bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒09‒03
thirty-two papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

  1. bioRxiv. 2023 Aug 19. pii: 2023.08.18.553856. [Epub ahead of print]
      Gene variants that hyperactivate PI3K-mTOR signaling in the brain lead to epilepsy and cortical malformations in humans. Some gene variants associated with these pathologies only hyperactivate mTORC1, but others, such as PTEN, PIC3CA , and AKT , hyperactivate both mTORC1- and mTORC2-dependent signaling. Previous work has established a key role for mTORC1 hyperactivity in mTORopathies, however, whether mTORC2 hyperactivity contributes is not clear. To test this, we inactivated mTORC1 and/or mTORC2 downstream of early Pten deletion in a new model of somatic Pten LOF in the cortex. Spontaneous seizures and epileptiform activity persisted despite mTORC1 or mTORC2 inactivation alone, but inactivating both mTORC1 and mTORC2 normalized pathology. These results suggest that hyperactivity of both mTORC1 and mTORC2 are sufficient to cause epilepsy, and that targeted therapies should aim to reduce activity of both complexes.
  2. Nat Commun. 2023 Aug 29. 14(1): 5252
      The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.
  3. Genet Med. 2023 Aug 24. pii: S1098-3600(23)00985-1. [Epub ahead of print] 100969
      PURPOSE: PROS encompasses several rare conditions resulting from activating variants in PIK3CA. Alpelisib, a PI3Kα-selective inhibitor, targets the underlying etiology of PROS, offering a novel therapeutic approach to current management strategies. This study evaluated the safety and efficacy of alpelisib in pediatric and adult patients with PROS.METHODS: EPIK-P1 (NCT04285723) was a non-interventional, retrospective chart review of 57 patients with PROS (≥2 years) treated with alpelisib through compassionate use. Patients had severe/life-threatening PROS-related conditions and confirmed PIK3CA pathogenic variant. The primary endpoint assessed patient response to treatment at Week 24 (6 months).
    RESULTS: Twenty-four weeks (6 months) after treatment initiation, 12/32 (37.5%) patients with complete case records included in the analysis of the primary endpoint experienced a ≥20% reduction in target lesion(s) volume. Additional clinical benefit independent from lesion volume reduction was observed across the full study population. Adverse events (AEs) and treatment-related AEs were experienced by 82.5% (47/57) and 38.6% (22/57) of patients, respectively; the most common treatment-related AEs were hyperglycemia (12.3%) and aphthous ulcer (10.5%). No deaths occurred.
    CONCLUSIONS: EPIK-P1 provides real-world evidence of alpelisib effectiveness and safety in patients with PROS and confirms PI3Kα as a valid therapeutic target for PROS symptom management.
    Keywords:  PIK3CA; PROS; alpelisib; overgrowth
  4. Proc Natl Acad Sci U S A. 2023 Sep 05. 120(36): e2306414120
      Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.
    Keywords:  BET-bromodomains; cMYC; chimeric small molecules; phosphatidylinositol-3-kinase
  5. PLoS Genet. 2023 Aug 28. 19(8): e1010904
      The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites in healthy tissues, is disrupted across many human cancers. Deregulated expression of the MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. It remains unclear what benefit cancer cells gain from suppressing clock oscillation, and how this loss of molecular clock oscillation impacts global gene expression and metabolism in cancer. We hypothesized that MYC or its paralog N-MYC (collectively termed MYC herein) suppress oscillation of gene expression and metabolism to upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC were examined, using time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression pathways, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter proteins while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.
  6. J Exp Med. 2023 Nov 06. pii: e20211743. [Epub ahead of print]220(11):
      Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies.
  7. Dev Cell. 2023 Aug 19. pii: S1534-5807(23)00385-4. [Epub ahead of print]
      The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.
    Keywords:  Cre/lox; conditional genetics; foxc1a; gata2a; rasa1a; ruvbl1; zebrafish
  8. Mol Cell Proteomics. 2023 Aug 30. pii: S1535-9476(23)00150-0. [Epub ahead of print] 100639
      Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes, while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities (ABRF) umbrella, the Proteomics Standards Research Group (sPRG) has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/IGF-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both EGF and IGF-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically-relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations, enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.
