bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒03‒26
thirty papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. JCI Insight. 2023 Mar 21. pii: e151933. [Epub ahead of print]
      Phosphoinositides (PI) are membrane lipids that regulate signal transduction and vesicular trafficking. X-linked centronuclear myopathy (XLCNM), also called myotubular myopathy, results from loss-of-function mutations in the Mtm1 gene, which encodes the myotubularin phosphatidylinositol 3-phosphate (PtdIns3P) lipid phosphatase. No therapy for this disease is currently available. Previous studies showed that loss of expression of the class II phosphoinositide 3-kinase (PI3K) PI3K-C2β protein improved the phenotypes of a XLCNM mouse model. PI3Ks are well known to have extensive scaffolding functions and the importance of the catalytic activity of this PI3K for rescue remains unclear. Here, using PI3K-C2β kinase-dead mice, we show that the selective inactivation of PI3K-C2β kinase activity is sufficient to fully prevent muscle atrophy and weakness, histopathology, and sarcomere and triad disorganization in Mtm1 knockout mice. This rescue correlates with normalization of PtdIns3P level and mTORC1 activity, a key regulator of protein synthesis and autophagy. Conversely, lack of PI3K-C2β kinase activity did not rescue the histopathology of the BIN1 autosomal centronuclear myopathy mouse model. Overall, these findings support the development of specific PI3K-C2β kinase inhibitors to cure myotubular myopathy.
    Keywords:  Inositol phosphates; Muscle Biology; Protein kinases; Skeletal muscle
  2. Cell Calcium. 2023 Mar 15. pii: S0143-4160(23)00030-1. [Epub ahead of print]111 102718
      Activation of phosphatidylinositol 3-kinase (PI3K) by lipid oxidation products, including lysophosphatidylcholine (lysoPC), increases the externalization of canonical transient receptor potential 6 (TRPC6) channels leading to a subsequent increase in intracellular calcium that contributes to cytoskeletal changes which inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo. The PI3K p110α and p110δ catalytic subunit isoforms regulate lysoPC-induced TRPC6 externalization in vitro, but have many other functions. The goal of the current study is to identify the PI3K regulatory subunit isoform involved in TRPC6 externalization to potentially identify a more specific treatment regimen to improve EC migration and arterial healing, while minimizing off-target effects. Decreasing the p85α regulatory subunit isoform protein levels, but not the p85β and p55γ regulatory subunit isoforms, with small interfering RNA inhibits lysoPC-induced translocation of the PI3K catalytic subunit to the plasma membrane, dramatically decreased phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production and TRPC6 externalization, and significantly improves EC migration in the presence of lysoPC. These results identify the important and specific role of p85α in controlling translocation of PI3K from the cytosol to the plasma membrane and PI3K-mediated TRPC externalization by oxidized lipids. Current PI3K inhibitors block the catalytic subunit, but our data suggest that the regulatory subunit is a novel therapeutic target to promote EC migration and healing after arterial injuries that occur with angioplasty.
    Keywords:  Endothelial cell migration; Phosphatidylinositol 3-kinase; TRPC6; p85α
  3. Sci Rep. 2023 Mar 18. 13(1): 4467
      There is little data concerning the implications of PIK3CA mutations outside of the known hotspots described in ER+/HER2- metastatic breast cancer (mBC). Similarly, PIK3R1 mutations could also lead to activation of PI3K pathway, but are poorly described. We determined the incidence and type of all somatic PIK3CA and PIK3R1 mutations by whole exome sequencing (WES) in a pan-cancer cohort of 1200 patients. Activation of the PI3K pathway was studied using phospho-AKT immunohistochemistry. Associations between PIK3CA/PIK3R1 mutations and response to chemotherapy were studied in mBC cases. We found 141 patients (11.8%) with a PIK3CA and/or PIK3R1 mutation across 20 different cancer types. The main cancer subtype was mBC (45.4%). Eighty-four mutations (62.2%) occurred in the three described hotspots; 51 mutations occurred outside of these hotspots. In total, 78.4% were considered activating or probably activating. Among PIK3R1 mutations, 20% were loss of function mutations, leading to a constitutional activation of the pathway. Phospho-AKT quantification in tumor samples was in favor of activation of the PI3K pathway in the majority of mutated tumors, regardless of mutation type. In ER+/HER2- mBC, first line chemotherapy efficacy was similar for PIK3CA-mutated and PIK3CA-WT tumors, whereas in triple negative mBC, chemotherapy appeared to be more effective in PIK3CA-WT tumors. In this large, real-life pan-cancer patient cohort, our results indicate that PIK3CA/PIK3R1 mutations are widely spread, and plead in favour of evaluating the efficacy of PI3K inhibitors outside of ER+/HER2- mBC and outside of hotspot mutations.
