bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021–11–07
fourteen papers selected by
Ralitsa Radostinova Madsen, University College London
  1. Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.
  2. Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation.
  3. Fasting-mimicking diet blocks triple-negative breast cancer and cancer stem cell escape.
  4. A synthetic gene circuit for imaging-free detection of signaling pulses.
  5. An analytical framework for interpretable and generalizable single-cell data analysis.
  6. The context-specific role of germline pathogenicity in tumorigenesis.
  7. The Role of PI3'-Lipid Signaling in Melanoma Initiation, Progression and Maintenance.
  8. Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.
  9. Protein kinase N2 connects blood flow with NO production in a double AKT.
  10. Single-cell normalization and association testing unifying CRISPR screen and gene co-expression analyses with Normalisr.
  11. Benchmarking pipelines for subclonal deconvolution of bulk tumour sequencing data.
  12. A multi-omic single-cell landscape of human gynecologic malignancies.
  13. From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control.
  14. Synonymous mutations reveal genome-wide levels of positive selection in healthy tissues.
  1. Nat Commun. 2021 Nov 04. 12(1): 6409
    Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.
    Chih-Yao Chung, Kritarth Singh, Vassilios N Kotiadis, Gabriel E Valdebenito, Jee Hwan Ahn, Emilie Topley, Joycelyn Tan, William D Andrews, Benoit Bilanges, Robert D S Pitceathly, Gyorgy Szabadkai, Mariia Yuneva, Michael R Duchen.
      Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.
    DOI:  https://doi.org/10.1038/s41467-021-26746-2
  2. Proc Natl Acad Sci U S A. 2021 Nov 09. pii: e2109327118. [Epub ahead of print]118(45):
    Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation.
    Xiao Liu, Su Yang, Jonathan R Hart, Yingna Xu, Xinyu Zou, Huibing Zhang, Qingtong Zhou, Tian Xia, Yan Zhang, Dehua Yang, Ming-Wei Wang, Peter K Vogt.
      Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.
    Keywords:  activation; activity-dependent conformational changes; inhibition; phosphoinositide 3-kinase (PI3K)
    DOI:  https://doi.org/10.1073/pnas.2109327118
  3. Cell Metab. 2021 Nov 02. pii: S1550-4131(21)00486-1. [Epub ahead of print]33(11): 2247-2259.e6
    Fasting-mimicking diet blocks triple-negative breast cancer and cancer stem cell escape.
    Giulia Salvadori, Federica Zanardi, Fabio Iannelli, Riccardo Lobefaro, Claudio Vernieri, Valter D Longo.
      Metastatic tumors remain lethal due to primary/acquired resistance to therapy or cancer stem cell (CSC)-mediated repopulation. We show that a fasting-mimicking diet (FMD) activates starvation escape pathways in triple-negative breast cancer (TNBC) cells, which can be identified and targeted by drugs. In CSCs, FMD lowers glucose-dependent protein kinase A signaling and stemness markers to reduce cell number and increase mouse survival. Accordingly, metastatic TNBC patients with lower glycemia survive longer than those with higher baseline glycemia. By contrast, in differentiated cancer cells, FMD activates PI3K-AKT, mTOR, and CDK4/6 as survival/growth pathways, which can be targeted by drugs to promote tumor regression. FMD cycles also prevent hyperglycemia and other toxicities caused by these drugs. These data indicate that FMD has wide and differential effects on normal, cancer, and CSCs, allowing the rapid identification and targeting of starvation escape pathways and providing a method potentially applicable to many malignancies.
    Keywords:  CDK4/6; PI3K/AKT; PKA; cancer stem cells; fasting; fasting-mimicking diet; glucose; mTOR; starvation escape pathways; triple-negative breast cancer
    DOI:  https://doi.org/10.1016/j.cmet.2021.10.008
  4. Cell Syst. 2021 Nov 01. pii: S2405-4712(21)00413-0. [Epub ahead of print]
    A synthetic gene circuit for imaging-free detection of signaling pulses.
    Pavithran T Ravindran, Sarah McFann, Richard H Thornton, Jared E Toettcher.
      Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution. Here, we describe an approach to infer pulsatile signaling dynamics from a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuits with the capability to selectively detect signaling pulses, revealing an incoherent feedforward topology that robustly performs this computation. We implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our "recorder of Erk activity dynamics" (READer) responds sensitively to spontaneous and stimulus-driven Erk pulses. READer circuits open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.
    Keywords:  cell signaling; network motifs; signaling dynamics; synthetic biology
    DOI:  https://doi.org/10.1016/j.cels.2021.10.002
  5. Nat Methods. 2021 Nov;18(11): 1317-1321
    An analytical framework for interpretable and generalizable single-cell data analysis.
    Jian Zhou, Olga G Troyanskaya.
      The scaling of single-cell data exploratory analysis with the rapidly growing diversity and quantity of single-cell omics datasets demands more interpretable and robust data representation that is generalizable across datasets. Here, we have developed a 'linearly interpretable' framework that combines the interpretability and transferability of linear methods with the representational power of non-linear methods. Within this framework we introduce a data representation and visualization method, GraphDR, and a structure discovery method, StructDR, that unifies cluster, trajectory and surface estimation and enables their confidence set inference.
    DOI:  https://doi.org/10.1038/s41592-021-01286-1
  6. Nat Genet. 2021 Nov;53(11): 1577-1585
    The context-specific role of germline pathogenicity in tumorigenesis.
    Preethi Srinivasan, Chaitanya Bandlamudi, Philip Jonsson, Yelena Kemel, Shweta S Chavan, Allison L Richards, Alexander V Penson, Craig M Bielski, Christopher Fong, Aijazuddin Syed, Gowtham Jayakumaran, Meera Prasad, Jason Hwee, Selcuk Onur Sumer, Ino de Bruijn, Xiang Li, JianJiong Gao, Nikolaus Schultz, Roy Cambria, Jesse Galle, Semanti Mukherjee, Joseph Vijai, Karen A Cadoo, Maria I Carlo, Michael F Walsh, Diana Mandelker, Ozge Ceyhan-Birsoy, Jinru Shia, Ahmet Zehir, Marc Ladanyi, David M Hyman, Liying Zhang, Kenneth Offit, Mark E Robson, David B Solit, Zsofia K Stadler, Michael F Berger, Barry S Taylor.
      Human cancers arise from environmental, heritable and somatic factors, but how these mechanisms interact in tumorigenesis is poorly understood. Studying 17,152 prospectively sequenced patients with cancer, we identified pathogenic germline variants in cancer predisposition genes, and assessed their zygosity and co-occurring somatic alterations in the concomitant tumors. Two major routes to tumorigenesis were apparent. In carriers of pathogenic germline variants in high-penetrance genes (5.1% overall), lineage-dependent patterns of biallelic inactivation led to tumors exhibiting mechanism-specific somatic phenotypes and fewer additional somatic oncogenic drivers. Nevertheless, 27% of cancers in these patients, and most tumors in patients with pathogenic germline variants in lower-penetrance genes, lacked particular hallmarks of tumorigenesis associated with the germline allele. The dependence of tumors on pathogenic germline variants is variable and often dictated by both penetrance and lineage, a finding with implications for clinical management.
    DOI:  https://doi.org/10.1038/s41588-021-00949-1
  7. Exp Dermatol. 2021 Oct 30.
    The Role of PI3'-Lipid Signaling in Melanoma Initiation, Progression and Maintenance.
    Gennie L Parkman, Mona Foth, David A Kircher, Sheri L Holmen, Martin McMahon.
      Phosphatidylinositol-3'-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3' hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5)P3 and PI(3,4)P2 regulate myriad biochemical and biological processes in both normal and cancer cells including: responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3'-kinases (PI3Kα, β, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K--lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3'-kinase signaling in cells. Indeed, in preclinical models, activation of the PI3K→AKT signaling axis cooperates with alterations such as expression of the BRAFV600E oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumor microenvironment, melanoma maintenance, and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3K→AKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.
