bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒10‒10
forty papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Mol Cell Oncol. 2021 ;8(4): 1954470
      AKT is the central phosphoinositide 3-kinase (PI3K) signaling effector, however, PIK3CA (p110α subunit of PI3Kα)-mutant estrogen receptor-positive (ER+) breast cancers exhibit minimal AKT activation and the downstream signaling is poorly characterized. We discovered that a subset of PIK3CA-mutant ER+ breast cancers exhibit increased inositol polyphosphate 4-phosphatase type II (INPP4B) expression, which promotes late endosome formation and glycogen synthase kinase 3 beta (GSK3β) trafficking, leading to enhanced Wingless-related integration site (WNT)/catenin beta 1 (β-catenin) activation.
    Keywords:  INPP4B; PIK3CA; WNT/β-catenin; cell proliferation; late endosome
  2. Nat Struct Mol Biol. 2021 Oct;28(10): 858-868
      Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.
  3. Trends Neurosci. 2021 Oct 05. pii: S0166-2236(21)00166-1. [Epub ahead of print]
      The lack of effective treatments for autism spectrum disorder (ASD) and congenital hydrocephalus (CH) reflects the limited understanding of the biology underlying these common neurodevelopmental disorders. Although ASD and CH have been extensively studied as independent entities, recent human genomic and preclinical animal studies have uncovered shared molecular pathophysiology. Here, we review and discuss phenotypic, genomic, and molecular similarities between ASD and CH, and identify the PTEN-PI3K-mTOR (phosphatase and tensin homolog-phosphoinositide 3-kinase-mammalian target of rapamycin) pathway as a common underlying mechanism that holds diagnostic, prognostic, and therapeutic promise for individuals with ASD and CH.
    Keywords:  mTOR; macrocephaly; neurodevelopmental disorders; rapamycin; ventriculomegaly
  4. Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2102560118. [Epub ahead of print]118(41):
      Understanding kinetic control of biological processes is as important as identifying components that constitute pathways. Insulin signaling is central for almost all metazoans, and its perturbations are associated with various developmental disorders, metabolic diseases, and aging. While temporal phosphorylation changes and kinetic constants have provided some insights, constant or variable parameters that establish and maintain signal topology are poorly understood. Here, we report kinetic parameters that encode insulin concentration and nutrient-dependent flow of information using iterative experimental and mathematical simulation-based approaches. Our results illustrate how dynamics of distinct phosphorylation events collectively contribute to selective kinetic gating of signals and maximum connectivity of the signaling cascade under normo-insulinemic but not hyper-insulinemic states. In addition to identifying parameters that provide predictive value for maintaining the balance between metabolic and growth-factor arms, we posit a kinetic basis for the emergence of insulin resistance. Given that pulsatile insulin secretion during a fasted state precedes a fed response, our findings reveal rewiring of insulin signaling akin to memory and anticipation, which was hitherto unknown. Striking disparate temporal behavior of key phosphorylation events that destroy the topology under hyper-insulinemic states underscores the importance of unraveling regulatory components that act as bandwidth filters. In conclusion, besides providing fundamental insights, our study will help in identifying therapeutic strategies that conserve coupling between metabolic and growth-factor arms, which is lost in diseases and conditions of hyper-insulinemia.
    Keywords:  fed–fast; insulin resistance; kinetic insulation; memory; signaling topology
  5. Cell Death Dis. 2021 Oct 08. 12(10): 920
      Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.
  6. Cell Rep. 2021 Oct 05. pii: S2211-1247(21)01244-4. [Epub ahead of print]37(1): 109785
      A hallmark of type 2 diabetes (T2D) is hepatic resistance to insulin's glucose-lowering effects. The serum- and glucocorticoid-regulated family of protein kinases (SGK) is activated downstream of mechanistic target of rapamycin complex 2 (mTORC2) in response to insulin in parallel to AKT. Surprisingly, despite an identical substrate recognition motif to AKT, which drives insulin sensitivity, pathological accumulation of SGK1 drives insulin resistance. Liver-specific Sgk1-knockout (Sgk1Lko) mice display improved glucose tolerance and insulin sensitivity and are protected from hepatic steatosis when fed a high-fat diet. Sgk1 promotes insulin resistance by inactivating AMP-activated protein kinase (AMPK) via phosphorylation on inhibitory site AMPKαSer485/491. We demonstrate that SGK1 is dominant among SGK family kinases in regulation of insulin sensitivity, as Sgk1, Sgk2, and Sgk3 triple-knockout mice have similar increases in hepatic insulin sensitivity. In aggregate, these data suggest that targeting hepatic SGK1 may have therapeutic potential in T2D.
