bims-pideca 2024-07-07 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2024‒07‒07
29 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.
quantms: a cloud-based pipeline for quantitative proteomics enables the reanalysis of public proteomics data.
Single-nucleus proteomics identifies regulators of protein transport.
Proteome-scale tagging and functional screening in mammalian cells by ORFtag.
Hyperactive mTORC1 disrupts habenula function and light preference in zebrafish model of Tuberous sclerosis complex.
Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.
mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.
A proximity proteomics pipeline with improved reproducibility and throughput.
Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.
Mitogen signaling strength and duration can control cell cycle decisions.
A data science roadmap for open science organizations engaged in early-stage drug discovery.
Neutrophils actively swell to potentiate rapid migration.
A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens.
Revealing gene function with statistical inference at single-cell resolution.
PI3K inhibitors in hematology: When one door closes….
Assessing the impact of transcriptomics data analysis pipelines on downstream functional enrichment results.
msproteomics sitereport: reporting DIA-MS phosphoproteomics experiments at site level with ease.
Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.
A coupled model between circadian, cell-cycle, and redox rhythms reveals their regulation of oxidative stress.
LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.
FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer.
ezSingleCell: an integrated one-stop single-cell and spatial omics analysis platform for bench scientists.
PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.
A unified model-based framework for doublet or multiplet detection in single-cell multiomics data.
Neural progenitor-derived Apelin controls tip cell behavior and vascular patterning.
GRminusRD: A Sensitive Assay to Detect Activation Processes at the Plasma Membrane in Living Cells.
Polarized endoplasmic reticulum-plasma membrane contacts in cell migration.
A model for accurate quantification of CRISPR effects in pooled FACS screens.
Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer.

J Bone Miner Res. 2024 Jul 03. pii: zjae102. [Epub ahead of print]

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Natalie K Y Wee, Narelle E McGregor, Emma C Walker, Ingrid J Poulton, Michelle Kieu Mi Dang, Jonathan H Gooi, Wayne A Phillips, Natalie A Sims.

  Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. Bone formation is increased by removing the PI3K inhibitor PTEN, but the effect of direct PI3K in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 weeks of age. Both sexes had smaller marrow areas from 6 weeks of age. Female mice also exhibited greater cross sectional area, which continued to increase until 24 weeks of age, resulting in a further increase in bone strength. While both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was a greater extent of lamellar bone formation with highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Keywords:   Osteoblast; bone strength; cortical bone; osteocyte; sex differences

DOI:  https://doi.org/10.1093/jbmr/zjae102
Nat Methods. 2024 Jul 04.

quantms: a cloud-based pipeline for quantitative proteomics enables the reanalysis of public proteomics data.

Chengxin Dai, Julianus Pfeuffer, Hong Wang, Ping Zheng, Lukas Käll, Timo Sachsenberg, Vadim Demichev, Mingze Bai, Oliver Kohlbacher, Yasset Perez-Riverol.

The volume of public proteomics data is rapidly increasing, causing a computational challenge for large-scale reanalysis. Here, we introduce quantms ( https://quant,ms.org/ ), an open-source cloud-based pipeline for massively parallel proteomics data analysis. We used quantms to reanalyze 83 public ProteomeXchange datasets, comprising 29,354 instrument files from 13,132 human samples, to quantify 16,599 proteins based on 1.03 million unique peptides. quantms is based on standard file formats improving the reproducibility, submission and dissemination of the data to ProteomeXchange.

DOI: https://doi.org/10.1038/s41592-024-02343-1
bioRxiv. 2024 Jun 18. pii: 2024.06.17.599449. [Epub ahead of print]

Single-nucleus proteomics identifies regulators of protein transport.

Jason Derks, Tobias Jonson, Andrew Leduc, Saad Khan, Luke Khoury, Mahmoud-Reza Rafiee, Nikolai Slavov.