  4. J Exp Med. 2023 Jun 05. pii: e20221020. [Epub ahead of print]220(6):
      Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.
  5. bioRxiv. 2023 Mar 06. pii: 2023.03.03.531032. [Epub ahead of print]
      Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP 2 ) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K + (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP 2 by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.One-sentence summary: PI3K inhibition rescues neurovascular coupling defects in cerebral small vessel disease.
  6. Nat Commun. 2023 Mar 22. 14(1): 1591
      In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. An integrated analysis of patient HNC subtypes using single-cell sequencing and proteome profiles reveals an epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature coincides with PI3K/mTOR inactivation in the mesenchymal subtype. Conversely, the signature is suppressed in epithelial cells of the basal subtype which exhibits hyperactive PI3K/mTOR signalling. We further identify YBX1 phosphorylation, downstream of the PI3K/mTOR pathway, restraining basal-like cancer cell proliferation. In contrast, YBX1 acts as a safeguard against the proliferation-to-invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuates partial-EMT and in vivo invasion. Interestingly, phospho-YBX1 that is mutually exclusive to partial-EMT, emerges as a prognostic marker for overall patient outcomes. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype as a promising therapeutic approach against HNC.
  7. Nat Genet. 2023 Mar 23.
      Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.
  8. Elife. 2023 Mar 24. pii: e75191. [Epub ahead of print]12
      Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.
    Keywords:  biochemistry; cancer biology; chemical biology; human
  9. bioRxiv. 2023 Mar 13. pii: 2023.03.07.531554. [Epub ahead of print]
      Channel capacity of signaling networks quantifies their fidelity in sensing extracellular inputs. Low estimates of channel capacities for several mammalian signaling networks suggest that cells can barely detect the presence/absence of environmental signals. However, given the extensive heterogeneity in cell states, we hypothesize that the sensing ability itself varies from cell to cell in a cell state dependent manner. In this work, we present an information theoretic framework to quantify the distribution of sensing abilities from single cell data. Using data on two mammalian pathways, we show that sensing abilities are widely distributed in the population and most cells achieve better resolution of inputs than what is implied by traditional cell state agnostic estimates. We verify these predictions using live cell imaging data on the IGFR/FoxO pathway. Importantly, we identify cell state variables that correlate with cells’ sensing abilities. This information theoretic framework will significantly improve our understanding of how cells sense in their environment.
  10. PLoS One. 2023 ;18(3): e0283203
      Several studies have highlighted the frequent alterations of the PI3K pathway in endometrial cancer leading to increased signaling activation with potential for targeted treatment. The objective of this meta-study was to evaluate how PIK3CA exon 9/20 mutations affect survival in endometrial cancer patients, based on available literature. Topic-based search strategies were applied to databases including CENTRAL, MEDLINE, Embase, Web of Science and COSMIC. All studies assessing the impact of mutations in exon 9 and exon 20 of PIK3CA on survival rates of endometrial cancer patients were selected for inclusion. Statistical meta-analysis was performed with the 'meta' package in RStudio. Overall, 7 of 612 screened articles were included in the present study, comprising 1098 women with endometrial cancer. Meta-analysis revealed a tendency of impaired survival for patients with PIK3CA exon 9 and/or exon 20 mutations (RR 1.28; 95% CI 0.84, 1.94; p = 0.25). This tendency was consistent in subgroup analyses stratified by histologic type or -grade, with the most prominent effect in low-grade endometrial cancers (RR 2.04; 95% CI 0.90, 4.62; p = 0.09). In summary, these results suggest that PIK3CA mutations negatively influence survival outcomes of patients with endometrial cancer, including those with low-grade tumors.
