bims-pideca 2024-10-27 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2024–10–27
twenty-one papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

AKT and the Hallmarks of Cancer.
Hyperglycemia secondary to phosphatidylinositol-3 kinase (PI3K) inhibition.
Protocol for CRISPR-based endogenous protein tagging in mammalian cells.
Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence.
Conditional Pten inactivation in pituitary results in sex-specific prolactinoma formation.
DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity.
Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells.
A structurally informed human protein-protein interactome reveals proteome-wide perturbations caused by disease mutations.
Mapping lineage-traced cells across time points with moslin.
Zinc ions activate AKT and promote prostate cancer cell proliferation via disrupting AKT intramolecular interaction.
Nuclear release of eIF1 restricts start-codon selection during mitosis.
Human stem cell-specific epigenetic signatures control transgene expression.
FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia.
Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.
Mitochondrial fatty acid oxidation drives senescence.
Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling.
CHF6523 data suggest that the phosphoinositide 3-kinase delta isoform is not a suitable target for the management of COPD.
Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns.
Adipocyte-derived glutathione promotes obesity-related breast cancer by regulating the SCARB2-ARF1-mTORC1 complex.
A protein phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome.
Mitochondrial mechanotransduction through MIEF1 coordinates the nuclear response to forces.

Cancer Res. 2024 Oct 22.

AKT and the Hallmarks of Cancer.

Eleonora Sementino, Dalal Hassan, Alfonso Bellacosa, Joseph R Testa.

Nearly a quarter century ago, Hanahan and Weinberg conceived six unifying principles explaining how normal cells transform into malignant tumors. Their provisional set of biological capabilities acquired during tumor development  cancer hallmarks  would evolve to fourteen tenets as knowledge of cancer genomes, molecular mechanisms, and the tumor microenvironment expanded, most recently adding four emerging enabling characteristics: phenotypic plasticity, epigenetic reprogramming, polymorphic microbiomes, and senescent cells. AKT kinases are critical signaling molecules that regulate cellular physiology upon receptor tyrosine kinases and phosphatidylinositol 3-kinase activation. The complex branching of the AKT signaling network involves several critical downstream nodes that significantly magnify its functional impact, such that nearly every organ system and cell in the body may be affected by AKT activity. Conversely, tumor intrinsic dysregulation of AKT can have numerous adverse cellular and pathological ramifications, particularly in oncogenesis, as multiple tumor suppressors and oncogenic proteins regulate AKT signaling. Herein, we review the mounting evidence implicating the AKT pathway in the aggregate of currently recognized hallmarks of cancer underlying the complexities of human malignant diseases. The challenges, recent successes, and likely areas for exciting future advances in targeting this complex pathway are also discussed.

DOI: https://doi.org/10.1158/0008-5472.CAN-24-1846
Endocrinol Diabetes Metab Case Rep. 2024 Oct 01. pii: 24-0040. [Epub ahead of print]2024(4):

Hyperglycemia secondary to phosphatidylinositol-3 kinase (PI3K) inhibition.

Arunan Sriravindrarajah, Joshua Hurwitz, Elgene Lim, Jerry R Greenfield.

   Summary: Phosphatidylinositol-3 kinase (PI3K) is a critical intracellular pathway that regulates cell growth, metabolism, and survival and has been implicated in most human cancers. Targeting this pathway has been approved as a therapeutic option for breast cancer and lymphoma (e.g. alpelisib, idelalisib), and there are several clinical trials underway in additional types of cancer. However, PI3K is an important mediator of the action of insulin, and the use of PI3K inhibitors has been associated with hyperglycemia. We report the case of a 53-year-old female with metastatic breast cancer who developed acute grade 3 hyperglycemia from a novel PI3K inhibitor, inavolisib. We review the treatment options for PI3K inhibitor-associated hyperglycemia. Treatment strategies that minimize hyperinsulinemia may be preferable considering animal models have demonstrated that hyperinsulinemia may result in partial reactivation of the PI3K pathway and counter the anti-cancer effectiveness of PI3K inhibitors.
Learning points: Phosphatidylinositol-3 kinase (PI3K) is an intracellular pathway that regulates a range of physiological functions, including cell growth, metabolism, survival, and angiogenesis. Hyperactivation of the PI3K pathway is associated with almost all human cancers, and thus PI3K inhibition has been proposed as a treatment option for selected cancers. The action of insulin after binding to the insulin receptor on the cell surface (e.g. glucose uptake in skeletal muscle, inhibition of glycogenolysis and gluconeogenesis) is mediated by the intracellular PI3K pathway, and thus PI3K inhibition may lead to hyperinsulinemic hyperglycemia. All patients treated with PI3K inhibitors should receive pre-treatment screening for hyperglycemia, lifestyle advice, and a glucometer to measure fasting BGL and 2-h post-dinner BGL levels twice per week for at least the first 30 days of treatment. Insulin or insulin secretagogues (e.g. sulfonylurea) may inhibit the anti-tumor activity of PI3K inhibitors, and thus treatment of PI3K inhibitor-associated hyperglycemia should prefer alternative approaches such as a low carbohydrate diet, metformin, SGLT2i, or dose reduction of the PI3K inhibitor.