    Keywords:  data-independent acquisition; mass spectrometry; phosphopeptide; phosphorylation; proteomics; stable isotope label; targeted
  9. ArXiv. 2023 Aug 14. pii: arXiv:2308.07465v1. [Epub ahead of print]
      Biological functions stem from coordinated interactions among proteins, nucleic acids and small molecules. Mass spectrometry technologies for reliable, high throughput single-cell proteomics will add a new modality to genomics and enable data-driven modeling of the molecular mechanisms coordinating proteins and nucleic acids at single-cell resolution. This promising potential requires estimating the reliability of measurements and computational analysis so that models can distinguish biological regulation from technical artifacts. We discuss approaches for developing both abstract and mechanistic models that aim to biologically interpret the measured differences across modalities. Mechanistic models of direct molecular interactions will provide generalizable and predictive representations of biological systems.
  10. Orphanet J Rare Dis. 2023 Sep 01. 18(1): 261
      BACKGROUND: Somatic mutations of cancer driver genes are found to be responsible for vascular malformations with clinical manifestations ranging from cutaneous birthmarks to life-threatening systemic anomalies. Till now, only a limited number of cases and mutations were reported in Chinese population. The purpose of this study was to describe the somatic mutation spectrum of a cohort of Chinese pediatrics with vascular malformations.METHODS: Pediatrics diagnosed with various vascular malformations were collected between May 2019 and October 2020 from Beijing Children's Hospital. Genomic DNA of skin lesion of each patient was extracted and sequenced by whole-exome sequencing to identify pathogenic somatic mutations. Mutations with variant allele frequency less than 5% were validated by ultra-deep sequencing.
    RESULTS: A total of 67 pediatrics (33 males, 34 females, age range: 0.1-14.8 years) were analyzed. Exome sequencing identified somatic mutations of corresponding genes in 53 patients, yielding a molecular diagnosis rate of 79.1%. Among 29 PIK3CA mutations, 17 were well-known hotspot p.E542K, p.E545K and p.H1047R/L. Non-hotspot mutations were prevalent in patients with PIK3CA-related overgrowth spectrum, accounting for 50.0% (11/22) of detected mutations. The hotspot GNAQ p.R183Q and TEK p.L914F mutations were responsible for the majority of port-wine stain/Sturge-Weber syndrome and venous malformation, respectively. In addition, we identified a novel AKT1 p.Q79K mutation in Proteus syndrome and MAP3K3 p.E387D mutation in verrucous venous malformation.
    CONCLUSIONS: The somatic mutation spectrum of vascular malformations in Chinese population is similar to that reported in other populations, but non-hotspot PIK3CA mutations may also be prevalent. Molecular diagnosis may help the clinical diagnosis, treatment and management of these pediatric patients with vascular malformations.
    Keywords:  Children; GNAQ; PIK3CA; Somatic mutation; Vascular malformations
  11. Open Res Eur. 2023 ;3 59
      With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.
    Keywords:  Clinical proteomics; clinical research; ethical challenges
  12. bioRxiv. 2023 Aug 15. pii: 2023.08.14.553324. [Epub ahead of print]
      CRISPR prime editing ( PE ) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA ( pegRNA ), an extended version of a standard guide RNA ( gRNA ) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as 6-fold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.
  13. Cell Rep. 2023 Aug 24. pii: S2211-1247(23)01039-2. [Epub ahead of print]42(9): 113028
      Biliary epithelial cells (BECs) are a potential source to repair the damaged liver when hepatocyte proliferation is compromised. Promotion of BEC-to-hepatocyte transdifferentiation could be beneficial to the clinical therapeutics of patients with end-stage liver diseases. However, mechanisms underlying the initiation of BEC transdifferentiation remain largely unknown. Here, we show that upon extreme hepatocyte injury, vegfaa and vegfr2/kdrl are notably induced in hepatic stellate cells and BECs, respectively. Pharmacological and genetic inactivation of vascular endothelial growth factor (VEGF) signaling would disrupt BEC dedifferentiation and proliferation, thus restraining hepatocyte regeneration. Mechanically, VEGF signaling regulates the activation of the PI3K-mammalian target of rapamycin complex 1 (mTORC1) axis, which is essential for BEC-to-hepatocyte transdifferentiation. In mice, VEGF signaling exerts conserved roles in oval cell activation and BEC-to-hepatocyte differentiation. Taken together, this study shows VEGF signaling as an initiator of biliary-mediated liver regeneration through activating the PI3K-mTORC1 axis. Modulation of VEGF signaling in BECs could be a therapeutic approach for patients with end-stage liver diseases.
