bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒09‒11
27 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Int J Mol Sci. 2022 Aug 29. pii: 9787. [Epub ahead of print]23(17):
      Phospholipid scramblase 4 (PLSCR4) is a member of a conserved enzyme family with high relevance for the remodeling of phospholipid distribution in the plasma membrane and the regulation of cellular signaling. While PLSCR1 and -3 are involved in the regulation of adipose-tissue expansion, the role of PLSCR4 is so far unknown. PLSCR4 is significantly downregulated in an adipose-progenitor-cell model of deficiency for phosphatase and tensin homolog (PTEN). PTEN acts as a tumor suppressor and antagonist of the growth and survival signaling phosphoinositide 3-kinase (PI3K)/AKT cascade by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP3). Patients with PTEN germline deletion frequently develop lipomas. The underlying mechanism for this aberrant adipose-tissue growth is incompletely understood. PLSCR4 is most highly expressed in human adipose tissue, compared with other phospholipid scramblases, suggesting a specific role of PLSCR4 in adipose-tissue biology. In cell and mouse models of lipid accumulation, we found PLSCR4 to be downregulated. We observed increased adipogenesis in PLSCR4-knockdown adipose progenitor cells, while PLSCR4 overexpression attenuated lipid accumulation. PLSCR4 knockdown was associated with increased PIP3 levels and the activation of AKT. Our results indicated that PLSCR4 is a regulator of PI3K/AKT signaling and adipogenesis and may play a role in PTEN-associated adipose-tissue overgrowth and lipoma formation.
    Keywords:  PIP3; PLSCR4; PTEN; adipogenesis; lipoma; scramblase
  2. Cells. 2022 Aug 30. pii: 2708. [Epub ahead of print]11(17):
      We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.
    Keywords:  Akt; PI3K; PTEN; knockout; lens; mouse
  3. Molecules. 2022 Aug 24. pii: 5414. [Epub ahead of print]27(17):
      Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.
    Keywords:  FOXO; aging; cancer; chemical biology; high content screening; kinases; mTOR
  4. Genet Med. 2022 Sep 06. pii: S1098-3600(22)00891-7. [Epub ahead of print]
      PURPOSE: PIK3CA-related overgrowth spectrum (PROS) conditions of the head and neck are treatment challenges. Traditionally, these conditions require multiple invasive interventions, with incomplete malformation removal, disfigurement, and possible dysfunction. Use of the PI3K inhibitor alpelisib, previously shown to be effective in PROS, has not been reported in PIK3CA-associated head and neck lymphatic malformations (HNLMs) or facial infiltrating lipomatosis (FIL). We describe prospective treatment of 5 children with PIK3CA-associated HNLMs or head and neck FIL with alpelisib monotherapy.METHODS: A total of 5 children with PIK3CA-associated HNLMs (n = 4) or FIL (n = 1) received alpelisib monotherapy (aged 2-12 years). Treatment response was determined by parental report, clinical evaluation, diary/questionnaire, and standardized clinical photography, measuring facial volume through 3-dimensional photos and magnetic resonance imaging.
    RESULTS: All participants had reduction in the size of lesion, and all had improvement or resolution of malformation inflammation/pain/bleeding. Common invasive therapy was avoided (ie, tracheotomy). After 6 or more months of alpelisib therapy, facial volume was reduced (range 1%-20%) and magnetic resonance imaging anomaly volume (range 0%-23%) were reduced, and there was improvement in swallowing, upper airway patency, and speech clarity.
    CONCLUSION: Individuals with head and neck PROS treated with alpelisib had decreased malformation size and locoregional overgrowth, improved function and symptoms, and fewer invasive procedures.
    Keywords:  Alpelisib; Infiltrating lipomatosis; Lymphatic malformation; PIK3CA-related overgrowth spectrum (PROS); Precision medicine
  5. J Pathol. 2022 Sep 08.
      PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underly the PTEN Hamartoma Tumour Syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI-3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralize the impact of PTEN loss, rendering this a more druggable context compared to those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6-weeks onwards to mice with heterozygous germline PTEN loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well-tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay, but not full blockade of the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor This article is protected by copyright. All rights reserved.
