bims-pideca 2023-02-05 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2023–02–05
27 papers selected by
Ralitsa Radostinova Madsen, University College London

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis.
Updates on Diagnosis and Treatment of PIK3CA-Related Overgrowth Spectrum.
Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.
AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation.
FOXO Nuclear Shuttling Dynamics are Stimulus Dependent and Correspond with Cell Fate.
Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells.
A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.
Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.
Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.
The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation.
Targeting pleckstrin-2-Akt signaling reduces proliferation in myeloproliferative neoplasm models.
Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.
Biologically informed deep learning to query gene programs in single-cell atlases.
Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.
Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.
Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines.
A cell fate reprogramming strategy reverses epithelial-to-mesenchymal transition of lung cancer cells while avoiding hybrid states.
Slow TCA flux and ATP production in primary solid tumours but not metastases.
Cost-effective DNA methylation profiling by FML-seq.
Models that matter.
Regulation of mTOR by phosphatidic acid.
Genomic characterisation of hormone receptor-positive breast cancer arising in very young women.
Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC.
mTORC1 syndrome (TorS): unified paradigm for diabetes/metabolic syndrome.
MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis.
Arf5 mediated regulation of mTORC1 at the plasma membrane.
O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

bioRxiv. 2023 Jan 02. pii: 2022.12.31.522383. [Epub ahead of print]

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis.

Hideaki T Matsubayashi, Jack Mountain, Tony Yao, Amy F Peterson, Abhijit Deb Roy, Takanari Inoue.

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable multifaceted roles, the catalytic subunit p110 utilizes a multi- domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, their product PI(3,4,5)P 3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and its relationship to PI(3,4,5)P 3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains previously uncharacterized AP-2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP-2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and upregulate both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non- catalytic, arising distinctly from juxtaposed regions within iSH2.

DOI: https://doi.org/10.1101/2022.12.31.522383
Ann Plast Surg. 2022 Dec 22.

Updates on Diagnosis and Treatment of PIK3CA-Related Overgrowth Spectrum.

Hongrui Chen, Wei Gao, Hongyuan Liu, Bin Sun, Chen Hua, Xiaoxi Lin.

ABSTRACT: Hyperactivation of the PI3K/AKT/mTOR signaling pathway caused by PIK3CA mutations is associated with a category of overgrowth syndromes that are defined as PIK3CA-related overgrowth spectrum (PROS). The clinical features of PROS are highly heterogeneous and usually present as vascular malformations, bone and soft tissue overgrowth, and neurological and visceral abnormalities. Detection of PIK3CA variants is necessary for diagnosis and provides the basis for targeted therapy for PROS. Drugs that inhibit the PI3K pathway offer alternatives to conventional therapies. This article reviews the current knowledge of PROS and summarizes the latest progress in precise treatment, providing new insights into future therapies and research goals.

DOI: https://doi.org/10.1097/SAP.0000000000003389
Mol Cell Biol. 2023 Jan;43(1): 1-21

Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.

Chi-Chun Yang, Hisao Masai.

  Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

Keywords:  Claspin; PDK1; PI3 kinase; mTOR; serum stimulation

DOI:  https://doi.org/10.1080/10985549.2022.2160598
Nat Metab. 2023 Feb 02.

AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation.

Xiaoming Dai, Cong Jiang, Qiwei Jiang, Lan Fang, Haihong Yu, Jinhe Guo, Peiqiang Yan, Fangtao Chi, Tao Zhang, Hiroyuki Inuzuka, John M Asara, Ping Wang, Jianping Guo, Wenyi Wei.

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth in response to amino acid and glucose levels. However, how mTORC1 senses glucose availability to regulate various downstream signalling pathways remains largely elusive. Here we report that AMP-activated protein kinase (AMPK)-mediated phosphorylation of WDR24, a core component of the GATOR2 complex, has a role in the glucose-sensing capability of mTORC1. Mechanistically, glucose deprivation activates AMPK, which directly phosphorylates WDR24 on S155, subsequently disrupting the integrity of the GATOR2 complex to suppress mTORC1 activation. Phosphomimetic Wdr24S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity. On the other hand, compared to wild-type littermates, phospho-deficient Wdr24S155A knock-in mice are more resistant to fasting and display elevated mTORC1 activity. Our findings reveal that AMPK-mediated phosphorylation of WDR24 modulates glucose-induced mTORC1 activation, thereby providing a rationale for targeting AMPK-WDR24 signalling to fine-tune mTORC1 activation as a potential therapeutic means to combat human diseases with aberrant activation of mTORC1 signalling including cancer.