  11. Biochem J. 2023 Mar 31. 480(6): 403-420
      Phosphorylation constitutes the most common and best-studied regulatory post-translational modification in biological systems and archetypal signalling pathways driven by protein and lipid kinases are disrupted in essentially all cancer types. Thus, the study of the phosphoproteome stands to provide unique biological information on signalling pathway activity and on kinase network circuitry that is not captured by genetic or transcriptomic technologies. Here, we discuss the methods and tools used in phosphoproteomics and highlight how this technique has been used, and can be used in the future, for cancer research. Challenges still exist in mass spectrometry phosphoproteomics and in the software required to provide biological information from these datasets. Nevertheless, improvements in mass spectrometers with enhanced scan rates, separation capabilities and sensitivity, in biochemical methods for sample preparation and in computational pipelines are enabling an increasingly deep analysis of the phosphoproteome, where previous bottlenecks in data acquisition, processing and interpretation are being relieved. These powerful hardware and algorithmic innovations are not only providing exciting new mechanistic insights into tumour biology, from where new drug targets may be derived, but are also leading to the discovery of phosphoproteins as mediators of drug sensitivity and resistance and as classifiers of disease subtypes. These studies are, therefore, uncovering phosphoproteins as a new generation of disruptive biomarkers to improve personalised anti-cancer therapies.
    Keywords:  kinase biology; mass spectrometry; proteomics
  12. Nat Commun. 2023 Mar 21. 14(1): 1570
      Integration of single-cell RNA sequencing data between different samples has been a major challenge for analyzing cell populations. However, strategies to integrate differential expression analysis of single-cell data remain underinvestigated. Here, we benchmark 46 workflows for differential expression analysis of single-cell data with multiple batches. We show that batch effects, sequencing depth and data sparsity substantially impact their performances. Notably, we find that the use of batch-corrected data rarely improves the analysis for sparse data, whereas batch covariate modeling improves the analysis for substantial batch effects. We show that for low depth data, single-cell techniques based on zero-inflation model deteriorate the performance, whereas the analysis of uncorrected data using limmatrend, Wilcoxon test and fixed effects model performs well. We suggest several high-performance methods under different conditions based on various simulation and real data analyses. Additionally, we demonstrate that differential expression analysis for a specific cell type outperforms that of large-scale bulk sample data in prioritizing disease-related genes.
  13. bioRxiv. 2023 Mar 10. pii: 2023.03.08.531744. [Epub ahead of print]
      Comparing molecular features, including the identification of genes with differential expression (DE) between conditions, is a powerful approach for characterising disease-specific phenotypes. When testing for DE in single-cell RNA sequencing data, current pipelines first assign cells into discrete clusters (or cell types), followed by testing for differences within each cluster. Consequently, the sensitivity and specificity of DE testing are limited and ultimately dictated by the granularity of the cell type annotation, with discrete clustering being especially suboptimal for continuous trajectories. To overcome these limitations, we present miloDE - a cluster-free framework for differential expression testing. We build on the Milo approach, introduced for differential cell abundance testing, which leverages the graph representation of single-cell data to assign relatively homogenous, neighbouring cells into overlapping neighbourhoods. We address key differences between differential abundance and expression testing at the level of neighbourhood assignment, statistical testing, and multiple testing correction. To illustrate the performance of miloDE we use both simulations and real data, in the latter case identifying a transient haemogenic endothelia-like state in chimeric mouse embryos lacking Tal1 as well as uncovering distinct transcriptional programs that characterise changes in macrophages in patients with Idiopathic Pulmonary Fibrosis. miloDE is available as an open-source R package at
  14. bioRxiv. 2023 Mar 06. pii: 2023.03.05.531161. [Epub ahead of print]
      The genetic code determines how the precise amino acid sequence of proteins is specified by genomic information in cells. But what specifies the precise histologic organization of cells in plant and animal tissues is unclear. We now hypothesize that another code, the tissue code , exists at an even higher level of complexity which determines how tissue organization is dynamically maintained. Accordingly, we modeled spatial and temporal asymmetries of cell division and established that five simple mathematical laws ("the tissue code") convey a set of biological rules that maintain the specific organization and continuous self-renewal dynamics of cells in tissues. These laws might even help us understand wound healing, and how tissue disorganization leads to birth defects and tissue pathology like cancer.