    Keywords:  PHTS; PI 3-kinase; PTEN; cancer prevention; drug; hamartoma; kinase inhibitor; mTORC1; rapamycin; rare disease; syndrome
  6. Nat Commun. 2022 Sep 07. 13(1): 5258
      CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.
  7. Heliyon. 2022 Aug;8(8): e10371
      Cardiomyopathy is a primary cause of death in Friedreich ataxia (FRDA) patients with defective iron-sulfur cluster (ISC) biogenesis due to loss of functional frataxin and in rare patients with functional loss of other ISC biogenesis factors. The mechanistic target of rapamycin (mTOR) and AKT signaling cascades that coordinate eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors, are crucial regulators of cardiovascular growth and homeostasis. We observed increased phosphorylation of AKT and dysregulation of multiple downstream effectors of mTORC1, including S6K1, S6, ULK1 and 4EBP1, in a cardiac/skeletal muscle specific FRDA conditional knockout (cKO) mouse model and in human cell lines depleted of ISC biogenesis factors. Knockdown of several mitochondrial metabolic proteins that are downstream targets of ISC biogenesis, including lipoyl synthase and subunit B of succinate dehydrogenase, also resulted in activation of mTOR and AKT signaling, suggesting that mTOR and AKT hyperactivations are part of the metabolic stress response to ISC deficiencies. Administration of rapamycin, a specific inhibitor of mTOR signaling, enhanced the survival of the Fxn cKO mice, providing proof of concept for the potential of mTOR inhibition to ameliorate cardiac disease in patients with defective ISC biogenesis. However, AKT phosphorylation remained high in rapamycin-treated Fxn cKO hearts, suggesting that parallel mTOR and AKT inhibition might be necessary to further improve the lifespan and healthspan of ISC deficient individuals.
    Keywords:  AKT; Cardiac hypertrophy; FXN; Frataxin; Friedreich ataxia; ISCU; Iron-sulfur cluster biogenesis; Metabolic stress; mTOR
  8. Front Immunol. 2022 ;13 990874
      Akt is a PI3K-activated serine-threonine kinase that exists in three distinct isoforms. Akt's expression in most immune cells, either at baseline or upon activation, reflects its importance in the immune system. While Akt is most highly expressed in innate immune cells, it plays crucial roles in both innate and adaptive immune cell development and/or effector functions. In this review, we explore what's known about the role of Akt in innate and adaptive immune cells. Wherever possible, we discuss the overlapping and distinct role of the three Akt isoforms, namely Akt1, Akt2, and Akt3, in immune cells.
    Keywords:  Akt; Akt1; Akt2; Akt3; immune cells
  9. Sci Rep. 2022 Sep 05. 12(1): 15095
      Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell-cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.
  10. Elife. 2022 Sep 07. pii: e75715. [Epub ahead of print]11
      Despite multiple possible oncogenic mutations in the proto-oncogene KRAS, unique subsets of these mutations are detected in different cancer types. As KRAS mutations occur early, if not being the initiating event, these mutational biases are ostensibly a product of how normal cells respond to the encoded oncoprotein. Oncogenic mutations can impact not only the level of active oncoprotein, but also engagement with proteins. To attempt to separate these two effects, we generated four novel Cre-inducible (LSL) Kras alleles in mice with the biochemically distinct G12D or Q61R mutations and encoded by native (nat) rare or common (com) codons to produce low or high protein levels. While there were similarities, each allele also induced a distinct transcriptional response shortly after activation in vivo. At one end of the spectrum, activating the KrasLSL-natG12D allele induced transcriptional hallmarks suggestive of an expansion of multipotent cells, while at the other end, activating the KrasLSL-comQ61R allele led to hallmarks of hyperproliferation and oncogenic stress. Evidence suggests that these changes may be a product of signaling differences due to increased protein expression as well as the specific mutation. To determine the impact of these distinct responses on RAS mutational patterning in vivo, all four alleles were globally activated, revealing that hematolymphopoietic lesions were permissive to the level of active oncoprotein, squamous tumors were permissive to the G12D mutant, while carcinomas were permissive to both these features. We suggest that different KRAS mutations impart unique signaling properties that are preferentially capable of inducing tumor initiation in a distinct cell-specific manner.