    Keywords:  AMPK; SGK; glucose homeostasis; hepatic insulin resistance; lipid homeostasis; mTORC1; mTORC2; obesity; type 2 diabetes
  7. Anal Chim Acta. 2021 Oct 16. pii: S0003-2670(21)00769-8. [Epub ahead of print]1182 338943
      PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.
    Keywords:  Allelic specific PCR (AS-PCR); CRISPR/Cas12a; Fluorescence; PIK3CA (H1047R) mutation Gene; Rolling circle amplification (RCA); Single-nucleotide variant (SNV)
  8. Ann N Y Acad Sci. 2021 Oct 03.
      Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.
    Keywords:  development; differentiation; lineage tracing; reprogramming; single cell sequencing; spatial transcriptomics
  9. Dev Cell. 2021 Sep 29. pii: S1534-5807(21)00731-0. [Epub ahead of print]
      The metabolic coupling of Schwann cells (SCs) and peripheral axons is poorly understood. Few molecules in SCs are known to regulate axon stability. Using SC-specific Rheb knockout mice, we demonstrate that Rheb-regulated mitochondrial pyruvate metabolism is critical for SC-mediated non-cell-autonomous regulation of peripheral axon stability. Rheb knockout suppresses pyruvate dehydrogenase (PDH) activity (independently of mTORC1) and shifts pyruvate metabolism toward lactate production in SCs. The increased lactate causes age-dependent peripheral axon degeneration, affecting peripheral nerve function. Lactate, as an energy substrate and a potential signaling molecule, enhanced neuronal mitochondrial metabolism and energy production of peripheral nerves. Albeit beneficial to injured peripheral axons in the short term, we show that persistently increased lactate metabolism of neurons enhances ROS production, eventually damaging mitochondria, neuroenergetics, and axon stability. This study highlights the complex roles of lactate metabolism to peripheral axons and the importance of lactate homeostasis in preserving peripheral nerves.
    Keywords:  ROS; Rheb; Schwann cells; axon degeneration; lactate shuttle; mTORC1; metabolic coupling; oxidative stress; peripheral axons; pyruvate metabolism
  10. Arterioscler Thromb Vasc Biol. 2021 Oct 07. ATVBAHA121316969
    Keywords:  Editorials; amino acids; angiogenic proteins; integrins; morphogenesis; proto-oncogene proteins c-akt
  11. J Biol Chem. 2021 Oct 05. pii: S0021-9258(21)00974-1. [Epub ahead of print] 101172
      The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.
    Keywords:  LLGL1; Lgl1; PREX1; TIAM1; cell differentiation; cell motility; cell polarity; glioblastoma; glioma; lethal giant larvae; phosphatidylinositol signaling
  12. Arthritis Res Ther. 2021 Oct 07. 23(1): 253
      BACKGROUND: The objective of this study was to determine if mechanistic target of rapamycin (mTOR) inhibition with or without AMP-activated protein kinase (AMPK) activation can protect against primary, age-related OA.DESIGN: Dunkin-Hartley guinea pigs develop mild primary OA pathology by 5 months of age that progresses to moderate OA by 8 months of age. At 5 months, guinea pigs served as young control (n = 3) or were fed either a control diet (n = 8), a diet enriched with the mTOR-inhibitor rapamycin (Rap, 14 ppm, n = 8), or Rap with the AMPK-activator metformin (Rap+Met, 1000 ppm, n = 8) for 12 weeks. Knee joints were evaluated by OARSI scoring, micro-computed tomography, and immunohistochemistry. Glenohumeral articular cartilage was collected for western blotting.