Keywords:  AUSTRALIA; Diabetes; PI3K inhibitor; Please select country of treatment for each patient reported; hyperglycemia

DOI:  https://doi.org/10.1530/EDM-24-0040
STAR Protoc. 2024 Oct 19. pii: S2666-1667(24)00569-0. [Epub ahead of print]5(4): 103404

Protocol for CRISPR-based endogenous protein tagging in mammalian cells.

Yu-Xi Xiao, Jiarun Wei, Jason Moffat.

  Tracking the localization and proximal interaction partners of endogenous proteins provides valuable functional insight. Here, we present a protocol for CRISPR-based endogenous protein tagging in mammalian cells. We describe steps for endogenously tagging human TSC22D2 and MAP4, including designing Cas9 and Cas12a guides for knockin, modularized repair template design and cloning, and procedures for lipid transfection and electroporation. This protocol accommodates Cas nucleases in plasmid expression or ribonucleoprotein complex (RNP) formats. This "endo-tagging" approach offers flexibility and broad applicability. For complete details on the use and execution of this protocol, please refer to Xiao et al.1.

Keywords:  CRISPR; Cell Biology; Genetics; High-Throughput Screening; Microscopy; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2024.103404
Nat Commun. 2024 Oct 24. 15(1): 9181

Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence.

Leighton H Daigh, Debarya Saha, David L Rosenthal, Katherine R Ferrick, Tobias Meyer.

DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes.

DOI: https://doi.org/10.1038/s41467-024-52820-6
Biochim Biophys Acta Mol Basis Dis. 2024 Oct 18. pii: S0925-4439(24)00537-4. [Epub ahead of print]1871(1): 167543

Conditional Pten inactivation in pituitary results in sex-specific prolactinoma formation.

Álvaro Flores-Martínez, Víctor Darío Ramos-Herrero, Alexia Barroso, Alicia Moreno, Miguel E G-García, Eva Venegas-Moreno, Elena Dios, Juan Pedro Martínez-Barberá, Raúl M Luque, Alfonso Soto-Moreno, David A Cano.

  Pituitary tumors, including prolactinomas, present significant clinical challenges that require a deeper understanding of their molecular roots for improved diagnostics and therapies. Here, we investigate the role of the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K) pathway in pituitary tumorigenesis using a mouse model. Conditional knockout of Pten in all pituitary cell lineages resulted in prolactinoma formation exclusively in female mice, demonstrating the critical role of PTEN in pituitary homeostasis. While Pten inactivation induced Akt activation in all pituitary cells, only prolactin-producing cells exhibited tumorigenic changes, suggesting specific cell-type effects. Histological and molecular analyses of prolactinomas revealed similarities with human pituitary tumors, such as decreased vascularization and cell adhesion proteins and increased accumulation of cell cycle proteins. Notably, prolactinomas displayed diminished levels of phosphorylated extracellular signal-regulated kinase (ERK), implicating downregulation of ERK in tumorigenesis. Finally, we analyzed PTEN/PI3K activation in a collection of human pituitary tumors. Overall, our study delineates the intricate interplay between the PTEN and ERK signaling pathways, providing insights into sex-specific mechanisms of pituitary tumorigenesis and potential therapeutic strategies for prolactinomas.

Keywords:  Pituitary tumors; Prolactin; Pten

DOI:  https://doi.org/10.1016/j.bbadis.2024.167543
Elife. 2024 Oct 22. pii: RP88318. [Epub ahead of print]12

DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity.