    Keywords:  RAS; cancer; cancer biology; genetically engineered mice; mouse; oncogenes
  11. Nat Cell Biol. 2022 Sep 08.
      Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.
  12. Front Cell Dev Biol. 2022 ;10 920683
      Osteoclasts are bone-resorbing cells that undergo extensive changes in morphology throughout their differentiation. Altered osteoclast differentiation and activity lead to changes in pathological bone resorption. The mammalian target of rapamycin (mTOR) is a kinase, and aberrant mTOR complex 1 (mTORC1) signaling is associated with altered bone homeostasis. The activation of mTORC1 is biphasically regulated during osteoclastogenesis; however, the mechanism behind mTORC1-mediated regulation of osteoclastogenesis and bone resorption is incompletely understood. Here, we found that MYC coordinates the dynamic regulation of mTORC1 activation during osteoclastogenesis. MYC-deficiency blocked the early activation of mTORC1 and also reversed the decreased activity of mTORC1 at the late stage of osteoclastogenesis. The suppression of mTORC1 activity by rapamycin in mature osteoclasts enhances bone resorption activity despite the indispensable role of high mTORC1 activation in osteoclast formation in both mouse and human cells. Mechanistically, MYC induces Growth arrest and DNA damage-inducible protein (GADD34) expression and suppresses mTORC1 activity at the late phase of osteoclastogenesis. Taken together, our findings identify a MYC-GADD34 axis as an upstream regulator of dynamic mTORC1 activation in osteoclastogenesis and highlight the interplay between MYC and mTORC1 pathways in determining osteoclast activity.
    Keywords:  GADD34 (PPP1R15A); MYC (c-myc); bone resorption; mTORC1 (mechanistic target of rapamycin complex 1); osteoclast (OC)
  13. Nat Commun. 2022 Sep 09. 13(1): 5317
      Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.
  14. Genome Biol. 2022 Sep 05. 23(1): 186
      BACKGROUND: Current methods for analyzing single-cell datasets have relied primarily on static gene expression measurements to characterize the molecular state of individual cells. However, capturing temporal changes in cell state is crucial for the interpretation of dynamic phenotypes such as the cell cycle, development, or disease progression. RNA velocity infers the direction and speed of transcriptional changes in individual cells, yet it is unclear how these temporal gene expression modalities may be leveraged for predictive modeling of cellular dynamics.RESULTS: Here, we present the first task-oriented benchmarking study that investigates integration of temporal sequencing modalities for dynamic cell state prediction. We benchmark ten integration approaches on ten datasets spanning different biological contexts, sequencing technologies, and species. We find that integrated data more accurately infers biological trajectories and achieves increased performance on classifying cells according to perturbation and disease states. Furthermore, we show that simple concatenation of spliced and unspliced molecules performs consistently well on classification tasks and can be used over more memory intensive and computationally expensive methods.
    CONCLUSIONS: This work illustrates how integrated temporal gene expression modalities may be leveraged for predicting cellular trajectories and sample-associated perturbation and disease phenotypes. Additionally, this study provides users with practical recommendations for task-specific integration of single-cell gene expression modalities.
