bims-pideca 2022-08-28 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2022–08–28
27 papers selected by
Ralitsa Radostinova Madsen, University College London

Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade.
Identifying mutant-specific multi-drug combinations using comparative network reconstruction.
Functional testing to characterize and stratify PI3K inhibitor responses in chronic lymphocytic leukemia.
The adipocyte supersystem of insulin and cAMP signaling.
ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance.
Determining drug dose in the era of targeted therapies: playing it (un)safe?
Targeting Akt/PKB in pediatric tumors: A review from preclinical to clinical trials.
A challenging diagnosis of the PIK3CA-related overgrowth spectrum.
Design, Synthesis, and Development of pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part I-Indole Derivatives.
High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.
Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II-Benzimidazole Derivatives.
Midkine expression by stem-like tumor cells drives persistence to mTOR inhibition and an immune-suppressive microenvironment.
Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.
Protocol for stratification of triple-negative breast cancer patients using in silico signaling dynamics.
A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.
The ClinGen Brain Malformation Variant Curation Expert Panel: Rules for somatic variants in AKT3, MTOR, PIK3CA, and PIK3R2.
Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.
Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters.
mBeRFP: a versatile fluorescent tool to enhance multichannel live imaging and its applications.
RING-finger E3 ligases regulatory network in PI3K/AKT-mediated glucose metabolism.
Real-life efficacy and safety of idelalisib in 55 double-refractory follicular lymphoma patients.
PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt-β-catenin signaling.
Long non-coding RNA SNHG6 couples cholesterol sensing with mTORC1 activation in hepatocellular carcinoma.
A human model of arteriovenous malformation (AVM)-on-a-chip reproduces key disease hallmarks and enables drug testing in perfused human vessel networks.
Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.
Mathematical modeling reveals differential dynamics of insulin action models on glycerol and glucose in adolescent girls with obesity.
Increasing cell size remodels the proteome and promotes senescence.

Nucleic Acids Res. 2022 Aug 22. pii: gkac704. [Epub ahead of print]

Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade.

Hsin-Wei Tseng, Anthony Mota-Sydor, Rania Leventis, Predrag Jovanovic, Ivan Topisirovic, Thomas F Duchaine.

Precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3'UTR. We previously reported the important diversity of 3'UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

DOI: https://doi.org/10.1093/nar/gkac704
iScience. 2022 Aug 19. 25(8): 104760

Identifying mutant-specific multi-drug combinations using comparative network reconstruction.

Evert Bosdriesz, João M Fernandes Neto, Anja Sieber, René Bernards, Nils Blüthgen, Lodewyk F A Wessels.

  Targeted inhibition of aberrant signaling is an important treatment strategy in cancer, but responses are often short-lived. Multi-drug combinations have the potential to mitigate this, but to avoid toxicity such combinations must be selective and given at low dosages. Here, we present a pipeline to identify promising multi-drug combinations. We perturbed an isogenic PI3K mutant and wild-type cell line pair with a limited set of drugs and recorded their signaling state and cell viability. We then reconstructed their signaling networks and mapped the signaling response to changes in cell viability. The resulting models, which allowed us to predict the effect of unseen combinations, indicated that no combination selectively reduces the viability of the PI3K mutant cells. However, we were able to validate 25 of the 30 combinations that we predicted to be anti-selective. Our pipeline enables efficient prioritization of multi-drug combinations from the enormous search space of possible combinations.

Keywords:  Bioinformatics; In silico biology; Pharmacoinformatics; Systems biology

DOI:  https://doi.org/10.1016/j.isci.2022.104760
Clin Cancer Res. 2022 Aug 23. pii: CCR-22-1221. [Epub ahead of print]

Functional testing to characterize and stratify PI3K inhibitor responses in chronic lymphocytic leukemia.

Yanping Yin, Paschalis Athanasiadis, Linda Karlsen, Aleksandra Urban, Haifeng Xu, Ishwarya Murali, Stacey M Fernandes, Alberto J Arribas, Abdul K Hilli, Kjetil Taskén, Francesco Bertoni, Anthony R Mato, Emmanuel Normant, Jennifer R Brown, Geir E Tjønnfjord, Tero Aittokallio, Sigrid S Skånland.