    RESULTS: Rap- and Rap+Met-treated guinea pigs displayed lower body weight than control. Rap and Rap+Met inhibited articular cartilage mTORC1 but not mTORC2 signaling. Rap+Met, but not Rap alone, stimulated AMPK. Despite lower body weight and articular cartilage mTORC1 inhibition, Rap- and Rap+Met-treated guinea pigs had greater OA severity in the medial tibial plateau due to articular cartilage structural damage and/or proteoglycan loss. Rap and Rap+Met increased plasma glucose compared to control. Plasma glucose concentration was positively correlated with proteoglycan loss, suggesting hyperglycemic stress after Rap treatment was related to worsened OA.
    CONCLUSIONS: This is the first study to show that Rap induced increase in plasma glucose was associated with greater OA severity. Further, articular cartilage mTORC1 inhibition and bodyweight reduction by dietary Rap and Rap+Met did not appear to protect against primary OA during the prevailing hyperglycemia.
    Keywords:  AMPK; Aging; Dunkin Hartley guinea pig; Primary osteoarthritis; mTOR
  13. Gen Comp Endocrinol. 2021 Oct 01. pii: S0016-6480(21)00215-X. [Epub ahead of print] 113922
      The size of an organ is proportional to the other body parts or the whole body. This relationship is known as allometry. Understanding how allometry is determined is a fundamental question in biology. Here we tested the hypothesis that local insulin-like growth factor (Igf) signaling is critical in regulating organ size and its allometric scaling by organ-specific expression of Igf binding proteins (Igfbp). Overexpression of Igfbp2a or 5b in the developing zebrafish eye, heart, and inner ear resulted in a disproportional reduction in their growth relative to the body. Stable transgenic zebrafish with lens-specific Igfbp5b expression selectively reduced adult eye size. The action is Igf-dependent because an Igf-binding deficient Igfbp5b mutant had no effect. Targeted expression of a dominant-negative Igf1 receptor (dnIgf1r) in the lens caused a similar reduction in relative eye growth. Furthermore, co-expression of IGF-1 with an Igfbp restored the eye size. Finally, co-expression of a constitutively active form of Akt with Igfbp or dnIgf1r restored the relative eye growth. These data suggest that local Igf availability and Igf signaling activity are critical determinants of organ size and allometric scaling in zebrafish.
    Keywords:  Akt; Eye; Heart; Inner ear; Insulin-like growth factor 1 receptor; Insulin-like growth factor binding protein; Organ growth; Zebrafish
  14. Nat Methods. 2021 Oct;18(10): 1204-1212
      Identifying gene-regulatory targets of nuclear proteins in tissues is a challenge. Here we describe intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq), a scalable method that measures multiplexed intranuclear protein levels and the transcriptome in parallel across thousands of nuclei, enabling joint analysis of transcription factor (TF) levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed genome-wide associations of each TF and recovered known gene targets. TF-associated genes were coexpressed as distinct modules that each reflected positive or negative TF levels, showing that our approach can disentangle relative putative contributions of TFs to gene expression and add interpretability to inferred gene networks. inCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.
  15. Nat Commun. 2021 Oct 06. 12(1): 5854
      The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.
  16. Cir Pediatr. 2021 Oct 01. 34(4): 200-206
      INTRODUCTION: Congenital muscular hyperplasia of the hand is a rarely described entity, characterized by the presence of aberrant or accessory muscles in the hypothenar eminence, which has been always reported as sporadic anomaly in the medical literature. The aim of this paper is to report a series of cases with a phenotype of aberrant muscle hyperplasia associated with PIK3CA mutations.MATERIAL AND METHODS: We describe a retrospective series of cases followed at our institution between 2008 and 2020, with a unique phenotypein the context of PIK3CA mutations.
    RESULTS: A total of 6 patients were included (4 males and 2 females), who presented with an enlargement of the hypothenar eminence of the hand and peculiar wrinkling of the overlying skin, associated with ulnar deviation of the metacarpo-phalangeal joints. In all cases, variable degrees of congenital overgrowth of the ipsilateral limb were observed. Skin punch biopsy (5 mm diameter and 3-5 mm depth) was performed on 4 patients, where striated muscle hyperplasia and the presence of striated muscular fibers in the dermis and hypodermis were observed in all of them. Genetic studies revealed PIK3CA mutation in 3 of the 4 patients whose affected tissue was analyzed, in a mosaic state ranging from 3 to 8% (His1047Arg, Glu542Lys, and Cys420Arg, 1 case each).