The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated single cells can be associatively linked with their response to stimulation. Here we developed an approach that leverages this association across individual cells and nuclei to systematically identify potential regulators of biological processes, followed by targeted validation. Specifically, we applied this approach to identify regulators of nucleocytoplasmic protein transport in macrophages stimulated with lipopolysaccharide (LPS). To this end, we quantified the proteomes of 3,412 individual nuclei, sampling the dynamic response to LPS treatment, and linking functional variability to proteomic variability. Minutes after the stimulation, the protein transport in individual nuclei correlated strongly with the abundance of known protein transport regulators, thus revealing the impact of natural protein variability on functional cellular response. We found that simple biophysical constraints, such as the quantity of nuclear pores, partially explain the variability in LPS-induced nucleocytoplasmic transport. Among the many proteins newly identified to be associated with the response, we selected 16 for targeted validation by knockdown. The knockdown phenotypes confirmed the inferences derived from natural protein and functional variation of single nuclei, thus demonstrating the potential of (sub-)single-cell proteomics to infer functional regulation. We expect this approach to generalize to broad applications and enhance the functional interpretability of single-cell omics data.

DOI: https://doi.org/10.1101/2024.06.17.599449
Nat Methods. 2024 Jul 05.

Proteome-scale tagging and functional screening in mammalian cells by ORFtag.

Filip Nemčko, Moritz Himmelsbach, Vincent Loubiere, Ramesh Yelagandula, Michaela Pagani, Nina Fasching, Julius Brennecke, Ulrich Elling, Alexander Stark, Stefan L Ameres.

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

DOI: https://doi.org/10.1038/s41592-024-02339-x
iScience. 2024 Jun 21. 27(6): 110149

Hyperactive mTORC1 disrupts habenula function and light preference in zebrafish model of Tuberous sclerosis complex.

Olga Doszyn, Magdalena Kedra, Justyna Zmorzynska.

  Mechanistic target of rapamycin complex 1 (mTORC1) is an integration hub for extracellular and intracellular signals necessary for brain development. Hyperactive mTORC1 is found in autism spectrum disorder (ASD) characterized by atypical reactivity to sensory stimuli, among other symptoms. In Tuberous sclerosis complex (TSC) inactivating mutations in the TSC1 or TSC2 genes result in hyperactivation of the mTORC1 pathway and ASD. Here, we show that lack of light preference of the TSC zebrafish model, tsc2 vu242/vu242 is caused by aberrant processing of light stimuli in the left dorsal habenula and tsc2 vu242/vu242 fish have impaired function of the left dorsal habenula, in which neurons exhibited higher activity and lacked habituation to the light stimuli. These characteristics were rescued by rapamycin. We thus discovered that hyperactive mTorC1 caused aberrant habenula function resulting in lack of light preference. Our results suggest that mTORC1 hyperactivity contributes to atypical reactivity to sensory stimuli in ASD.

Keywords:  Molecular neuroscience; Neurogenetics; behavioral neuroscience; neuroscience; sensory neuroscience

DOI:  https://doi.org/10.1016/j.isci.2024.110149
bioRxiv. 2024 Jun 22. pii: 2024.06.21.600132. [Epub ahead of print]

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.

Joseph H Lobel, Nicholas T Ingolia.

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

DOI: https://doi.org/10.1101/2024.06.21.600132
Circ Res. 2024 Jul 03.

mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

Wang Min, Lingfeng Qin, Haifeng Zhang, Francesc López-Giráldez, Ning Jiang, Yeaji Kim, Varsha K Mohan, Minhong Su, Katie N Murray, Jaime Grutzendler, Jenny Huanjiao Zhou.

  BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear.METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches.
RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins.
CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Keywords:  brain parenchyma; cerebral cavernous malformation; mechanistic target of rapamycin; single-cell RNA-seq; trafficking

DOI:  https://doi.org/10.1161/CIRCRESAHA.123.324015
Mol Syst Biol. 2024 Jul 01.

A proximity proteomics pipeline with improved reproducibility and throughput.