  15. Nat Chem Biol. 2023 Mar 23.
      The global regulation of cell growth rate on gene expression perturbs the performance of gene networks, which would impose complex variations on the cell-fate decision landscape. Here we use a simple synthetic circuit of mutual repression that allows a bistable landscape to examine how such global regulation would affect the stability of phenotypic landscape and the accompanying dynamics of cell-fate determination. We show that the landscape experiences a growth-rate-induced bifurcation between monostability and bistability. Theoretical and experimental analyses reveal that this bifurcating deformation of landscape arises from the unbalanced response of gene expression to growth variations. The path of growth transition across the bifurcation would reshape cell-fate decisions. These results demonstrate the importance of growth regulation on cell-fate determination processes, regardless of specific molecular signaling or regulation.
  16. Mol Metab. 2023 Mar 16. pii: S2212-8778(23)00041-8. [Epub ahead of print] 101707
      Litter size is a biological variable that strongly influences adult physiology in rodents. Despite evidence from previous decades [4] and recent studies highlighting its major impact on metabolism, information about litter size is currently underreported in the scientific literature. Here, we urge that this important biological variable should be explicitly stated in research articles. Below, we briefly describe the scientific evidence supporting the impact of litter size on adult physiology and outline a series of recommendations and guidelines to be implemented by investigators, funding agencies, editors in scientific journals, and animal suppliers to fill this important gap.
    Keywords:  Childhood obesity; Experimental models of physiology; Litter size reduction; Neonatal growth
  17. J Biol Chem. 2023 Mar 22. pii: S0021-9258(23)00277-6. [Epub ahead of print] 104635
      Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction (SVF) cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mTORC1 is activated by purine accumulation, mTORC1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, PPARγ and C/EBPα to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation.
    Keywords:  adipocytes; adipogenesis; lipid droplets; metabolism; nucleotides; purine; pyrimidine
  18. Anal Chem. 2023 Mar 23.
      Imaging defined aspects of functional tumor biology with bioluminescent reporter transgenes is a popular approach in preclinical drug development as it is sensitive, relatively high-throughput and low cost. However, the lack of internal controls subject functional bioluminescence to a number of unpredictable variables that reduce this powerful tool to semi-quantitative interpretation of large-scale effects. Here, we report the generation of sensitive and quantitative live reporters for two key measures of functional cancer biology and pharmacologic stress: the cell cycle and oxidative stress. We developed a two-colored readout, where two independent enzymes convert a common imaging substrate into spectrally distinguishable light. The signal intensity of one color is dependent upon the biological state, whereas the other color is constitutively expressed. The ratio of emitted colored light corrects the functional signal for independent procedural variables, substantially improving the robustness and interpretation of relatively low-fold changes in functional signal intensity after drug treatment. The application of these readouts in vitro is highly advantageous, as peak cell response to therapy can now be readily visualized for single or combination treatments and not simply assessed at an arbitrary and destructive timepoint. Spectral imaging in vivo can be challenging, but we also present evidence to show that the reporters can work in this context as well. Collectively, the development and validation of these internally controlled reporters allow researchers to robustly and dynamically visualize tumor cell biology in response to treatment. Given the prevalence of bioluminescence imaging, this presents significant and much needed opportunities for preclinical therapeutic development.
