle cells proliferating in hypoxia. Unexpectedly, we found that hypoxia decreased glycolysis despite activation of hypoxia-inducible factor 1α (HIF-1α) and increased glycolytic enzyme expression. While HIF-1α activation in normoxia by prolyl hydroxylase (PHD) inhibition did increase glycolysis, hypoxia blocked this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and PHD inhibition, and suggested a critical role for MYC in modulating HIF-1α responses to hypoxia. Consistent with this hypothesis, MYC knockdown in hypoxia increased glycolysis and MYC over-expression in normoxia decreased glycolysis stimulated by PHD inhibition. These data suggest that MYC signaling in hypoxia uncouples an increase in HIF-dependent glycolytic gene transcription from glycolytic flux.
    Keywords:  MYC; biochemistry; cell biology; chemical biology; human; hypoxia; hypoxia-inducible factor; metabolic flux analysis; prolyl hydroxylase
    DOI:  https://doi.org/10.7554/eLife.82597
  10. iScience. 2023 Jul 21. 26(7): 107126
    Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24.
    Lalit R Patel, Sabrina A Stratton, Megan McLaughlin, Patrick Krause, Kendra Allton, Andrés López Rivas, Daniela Barbosa, Traver Hart, Michelle C Barton.
      TRIM24 is an oncogenic chromatin reader that is frequently overexpressed in human tumors and associated with poor prognosis. However, TRIM24 is rarely mutated, duplicated, or rearranged in cancer. This raises questions about how TRIM24 is regulated and what changes in its regulation are responsible for its overexpression. Here, we perform a genome-wide CRISPR-Cas9 screen by fluorescence-activated cell sorting (FACS) that nominated 220 negative regulators and elucidated a regulatory network that includes the KAP1 corepressor, CNOT deadenylase, and GID/CTLH E3 ligase. Knocking out required components of these three complexes caused TRIM24 overexpression, confirming their negative regulation of TRIM24. Our findings identify regulators of TRIM24 that nominate previously unexplored contexts for this oncoprotein in biology and disease. These findings were enabled by SLIDER, a new scoring system designed and vetted in our study as a broadly applicable tool for analysis of CRISPR screens performed by FACS.
    Keywords:  Biochemical classification methods; Biocomputational method; Cell biology; Functional aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2023.107126
  11. Sci Adv. 2023 Jul 14. 9(28): eadf4766
    RAS-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy.
    Antonio Cuevas-Navarro, Morgan Wagner, Richard Van, Monalisa Swain, Stephanie Mo, John Columbus, Madeline R Allison, Alice Cheng, Simon Messing, Thomas J Turbyville, Dhirendra K Simanshu, Matthew J Sale, Frank McCormick, Andrew G Stephen, Pau Castel.
      RIT1 is a RAS guanosine triphosphatase (GTPase) that regulates different aspects of signal transduction and is mutated in lung cancer, leukemia, and in the germline of individuals with Noonan syndrome. Pathogenic RIT1 proteins promote mitogen-activated protein kinase (MAPK) hyperactivation; however, this mechanism remains poorly understood. Here, we show that RAF kinases are direct effectors of membrane-bound mutant RIT1 necessary for MAPK activation. We identify critical residues in RIT1 that facilitate interaction with membrane lipids and show that these are necessary for association with RAF kinases and MAPK activation. Although mutant RIT1 binds to RAF kinases directly, it fails to activate MAPK signaling in the absence of classical RAS proteins. Consistent with aberrant RAF/MAPK activation as a driver of disease, we show that pathway inhibition alleviates cardiac hypertrophy in a mouse model of RIT1 mutant Noonan syndrome. These data shed light on the function of pathogenic RIT1 and identify avenues for therapeutic intervention.
    DOI:  https://doi.org/10.1126/sciadv.adf4766
  12. Sci Transl Med. 2023 07 12. 15(704): eadd7464
    PTEN deficiency facilitates gemcitabine efficacy in cancer by modulating the phosphorylation of PP2Ac and DCK.