    CONCLUSION: Aberrant muscular hyperplasia of the hand is a well-recognized entity scarcely described in association with PIK3CA spectrum disorder in the pediatric population. The role of PIK3CA in muscle overgrowth is not yet well understood.
    Keywords:  Aberrant tissue; Congenital hand deformity; Human; Hyperplasia; PIK3CA protein
  17. Clin Cancer Res. 2021 Oct 06. pii: clincanres.CCR-21-2183-A.2021. [Epub ahead of print]
      PURPOSE: Activation of Bruton's tyrosine kinase (BTK) and phosphatidylinositol-3-kinase (PI3K) represent parallel, synergistic pathways in lymphoma pathogenesis. As predominant PI3Kδ inhibition is a possible mechanism of tumor escape, we proposed a clinical trial of dual BTK and pan-PI3K inhibition.PATIENTS AND METHODS: We conducted a single-center phase I/Ib trial combining a BTK inhibitor (ibrutinib) and a pan-PI3K inhibitor (buparlisib) in 37 patients with relapsed/refractory (R/R) B-cell lymphoma. Buparlisib and ibrutinib were administered orally, once daily in 28 day cycles until progression or unacceptable toxicity. The clinical trial is registered with, NCT02756247 Results: Patients with mantle cell lymphoma (MCL) receiving the combination had a 94% overall response rate (ORR) and 33-month median progression-free survival; ORR of 31% and 20% were observed in patients with diffuse large B-cell lymphoma and follicular lymphoma, respectively. The maximum tolerated dose was ibrutinib 560 mg plus buparlisib 100 mg and the recommended phase II dose was ibrutinib 560 mg plus buparlisib 80 mg. The most common grade 3 adverse events were rash/pruritis/dermatitis (19%), diarrhea (11%), hyperglycemia (11%), and hypertension (11%). All grade mood disturbances ranging from anxiety, depression, to agitation were observed in 22% of patients. Results from serial monitoring of cell-free DNA samples corresponded to radiographic resolution of disease and tracked the emergence of mutations known to promote BTK inhibitor resistance.
    CONCLUSIONS: BTK and pan-PI3K inhibition in mantle cell lymphoma demonstrates a promising efficacy signal. Addition of BCL2 inhibitors to a BTK and pan-PI3K combination remain suitable for further development in mantle cell lymphoma.
  18. Dev Cell. 2021 Sep 30. pii: S1534-5807(21)00732-2. [Epub ahead of print]
      Neuroblastoma (NB), the most common cancer in the first year of life, presents almost exclusively in the trunk. To understand why an early-onset cancer would have such a specific localization, we xenotransplanted human NB cells into discrete neural crest (NC) streams in zebrafish embryos. Here, we demonstrate that human NB cells remain in an undifferentiated, tumorigenic state when comigrating posteriorly with NC cells but, upon comigration into the head, differentiate into neurons and exhibit decreased survival. Furthermore, we demonstrate that this in vivo differentiation requires retinoic acid and brain-derived neurotrophic factor signaling from the microenvironment, as well as cell-autonomous intersectin-1-dependent phosphoinositide 3-kinase-mediated signaling, likely via Akt kinase activation. Our findings suggest a microenvironment-driven explanation for NB's trunk-biased localization and highlight the potential for induced differentiation to promote NB resolution in vivo.
    Keywords:  differentiating microenvironment; live imaging; neuroblastoma; neuronal differentiation; pediatric cancer
  19. Nat Med. 2021 Oct 04.
      Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter the cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature indicative of an active cell cycle program. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. Chronic hyperinsulinemia in vitro or in humans, however, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can activate a cell cycle program, we define a mechanism whereby mature human adipocytes senesce. We further show that by targeting the adipocyte cell cycle program using metformin, it is possible to influence adipocyte senescence and obesity-associated adipose tissue inflammation.
  20. Oncogene. 2021 Oct 04.
      Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers.