  19. J Comput Biol. 2023 Mar 24. 1-6
      Integration of multi-omics data can provide a more complex view of the biological system consisting of different interconnected molecular components. We present a new comprehensive R/Bioconductor-package, IntOMICS, which implements a Bayesian framework for multi-omics data integration. IntOMICS adopts a Markov Chain Monte Carlo sampling scheme to systematically analyze gene expression, copy number variation, DNA methylation, and biological prior knowledge to infer regulatory networks. The unique feature of IntOMICS is an empirical biological knowledge estimation from the available experimental data, which complements the missing biological prior knowledge. IntOMICS has the potential to be a powerful resource for exploratory systems biology.
    Keywords:  Bayesian networks; integrative analysis; multi-omics; regulatory network
  20. Nat Biotechnol. 2023 Mar 23.
      An average shotgun proteomics experiment detects approximately 10,000 human proteins from a single sample. However, individual proteins are typically identified by peptide sequences representing a small fraction of their total amino acids. Hence, an average shotgun experiment fails to distinguish different protein variants and isoforms. Deeper proteome sequencing is therefore required for the global discovery of protein isoforms. Using six different human cell lines, six proteases, deep fractionation and three tandem mass spectrometry fragmentation methods, we identify a million unique peptides from 17,717 protein groups, with a median sequence coverage of approximately 80%. Direct comparison with RNA expression data provides evidence for the translation of most nonsynonymous variants. We have also hypothesized that undetected variants likely arise from mutation-induced protein instability. We further observe comparable detection rates for exon-exon junction peptides representing constitutive and alternative splicing events. Our dataset represents a resource for proteoform discovery and provides direct evidence that most frame-preserving alternatively spliced isoforms are translated.
  21. Mol Syst Biol. 2023 Mar 20. e11325
      The analysis of omic data depends on machine-readable information about protein interactions, modifications, and activities as found in protein interaction networks, databases of post-translational modifications, and curated models of gene and protein function. These resources typically depend heavily on human curation. Natural language processing systems that read the primary literature have the potential to substantially extend knowledge resources while reducing the burden on human curators. However, machine-reading systems are limited by high error rates and commonly generate fragmentary and redundant information. Here, we describe an approach to precisely assemble molecular mechanisms at scale using multiple natural language processing systems and the Integrated Network and Dynamical Reasoning Assembler (INDRA). INDRA identifies full and partial overlaps in information extracted from published papers and pathway databases, uses predictive models to improve the reliability of machine reading, and thereby assembles individual pieces of information into non-redundant and broadly usable mechanistic knowledge. Using INDRA to create high-quality corpora of causal knowledge we show it is possible to extend protein-protein interaction databases and explain co-dependencies in the Cancer Dependency Map.
    Keywords:  curation; databases; modeling; networks; text mining
  22. bioRxiv. 2023 Mar 11. pii: 2023.03.08.531782. [Epub ahead of print]
      In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.
  23. Oncologist. 2023 Mar 21. pii: oyad024. [Epub ahead of print]
      BACKGROUND: Hyperglycemia is recognized as a common adverse event for patients receiving alpelisib but has been little studied outside of clinical trials. We report the frequency of alpelisib-associated hyperglycemia in a real-world setting and evaluate proposed risk factors.PATIENTS AND METHODS: We retrospectively identified patients with PIK3CA-mutated, hormone receptor-positive, metastatic breast cancer who initiated treatment with alpelisib plus fulvestrant between August 2019 and December 2021. Ordinal logistic regression evaluated 5 characteristics (diabetes, prediabetes, body mass index [BMI], age, and Asian ancestry) as independent risk factors for ALP-associated hyperglycemia grades 2-4. Risk of error from multiple hypothesis testing was controlled using the false discovery rate method.