Pinhua Wang, Sunayana Sarkar, Menghuan Zhang, Tingting Xiao, Fenhua Kong, Zhe Zhang, Deepa Balasubramanian, Nandan Jayaram, Sayantan Datta, Ruyu He, Ping Wu, Peng Chao, Ying Zhang, Michael Washburn, Laurence A Florens, Sonal Nagarkar-Jaiswal, Manish Jaiswal, Man Mohan.

  DYRK1A, a ubiquitously expressed kinase, is linked to the dominant intellectual developmental disorder, microcephaly, and Down syndrome in humans. It regulates numerous cellular processes such as cell cycle, vesicle trafficking, and microtubule assembly. DYRK1A is a critical regulator of organ growth; however, how it regulates organ growth is not fully understood. Here, we show that the knockdown of DYRK1A in mammalian cells results in reduced cell size, which depends on mTORC1. Using proteomic approaches, we found that DYRK1A interacts with the tuberous sclerosis complex (TSC) proteins, namely TSC1 and TSC2, which negatively regulate mTORC1 activation. Furthermore, we show that DYRK1A phosphorylates TSC2 at T1462, a modification known to inhibit TSC activity and promote mTORC1 activity. We also found that the reduced cell growth upon knockdown of DYRK1A can be rescued by overexpression of RHEB, an activator of mTORC1. Our findings suggest that DYRK1A inhibits TSC complex activity through inhibitory phosphorylation on TSC2, thereby promoting mTORC1 activity. Furthermore, using the Drosophila neuromuscular junction as a model, we show that the mnb, the fly homologs of DYRK1A, is rescued by RHEB overexpression, suggesting a conserved role of DYRK1A in TORC1 regulation.

Keywords:  D. melanogaster; DYRK1A; Drosophila melanogaster; biochemistry; cell biology; cell growth; chemical biology; microcephaly

DOI:  https://doi.org/10.7554/eLife.88318
Sci Signal. 2024 Oct 22. 17(859): eadi8753

Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells.

Teresa Rosenlehner, Stefanie Pennavaria, Batuhan Akçabozan, Shiva Jahani, Thomas J O'Neill, Daniel Krappmann, Tobias Straub, Jan Kranich, Reinhard Obst.

Ribosomal biosynthesis in nucleoli is an energy-demanding process driven by all RNA polymerases and hundreds of auxiliary proteins. We investigated how this process is regulated in activated T lymphocytes by T cell receptor (TCR) signals and the multiprotein complexes mTORC1 and mTORC2, both of which contain the kinase mTOR. Deficiency in mTORC1 slowed the proliferation of T cells, with further delays in each consecutive division, an effect not seen with deficiency in mTORC2. mTORC1 signaling was stimulated by components of conventional TCR signaling, and, reciprocally, TCR sensitivity was decreased by mTORC1 inhibition. The substantial increase in the amount of RNA per cell induced by TCR activation was reduced by 50% by deficiency in mTORC1, but not in mTORC2 or in S6 kinases 1 and 2, which are activated downstream of mTORC1. RNA-seq data showed that mTORC1 deficiency reduced the abundance of all RNA biotypes, although rRNA processing was largely intact in activated T cells. Imaging cytometry with FISH probes for nascent pre-rRNA revealed that deletion of mTORC1, but not that of mTORC2, reduced the number and expansion of nucleolar sites of active transcription. Protein translation was consequently decreased by 50% in the absence of mTORC1. Inhibiting RNA polymerase I blocked not only proliferation but also mTORC1 signaling. Our data show that TCR signaling, mTORC1 activity, and ribosomal biosynthesis in the nucleolus regulate each other during biomass production in clonally expanding T cells.

DOI: https://doi.org/10.1126/scisignal.adi8753
Nat Biotechnol. 2024 Oct 24.

A structurally informed human protein-protein interactome reveals proteome-wide perturbations caused by disease mutations.

Dapeng Xiong, Yunguang Qiu, Junfei Zhao, Yadi Zhou, Dongjin Lee, Shobhita Gupta, Mateo Torres, Weiqiang Lu, Siqi Liang, Jin Joo Kang, Charis Eng, Joseph Loscalzo, Feixiong Cheng, Haiyuan Yu.

