bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2025–07–20
ten papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition.
  2. Cell barcoding with tandem fluorescent proteins enables high-throughput signaling dynamics analysis.
  3. Systems-level Consequences of Low RAF Abundance for EGFR-ERK Signaling.
  4. The constitutive oncogenic and signaling activities of phosphatidylinositol 3-kinase (PI3K) isoforms p110β and p110δ.
  5. Growth Factor-Independent mTORC1 Signaling Promotes Primary Cilia Length via Suppression of Autophagy.
  6. Multiplexed, scalable analog recording of gene regulation dynamics over weeks using intracellular protein tapes.
  7. A clinical road map for single-cell omics.
  8. Inactivation of PI3K-C2α deregulates cell death pathways and sensitizes to endotoxic shock.
  9. In cellulo DNA assembly for targeted genomic integration and rearrangement in human cells.
  10. The proteostatic landscape of healthy human oocytes.
  1. bioRxiv. 2025 Jun 25. pii: 2025.06.20.660715. [Epub ahead of print]
    Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition.
    Xiangyu Ge, Jaffarguriqbal Singh, Wenxue Li, Cassandra S Markham, Christian Felipe Ruiz, Moitrayee Bhattacharyya, Yansheng Liu, Mandar Deepak Muzumdar.
      Despite the availability of RAS inhibitors and the dependence of >90% of pancreatic ductal adenocarcinomas (PDAC) on oncogenic KRAS mutations, resistance to KRAS inhibition remains a serious obstacle. We show here that phosphoinositide 3-kinase (PI3K) plays a major role in this resistance through upstream activation of wild-type RAS signaling - beyond its known KRAS effector function. Combining proximity labeling, CRISPR screens, live-cell imaging, and functional assays we found that PI3K orchestrates phosphoinositide-mediated GAB1 recruitment to the plasma membrane, nucleating assembly of RAS signaling complexes that activate mitogen-activated protein kinase (MAPK) in an EGFR/SHP2/SOS1-dependent manner. We further demonstrate that inhibiting PI3K enhances sensitivity to mutant-specific KRAS inhibitors in PDAC cells, including cells with clinically identified PIK3CA mutations. Our findings refine RAS-PI3K signaling paradigms, reveal that PI3K-driven wild-type RAS activation drives resistance to KRAS inhibition, and illuminate new avenues for augmenting KRAS-targeted therapies in PDAC.
    DOI:  https://doi.org/10.1101/2025.06.20.660715
  2. bioRxiv. 2025 Jun 20. pii: 2025.06.17.660155. [Epub ahead of print]
    Cell barcoding with tandem fluorescent proteins enables high-throughput signaling dynamics analysis.
    Jhen-Wei Wu, Jr-Ming Yang, Suyang Wang, Yun Chen, Chuan-Hsiang Huang.
      Cell barcodes are essential for a wide array of experimental applications, including lineage tracing, genetic screening, and single-cell analysis. An optimal barcode library would provide high diversity, live-cell compatible identification, and simple readout. In this work, we introduce single chain tandem fluorescent protein (sctFP) barcodes, constructed by linking different fluorescent proteins (FPs) into a single polypeptide chain with varied copy numbers. We found that the fluorescence signal intensity ratio at different wavelengths can reliably differentiate sctFPs generated using cnidarian FPs, but not prokaryotic FPs that require exogenous cofactors. The sctFPs enable the multiplexing of genetically encoded fluorescent biosensors, enhancing current biosensor multiplexing methods through a simplified imaging and analysis pipeline that support high-throughput applications. Their robust spectral profiles are compatible with a broad range of biosensor types. Using sctFPs, we demonstrate simultaneous tracking of various signaling activities with biosensors of different spectral properties. Together, this strategy provides a robust and scalable method for barcoding cells across diverse experimental contexts.
    DOI:  https://doi.org/10.1101/2025.06.17.660155
  3. bioRxiv. 2025 Jun 17. pii: 2025.06.12.659034. [Epub ahead of print]
    Systems-level Consequences of Low RAF Abundance for EGFR-ERK Signaling.
