bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–11–05
twenty-six papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Successful Treatment of Hypoglycemia With Alpelisib in Pediatric Patients With PIK3CA-Related Overgrowth Spectrum.
  2. Cholesterol regulates insulin-induced mTORC1 signaling.
  3. A New Wave of PI3Kα Inhibitors.
  4. The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra) - phosphoinositide 3-kinase (PI3K) signaling cascade to steer towards the midline during zebrafish heart tube formation.
  5. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations.
  6. Medium Depth Influences O2 Availability and Metabolism in Human RPE Cultures.
  7. Fluorescent biosensors illuminate the spatial regulation of cell signaling across scales.
  8. Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1.
  9. Differential cell cycle checkpoint evasion by PTEN germline mutations associated with dichotomous phenotypes of cancer versus autism spectrum disorder.
  10. Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.
  11. Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations.
  12. Discovery and clinical proof-of-concept of RLY-2608, a first-in-class mutant-selective allosteric PI3Ka inhibitor that decouples anti-tumor activity from hyperinsulinemia.
  13. AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.
  14. A protocol for simultaneous high-sensitivity genotyping and chromatin accessibility profiling in single cells.
  15. Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.
  16. Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.
  17. Collective signalling drives rapid jumping between cell states.
  18. A framework for ultra-low-input spatial tissue proteomics.
  19. Causal identification of single-cell experimental perturbation effects with CINEMA-OT.
  20. Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors.
  21. Imagining the future of optical microscopy: everything, everywhere, all at once.
  22. Live-cell imaging in the deep learning era.
  23. Best practices to promote rigor and reproducibility in the era of sex-inclusive research.
  24. scDiffCom: a tool for differential analysis of cell-cell interactions provides a mouse atlas of aging changes in intercellular communication.
  25. Is N-Hacking Ever OK? The consequences of collecting more data in pursuit of statistical significance.
  26. LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase.
  1. JCEM Case Rep. 2023 Mar;1(2): luad027
    Successful Treatment of Hypoglycemia With Alpelisib in Pediatric Patients With PIK3CA-Related Overgrowth Spectrum.
    Nat Nasomyont, Meilan M Rutter, Philippe F Backeljauw.
      Activating mutations in the PIK3CA gene, causing phosphoinositide 3-kinase (PI3K) hyperactivation, are rare causes of hypoglycemia. We report the novel use of alpelisib (a PI3K inhibitor) for the treatment of hypoketotic, hypoinsulinemic hypoglycemia in 2 children with PIK3CA-related overgrowth spectrum (PROS). Patient 1 was a 7-month-old girl who presented with a hypoglycemic seizure. Despite nutritional management including continuous feeds, she continued to have frequent hypoglycemia. At age 2.8 years, alpelisib was started at 50 mg daily and titrated to 100 mg daily. She was weaned off nocturnal continuous feeds by 8 months. She developed colitis when the alpelisib dose was increased to 125 mg, but this resolved with a dose decrease and medical management. At age 5.3 years, she was doing well with rare hypoglycemia. Her accelerated growth stabilized. Patient 2 was a 3-year-old boy who developed hypoglycemia in early infancy. Alpelisib 50 mg daily was started due to recurrent hypoglycemia despite nutritional management. He came off continuous feeds after 4 months, with decreased hypoglycemia frequency. At age 4.5 years, he had not experienced side effects from treatment. In conclusion, alpelisib appears to be effective in decreasing PROS-related hypoglycemia frequency and severity and should be considered for refractory hypoglycemia in this condition.
    Keywords:  PIK3CA; PROS; alpelisib; hypoglycemia; pediatrics
    DOI:  https://doi.org/10.1210/jcemcr/luad027
  2. J Cell Sci. 2023 Nov 03. pii: jcs.261402. [Epub ahead of print]
    Cholesterol regulates insulin-induced mTORC1 signaling.