DOI: https://doi.org/10.1038/s42255-022-00732-4
Mol Biol Cell. 2023 Feb 03. mbcE22050193

FOXO Nuclear Shuttling Dynamics are Stimulus Dependent and Correspond with Cell Fate.

Kathleen A Lasick, Elizabeth Jose, Allison M Samayoa, Lisa Shanks, Kelvin W Pond, Curtis A Thorne, Andrew L Paek.

FOXO transcription factors are regulators of cellular homeostasis linked to increased lifespan and tumor suppression. FOXOs are activated by diverse cell stresses including serum starvation and oxidative stress. FOXO activity is regulated through post-translational modifications that control shuttling of FOXO proteins to the nucleus. In the nucleus, FOXOs upregulate genes in multiple, often conflicting pathways including cell-cycle arrest and apoptosis. How cells control FOXO activity to ensure the proper response for a given stress is an open question. Using quantitative immunofluorescence and live-cell imaging we found that the dynamics of FOXO nuclear shuttling are stimulus dependent and correspond with cell fate. H2O2 treatment leads to an all-or-none response where some cells show no nuclear FOXO accumulation, while other cells show strong nuclear FOXO signal. The time that FOXO remains in the nucleus increases with dose and is linked with cell death. In contrast, serum starvation causes low amplitude pulses of nuclear FOXO and predominantly results in cell-cycle arrest. The accumulation of FOXO in the nucleus is linked with low AKT activity for both H2O2 and serum starvation. Our findings suggest the dynamics of FOXO nuclear shuttling is one way in which the FOXO pathway dictates different cellular outcomes. [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E22-05-0193
bioRxiv. 2023 Jan 03. pii: 2023.01.03.522657. [Epub ahead of print]

Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells.

Oriana Y Teran, Matthew R Zanotelli, Miao-Chong Joy Lin, Richard A Cerione, Kristin F Wilson.

Cancer cells, both within a developing tumor and during metastatic spread, encounter many stresses that require adaptive mechanisms to survive and maintain malignant progression. Here we describe a signaling complex involving the small GTPase Cdc42 and Dock7, a Cdc42/Rac GEF and unique Cdc42-effector, that has a previously unappreciated role in regulating AKT, mTOR, and other mTOR signaling and regulatory partners including the TSC1/TCS2 complex and S6K during serum starvation. Dock7 is highly expressed in triple-negative breast cancers and is essential for the malignant properties in nutrient-deprived growth conditions of several cancer cell lines. We find that Dock7 interacts with phosphorylated AKT to maintain a low, but critical activation of a rapamycin-sensitive and Raptor-independent mTORC1-like activity required for survival during nutrient stress. Following the knock-out of Dock7 from cancer cells, interactions between AKT and the phosphatase PHLPP increased while phosphorylation of AKT at Ser473 decreased, suggesting Dock7 protects AKT from dephosphorylation. The DHR1 domain of Dock7, previously of unknown function, is responsible for maintaining AKT Ser473 phosphorylation during serum starvation through an interaction requiring its C2-like motif. Together, these findings indicate that Dock7 protects and maintains the phosphorylation of AKT to sustain a tonic mTOR/S6K activity in cancer cells necessary for their resistance to anoikis and to prevent them from undergoing apoptosis during stressful conditions.

DOI: https://doi.org/10.1101/2023.01.03.522657
PLoS Genet. 2023 Feb 02. 19(2): e1010619

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.

Deborah D Tsao, Kathleen R Chang, Lutz Kockel, Sangbin Park, Seung K Kim.

Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses.

DOI: https://doi.org/10.1371/journal.pgen.1010619
Angiogenesis. 2023 Jan 31.

Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.

Ran Huo, Yingxi Yang, Yingfan Sun, Qiuxia Zhou, Shaozhi Zhao, Zongchao Mo, Hongyuan Xu, Jie Wang, Jiancong Weng, Yuming Jiao, Junze Zhang, Qiheng He, Shuo Wang, Jizong Zhao, Jiguang Wang, Yong Cao.