    Keywords:  Data integration; Multi-omics; RNA velocity; Single-cell prediction; Trajectory inference
  15. Chembiochem. 2022 Sep 08.
      Gene therapy offers an alternative and promising avenue to lung cancer treatment. Here, we used dibenzocyclooctyne (DBCO)-branched primers to construct a kind of PTEN gene nanovector (NP-PTEN) through branch-PCR. NP-PTEN showed the nanoscale structure with the biocompatible size (84.7 ± 11.2 nm) and retained the improved serum stability compared to linear DNA. When transfected into NCI-H1299 cancer cells, NP-PTEN could overexpress PTEN protein to restore PTEN function through the deactivation of PI3K-AKT-mTOR signaling pathway to inhibit cell proliferation and induce cell apoptosis. The apoptosis rate of NCI-H1299 cancer cells could reach up to 54.5% ± 4.6% when the transfection concentration of NP-PTEN was 4.0 μg/mL. In mice bearing NCI-H1299 tumor xenograft intratumorally administrated with NP-PTEN, the average tumor volume and tumor weight was separately reduced by 61.7% and 63.9% compared with the PBS group on the 18 th day of administration. The anticancer efficacy of NP-PTEN in NCI-H1299 tumor xenograft suggested the promising therapeutic potential of this branch-PCR assembled PTEN gene nanovectors in lung cancer gene therapy and also provided more opportunities to introduce two or more tumor suppressor genes as the all-in-one gene nanovectors for multiple gene-based cancer gene therapy.
    Keywords:  Gene therapy, PTEN, NSCLC, Branch-PCR
  16. Nat Commun. 2022 Sep 08. 13(1): 5295
      DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.
  17. Nat Rev Endocrinol. 2022 Sep 07.
      Metabolic diseases, including obesity, diabetes mellitus and cardiovascular disease, are a major threat to health in the modern world, but efforts to understand the underlying mechanisms and develop rational treatments are limited by the lack of appropriate human model systems. Notably, advances in stem cell and organoid technology allow the generation of cellular models that replicate the histological, molecular and physiological properties of human organs. Combined with marked improvements in gene editing tools, human stem cells and organoids provide unprecedented systems for studying mechanisms of metabolic diseases. Here, we review progress made over the past decade in the generation and use of stem cell-derived metabolic cell types and organoids in metabolic disease research, especially obesity and liver diseases. In particular, we discuss the limitations of animal models and the advantages of stem cells and organoids, including their application to metabolic diseases. We also discuss mechanisms of drug action, understanding the efficacy and toxicity of existing therapies, screening for new treatments and pursuing personalized therapies. We highlight the potential of combining stem cell-derived organoids with gene editing and functional genomics to revolutionize the approach to finding treatments for metabolic diseases.
  18. Pediatr Blood Cancer. 2022 Aug;69 Suppl 3 e29273
      Vascular anomalies, both vascular tumors and vascular malformations, can occur in isolation or as part of syndromes including those which feature phenotypic overgrowth. To update what is known about vascular anomalies associated with overgrowth, PubMed was searched for "overgrowth syndromes and vascular anomalies or malformations." PubMed, OMIM, and the Rare Disease Database also were searched for specific diagnoses. We review individual overgrowth syndromes, provide a case-based approach to the clinical, radiographic, pathologic, and genetic basis for diagnosis, to complications of both the vascular anomalies and the overgrowth, and emphasize the need for a multidisciplinary approach to care.
    Keywords:  PIK3CA; overgrowth; vascular malformations
  19. Cell Death Dis. 2022 Sep 08. 13(9): 777
      Understanding prostate cancer onset and progression in order to rationally treat this disease has been critically limited by a dire lack of relevant pre-clinical animal models. We have generated a set of genetically engineered mice that mimic human prostate cancer, initiated from the gland epithelia. We chose driver gene mutations that are specifically relevant to cancers of young men, where aggressive disease poses accentuated survival risks. An outstanding advantage of our models are their intact repertoires of immune cells. These mice provide invaluable insight into the importance of immune responses in prostate cancer and offer scope for studying treatments, including immunotherapies. Our prostate cancer models strongly support the role of tumour suppressor p53 in functioning to critically restrain the emergence of cancer pathways that drive cell cycle progression; alter metabolism and vasculature to fuel tumour growth; and mediate epithelial to mesenchymal-transition, as vital to invasion. Importantly, we also discovered that the type of p53 alteration dictates the specific immune cell profiles most significantly disrupted, in a temporal manner, with ramifications for disease progression. These new orthotopic mouse models demonstrate that each of the isogenic hotspot p53 amino acid mutations studied (R172H and R245W, the mouse equivalents of human R175H and R248W respectively), drive unique cellular changes affecting pathways of proliferation and immunity. Our findings support the hypothesis that individual p53 mutations confer their own particular oncogenic gain of function in prostate cancer.