To assist the translation of genetic findings to disease pathobiology and therapeutics discovery, we present an ensemble deep learning framework, termed PIONEER (Protein-protein InteractiOn iNtErfacE pRediction), that predicts protein-binding partner-specific interfaces for all known protein interactions in humans and seven other common model organisms to generate comprehensive structurally informed protein interactomes. We demonstrate that PIONEER outperforms existing state-of-the-art methods and experimentally validate its predictions. We show that disease-associated mutations are enriched in PIONEER-predicted protein-protein interfaces and explore their impact on disease prognosis and drug responses. We identify 586 significant protein-protein interactions (PPIs) enriched with PIONEER-predicted interface somatic mutations (termed oncoPPIs) from analysis of approximately 11,000 whole exomes across 33 cancer types and show significant associations of oncoPPIs with patient survival and drug responses. PIONEER, implemented as both a web server platform and a software package, identifies functional consequences of disease-associated alleles and offers a deep learning tool for precision medicine at multiscale interactome network levels.

DOI: https://doi.org/10.1038/s41587-024-02428-4
Genome Biol. 2024 Oct 21. 25(1): 277

Mapping lineage-traced cells across time points with moslin.

Marius Lange, Zoe Piran, Michal Klein, Bastiaan Spanjaard, Dominik Klein, Jan Philipp Junker, Fabian J Theis, Mor Nitzan.

  Simultaneous profiling of single-cell gene expression and lineage history holds enormous potential for studying cellular decision-making. Recent computational approaches combine both modalities into cellular trajectories; however, they cannot make use of all available lineage information in destructive time-series experiments. Here, we present moslin, a Gromov-Wasserstein-based model to couple cellular profiles across time points based on lineage and gene expression information. We validate our approach in simulations and demonstrate on Caenorhabditis elegans embryonic development how moslin predicts fate probabilities and putative decision driver genes. Finally, we use moslin to delineate lineage relationships among transiently activated fibroblast states during zebrafish heart regeneration.

Keywords:  Cellular dynamics; Fate decisions; Lineage tracing; Optimal transport; Regeneration

DOI:  https://doi.org/10.1186/s13059-024-03422-4
Oncogene. 2024 Oct 23.

Zinc ions activate AKT and promote prostate cancer cell proliferation via disrupting AKT intramolecular interaction.

Kangjunjie Wang, Min Chen, Shukun Yan, Ying Han, Huairui Yuan, Qiuli Liu, Dayun Lu, Long Li, Kaihua Wang, Fen Liu, Qianqian Li, Dakui Luo, Jun Jiang, Hu Zhou, Yong Chen, Jun Qin, Daming Gao.

Prostate is a zinc rich organ and the physiological function of the abundant zinc ions is relatively less understood. AKT kinase is a pivotal regulator downstream of cytokines, growth factors and other extracellular stimuli, and the attachment of its PH domain to PtdIns-3,4,5-P3 (PIP3) and the subsequent phosphorylation of its kinase domain by PDPK1 are considered important for its activation. Herein, we report a regulatory mechanism of AKT kinase by zinc ions. Mechanistically, zinc ions directly bind to AKT and facilitate AKT activation through disrupting the interaction between PH and kinase domains within AKT molecule. Consistently, AKT1-H89A/E91A mutant (zinc-binding-deficient) fails to respond to zinc ions and exhibits strong interaction between PH and kinase domains, and it is less oncogenic in orthotopic xenograft model of prostate cancer. On the other hand, the AKT1-W80L mutant with minimum intra-molecular interaction between PH and kinase domains shows strong tumor promoting capacity although it could not be further stimulated by zinc ions. Overall, this study reveals a distinctive regulatory mechanism of AKT activation and implies a tumor promoting role of the zinc ions in prostate cancer.

DOI: https://doi.org/10.1038/s41388-024-03195-x
Nature. 2024 Oct 23.

Nuclear release of eIF1 restricts start-codon selection during mitosis.

Jimmy Ly, Kehui Xiang, Kuan-Chung Su, Gunter B Sissoko, David P Bartel, Iain M Cheeseman.

Regulated start-codon selection has the potential to reshape the proteome through the differential production of upstream open reading frames, canonical proteins, and alternative translational isoforms1-3. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation-initiation-site profiling4, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start-codon selection during mitosis results from increased association between the 40S ribosome and the key regulator of start-codon selection, eIF1. We find that increased eIF1-40S ribosome interaction during mitosis is mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the change to translational stringency during mitosis, resulting in altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapies. Thus, cells globally control stringency of translation initiation, which has critical roles during the mammalian cell cycle in preserving mitotic cell physiology.