    Tian-Yi Jiang, Xiao-Wen Cui, Tian-Mei Zeng, Yu-Fei Pan, Yun-Kai Lin, Xiao-Fan Feng, Ye-Xiong Tan, Zhen-Gang Yuan, Li-Wei Dong, Hong-Yang Wang.
      Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line-derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.
    DOI:  https://doi.org/10.1126/scitranslmed.add7464
  13. Nat Methods. 2023 Jul 12.
    Addgene AAV Data Hub: a platform for sharing AAV experimental data.
    Jason S Nasse, Rachel Leeson, Heather Zirkle, Daniela Bourges-Waldegg, Brook Pyhtila, Melina Fan.
      
    DOI:  https://doi.org/10.1038/s41592-023-01963-3
  14. Elife. 2023 Jul 14. pii: RP84364. [Epub ahead of print]12
    Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.
    Eike K Mahlandt, Sebastián Palacios Martínez, Janine J G Arts, Simon Tol, Jaap D van Buul, Joachim Goedhart.
      The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.
    Keywords:  Rho GTPase; RhoGEF; VE-cadherin; cell biology; endothelium; human; optogenetics; vascular barrier
    DOI:  https://doi.org/10.7554/eLife.84364
  15. Brain. 2023 Jul 12. pii: awad231. [Epub ahead of print]
    Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.
    Maria O Levitin, Lettie E Rawlins, Gabriela Sanchez-Andrade, Osama A Arshad, Stephan C Collins, Stephen J Sawiak, Phillip H Iffland, Malin H L Andersson, Caleb Bupp, Emma L Cambridge, Eve L Coomber, Ian Ellis, Johanna C Herkert, Holly Ironfield, Logan Jory, Perrine F Kretz, Sarina G Kant, Alexandra Neaverson, Esther Nibbeling, Christine Rowley, Emily Relton, Mark Sanderson, Ethan M Scott, Helen Stewart, Andrew Y Shuen, John Schreiber, Liz Tuck, James Tonks, Thorkild Terkelsen, Conny van Ravenswaaij-Arts, Pradeep Vasudevan, Olivia Wenger, Michael Wright, Andrew Day, Adam Hunter, Minal Patel, Christopher J Lelliott, Peter B Crino, Binnaz Yalcin, Andrew Crosby, Emma L Baple, Darren W Logan, Matthew E Hurles, Sebastian S Gerety.
      KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn-/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n=6) and postnatal onset of brain overgrowth (n=19). By analysing head size data from their parents (n=24), we have identified a previously unrecognised KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape, and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated iPSC models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we find that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1 related disorders affecting brain structure, cognitive function, and network integrity.
    Keywords:  IPSC; animal model; mTOR; macrocephaly; neurodevelopmental disorders; recessive
    DOI:  https://doi.org/10.1093/brain/awad231
  16. Nat Rev Cancer. 2023 Jul 12.
    Pathogenesis of cancers derived from thyroid follicular cells.
    James A Fagin, Gnana P Krishnamoorthy, Iñigo Landa.
      The genomic simplicity of differentiated cancers derived from thyroid follicular cells offers unique insights into how oncogenic drivers impact tumour phenotype. Essentially, the main oncoproteins in thyroid cancer activate nodes in the receptor tyrosine kinase-RAS-BRAF pathway, which constitutively induces MAPK signalling to varying degrees consistent with their specific biochemical mechanisms of action. The magnitude of the flux through the MAPK signalling pathway determines key elements of thyroid cancer biology, including differentiation state, invasive properties and the cellular composition of the tumour microenvironment. Progression of disease results from genomic lesions that drive immortalization, disrupt chromatin accessibility and cause cell cycle checkpoint dysfunction, in conjunction with a tumour microenvironment characterized by progressive immunosuppression. This Review charts the genomic trajectories of these common endocrine tumours, while connecting them to the biological states that they confer.
    DOI:  https://doi.org/10.1038/s41568-023-00598-y
  17. Genome Biol. 2023 07 11. 24(1): 163
    CMOT: Cross-Modality Optimal Transport for multimodal inference.