  20. Nat Commun. 2022 Sep 09. 13(1): 5304
      Biomedical data is accumulating at a fast pace and integrating it into a unified framework is a major challenge, so that multiple views of a given biological event can be considered simultaneously. Here we present the Bioteque, a resource of unprecedented size and scope that contains pre-calculated biomedical descriptors derived from a gigantic knowledge graph, displaying more than 450 thousand biological entities and 30 million relationships between them. The Bioteque integrates, harmonizes, and formats data collected from over 150 data sources, including 12 biological entities (e.g., genes, diseases, drugs) linked by 67 types of associations (e.g., 'drug treats disease', 'gene interacts with gene'). We show how Bioteque descriptors facilitate the assessment of high-throughput protein-protein interactome data, the prediction of drug response and new repurposing opportunities, and demonstrate that they can be used off-the-shelf in downstream machine learning tasks without loss of performance with respect to using original data. The Bioteque thus offers a thoroughly processed, tractable, and highly optimized assembly of the biomedical knowledge available in the public domain.
  21. Elife. 2022 Sep 07. pii: e79208. [Epub ahead of print]11
      The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multi-component editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair (HDR) of site-specific nuclease-induced double-strand breaks (DSBs). Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs (ngRNAs) improved editing efficiencies up to 13-fold compared to transfecting the prime editing components as plasmids or ribonucleoprotein particles (RNPs). Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
    Keywords:  genetics; genomics; human; regenerative medicine; stem cells
  22. Biomaterials. 2022 Sep 02. pii: S0142-9612(22)00423-9. [Epub ahead of print]289 121783
      While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy.
    Keywords:  Bystander killing; Caspase-3; Macropinocytosis; Metastatic triple-negative breast cancer; PTEN-Loss; Peptide-drug conjugate
  23. Cell Syst. 2022 Sep 01. pii: S2405-4712(22)00330-1. [Epub ahead of print]
      Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.
    Keywords:  Hippo/Yap1 pathway; computational drug discovery; drug screening; image-based profiling; virtual screening
  24. F1000Res. 2020 ;9 1263
    Tumor Profiler Consortium
      Mass cytometry (CyTOF) has become a method of choice for in-depth characterization of tissue heterogeneity in health and disease, and is currently implemented in multiple clinical trials, where higher quality standards must be met. Currently, preprocessing of raw files is commonly performed in independent standalone tools, which makes it difficult to reproduce. Here, we present an R pipeline based on an updated version of CATALYST that covers all preprocessing steps required for downstream mass cytometry analysis in a fully reproducible way. This new version of CATALYST is based on Bioconductor's SingleCellExperiment class and fully unit tested. The R-based pipeline includes file concatenation, bead-based normalization, single-cell deconvolution, spillover compensation and live cell gating after debris and doublet removal. Importantly, this pipeline also includes different quality checks to assess machine sensitivity and staining performance while allowing also for batch correction. This pipeline is based on open source R packages and can be easily be adapted to different study designs. It therefore has the potential to significantly facilitate the work of CyTOF users while increasing the quality and reproducibility of data generated with this technology.
    Keywords:  Batch correction; Compensation; CyTOF; Debarcoding; Gating; Normalization; Preprocessing; Reproducibility
  25. Elife. 2022 09 05. pii: e78383. [Epub ahead of print]11
      Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.
    Keywords:  INPP5E; Joubert syndrome; biochemistry; cell biology; chemical biology; cilia; ciliopathies; none; phosphatase; phosphoinositides