bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–05–05
fiveteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Single-Cell Proteomics Accelerates toward Proteoforms.
  2. Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput.
  3. Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.
  4. Kernel-based testing for single-cell differential analysis.
  5. SynDesign: web-based prime editing guide RNA design and evaluation tool for saturation genome editing.
  6. Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.
  7. Branching topology of the human embryo transcriptome revealed by entropy sort feature weighting.
  8. Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.
  9. FlexStat: combinatory differentially expressed protein extraction.
  10. High-throughput tagging of endogenous loci for rapid characterization of protein function.
  11. A reverse brake for the cell cycle.
  12. 3D genomic mapping reveals multifocality of human pancreatic precancers.
  13. CDK4/6 activity is required during G2 arrest to prevent stress-induced endoreplication.
  14. Hallmarks of cancer stemness.
  15. Epithelial-mesenchymal transition in tissue repair and degeneration.
  1. J Proteome Res. 2024 May 03. 23(5): 1545-1546
    Single-Cell Proteomics Accelerates toward Proteoforms.
    Sarah N Sipe, Nikolai Slavov.
      
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00290
  2. Nat Methods. 2024 Apr 29.
    Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput.
    Patrick H Kennedy, Amin Alborzian Deh Sheikh, Matthew Balakar, Alexander C Jones, Meagan E Olive, Mudra Hegde, Maria I Matias, Natan Pirete, Rajan Burt, Jonathan Levy, Tamia Little, Patrick G Hogan, David R Liu, John G Doench, Alexandra C Newton, Rachel A Gottschalk, Carl G de Boer, Suzie Alarcón, Gregory A Newby, Samuel A Myers.
      Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
    DOI:  https://doi.org/10.1038/s41592-024-02256-z
  3. Cell Rep. 2024 May 02. pii: S2211-1247(24)00502-3. [Epub ahead of print]43(5): 114174
    Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.
    Ryan Blawski, Bujamin H Vokshi, Xinyu Guo, Srushti Kittane, Mirna Sallaku, Wanlu Chen, Martina Gjyzari, Tony Cheung, Yuhan Zhang, Christopher Simpkins, Weiqiang Zhou, Amanda Kulick, Peihua Zhao, Meihan Wei, Pranavkrishna Shivashankar, Tatiana Prioleau, Pedram Razavi, Richard Koche, Vito W Rebecca, Elisa de Stanchina, Pau Castel, Ho Man Chan, Maurizio Scaltriti, Emiliano Cocco, Hongkai Ji, Minkui Luo, Eneda Toska.
      Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.
    Keywords:  AKT; CP: Cancer; CP: Molecular biology; KMT2D; PI3K pathway; SMYD2; alpelisib; breast cancer; chromatin regulation; endocrine therapy; estrogen receptor
    DOI:  https://doi.org/10.1016/j.celrep.2024.114174
  4. Genome Biol. 2024 May 03. 25(1): 114
    Kernel-based testing for single-cell differential analysis.
    A Ozier-Lafontaine, C Fourneaux, G Durif, P Arsenteva, C Vallot, O Gandrillon, S Gonin-Giraud, B Michel, F Picard.
      Single-cell technologies offer insights into molecular feature distributions, but comparing them poses challenges. We propose a kernel-testing framework for non-linear cell-wise distribution comparison, analyzing gene expression and epigenomic modifications. Our method allows feature-wise and global transcriptome/epigenome comparisons, revealing cell population heterogeneities. Using a classifier based on embedding variability, we identify transitions in cell states, overcoming limitations of traditional single-cell analysis. Applied to single-cell ChIP-Seq data, our approach identifies untreated breast cancer cells with an epigenomic profile resembling persister cells. This demonstrates the effectiveness of kernel testing in uncovering subtle population variations that might be missed by other methods.
    Keywords:  Differential analysis; Kernel methods; Single cell epigenomics; Single cell transcriptomics
    DOI:  https://doi.org/10.1186/s13059-024-03255-1
  5. Nucleic Acids Res. 2024 Apr 29. pii: gkae304. [Epub ahead of print]
    SynDesign: web-based prime editing guide RNA design and evaluation tool for saturation genome editing.
    Jinman Park, Goosang Yu, Sang-Yeon Seo, Jinyeong Yang, Hyongbum Henry Kim.