  Cerebral cavernous malformations (CCMs) refer to a common vascular abnormality that affects up to 0.5% of the population. A somatic gain-of-function mutation in MAP3K3 (p.I441M) was recently reported in sporadic CCMs, frequently accompanied by somatic activating PIK3CA mutations in diseased endothelium. However, the molecular mechanisms of these driver genes remain elusive. In this study, we performed whole-exome sequencing and droplet digital polymerase chain reaction to analyze CCM lesions and the matched blood from sporadic patients. 44 of 94 cases harbored mutations in KRIT1/CCM2 or MAP3K3, of which 75% were accompanied by PIK3CA mutations (P = 0.006). AAV-BR1-mediated brain endothelial-specific MAP3K3I441M overexpression induced CCM-like lesions throughout the brain and spinal cord in adolescent mice. Interestingly, over half of lesions disappeared at adulthood. Single-cell RNA sequencing found significant enrichment of the apoptosis pathway in a subset of brain endothelial cells in MAP3K3I441M mice compared to controls. We then demonstrated that MAP3K3I441M overexpression activated p38 signaling that is associated with the apoptosis of endothelial cells in vitro and in vivo. In contrast, the mice simultaneously overexpressing PIK3CA and MAP3K3 mutations had an increased number of CCM-like lesions and maintained these lesions for a longer time compared to those with only MAP3K3I441M. Further in vitro and in vivo experiments showed that activating PI3K signaling increased proliferation and alleviated apoptosis of endothelial cells. By using AAV-BR1, we found that MAP3K3I441M mutation can provoke CCM-like lesions in mice and the activation of PI3K signaling significantly enhances and maintains these lesions, providing a preclinical model for the further mechanistic and therapeutic study of CCMs.

Keywords:  Cerebral Cavernous Malformations; MAP3K3 p.I441M; Mouse model; PIK3CA mutation

DOI:  https://doi.org/10.1007/s10456-023-09866-9
Sci Rep. 2023 Feb 02. 13(1): 1889

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.

Mohamed Ismail, Stephen R Martin, Roger George, Francesca Houghton, Geoff Kelly, Raphaël A G Chaleil, Panayiotis Anastasiou, Xinyue Wang, Nicola O'Reilly, Stefania Federico, Dhira Joshi, Hemavathi Nagaraj, Rachel Cooley, Ning Sze Hui, Miriam Molina-Arcas, David C Hancock, Ali Tavassoli, Julian Downward.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110α-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a "split-intein cyclisation of peptides and proteins" (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110α-RBD (Kd about 360 µM). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110α-RBD in the low µM (Kd 3 µM). We show that cyclo-CRVLIR binds selectively to the p110α-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110α/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.

DOI: https://doi.org/10.1038/s41598-023-28756-0
bioRxiv. 2023 Jan 03. pii: 2023.01.03.522612. [Epub ahead of print]

The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation.

Rabina Shrestha, Tess McCann, Harini Saravanan, Jaret Lieberth, Prashanna Koirala, Joshua Bloomekatz.

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move towards the midline (cardiac fusion) to form the primitive heart tube. Along with extrinsic influences such as the adjacent anterior endoderm which are known to be required for cardiac fusion, we previously showed that the platelet-derived growth factor receptor alpha (Pdgfra) is also required. However, an intrinsic mechanism that regulates myocardial movement remains to be elucidated. Here, we uncover an essential intrinsic role in the myocardium for the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway in directing myocardial movement towards the midline. In vivo imaging reveals that in PI3K-inhibited zebrafish embryos myocardial movements are misdirected and slower, while midline-oriented dynamic myocardial membrane protrusions become unpolarized. Moreover, PI3K activity is dependent on and genetically interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.

DOI: https://doi.org/10.1101/2023.01.03.522612
J Clin Invest. 2023 Jan 31. pii: e159638. [Epub ahead of print]

Targeting pleckstrin-2-Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Xu Han, Yang Mei, Rama K Mishra, Honghao Bi, Atul D Jain, Gary E Schiltz, Baobing Zhao, Madina Sukhanova, Pan Wang, Arabela A Grigorescu, Patricia C Weber, John J Piwinski, Miguel A Prado, Joao A Paulo, Len Stephens, Karen E Anderson, Charles S Abrams, Jing Yang, Peng Ji.

  Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2-STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2-STAT pathway and overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2 mutated MPNs. The non-essential roles of Plek2 under physiologic conditions makes it an ideal target for MPN therapy. Here we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening and cell-based assays followed by the synthesis of analogs. The Plek2 specific small molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2 signaling complex also includes Hsp72 that protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to Plek2 DEP domain. The role of Plek2 in activating the Akt signaling was further confirmed in vivo using a hematopoietic specific Pten knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34 positive cells from MPN patients. Our studies reveal a Plek2-Akt complex that drives cell proliferation and can be targeted by a new class of anti-proliferative compounds for MPN therapy.

Keywords:  Bone marrow; Cancer; Drug screens; Hematology; Oncology

DOI:  https://doi.org/10.1172/JCI159638
Nat Immunol. 2023 Feb 02.

Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.

Joy Edwards-Hicks, Petya Apostolova, Joerg M Buescher, Hannes Maib, Michal A Stanczak, Mauro Corrado, Ramon I Klein Geltink, Maria Elena Maccari, Matteo Villa, Gustavo E Carrizo, David E Sanin, Francesc Baixauli, Beth Kelly, Jonathan D Curtis, Fabian Haessler, Annette Patterson, Cameron S Field, George Caputa, Ryan L Kyle, Melanie Soballa, Minsun Cha, Harry Paul, Jacob Martin, Katarzyna M Grzes, Lea Flachsmann, Michael Mitterer, Liang Zhao, Frances Winkler, David Ali Rafei-Shamsabadi, Frank Meiss, Bertram Bengsch, Robert Zeiser, Daniel J Puleston, David O'Sullivan, Edward J Pearce, Erika L Pearce.

How lipidome changes support CD8+ effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPn with 3-4 double bonds), Teff cells have unique PIPn marked by saturated fatty acyl chains (0-2 double bonds). PIPn are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teff cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2 into downstream mediators, waned, and saturated PIPn became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2 with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPn impaired Teff cell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPn synthesis, and was rapidly utilized for saturated PIP2 generation. Thus, separate PIPn pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.

DOI: https://doi.org/10.1038/s41590-023-01419-y
Nat Cell Biol. 2023 Feb 02.

Biologically informed deep learning to query gene programs in single-cell atlases.

Mohammad Lotfollahi, Sergei Rybakov, Karin Hrovatin, Soroor Hediyeh-Zadeh, Carlos Talavera-López, Alexander V Misharin, Fabian J Theis.

The increasing availability of large-scale single-cell atlases has enabled the detailed description of cell states. In parallel, advances in deep learning allow rapid analysis of newly generated query datasets by mapping them into reference atlases. However, existing data transformations learned to map query data are not easily explainable using biologically known concepts such as genes or pathways. Here we propose expiMap, a biologically informed deep-learning architecture that enables single-cell reference mapping. ExpiMap learns to map cells into biologically understandable components representing known 'gene programs'. The activity of each cell for a gene program is learned while simultaneously refining them and learning de novo programs. We show that expiMap compares favourably to existing methods while bringing an additional layer of interpretability to integrative single-cell analysis. Furthermore, we demonstrate its applicability to analyse single-cell perturbation responses in different tissues and species and resolve responses of patients who have coronavirus disease 2019 to different treatments across cell types.

DOI: https://doi.org/10.1038/s41556-022-01072-x
bioRxiv. 2023 Jan 06. pii: 2023.01.05.522335. [Epub ahead of print]

Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Alexis Chang, Mario Leutert, Ricard A Rodriguez-Mias, Judit Vill N.

Phosphotyrosine (pY) enrichment is critical for expanding fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY handles 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic and tyrosine specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

DOI: https://doi.org/10.1101/2023.01.05.522335
Mol Cell. 2023 Jan 31. pii: S1097-2765(23)00038-2. [Epub ahead of print]

Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.

Martin Golkowski, Andrea Lius, Tanmay Sapre, Ho-Tak Lau, Taylor Moreno, Dustin J Maly, Shao-En Ong.

  Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease.

Keywords:  kinome; native complex; protein interactions; proteomics; signaling pathways

DOI:  https://doi.org/10.1016/j.molcel.2023.01.015
iScience. 2023 Feb 17. 26(2): 105931

Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines.