DOI: https://doi.org/10.1038/s41586-024-08088-3
Biochim Biophys Acta Gene Regul Mech. 2024 Oct 20. pii: S1874-9399(24)00059-2. [Epub ahead of print]1867(4): 195063

Human stem cell-specific epigenetic signatures control transgene expression.

Chulhwan S Kwak, Furkan E Oflaz, Jiamin Qiu, Xinnan Wang.

  Human stem cell-derived models have emerged as an important platform to study tissue differentiation and disease mechanisms. Those models could capitalize on biochemical and cell biological methodologies such as omics, autophagy, and organelle dynamics. However, epigenetic silencing in stem cells creates a barrier to apply genetically encoded tools. Here we investigate the molecular mechanisms underlying exogenously expressed gene silencing by employing multiple commonly used promoters in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK). We discover that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs, as compared to non-iPSCs. Elongation factor 1 alpha short (EF1α short or EFS) promoter, which has fewer CpG island number compared to the other promoters, can drive relatively higher gene expression in iPSCs, despite CpG methylation. Adding a minimal A2 ubiquitous chromatin opening element (minimal A2 UCOE or miniUCOE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs. Our results demonstrate stem cell type-specific epigenetic modification of transgenic promoter region and provide useful information for designing anti-silencing strategies to increase transgene expression in iPSCs.

Keywords:  Gene expression; Methylation; Promoter; Silence; iPSCs

DOI:  https://doi.org/10.1016/j.bbagrm.2024.195063
J Clin Invest. 2024 Oct 22. pii: e173770. [Epub ahead of print]

FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia.

Laura Ondrisova, Vaclav Seda, Krystof Hlavac, Petra Pavelkova, Eva Hoferkova, Giorgia Chiodin, Lenka Kostalova, Gabriela Mladonicka Pavlasova, Daniel Filip, Josef Vecera, Pedro Faria Zeni, Jan Oppelt, Zuzana Kahounova, Rachel Vichova, Karel Soucek, Anna Panovska, Karla Plevova, Sarka Pospisilova, Martin Simkovic, Filip Vrbacky, Daniel Lysak, Stacey M Fernandes, Matthew S Davids, Alba Maiques-Diaz, Stella Charalampopoulou, Jose I Martin-Subero, Jennifer R Brown, Michael Doubek, Francesco Forconi, Jiri Mayer, Marek Mraz.

  BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21).

Keywords:  Drug therapy; Hematology; Leukemias; Oncology; Signal transduction

DOI:  https://doi.org/10.1172/JCI173770
CRISPR J. 2024 Oct;7(5): 293-304

Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.

Ryo Niwa, Tomoko Matsumoto, Alexander Y Liu, Maki Kawato, Takayuki Kondo, Kayoko Tsukita, Peter Gee, Haruhisa Inoue, Thomas L Maurissen, Knut Woltjen.

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.

DOI: https://doi.org/10.1089/crispr.2024.0028
Sci Adv. 2024 Oct 25. 10(43): eado5887

Mitochondrial fatty acid oxidation drives senescence.

Shota Yamauchi, Yuki Sugiura, Junji Yamaguchi, Xiangyu Zhou, Satoshi Takenaka, Takeru Odawara, Shunsuke Fukaya, Takao Fujisawa, Isao Naguro, Yasuo Uchiyama, Akiko Takahashi, Hidenori Ichijo.

Cellular senescence is a stress-induced irreversible cell cycle arrest involved in tumor suppression and aging. Many stresses, such as telomere shortening and oncogene activation, induce senescence by damaging nuclear DNA. However, the mechanisms linking DNA damage to senescence remain unclear. Here, we show that DNA damage response (DDR) signaling to mitochondria triggers senescence. A genome-wide small interfering RNA screen implicated the outer mitochondrial transmembrane protein BNIP3 in senescence induction. We found that BNIP3 is phosphorylated by the DDR kinase ataxia telangiectasia mutated (ATM) and contributes to an increase in the number of mitochondrial cristae. Stable isotope labeling metabolomics indicated that the increase in cristae enhances fatty acid oxidation (FAO) to acetyl-coenzyme A (acetyl-CoA). This promotes histone acetylation and expression of the cyclin-dependent kinase inhibitor p16INK4a. Notably, pharmacological activation of FAO alone induced senescence both in vitro and in vivo. Thus, mitochondrial energy metabolism plays a critical role in senescence induction and is a potential intervention target to control senescence.