    Kolaparamba V Navyasree, Shikha T Ramesh, Perunthottathu K Umasankar.
      The rapid activation of the critical kinase, mechanistic target of rapamycin complex-1 (mTORC1) by insulin is key to cell growth in mammals, but the regulatory factors remain unclear. Here, we demonstrate that cholesterol plays a crucial role in the regulation of insulin-stimulated mTORC1 signaling. The rapid progression of insulin-induced mTORC1 signaling declines in sterol-depleted cells and restores in cholesterol-repleted cells. In insulin-stimulated cells, cholesterol promotes recruitment of mTORC1 onto lysosomes without affecting insulin-induced dissociation of the TSC complex from lysosomes, thereby enabling complete activation of mTORC1. We also show that under prolonged starvation conditions, cholesterol coordinates with autophagy to support mTORC1 reactivation on lysosomes thereby restoring insulin-responsive mTORC1 signaling. Further, we identify that Smith-Lemli-Opitz Syndrome (SLOS) patient fibroblasts and HeLa-SLOS model which are deficient in cholesterol biosynthesis exhibit defects in insulin-mTORC1 growth axis. These defects are rescued by supplementation of exogenous cholesterol or by expression of constitutively active Rag GTPase, a downstream activator of mTORC1. Overall, our findings propose novel signal integration mechanisms to achieve spatial and temporal control of mTORC1-dependent growth signaling and their aberrations in disease.
    Keywords:  Cholesterol; Insulin; Lysosome; Signaling; mTORC1
    DOI:  https://doi.org/10.1242/jcs.261402
  3. Cancer Discov. 2023 11 01. 13(11): 2313-2315
    A New Wave of PI3Kα Inhibitors.
    Alison L Kearney, Neil Vasan.
       SUMMARY: This is the first peer-reviewed report of an allosteric, mutant-selective PI3Kα inhibitor, STX-478, that reduces PIK3CA-mutant tumor growth in mice. However, in contrast to the FDA-approved PI3Kα isoform-selective inhibitor alpelisib, STX-478 does not induce hyperglycemia or other metabolic dysfunctions. See related article by Buckbinder et al., p. 2432 (7).
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-0945
  4. Elife. 2023 Nov 03. pii: e85930. [Epub ahead of print]12
    The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra) - phosphoinositide 3-kinase (PI3K) signaling cascade to steer towards the midline during zebrafish heart tube formation.
    Rabina Shrestha, Tess McCann, Harini Saravanan, Jaret Lieberth, Prashanna Koirala, Joshua Bloomekatz.
      Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move towards the midline (cardiac fusion) to form the primitive heart tube. Extrinsic influences such as the adjacent anterior endoderm are known to be required for cardiac fusion. We previously showed however, that the platelet-derived growth factor receptor alpha (Pdgfra) is also required for cardiac fusion (Bloomekatz et al. 2017). Nevertheless, an intrinsic mechanism that regulates myocardial movement has not been elucidated. Here, we show that the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway has an essential intrinsic role in the myocardium directing movement towards the midline. In vivo imaging further reveals midline-oriented dynamic myocardial membrane protrusions that become unpolarized in PI3K-inhibited zebrafish embryos where myocardial movements are misdirected and slower. Moreover, we find that PI3K activity is dependent on and interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.
    Keywords:  developmental biology; zebrafish
    DOI:  https://doi.org/10.7554/eLife.85930
  5. Cancer Discov. 2023 Nov 02.
    Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations.
    Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth E Martin, Bryanna L Norden, Nicholas Chevalier, Allison M Kehlmann, Ignaty Leshchiner, Haley Barnes, Sara Ehnstrom, Anastasia-Maria Stavridi, Xin Yuan, Janice S Kim, Haley Ellis, Alkistis Papatheodoridi, Hakan Gunaydin, Brian P Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg M Wulf, Aditya Bardia, Laura M Spring, Steven J Isakoff, Jochen K Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan B Corcoran, Dejan Juric.