Thomas Sevrin, Lisa Strasser, Camille Ternet, Philipp Junk, Miriam Caffarini, Stella Prins, Cian D'Arcy, Simona Catozzi, Giorgio Oliviero, Kieran Wynne, Christina Kiel, Philip J Luthert.

  Cellular utilization of available energy flows to drive a multitude of forms of cellular "work" is a major biological constraint. Cells steer metabolism to address changing phenotypic states but little is known as to how bioenergetics couples to the richness of processes in a cell as a whole. Here, we outline a whole-cell energy framework that is informed by proteomic analysis and an energetics-based gene ontology. We separate analysis of metabolic supply and the capacity to generate high-energy phosphates from a representation of demand that is built on the relative abundance of ATPases and GTPases that deliver cellular work. We employed mouse embryonic fibroblast cell lines that express wild-type KRAS or oncogenic mutations and with distinct phenotypes. We observe shifts between energy-requiring processes. Calibrating against Seahorse analysis, we have created a whole-cell energy budget with apparent predictive power, for instance in relation to protein synthesis.

Keywords:  Cellular physiology; Protein

DOI:  https://doi.org/10.1016/j.isci.2023.105931
Cancer Res. 2023 Sep 13. pii: CAN-22-1559. [Epub ahead of print]

A cell fate reprogramming strategy reverses epithelial-to-mesenchymal transition of lung cancer cells while avoiding hybrid states.

Namhee Kim, Chae Young Hwang, Taeyoung Kim, Hyunjin Kim, Kwang-Hyun Cho.

The epithelial-to-mesenchymal transition (EMT) of primary cancer contributes to the acquisition of lethal properties, including metastasis and drug resistance. Blocking or reversing EMT could be an effective strategy to improve cancer treatment. However, it is still unclear how to achieve complete EMT reversal (rEMT) as cancer cells often transition to hybrid EMT states with high metastatic potential. To tackle this problem, we employed a systems biology approach and identified a core-regulatory circuit that plays the primary role in driving rEMT without hybrid properties. Perturbation of any single-node was not sufficient to completely revert EMT. Inhibition of both SMAD4 and ERK signaling along with p53 activation could induce rEMT in cancer cells even with TGF-β stimulation, a primary inducer of EMT. Induction of rEMT in lung cancer cells with the triple combination approach restored chemosensitivity. This cell-fate reprogramming strategy based on attractor landscapes revealed potential therapeutic targets that can eradicate metastatic potential by subverting EMT while avoiding hybrid states.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-1559
Nature. 2023 Feb 01.

Slow TCA flux and ATP production in primary solid tumours but not metastases.

Caroline R Bartman, Daniel R Weilandt, Yihui Shen, Won Dong Lee, Yujiao Han, Tara TeSlaa, Connor S R Jankowski, Laith Samarah, Noel R Park, Victoria da Silva-Diz, Maya Aleksandrova, Yetis Gultekin, Argit Marishta, Lin Wang, Lifeng Yang, Asael Roichman, Vrushank Bhatt, Taijin Lan, Zhixian Hu, Xi Xing, Wenyun Lu, Shawn Davidson, Martin Wühr, Matthew G Vander Heiden, Daniel Herranz, Jessie Yanxiang Guo, Yibin Kang, Joshua D Rabinowitz.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

DOI: https://doi.org/10.1038/s41586-022-05661-6
bioRxiv. 2023 Jan 14. pii: 2023.01.13.523849. [Epub ahead of print]

Cost-effective DNA methylation profiling by FML-seq.

Joseph W Foley, Shirley X Zhu, Robert B West.

Current methods for profiling DNA methylation require costly reagents, sequencing, or labor time. We introduce FML-seq, a sequencing library protocol that greatly reduces all these costs. Relative to other techniques tested on the same human cell lines, FML-seq produces similar measurements of absolute and differential cytosine methylation at a fraction of the price. FML-seq enables inexpensive, high-throughput experimental designs for large-scale epigenetics research projects.

DOI: https://doi.org/10.1101/2023.01.13.523849
Mol Cell. 2023 Feb 02. pii: S1097-2765(23)00001-1. [Epub ahead of print]83(3): 315-316

Models that matter.

Molecular Cell editorial team

Models, like the Central Dogma, are a core feature of many papers we publish, yet their utility can be undermined when they are either overly simplistic or overly complicated, and it can contribute to misunderstandings when key details or limitations are left out. In this editorial, we connect different versions of the Central Dogma and how we think about scientific models more generally.