PURPOSE: Phosphatidylinositol 3-kinase inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response to these therapies, development of treatment intolerance or resistance remains clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of ten PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki.
EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell (PBMC) samples (n=51 in total) from treatment naïve and PI3Ki-treated CLL patients were studied. Cells were profiled against ten PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.
RESULTS: pan-PI3Ki were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110d inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively.
CONCLUSIONS: Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

DOI: https://doi.org/10.1158/1078-0432.CCR-22-1221
Trends Cell Biol. 2022 Aug 18. pii: S0962-8924(22)00190-8. [Epub ahead of print]

The adipocyte supersystem of insulin and cAMP signaling.

Adilson Guilherme, Leslie A Rowland, Hui Wang, Michael P Czech.

  Adipose tissue signals to brain, liver, and muscles to control whole body metabolism through secreted lipid and protein factors as well as neurotransmission, but the mechanisms involved are incompletely understood. Adipocytes sequester triglyceride (TG) in fed conditions stimulated by insulin, while in fasting catecholamines trigger TG hydrolysis, releasing glycerol and fatty acids (FAs). These antagonistic hormone actions result in part from insulin's ability to inhibit cAMP levels generated through such G-protein-coupled receptors as catecholamine-activated β-adrenergic receptors. Consistent with these antagonistic signaling modes, acute actions of catecholamines cause insulin resistance. Yet, paradoxically, chronically activating adipocytes by catecholamines cause increased glucose tolerance, as does insulin. Recent results have helped to unravel this conundrum by revealing enhanced complexities of these hormones' signaling networks, including identification of unexpected common signaling nodes between these canonically antagonistic hormones.

Keywords:  adipose tissue; lipogenesis; lipolysis; thermogenesis; type 2 diabetes

DOI:  https://doi.org/10.1016/j.tcb.2022.07.009
Am J Physiol Heart Circ Physiol. 2022 Aug 26.

ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance.

Thaysa Ghiarone, Jorge A Castorena-Gonzalez, Christopher A Foote, Francisco I Ramirez-Perez, Larissa Ferreira-Santos, Francisco J Cabral-Amador, Roger de la Torre, Rama R Ganga, Andrew A Wheeler, Camila Manrique-Acevedo, Jaume Padilla, Luis A Martinez-Lemus.

  Inflammation and vascular insulin resistance are hallmarks of type 2 diabetes (T2D). However, the specific mechanisms that cause abnormal endothelial insulin signaling in T2D remain largely unknown. Evidence indicates that the activity of ADAM17 (a disintegrin and metalloproteinase-17) and the presence of insulin receptor (IR) in plasma are increased in T2D subjects. Accordingly, we hypothesized that, in T2D, increased ADAM17 activity sheds the IR ectodomain from endothelial cells and impairs insulin-induced vasodilation. We used small visceral arteries isolated from a cross-sectional study of T2D and non-T2D subjects undergoing bariatric surgery, human cultured endothelial cells, and recombinant proteins to test our hypothesis. Here, we demonstrate that arteries from T2D subjects had increased ADAM17 expression, reduced presence of tissue inhibitor of metalloproteinase-3 (TIMP3), decreased extracellular IRα, and impaired insulin-induced vasodilation vs. those from non-T2D subjects. In vitro, active ADAM17 cleaved the ectodomain of the IRbsubunit. Endothelial cells with ADAM17 overexpression or exposed to the protein kinase-C activator, PMA, hadincreased ADAM17 activity, decreased IRα presence on the cell surface, and increased IR shedding. Moreover, pharmacological inhibition of ADAM17 with TAPI-0 rescued PMA-induced impairments in endothelial insulin signaling and insulin-stimulated vasodilation in human arteries. In aggregate, our findings suggest that ADAM17-mediated shedding of IR from the endothelial surface impairs insulin-mediated vasodilation. Thus, we propose that inhibition of ADAM17 sheddase activity should be considered a strategy to restore vascular insulin sensitivity in T2D.

Keywords:  Endothelium; cardiovascular disease; endothelial dysfunction; insulin-induced vasodilation; type 2 diabetes

DOI:  https://doi.org/10.1152/ajpheart.00039.2022
Blood Cancer J. 2022 Aug 23. 12(8): 123

Determining drug dose in the era of targeted therapies: playing it (un)safe?