      PIK3CA mutations occur in ~8% of cancers, including ~40% of HR-positive breast cancers, where the PI3K-alpha (PI3Ka)-selective inhibitor alpelisib is FDA-approved in combination with fulvestrant. Although prior studies have identified resistance mechanisms, such as PTEN loss, clinical acquired resistance to PI3Ka inhibitors remains poorly understood. Through serial liquid biopsies and rapid autopsies in 39 patients with advanced breast cancer developing acquired resistance to PI3Ka-inhibitors, we observe that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers.
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-0704
  6. Invest Ophthalmol Vis Sci. 2023 Nov 01. 64(14): 4
    Medium Depth Influences O2 Availability and Metabolism in Human RPE Cultures.
    Daniel T Hass, Qitao Zhang, Gillian A Autterson, Richard A Bryan, James B Hurley, Jason M L Miller.
       Purpose: Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism.
    Methods: We altered the availability of O2 to human primary and induced pluripotent stem cell-derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, 13C6-glucose and 13C5-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation.
    Results: Medium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions.
    Conclusions: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood." As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.
    DOI:  https://doi.org/10.1167/iovs.64.14.4
  7. Biochem J. 2023 Oct 31. 480(20): 1693-1717
    Fluorescent biosensors illuminate the spatial regulation of cell signaling across scales.
    Anne C Lyons, Sohum Mehta, Jin Zhang.
      As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed.
    Keywords:  cellular localization; fluorescent biosensors; intracellular signaling; kinases; organelles
    DOI:  https://doi.org/10.1042/BCJ20220223
  8. J Clin Invest. 2023 Nov 01. pii: e167861. [Epub ahead of print]133(21):
    Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1.
    Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John M Asara, Kwok-Kin Wong, Elizabeth P Henske, Mallika Singh, David J Kwiatkowski.
      The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation.
    Keywords:  Cancer; Oncology; Therapeutics
    DOI:  https://doi.org/10.1172/JCI167861
  9. Oncogene. 2023 Nov 01.
    Differential cell cycle checkpoint evasion by PTEN germline mutations associated with dichotomous phenotypes of cancer versus autism spectrum disorder.
    Masahiro Hitomi, Juan Venegas, Shin Chung Kang, Charis Eng.
      Individuals with a PTEN germline mutation receive the molecular diagnosis of PTEN hamartoma tumor syndrome (PHTS). PHTS displays a complex spectrum of clinical phenotypes including harmartomas, predisposition to cancers, and autism spectrum disorder (ASD). Clear-cut genotype-phenotype correlations are yet to be established due to insufficient information on the PTEN function being impacted by mutations. To fill this knowledge gap, we compared functional impacts of two selected missense PTEN mutant alleles, G132D and M134R, each respectively being associated with distinct clinical phenotype, ASD or thyroid cancer without ASD using gene-edited human induced pluripotent stem cells (hiPSCs). In homozygous hiPSCs, PTEN expression was severely reduced by M134R mutation due to shortened protein half-life. G132D suppressed PTEN expression to a lesser extent than Μ134R mutation without altering protein half-life. When challenged with γ-irradiation, G132D heterozygous cells exited radiation-induced G2 arrest earlier than wildtype and M134R heterozygous hiPSCs despite the similar DNA damage levels as the latter two. Immunoblotting analyses suggested that γ-irradiation induced apoptosis in G132D heterozygous cells to lesser degrees than in the hiPSCs of other genotypes. These data suggest that ASD-associated G132D allele promotes genome instability by premature cell cycle reentry with incomplete DNA repair.
    DOI:  https://doi.org/10.1038/s41388-023-02867-4
  10. Bio Protoc. 2023 Oct 20. 13(20): e4853
    Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.
    Wenjie Han, Haojun Liang, Jianqiang Bao.
      An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3'-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3'-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1-3 kb) KI in mammalian cells.