Xiaofang Zhong, Qiongyu Li, Benjamin J Polacco, Trupti Patil, Aaron Marley, Helene Foussard, Prachi Khare, Rasika Vartak, Jiewei Xu, Jeffrey F DiBerto, Bryan L Roth, Manon Eckhardt, Mark von Zastrow, Nevan J Krogan, Ruth Hüttenhain.

  Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Keywords:  APEX2-based Proximity Labeling; G Protein-Coupled Receptor; Protein–Protein Interaction; Proximity Proteomics; Subcellular Proteomics

DOI:  https://doi.org/10.1038/s44320-024-00049-2
Stem Cell Reports. 2024 Jun 18. pii: S2213-6711(24)00183-8. [Epub ahead of print]

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Dylan Stavish, Christopher J Price, Gabriele Gelezauskaite, Haneen Alsehli, Kimberly A Leonhard, Seth M Taapken, Erik M McIntire, Owen Laing, Bethany M James, Jack J Riley, Johanna Zerbib, Duncan Baker, Amy L Harding, Lydia H Jestice, Thomas F Eleveld, Ad J M Gillis, Sanne Hillenius, Leendert H J Looijenga, Paul J Gokhale, Uri Ben-David, Tenneille E Ludwig, Ivana Barbaric.

  Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Keywords:  MDM4; culture conditions; genetic changes; genome damage; human pluripotent stem cells

DOI:  https://doi.org/10.1016/j.stemcr.2024.06.003
Sci Adv. 2024 Jul 05. 10(27): eadm9211

Mitogen signaling strength and duration can control cell cycle decisions.

Ruth Nussinov, Wengang Zhang, Yonglan Liu, Hyunbum Jang.

Decades ago, mitogen-promoted signaling duration and strength were observed to be sensed by the cell and to be critical for its decisions: to proliferate or differentiate. Landmark publications established the importance of mitogen signaling not only in the G1 cell cycle phase but also through the S and the G2/M transition. Despite these early milestones, how mitogen signal duration and strength, short and strong or weaker and sustained, control cell fate has been largely unheeded. Here, we center on cardinal signaling-related questions, including (i) how fluctuating mitogenic signals are converted into cell proliferation-differentiation decisions and (ii) why extended duration of weak signaling is associated with differentiation, while bursts of strong and short induce proliferation but, if too strong and long, induce irreversible senescence. Our innovative broad outlook harnesses cell biology and protein conformational ensembles, helping us to define signaling strength, clarify cell cycle decisions, and thus cell fate.

DOI: https://doi.org/10.1126/sciadv.adm9211
Nat Commun. 2024 Jul 05. 15(1): 5640

A data science roadmap for open science organizations engaged in early-stage drug discovery.

Kristina Edfeldt, Aled M Edwards, Ola Engkvist, Judith Günther, Matthew Hartley, David G Hulcoop, Andrew R Leach, Brian D Marsden, Amelie Menge, Leonie Misquitta, Susanne Müller, Dafydd R Owen, Kristof T Schütt, Nicholas Skelton, Andreas Steffen, Alexander Tropsha, Erik Vernet, Yanli Wang, James Wellnitz, Timothy M Willson, Djork-Arné Clevert, Benjamin Haibe-Kains, Lovisa Holmberg Schiavone, Matthieu Schapira.

The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design.

DOI: https://doi.org/10.1038/s41467-024-49777-x
Elife. 2024 Jul 02. pii: RP90551. [Epub ahead of print]12

Neutrophils actively swell to potentiate rapid migration.

Tamas L Nagy, Evelyn Strickland, Orion D Weiner.

  While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Keywords:  cell biology; cell migration; cell size; cell volume; human; neutrophil; physical forces; physics of living systems

DOI:  https://doi.org/10.7554/eLife.90551
Clin Proteomics. 2024 Jul 05. 21(1): 49

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens.