  21. Nat Methods. 2021 Oct;18(10): 1169-1180
      Deep learning using neural networks relies on a class of machine-learnable models constructed using 'differentiable programs'. These programs can combine mathematical equations specific to a particular domain of natural science with general-purpose, machine-learnable components trained on experimental data. Such programs are having a growing impact on molecular and cellular biology. In this Perspective, we describe an emerging 'differentiable biology' in which phenomena ranging from the small and specific (for example, one experimental assay) to the broad and complex (for example, protein folding) can be modeled effectively and efficiently, often by exploiting knowledge about basic natural phenomena to overcome the limitations of sparse, incomplete and noisy data. By distilling differentiable biology into a small set of conceptual primitives and illustrative vignettes, we show how it can help to address long-standing challenges in integrating multimodal data from diverse experiments across biological scales. This promises to benefit fields as diverse as biophysics and functional genomics.
  22. Nat Biotechnol. 2021 Oct 04.
      Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.
  23. Nat Commun. 2021 Oct 08. 12(1): 5906
      Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.
  24. J Biol Chem. 2021 Sep 30. pii: S0021-9258(21)01066-8. [Epub ahead of print] 101263
      Autophagy is a major cellular quality control system responsible for degradation of proteins and organelles in response to stress and damage in order to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase USP11 restricts autophagy and that knockout (KO) of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human β-amyloid peptide 42 aggregation-induced paralysis. USP11 co-precipitated with the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) and limited its interaction with nuclear receptor-binding factor 2 (NRBF2), thus decreasing lipid kinase activity of the PI3KC3-C1 and subsequent recruitment of effectors like WIPI proteins to the autophagosomal membrane. Accordingly, more WIPI2-puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mTOR, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.
    Keywords:  Autophagy; PI3KC3-C1; deubiquitinase (DUB); mTORC1; proteostasis; ubiquitin
  25. Pediatr Neurol. 2021 Sep 25. pii: S0887-8994(21)00207-1. [Epub ahead of print]125 26-31
      BACKGROUND: In tuberous sclerosis, most cardiac rhabdomyomas regress spontaneously. In some cases, the tumors can cause life-threatening hemodynamic compromise requiring subsequent surgical resection. The mechanistic target of rapamycin inhibitors everolimus and sirolimus have shown to be effective treatments for multiple conditions. There are four reports of off-label treatment with transplacental sirolimus for fetal rhabdomyomas due to tuberous sclerosis complex. The optimal dosing regimen is unknown.METHODS: We reviewed the medical records of all patients treated prenatally with sirolimus for rhabdomyomas. All fetuses had a clinical and molecular diagnosis of tuberous sclerosis complex (2012 Consensus Diagnostic Criteria, including a positive genetic test). Clinical history, mechanistic target of rapamycin inhibitor dosing and levels, outcome, and adverse events were reviewed after initiation of sirolimus treatment.
    RESULTS: Three fetuses were treated with maternal sirolimus. Dosing regimens and subsequent trough levels differed from 1 mg/day to 6 mg/day and <1.0 ng/mL to 12.2 ng/mL. Cardiac rhabdomyomas gradually shrank in all patients. Growth restriction was noted in one patient. No severe adverse events occurred during the treatment period.
    CONCLUSIONS: Maternal sirolimus appears to be a safe treatment option in prenatally detected rhabdomyomas with possible need for intervention. Follow-up visits with fetal ultrasound, echocardiography, and laboratory work should be performed weekly during the treatment period. The optimal dosing and trough level timepoints remain unclear. Based on our results, we recommend a sirolimus starting dose of at least 2 mg/m2/day, preferably 3-3.5 mg/m2/day to achieve a target trough level of 10-12 ng/mL.
    Keywords:  Cardiac tumor; Fetal; Maternal; Rapamycin; Sirolimus; Transplacental; Tuberous sclerosis; mTOR inhibitor
  26. Immunometabolism. 2021 Sep 24. 3(4): e210030
      Immunotherapy has underscored a revolution in cancer treatment. Yet, many patients fail to respond due to T cell exhaustion. Here, an intervention that restores mitochondrial function reversed the exhausted T cell phenotype to promote cytotoxicity and durable anti-tumour responses in vivo.
