bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024‒08‒25
twelve papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.
  2. Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies.
  3. Improved Production of Class I Phosphatidylinositol 4,5-bisphosphate 3-kinase.
  4. Rag-Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway.
  5. Organismal metabolism regulates the expansion of oncogenic PIK3CA mutant clones in normal esophagus.
  6. A statistical simulation model to guide the choices of analytical methods in arrayed CRISPR screen experiments.
  7. Benchmarking computational methods for single-cell chromatin data analysis.
  8. PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α.
  9. Cooperative genomic lesions in HRAS-mutant cancers predict resistance to farnesyltransferase inhibitors.
  10. Membrane-anchored intracellular insulin receptor or insulin-like growth factor-1 receptor elicits ligand-independent downstream signaling.
  11. Sirolimus for vascular anomalies associated with PTEN hamartoma tumor syndrome.
  12. Transcriptome data are insufficient to control false discoveries in regulatory network inference.
  1. Elife. 2024 Aug 20. pii: RP91012. [Epub ahead of print]12
    Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.
    Duk-Su Koh, Anastasiia Stratiievska, Subhashis Jana, Shauna C Otto, Teresa M Swanson, Anthony Nhim, Sara Carlson, Marium Raza, Ligia Araujo Naves, Eric N Senning, Ryan A Mehl, Sharona E Gordon.
      Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.
    Keywords:  biochemistry; chemical biology; click chemistry; human; membrane protein; molecular biophysics; rat; structural biology; trafficking
    DOI:  https://doi.org/10.7554/eLife.91012
  2. Blood Adv. 2024 Aug 16. pii: bloodadvances.2024013205. [Epub ahead of print]
    Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies.
    Nardjas Bettazová, Jana Senavova, Kristyna Kupcova, Dana Sovilj, Anezka Rejmonova, Ladislav Anděra, Karla Svobodová, Adela Berkova, Zuzana Zemanova, Lenka Daumova, Vaclav Herman, Alexandra Dolnikova, Richard Eric Davis, Marek Trněný, Pavel Klener, Ondrej Havranek.
      Besides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout/knockdown of PTEN gene (PTEN KO / KD) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged AKT phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might impact MCL cells in AKT independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, BTK inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL.
    DOI:  https://doi.org/10.1182/bloodadvances.2024013205
  3. Protein Expr Purif. 2024 Aug 20. pii: S1046-5928(24)00154-2. [Epub ahead of print] 106582
    Improved Production of Class I Phosphatidylinositol 4,5-bisphosphate 3-kinase.
    Simon Messing, Stephanie R T Widmeyer, John-Paul Denson, Jennifer Mehalko, Vanessa E Wall, Matthew Drew, Kelly Snead, Min Hong, Carissa Grose, Dominic Esposito, William Gillette.
      Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.
    Keywords:  PI3K; PIK3CA; Phosphoinositide 3-kinase; p110α; p110β; p110δ protein production; p85α
    DOI:  https://doi.org/10.1016/j.pep.2024.106582
  4. Proc Natl Acad Sci U S A. 2024 Aug 27. 121(35): e2322755121
    Rag-Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway.
    Max L Valenstein, Pranav V Lalgudi, Xin Gu, Jibril F Kedir, Martin S Taylor, Raghu R Chivukula, David M Sabatini.
      The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.
    Keywords:  biochemistry; mTOR signaling; nutrient sensing
    DOI:  https://doi.org/10.1073/pnas.2322755121
  5. Nat Genet. 2024 Aug 21.
    Organismal metabolism regulates the expansion of oncogenic PIK3CA mutant clones in normal esophagus.
    Albert Herms, Bartomeu Colom, Gabriel Piedrafita, Argyro Kalogeropoulou, Ujjwal Banerjee, Charlotte King, Emilie Abby, Kasumi Murai, Irene Caseda, David Fernandez-Antoran, Swee Hoe Ong, Michael W J Hall, Christopher Bryant, Roshan K Sood, Joanna C Fowler, Albert Pol, Christian Frezza, Bart Vanhaesebroeck, Philip H Jones.
      Oncogenic PIK3CA mutations generate large clones in aging human esophagus. Here we investigate the behavior of Pik3ca mutant clones in the normal esophageal epithelium of transgenic mice. Expression of a heterozygous Pik3caH1047R mutation drives clonal expansion by tilting cell fate toward proliferation. CRISPR screening and inhibitor treatment of primary esophageal keratinocytes confirmed the PI3K-mTOR pathway increased mutant cell competitive fitness. The antidiabetic drug metformin reduced mutant cell advantage in vivo and in vitro. Conversely, metabolic conditions such as type 1 diabetes or diet-induced obesity enhanced the competitive fitness of Pik3caH1047R cells. Consistently, we found a higher density of PIK3CA gain-of-function mutations in the esophagus of individuals with high body mass index compared with those with normal weight. We conclude that the metabolic environment selectively influences the evolution of the normal epithelial mutational landscape. Clinically feasible interventions to even out signaling imbalances between wild-type and mutant cells may limit the expansion of oncogenic mutants in normal tissues.