Sigrid S Skånland, Geir E Tjønnfjord.

Targeted therapies against phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (BTK), and B-cell lymphoma-2 (BCL-2) are approved for chronic lymphocytic leukemia (CLL). Since approval of the first-in-class drugs, next-generation agents have become available and are continuously under development. While these therapies act on well-characterized molecular targets, this knowledge is only to some extent taken into consideration when determining their dose in phase I trials. For example, BTK occupancy has been assessed in dose-finding studies of various BTK inhibitors, but the minimum doses that result in full BTK occupancy were not determined. Although targeted agents have a different dose-response relationship than cytotoxic agents, which are more effective near the maximum tolerated dose, the traditional 3 + 3 toxicity-driven trial design remains heavily used in the era of targeted therapies. If pharmacodynamic biomarkers were more stringently used to guide dose selection, the recommended phase II dose would likely be lower as compared to the toxicity-driven selection. Reduced drug doses may lower toxicity, which in some cases is severe for these agents, and are supported by retrospective studies demonstrating non-inferior outcomes for patients with clinically indicated dose reductions. Here, we review strategies that were used for dose selection in phase I studies of currently approved and select investigational targeted therapies in CLL, and discuss how our initial clinical experience with targeted therapies have pointed to dose reductions, intermittent dosing, and drug combinations as strategies to overcome treatment intolerance and resistance.

DOI: https://doi.org/10.1038/s41408-022-00720-7
Pharmacol Res. 2022 Aug 18. pii: S1043-6618(22)00348-6. [Epub ahead of print]183 106403

Targeting Akt/PKB in pediatric tumors: A review from preclinical to clinical trials.

Bruno Toson, Isadora S Fortes, Rafael Roesler, Saulo F Andrade.

  The serine/threonine kinase Akt is a major player in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and its modulation impacts multiple cellular processes such as growth, proliferation, and survival. Several abnormalities in this pathway have been documented over the years, and these alterations were shown to have great implications in tumorigenesis and resistance to chemotherapy. Thus, multiple Akt inhibitors have been developed and tested in adult tumors, and some of them are currently undergoing phase I, II, and III clinical trials for distinct cancers that arise during adulthood. Despite that, the impact of these inhibitors is still not fully understood in pediatric tumors, and Akt-specific targeting seems to be a promising approach to treat children affected by cancers. This review summarizes recent available evidence of Akt inhibitors in pediatric cancers, from both preclinical and clinical studies. In short, we demonstrate the impact that Akt inhibition provides in tumorigenesis, and we suggest targeting the PI3K/Akt/mTOR signaling pathway, alone or in combination with other inhibitors, is a feasible tool to achieve better outcomes in pediatric tumors.

Keywords:  AZD5363 (PubChem CID 25227436); Akt inhibitor; CCT128930 (PubChem CID17751819); GDC-0068 (PubChem CID 24788740); GSK2110183 (PubChem CID 92044396); GSK2141795 (PubChem CID 51042438); GSK690693 (PubChem CID 16725726); MK-2206 (PubChem CID 24964624); PI3K/Akt/mTOR pathway; Pediatric cancer; Perifosine (PubChem CID 148177); TAS-117 (PubChem CID 66555816); Targeted therapy; Triciribine (PubChem CID 65399)

DOI:  https://doi.org/10.1016/j.phrs.2022.106403
Clin Dysmorphol. 2022 Aug 05.

A challenging diagnosis of the PIK3CA-related overgrowth spectrum.

Ataf Hussain Sabir, Alessandra Cocca, Moira Cheung, Melita Irving.

DOI: https://doi.org/10.1097/MCD.0000000000000425
Pharmaceuticals (Basel). 2022 Jul 30. pii: 949. [Epub ahead of print]15(8):

Design, Synthesis, and Development of pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part I-Indole Derivatives.

Mariola Stypik, Marcin Zagozda, Stanisław Michałek, Barbara Dymek, Daria Zdżalik-Bielecka, Maciej Dziachan, Nina Orłowska, Paweł Gunerka, Paweł Turowski, Joanna Hucz-Kalitowska, Aleksandra Stańczak, Paulina Stańczak, Krzysztof Mulewski, Damian Smuga, Filip Stefaniak, Lidia Gurba-Bryśkiewicz, Arkadiusz Leniak, Zbigniew Ochal, Mateusz Mach, Karolina Dzwonek, Monika Lamparska-Przybysz, Krzysztof Dubiel, Maciej Wieczorek.