    Keywords:  IL-10; T cell; exhaustion; immunotherapy; metabolism; mitochondria
  27. Nat Commun. 2021 Oct 06. 12(1): 5855
      Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.
  28. Dev Cell. 2021 Oct 01. pii: S1534-5807(21)00728-0. [Epub ahead of print]
      Animals have developed various nutrient-sensing mechanisms for survival under fluctuating environmental conditions. Although extensive cell-culture-based analyses have identified diverse mediators of amino acid sensing upstream of mTOR, studies using animal models to examine intestine-initiated amino acid sensing mechanisms under specific physiological conditions are lacking. Here, we developed a Caenorhabditis elegans model to examine the impact of amino acid deficiency on development. We discovered a leucine-derived monomethyl branched-chain fatty acid and its downstream metabolite, glycosphingolipid, which critically mediates the overall amino acid sensing by intestinal and neuronal mTORC1, which in turn regulates postembryonic development at least partly by controlling protein translation and ribosomal biogenesis. Additional data suggest that a similar mechanism may operate in mammals. This study uncovers an amino-acid-sensing mechanism mediated by a lipid biosynthesis pathway.
    Keywords:  C. elegans; amino acid sensing; developmental arrest; developmental control; glucosylceramide; mTOR; mTORC1; mmBCFA; nutrient sensing; sphingolipid
  29. J Med Chem. 2021 Oct 04.
      The protein homeostasis (proteostasis) network is composed of multiple pathways that work together to balance protein folding, stability, and turnover. Cancer cells are particularly reliant on this network; however, it is hypothesized that inhibition of one node might lead to compensation. To better understand these connections, we dosed 22Rv1 prostate cancer cells with inhibitors of four proteostasis targets (Hsp70, Hsp90, proteasome, and p97), either alone or in binary combinations, and measured the effects on cell growth. The results reveal a series of additive, synergistic, and antagonistic relationships, including strong synergy between inhibitors of p97 and the proteasome and striking antagonism between inhibitors of Hsp90 and the proteasome. Based on RNA-seq, these relationships are associated, in part, with activation of stress pathways. Together, these results suggest that cocktails of proteostasis inhibitors might be a powerful way of treating some cancers, although antagonism that blunts the efficacy of both molecules is also possible.
  30. Am J Pathol. 2021 Oct 04. pii: S0002-9440(21)00433-8. [Epub ahead of print]
      High circulating lipids occurring in obese individuals and insulin resistant patients are considered a contributing factor to Type 2 Diabetes (T2D). Exposure to high lipids is proposed to both protect and damage beta-cells under different circumstances. Here, by feeding mice high fat diet (HFD) for 2 weeks up to 14 months, we showed that HFD initially causes the beta-cells to expand in population whereas long-term exposure to HFD is associated with failure of beta-cells and the inability of animals to respond to glucose challenge. To prevent the failure of beta-cells and the development of T2D, we explored the molecular mechanisms that underlie this biphasic response of beta-cells to lipid exposure. Using palmitic acid (PA) in cultured beta-cells and islets, we demonstrated that chronic exposure to lipids leads to reduced viability and inhibition of cell cycle progression concurrent with downregulation of a pro-growth/survival kinase AKT, independent of glucose. This AKT downregulation by PA is correlated with the induction of mTOR/S6K activity. Inhibiting mTOR activity with rapamycin induces raptor and restores AKT activity, allowing beta-cells to gain proliferation capacity that are lost after HFD exposure. In summary, we elucidated a novel mechanism for which lipid exposure may cause the dipole effects on beta-cell growth, where mTOR acts as a lipid sensor. These mechanisms can be novel targets for future therapeutic developments.
    Keywords:  Beta-cells; Free Fatty Acid (FFA); High Fat Diet (HFD); Islet; Palmitic Acid; Rapamycin; S6K; mTOR; p-AKT
  31. Curr Opin Immunol. 2021 Oct 04. pii: S0952-7915(21)00120-5. [Epub ahead of print]74 25-31
      Certain aspects of experimental tumor models in mice most accurately reflect the biology and immunology of cancer in patients. A survey of experimental cancer immunotherapy papers published in 2020 shows most do not achieve cancer shrinkage although treatment is initiated at an early time point after cancer cell injection, which does not reflect cancer immunotherapy in patients. Even then, few current experimental approaches eradicate the injected malignant cells, most only delay outgrowth. The value of targeting mutation-encoded tumor-specific antigens becomes increasingly evident while problems of finding normal gene-encoded tumor-associated antigens as safe, effective targets persist. It might be time to refocus on realistic experimental settings and truly cancer-specific targets. These antigens are associated with the least risk of side effects.