    Sung Hyun Lee, Paul J Myers, Kevin S Brown, Leslie M Loew, Alexander Sorkin, Matthew J Lazzara.
      The RAF kinases are central links between RAS, once activated by receptor tyrosine kinases (RTKs), and the extracellular signal-regulated kinases (ERK). In many cancer cells, RAFs are the least abundantly expressed RTK-ERK pathway proteins and can be present at just hundreds of copies per cell at the plasma membrane, but the consequences of limited RAF expression are unclear. By developing continuum and stochastic computational models of the epidermal growth factor receptor (EGFR)-ERK pathway, we showed that low RAF abundance creates stoichiometric bottlenecks between RTKs and ERK with concomitant stochastic RAF dynamics that propagate to weakly expressed downstream pathway proteins. Advanced sensitivity and Sloppiness analyses identified RAS activation and RAS-RAF interactions as strong determinants of signaling in low-RAF settings and revealed an efficient model fitting approach. RAF bottlenecks were predicted to impede ERK activation by oncogenic RAS mutants and explained a tendency for RAF1 membrane localization to be noisy. This work provides quantitative insight into a common, yet unexplored, regime for EGFR-ERK signaling and a systematic approach to develop and characterize dynamic models of receptor-mediated signaling.
    STATEMENT OF SIGNIFICANCE: RAF kinases connect receptors to the mitogenic ERK signaling pathway by translocating to the plasma membrane, but in a substantial fraction of cancer cell contexts RAFs are greatly outnumbered by other pathway proteins, potentially creating an unrecognized and consequential signaling bottleneck. We trained a novel computational model of EGFR-ERK signaling and characterized it comprehensively using integrated multivariate sensitivity analyses and Sloppiness analysis. The results revealed that low RAF abundance suppresses EGFR-mediated ERK activation, limits the effects of upstream oncogenic RAS mutants, and creates stochastic RAF dynamics that can propagate downstream. Thus, the canonical EGFR-ERK pathway exhibits divergent behaviors in a parameter space representative of a substantial fraction of cancer cell settings.
    DOI:  https://doi.org/10.1101/2025.06.12.659034
  4. bioRxiv. 2025 Jun 26. pii: 2025.06.26.659600. [Epub ahead of print]
    The constitutive oncogenic and signaling activities of phosphatidylinositol 3-kinase (PI3K) isoforms p110β and p110δ.
    Yoshihiro Ito, Petra Pavlickova, Claire S Levy, Sohye Kang, Jonathan R Hart, Lynn Ueno, Peter K Vogt.
      The p110β and p110δ isoforms of the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) show enhanced oncogenic and signaling activities as compared with the p110α protein. The adapter binding domains (ABDs) of p110β and p110δ contain an isoform-specific PXXP mo- tif. Mutations of this PXXP to AXXA diminish the oncogenic and signaling activities. This loss of function can be compensated by placing a gain-of-function mutation in the helical domain of the P/A mutants. The P/A mutants still associate with the regulatory p85 subunit, but the affinity of this interaction is decreased. p110α with the ABD of either p110β or p110δ shows an increase of oncogenic and signaling activities, whereas p110β or p110δ with the ABD of p110α have greatly reduced oncogenic and signaling activities. Introducing the PXXP motif in the ABD of p110α re- sults in a significant gain of function. We conclude that the PXXP motif in the ABD of p110β and p110δ is essential for the elevated oncogenic and signaling activities of these isoforms. We pro- pose that the PXXP motif in the ABD of p110β and p110δ affects the interaction with the iSH2 domain of p85, shifting the regulatory SH2 domains into less effective inhibitory conformations.
    DOI:  https://doi.org/10.1101/2025.06.26.659600
  5. bioRxiv. 2025 May 07. pii: 2025.05.07.652626. [Epub ahead of print]
    Growth Factor-Independent mTORC1 Signaling Promotes Primary Cilia Length via Suppression of Autophagy.