    Keywords:  CRISPR/Cas9; Cas9-PCV2 fusion; Gene knock-in (KI); Genome editing; Homologous recombination; dsDNA donor; odsDNA donor; ssDNA donor
    DOI:  https://doi.org/10.21769/BioProtoc.4853
  11. Nat Commun. 2023 Nov 02. 14(1): 7009
    Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations.
    Andrew K Ressler, Daniel A Snellings, Romuald Girard, Carol J Gallione, Rhonda Lightle, Andrew S Allen, Issam A Awad, Douglas A Marchuk.
      Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations.
    DOI:  https://doi.org/10.1038/s41467-023-42908-w
  12. Cancer Discov. 2023 Nov 02.
    Discovery and clinical proof-of-concept of RLY-2608, a first-in-class mutant-selective allosteric PI3Ka inhibitor that decouples anti-tumor activity from hyperinsulinemia.
    Andreas Varkaris, Ermira Pazolli, Hakan Gunaydin, Qi Wang, Levi Pierce, Alessandro A Boezio, Lucian DiPietro, Adam Frost, Fabrizio Giordanetto, Erika P Hamilton, Katherine Harris, Michael Holliday, Tamieka L Hunter, Amanda Iskandar, Yongli Ji, Alexandre Larivée, Jonathan R LaRochelle, Andre Lescarbeau, Fabien Llambi, Brenda Lormil, Mary M Mader, Brenton G Mar, Iain Martin, Thomas H McLean, Klaus Michelsen, Yakov Pechersky, Erika Puente-Poushnejad, Ramin Samadani, Alison M Schram, Kelley Shortsleeves, Sweta Swaminathan, Shahein Tajmir, Gege Tan, Yong Tang, Roberto Valverde, Bryan Wehrenberg, Jeremy Wilbur, Bret R Williams, Hongtao Zeng, W Patrick Walters, Beni B Wolf, David E Shaw, Donald A Bergstrom, James Watters, James S Fraser, Pascal D Fortin, D Randal Kipp.
      PIK3CA (PI3Ka) is a lipid kinase commonly mutated in cancer, including ~40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Ka inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Ka. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Ka activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Ka. RLY-2608 inhibited tumor growth in PIK3CA mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Ka-related toxicities.
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-0944
  13. BMC Res Notes. 2023 Nov 03. 16(1): 309
    AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.
    Marcos Gabriel Francia, Paula Verneri, Camila Oses, Camila Vazquez Echegaray, Mora Reneé Garcia, Ayelen Toro, Valeria Levi, Alejandra Sonia Guberman.
      AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.
    Keywords:  AKT1 SUMO conjugation; E17K AKT1 mutant; Embryonic stem cells; Induced pluripotent stem cells; MEF; U-2 OS; UBC9(C93S)
    DOI:  https://doi.org/10.1186/s13104-023-06598-3
  14. STAR Protoc. 2023 Oct 26. pii: S2666-1667(23)00608-1. [Epub ahead of print]4(4): 102641
    A protocol for simultaneous high-sensitivity genotyping and chromatin accessibility profiling in single cells.
    Sven Turkalj, Niels Asger Jakobsen, Angus Groom, Felix A Radtke, Paresh Vyas.
      Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) resolves the heterogeneity of epigenetic states across cells but does not typically capture exonic mutations, which limits our knowledge of how somatic mutations alter chromatin landscapes. Here, we present a plate-based approach coupling high-sensitivity genotyping of genomic loci with high-content scATAC-seq libraries from the same single cells. We first describe steps for optimization of genotyping primers, followed by detailed guidance on the preparation of both scATAC-seq and single-cell genotyping libraries, fully automated on high-throughput liquid handling platforms. For complete details on the use and execution of this protocol, please refer to Turkalj, Jakobsen et al.1.