  32. Cell Rep. 2021 Oct 05. pii: S2211-1247(21)01247-X. [Epub ahead of print]37(1): 109788
      Functional precision medicine aims to match individual cancer patients to optimal treatment through ex vivo drug susceptibility testing on patient-derived cells. However, few functional diagnostic assays have been validated against patient outcomes at scale because of limitations of such assays. Here, we describe a high-throughput assay that detects subtle changes in the mass of individual drug-treated cancer cells as a surrogate biomarker for patient treatment response. To validate this approach, we determined ex vivo response to temozolomide in a retrospective cohort of 69 glioblastoma patient-derived neurosphere models with matched patient survival and genomics. Temozolomide-induced changes in cell mass distributions predict patient overall survival similarly to O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and may aid in predictions in gliomas with mismatch-repair variants of unknown significance, where MGMT is not predictive. Our findings suggest cell mass is a promising functional biomarker for cancers and drugs that lack genomic biomarkers.
    Keywords:  cancer; cell mass; cell size; functional drug susceptibility testing; glioblastoma
  33. Diabetes. 2021 Oct 05. pii: db210147. [Epub ahead of print]
      Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects are persistent challenges. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination; beta cell architecture; in vitro and in vivo glucose-stimulated insulin secretion; and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with beta cells and highlights the need for consistent and comprehensive controls when using the Tet-On or Tet-Off systems for genome editing.
  34. Mol Cell. 2021 Sep 28. pii: S1097-2765(21)00744-9. [Epub ahead of print]
      Cell signaling is a complex process. The faithful transduction of information into specific cellular actions depends on the synergistic effects of many regulatory molecules, nurtured by their strict spatiotemporal regulation. Over the years, we have gained copious insights into the subcellular architecture supporting this spatiotemporal control, including the roles of membrane-bound organelles and various signaling nanodomains. Recently, liquid-liquid phase separation (LLPS) has been recognized as another potentially ubiquitous framework for organizing signaling molecules with high specificity and precise spatiotemporal control in cells. Here, we review the pervasive role of LLPS in signal transduction, highlighting several key pathways that intersect with LLPS, including examples in which LLPS is controlled by signaling events. We also examine how LLPS orchestrates signaling by compartmentalizing signaling molecules, amplifying signals non-linearly, and moderating signaling dynamics. We focus on the specific molecules that drive LLPS and highlight the known functional and pathological consequences of LLPS in each pathway.
    Keywords:  Compartmentation; biomolecular condensates; membraneless organelle
  35. Nat Commun. 2021 Oct 08. 12(1): 5897
      Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.
  36. Elife. 2021 Oct 06. pii: e67886. [Epub ahead of print]10
      Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag' our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly-targeted cells. Our toolbox also incorporates conditional gene knock-down or overexpression using tightly-inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many on-going descriptive studies, such as the Human Cell Atlas Project.
    Keywords:  developmental biology; human
  37. Mol Cell. 2021 Oct 07. pii: S1097-2765(21)00743-7. [Epub ahead of print]81(19): 3886-3887
      Gong et al. (2021) demonstrate that MYC-induced proteotoxic stress could be relieved by inactivating RNA helicase DDX3X for tumor initiation, and in male MYC-driven lymphomas, the homologous helicase DDX3Y, encoded on the Y chromosome, is subsequently induced for disease progression.
  38. Nat Commun. 2021 Oct 08. 12(1): 5909
      Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we introduce an unsupervised and annotation-free framework, termed ClusterMap, which incorporates the physical location and gene identity of RNAs, formulates the task as a point pattern analysis problem, and identifies biologically meaningful structures by density peak clustering (DPC). Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and performs consistently on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell niche, and tissue organization principles from images with high-dimensional transcriptomic profiles.