    Jong Shin, Yann Cormerais, Khaled Tighanimine, Pratima Niroula, Madi Y Cisse, Samuel C Lapp, Krystle C Kalafut, Sandra Schrötter, Brendan D Manning.
      The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), as a sensor of growth signals that subsequently controls cell growth, has been predominantly studied in actively proliferating cells. Primary cilia are sensory organelles present on most quiescent cells, where they play essential roles in receiving environmental and developmental signals. Given that ciliated cells are non-proliferative, we investigated whether mTORC1 signaling influences the growth of primary cilia. Here, we show that mTORC1 promotes primary cilia elongation, without effects on ciliogenesis or cell growth, by suppressing autophagy. Inhibition of mTORC1 signaling through pharmacological, nutritional, or genetic interventions gave rise to shortened primary cilia, while activation of the pathway resulted in elongation. Furthermore, pharmacological or genetic inhibition of autophagy, a key downstream process blocked by mTORC1, also elongated primary cilia and rendered them resistant to mTORC1 inhibition. Notably, these mTORC1-mediated effects on primary cilia extend to mouse neurons ex vivo and in vivo. These findings highlight a previously unrecognized role for mTORC1 signaling in the control of primary cilia length that may contribute to diseases where ciliary function is altered, referred to as ciliopathies.
    DOI:  https://doi.org/10.1101/2025.05.07.652626
  6. bioRxiv. 2025 May 10. pii: 2025.05.10.653182. [Epub ahead of print]
    Multiplexed, scalable analog recording of gene regulation dynamics over weeks using intracellular protein tapes.
    Lirong Zheng, Yixiao Yan, Bingxin Zhou, Jormay Lim, Dongqing Shi, Bobae An, BumJin Ko, Emily Klyder, Sethuramasundaram Pitchiaya, Denise J Cai, Edward S Boyden, Donglai Wei, Pietro Lio, Changyang Linghu.
      Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape-a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed in situ recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-fos, Arc, Egr1, and Npas4 activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.
    DOI:  https://doi.org/10.1101/2025.05.10.653182
  7. Cell. 2025 Jul 10. pii: S0092-8674(25)00676-2. [Epub ahead of print]188(14): 3633-3647
    A clinical road map for single-cell omics.
    Michael A Skinnider, Gregoire Courtine, Jocelyne Bloch, Jordan W Squair.
      In a matter of years, single-cell omics has matured from a pioneering technique employed by just a handful of specialized laboratories to become a ubiquitous feature of biological research and a key driver of scientific discovery. The widespread adoption and development of single-cell omic assays has sparked mounting enthusiasm that these technologies are poised to also enhance the precision of diagnosis, the monitoring of disease progression, and the personalization of therapeutic strategies. Despite initial forays into clinical settings, however, single-cell technologies are not yet routinely used to inform medical or surgical decision-making. Here, we identify and categorize key experimental, computational, and conceptual barriers that currently hinder the clinical deployment of single-cell omics. We focus on the potential for single-cell transcriptomics to guide clinical decision-making through the development of combinatorial biomarkers that simultaneously quantify multiple cell-type-specific pathophysiological processes. We articulate a framework to identify patient subpopulations that stand to benefit from such biomarkers, and we outline the experimental and computational requirements to derive reproducible and actionable clinical readouts from single-cell omics.
    Keywords:  machine learning; patient cohorts; personalized medicine; single-cell; spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.cell.2025.06.009
  8. Proc Natl Acad Sci U S A. 2025 Jul 22. 122(29): e2423358122
    Inactivation of PI3K-C2α deregulates cell death pathways and sensitizes to endotoxic shock.
    York Posor, Sarah E Conduit, Wayne Pearce, Daniele Morelli, Georgia Constantinou, Maria Whitehead, Neil J Sebire, Cheryl L Scudamore, Nieves Peltzer, Henning Walczak, Bart Vanhaesebroeck.