Hagen M Gegner, Thomas Naake, Karim Aljakouch, Aurelien Dugourd, Georg Kliewer, Torsten Müller, Dustin Schilling, Marc A Schneider, Nina Kunze-Rohrbach, Thomas G P Grünewald, Rüdiger Hell, Julio Saez-Rodriguez, Wolfgang Huber, Gernot Poschet, Jeroen Krijgsveld.

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.

DOI: https://doi.org/10.1186/s12014-024-09501-9
Nat Rev Genet. 2024 Jul 01.

Revealing gene function with statistical inference at single-cell resolution.

Cole Trapnell.

Single-cell and spatial molecular profiling assays have shown large gains in sensitivity, resolution and throughput. Applying these technologies to specimens from human and model organisms promises to comprehensively catalogue cell types, reveal their lineage origins in development and discern their contributions to disease pathogenesis. Moreover, rapidly dropping costs have made well-controlled perturbation experiments and cohort studies widely accessible, illuminating mechanisms that give rise to phenotypes at the scale of the cell, the tissue and the whole organism. Interpreting the coming flood of single-cell data, much of which will be spatially resolved, will place a tremendous burden on existing computational pipelines. However, statistical concepts, models, tools and algorithms can be repurposed to solve problems now arising in genetic and molecular biology studies of development and disease. Here, I review how the questions that recent technological innovations promise to answer can be addressed by the major classes of statistical tools.

DOI: https://doi.org/10.1038/s41576-024-00750-w
Clin Cancer Res. 2024 Jul 05.

PI3K inhibitors in hematology: When one door closes….

Sigrid S Skånland, Klaus Okkenhaug, Matthew S Davids.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates key cellular processes and is one of the most aberrantly activated pathways in cancer. The class I PI3K catalytic subunits p110g and p110d are highly enriched in leukocytes, providing additional rationale for targeting these PI3Ks in hematologic malignancies. In 2014, the PI3Kd inhibitor idelalisib was the first of four PI3K inhibitors to receive regulatory approval for relapsed B-cell malignancies. This was followed by approvals of the pan-class I inhibitor copanlisib (2017), the dual PI3Kg/d inhibitor duvelisib (2018), and the PI3Kd and casein kinase-1e inhibitor umbralisib (2021). Copanlisib and umbralisib received accelerated approvals, while idelalisib and duvelisib received initial accelerated approvals followed by full approvals. The accelerated approvals were based on overall response rates, however follow-up studies showed increased risk of death and serious side effects. Furthermore, the confirmatory trial with copanlisib failed to show an improvement in progression free survival when compared to chemoimmunotherapy. These developments led to black box warnings for idelalisib and duvelisib, and withdrawal of copanlisib and umbralisib from the market by their manufacturers. Given the uncertain future of this drug class, additional manufacturers terminated ongoing phase III trials with novel PI3K inhibitors. Here, we review the development and current status of PI3K inhibitors in hematology, limitations to their use, and our perspective on whether there is a future for PI3K inhibitors in hematology.

DOI: https://doi.org/10.1158/1078-0432.CCR-24-0967
Nucleic Acids Res. 2024 Jun 29. pii: gkae552. [Epub ahead of print]

Assessing the impact of transcriptomics data analysis pipelines on downstream functional enrichment results.

Victor Paton, Ricardo Omar Ramirez Flores, Attila Gabor, Pau Badia-I-Mompel, Jovan Tanevski, Martin Garrido-Rodriguez, Julio Saez-Rodriguez.

Transcriptomics is widely used to assess the state of biological systems. There are many tools for the different steps, such as normalization, differential expression, and enrichment. While numerous studies have examined the impact of method choices on differential expression results, little attention has been paid to their effects on further downstream functional analysis, which typically provides the basis for interpretation and follow-up experiments. To address this, we introduce FLOP, a comprehensive nextflow-based workflow combining methods to perform end-to-end analyses of transcriptomics data. We illustrate FLOP on datasets ranging from end-stage heart failure patients to cancer cell lines. We discovered effects not noticeable at the gene-level, and observed that not filtering the data had the highest impact on the correlation between pipelines in the gene set space. Moreover, we performed three benchmarks to evaluate the 12 pipelines included in FLOP, and confirmed that filtering is essential in scenarios of expected moderate-to-low biological signal. Overall, our results underscore the impact of carefully evaluating the consequences of the choice of preprocessing methods on downstream enrichment analyses. We envision FLOP as a valuable tool to measure the robustness of functional analyses, ultimately leading to more reliable and conclusive biological findings.