    Keywords:  Bioinformatics; Cancer; Genetics; Genomics; Molecular Biology; Sequence Analysis; Sequencing; Single Cell; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2023.102641
  15. bioRxiv. 2023 Oct 26. pii: 2023.10.16.562558. [Epub ahead of print]
    Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.
    Michael C Lanz, Shuyuan Zhang, Matthew P Swaffer, Luisa Hernández Götz, Frank McCarty, Inbal Ziv, Daniel F Jarosz, Joshua E Elias, Jan M Skotheim.
      Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how size influences cell physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be biochemically different. Here, we corroborate these results in mouse hepatocytes and extend our analysis using yeast. We find that size-dependent proteome changes are highly conserved and mostly independent of metabolic state. As eukaryotic cells grow larger, the dilution of the genome elicits a starvation-like proteome phenotype, suggesting that growth in large cells is limited by the genome in a manner analogous to a limiting nutrient. We also demonstrate that the proteomes of replicatively-aged yeast are primarily determined by their large size. Overall, our data suggest that genome concentration is a universal determinant of proteome content in growing cells.
    DOI:  https://doi.org/10.1101/2023.10.16.562558
  16. Mol Syst Biol. 2023 Nov 02. e11782
    Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.
    Jamin B Hein, Hieu T Nguyen, Dimitriya H Garvanska, Isha Nasa, Thomas Kruse, Yinnian Feng, Blanca Lopez Mendez, Norman Davey, Arminja N Kettenbach, Polly M Fordyce, Jakob Nilsson.
      Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A-B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.
    Keywords:  MRBLE-Pep; MRBLE:Dephos; mitotic exit; protein phosphatase; substrates
    DOI:  https://doi.org/10.15252/msb.202311782
  17. Development. 2023 Nov 03. pii: dev.201946. [Epub ahead of print]
    Collective signalling drives rapid jumping between cell states.
    Elizabeth R Westbrook, Tchern Lenn, Jonathan R Chubb, Vlatka Antolović.
      Development can proceed in "fits and starts", with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp "jump" in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, while transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.
    Keywords:   in vivo imaging; Cell state transition; Collective behaviour; Excitable signalling; MS2 imaging; Optogenetics; Oscillation; Positive feedback; Stem cell niche; Transcriptional noise
    DOI:  https://doi.org/10.1242/dev.201946
  18. Cell Syst. 2023 Oct 26. pii: S2405-4712(23)00287-9. [Epub ahead of print]
    A framework for ultra-low-input spatial tissue proteomics.
    Anuar Makhmut, Di Qin, Sonja Fritzsche, Jose Nimo, Janett König, Fabian Coscia.
      Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell function and heterogeneity in (patho)physiology. However, optimized workflows that preserve morphological information for phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected tissue are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low-input archival material. Benchmarking in murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions. Applied to human tonsil, we profiled 146 microregions including T and B lymphocyte niches and quantified cell-type-specific markers, cytokines, and transcription factors. These data also highlighted proteome dynamics within activated germinal centers, illuminating sites undergoing B cell proliferation and somatic hypermutation. This approach has broad implications in biomedicine, including early disease profiling and drug target and biomarker discovery. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  FFPE; deep visual proteomics; histopathology; mass spectrometry; proteomics; single-cell proteomics; spatial proteomics
    DOI:  https://doi.org/10.1016/j.cels.2023.10.003
  19. Nat Methods. 2023 Nov 02.
    Causal identification of single-cell experimental perturbation effects with CINEMA-OT.
    Mingze Dong, Bao Wang, Jessica Wei, Antonio H de O Fonseca, Curtis J Perry, Alexander Frey, Feriel Ouerghi, Ellen F Foxman, Jeffrey J Ishizuka, Rahul M Dhodapkar, David van Dijk.