    Sayali Anil Alatkar, Daifeng Wang.
      Multimodal measurements of single-cell sequencing technologies facilitate a comprehensive understanding of specific cellular and molecular mechanisms. However, simultaneous profiling of multiple modalities of single cells is challenging, and data integration remains elusive due to missing modalities and cell-cell correspondences. To address this, we developed a computational approach, Cross-Modality Optimal Transport (CMOT), which aligns cells within available multi-modal data (source) onto a common latent space and infers missing modalities for cells from another modality (target) of mapped source cells. CMOT outperforms existing methods in various applications from developing brain, cancers to immunology, and provides biological interpretations improving cell-type or cancer classifications.
    Keywords:  Cross-modal inference; Multimodal data alignment; Optimal transport; Probabilistic coupling; Single-cell multi-modality; Weighted nearest neighbor
    DOI:  https://doi.org/10.1186/s13059-023-02989-8
  18. NAR Genom Bioinform. 2023 Sep;5(3): lqad069
    Transmorph: a unifying computational framework for modular single-cell RNA-seq data integration.
    Aziz Fouché, Loïc Chadoutaud, Olivier Delattre, Andrei Zinovyev.
      Data integration of single-cell RNA-seq (scRNA-seq) data describes the task of embedding datasets gathered from different sources or experiments into a common representation so that cells with similar types or states are embedded close to one another independently from their dataset of origin. Data integration is a crucial step in most scRNA-seq data analysis pipelines involving multiple batches. It improves data visualization, batch effect reduction, clustering, label transfer, and cell type inference. Many data integration tools have been proposed during the last decade, but a surge in the number of these methods has made it difficult to pick one for a given use case. Furthermore, these tools are provided as rigid pieces of software, making it hard to adapt them to various specific scenarios. In order to address both of these issues at once, we introduce the transmorph framework. It allows the user to engineer powerful data integration pipelines and is supported by a rich software ecosystem. We demonstrate transmorph usefulness by solving a variety of practical challenges on scRNA-seq datasets including joint datasets embedding, gene space integration, and transfer of cycle phase annotations. transmorph is provided as an open source python package.
    DOI:  https://doi.org/10.1093/nargab/lqad069
  19. Cell. 2023 Jul 10. pii: S0092-8674(23)00689-X. [Epub ahead of print]
    Engineering RNA export for measurement and manipulation of living cells.
    Felix Horns, Joe A Martinez, Chengcheng Fan, Mehernaz Haque, James M Linton, Victoria Tobin, Leah Santat, Ailiena O Maggiolo, Pamela J Bjorkman, Carlos Lois, Michael B Elowitz.
      A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
    Keywords:  RNA; delivery; export; extracellular vesicles; monitoring; non-destructive; reporter; virus-like particles
    DOI:  https://doi.org/10.1016/j.cell.2023.06.013
  20. Methods Mol Biol. 2023 ;2690 335-354
    A Data-Driven Signaling Network Inference Approach for Phosphoproteomics.
    Imani Madison, Fin Amin, Kuncheng Song, Rosangela Sozzani, Lisa Van den Broeck.
      Proteins are rapidly and dynamically post-transcriptionally modified as cells respond to changes in their environment. For example, protein phosphorylation is mediated by kinases while dephosphorylation is mediated by phosphatases. Quantifying and predicting interactions between kinases, phosphatases, and target proteins over time will aid the study of signaling cascades under a variety of environmental conditions. Here, we describe methods to statistically analyze label-free phosphoproteomic data and infer posttranscriptional regulatory networks over time. We provide an R-based method that can be used to normalize and analyze label-free phosphoproteomic data using variance stabilizing normalization and a linear mixed model across multiple time points and conditions. We also provide a method to infer regulator-target interactions over time using a discretization scheme followed by dynamic Bayesian modeling computations to validate our conclusions. Overall, this pipeline is designed to perform functional analyses and predictions of phosphoproteomic signaling cascades.