      The organismal roles of the class II PI3K isoform PI3K-C2α remain poorly understood. Recent studies have found PI3K-C2α to promote arterial thrombosis and breast cancer metastasis, generating interest in this kinase as a drug target, with small molecule PI3K-C2α inhibitors now available. However, the consequences of systemic PI3K-C2α inactivation in the nondiseased, postnatal state are largely unknown. Here, we show that induction of genetic PI3K-C2α inactivation in adult mice is well tolerated, without adverse effects on normal physiology. Surprisingly, however, mice with inactive PI3K-C2α display strong sensitization to challenge with bacterial lipopolysaccharide (LPS), a model of endotoxic shock. This sensitization is recapitulated by vascular endothelial-specific deletion of PI3K-C2α. Furthermore, sensitization to LPS can be fully rescued by disabling extrinsic induction of cell death by combined caspase-8- and RIPK3 deficiency. These observations validate the tolerability of systemic PI3K-C2α inhibition in principle but reveal an unexpected role for PI3K-C2α in the regulation of extrinsic cell death pathways.
    Keywords:  PI3K; endotoxic shock; phosphoinositide; regulated cell death; vascular endothelia
    DOI:  https://doi.org/10.1073/pnas.2423358122
  9. bioRxiv. 2025 Jun 16. pii: 2025.06.16.659926. [Epub ahead of print]
    In cellulo DNA assembly for targeted genomic integration and rearrangement in human cells.
    Sébastien Levesque, Nozomu Kawashima, GueHo Hwang, Basheer Becerra, Vivien A C Schoonenberg, William Mannherz, Luke Homfeldt, Luca Pinello, Suneet Agarwal, Daniel E Bauer.
      Although therapeutic genome editing holds great potential to remedy diverse inherited and acquired disorders, targeted installation of medium to large sized genomic modifications in therapeutically relevant cells remains challenging. We have developed an approach that permits DNA sequence assembly and integration in human cells leveraging CRISPR-targeted dual flap synthesis. This method, named prime assembly, allows for RNA-programmable site-specific integration of single- or double-stranded DNA fragments. Unlike homology-directed repair, prime assembly was similarly active in dividing and non-dividing cells. We applied prime assembly to perform targeted exon recoding, transgene integration, and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells. Prime assembly expands the capabilities of genome engineering by enabling the targeted integration of medium to large sized DNA sequences without relying on double-stranded DNA donors, nuclease-driven double strand breaks, or cell cycle progression.
    DOI:  https://doi.org/10.1101/2025.06.16.659926
  10. EMBO J. 2025 Jul 16.
    The proteostatic landscape of healthy human oocytes.
    Gabriele Zaffagnini, Miquel Solé, Juan Manuel Duran, Nikolaos P Polyzos, Elvan Böke.
      Oocytes, female germ cells that develop into eggs, are among the longest-lived cells in the animal body. Recent studies on mouse oocytes highlight unique adaptations in protein homeostasis (proteostasis) within these cells. However, the mechanisms of proteostasis in human oocytes remain virtually unstudied. We present the first large-scale study of proteostatic activity in human oocytes using over 100 freshly donated oocytes from 21 healthy women aged 19-34 years. We analysed the activity and distribution of lysosomes, proteasomes, and mitochondria in both immature and mature oocytes. Notably, human oocytes exhibit nearly twofold lower proteolytic activity than surrounding somatic cells, with further decreases as oocytes mature. Oocyte maturation is also coupled with lysosomal exocytosis and a decrease in mitochondrial membrane potential. We propose that reduced organelle activity preserves key cellular components critical for early embryonic development during the prolonged maturation of human oocytes. Our findings highlight the distinctive biology of human oocytes and the need to investigate human-specific reproductive biology to address challenges in female fertility.
    Keywords:  Female Fertility; Human Oocytes; Lysosomes; Mitochondria; Proteostasis
    DOI:  https://doi.org/10.1038/s44318-025-00493-2
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