    DOI:  https://doi.org/10.1038/s41588-024-01891-8
  6. PLoS One. 2024 ;19(8): e0307445
    A statistical simulation model to guide the choices of analytical methods in arrayed CRISPR screen experiments.
    Chang Sik Kim, Jonathan Cairns, Valentina Quarantotti, Bogumil Kaczkowski, Yinhai Wang, Peter Konings, Xiang Zhang.
      An arrayed CRISPR screen is a high-throughput functional genomic screening method, which typically uses 384 well plates and has different gene knockouts in different wells. Despite various computational workflows, there is currently no systematic way to find what is a good workflow for arrayed CRISPR screening data analysis. To guide this choice, we developed a statistical simulation model that mimics the data generating process of arrayed CRISPR screening experiments. Our model is flexible and can simulate effects on phenotypic readouts of various experimental factors, such as the effect size of gene editing, as well as biological and technical variations. With two examples, we showed that the simulation model can assist making principled choice of normalization and hit calling method for the arrayed CRISPR data analysis. This simulation model is implemented in an R package and can be downloaded from Github.
    DOI:  https://doi.org/10.1371/journal.pone.0307445
  7. Genome Biol. 2024 Aug 16. 25(1): 225
    Benchmarking computational methods for single-cell chromatin data analysis.
    Siyuan Luo, Pierre-Luc Germain, Mark D Robinson, Ferdinand von Meyenn.
      BACKGROUND: Single-cell chromatin accessibility assays, such as scATAC-seq, are increasingly employed in individual and joint multi-omic profiling of single cells. As the accumulation of scATAC-seq and multi-omics datasets continue, challenges in analyzing such sparse, noisy, and high-dimensional data become pressing. Specifically, one challenge relates to optimizing the processing of chromatin-level measurements and efficiently extracting information to discern cellular heterogeneity. This is of critical importance, since the identification of cell types is a fundamental step in current single-cell data analysis practices.RESULTS: We benchmark 8 feature engineering pipelines derived from 5 recent methods to assess their ability to discover and discriminate cell types. By using 10 metrics calculated at the cell embedding, shared nearest neighbor graph, or partition levels, we evaluate the performance of each method at different data processing stages. This comprehensive approach allows us to thoroughly understand the strengths and weaknesses of each method and the influence of parameter selection.
    CONCLUSIONS: Our analysis provides guidelines for choosing analysis methods for different datasets. Overall, feature aggregation, SnapATAC, and SnapATAC2 outperform latent semantic indexing-based methods. For datasets with complex cell-type structures, SnapATAC and SnapATAC2 are preferred. With large datasets, SnapATAC2 and ArchR are most scalable.
    Keywords:  Benchmark; Clustering; Dimensional reduction; Feature engineering; ScATAC-seq
    DOI:  https://doi.org/10.1186/s13059-024-03356-x
  8. Cell Mol Life Sci. 2024 Aug 19. 81(1): 358
    PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α.
    Sergio López-García, Esperanza López-Merino, Alba Fernández-Rodrigo, Pablo Zamorano-González, Silvia Gutiérrez-Eisman, Raquel Jiménez-Sánchez, José A Esteban.
      Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.
    Keywords:  LTD; LTP; Rac1; Spine; Structural plasticity; p85
    DOI:  https://doi.org/10.1007/s00018-024-05394-x
  9. Oncogene. 2024 Aug 16.
    Cooperative genomic lesions in HRAS-mutant cancers predict resistance to farnesyltransferase inhibitors.
    Aradhya Nigam, Gnana P Krishnamoorthy, Walid K Chatila, Katherine Berman, Mahesh Saqcena, Henry Walch, Mandakini Venkatramani, Alan L Ho, Nikolaus Schultz, James A Fagin, Brian R Untch.