DOI: https://doi.org/10.1126/sciadv.ado5887
Nat Commun. 2024 Oct 23. 15(1): 9146

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling.

Bing Wu, Hayley M Bennett, Xin Ye, Akshayalakshmi Sridhar, Celine Eidenschenk, Christine Everett, Evgeniya V Nazarova, Hsu-Hsin Chen, Ivana K Kim, Margaret Deangelis, Leah A Owen, Cynthia Chen, Julia Lau, Minyi Shi, Jessica M Lund, Ana Xavier-Magalhães, Neha Patel, Yuxin Liang, Zora Modrusan, Spyros Darmanis.

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK's versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK's performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK's ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells.

DOI: https://doi.org/10.1038/s41467-024-53227-z
Respir Res. 2024 Oct 19. 25(1): 380

CHF6523 data suggest that the phosphoinositide 3-kinase delta isoform is not a suitable target for the management of COPD.

Mirco Govoni, Michele Bassi, Luca Girardello, Germano Lucci, François Rony, Rémi Charretier, Dmitry Galkin, Maria Laura Faietti, Barbara Pioselli, Gloria Modafferi, Rui Benfeitas, Martina Bonatti, Daniela Miglietta, Jonathan Clark, Frauke Pedersen, Anne-Marie Kirsten, Kai-Michael Beeh, Oliver Kornmann, Stephanie Korn, Andrea Ludwig-Sengpiel, Henrik Watz.

   BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition. Given patients with COPD continue to experience exacerbations despite the availability of effective therapies, anti-inflammatory treatments targeting novel pathways are needed. Kinases, notably the phosphoinositide 3-kinases (PI3K), are thought to be involved in chronic airway inflammation, with this pathway proposed as a critical regulator of inflammation and oxidative stress response in COPD. CHF6523 is an inhaled PI3Kδ inhibitor that has shown positive preclinical results. This manuscript reports the results of a study of CHF6523 in patients with stable COPD (chronic bronchitis phenotype), and who had evidence of type-2 inflammation.
METHODS: This randomised, double-blind, placebo-controlled, two-way crossover study comprised two 28-day treatment periods separated by a 28-day washout. Patients (N = 44) inhaled CHF6523 in one period, and placebo in the other, both twice daily. The primary objective was to assess the safety and tolerability of CHF6523; the secondary objective was to assess CHF6523 pharmacokinetics. Exploratory endpoints included target engagement (the relative reduction in phosphatidylinositol (3,4,5)-trisphosphate [PIP3]), pharmacodynamic evaluations such as airflow obstruction, and hyperinflation, and to identify biomarker(s) of drug response using proteomics and transcriptomics.
RESULTS: CHF6523 plasma pharmacokinetics were characterised by an early maximum concentration (Cmax), reached 15 and 10 min after dosing on Days 1 and 28, respectively, followed by a rapid decline. Systemic exposure on Day 28 showed limited accumulation, with ratios < 1.6 for Cmax and area under the curve from 0 to 12 h post-dose, and with steady state achieved on Day 20. Target engagement was confirmed by a significant 29.7% reduction from baseline in induced sputum PIP3 (29.5% reduction vs. placebo; adjusted ratio 0.705 [0.580, 0.856]; p = 0.001), but this did not translate into an anti-inflammatory pharmacodynamic effect, as assessed through measures including biomarkers and multi-omics. Additionally, although CHF6523 was generally well-tolerated, 95.2% of patients reported cough as an adverse event, most mild to moderate and resolving within one-hour post-dose.
CONCLUSIONS: These data, together with those from other PI3K inhibitors, suggest that PI3Kδ is not a suitable pathway for the management of COPD, as the achieved target engagement did not translate into any pharmacodynamic anti-inflammatory effect.
TRIAL REGISTRATION: ClinicalTrials.gov (NCT04032535); posted 23rd July 2019.

Keywords:  Gene expression profiling; Multi-omics; Phosphatidylinositol 3-kinases; Proteomics; Therapeutics

DOI:  https://doi.org/10.1186/s12931-024-02999-5
Nat Commun. 2024 Oct 25. 15(1): 9209

Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns.