    Keywords:  BIOMARKERS; CANCER; MELANOGENESIS; MELANOMA
    DOI:  https://doi.org/10.1111/exd.14489
  8. Sci Rep. 2021 Nov 04. 11(1): 21689
    Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.
    Thanh-Trang Vo, Lee-Or Herzog, Roberta Buono, Jong-Hoon Scott Lee, Sharmila Mallya, Madeleine R Duong, Joshua Thao, Moran Gotesman, David A Fruman.
      The mechanistic target of rapamycin (mTOR) is a kinase whose activation is associated with poor prognosis in pre-B cell acute lymphoblastic leukemia (B-ALL). These and other findings have prompted diverse strategies for targeting mTOR signaling in B-ALL and other B-cell malignancies. In cellular models of Philadelphia Chromosome-positive (Ph+) B-ALL, mTOR kinase inhibitors (TOR-KIs) that inhibit both mTOR-complex-1 (mTORC1) and mTOR-complex-2 (mTORC2) enhance the cytotoxicity of tyrosine kinase inhibitors (TKIs) such as dasatinib. However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or downstream effectors provides alternative strategies that may improve selectivity towards leukemia cells. Of particular interest is the eukaryotic initiation factor 4F (eIF4F) complex that mediates cap-dependent translation. Here we use novel chemical and genetic approaches to show that selective targeting of either mTORC1 kinase activity or components of the eIF4F complex sensitizes murine BCR-ABL-dependent pre-B leukemia cells to dasatinib. SBI-756, a small molecule inhibitor of eIF4F assembly, sensitizes human Ph+ and Ph-like B-ALL cells to dasatinib cytotoxicity without affecting survival of T lymphocytes or natural killer cells. These findings support the further evaluation of eIF4F-targeted molecules in combination therapies with TKIs in B-ALL and other blood cancers.
    DOI:  https://doi.org/10.1038/s41598-021-00950-y
  9. J Clin Invest. 2021 Nov 01. pii: e154256. [Epub ahead of print]131(21):
    Protein kinase N2 connects blood flow with NO production in a double AKT.
    David Jr Fulton, David W Stepp.
      Shear stress is an important regulator of blood flow, and luminal endothelial cells (ECs) sense increases in frictional forces and respond with an appropriate release of vasoactive mediators. In this issue of the JCI, Jin et al. identified a mechanism by which ECs respond to shear stress with endothelial NOS (eNOS) activation and NO release. The authors showed that PKN2 was activated by fluid shear stress and contributed to eNOS activation via a double play - indirect phosphorylation at serine 1177 (S1177) via AKT and direct phosphorylation of the S1179 site. Phosphorylation of both sites individually increased eNOS activity, but together they had an additive effect. In sum, these findings reveal exciting details about how shear stress regulates eNOS and have important implications for blood flow and blood pressure.
    DOI:  https://doi.org/10.1172/JCI154256
  10. Nat Commun. 2021 Nov 04. 12(1): 6395
    Single-cell normalization and association testing unifying CRISPR screen and gene co-expression analyses with Normalisr.
    Lingfei Wang.
      Single-cell RNA sequencing (scRNA-seq) provides unprecedented technical and statistical potential to study gene regulation but is subject to technical variations and sparsity. Furthermore, statistical association testing remains difficult for scRNA-seq. Here we present Normalisr, a normalization and statistical association testing framework that unifies single-cell differential expression, co-expression, and CRISPR screen analyses with linear models. By systematically detecting and removing nonlinear confounders arising from library size at mean and variance levels, Normalisr achieves high sensitivity, specificity, speed, and generalizability across multiple scRNA-seq protocols and experimental conditions with unbiased p-value estimation. The superior scalability allows us to reconstruct robust gene regulatory networks from trans-effects of guide RNAs in large-scale single cell CRISPRi screens. On conventional scRNA-seq, Normalisr recovers gene-level co-expression networks that recapitulated known gene functions.
    DOI:  https://doi.org/10.1038/s41467-021-26682-1
  11. Nat Commun. 2021 Nov 04. 12(1): 6396
    Benchmarking pipelines for subclonal deconvolution of bulk tumour sequencing data.