  Phosphoinositide 3-kinase δ (PI3Kδ), a member of the class I PI3K family, is an essential signaling biomolecule that regulates the differentiation, proliferation, migration, and survival of immune cells. The overactivity of this protein causes cellular dysfunctions in many human disorders, for example, inflammatory and autoimmune diseases, including asthma or chronic obstructive pulmonary disease (COPD). In this work, we designed and synthesized a new library of small-molecule inhibitors based on indol-4-yl-pyrazolo[1,5-a]pyrimidine with IC50 values in the low nanomolar range and high selectivity against the PI3Kδ isoform. CPL302253 (54), the most potent compound of all the structures obtained, with IC50 = 2.8 nM, is a potential future candidate for clinical development as an inhaled drug to prevent asthma.

Keywords:  5-indole-pyrazolo[1,5-a]pyrimidine; Asthma; COPD; CPL302253; PI3Kδ inhibitors

DOI:  https://doi.org/10.3390/ph15080949
Nat Biotechnol. 2022 Aug 25.

High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.

Brian R Shy, Vivasvan S Vykunta, Alvin Ha, Alexis Talbot, Theodore L Roth, David N Nguyen, Wolfgang G Pfeifer, Yan Yi Chen, Franziska Blaeschke, Eric Shifrut, Shane Vedova, Murad R Mamedov, Jing-Yi Jing Chung, Hong Li, Ruby Yu, David Wu, Jeffrey Wolf, Thomas G Martin, Carlos E Castro, Lumeng Ye, Jonathan H Esensten, Justin Eyquem, Alexander Marson.

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.

DOI: https://doi.org/10.1038/s41587-022-01418-8
Pharmaceuticals (Basel). 2022 Jul 27. pii: 927. [Epub ahead of print]15(8):

Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II-Benzimidazole Derivatives.

Mariola Stypik, Stanisław Michałek, Nina Orłowska, Marcin Zagozda, Maciej Dziachan, Martyna Banach, Paweł Turowski, Paweł Gunerka, Daria Zdżalik-Bielecka, Aleksandra Stańczak, Urszula Kędzierska, Krzysztof Mulewski, Damian Smuga, Wioleta Maruszak, Lidia Gurba-Bryśkiewicz, Arkadiusz Leniak, Wojciech Pietruś, Zbigniew Ochal, Mateusz Mach, Beata Zygmunt, Jerzy Pieczykolan, Krzysztof Dubiel, Maciej Wieczorek.

  Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, β, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC50 values ranging from 1.892 to 0.018 μM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC50 value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kβ/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.

Keywords:  5-benzimidazole-pyrazolo[1,5-a]pyrimidine; CPL302415; PI3Kδ inhibitors; anti-inflammatory therapy

DOI:  https://doi.org/10.3390/ph15080927
Nat Commun. 2022 Aug 26. 13(1): 5018

Midkine expression by stem-like tumor cells drives persistence to mTOR inhibition and an immune-suppressive microenvironment.

Yan Tang, David J Kwiatkowski, Elizabeth P Henske.

mTORC1 is hyperactive in multiple cancer types1,2. Here, we performed integrative analysis of single cell transcriptomic profiling, paired T cell receptor (TCR) sequencing, and spatial transcriptomic profiling on Tuberous Sclerosis Complex (TSC) associated tumors with mTORC1 hyperactivity, and identified a stem-like tumor cell state (SLS) linked to T cell dysfunction via tumor-modulated immunosuppressive macrophages. Rapamycin and its derivatives (rapalogs) are the primary treatments for TSC tumors, and the stem-like tumor cells showed rapamycin resistance in vitro, reminiscent of the cytostatic effects of these drugs in patients. The pro-angiogenic factor midkine (MDK) was highly expressed by the SLS population, and associated with enrichment of endothelial cells in SLS-dominant samples. Inhibition of MDK showed synergistic benefit with rapamycin in reducing the growth of TSC cell lines in vitro and in vivo. In aggregate, this study suggests an autocrine rapamycin resistance mechanism and a paracrine tumor survival mechanism via immune suppression adopted by the stem-like state tumor cells with mTORC1 hyperactivity.