DOI: https://doi.org/10.1093/nar/gkae552
Bioinformatics. 2024 Jun 29. pii: btae432. [Epub ahead of print]

msproteomics sitereport: reporting DIA-MS phosphoproteomics experiments at site level with ease.

Thang V Pham, Alex A Henneman, Nam X Truong, Connie R Jimenez.

  SUMMARY: Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools.AVAILABILITY AND IMPLEMENTATION: sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Keywords:  DIA-MS; MaxLFQ quantification; identification; mass spectrometry; phosphopeptide; phosphoproteomics; phosphosite

DOI:  https://doi.org/10.1093/bioinformatics/btae432
bioRxiv. 2024 Jun 17. pii: 2024.06.14.599022. [Epub ahead of print]

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

Valerie Erhardt, Elli Hartig, Kristian Lorenzo, Hannah R Megathlin, Basile Tarchini, Vishnu Hosur.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter- loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter- loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter- loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

DOI: https://doi.org/10.1101/2024.06.14.599022
Sci Rep. 2024 Jul 05. 14(1): 15479

A coupled model between circadian, cell-cycle, and redox rhythms reveals their regulation of oxidative stress.

Kosaku Masuda, Takeshi Sakurai, Arisa Hirano.

  Most organisms possess three biological oscillators, circadian clock, cell cycle, and redox rhythm, which are autonomous but interact each other. However, whether their interactions and autonomy are beneficial for organisms remains unclear. Here, we modeled a coupled oscillator system where each oscillator affected the phase of the other oscillators. We found that multiple types of coupling prevent a high H2O2 level in cells at M phase. Consequently, we hypothesized a high H2O2 sensitivity at the M phase and found that moderate coupling reduced cell damage due to oxidative stress by generating appropriate phase relationships between three rhythms, whereas strong coupling resulted in an elevated cell damage by increasing the average H2O2 level and disrupted the cell cycle. Furthermore, the multicellularity model revealed that phase variations among cells confer flexibility in synchronization with environments at the expense of adaptability to the optimal environment. Thus, both autonomy and synchrony among the oscillators are important for coordinating their phase relationships to minimize oxidative stress, and couplings balance them depending on environments.

Keywords:  Cell cycle; Circadian clock; Multicellularity; Oxidative stress; Redox rhythm; Synchronization

DOI:  https://doi.org/10.1038/s41598-024-66347-9
J Cell Mol Med. 2024 Jul;28(13): e18509

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Yanping Hao, Liyan Wu, Yuhui Wang, Dongmei Shan, Yifei Liu, Jing Feng, Yi Chang, Ting Wang.

  Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Keywords:  LPS; PI3K; TLR4; TRPV4; itch sensitization

DOI:  https://doi.org/10.1111/jcmm.18509
EMBO Mol Med. 2024 Jul 02.

FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer.

Yifan Tai, Angela Chow, Seoyoung Han, Courtney Coker, Wanchao Ma, Yifan Gu, Valeria Estrada Navarro, Manoj Kandpal, Hanina Hibshoosh, Kevin Kalinsky, Katia Manova-Todorova, Anton Safonov, Elaine M Walsh, Mark Robson, Larry Norton, Richard Baer, Taha Merghoub, Anup K Biswas, Swarnali Acharyya.

  Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

Keywords:  Breast Cancer; FLT1; PARP-Inhibitor-Resistance; VEGFR1

DOI:  https://doi.org/10.1038/s44321-024-00094-2
Nat Commun. 2024 Jul 03. 15(1): 5600

ezSingleCell: an integrated one-stop single-cell and spatial omics analysis platform for bench scientists.

Raman Sethi, Kok Siong Ang, Mengwei Li, Yahui Long, Jingjing Ling, Jinmiao Chen.

ezSingleCell is an interactive and easy-to-use application for analysing various single-cell and spatial omics data types without requiring prior programing knowledge. It combines the best-performing publicly available methods for in-depth data analysis, integration, and interactive data visualization. ezSingleCell consists of five modules, each designed to be a comprehensive workflow for one data type or task. In addition, ezSingleCell allows crosstalk between different modules within a unified interface. Acceptable input data can be in a variety of formats while the output consists of publication ready figures and tables. In-depth manuals and video tutorials are available to guide users on the analysis workflows and parameter adjustments to suit their study aims. ezSingleCell's streamlined interface can analyse a standard scRNA-seq dataset of 3000 cells in less than five minutes. ezSingleCell is available in two forms: an installation-free web application ( https://immunesinglecell.org/ezsc/ ) or a software package with a shinyApp interface ( https://github.com/JinmiaoChenLab/ezSingleCell2 ) for offline analysis.

DOI: https://doi.org/10.1038/s41467-024-48188-2
Nat Immunol. 2024 Jul 03.

PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.

Stephen M Lanahan, Lucas Yang, Kate M Jones, Zhihong Qi, Emylette Cruz Cabrera, Lauren Y Cominsky, Anjali Ramaswamy, Anis Barmada, Gisela Gabernet, Dinesh Babu Uthaya Kumar, Lan Xu, Peiying Shan, Matthias P Wymann, Steven H Kleinstein, V Koneti Rao, Peter Mustillo, Neil Romberg, Roshini S Abraham, Carrie L Lucas.

The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.

DOI: https://doi.org/10.1038/s41590-024-01890-1
Nat Commun. 2024 Jul 02. 15(1): 5562

A unified model-based framework for doublet or multiplet detection in single-cell multiomics data.

Haoran Hu, Xinjun Wang, Site Feng, Zhongli Xu, Jing Liu, Elisa Heidrich-O'Hare, Yanshuo Chen, Molin Yue, Lang Zeng, Ziqi Rong, Tianmeng Chen, Timothy Billiar, Ying Ding, Heng Huang, Richard H Duerr, Wei Chen.

Droplet-based single-cell sequencing techniques rely on the fundamental assumption that each droplet encapsulates a single cell, enabling individual cell omics profiling. However, the inevitable issue of multiplets, where two or more cells are encapsulated within a single droplet, can lead to spurious cell type annotations and obscure true biological findings. The issue of multiplets is exacerbated in single-cell multiomics settings, where integrating cross-modality information for clustering can inadvertently promote the aggregation of multiplet clusters and increase the risk of erroneous cell type annotations. Here, we propose a compound Poisson model-based framework for multiplet detection in single-cell multiomics data. Leveraging experimental cell hashing results as the ground truth for multiplet status, we conducted trimodal DOGMA-seq experiments and generated 17 benchmarking datasets from two tissues, involving a total of 280,123 droplets. We demonstrated that the proposed method is an essential tool for integrating cross-modality multiplet signals, effectively eliminating multiplet clusters in single-cell multiomics data-a task at which the benchmarked single-omics methods proved inadequate.

DOI: https://doi.org/10.1038/s41467-024-49448-x
Sci Adv. 2024 Jul 05. 10(27): eadk1174

Neural progenitor-derived Apelin controls tip cell behavior and vascular patterning.

Julian Malchow, Jean Eberlein, Wei Li, Benjamin M Hogan, Kazuhide S Okuda, Christian S M Helker.