      Recent advancements in single-cell technologies allow characterization of experimental perturbations at single-cell resolution. While methods have been developed to analyze such experiments, the application of a strict causal framework has not yet been explored for the inference of treatment effects at the single-cell level. Here we present a causal-inference-based approach to single-cell perturbation analysis, termed CINEMA-OT (causal independent effect module attribution + optimal transport). CINEMA-OT separates confounding sources of variation from perturbation effects to obtain an optimal transport matching that reflects counterfactual cell pairs. These cell pairs represent causal perturbation responses permitting a number of novel analyses, such as individual treatment-effect analysis, response clustering, attribution analysis, and synergy analysis. We benchmark CINEMA-OT on an array of treatment-effect estimation tasks for several simulated and real datasets and show that it outperforms other single-cell perturbation analysis methods. Finally, we perform CINEMA-OT analysis of two newly generated datasets: (1) rhinovirus and cigarette-smoke-exposed airway organoids, and (2) combinatorial cytokine stimulation of immune cells. In these experiments, CINEMA-OT reveals potential mechanisms by which cigarette-smoke exposure dulls the airway antiviral response, as well as the logic that governs chemokine secretion and peripheral immune cell recruitment.
    DOI:  https://doi.org/10.1038/s41592-023-02040-5
  20. Nat Chem Biol. 2023 Oct 30.
    Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors.
    Maria Reinecke, Paul Brear, Larsen Vornholz, Benedict-Tilmann Berger, Florian Seefried, Stephanie Wilhelm, Patroklos Samaras, Laszlo Gyenis, David William Litchfield, Guillaume Médard, Susanne Müller, Jürgen Ruland, Marko Hyvönen, Mathias Wilhelm, Bernhard Kuster.
      Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.
    DOI:  https://doi.org/10.1038/s41589-023-01459-3
  21. Commun Biol. 2023 10 28. 6(1): 1096
    Imagining the future of optical microscopy: everything, everywhere, all at once.
    Harikrushnan Balasubramanian, Chad M Hobson, Teng-Leong Chew, Jesse S Aaron.
      The optical microscope has revolutionized biology since at least the 17th Century. Since then, it has progressed from a largely observational tool to a powerful bioanalytical platform. However, realizing its full potential to study live specimens is hindered by a daunting array of technical challenges. Here, we delve into the current state of live imaging to explore the barriers that must be overcome and the possibilities that lie ahead. We venture to envision a future where we can visualize and study everything, everywhere, all at once - from the intricate inner workings of a single cell to the dynamic interplay across entire organisms, and a world where scientists could access the necessary microscopy technologies anywhere.
    DOI:  https://doi.org/10.1038/s42003-023-05468-9
  22. Curr Opin Cell Biol. 2023 Oct 26. pii: S0955-0674(23)00120-5. [Epub ahead of print]85 102271
    Live-cell imaging in the deep learning era.
    Joanna W Pylvänäinen, Estibaliz Gómez-de-Mariscal, Ricardo Henriques, Guillaume Jacquemet.
      Live imaging is a powerful tool, enabling scientists to observe living organisms in real time. In particular, when combined with fluorescence microscopy, live imaging allows the monitoring of cellular components with high sensitivity and specificity. Yet, due to critical challenges (i.e., drift, phototoxicity, dataset size), implementing live imaging and analyzing the resulting datasets is rarely straightforward. Over the past years, the development of bioimage analysis tools, including deep learning, is changing how we perform live imaging. Here we briefly cover important computational methods aiding live imaging and carrying out key tasks such as drift correction, denoising, super-resolution imaging, artificial labeling, tracking, and time series analysis. We also cover recent advances in self-driving microscopy.
    DOI:  https://doi.org/10.1016/j.ceb.2023.102271
  23. Elife. 2023 Nov 02. pii: e90623. [Epub ahead of print]12
    Best practices to promote rigor and reproducibility in the era of sex-inclusive research.
    Janet W Rich-Edwards, Donna L Maney.