    Keywords:  CP: Cell biology
  14. Sci Adv. 2023 Sep;9(35): eadj1568
      CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes. Here, we develop optimized C-to-T base editing systems for the generation of precise loss- or gain-of-function alleles in Drosophila and identify temperature as a crucial parameter for efficiency. We find that a variant of the widely used APOBEC1 deaminase has attenuated activity at 18° to 29°C and shows considerable dose-dependent toxicity. In contrast, the temperature-tolerant evoCDA1 domain mediates editing of typically more than 90% of alleles and is substantially better tolerated. Furthermore, formation of undesired mutations is exceptionally rare in Drosophila compared to other species. The predictable editing outcome, high efficiency, and product purity enables near homogeneous induction of STOP codons or alleles encoding protein variants in vivo. Last, we demonstrate how optimized expression enables conditional base editing in marked cell populations. This work substantially facilitates creation of precise alleles in Drosophila and provides key design parameters for developing efficient base editing systems in other ectothermic species.
  15. Cell. 2023 Aug 31. pii: S0092-8674(23)00854-1. [Epub ahead of print]186(18): 3983-4002.e26
      Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.
    Keywords:  CRISPR-Cas9; directed evolution; genome editing; guide RNAs; pegRNAs; phage-assisted continuous evolution; prime editing; protein engineering
  16. Cell Syst. 2023 Aug 25. pii: S2405-4712(23)00215-6. [Epub ahead of print]
      Cooperative DNA binding of transcription factors (TFs) integrates the cellular context to support cell specification during development. Naive mouse embryonic stem cells are derived from early development and can sustain their pluripotent identity indefinitely. Here, we ask whether TFs associated with pluripotency evolved to directly support this state or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is absent, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB allows quantitative titration of this differentiation, and in silico modeling of bipartite ESRRB activity suggests it safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of immortal stem cells. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  ESCs; cooperativity; plasticity; pluripotency; pre-implantation development; primitive endoderm; regulative development; self-renewal; transcription
  17. Nat Chem Biol. 2023 Aug 31.
      The cullin-RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs.
  18. Cell Rep. 2023 Aug 31. pii: S2211-1247(23)01090-2. [Epub ahead of print]42(9): 113079
      Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.
    Keywords:  CDK4/6; CP: Cell biology; MEK; MYC; cell cycle; palbociclib; pre-malignant lesions; senescence; time-lapse imaging; trametinib
  19. Cell Chem Biol. 2023 Aug 28. pii: S2451-9456(23)00274-X. [Epub ahead of print]
      Target deconvolution is a crucial but costly and time-consuming task that hinders large-scale profiling for drug discovery. We present a matrix-augmented pooling strategy (MAPS) which mixes multiple drugs into samples with optimized permutation and delineates targets of each drug simultaneously with mathematical processing. We validated this strategy with thermal proteome profiling (TPP) testing of 15 drugs concurrently, increasing experimental throughput by 60x while maintaining high sensitivity and specificity. Benefiting from the lower cost and higher throughput of MAPS, we performed target deconvolution of the 15 drugs across 5 cell lines. Our profiling revealed that drug-target interactions can differ vastly in targets and binding affinity across cell lines. We further validated BRAF and CSNK2A2 as potential off-targets of bafetinib and abemaciclib, respectively. This work represents the largest thermal profiling of structurally diverse drugs across multiple cell lines to date.
    Keywords:  cellular thermal shift assay; chemical biology; chemical proteomics; drug development; drug discovery; protein mass spectrometry; proteome thermal profiling; target deconvolution
  20. Trends Cell Biol. 2023 Aug 28. pii: S0962-8924(23)00157-5. [Epub ahead of print]
      The multistep process of in vivo reprogramming, mediated by the transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc (OSKM), holds great promise for the development of rejuvenating and regenerative strategies. However, most of the approaches developed so far are accompanied by a persistent risk of tumorigenicity. Here, we review the groundbreaking effects of in vivo reprogramming with a particular focus on rejuvenation and regeneration. We discuss how the activity of pioneer TFs generates cellular plasticity that may be critical for inducing not only reprogramming and regeneration, but also cancer initiation. Finally, we highlight how a better understanding of the uncoupled control of cellular identity, plasticity, and aging during reprogramming might pave the way to the development of rejuvenating/regenerating strategies in a nontumorigenic manner.