During angiogenesis, vascular tip cells guide nascent vascular sprouts to form a vascular network. Apelin, an agonist of the G protein-coupled receptor Aplnr, is enriched in vascular tip cells, and it is hypothesized that vascular-derived Apelin regulates sprouting angiogenesis. We identify an apelin-expressing neural progenitor cell population in the dorsal neural tube. Vascular tip cells exhibit directed elongation and migration toward and along the apelin-expressing neural progenitor cells. Notably, restoration of neural but not vascular apelin expression in apelin mutants remedies the angiogenic defects of mutants. By functional analyses, we show the requirement of Apelin signaling for tip cell behaviors, like filopodia formation and cell elongation. Through genetic interaction studies and analysis of transgenic activity reporters, we identify Apelin signaling as a modulator of phosphoinositide 3-kinase and extracellular signal-regulated kinase signaling in tip cells in vivo. Our results suggest a previously unidentified neurovascular cross-talk mediated by Apelin signaling that is important for tip cell function during sprouting angiogenesis.

DOI: https://doi.org/10.1126/sciadv.adk1174
Methods Mol Biol. 2024 ;2814 133-147

GRminusRD: A Sensitive Assay to Detect Activation Processes at the Plasma Membrane in Living Cells.

Peter J M van Haastert, Ineke Keizer-Gunnink, Arjan Kortholt.

  Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.

Keywords:  Activation membrane proteins; Cytosolic-RFP; Dictyostelium; GFP-sensor; PIP3; Rap; Ras

DOI:  https://doi.org/10.1007/978-1-0716-3894-1_10
Nat Rev Mol Cell Biol. 2024 Jul 01.

Polarized endoplasmic reticulum-plasma membrane contacts in cell migration.

Lisa Heinke.

DOI: https://doi.org/10.1038/s41580-024-00759-3
bioRxiv. 2024 Jun 18. pii: 2024.06.17.599448. [Epub ahead of print]

A model for accurate quantification of CRISPR effects in pooled FACS screens.

Harold Pimentel, Jacob W Freimer, Maya M Arce, Christian M Garrido, Alexander Marson, Jonathan K Pritchard.

CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of marker genes, followed by guide RNA (gRNA) sequencing. Analysis of these data presents several statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our experimental design model findings that, when using Waterbear for analysis, high power is maintained even at low cell coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.

DOI: https://doi.org/10.1101/2024.06.17.599448
Cell Death Dis. 2024 Jul 03. 15(7): 474

Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer.

Ya-Nan Zheng, Si-Yue Lou, Jun Lu, Fan-Li Zheng, Yong-Mei Tang, En-Jun Zhang, Sun-Liang Cui, Hua-Jun Zhao.

Colorectal cancer (CRC) is one of the most common tumors of the digestive system worldwide. KRAS mutations limit the use of anti-EGFR antibodies in combination with chemotherapy for the treatment of CRC. Therefore, novel targeted therapies are needed to overcome the KRAS-induced oncogenesis. Recent evidence suggests that inhibition of PI3K led to ferroptosis, a nonapoptotic cell death closely related to KRAS-mutant cells. Here, we showed that a selective PI3Kδ inhibitor TYM-3-98 can suppress the AKT/mTOR signaling and activate the ferroptosis pathway in KRAS-mutant CRC cells in a concentration-dependent manner. This was evidenced by the lipid peroxidation, iron accumulation, and depletion of GSH. Moreover, the overexpression of the sterol regulatory element-binding protein 1 (SREBP1), a downstream transcription factor regulating lipid metabolism, conferred CRC cells greater resistance to ferroptosis induced by TYM-3-98. In addition, the effect of TYM-3-98 was confirmed in a xenograft mouse model, which demonstrated significant tumor suppression without obvious hepatoxicity or renal toxicity. Taken together, our work demonstrated that the induction of ferroptosis contributed to the PI3Kδ inhibitor-induced cell death via the suppression of AKT/mTOR/SREBP1-mediated lipogenesis, thus displaying a promising therapeutic effect of TYM-3-98 in CRC treatment.

DOI: https://doi.org/10.1038/s41419-024-06848-7