Avantika Ghosh, Marisa Riester, Jagriti Pal, Kadri-Ann Lainde, Carla Tangermann, Angela Wanninger, Ursula K Dueren, Sonam Dhamija, Sven Diederichs.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3' untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

DOI: https://doi.org/10.1038/s41467-024-52779-4
Cell Metab. 2024 Oct 18. pii: S1550-4131(24)00395-4. [Epub ahead of print]

Adipocyte-derived glutathione promotes obesity-related breast cancer by regulating the SCARB2-ARF1-mTORC1 complex.

Chenxi Zhao, Tingting Zhang, Si-Tu Xue, Peitao Zhang, Feng Wang, Yunxuan Li, Ying Liu, Luyao Zhao, Jie Wu, Yechao Yan, Xiaoyun Mao, Yuping Chen, Jian Yuan, Zhuorong Li, Ke Li.

  Obesity is a major risk factor for poor breast cancer outcomes, but the impact of obesity-induced tumor microenvironment (TME) metabolites on breast cancer growth and metastasis remains unclear. Here, we performed TME metabolomic analysis in high-fat diet (HFD) mouse models and found that glutathione (GSH) levels were elevated in the TME of obesity-accelerated breast cancer. The deletion of glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH biosynthesis, in adipocytes but not tumor cells reduced obesity-related tumor progression. Mechanistically, we identified that GSH entered tumor cells and directly bound to lysosomal integral membrane protein-2 (scavenger receptor class B, member 2 [SCARB2]), interfering with the interaction between its N and C termini. This, in turn, recruited mTORC1 to lysosomes through ARF1, leading to the activation of mTOR signaling. Overall, we demonstrated that GSH links obesity and breast cancer progression by acting as an activator of mTOR signaling. Targeting the GSH/SCARB2/mTOR axis could benefit breast cancer patients with obesity.

Keywords:  ARF1; GSH; SCARB2; adipocyte; breast cancer; glutathione; lysosomal integral membrane protein-2; mTORC1; mammalian target of rapamycin complex 1; obesity

DOI:  https://doi.org/10.1016/j.cmet.2024.09.013
Proc Natl Acad Sci U S A. 2024 Oct 29. 121(44): e2415383121

A protein phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome.

Meng S Choy, Hieu T Nguyen, Ganesan S Kumar, Wolfgang Peti, Arminja N Kettenbach, Rebecca Page.

  Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

Keywords:  phosphatase targeting peptide (PhosTAP); phosphoprotein phosphatases (PPP); protein engineering; protein phosphatase 1 (PP1); protein–protein interactions

DOI:  https://doi.org/10.1073/pnas.2415383121
Nat Cell Biol. 2024 Oct 21.

Mitochondrial mechanotransduction through MIEF1 coordinates the nuclear response to forces.

Patrizia Romani, Giada Benedetti, Martina Cusan, Mattia Arboit, Carmine Cirillo, Xi Wu, Georgia Rouni, Vassiliki Kostourou, Mariaceleste Aragona, Costanza Giampietro, Paolo Grumati, Graziano Martello, Sirio Dupont.

Tissue-scale architecture and mechanical properties instruct cell behaviour under physiological and diseased conditions, but our understanding of the underlying mechanisms remains fragmentary. Here we show that extracellular matrix stiffness, spatial confinements and applied forces, including stretching of mouse skin, regulate mitochondrial dynamics. Actomyosin tension promotes the phosphorylation of mitochondrial elongation factor 1 (MIEF1), limiting the recruitment of dynamin-related protein 1 (DRP1) at mitochondria, as well as peri-mitochondrial F-actin formation and mitochondrial fission. Strikingly, mitochondrial fission is also a general mechanotransduction mechanism. Indeed, we found that DRP1- and MIEF1/2-dependent fission is required and sufficient to regulate three transcription factors of broad relevance-YAP/TAZ, SREBP1/2 and NRF2-to control cell proliferation, lipogenesis, antioxidant metabolism, chemotherapy resistance and adipocyte differentiation in response to mechanical cues. This extends to the mouse liver, where DRP1 regulates hepatocyte proliferation and identity-hallmark YAP-dependent phenotypes. We propose that mitochondria fulfil a unifying signalling function by which the mechanical tissue microenvironment coordinates complementary cell functions.

DOI: https://doi.org/10.1038/s41556-024-01527-3