      Saturation genome editing (SGE) enables in-depth functional evaluation of disease-associated genes and variants by generating all possible single nucleotide variants (SNVs) within a given coding region. Although prime editing can be employed for inducing these SNVs, designing efficient prime editing guide RNAs (pegRNAs) can be challenging and time-consuming. Here, we present SynDesign, an easy-to-use webtool for the design, evaluation, and construction precision pegRNA libraries for SGE with synonymous mutation markers. SynDesign offers a simple yet powerful interface that automates the generation of all feasible pegRNA designs for a target gene or variant of interest. The pegRNAs are selected using the state-of-the-art models to predict prime editing efficiencies for various prime editors and cell types. Top-scoring pegRNA designs are further enhanced using synonymous mutation markers which improve pegRNA efficiency by diffusing the cellular mismatch repair mechanism and serve as sequence markers for improved identification of intended edits following deep sequencing. SynDesign is expected to facilitate future research using SGE to investigate genes or variants of interest associated with human diseases. SynDesign is freely available at https://deepcrispr.info/SynDesign without a login process.
    DOI:  https://doi.org/10.1093/nar/gkae304
  6. Cell Rep. 2024 May 02. pii: S2211-1247(24)00501-1. [Epub ahead of print]43(5): 114173
    Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.
    Navroop K Dhaliwal, Octavia Yifang Weng, Xiaoxue Dong, Afrin Bhattacharya, Mai Ahmed, Haruka Nishimura, Wendy W Y Choi, Aditi Aggarwal, Bryan W Luikart, Qiang Shu, Xuekun Li, Michael D Wilson, Jason Moffat, Lu-Yang Wang, Julien Muffat, Yun Li.
      Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.
    Keywords:  CP: Cell biology; CP: Neuroscience; PTEN; autism; brain organoids; epilepsy; mTOR; neurodevelopmental disorders; neurons
    DOI:  https://doi.org/10.1016/j.celrep.2024.114173
  7. Development. 2024 May 01. pii: dev.202832. [Epub ahead of print]
    Branching topology of the human embryo transcriptome revealed by entropy sort feature weighting.
    Arthur Radley, Stefan Boeing, Austin Smith.
      Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after sub-setting to highly variable genes (HVGs). Here we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous entropy sort feature weighting (cESFW) outperforms HVG selection in distinguishing cell state specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from 8-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression we provide markers for staging progression from morula to blastocyst.
    Keywords:  Feature selection; Human development; Single cell
    DOI:  https://doi.org/10.1242/dev.202832
  8. Nat Cell Biol. 2024 Apr 30.
    Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.
    Tianchi Xin, Sara Gallini, Haoyang Wei, David G Gonzalez, Catherine Matte-Martone, Hiroki Machida, Hironobu Fujiwara, H Amalia Pasolli, Kathleen C Suozzi, Lauren E Gonzalez, Sergi Regot, Valentina Greco.
      Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.
    DOI:  https://doi.org/10.1038/s41556-024-01413-y
  9. Bioinform Adv. 2024 ;4(1): vbae056
    FlexStat: combinatory differentially expressed protein extraction.
    Senuri De Silva, Asfa Alli-Shaik, Jayantha Gunaratne.
       Motivation: Mass spectrometry-based system proteomics allows identification of dysregulated protein hubs and associated disease-related features. Obtaining differentially expressed proteins (DEPs) is the most important step of downstream bioinformatics analysis. However, the extraction of statistically significant DEPs from datasets with multiple experimental conditions or disease types through currently available tools remains a laborious task. More often such an analysis requires considerable bioinformatics expertise, making it inaccessible to researchers with limited computational analytics experience.
    Results: To uncover the differences among the many conditions within the data in a user-friendly manner, here we introduce FlexStat, a web-based interface that extracts DEPs through combinatory analysis. This tool accepts a protein expression matrix as input and systematically generates DEP results for every conceivable combination of various experimental conditions or disease types. FlexStat includes a suite of robust statistical tools for data preprocessing, in addition to DEP extraction, and publication-ready visualization, which are built on established R scientific libraries in an automated manner. This analytics suite was validated in diverse public proteomic datasets to showcase its high performance of rapid and simultaneous pairwise comparisons of comprehensive datasets.
    Availability and implementation: FlexStat is implemented in R and is freely available at https://jglab.shinyapps.io/flexstatv1-pipeline-only/. The source code is accessible at https://github.com/kts-desilva/FlexStat/tree/main.
    DOI:  https://doi.org/10.1093/bioadv/vbae056
  10. Sci Adv. 2024 May 03. 10(18): eadg8771
    High-throughput tagging of endogenous loci for rapid characterization of protein function.