DOI: https://doi.org/10.1016/j.molcel.2023.01.001
Trends Endocrinol Metab. 2023 Jan 31. pii: S1043-2760(23)00015-2. [Epub ahead of print]

Regulation of mTOR by phosphatidic acid.

Maria A Frias, Ahmet Hatipoglu, David A Foster.

  mTORC1, the mammalian target of rapamycin complex 1, is a key regulator of cellular physiology. The lipid metabolite phosphatidic acid (PA) binds to and activates mTORC1 in response to nutrients and growth factors. We review structural findings and propose a model for PA activation of mTORC1. PA binds to a highly conserved sequence in the α4 helix of the FK506 binding protein 12 (FKBP12)/rapamycin-binding (FRB) domain of mTOR. It is proposed that PA binding to two adjacent positively charged amino acids breaks and shortens the C-terminal region of helix α4. This has profound consequences for both substrate binding and the catalytic activity of mTORC1.

Keywords:  FRB; Rheb; mTOR; phosphatidic acid; phospholipase D; structure

DOI:  https://doi.org/10.1016/j.tem.2023.01.004
Ann Oncol. 2023 Jan 25. pii: S0923-7534(23)00047-9. [Epub ahead of print]

Genomic characterisation of hormone receptor-positive breast cancer arising in very young women.

S J Luen, G Viale, S Nik-Zainal, P Savas, R Kammler, P Dell'Orto, O Biasi, A Degasperi, L C Brown, I Láng, G MacGrogan, C Tondini, M Bellet, F Villa, A Bernardo, E Ciruelos, P Karlsson, P Neven, M Climent, B Müller, W Joshum, H Bonnefoi, S Martino, N E Davidson, C Geyer, S K Chia, J N Ingle, R Coleman, C Solbach, B Thürlimann, M Colleoni, A S Coates, A Goldhirsch, G F Fleming, P A Francis, T P Speed, M M Regan, S Loi.

   BACKGROUND: Very young premenopausal women diagnosed with hormone receptor-positive, HER2-negative (HR+HER2-) early breast cancer (EBC) have higher rates of recurrence and death for reasons that remain largely unexplained.
PATIENTS AND METHODS: Genomic sequencing was applied to HR+HER2- tumours from patients enrolled in the SOFT clinical trial to determine genomic drivers that are enriched in young premenopausal women. Genomic alterations were characterised using next-generation sequencing from a subset of 1,276 patients (deep targeted sequencing, N=1258; whole-exome sequencing in a young-age, case-control subsample, N=82). We defined copy number (CN) subgroups and assessed for features suggestive of homologous recombination deficiency (HRD). Genomic alteration frequencies were compared between young premenopausal women (<40 years) and older premenopausal women (≥40 years), and assessed for associations with distant recurrence-free interval (DRFI), and overall survival (OS).
RESULTS: Younger women (<40 years, N=359) compared with older women (≥40 years, N=917) had significantly higher frequencies of mutations in GATA3 (19%vs16%) and CN-amplifications (47%vs26%), but significantly lower frequencies of mutations in PIK3CA (32%vs47%), CDH1 (3%vs9%), and MAP3K1 (7%vs12%). Additionally, significantly higher frequencies of features suggestive of HRD (27%vs21%), and a higher proportion of PIK3CA mutations with concurrent CN-amplifications (23%vs11%).Genomic features suggestive of HRD, PIK3CA mutations with CN-amplifications, and CN-amplifications associated with significantly worse DRFI and OS compared with those without these features. These poor prognostic features were enriched in younger patients: present in 72% of patients aged <35 years, 54% aged 35-39 years, and 40% ≥40 years. Poor prognostic features (N=584[46%]) vs none (N=692[54%]) had an 8-year DRFI of 84%vs94% and OS of 88%vs96%. Younger women (<40) had the poorest outcomes: 8-year DRFI 74%vs85% and OS of 80%vs93% respectively.
CONCLUSION: These results provide insights into genomic alterations that are enriched in young women with HR+HER2-EBC, provide rationale for genomic subgrouping, and highlight priority molecular targets for future clinical trials.

Keywords:  Breast cancer; genomics; hormone receptor positive; prognosis; young women

DOI:  https://doi.org/10.1016/j.annonc.2023.01.009
Sci Rep. 2023 Jan 31. 13(1): 1719

Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC.