    RESULTS: The study included n = 92 subjects, all but 1 female, mean age 59.9 (+11.9) years with 50% non-Hispanic White, 15% Hispanic/Latino, 13% Asian, 9% African/Black, and 13% other/unknown. In total 34% of patients had diabetes, 10% had pre-diabetes, and 56% had normoglycemia. Thirty-six percent were obese, 32% were overweight, 25% were normal weight, and 7% were lean. Frequency of grades 1-4 hyperglycemia in current subjects (64.1%) was similar to hyperglycemia reported in the SOLAR-1 trial (63.7%). Our subjects' risk of grades 2-4 hyperglycemia was independently increased by pre-existing diabetes (Odds ratio 3.75, 95% CI, 1.40-10.01), pre-diabetes (6.22, 1.12-34.47), Asian ancestry (7.10, 1.75-28.84), and each unit of BMI above 20 (1.17, 1.07-1.28).
    CONCLUSION: While receiving alpelisib, patients of Asian ancestry, as well as patients with pre-existing hyperglycemia and/or BMI above 20, should be closely monitored for hyperglycemia. The mechanism underlying the current association of alpelisib-associated hyperglycemia with Asian ancestry is independent of BMI and merits further study. The high incidence of hyperglycemia resulted in a change in practice to include consultation with a diabetes nurse educator or endocrinologist at the start of alpelisib.
    Keywords:  alpelisib; breast cancer; fulvestrant; hyperglycemia; quality of life
  24. Cell Genom. 2023 Mar 08. 3(3): 100250
      Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.
    Keywords:  IP-MS; autism spectrum disorders; exome sequencing; induced excitatory neurons; protein-protein interactions
  25. Mol Cancer Res. 2023 Mar 24. pii: MCR-22-0843. [Epub ahead of print]
      Protein homeostasis (proteostasis) regulates tumor growth and proliferation when cells are exposed to proteotoxic stress, such as during treatment with certain chemotherapeutics. Consequently, cancer cells depend to a greater extent on stress signaling, and require the integrated stress response (ISR), amino acid metabolism, and efficient protein folding and degradation pathways to survive. To define how these interconnected pathways are wired when cancer cells are challenged with proteotoxic stress, we investigated how amino acid abundance influences cell survival when Hsp70, a master proteostasis regulator, is inhibited. We previously demonstrated that cancer cells exposed to a specific Hsp70 inhibitor induce the ISR via the action of two sensors, GCN2 and PERK, in stress-resistant and sensitive cells, respectively. In resistant cells, the induction of GCN2 and autophagy supported resistant cell survival, yet the mechanism by which these events were induced remained unclear. We now report that amino acid availability reconfigures the proteostasis network. Amino acid supplementation, and in particular arginine addition, triggered cancer cell death by blocking autophagy. Consistent with the importance of amino acid availability, which when limited activates GCN2, resistant cancer cells succumbed when challenged with a potentiator for another amino acid sensor, mTORC1, in conjunction with Hsp70 inhibition. Implications: These data position amino acid abundance, GCN2, mTORC1, and autophagy as integrated therapeutic targets whose coordinated modulation regulates the survival of proteotoxic-resistant breast cancer cells.
  26. bioRxiv. 2023 Mar 10. pii: 2023.03.09.531972. [Epub ahead of print]
      Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn . We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
  27. J Cell Physiol. 2023 Mar 22.
      In vitro cultivation conditions play a crucial role in cell physiology and the cellular response to external stimuli. Oxygen concentrations represent an essential microenvironmental factor influencing cell physiology and behaviour both in vivo and in vitro. Therefore, new approaches are urgently needed to monitor and control oxygen concentrations in 2D and 3D cultures, as well as cell reactions to these concentrations. In this work, we modified two types of human endothelial cells-human microvascular (huMECs) and umbilical vein endothelial cells (huVECs) with genetically encoded hypoxia biosensors and monitored cell reactions in 2D to different oxygen concentrations. Moreover, we fabricated 3D cell spheroids of different cell numbers and sizes to reveal the onset of hypoxia in huVECs and huMECs. We could demonstrate a quantitative sensor response of two cell types to reduced oxygen supply in 2D and reveal different thresholds for hypoxic response. In 3D cell spheroids we could estimate critical construct sizes for the appearance of a hypoxic core. This work for the first time directly demonstrates different hypoxic signatures for huVECs and huMECs in 2D and 3D cell culture systems.