    Keywords:  cancer; cellular identity and plasticity; pioneer transcription factor; pluripotent reprogramming; regeneration; rejuvenation
  21. Nature. 2023 Sep;621(7977): 47-48
    Keywords:  Biochemistry; Cell biology; Metabolism
  22. Mol Cell. 2023 Aug 25. pii: S1097-2765(23)00649-4. [Epub ahead of print]
      N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.
    Keywords:  CUT&Tag; RNA modification; droplet-based; embryonic stem cell; epitranscriptomics; in situ; m(6)A; multimodal; single nucleus
  23. Cytotechnology. 2023 Oct;75(5): 381-390
      Cell proliferation can be measured directly by counting cells or indirectly using assays that quantitate total protein or metabolic activity. However, for comparing cell proliferation under varying oxygen conditions it is not clear that these assays are appropriate surrogates for cell counting as cell metabolism and protein synthesis may vary under different oxygen environments. We used permeable bottom tissue culture ware to compare proliferation assays as a function of static oxygen concentrations under oxygen partial pressure (pO2) levels ranging from 2 to 139 mmHg. Cell proliferation was measured by cell counting and compared to surrogate methods measuring cell metabolism (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) and total protein (sulforhodamine B) assays under these different environments in Caco-2, MCF-7, MCF-10A and PANC-1 human cell lines. We found that the MTT readings do not correlate with cell number for the Caco-2 and PANC-1 cell lines under different oxygen conditions, whereas the sulforhodamine B protein assays perform well under all conditions. However, within a given oxygen environment, both proliferation assays show a correlation with cell number. Therefore, the MTT assay must be used with caution when comparing cell growth or drug response for cells grown in different oxygen environments.Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00584-0.
    Keywords:  Cancer; Hypoxia; MTT assay; Oxygen partial pressure; Proliferation; SRB
  24. Lett Biomath. 2023 Jan 10. 10(1): 87-103
      Stochastic modeling has become an essential tool for studying biochemical reaction networks. There is a growing need for user-friendly and feature-complete software for model design and simulation. To address this need, we present GillesPy2, an open-source framework for building and simulating mathematical and biochemical models. GillesPy2, a major upgrade from the original GillesPy package, is now a stand-alone Python 3 package. GillesPy2 offers an intuitive interface for robust and reproducible model creation, facilitating rapid and iterative development. In addition to expediting the model creation process, GillesPy2 offers efficient algorithms to simulate stochastic, deterministic, and hybrid stochastic-deterministic models.
    Keywords:  Hybrid; Modeling; Simulation; Stochastic
  25. BMC Bioinformatics. 2023 Aug 31. 24(1): 326
      BACKGROUND: Here we present scSNPdemux, a sample demultiplexing pipeline for single-cell RNA sequencing data using natural genetic variations in humans. The pipeline requires alignment files from Cell Ranger (10× Genomics), a population SNP database and genotyped single nucleotide polymorphisms (SNPs) per sample. The tool works on sparse genotyping data in VCF format for sample identification.RESULTS: The pipeline was tested on both single-cell and single-nuclei based RNA sequencing datasets and showed superior demultiplexing performance over the lipid-based CellPlex and Multi-seq sample multiplexing technique which incurs additional single cell library preparation steps. Specifically, our pipeline demonstrated superior sensitivity and specificity in cell-identity assignment over CellPlex, especially on immune cell types with low RNA content.
    CONCLUSIONS: We designed a streamlined pipeline for single-cell sample demultiplexing, aiming to overcome common problems in multiplexing samples using single cell libraries which might affect data quality and can be costly.
    Keywords:  Sample demultiplexing; Sample pooling; Single nucleotide polymorphisms; Single-cell
  26. bioRxiv. 2023 Aug 19. pii: 2023.07.20.549945. [Epub ahead of print]
      Exploratory spatial data analysis (ESDA) can be a powerful approach to understanding single-cell genomics datasets, but it is not yet part of standard data analysis workflows. In particular, geospatial analyses, which have been developed and refined for decades, have yet to be fully adapted and applied to spatial single-cell analysis. We introduce the Voyager platform, which systematically brings the geospatial ESDA tradition to (spatial) -omics, with local, bivariate, and multivariate spatial methods not yet commonly applied to spatial -omics, united by a uniform user interface. Using Voyager, we showcase biological insights that can be derived with its methods, such as biologically relevant negative spatial autocorrelation. Underlying Voyager is the SpatialFeatureExperiment data structure, which combines Simple Feature with SingleCellExperiment and AnnData to represent and operate on geometries bundled with gene expression data. Voyager has comprehensive tutorials demonstrating ESDA built on GitHub Actions to ensure reproducibility and scalability, using data from popular commercial technologies. Voyager is implemented in both R/Bioconductor and Python/PyPI, and features compatibility tests to ensure that both implementations return consistent results.