DOI: https://doi.org/10.1038/s41467-022-32673-7
Science. 2022 Aug 25. eabg6621

Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.

Hijai R Shin, Y Rose Citron, Lei Wang, Laura Tribouillard, Claire S Goul, Robin Stipp, Yusuke Sugasawa, Aakriti Jain, Nolwenn Samson, Chun-Yan Lim, Oliver B Davis, David Castaneda-Carpio, Mingxing Qian, Daniel K Nomura, Rushika M Perera, Eunyong Park, Douglas F Covey, Mathieu Laplante, Alex S Evers, Roberto Zoncu.

Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified LYsosomal CHOlesterol Signaling (LYCHOS, previously annotated as G-protein coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1). Cholesterol bound to the N-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a GTPase-activating protein for the Rag guanosine triphosphatases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing, and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.

DOI: https://doi.org/10.1126/science.abg6621
STAR Protoc. 2022 Sep 16. 3(3): 101619

Protocol for stratification of triple-negative breast cancer patients using in silico signaling dynamics.

Hiroaki Imoto, Sawa Yamashiro, Ken Murakami, Mariko Okada.

  Personalized kinetic models can predict potential biomarkers and drug targets. Here, we provide a step-by-step approach for building an executable mathematical model from text and integrating transcriptomic datasets. We additionally describe the steps to personalize the mechanistic model and to stratify patients with triple-negative breast cancer (TNBC) based on in silico signaling dynamics. This protocol can also be applied to any signaling pathway for patient-specific modeling. For complete details on the use and execution of this protocol, please refer to Imoto et al. (2022).

Keywords:  Bioinformatics; Cancer; Computer sciences; Gene expression; Genomics; Signal transduction; Systems biology

DOI:  https://doi.org/10.1016/j.xpro.2022.101619
Mol Cell. 2022 Aug 20. pii: S1097-2765(22)00756-0. [Epub ahead of print]

A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.

Vishnu Balaji, Leonie Müller, Robin Lorenz, Éva Kevei, William H Zhang, Ulises Santiago, Jan Gebauer, Ernesto Llamas, David Vilchez, Carlos J Camacho, Wojciech Pokrzywa, Thorsten Hoppe.

  The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

Keywords:  C. elegans; CHIP; DAF-2; E3 ligase; aging; chaperones; insulin signaling; longevity; proteostasis; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2022.08.003
Genet Med. 2022 Aug 22. pii: S1098-3600(22)00871-1. [Epub ahead of print]

The ClinGen Brain Malformation Variant Curation Expert Panel: Rules for somatic variants in AKT3, MTOR, PIK3CA, and PIK3R2.

ClinGen Brain Malformation Variant Curation Expert Panel

   PURPOSE: Postzygotic (somatic) variants in the mTOR pathway genes cause a spectrum of distinct developmental abnormalities. Accurate classification of somatic variants in this group of disorders is crucial for affected individuals and their families.
METHODS: The ClinGen Brain Malformation Variant Curation Expert Panel was formed to curate somatic variants associated with developmental brain malformations. We selected the genes AKT3, MTOR, PIK3CA, and PIK3R2 as the first set of genes to provide additional specifications to the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) sequence variant interpretation guidelines, which currently focus solely on germline variants.
RESULTS: A total of 24 of the original 28 ACMG/AMP criteria required modification. Several modifications used could be applied to other genes and disorders in which somatic variants play a role: 1) using variant allele fraction differences as evidence that somatic mutagenesis occurred as a proxy for de novo variation, 2) incorporating both somatic and germline evidence, and 3) delineating phenotype on the basis of variable tissue expression.
CONCLUSION: We have established a framework for rigorous interpretation of somatic mosaic variants, addressing issues unique to somatic variants that will be applicable to many genes and conditions.

Keywords:  AKT3; MTOR; PIK3CA; PIK3R2; Somatic mosaicism

DOI:  https://doi.org/10.1016/j.gim.2022.07.020
Cell Rep. 2022 Aug 23. pii: S2211-1247(22)01077-4. [Epub ahead of print]40(8): 111259

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

Yating Wang, Stefano Spolitu, John A Zadroga, Amesh K Sarecha, Lale Ozcan.

  Excessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a's membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Keywords:  CP: Metabolism; Rap1a; geranylgeranylation; gluconeogenesis; hepatic glucose production; hyperglycemia; obesity; statins; type 2 diabetes

DOI:  https://doi.org/10.1016/j.celrep.2022.111259
PLoS One. 2022 ;17(8): e0261801

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters.

Lucas J D'Souza, Stephen H Wright, Deepta Bhattacharya.

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by primary plasma cells into the cytoplasm suggesting a specific mechanism that is unlinked from glucose import and that of chemically similar compounds. Neither excess glucose nor pharmacological inhibition of GLUT1 impacted 2NBDG uptake in myeloma cells or primary splenocytes. Genetic ablation of other expressed hexose transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism.

DOI: https://doi.org/10.1371/journal.pone.0261801
Development. 2022 Aug 15. pii: dev200495. [Epub ahead of print]149(16):

mBeRFP: a versatile fluorescent tool to enhance multichannel live imaging and its applications.

Emmanuel Martin, Magali Suzanne.

  Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins offers interesting new perspectives to bypass this limitation. Here, we provide a detailed characterization of mBeRFP for in vivo live imaging and its applications in Drosophila. Briefly, we show that a single illumination source is sufficient to stimulate mBeRFP and GFP simultaneously. We demonstrate that mBeRFP can be easily combined with classical green and red fluorescent proteins without any crosstalk. We also show that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications, such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano-biologists in their current research.

Keywords:   Drosophila ; FRAP; Fluorescent protein; Laser ablation; Live imaging; mBeRFP

DOI:  https://doi.org/10.1242/dev.200495
Cell Death Discov. 2022 Aug 24. 8(1): 372

RING-finger E3 ligases regulatory network in PI3K/AKT-mediated glucose metabolism.

Wenke Wang, Bei Shi, Ruiting Cong, Mingjun Hao, Yuanyuan Peng, Hongyue Yang, Jiahui Song, Di Feng, Naijin Zhang, Da Li.

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway plays an essential role in glucose metabolism, promoting glycolysis and resisting gluconeogenesis. PI3K/AKT signaling can directly alter glucose metabolism by phosphorylating several metabolic enzymes or regulators of nutrient transport. It can indirectly promote sustained aerobic glycolysis by increasing glucose transporters and glycolytic enzymes, which are mediated by downstream transcription factors. E3 ubiquitin ligase RING-finger proteins are mediators of protein post-translational modifications and include the cullin-RING ligase complexes, the tumor necrosis factor receptor-associated family, the tripartite motif family and etc. Some members of the RING family play critical roles in regulating cell signaling and are involved in the development and progression of various metabolic diseases, such as cancer, diabetes, and dyslipidemia. And with the progression of modern research, as a negative or active regulator, the RING-finger adaptor has been found to play an indispensable role in PI3K/AKT signaling. However, no reviews have comprehensively clarified the role of RING-finger E3 ligases in PI3K/AKT-mediated glucose metabolism. Therefore, in this review, we focus on the regulation and function of RING ligases in PI3K/AKT-mediated glucose metabolism to establish new insights into the prevention and treatment of metabolic diseases.

DOI: https://doi.org/10.1038/s41420-022-01162-7
Br J Haematol. 2022 Aug 24.

Real-life efficacy and safety of idelalisib in 55 double-refractory follicular lymphoma patients.

Alessandro Isidori, Federica Loscocco, Giuseppe Visani, Sara Paolasini, Potito Scalzulli, Pellegrino Musto, Tommasina Perrone, Attilio Guarini, Domenico Pastore, Patrizio Mazza, Lorenzo Tonialini, Vincenzo Pavone, Gaetano De Santis, Giuseppe Tarantini.

  Idelalisib, a reversible inhibitor of PI3Kδ (phosphoinositide-3 kinase delta), showed remarkable activity in the phase II DELTA trial, leading to its approval by the European Medicines Agency (EMA) in patients with relapsed/refractory (R/R) follicular lymphoma (FL). However, real-life data on idelalisib are scarce. We treated 55 double-refractory FL patients with idelalisib in a real-life setting. With a median exposure to idelalisib of 10 months (range 1-43), overall response rate was 73%, the highest ever reported. Non-haematological toxicities were mild and manageable. At 12 months, 80% of patients were alive, and 72% disease-free. The efficacy and safety of idelalisib was confirmed in a real-life setting.