Sameer Singh, Anoosha Banerjee, Arnaud Vanden Broeck, Sebastian Klinge.

One of the challenges faced by current CRISPR/Cas9 editing strategies is the difficulty in rapidly selecting clonal populations of biallelically edited cells. Here we present Surface engiNeered fluorEscence Assisted Kit with Protein Epitope Enhanced Capture (SNEAK PEEC), a platform that combines human genome editing with cell-surface display, which enables the direct identification of biallelically edited clones with minimal screening.

DOI: https://doi.org/10.1038/s41598-023-28732-8
Trends Endocrinol Metab. 2023 Jan 28. pii: S1043-2760(23)00012-7. [Epub ahead of print]

mTORC1 syndrome (TorS): unified paradigm for diabetes/metabolic syndrome.

Jacob Bar-Tana.

  'Glucolipotoxicity' and 'insulin resistance' are claimed to drive type 2 diabetes (T2D) and the non-glycemic diseases of the metabolic syndrome (MetS) (obesity, dyslipidemia, hypertension). In line with that, glycemic and/or insulin control are considered to be primary goal in treating T2D/MetS. However, recent standard-of-care (SOC) treatments of T2D, initially designed to control T2D hyperglycemia, appear now to alleviate the cardio-renal and non-glycemic diseases of T2D/MetS independently of glucose lowering and insulin resistance, and in non-T2D patients altogether, calling for an alternative unifying pathophysiology/treatment paradigm for T2D/MetS. This opinion article proposes to replace the current 'glucolipotoxic/insulin-resistance' paradigm of T2D/MetS with an 'mammalian target of rapamycin complex 1 (mTORC1) syndrome' (TorS) paradigm, implying an exhaustive cohesive disease entity driven by an upstream hyperactive mTORC1, and which includes diabetic hyperglycemia, diabetic dyslipidemia, hypertension, diabetic macrovascular and microvascular disease, non-alcoholic fatty liver disease, some cancers, neurodegeneration, polycystic ovary syndrome (PCOS), psoriasis, and others. The TorS paradigm may account for the insulin-resistant glycemic context of TorS, combined with response to insulin of the non-glycemic diseases of TorS. The TorS paradigm may account for the efficacy of current antidiabetic SOC treatments in diabetic and nondiabetic patients. Most importantly, the TorS paradigm may generate novel treatments for TorS.

Keywords:  diabetes; mTORC1; metabolic syndrome

DOI:  https://doi.org/10.1016/j.tem.2023.01.001
bioRxiv. 2023 Jan 04. pii: 2023.01.03.522637. [Epub ahead of print]

MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis.

Rachel E DeRollo, Juliana Cazarin, Siti Noor Ain Binti Ahmad Shahidan, Jamison B Burchett, Daniel Mwangi, Saikumari Krishnaiah, Annie L Hsieh, Zandra E Walton, Rebekah Brooks, Stephano S Mello, Aalim M Weljie, Chi V Dang, Brian J Altman.

The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites, is disrupted across many human cancers. Deregulated expression of 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 suppresses oscillation of gene expression and metabolism to instead upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC or the closely related N-MYC were examined, using detailed time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression, programs, 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 glycosylation 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.

DOI: https://doi.org/10.1101/2023.01.03.522637
Mol Biol Cell. 2023 Feb 03. mbcE22070302

Arf5 mediated regulation of mTORC1 at the plasma membrane.

Christian Makhoul, Fiona J Houghton, Elizabeth Hinde, Paul A Gleeson.

The mechanistic target of rapamycin (mTOR) kinase regulates a major signalling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localised at other locations. However, little is known about the recruitment and activation of mTORC1 at non-lysosomal sites. To identify regulators of mTORC1 recruitment to non-lysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signalling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2 enriched membrane ruffles together the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor and Arf5 shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and re-feeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

DOI: https://doi.org/10.1091/mbc.E22-07-0302
J Biol Chem. 2023 Jan 30. pii: S0021-9258(23)00095-9. [Epub ahead of print] 102963

O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

Sadia Rahmani, Hafsa Ahmed, Osemudiamen Ibazebo, Eden Fussner-Dupas, Warren W Wakarchuk, Costin N Antonescu.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

DOI: https://doi.org/10.1016/j.jbc.2023.102963