  27. Trends Cell Biol. 2023 Aug 28. pii: S0962-8924(23)00156-3. [Epub ahead of print]
      The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's requirement in animal development, is tissue-specific, context-specific, and sometimes appears uncoupled from cell proliferation. Detailed new information about RB's genomic distribution provides a new perspective on the complexity of RB function, suggesting that some of its functional specificity results from context-specific RB association with chromatin. Here we summarize recent evidence showing that RB targets different types of chromatin regulatory elements at different cell cycle stages. RB controls traditional RB/E2F targets prior to S-phase, but, when cells proliferate, RB redistributes to cell type-specific chromatin loci. We discuss the broad implications of the new data for RB research.
    Keywords:  RB target genes; cell cycle control; conserved and cell type-specific RB targets; regulation of RB’s chromatin distribution; retinoblastoma protein (RB)
  28. iScience. 2023 Sep 15. 26(9): 107511
      Cell differentiation is associated with global changes in translational activity. Here, we characterize how mRNA poly(A) tail processing supports this dynamic. We observe that decreased translation during neuronal differentiation of P19 cells correlates with the downregulation of 5'-terminal oligopyrimidine (TOP) transcripts which encode the translational machinery. Despite their downregulation, TOP transcripts remain highly stable and show increased translation as cells differentiate. Changes in TOP mRNA metabolism are reflected by their accumulation with poly(A) tails ∼60-nucleotide (nt) long. The dynamic changes in poly(A) processing can be partially recapitulated by depleting LARP1 or activating the mTOR pathway in undifferentiated cells. Although mTOR-induced accumulation of TOP mRNAs with tails ∼60-nt long does not trigger differentiation, it is associated with reduced proliferation of neuronal progenitors. We propose that while TOP mRNAs are transcriptionally silenced, their post-transcriptional regulation mediated by a specific poly(A) processing ensures an adequate supply of ribosomes to complete differentiation.
    Keywords:  Cell biology; Developmental neuroscience; Molecular biology
  29. STAR Protoc. 2023 Aug 31. pii: S2666-1667(23)00504-X. [Epub ahead of print]4(3): 102537
      Pediatric cell line models are important for basic and translational research. However, their establishment has been hampered by low success rates and the lack of a unified approach. Here, we present a protocol to establish pediatric cancer cell lines from rare childhood tumors. We describe the requirements for successful establishment, including an optimized dissociation technique, and the appropriate media conditions necessary for several types of rare but lethal forms of childhood cancers. For complete details on the use and execution of this protocol, please refer to Sun et al.1.
    Keywords:  Cancer; Cell Biology; Cell Culture; Cell Isolation
  30. iScience. 2023 Sep 15. 26(9): 107529
      The CRISPR-Cas9 system has emerged as the dominant technology for gene editing and clinical applications. One major concern is its off-target effect after the introduction of exogenous CRISPR-Cas9 into cells. Several previous studies have investigated either Cas9 alone or CRISPR-Cas9 interactions with p53. Here, we reanalyzed previously reported data of p53-associated Cas9 activities and observed large significant sex differences between p53-wildtype and p53-mutant cells. To expand the impact of this finding, we further examined all protein-coding genes for sex-specific dependencies in a large-scale CRISPR-Cas9 screening dataset from the DepMap project. We highlighted the p53-dependent sex bias of gene knockouts (including MYC, PIK3CA, KAT2B, KDM4E, SUV39H1, FANCB, TLR7, and APC2) across cancer types and potential mechanisms (mediated by transcriptional factors, including SOX9, FOXO4, LEF1, and RYBP) underlying this phenomenon. Our results suggest that the p53-dependent sex bias may need to be considered in future clinical applications of CRISPR-Cas9, especially in cancer.
    Keywords:  Cancer; Genetics; Molecular genetics