Keywords:  PI3Kδ; follicular lymphoma; idelalisib; relapsed/refractory

DOI:  https://doi.org/10.1111/bjh.18426
Sci Signal. 2022 Aug 23. 15(748): eabo2820

PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt-β-catenin signaling.

Jacob P Mahoney, Elise S Bruguera, Mansi Vasishtha, Lauren B Killingsworth, Saw Kyaw, William I Weis.

In the Wnt-β-catenin pathway, Wnt binding to Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6) co-receptors inhibits the degradation of the transcriptional coactivator β-catenin by recruiting the cytosolic effector Dishevelled (Dvl). Polymerization of Dvl at the plasma membrane recruits the β-catenin destruction complex, enabling the phosphorylation of LRP5/6, a key step in inhibiting β-catenin degradation. Using purified Fzd proteins reconstituted in lipid nanodiscs, we investigated the factors that promote the recruitment of Dvl to the plasma membrane. We found that the affinity of Fzd for Dvl was not affected by Wnt ligands, in contrast to other members of the GPCR superfamily for which the binding of extracellular ligands affects the affinity for downstream transducers. Instead, Fzd-Dvl binding was enhanced by increased concentration of the lipid PI(4,5)P2, which is generated by Dvl-associated lipid kinases in response to Wnt and which is required for LRP5/6 phosphorylation. Moreover, binding to Fzd did not promote Dvl DEP domain dimerization, which has been proposed to be required for signaling downstream of Fzd. Our findings suggest a positive feedback loop in which Wnt-stimulated local PI(4,5)P2 production enhances Dvl recruitment and further PI(4,5)P2 production to support Dvl polymerization, LRP5/6 phosphorylation, and β-catenin stabilization.

DOI: https://doi.org/10.1126/scisignal.abo2820
Nat Metab. 2022 Aug;4(8): 1022-1040

Long non-coding RNA SNHG6 couples cholesterol sensing with mTORC1 activation in hepatocellular carcinoma.

Fangzhou Liu, Tian Tian, Zhen Zhang, Shanshan Xie, Jiecheng Yang, Linyu Zhu, Wen Wang, Chengyu Shi, Lingjie Sang, Kaiqiang Guo, Zuozhen Yang, Lei Qu, Xiangrui Liu, Jian Liu, Qingfeng Yan, Huai-Qiang Ju, Wenqi Wang, Hai-Long Piao, Jianzhong Shao, Tianhua Zhou, Aifu Lin.

Cholesterol contributes to the structural basis of biological membranes and functions as a signaling molecule, whose dysregulation has been associated with various human diseases. Here, we report that the long non-coding RNA (lncRNA) SNHG6 increases progression from non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) by modulating cholesterol-induced mTORC1 activation. Mechanistically, cholesterol binds ER-anchored FAF2 protein to promote the formation of a SNHG6-FAF2-mTOR complex. As a putative cholesterol effector, SNHG6 enhances cholesterol-dependent mTORC1 lysosomal recruitment and activation via enhancing FAF2-mTOR interaction at ER-lysosome contacts, thereby coordinating mTORC1 kinase cascade activation with cellular cholesterol biosynthesis in a self-amplified cycle to accelerate cholesterol-driven NAFLD-HCC development. Notably, loss of SNHG6 inhibits mTORC1 signaling and impairs growth of patient-derived xenograft liver cancer tumors, identifyifng SNHG6 as a potential target for liver cancer treatment. Together, our findings illustrate the crucial role of organelle-associated lncRNA in organelle communication, nutrient sensing, and kinase cascades.

DOI: https://doi.org/10.1038/s42255-022-00616-7
Biomaterials. 2022 Aug 15. pii: S0142-9612(22)00369-6. [Epub ahead of print] 121729

A human model of arteriovenous malformation (AVM)-on-a-chip reproduces key disease hallmarks and enables drug testing in perfused human vessel networks.

Kayla Soon, Mengyuan Li, Ruilin Wu, Angela Zhou, Negar Khosraviani, Williamson D Turner, Joshua D Wythe, Jason E Fish, Sara S Nunes.

Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

DOI: https://doi.org/10.1016/j.biomaterials.2022.121729
Front Endocrinol (Lausanne). 2022 ;13 949097

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.

Manuel Bulfoni, Costas Bouyioukos, Albatoul Zakaria, Fabienne Nigon, Roberta Rapone, Laurence Del Maestro, Slimane Ait-Si-Ali, Raphaël Scharfmann, Bertrand Cosson.

  Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.

Keywords:  Human beta cells; RNA co-regulation; eIF2; mRNA feature; mTOR; response to glucose; translation regulation

DOI:  https://doi.org/10.3389/fendo.2022.949097
Front Physiol. 2022 ;13 895118

Mathematical modeling reveals differential dynamics of insulin action models on glycerol and glucose in adolescent girls with obesity.

Griffin S Hampton, Kai Bartlette, Kristen J Nadeau, Melanie Cree-Green, Cecilia Diniz Behn.

  Under healthy conditions, the pancreas responds to a glucose challenge by releasing insulin. Insulin suppresses lipolysis in adipose tissue, thereby decreasing plasma glycerol concentration, and it regulates plasma glucose concentration through action in muscle and liver. Insulin resistance (IR) occurs when more insulin is required to achieve the same effects, and IR may be tissue-specific. IR emerges during puberty as a result of high concentrations of growth hormone and is worsened by youth-onset obesity. Adipose, liver, and muscle tissue exhibit distinct dose-dependent responses to insulin in multi-phase hyperinsulinemic-euglycemic (HE) clamps, but the HE clamp protocol does not address potential differences in the dynamics of tissue-specific insulin responses. Changes to the dynamics of insulin responses would alter glycemic control in response to a glucose challenge. To investigate the dynamics of insulin acting on adipose tissue, we developed a novel differential-equations based model that describes the coupled dynamics of glycerol concentrations and insulin action during an oral glucose tolerance test in female adolescents with obesity and IR. We compared these dynamics to the dynamics of insulin acting on muscle and liver as assessed with the oral minimal model applied to glucose and insulin data collected under the same protocol. We found that the action of insulin on glycerol peaks approximately 67 min earlier (p < 0.001) and follows the dynamics of plasma insulin more closely compared to insulin action on glucose as assessed by the parameters representing the time constants for insulin action on glucose and glycerol (p < 0.001). These findings suggest that the dynamics of insulin action show tissue-specific differences in our IR adolescent population, with adipose tissue responding to insulin more quickly compared to muscle and liver. Improved understanding of the tissue-specific dynamics of insulin action may provide novel insights into the progression of metabolic disease in patient populations with diverse metabolic phenotypes.

Keywords:  glucose; glycerol; insulin; insulin resisitance; lipolysis; mathematical model

DOI:  https://doi.org/10.3389/fphys.2022.895118
Mol Cell. 2022 Aug 15. pii: S1097-2765(22)00713-4. [Epub ahead of print]

Increasing cell size remodels the proteome and promotes senescence.

Michael C Lanz, Evgeny Zatulovskiy, Matthew P Swaffer, Lichao Zhang, Ilayda Ilerten, Shuyuan Zhang, Dong Shin You, Georgi Marinov, Patrick McAlpine, Joshua E Elias, Jan M Skotheim.

  Cell size is tightly controlled in healthy tissues, but it is unclear how deviations in cell size affect cell physiology. To address this, we measured how the cell's proteome changes with increasing cell size. Size-dependent protein concentration changes are widespread and predicted by subcellular localization, size-dependent mRNA concentrations, and protein turnover. As proliferating cells grow larger, concentration changes typically associated with cellular senescence are increasingly pronounced, suggesting that large size may be a cause rather than just a consequence of cell senescence. Consistent with this hypothesis, larger cells are prone to replicative, DNA-damage-induced, and CDK4/6i-induced senescence. Size-dependent changes to the proteome, including those associated with senescence, are not observed when an increase in cell size is accompanied by an increase in ploidy. Together, our findings show how cell size could impact many aspects of cell physiology by remodeling the proteome and provide a rationale for cell size control and polyploidization.

Keywords:  DNA damage; SA-beta-Gal; cell cycle; cell size; p16(INK4); palbociclib; polyploidy; proteomics; senescence; size-scaling

DOI:  https://doi.org/10.1016/j.molcel.2022.07.017