      In the clinical development of farnesyltransferase inhibitors (FTIs) for HRAS-mutant tumors, responses varied by cancer type. Co-occurring mutations may affect responses. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and to study their effect on sensitivity to FTIs. Using targeted sequencing data from the MSK-IMPACT and Dana-Farber Cancer Institute Genomic Evidence Neoplasia Information Exchange databases, we identified comutations that were observed predominantly in HRAS-mutant versus KRAS-mutant or NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of coaltered mutations (48.8%) in the MAPK, PI3K, or RTK pathway genes, compared with KRAS-mutant (41.4%) and NRAS-mutant (38.4%) cancers (p < 0.05). Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effects of comutations on sensitivity to FTIs, HrasG13R was transfected into "RASless" (Kraslox/lox/Hras-/-/Nras-/-/RERTert/ert) mouse embryonic fibroblasts (MEFs), which sensitized nontransfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion and Pik3caH1047R transduction led to resistance to tipifarnib in HrasG13R-transfected MEFs in the presence or absence of KrasWT, whereas BrafG466E transduction led to resistance to tipifarnib only in the presence of KrasWT. Combined treatment with tipifarnib and MEK inhibition sensitized cells to tipifarnib in all settings, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage-dependent MAPK or PI3K pathway alterations, which confer resistance to tipifarnib. The combined use of FTIs and MEK inhibition is a promising strategy for HRAS-mutant tumors.
    DOI:  https://doi.org/10.1038/s41388-024-03095-0
  10. Biochem Biophys Rep. 2024 Sep;39 101799
    Membrane-anchored intracellular insulin receptor or insulin-like growth factor-1 receptor elicits ligand-independent downstream signaling.
    Akiko Sotozono, Kazuhiko Namekata, Xiaoli Guo, Youichi Shinozaki, Chikako Harada, Takahiko Noro, Tadashi Nakano, Takayuki Harada.
      Neurodegenerative diseases including glaucoma affect insulin signaling, and insulin treatment has been shown to reverse the neurodegenerative loss of dendritic complexity in retinal ganglion cells. Therefore, strategies for enhancing or maintaining insulin signaling are worth pursuing to establish new therapies for these diseases. In the present study, we generated constitutively active insulin receptor (F-iIR) and insulin-like growth factor-1 receptor (F-iIGF1R) using a system that forces membrane localization of the intracellular domains of these receptors by farnesylation. Immunohistochemistry and Western blot analysis revealed that F-iIR and F-iIGF1R caused the activation of ERK and AKT in the absence of ligands in vitro. Our results suggest that in vivo effects of F-iIR and F-iIGF1R on the progression of neurodegenerative diseases should be investigated in the future.
    Keywords:  AKT; ERK; Farnesylation; Insulin receptor; Insulin-like growth factor-1 receptor
    DOI:  https://doi.org/10.1016/j.bbrep.2024.101799
  11. Pediatr Blood Cancer. 2024 Aug 21. e31282
    Sirolimus for vascular anomalies associated with PTEN hamartoma tumor syndrome.
    Alexandra Zabeida, Jack J Brzezinski, Jonathan D Wasserman, Cheryl Cytrynbaum, Rosanna Weksberg, Kelley Zwicker, Kevin Zbuk, Alessandro Gasparetto, Laura Willis, Michelle Fantauzzi, Manuel Carcao.
      Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is a rare condition associated with vascular anomalies and increased tumor risk. Sirolimus, an mTOR inhibitor used for managing vascular anomalies is underexplored in PHTS. A single-institution retrospective review of children with PHTS and vascular anomalies treated with sirolimus identified seven patients. Median age at sirolimus initiation was 10 years. After a median 2.5-year follow-up, six of seven patients (86%) showed significant clinical improvement. No significant adverse effects were observed, except mild buccal ulcers and acne. This study supports sirolimus as an effective and safe treatment for vascular anomalies in a small group of children with PHTS.
    Keywords:  PTEN hamartoma tumor syndrome; child; sirolimus; vascular anomalies
    DOI:  https://doi.org/10.1002/pbc.31282
  12. Cell Syst. 2024 Aug 21. pii: S2405-4712(24)00205-9. [Epub ahead of print]15(8): 709-724.e13
    Transcriptome data are insufficient to control false discoveries in regulatory network inference.
    Eric Kernfeld, Rebecca Keener, Patrick Cahan, Alexis Battle.
      Inference of causal transcriptional regulatory networks (TRNs) from transcriptomic data suffers notoriously from false positives. Approaches to control the false discovery rate (FDR), for example, via permutation, bootstrapping, or multivariate Gaussian distributions, suffer from several complications: difficulty in distinguishing direct from indirect regulation, nonlinear effects, and causal structure inference requiring "causal sufficiency," meaning experiments that are free of any unmeasured, confounding variables. Here, we use a recently developed statistical framework, model-X knockoffs, to control the FDR while accounting for indirect effects, nonlinear dose-response, and user-provided covariates. We adjust the procedure to estimate the FDR correctly even when measured against incomplete gold standards. However, benchmarking against chromatin immunoprecipitation (ChIP) and other gold standards reveals higher observed than reported FDR. This indicates that unmeasured confounding is a major driver of FDR in TRN inference. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  Markov random field; false discovery rate; gene regulatory network; knockoff filter; network inference; structure learning; transcription factor; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.cels.2024.07.006
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