    Keywords:  Bayesian modeling; Kinase regulatory networks; Label-free phosphoproteomics; Post-translational modifications
    DOI:  https://doi.org/10.1007/978-1-0716-3327-4_27
  21. JCI Insight. 2023 07 10. pii: e158098. [Epub ahead of print]8(13):
    Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth.
    Mi-Hyeon Jeong, Greg Urquhart, Cheryl Lewis, Zhikai Chi, Jenna L Jewell.
      The mammalian target of rapamycin complex 1 (mTORC1) senses multiple upstream stimuli to orchestrate anabolic and catabolic events that regulate cell growth and metabolism. Hyperactivation of mTORC1 signaling is observed in multiple human diseases; thus, pathways that suppress mTORC1 signaling may help to identify new therapeutic targets. Here, we report that phosphodiesterase 4D (PDE4D) promotes pancreatic cancer tumor growth by increasing mTORC1 signaling. GPCRs paired to Gαs proteins activate adenylyl cyclase, which in turn elevates levels of 3',5'-cyclic adenosine monophosphate (cAMP), whereas PDEs catalyze the hydrolysis of cAMP to 5'-AMP. PDE4D forms a complex with mTORC1 and is required for mTORC1 lysosomal localization and activation. Inhibition of PDE4D and the elevation of cAMP levels block mTORC1 signaling via Raptor phosphorylation. Moreover, pancreatic cancer exhibits an upregulation of PDE4D expression, and high PDE4D levels predict the poor overall survival of patients with pancreatic cancer. Importantly, FDA-approved PDE4 inhibitors repress pancreatic cancer cell tumor growth in vivo by suppressing mTORC1 signaling. Our results identify PDE4D as an important activator of mTORC1 and suggest that targeting PDE4 with FDA-approved inhibitors may be beneficial for the treatment of human diseases with hyperactivated mTORC1 signaling.
    Keywords:  Cancer; Cell Biology
    DOI:  https://doi.org/10.1172/jci.insight.158098
  22. J Vis Exp. 2023 06 23.
    Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.
    Katie B Grausam, Joshua J Breunig.
      Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.
    DOI:  https://doi.org/10.3791/65286
  23. Cell Metab. 2023 Jul 11. pii: S1550-4131(23)00217-6. [Epub ahead of print]35(7): 1195-1208.e6
    Fetal manipulation of maternal metabolism is a critical function of the imprinted Igf2 gene.
    Jorge Lopez-Tello, Hannah E J Yong, Ionel Sandovici, Georgina K C Dowsett, Efthimia R Christoforou, Esteban Salazar-Petres, Rebecca Boyland, Tina Napso, Giles S H Yeo, Brian Y H Lam, Miguel Constancia, Amanda N Sferruzzi-Perri.
      Maternal-offspring interactions in mammals involve both cooperation and conflict. The fetus has evolved ways to manipulate maternal physiology to enhance placental nutrient transfer, but the mechanisms involved remain unclear. The imprinted Igf2 gene is highly expressed in murine placental endocrine cells. Here, we show that Igf2 deletion in these cells impairs placental endocrine signaling to the mother, without affecting placental morphology. Igf2 controls placental hormone production, including prolactins, and is crucial to establish pregnancy-related insulin resistance and to partition nutrients to the fetus. Consequently, fetuses lacking placental endocrine Igf2 are growth restricted and hypoglycemic. Mechanistically, Igf2 controls protein synthesis and cellular energy homeostasis, actions dependent on the placental endocrine cell type. Igf2 loss also has additional long-lasting effects on offspring metabolism in adulthood. Our study provides compelling evidence for an intrinsic fetal manipulation system operating in placenta that modifies maternal metabolism and fetal resource allocation, with long-term consequences for offspring metabolic health.
    Keywords:  fetal programming; genomic imprinting; glucose; hormones; insulin-like growth factor 2; metabolism; placenta; pregnancy; prolactin
    DOI:  https://doi.org/10.1016/j.cmet.2023.06.007
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