    Keywords:  3D cell culture; cell spheroids; endothelial cells; huMECs; huVECs; hypoxia
  28. bioRxiv. 2023 Mar 06. pii: 2023.03.03.531019. [Epub ahead of print]
      Three-dimensional (3D) culture models, such as organoids, are flexible systems to interrogate cellular growth and morphology, multicellular spatial architecture, and cell interactions in response to drug treatment. However, new computational methods to segment and analyze 3D models at cellular resolution with sufficiently high throughput are needed to realize these possibilities. Here we report Cellos (Cell and Organoid Segmentation), an accurate, high throughput image analysis pipeline for 3D organoid and nuclear segmentation analysis. Cellos segments organoids in 3D using classical algorithms and segments nuclei using a Stardist-3D convolutional neural network which we trained on a manually annotated dataset of 3,862 cells from 36 organoids confocally imaged at 5 μm z-resolution. To evaluate the capabilities of Cellos we then analyzed 74,450 organoids with 1.65 million cells, from multiple experiments on triple negative breast cancer organoids containing clonal mixtures with complex cisplatin sensitivities. Cellos was able to accurately distinguish ratios of distinct fluorescently labelled cell populations in organoids, with <3% deviation from the seeding ratios in each well and was effective for both fluorescently labelled nuclei and independent DAPI stained datasets. Cellos was able to recapitulate traditional luminescence-based drug response quantifications by analyzing 3D images, including parallel analysis of multiple cancer clones in the same well. Moreover, Cellos was able to identify organoid and nuclear morphology feature changes associated with treatment. Finally, Cellos enables 3D analysis of cell spatial relationships, which we used to detect ecological affinity between cancer cells beyond what arises from local cell division or organoid composition. Cellos provides powerful tools to perform high throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging.
  29. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]
      Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.
  30. Front Oncol. 2023 ;13 1144056
      Modern cancer therapeutics are increasingly targeted, bringing the promise of new and improved activity, alongside better tolerability. However, while many are indeed resulting in dramatic improvements in disease control and patient survival, short- and long-term tolerability has not always accompanied it. The choice of dose and schedule is often in the upper range of the therapeutic window, driven by the maximum tolerated dose (MTD) model of previous cytotoxic agents. There is increasing recognition that this needs to change, by taking a more holistic approach to determine the optimal dose for desired biological effects and tolerability early in clinical development. In the US, the FDA's Oncology Centre of Excellence is addressing this via the Project Optimus initiative: aiming to reform dose optimisation studies so that they can demonstrate the most appropriate dose selection. Early clinical development will need to demonstrate the dose-exposure, -pharmacodynamic, -toxicity and -activity relationships, including randomised evaluations for dose selection. Regulatory agencies outside the US are similarly exploring this. Along with Australia, Brazil, Canada, Israel, Singapore and Switzerland, the UK participates in Project Orbis, a collaborative program with the FDA to accelerate patient access to new cancer medicines through coordinated regulatory review. Close alignment with Project Optimus will be important internationally and will require changes across industry, including for academic units and small biotech. We discuss our perspective on the implications, and opportunities, for early phase oncology trials as a uniquely charity-funded drug development facility, the Centre for Drug Development within the Cancer Research UK charity.
    Keywords:  FDA – Food and Drug Administration; Project Optimus; United Kingdom; academic; cancer; drug development [MeSH]; international; oncology