      To enhance inclusivity and rigor, many funding agencies and journals now mandate the inclusion of females as well as males in biomedical studies. These mandates have enhanced generalizability and created unprecedented opportunities to discover sex differences. However, education in sound methods to consider sex as a subgroup category has lagged behind, resulting in a problematic literature in which study designs, analyses, and interpretations of results are often flawed. Here, we outline best practices for complying with sex-inclusive mandates, both for studies in which sex differences are a primary focus and for those in which they are not. Our recommendations are organized within the "4 Cs of Studying Sex to Strengthen Science: Consideration, Collection, Characterization and Communication," a framework developed by the Office of Research on Women's Health at the National Institutes of Health in the United States. Following these guidelines should help researchers include females and males in their studies while at the same time upholding high standards of rigor.
    Keywords:  NIH policy; data analysis; epidemiology; global health; medicine; none; science forum; sex as a biological variable; sex differences; statistics
    DOI:  https://doi.org/10.7554/eLife.90623
  24. Nat Aging. 2023 Nov 02.
    scDiffCom: a tool for differential analysis of cell-cell interactions provides a mouse atlas of aging changes in intercellular communication.
    Cyril Lagger, Eugen Ursu, Anaïs Equey, Roberto A Avelar, Angela Oliveira Pisco, Robi Tacutu, João Pedro de Magalhães.
      Dysregulation of intercellular communication is a hallmark of aging. To better quantify and explore changes in intercellular communication, we present scDiffCom and scAgeCom. scDiffCom is an R package, relying on approximately 5,000 curated ligand-receptor interactions, that performs differential intercellular communication analysis between two conditions from single-cell transcriptomics data. Built upon scDiffCom, scAgeCom is an atlas of age-related cell-cell communication changes covering 23 mouse tissues from 58 single-cell RNA sequencing datasets from Tabula Muris Senis and the Calico murine aging cell atlas. It offers a comprehensive resource of tissue-specific and sex-specific aging dysregulations and highlights age-related intercellular communication changes widespread across the whole body, such as the upregulation of immune system processes and inflammation, the downregulation of developmental processes, angiogenesis and extracellular matrix organization and the deregulation of lipid metabolism. Our analysis emphasizes the relevance of the specific ligands, receptors and cell types regulating these processes. The atlas is available online ( https://scagecom.org ).
    DOI:  https://doi.org/10.1038/s43587-023-00514-x
  25. PLoS Biol. 2023 Nov;21(11): e3002345
    Is N-Hacking Ever OK? The consequences of collecting more data in pursuit of statistical significance.
    Pamela Reinagel.
      Upon completion of an experiment, if a trend is observed that is "not quite significant," it can be tempting to collect more data in an effort to achieve statistical significance. Such sample augmentation or "N-hacking" is condemned because it can lead to an excess of false positives, which can reduce the reproducibility of results. However, the scenarios used to prove this rule tend to be unrealistic, assuming the addition of unlimited extra samples to achieve statistical significance, or doing so when results are not even close to significant; an unlikely situation for most experiments involving patient samples, cultured cells, or live animals. If we were to examine some more realistic scenarios, could there be any situations where N-hacking might be an acceptable practice? This Essay aims to address this question, using simulations to demonstrate how N-hacking causes false positives and to investigate whether this increase is still relevant when using parameters based on real-life experimental settings.
    DOI:  https://doi.org/10.1371/journal.pbio.3002345
  26. bioRxiv. 2023 Oct 20. pii: 2023.10.19.563158. [Epub ahead of print]
    LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase.
    John R Ferrarone, Jerin Thomas, Arun M Unni, Yuxiang Zheng, Michal J Nagiec, Eric E Gardner, Oksana Mashadova, Kate Li, Nikos Koundouros, Antonino Montalbano, Meer Mustafa, Lewis C Cantley, John Blenis, Neville E Sanjana, Harold Varmus.
      The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1 -mutant LUAD.
    DOI:  https://doi.org/10.1101/2023.10.19.563158
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