    Georgette Tanner, David R Westhead, Alastair Droop, Lucy F Stead.
      Intratumour heterogeneity provides tumours with the ability to adapt and acquire treatment resistance. The development of more effective and personalised treatments for cancers, therefore, requires accurate characterisation of the clonal architecture of tumours, enabling evolutionary dynamics to be tracked. Many methods exist for achieving this from bulk tumour sequencing data, involving identifying mutations and performing subclonal deconvolution, but there is a lack of systematic benchmarking to inform researchers on which are most accurate, and how dataset characteristics impact performance. To address this, we use the most comprehensive tumour genome simulation tool available for such purposes to create 80 bulk tumour whole exome sequencing datasets of differing depths, tumour complexities, and purities, and use these to benchmark subclonal deconvolution pipelines. We conclude that i) tumour complexity does not impact accuracy, ii) increasing either purity or purity-corrected sequencing depth improves accuracy, and iii) the optimal pipeline consists of Mutect2, FACETS and PyClone-VI. We have made our benchmarking datasets publicly available for future use.
    DOI:  https://doi.org/10.1038/s41467-021-26698-7
  12. Mol Cell. 2021 Nov 01. pii: S1097-2765(21)00842-X. [Epub ahead of print]
    A multi-omic single-cell landscape of human gynecologic malignancies.
    Matthew J Regner, Kamila Wisniewska, Susana Garcia-Recio, Aatish Thennavan, Raul Mendez-Giraldez, Venkat S Malladi, Gabrielle Hawkins, Joel S Parker, Charles M Perou, Victoria L Bae-Jump, Hector L Franco.
      Deconvolution of regulatory mechanisms that drive transcriptional programs in cancer cells is key to understanding tumor biology. Herein, we present matched transcriptome (scRNA-seq) and chromatin accessibility (scATAC-seq) profiles at single-cell resolution from human ovarian and endometrial tumors processed immediately following surgical resection. This dataset reveals the complex cellular heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression. We show that malignant cells acquire previously unannotated regulatory elements to drive hallmark cancer pathways. Moreover, malignant cells from within the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intratumoral heterogeneity. Finally, we infer the malignant cell type-specific activity of transcription factors. By defining the regulatory logic of cancer cells, this work reveals an important reliance on oncogenic regulatory elements and highlights the ability of matched scRNA-seq/scATAC-seq to uncover clinically relevant mechanisms of tumorigenesis in gynecologic cancers.
    Keywords:  chromatin accessibility; endometrial cancer; enhancer elements; gastro-intestinal stromal tumors; gene regulation; intratumoral heterogeneity; ovarian cancer; scATAC-seq; scRNA-seq; single-cell genomics
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.013
  13. PLoS Comput Biol. 2021 Nov 01. 17(11): e1009543
    From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control.
    Dharsan K Soundarrajan, Francisco J Huizar, Ramezan Paravitorghabeh, Trent Robinett, Jeremiah J Zartman.
      Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. We term these two subpopulations "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. We found that the type and strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. Our computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+ signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009543
  14. Nat Genet. 2021 Nov;53(11): 1597-1605
    Synonymous mutations reveal genome-wide levels of positive selection in healthy tissues.
    Gladys Y P Poon, Caroline J Watson, Daniel S Fisher, Jamie R Blundell.
      Genetic alterations under positive selection in healthy tissues have implications for cancer risk. However, total levels of positive selection across the genome remain unknown. Passenger mutations are influenced by all driver mutations, regardless of type or location in the genome. Therefore, the total number of passengers can be used to estimate the total number of drivers-including unidentified drivers outside of cancer genes that are traditionally missed. Here we analyze the variant allele frequency spectrum of synonymous mutations from healthy blood and esophagus to quantify levels of missing positive selection. In blood, we find that only 30% of passengers can be explained by single-nucleotide variants in driver genes, suggesting high levels of positive selection for mutations elsewhere in the genome. In contrast, more than half of all passengers in the esophagus can be explained by just the two driver genes NOTCH1 and TP53, suggesting little positive selection elsewhere.
    DOI:  https://doi.org/10.1038/s41588-021-00957-1
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