    Joonwon Kim, Alexander F Kratz, Shiye Chen, Jenny Sheng, Hark Kyun Kim, Liudeng Zhang, Brijesh Kumar Singh, Alejandro Chavez.
      To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.
    DOI:  https://doi.org/10.1126/sciadv.adg8771
  11. Science. 2024 May 03. 384(6695): 512-513
    A reverse brake for the cell cycle.
    Bart Westendorp.
      Mitogenic signaling acts beyond S-phase entry to prevent whole-genome duplications.
    DOI:  https://doi.org/10.1126/science.adp1866
  12. Nature. 2024 May 01.
    3D genomic mapping reveals multifocality of human pancreatic precancers.
    Alicia M Braxton, Ashley L Kiemen, Mia P Grahn, André Forjaz, Jeeun Parksong, Jaanvi Mahesh Babu, Jiaying Lai, Lily Zheng, Noushin Niknafs, Liping Jiang, Haixia Cheng, Qianqian Song, Rebecca Reichel, Sarah Graham, Alexander I Damanakis, Catherine G Fischer, Stephanie Mou, Cameron Metz, Julie Granger, Xiao-Ding Liu, Niklas Bachmann, Yutong Zhu, YunZhou Liu, Cristina Almagro-Pérez, Ann Chenyu Jiang, Jeonghyun Yoo, Bridgette Kim, Scott Du, Eli Foster, Jocelyn Y Hsu, Paula Andreu Rivera, Linda C Chu, Fengze Liu, Elliot K Fishman, Alan Yuille, Nicholas J Roberts, Elizabeth D Thompson, Robert B Scharpf, Toby C Cornish, Yuchen Jiao, Rachel Karchin, Ralph H Hruban, Pei-Hsun Wu, Denis Wirtz, Laura D Wood.
      Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.
    DOI:  https://doi.org/10.1038/s41586-024-07359-3
  13. Science. 2024 May 03. 384(6695): eadi2421
    CDK4/6 activity is required during G2 arrest to prevent stress-induced endoreplication.
    Connor McKenney, Yovel Lendner, Adler Guerrero Zuniga, Niladri Sinha, Benjamin Veresko, Timothy J Aikin, Sergi Regot.
      Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling.
    DOI:  https://doi.org/10.1126/science.adi2421
  14. Cell Stem Cell. 2024 May 02. pii: S1934-5909(24)00131-0. [Epub ahead of print]31(5): 617-639
    Hallmarks of cancer stemness.
    Jia-Jian Loh, Stephanie Ma.
      Cancer stemness is recognized as a key component of tumor development. Previously coined "cancer stem cells" (CSCs) and believed to be a rare population with rigid hierarchical organization, there is good evidence to suggest that these cells exhibit a plastic cellular state influenced by dynamic CSC-niche interplay. This revelation underscores the need to reevaluate the hallmarks of cancer stemness. Herein, we summarize the techniques used to identify and characterize the state of these cells and discuss their defining and emerging hallmarks, along with their enabling and associated features. We also highlight potential future directions in this field of research.
    Keywords:  cancer cell plasticity; cancer stem cells; cancer stemness; immunosuppression; metastasis; self-renewal; tumor microenvironment; tumor recurrence; tumor-initiating cells
    DOI:  https://doi.org/10.1016/j.stem.2024.04.004
  15. Nat Rev Mol Cell Biol. 2024 Apr 29.
    Epithelial-mesenchymal transition in tissue repair and degeneration.
    Khalil Kass Youssef, M Angela Nieto.
      Epithelial-mesenchymal transitions (EMTs) are the epitome of cell plasticity in embryonic development and cancer; during EMT, epithelial cells undergo dramatic phenotypic changes and become able to migrate to form different tissues or give rise to metastases, respectively. The importance of EMTs in other contexts, such as tissue repair and fibrosis in the adult, has become increasingly recognized and studied. In this Review, we discuss the function of EMT in the adult after tissue damage and compare features of embryonic and adult EMT. Whereas sustained EMT leads to adult tissue degeneration, fibrosis and organ failure, its transient activation, which confers phenotypic and functional plasticity on somatic cells, promotes tissue repair after damage. Understanding the mechanisms and temporal regulation of different EMTs provides insight into how some tissues heal and has the potential to open new therapeutic avenues to promote repair or regeneration of tissue damage that is currently irreversible. We also discuss therapeutic strategies that modulate EMT that hold clinical promise in ameliorating fibrosis, and how precise EMT activation could be harnessed to enhance tissue repair.
    DOI:  https://doi.org/10.1038/s41580-024-00733-z
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